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DOI : 10.2478/9788367405256-fm. CBS22. 2022. I- VIII

Proceeding of
1st INTERNATIONAL CONFERENCE ON CHIMICAL AND
BIOLOGICAL SCIENCES (CBS'22)
27th-28th May 2022, Istanbul, Turkey

I
II
Presentation

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acceptable, provided they fit in one of the following main topic areas:

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AREA 1: CHEMICAL SCIENCES

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III
  Surface Chemistry
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AREA 2: BIOLOGICAL SCIENCES

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V
 COMMITTEES
1st INTERNATIONAL CONFERENCE ON CHIMICAL AND BIOLOGICAL
SCIENCES (CBS'22)

General Chair

 BANACER Himmi, ISPITS-RABAT, Morocco

 JAMAL EDDINE Oumaima, Faculty of Sciences of Ben M’sick, Hassan II University,
Casablanca, Morocco
 DAGHOR Lamia, Private University of Marrakech, Marrakech, Morocco

INTERNATIONAL SCIENTIFIC COMMITTEE

 CHAKHCHAOUI Nabil, University of Sherbrooke, Sherbrooke, Canada
 FARHAN Rida, University of Sherbrooke, Sherbrooke, Canada
 François Xavier, Nshimiyimana University of Lay Adiventists of Kigali –Rwanda
 Hermann-Desire Nguessan Maximilien Lallie, Université Peleforo GON COULIBALY, Ivory
Coast
 KHALED Amina, Clermont Auvergne University, Clermont-Ferrand, France
 MEGARBANE Bruno Lariboisière Hospital Paris – France
 SALMI Louis Rachid University of Bordeaux, – France
 Sanou Khô Coulibaly Faculty of Medicine and Odontostomatology,Bamako, Mali
 Tidiane DIALLO Faculty of Pharmacy, Bamako, Mali
 Wafik Noseir, Modern Center, Egypt
 Sylvia Agostini, University of Corsica, France
 Tugba Topal,University of Usak, Turkey
 Seyda Cengiz,Atatürk University, Turkey
 Nese Aysit,Medipol University, Turkey
 Aslı Şalcıoğlu,Bosphorus University, Turkey
 Muhammad Safdar,Gaziantep University, Turkey
 Ozkan Cigdem,Eastern Mediterranean University, Turkey
 Sukru Torun,Anadolu University, Faculty of Health Sciences, Turkey
 Oznur Ozge Ozcan,Uskudar University, Turkey
 Allah Bakhsh,Nigde Omer Halisdemir University, Turkey
 Dineshen Chuckravanen,Necmettin Erbakan University Meram Faculty of Medicine,
Turkey
 Ozhan Simsek,Cukurova University, Turkey
 Çiğdem Gökçek Saraç,Akdeniz University, Turkey

VI
  Emre Aksoy,Omer Halisdemir University, Turkey
 Fatma Keskin Krzan,Üsküdar University, Turkey
 Manish Kohli, Division of Medical Oncology, Mayo Clinic, Rochester, USA
 Kifayat Ullah Shah, Quaid-i-Azam University,Islamabad
 Salman alfadhel King Abdullah, International Medical Reasearch Center,Saudi Arabia
 Leonard M. Klein, Illinois Cancer Specialists, United States
 Robin Jones, Sarcoma Clinic, Royal Marsden Hospital, UK
 Meenu Gupta, Swami Rama Himalayan University Dehradun, India
 BOYER Damien, University of Clermont Auvergne, France

NATIONAL SCIENTIFIC COMMITTEE

 ACHOUR Sanae, Faculty of Médecine and Pharmacie of Fès – Morocco
 Ait Mhand Rajae FST, Mohammedia, Morocco
 AMARDO Nadia, Faculty of Sciences of Ben M’sik, Hassan II University, Casablanca,
Morocco
 Amrousse Rachid, Chouaib doukkali University; Morocco
 Assia Kettani FMPC-Casablanca, Morocco
 AZELMAT Souad, Military Hospital of Instruction Mohamed 5, Morocco
 AZIDANE Hind, Ibn Tofail University, Kenitra, Morocco
 Badrane Narjis, Anti Poison and Pharmacovigilance Center Morocco
 BELOUAFA Soumia, Faculty of Sciences of Ben M’sik, Hassan II University, Casablanca,
Morocco
 BENAJI Brahim, National School of Arts and Crafts/Rabat – Morocco
 BENAZZOUZ BOUCHRA, University of Ibn Tofail – Morocco
 BENKIRANE HANSAA, University of Ibn Tofail – Morocco
 CHAFI HAFID, University of Mohamed 1er- Morocco
 CHAOUI Hanane, Anti Poison and Pharmacovigilance Center Morocco
 CHOULLI Mohamed Khaled, University of Caddi Ayyad Morocco
 Diawara Idrissa UM6SS, Casablanca, Morocco
 Dihaj Barguiga FS, Sultan Moulay slimane University, Benimellal
 ECHCHELH Adil, University of Ibn Tofail – Morocco
 EL BOUARI Abdeslam, Faculty of Sciences of Ben M’sik, Hassan II University, Casablanca,
Morocco
 Fatima Zahra Laasri, Faculty of Sciences of Kenitra, Ibn Tofail University, Kenitra,
Morocco
 FEKHAOUI Mohammed, Scientific Institute of Rabat – Morocco
 HADRYA FATINE, Université of Hassan 1 – Morocco
 HAMI Hinde, University of Ibn Tofail – Morocco
 HANNACHE Hassan, Faculty of Sciences of Ben M’sik, Hassan II University, Casablanca,
Morocco
 Himmi Banacer ISPITS-RABAT, Morocco
 Imane Barakate ISPITS-RABAT, Morocco
 JAMAL EDDINE Oumaima, Faculty of Sciences of Ben M’sik, Hassan II University,
Casablanca, Morocco

VII
 KAFSAOUI Sanaa, Military Hospital of Instruction Mohamed 5
 Kettani halabi mohamed UM6SS, Casablanca, Morocco
 Khassouani Chems Eddouha, Ministry of Health – Morocco
 KHATTABI Asmaa, National School of Public Health – Morocco
 MADHI YOUSSEF, Regional Center for Education and Training Professions Morrocco
 MERIMI Imane, University of Mohamed 1 Morocco
 MOKHTARI Abdelrhani, University of Ibn Tofail – Morocco
 Mouallif Moustapha ISSS, Hassan I University, Settat, Morocco
 MOUGHAOUI Fatiha, Faculty of Sciences of Ben M’sik, Hassan II University, Casablanca,
Morocco
 Nabil Khélifi, University of Kiel, Kiel, Germany
 OUBEJJA Houda, Faculty of Medicine and Pharmacy of Rabat – Morocco
 OUBLA M’hamed, Mohamed V University, Rabat, Morocco
 Oudghiri Amal, National Center for Energy, Nuclear Sciences and Techniques, Rabat,
Morocco
 Oudghiri Mouna FS-Ain chock, Casablanca, Morocco
 OUMAM Mina, Faculty of Sciences of Ben M’sik, Hassan II University, Casablanca,
Morocco
 Ouzir Mounir ISPITS-Benimellal, Morocco
 RHALEM Naima, Anti Poison and Pharmacovigilance Center Morocco
 RHARRABE Kacem, University of Abdelmalek Essaadi – Morocco
 RHAZI Naima, Faculty of Sciences of Ben M’sik, Hassan II University, Casablanca,
Morocco
 SEFFIANI Houda, Anti Poison and Pharmacovigilance Center Morocco
 SEFIANI MAJIDA, Université Moulay Ismail – Maroc
 Souad Skalli, Faculty of Sciences University Mohammed V Rabat Morocco
 SOULAYMANI Abdelmajid, University of Ibn Tofail – Morocco
 SOULAYMANI Rachida, Centre AntiPoison et de Pharmacovigilance Morocco
 Tebaa Amina, Anti Poison and Pharmacovigilance Center Morocco
 Ziad Abdelmajid FS, Sultan Moulay slimane University, Benimellal, Morocco

VIII
DOI : 10.2478/9788367405256-toc. CBS22. 2022. IX-X

Proceeding of
1st INTERNATIONAL CONFERENCE ON CHIMICAL AND
BIOLOGICAL SCIENCES (CBS'22)
27th-28th May 2022, Istanbul, Turkey

Contents
The impact of socio-demographic factors on access to treatment for patients with multiple sclerosis in
Beni Mellal-Khenifra, Morocco…………………………………………………………………………………………………………………1

Evaluation of Antibacterial Activity of the Filamentous Cyanobacterium Arthrospira Jenneri

Extracts……………………………………………………………………………………………………………………………………………………8

The Coordination of Care in the Management of Breast Cancer: a Qualitative Insight into Patients and
Healthcare Professionals’ Perspectives……………………………………………………..…………………………………………..15

Chitosan Effect on Fungal Strains Responsible for Strawberries Post-harvest

Losses……………………………………………………………………………………………………………………………………………………26
Phosphate Solubilizing Bacteria: Value and Response in Two Species of Papilionaceae
Seeds…………………………………………………………………………………………………………………………………………………….33

Comparison of automatic and manual methods for analyzing heart rate variability
parameters…………………………………….………………………………………………………………………………………………………39
Dietary Transition and Colorectal Cancer: A Case-Control Study of Dietary Factors in the Region
Marrakech-Safi, Morocco………………………………………………………..…………………………………………………………….45

Potability of drinking water in rural areas of Tiznit in southern Morocco…………………………………….………..52

The effect of physicochemical parameters and enzyme concentration on the clotting and cutting times
properties of Alpine breed goat's milk…………………………………………………………………………………………………..59

Effect of Cooking and Washing on Sulfites (SO2) Content of Dried

Fruits………………………………………………………………………………………………..……………………………………………………68

Citrus Polymethoxyflavones as Corrosion Inhibitor for Mild Steel in HCl

medium…………………………………………………………………………………………………………………………………………………73
Screening and risk factors for nasal carriage of Staphylococcus aureus in a sample of Moroccan
haemodialysis patients……….……………………………………………………………………………………………………………..….78

IX
Hormonal Contraception and Breast Cancer Among Women in the Region Marrakech-Safi,
Morocco………………………………………………………………………………………………………………………………………………..87

Contribution to the study of the acute and subacute toxicity of aqueous extract of Lawsonia Inermis L.
leaves in Wistar rats……………………………………………………………………………………………………………..………….……97

Photocatalytic degradation of dye using ZnO and 1%Al-Doped ZnO: a comparative investigation of
nanocatalysts properties andoperating conditions………………………………………………………………......……….106

Association of aldosterone synthase gene -344C/T polymorphism with type 2 diabetes in Moroccan
population……………………………………………………………………………………………………………………………………………113

Optimizing Fitness in Obesity: A Program Combining Various Exercise Techniques…………………………....121

Study of Didactic Obstacles Related to the Learning of Basic Concepts in Organic Chemistry: Case of
Undergraduate Teaching University Cycle……………………………………………………………………………………………134

The Contribution of Morocco to the Global Traumatic Brain Injury Research Study of Moroccan cumin
seeds (Cuminum cyminum L.) essential oils…………………………………………………………………….………………..…140

Study of Moroccan cumin seeds (Cuminum cyminum L.) essential oilsResults of an Entomological …….144

Results of an Entomological Survey on Phlebotomine Sand flies (Diptera: Psychodidae) Fauna of affected
area of leishmaniasis in central Morocco………………………………………………………………………………………….….149

Monitoring system for Acute Flaccid Paralysis in Children: the case of Kénitra Province, Morocco;
between 1994 and 2018……………………………………………………………………………………………………………………...157

Evaluation of dietary habits in type 2 diabetic subjects………………………………………………………………….…...165

First national training in public health emergency for medical students in Morocco: A mixed method
study……………………………………………………………………………………………………………………………………………………174

X
CONFERENCE PAPER Rachid Lotfi et al., 2022, 1-7

The impact of socio-demographic factors on access to treatment for patients

with multiple sclerosis in Beni Mellal-Khenifra, Morocco
Rachid Lotfi, Mohamed Najimi, and Fatiha Chigr
Biological Engineering laboratory, Faculty of Sciences and Techniques, Beni-Mellal, Morocco.

This study aims to determine the association between socio-demographic

factors and access to disease-modifying therapies (DMTs) for patients with
multiple sclerosis. This study concerned a sample of 125 patients
representing the five provinces of the Beni Mellal-Khenifra region. In this
study, we used the following methods: a survey through an anonymous
questionnaire intended and distributed individually to patients. The
collection of data through the admission registers and files of patients who
had a consultation with neurologists. The findings show that patients with
multiple sclerosis are characterized by female dominance (64.5%). In
addition, this study showed that patients with this disease are generally
young: their average age is 36.39 years. The results also showed that nearly
82.4% of patients are unemployed, more than 92 % have no medical
coverage, and 89.6% of the patients surveyed live without DMTs. There are
statistically significant associations between access to DMTs for patients
with multiple sclerosis and several socio-demographic factors, including
patient profession (p = 0,037), medical coverage (p ≤ 0.001), and sex (p =
0.025). Access to care is closely related to specific socio-demographic
factors such as medical coverage, patient occupation, and gender.

Corresponding Author
INTRODUCTION
Name: Rachid Lotfi
Email: r.lotfi@usms.ma Phone:
Multiple sclerosis is a potentially disabling,
212600695013
progressive chronic disease with considerable
ISBN: 9788367405256 social impact and economic consequences
(Béneton & Rumbach, 1999). It is an acquired
DOI: 10.2478/9788367405256-001 autoimmune, inflammatory, and
neurodegenerative disease of the central nervous
Published by Sciendo. © 2022 Rachid Lotfi et al. This is an
system. MS is characterized by inflammation,
open access article licensed under the Attribution 4.0
International demyelination, and primary or secondary axonal
(https://creativecommons.org/licenses/by/4.0/) degeneration (Hirsch, 2002; Lassmann, 2018;
Raymond et al., 2017). The findings showed a
significant association between inflammation and
neurodegeneration in all lesions and stages of MS
disease plaques (Frischer et al., 2009).

1
MS generally affects young people between 20 and
40 years old, with a median age of 32 (Charil et al.,
2006) and a female predominance. There are at
least twice as many females (Multiple Sclerosis
International Federation, 2020b). The reasons for
this predominance are unknown, but it may be
influenced by hormonal (Russi et al., 2018) and
genetic factors (Voskuhl et al., 2018). MS is the
most common cause of neurological disability in
young adults (Feigin et al., 2019), with
considerable social and economic consequences
affecting an estimated 2.8 million people
worldwide, or 1 in 3,000 people (Multiple
Sclerosis International Federation, 2020b). In
Morocco, according to the results of the 3rd
edition of the "Atlas of MS" published in 2020, the
prevalence rate was estimated at 20/100,000
(Atlas of MS, 2020).
We have conducted a descriptive study concerning
a sample of 125 persons with a definite diagnosis
of MS according to the prevailing diagnostic
criteria, representing the five provinces of the
Beni Mellal-Khenifra region (Figure 1).
Patients with MS provided information via a
questionnaire containing information on
sociodemographic characteristics: age, gender,
profession, marital status, school level, medical
coverage, province of residence, and access to
treatment. This questionnaire was developed
based on a validated questionnaire (H. Flemmen,
2019). Data collection was carried out through
admission registers inside public hospitals and the
study of the files of patients who had a
consultation with neurologists or had been
hospitalized.

In general, there is an association between the

health status of individuals and socioeconomic
status; individuals with higher socioeconomic
status have favorable health compared to those
with lower socioeconomic status (Mackenbach et
al., 2018). In addition, some studies showed an
association between low socioeconomic status
and a more accelerated disease progression to
disability (Calocer et al., 2020; Harding et al.,
2019).

DMTs are an essential element in MS management.

This class of medications is used to interrupt the
immune system’s attacks on the central nervous
system (Multiple Sclerosis Society, 2019).
According to, the Multiple Sclerosis International
Federation, there are significant international
differences in access to DMT (Multiple Sclerosis
International Federation, 2020a). Furthermore,
studies showed also the association between
access to DMT and specific sociodemographic
characteristics of patients, namely: sex, race,
education, employment, and health insurance (H.
Ø. Flemmen et al., 2022; Minden et al., 2008;
Poudel et al., 2021; Souliotis et al., 2015).
This study aims to determine the association
between socio-demographic factors and access to
disease-modifying therapies (DMTs) for patients
with multiple sclerosis in the region of Beni
Mellal-Khenifra (Morocco).
Figure 1: Distribution of the sample by
METHODS province

2
Furthermore, MS therapy is based on three types dependency between access to DMT and the
of treatments: acute relapse treatment, symptom patient's profession is significant (p=0.002)
treatment, and DMT. Our study analyzes patients' (table 2).
access to DTMs that slow down or stop these
attacks.
Table 1: Sociodemographic Characteristics
The development of the questionnaire and the
data analysis were carried out through SPHINX
PLUS2 version 5. We analyzed all variables Socio-demog
induced by our data, and the results are raphic No. of
%
presented as an odds ratio (OR) with a 95% Characterist Patients
confidence interval (CI) and a significance level ics
of 5%. The chi-square (χ2) test) was used to Total 125 100.0
determine the association between two
qualitative variables: sociodemographic Gender
characteristics (age, gender, profession, marital Men 45 36.0
status, school level, medical coverage, province
of residence, access to treatment) and access to Women 80 64.0
treatment). We use the Student and Fisher tests Age
to determine the association between a
qualitative variable, such as age, and a qualitative < 20 4 3.2
variable (sociodemographic characteristics), 20-29 35 28.0

We used ArcGIS version 10.1 to develop the map 30-39 47 37.6

containing the region that was the subject of our 40-49 20 16.0
study.
50 et plus 19 15.2
RESULTS Marital status
The obtained results indicate that the patients Married 50 41.3
with multiple sclerosis are characterized by Single 71 58.7
female dominance (64.5%). In addition, this
Medical coverage
study demonstrates that patients with this
disease are generally young: their average age is Yes 14 11.2
36.39 years. Moreover, the majority of our No 111 88.8
sample (over 84.8%) comprises age groups
Province
between 20 and 50 years old. Furthermore, the
Beni
results show that more than half (58.7%) are 40 32.0
Mellal
single, nearly 82.4% of patients are unemployed
Khouribga 29 32.2
and More than 88.8 % have no medical coverage
(table 1). Fquih Ben
27 21.6
Salah
More than 89.6% of patients do not have access Khenifra 23 18.4
to DMT: this result showed a statistically very Azilal 6 4.8
significant relationship between access to DMT
and Medical coverage (P≤0.001). Statistical Profession
analysis also shows a significant dependence Yes 22 12
between access to DMT and gender (p=0.025): No 103 88
15% of female patients have access to DMT
against 2.2% of males. In addition, the
3
 Table 2: sociodemographic characteristics and access to treatment

Socio-demographic Access to treatment

characteristics Yes (No. of Patients) No ( No. of Patients ) p-value
Gender
Men 1 44
p =0.025
Women 12 68
Âge
< 20 0 4
20-29 8 27
30-39 3 44 p=0.082
40-49 1 19
>50 1 18
Marital status
Married 3 47
p=0.157
Single 10 61
Province
Beni Mellal 6 34
Khouribga 2 27
Fquih Ben Salah 2 25 p=0.211
Khenifra 1 22
Azilal 2 4
Medical coverage
Yes 11 03
p ≤ 0.001
No 1 110
Profession
Yes 7 15
p=0.002
No 8 95

DISCUSSION
Another study found that 70-80% of adults with
The surveyed multiple sclerosis patients are multiple sclerosis are unemployed after their
characterized by female dominance (64.5%). diagnosis (Kornblith et al., 1986; Roessler &
These results are coherent with the outcomes of Rumrill, 2003) about 40% would like to return to
the 2020 report published by the International work (Larocca et al., 1985).
Multiple Sclerosis Federation, in which the
Our study showed that 88 % of patients are
percentage of females affected is 69% (Multiple
unable to access DMT. This result is consistent
Sclerosis International Federation, 2020b). In
with data reported in 2020 by the Multiple
addition, this study showed that patients with
Sclerosis International Federation, which showed
this disease are generally young, with an average
that 72% of countries cite barriers to DMT access.
age fixed at 36.39 years. Furthermore, our
Thus, inequality is a reality revealed by other
research revealed that almost 82.4 % of the
studies (Browne et al., 2014). In this sense, an
patients surveyed are unemployed. The
American study showed that a large proportion of
association between multiple sclerosis and
MS patients find it difficult to access expensive
professional status has been discovered by
DMT (Iezzoni et al., 2008). The high cost of drugs
several studies (Cambier-Langrand et al., 2016;
is a barrier to accessing treatment (Wang et al.,
Douma et al., 2018). In this sense, a
2016). And this is mainly due to the cost of these
cross-sectional study of 73 patients reports that
drugs for the government, the health system, or
69.7% either stopped working or never worked
medical insurance (Multiple Sclerosis
(Douma et al., 2018).
4
International Federation, 2020a). Regarding this
last factor, our study revealed that more than
79.2% do not have medical insurance to cover the
treatment costs.
In our study, we analyzed the association between
access to DMT and specific socio-demographic
characteristics related to the individual:
profession, educational level, gender, medical
coverage, age, marital status, and those related to
the province of belonging. We found differences
in access to DMT according to medical coverage
(P<0.001), gender (p=0.025), and profession
(p=0.002). In this context, a previous study
showed that 54% of families with MS patients
suffer from tragic health expenses (Gharibi et al.,
2021). Another study of a sample of 507 found
that nearly half of the respondents had difficulties
in getting a DMT in the past, and this was due to
several factors such as insurance company
authorization requirements (Simacek et al.,
2018).
Furthermore, socioeconomic factors may
indirectly influence access to DMT, as people from
less deprived areas are more likely to receive
early treatment, making their disease less severe
through early diagnosis (Owens et al., 2013).
Another study showed that a lower range of
available DMT is related to older age and lower
socioeconomic status (Roddam et al., 2019). In
the same sense, patients' consent to pay for their
DMT is influenced by several socio-demographic
characteristics namely: sex, race, education,
employment, and health insurance (Poudel et al.,
2021). Additionally, the probability of seeing a
neurologist is negatively associated with specific
sociodemographic characteristics such as lack of
health insurance, poverty, and rural location
(Minden et al., 2008). Also, Among the obstacles
preventing patients from gaining access to DMT
are the distance from pharmacies and the
difficulties in attaining a diagnosis from a doctor
(Souliotis et al., 2015).
Thus, access to DMT is associated with medical
coverage. But, even with medical coverage, a US
study indicated that many people with MS find it
hard to access specialists (Minden et al., 2007).
However, a recent study on a cohort of Norwegian
patients with MS showed that these patients are
treated globally equally with DMT in terms of
socioeconomic position (H. Ø. Flemmen et al.,
2022).
Finally, socio-demographic and economic factors
are not the only ones that determine access to
DMT. The latter may be influenced by other
factors, namely comorbidity, relapse rate, and
MRI findings. In addition, access to treatment may
be influenced by the health status of patients
(Iezzoni et al., 2008). Thus, determining the
factors that influence access to DTMs requires a
global and multidisciplinary study.
CONCLUSION
The present study showed that most patients who
have multiple sclerosis face many defies to access
to DMT. These difficulties are related to specific
socio-demographic factors such as gender,
professional activity, and medical coverage. A
generalization of “medical coverage” is one of the
solutions to improve access to DMT for this
category of patients.
ACKNOWLEDGMENTS
We are honored to extend our special thanks to
all members of the Moroccan Federation Union of
Multiple Sclerosis associations, without forgetting
other active associations in this field, for their
support in data collection.
REFERENCES
Atlas of MS. (2020). Morocco—Atlas of MS.
https://www.atlasofms.org/fact-sheet/morocco
Béneton, C., & Rumbach, L. (1999). [Social and
economic impact of multiple sclerosis]. La Revue
Du Praticien, 49(17), 1890‑1893.
Browne, P., Chandraratna, D., Angood, C., Tremlett,
H., Baker, C., Taylor, B. V., & Thompson, A. J.
(2014). Atlas of multiple sclerosis 2013 : A
growing global problem with widespread
inequity. Neurology, 83(11), 1022‑1024. Scopus.
https://doi.org/10.1212/WNL.00000000000007
68
Calocer, F., Dejardin, O., Kwiatkowski, A., Bourre, B.,
Vermersch, P., Hautecoeur, P., Launoy, G., & Defer,
G. (2020). Socioeconomic deprivation increases
the risk of disability in multiple sclerosis patients.
Multiple Sclerosis and Related Disorders, 40,
101930.
https://doi.org/10.1016/j.msard.2020.101930
Cambier-Langrand, E., Leroyer, A., Zéphir, H.,
Fernandes, C., Jougleux, C., Cabaret, M.,
Vermersch, P., & Fantoni-Quinton, S. (2016).
Impact socioprofessionnel précoce de la sclérose
en plaques : Une étude prospective d’une série de
5
 24 patients. Archives des Maladies Professionnelles
et de l’Environnement, 77(2), 165‑178.
https://doi.org/10.1016/j.admp.2015.09.004
Charil, A., Yousry, T. A., Rovaris, M., Barkhof, F.,
Stefano, N. D., Fazekas, F., Miller, D. H., Montalban,
X., Simon, J. H., Polman, C., & Filippi, M. (2006).
MRI and the diagnosis of multiple sclerosis :
Expanding the concept of “no better explanation”.
12.
Douma, R., Hassine, A., Zouari, N., Ben Amor, S.,
Salma, N., & Soufien, B. A. (2018). Statut
professionnel au cours de la sclérose en plaques.
Revue Neurologique, 174, S103.
https://doi.org/10.1016/j.neurol.2018.01.233
Feigin, V. L., Nichols, E., Alam, T., Bannick, M. S.,
Beghi, E., Blake, N., Culpepper, W. J., Dorsey, E. R.,
Elbaz, A., Ellenbogen, R. G., Fisher, J. L.,
Fitzmaurice, C., Giussani, G., Glennie, L., James, S.
L., Johnson, C. O., Kassebaum, N. J., Logroscino, G.,
Marin, B., … Vos, T. (2019). Global, regional, and
national burden of neurological disorders,
1990–2016 : A systematic analysis for the Global
Burden of Disease Study 2016. The Lancet
Neurology, 18(5), 459‑480.
https://doi.org/10.1016/S1474-4422(18)30499-
X Research.
Flemmen, H. (2019). Socioeconomic factors as
predictors for ms susceptibility and disease
progression—Validation of a new norwegian
questionnaire.
https://onlinelibrary.ectrims-congress.eu/ectrim
s/2019/stockholm/279125/
Flemmen, H. Ø., Simonsen, C. S., Broch, L.,
Brunborg, C., Berg-Hansen, P., Moen, S. M.,
Kersten, H., & Celius, E. G. (2022). The influence of
socioeconomic factors on access to disease
modifying treatment in a Norwegian multiple
sclerosis cohort. Multiple Sclerosis and Related
Disorders, 61.
https://doi.org/10.1016/j.msard.2022.103759
Frischer, J. M., Bramow, S., Dal-Bianco, A.,
Lucchinetti, C. F., Rauschka, H., Schmidbauer, M.,
Laursen, H., Sorensen, P. S., & Lassmann, H.
(2009). The relation between inflammation and
neurodegeneration in multiple sclerosis brains.
Brain: A Journal of Neurology, 132(Pt 5),
1175‑1189.
https://doi.org/10.1093/brain/awp070
Gharibi, F., Imani, A., & Koustuv, D. (2021). The
catastrophic out-of-pocket health expenditure of
multiple sclerosis patients in Iran. BMC Health
Services Research, 21(1).
https://doi.org/10.1186/s12913-021-06251-4
Harding, K. E., Wardle, M., Carruthers, R.,
Robertson, N., Zhu, F., Kingwell, E., & Tremlett, H.
(2019). Socioeconomic status and disability
progression in multiple sclerosis : A multinational
study. Neurology, 92(13), e1497‑e1506.
https://doi.org/10.1212/WNL.00000000000071
90
Hirsch, E. C. (2002). Inflammatory Changes and
Apoptosis in Parkinson’s Disease. In Y. Mizuno, A.
Fisher, & I. Hanin (Éds.), Mapping the Progress of
Alzheimer’s and Parkinson’s Disease (p. 259‑263).
Springer US.
https://doi.org/10.1007/978-0-306-47593-1_43
Iezzoni, L. I., Ngo, L. H., & Kinkel, R. P. (2008).
Working-age persons with multiple sclerosis and
access to disease-modifying medications. Multiple
Sclerosis Journal, 14(1), 112‑122.
https://doi.org/10.1177/1352458507080466
Kornblith, A., La Rocca, N., & Baum, H. (1986).
Employment in individuals with multiple
sclerosis. International Journal of Rehabilitation
Internationale Zeitschrift Fur
Rehabilitationsforschung. Revue Internationale de
Recherches de Readaptation, 9(2).
https://doi.org/10.1097/00004356-198606000-
00006
Larocca, N., Kalb, R., Scheinberg, L., & Kendall, P.
(1985). Factors associated with unemployment of
patients with multiple sclerosis. Journal of
Chronic Diseases, 38(2), 203‑210.
https://doi.org/10.1016/0021-9681(85)90093-1
Lassmann, H. (2018). Multiple Sclerosis Pathology.
Cold Spring Harbor Laboratory Press.
Mackenbach, J. P., Valverde, J. R., Artnik, B., Bopp,
M., Brønnum-Hansen, H., Deboosere, P., Kalediene,
R., Kovács, K., Leinsalu, M., Martikainen, P.,
Menvielle, G., Regidor, E., Rychtaříková, J.,
Rodriguez-Sanz, M., Vineis, P., White, C.,
Wojtyniak, B., Hu, Y., & Nusselder, W. J. (2018).
Trends in health inequalities in 27 European
countries. Proceedings of the National Academy of
Sciences, 115(25), 6440‑6445.
https://doi.org/10.1073/pnas.1800028115
Minden, S. L., Frankel, D., Hadden, L., & Hoaglin, D.
C. (2007). Access to health care for people with
multiple sclerosis. Multiple Sclerosis (Houndmills,
6
 Basingstoke, England), 13(4), 547‑558.
https://doi.org/10.1177/1352458506071306
Minden, S. L., Hoaglin, D. C., Hadden, L., Frankel, D.,
Robbins, T., & Perloff, J. (2008). Access to and
utilization of neurologists by people with multiple
sclerosis. Neurology, 70(13 Part 2), 1141‑1149.
https://doi.org/10.1212/01.wnl.0000306411.46
934.ef
Multiple Sclerosis International Federation.
(2020a). Clinical management of multiple sclerosis
around the world, key finding about the diagnosis
and clinical management of MS (3rd Edition; p.
45).
Multiple Sclerosis International Federation.
(2020b). Mapping multiple sclerosis around the
world key epidemiology findings (3rd Edition).
Multiple Sclerosis Society. (2019). Disease
modifying therapies booklet.
https://www.mssociety.org.uk/care-and-support
/resources-and-publications/publications-search
/disease-modifying-therapies-booklet
Owens, T., Evangelou, N., & Whynes, D. K. (2013).
Rationing and deprivation : Disease-modifying
therapies for multiple sclerosis in the United
Kingdom. The European Journal of Health
Economics, 14(2), 315‑321.
https://doi.org/10.1007/s10198-012-0378-7
Poudel, N., Banjara, B., Kamau, S., Frost, N., &
Ngorsuraches, S. (2021). Factors influencing
patients’ willingness-to-pay for
disease-modifying therapies for multiple
sclerosis. Multiple Sclerosis and Related Disorders,
48, 102720.
https://doi.org/10.1016/j.msard.2020.102720
Raymond, M. J., Ray, P., Kaur, G., Fredericks, M.,
Singh, A. V., & Wan, L. Q. (2017). Multiaxial
Polarity Determines Individual Cellular and
Nuclear Chirality. Cellular and Molecular
Bioengineering, 10(1), 63‑74.
https://doi.org/10.1007/s12195-016-0467-2
Roddam, H., Rog, D., Janssen, J., Wilson, N., Cross, L.,
Olajide, O., & Dey, P. (2019). Inequalities in access
to health and social care among adults with
multiple sclerosis : A scoping review of the
literature. Multiple Sclerosis and Related Disorders,
28, 290‑304.
https://doi.org/10.1016/j.msard.2018.12.043
Roessler, R. T., & Rumrill, P. D. (2003). Multiple
sclerosis and employment barriers : A systemic
perspective on diagnosis and intervention. Work
(Reading, Mass.), 21(1), 17‑23.
Russi, A. E., Ebel, M. E., Yang, Y., & Brown, M. A.
(2018). Male-specific IL-33 expression regulates
sex-dimorphic EAE susceptibility. Proceedings of
the National Academy of Sciences, 115(7),
E1520‑E1529.
https://doi.org/10.1073/pnas.1710401115
Simacek, K. F., Ko, J. J., Moreton, D., Varga, S.,
Johnson, K., & Katic, B. J. (2018). The Impact of
Disease-Modifying Therapy Access Barriers on
People With Multiple Sclerosis : Mixed-Methods
Study. Journal of Medical Internet Research,
20(10), e11168. https://doi.org/10.2196/11168
Souliotis, K., Alexopoulou, E., Papageorgiou, M.,
Politi, A., Litsa, P., & ContiadesX, X. (2015). Access
to Care for Multiple Sclerosis in Times of
Economic Crisis in Greece—The HOPE II Study.
International Journal of Health Policy and
Management, 5(2).
https://doi.org/10.15171/ijhpm.2015.173
Voskuhl, R. R., Sawalha, A. H., & Itoh, Y. (2018). Sex
chromosome contributions to sex differences in
multiple sclerosis susceptibility and progression.
Multiple Sclerosis Journal, 24(1), 22‑31.
https://doi.org/10.1177/1352458517737394
Wang, G., Marrie, R. A., Salter, A. R., Fox, R., Cofield,
S. S., Tyry, T., & Cutter, G. R. (2016). Health
insurance affects the use of disease-modifying
therapy in multiple sclerosis. Neurology, 87(4),
365‑374. Scopus.
https://doi.org/10.1212/WNL.00000000000028
87
7
 CONFERENCE PAPER FASSI FIHRI Reda et al., 2022, 8-14

Evaluation of Antibacterial Activity of the Filamentous Cyanobacterium

Arthrospira Jenneri Extracts
FASSI FIHRI Reda*1 , SLIMANI Chaimae1, RAIS Chaimae2, RACHIQ Saad1, EL GHADRAOUI Lahsen1, and
BOUCHAMMA El Ouazna1

1
Functional Ecology and Environment Engineering Laboratory, Faculty of Science and Technology, Sidi
Mohamed Ben Abdellah University, Imouzzer street, B.P. 2202, Fez, Morocco
2
Laboratory of Botany, National Agency of Medicinal and Aromatic Plants, 159 Taounate Principale,
Taounate 34000, Morocco

Antibioresistance is one of the major arising issues related to healthcare

globally. This study evaluated the antibacterial potential of a cyanobacterium
Arthrospira Jenneri isolated from a eutrophic artificial reservoir (Morocco).
Ethanolic, methanolic, and hexane microalgal extracts were obtained,
analyzed, and tested. Target organisms were both Gram-negative
(Pseudomonas aeruginosa and Escherichia coli) and Gram-positive (Bacillus
subtillis and Staphylococcus aureus) human pathogenic bacterial strains. To
evaluate the antibacterial activity, we used the broth microdilution method.
The results obtained reveal that Arthrospira Jenneri has a minimum
inhibitory concentration activity against Bacillus subtillis and Staphylococcus
aureus for all tested extracts. In contrast, the hexane extract showed the
minimum bactericidal concentration against Pseudomonas aeruginosa,
Bacillus subtillis, and Escherichia coli. These results emphasize the potential
use of microalgae extracts as a source of new antibiotic compounds.

Corresponding Author
Name: Fassi Fihri Reda
Email: Reda.Fassifihri@usmba.ac.ma
Phone: +212 633 97 91 91

ISBN : 9788367405256
DOI: 10.2478/9788367405256-002
Published by Sciendo. © 2022 FASSI FIHRI Reda et al. This
is an open access article licensed under the Attribution 4.0
International
(https://creativecommons.org/licenses/by/4.0/)

1
● https://orcid.org/0000-0001-7156-5680

8
INTRODUCTION
Antibiotic resistance in bacteria is one of the
most crucial developing healthcare challenges,
and it has emerged as the most severe problem
when it comes to resistant pathogenic bacteria
(Sieradzki et al., 1999). Discovering novel
antimicrobial agents is one of the approaches to
combating antibiotic bioresistance. (Desbois et
al., 2008, 2009).
Microalgae are a valuable potential source of
newer active compounds since they grow fast,
have a short production period, and are
environmentally friendly and renewable
(Lauritano et al., 2016). Amino acids, aromatic
organic acids, alcohols, phenolic compounds,
hydroquinones, lipids, pigments, polysaccharides,
sterols, and terpenes, among other active
substances, have been identified in microalgae.
(Marrez et al., 2019; Vikneshan et al., 2020;
Shaima et al., 2022). These compounds have been
linked to a range of pharmacological activities,
including antimicrobial, antioxidant, antiviral,
antitumor, anti-inflammatory, and antiallergic
(Lauritano et al., 2016; Shannon et al., 2016).
Because of their vast biodiversity and high
development rate, cyanobacteria have numerous
advantages (Abdel-Hameed, 2013). Furthermore,
these ubiquitous microbes are known for their
ecological plasticity and extraordinary
adaptability, having colonized the Earth for more
than 3.5 billion years (Büdel, 1999; Douglas,
MATERIAL AND METHODS
Isolation and culture of the studied strain
The cyanobacteria Arthrospira Jenneri used in this
work was sampled from the eutrophic artificial
reservoir SWANI (Fez-Meknes, Morocco). In all
experiments, we used a monospecific culture of
Arthrospira Jenneri, isolated and purified on Petri
dishes, followed by successive subcultures under
aseptic and controlled heliothermic conditions in
the laboratory of Functional Ecology and
Environmental Engineering of the Faculty of
Sciences and Techniques of Fez.
The medium used for the culture is the "Dauta
medium", entirely mineral, which considerably
reduces the risks of bacterial contamination
(Dauta, 1982).
Table 1. Culture medium’s chemical composition
(Dauta, 1982).
Chemical Concentration
Composition (mg. l-1)
Base medium MgSO4, 25
7H2O 1 the cultures was ensured by sterile compressed
FeSO4, 7H2O 25
CaCl2, 2H2O 1 purity of the microalgae colony was checked
Na2EDTA 50
2011; Lacap et al., 2011). The latter is reflected in
producing a wide variety of metabolites that can
have algicidal, antibiotic, cytotoxic, and
immunosuppressive properties in response to
their surrounding environment (Trabelsi et al.,
2010), ranging from low molecular weight
peptides to heavy lipopolysaccharides.
Furthermore, apart from the substances
mentioned above, cyanobacteria produce
cyanotoxins mainly obtained during the "bloom"
period when cell lysis occurs (Onderka, 2007).
The antibacterial activity of Arthrospira jenneri
has received comparatively with other microalgae
little attention. On the other hand, species in the
Arthrospira genus, particularly (Fox, 1996).
Several publishers have already evaluated
Arthrospira sp.’s pharmacological activities, such
as antibacterial activity (Ozdemir et al.,
2004). Screening for nonribosomal
peptides NRPs- and Polyketides PKs encoding
genes was used to assess the potential toxicity of
commercial Arthrospira strains, and none of the
Spirulina strains tested were positive for NRPs or
PKs genes, indicating that these strains do not
produce toxins (Kwei et al., 2011).
This work aims to evaluate the antibacterial
activity of hexane, methanolic and ethanolic
extracts of cyanobacterium Arthrospira Jenneri
against Gram-negative (Pseudomonas aeruginosa
and Escherichia coli) and Gram-positive (Bacillus
subtillis and Staphylococcus aureus) bacteria.
NaHCO3 5
Na2CO3
Trace ZnSO4, 7H2O 0.020
elements CuCl2, 2H2O 0.020
MnCl2, 4H2O 0.400
CoCl2, 6H2O 0.010
BO3H3 0.001
Na2MoO4 0.035
nutrients KNO3 200
K2HPO4 25
The experimental culture was maintained in the
exponential phase of growth by transplanting
every 2 to 3 days in an air-conditioned growth
chamber under suboptimal conditions of
temperature (25°C ± 1°C) and was subjected to
continuous illumination (24/24), provided by
fluorescent tubes (Philips TL-D 18W Snow
White 12000K). An ongoing homogenization of
air enriched with CO2 (1%). In addition, the
daily by optical microscopy.
9
 that inhibits microbial growth. For its
determination, the broth microdilution was
performed. The microplates were incubated at
37°C for 24 h.To reveal the microbial development,
10 µl of resazurin dye was applied after incubation.
(Abrini, 2006).

Determination of the MBC

3 µl of negative wells were poured onto the surface
of an LB agar plate and incubated at 37°C for 24
hours to determine the minimal bactericidal
concentrations (MBC). Following incubation, the
MBC was found to be the lowest concentration,
resulting in an evident negative growth. The
MBC/MIC ratio was determined to emphasize the
nature of the extracts' antimicrobial effect.
Figure 1 : Microscopic observation of
Arthrospira Jenneri. (Inverted microscope (Levison, 2004).
OLYMPUS IMT2) (× 400).
RESULTS
Extraction
MIC and MBC values of extracts against different
The culture was centrifuged at 5000 rpm for 20 bacterial strains studied are given in table 2. It was
minutes, and then the pellet was dried in an oven at found that the MIC obtained from methanolic,
(30°C) to a constant weight and used to extract ethanolic, and hexane extracts was 1.5 mg/mL
antimicrobial agents. One gram of dried biomass was against Staphylococcus aureus and Bacillus subtillis.
added to 10 ml of each of the following solvents
Also, it was found that CMB/MIC ratio for hexane
(Hexane, ethanol, and methanol), then incubated
overnight in the dark under stirring at room extract was less or equal to 1.
temperature. After 24h, the sample was centrifuged
at 5000rpm for 1min. Next, the supernatant was Therefore, this extract had a bactericidal
concentrated and dried in a rotary evaporator. The effect on B. subtillis, Pseudomonas
concentrated extracts were stored in vials, weighed, aeruginosa, and Escherichia coli.
and left to dry in a desiccator until they reached a
constant weight. All excerpts were stored at -4°C (Val Table 2. Antibacterial parameters of ethanolic,
et al., 2001 ; Alsenani et al., 2020; LewisOscar, et al., methanolic, and hexane extract of Arthrospira
2021).
jenneri for the four microbial strains tested.
Antibacterial activity
Methanolic extract
Bacterial strains MI MBC MBC/MIC Read
Two Gram-negative bacteria (Escherichia coli C
(ATCC 25922) and Pseudomonas aeruginosa (ATCC S. aureus 1,5 >1,5 NA NA
27853)) and two Gram-positive bacteria B. subtillis 1,5 >1,5 NA NA
(Staphylococcus aureus (ATCC 29213), Bacillus
subtillis (ATCC 3366)) were used for the P. NA NA NA NA
antibacterial activity test. aeruginosa
E. coli NA NA NA NA
Bacterial suspensions
Ethanolic extract
One to two colonies were transferred in sterile
MIC MBC MBC/MI Read
saline solution from a 24h old pure agar culture for
C
each strain. After that, the solutions were vortexed,
and their turbidity was set to 0.5°. McFarland. S. aureus 1,5 >1,5 NA NA
B. subtillis 1,5 >1,5 NA NA
Determination of the MIC P. NA NA NA NA
The minimal inhibitory concentration (MIC) was aeruginosa
defined as the lowest concentration of the extract E. coli NA NA NA NA

10
 Hexane extract were recorded by Senthil et al. in 2013, who
MIC MBC MBC/MI Read demonstrated that hexane extract of Chroococcus
C turgidus manifests significant inhibition against
S. aureus 1,5 >1,5 NA NA Escherichia coli, followed by ethanol extract of
B. subtillis 1,5 1,5 1 Bacteric Skeletonema costatum against Streptococcus
P. 1,5 1,5 1 Bacteric pyogenes. Moreover, the Cyanobacteria Phormidium
aeruginosa fragile methanolic extract showed promising
E. coli 1,5 1,5 1 Bacteric antibacterial activity against pseudomonas
Notes: NA (Not Active); (MIC and MBC (mg/ml)) aeruginosa and Staphylococcus aureus (Kumar et al.,
2013).
Discussion
The antibacterial activity has been identified in
Many investigations have focused on the active other microalgae. The study was done by Sushanth,
compounds released by cyanobacteria as a and Rajashekhar (Sushanth & Rajashekhar, 2015)
possible source of novel pharmacological and on the ethanolic, methanolic, and hexane extracts
medicinal products in the recent decade. The of four marine microalgae showed effective
antibacterial activity of Arthrospira jenneri have inhibitory activity against Staphylococcus aureus,
received comparatively little attention. Species Streptococcus pyogenes, and Bacillus subtilis.
belonging to the genus Arthrospira, particularly Furthermore. Olfat highlighted the antimicrobial
Arthrospira platensis, generally known as activity using extracts of cyanobacteria and algal
Spirulina, on the other hand, have been species. The methanol extract was more effective
extensively utilized on an industrial scale. (Fox, against Bacillus subtilis, Klebsiella pneumonia, and
1996). Staphylococcus aureus (Salem, 2014). As a result,
The present study indicated that the these findings were consistent with Prakash's work
antibacterial properties of Arthrospira jenneri on Oscillatoria sancta and Lyngbya birgei's
against the selected strains of bacteria vary antibacterial activity against Staphylococcus aureus
depending upon the organic solvent used for (Prakash et al., 2011). In methanol extracts,
extraction. Our results showed that methanolic Scenedesmus sp. showed antibacterial action
and ethanolic extract of Arthrospira jenneri against Staphylococcus aureus (Catarina Guedes et
revealed minimal inhibitory activity at al., 2011). Scenedesmus and Chlorella
1.5mg/ml against demonstrated antibacterial action against Bacillus
Staphylococcus aureus and Bacillus subtillis. At the subtilis and Staphylococcus aureus as well
same time, Pseudomonas aeruginosa and (Ördög et al., 2004 ; Ghasemi, et al., 2004 ;
Escherichia coli proved to be utterly resistant to Desbois et al., 2009). Staphylococcus aureus,
these extracts. On the other hand, the hexane Bacillus subtilis, and Klebsiella pneumoniae were
extract showed minimal inhibitory activity against all impacted by Nostoc, Microcystis, and
all the tested strains with a concentration of Oscillatoria methanol extract (Salem, 2014).
1,5mg/ml and a bactericidal activity with the same The presence or absence of antibacterial
concentration against Pseudomonas aeruginosa, activities and their intensity depend on the age
Escherichia coli, and Bacillus subtillis. Therefore, of the culture and, therefore, on the producing
this solvent showed only a bacteriostatic activity species' physiological state. Indeed, the maxima
against Staphylococcus aureus. It has been reported of metabolites synthesis are associated with the
that the compounds responsible for the stationary growth phase. This confirms that
antibacterial action seem likely to be the phenolic these compounds are secondary metabolites,
diterpenoids, which are the main compounds of the synthesized in case of stress to play a defensive
apolar fraction of microalgae extracts role, and would have no direct implication in the
(Fernández-López et al., 2005). These compounds reproduction and growth of the species
are highly lipophilic by nature and, therefore, (Trabelsi et al., 2010).
extracted by low polarity solvents such as hexane
(Albano & Miguel, 2011). This could explain the CONCLUSIONS
modest activity of methanolic and ethanolic The present study emphasizes the in vitro
extracts towards the tested bacteria. Similar results antibacterial properties of Arthrospira Jenneri.

11
The MIC obtained from methanol, ethanol, and
hexane extracts was 1.5 mg/mL against
Staphylococcus aureus and Bacillus subtillis.
Also, CMB/MIC ratio for hexane extract was less
or equal to 1. Therefore, this extract had a
bactericidal effect on Bacillus subtillis,
Pseudomonas aeruginosa, and Escherichia coli. It
concludes that this strain is a potential source of
new bioactive compounds, especially in the
pharmaceutical field. The characterization of
the different secondary metabolites needs to be
verified and refined in the different phases of
growth and under different stress conditions to
broaden the field of study concerning various
biological activities of Arthrospira jenneri.

12
ACKNOWLEDGEMENTS
The National Agency of Medicinal and Aromatic
Plants (ANPMA) and its staff are enormously
appreciated for their contribution to this work.
REFERENCES
Abdel-Hameed, M. (2013). Isolation and
characterization of antimicrobial active
compounds from the cyanobacterium Nostoc
commune Vauch. Journal of Pure and
Applied Microbiology.
Abrini, J. (2006). Thymus essential oils: Chemical
composition and in vitro antioxidant and
antibacterial activities. Congrs International
de Biochimie, 324.
Albano, S. M., & Miguel, M. G. (2011). Biological
activities of extracts of plants grown in
Portugal. Industrial Crops and Products,
33(2), 338–343.
https://doi.org/10.1016/j.indcrop.2010.11.
012
Alsenani, F., Tupally, K. R., Chua, E. T., Eltanahy, E.,
Alsufyani, H., Parekh, H. S., & Schenk, P. M.
(2020). Evaluation of microalgae and
cyanobacteria as potential sources of
antimicrobial compounds. Saudi
Pharmaceutical Journal, 28(12), 1834–1841.
https://doi.org/10.1016/j.jsps.2020.11.010
Büdel, B. (1999). Ecology and diversity of
rock-inhabiting cyanobacteria in tropical
regions. European Journal of Phycology,
34(4), 361–370.
https://doi.org/10.1080/09670269910001
736422
Catarina Guedes, A., Barbosa, C. R., Amaro, H. M.,
Pereira, C. I., & Xavier Malcata, F. (2011).
Microalgal and cyanobacterial cell extracts
for use as natural antibacterial additives
against food pathogens. International
Journal of Food Science & Technology, 46(4),
862–870.
https://doi.org/10.1111/j.1365-2621.2011.
02567.x
Dauta, A. (1982). Conditions de développement du
phytoplancton. Etude comparative du
comportement de huit espèces en culture. I.
Détermination des paramètres de croissance
en fonction de la lumière et de la
température. Annales de Limnologie, 18(3),
217–262.
https://doi.org/10.1051/limn/1982005
Desbois, A. P., Lebl, T., Yan, L., & Smith, V. J. (2008).
Isolation and structural characterisation of
two antibacterial free fatty acids from the
marine diatom, Phaeodactylum tricornutum.
Applied Microbiology and Biotechnology,
81(4), 755–764.
https://doi.org/10.1007/s00253-008-1714-
9
Desbois, A. P., Mearns-Spragg, A., & Smith, V. J.
(2009). A Fatty Acid from the Diatom
Phaeodactylum tricornutum is Antibacterial
Against Diverse Bacteria Including
Multi-resistant Staphylococcus aureus
(MRSA). Marine Biotechnology, 11(1),
45–52.
https://doi.org/10.1007/s10126-008-9118-
5
Douglas, S. (2011). Effects of alkaline earth metal
ions on the growth of Calothrix strain RC3, a
natural isolate from Rock Creek, British
Columbia. Canadian Journal of Microbiology.
https://doi.org/10.1139/w97-132
Fernández-López, J., Zhi, N., Aleson-Carbonell, L.,
Pérez-Alvarez, J. A., & Kuri, V. (2005).
Antioxidant and antibacterial activities of
natural extracts: Application in beef
meatballs. Meat Science, 69(3), 371–380.
https://doi.org/10.1016/j.meatsci.2004.08.
004
Fox, R. D. (1996). Spirulina: Production & potential.
Edisud.
http://catalog.hathitrust.org/api/volumes/o
clc/36758643.html
Ghasemi, Y., Yazdi, M. T., Shafiee, A., Amini, M.,
Shokravi, S., & Zarrini, G. (2004). Parsiguine,
A Novel Antimicrobial Substance from
Fischerella ambigua. Pharmaceutical
Biology, 42(4–5), 318–322.
https://doi.org/10.1080/13880200490511
918
Kumar, N. S. S., Sivasubramanian, V., & Mukund, S.
(2013). Antimicrobial and Antifungal activity
of extracts of Phormidium fragile Gomont. 6.
Kwei, K. A., Shain, A. H., Bair, R., Montgomery, K.,
Karikari, C. A., Rijn, M. van de, Hidalgo, M.,
Maitra, A., Bashyam, M. D., & Pollack, J. R.
(2011). SMURF1 Amplification Promotes
Invasiveness in Pancreatic Cancer. PLOS
ONE, 6(8), e23924.
https://doi.org/10.1371/journal.pone.0023
924
Lacap, D. C., Smith, G. J. D., Warren-Rhodes, K., &
Pointing, S. B. (2011). Community structure
of free-floating filamentous cyanobacterial
mats from the Wonder Lake geothermal
springs in the Philippines. Canadian Journal
of Microbiology.
https://doi.org/10.1139/w05-038
Lauritano, C., Andersen, J. H., Hansen, E., Albrigtsen,
M., Escalera, L., Esposito, F., Helland, K.,
Hanssen, K. Ø., Romano, G., & Ianora, A.
(2016). Bioactivity Screening of Microalgae
for Antioxidant, Anti-Inflammatory,
13
 Anticancer, Anti-Diabetes, and Antibacterial
Activities. Frontiers in Marine Science, 3.
https://www.frontiersin.org/article/10.338
9/fmars.2016.00068
Levison, M. E. (2004). Pharmacodynamics of
antimicrobial drugs. Infectious Disease
Clinics of North America, 18(3), 451–465,
vii.
https://doi.org/10.1016/j.idc.2004.04.012
LewisOscar, F., Nithya, C., Vismaya, S., Arunkumar, M.,
Pugazhendhi, A., Nguyen-Tri, P., Alharbi, S. A.,
Alharbi, N. S., & Thajuddin, N. (2021). In
vitro analysis of green fabricated silver
nanoparticles (AgNPs) against Pseudomonas
aeruginosa PA14 biofilm formation, their
application on urinary catheter. Progress in
Organic Coatings, 151, 106058.
https://doi.org/10.1016/j.porgcoat.2020.10
6058
Marrez, D. A., Naguib, M. M., Sultan, Y. Y., & Higazy, A.
M. (2019). Antimicrobial and anticancer
activities of Scenedesmus obliquus
metabolites. Heliyon, 5(3), e01404.
https://doi.org/10.1016/j.heliyon.2019.e01
404
Onderka, M. (2007). Correlations between several
environmental factors affecting the bloom
events of cyanobacteria in Liptovska Mara
reservoir (Slovakia)—A simple regression
model. Ecological Modelling, 209(2),
412–416.
https://doi.org/10.1016/j.ecolmodel.2007.0
7.028
Ördög, V., Stirk, W. A., Lenobel, R., Bancířová, M.,
Strnad, M., van Staden, J., Szigeti, J., &
Németh, L. (2004). Screening microalgae for
some potentially useful agricultural and
pharmaceutical secondary metabolites.
Journal of Applied Phycology, 16(4),
309–314.
https://doi.org/10.1023/B:JAPH.00000477
89.34883.aa
Ozdemir, G., Ulku Karabay, N., Dalay, M. C., &
Pazarbasi, B. (2004). Antibacterial activity of
volatile component and various extracts of
Spirulina platensis. Phytotherapy Research,
18(9), 754–757.
https://doi.org/10.1002/ptr.1541
Prakash, J. W., @ Antonisamy, J. M., & Jeeva, S. (2011).
Antimicrobial activity of certain fresh water
microalgae from Thamirabarani River, Tamil
Nadu, South India. Asian Pacific Journal of
Tropical Biomedicine, 1(2, Supplement),
S170–S173.
https://doi.org/10.1016/S2221-1691(11)6
0149-4
Salem, O. (2014). Antimicrobial activity of microalgal
extracts with special emphasize on Nostoc
sp. Life Science Journal.
Shaima, A. F., Mohd Yasin, N. H., Ibrahim, N., Takriff,
M. S., Gunasekaran, D., & Ismaeel, M. Y. Y.
(2022). Unveiling antimicrobial activity of
microalgae Chlorella sorokiniana (UKM2),
Chlorella sp. (UKM8) and Scenedesmus sp.
(UKM9). Saudi Journal of Biological Sciences,
29(2), 1043–1052.
https://doi.org/10.1016/j.sjbs.2021.09.069
Shannon, E., & Abu-Ghannam, N. (2016).
Antibacterial Derivatives of Marine Algae: An
Overview of Pharmacological Mechanisms
and Applications. Marine Drugs, 14(4),
Article 4.
https://doi.org/10.3390/md14040081
Sieradzki, K., Roberts, R. B., Haber, S. W., & Tomasz, A.
(1999). The Development of Vancomycin
Resistance in a Patient with
Methicillin-Resistant Staphylococcus aureus
Infection. New England Journal of Medicine,
340(7), 517–523.
https://doi.org/10.1056/NEJM1999021834
00704
Sushanth, V. R., & Rajashekhar, M. (2015).
Antioxidant and antimicrobial activities in
the four species of marine microalgae
isolated from Arabian Sea of Karnataka
Coast. IJMS Vol.44(01) [January 2015].
http://nopr.niscair.res.in/handle/12345678
9/34627
Trabelsi, L., Ben Ouada, H., & Bassa, H. (2010).
Activités biologiques des métabolites
excrétés par la cyanobactérie filamenteuse
Arthrospira platensis. Phytothérapie, 8(5),
282–289.
https://doi.org/10.1007/s10298-010-0577-
2
Val, A., Platas, G., Basilio, A., Cabello, A.,
Gorrochategui, J., Suay, I., Vicente, F., Portillo,
E., Río, M., Reina, G., & Peláez, F. (2001).
Screening of antimicrobial activities in red,
green and brown macroalgae from Gran
Canaria (Canary Islands, Spain).
International Microbiology, 4(1), 35–40.
https://doi.org/10.1007/s101230100006
Vikneshan, M., Saravanakumar, R., Mangaiyarkarasi,
R.,
Rajeshkumar, S., Samuel, S. R., Suganya, M., & Baskar,
G. (2020). Algal biomass as a source for
novel oral nano-antimicrobial agent. Saudi
Journal of Biological Sciences, 27(12),
3753–3758.
https://doi.org/10.1016/j.sjbs.2020.08.022
14
CONFERENCE PAPER Hicham El Mouaddib et al., 2022, 15-25

The Coordination of Care in the Management of Breast Cancer: a Qualitative

Insight into Patients and Healthcare Professionals’ Perspectives
Hicham El Mouaddib1* , Majda Sebbani1,2,3 , Latifa Adarmouch1,2,3 , and Mohamed Amine 1,2,3
1
Biosciences & Health Laboratory, Cadi Ayyad University, 40000 Marrakech, Morocco.
2
Clinical Research Unit, Mohammed VI University Hospital, 40000 Marrakech, Morocco.
3
Community Medicine and Public Health Department, School of Medicine, Cadi Ayyad University,
SidiAbbad, 40000 Marrakech, Morocco.

Breast cancer is the leading cause of cancer death in women worldwide. In

Morocco, it is the first cancer in women and the third of all cancer cases
recorded. The coordination between healthcare levels comes to promote
better care. Thus, the present research tends to describe the coordination of
care between the primary, secondary and tertiary levels in the management
of breast cancer at the prefecture of Marrakech. Semi-structured face to face
interviews were conducted with 10 patients followed for breast cancer and 8
healthcare professionals working at the three healthcare levels. The content
analysis of the verbatim focused on the dimensions of coordination of care; it
was carried out using the QSR Nvivo software (Version 10.0). The analysis
resulted in three respective dimensions for each perspective and another
common dimension which is communication. For healthcare professionals,
the dimensions are the relationship, information and training. For patients,
the dimensions are orientation, empathy and satisfaction. The coordination
of care between the three levels of breast cancer management requires the
improvement of dimensions such as communication, relationship,
information, orientation, empathy, satisfaction and training.
* against cancer between developing countries and
Corresponding Author
developed countries is at least obvious (Cazap et
Name: Hicham El Mouaddib al., 2016). The cost, the distance from the
Email: hiyazid@gmail.com healthcare settings, the availability of human
Phone: resources is mainly responsible for these
disparities (Ambroggi et al., 2015, Cazap et al.,
ISBN : 9788367405256 2016).
DOI: 10.2478/9788367405256-003 In Morocco, cancer mortality is at 12% (Boutayeb
et al., 2013). However, breast cancer represents a
Published by Sciendo. © 2022 Hicham El Mouaddib et al.
This is an open access article licensed under the Attribution major public health problem because of the lack of
4.0 International screening and early diagnosis. This is the first
(https://creativecommons.org/licenses/by/4.0/) cancer in women and the third of all cases of
registered cancer. According to the World Health
Organisation (WHO), 6.650 new cases of breast
INTRODUCTION cancer were diagnosed in the Kingdom in 2014
Breast cancer is a health problem around the (WHO, 2014). In this respect, the interest in the
world. It is the most often diagnosed cancer in study of breast cancer has increased significantly
women with approximately 2.1 million new cases over the last decade. Since 2005, the screeningand
diagnosed each year. In addition, it represents the treatment of breast cancer in Morocco has
24.2% of all cancer diagnoses in women. Breast improved and has become a public health priority
cancer accounts for 25% of cancers in women (Slaoui et al., 2014).
globally and contributes to 15% of mortality (Bray The diagnosis of breast cancer can have a
et al., 2018). However, disparities in access to care
15
psychological impact on women (Iddrisu et al.,
2020). However, according to a systematic review
of literature on research in 11 countries in Africa,
the management of breast cancer has several
barriers. Among these barriers, there is the delay
of protocols at the level of health facilities and the
poor circulation of information between them,
respectively, in 28% of researches and 54.5% of
countries (Salisu et al., 2021). In this sense, the
problems identified concern the administrative
errors of healthcare professionals (Du Plessis &
Apffelstaedt, 2015), the wrong and inadequate
advice, as well as an archaic appointment system
(Dedey et al., 2016, Obrist et al., 2014). Apart from
the bad reference pathways in primary care
(Mutebi et al., 2018, Schleimer et al., 2019),
hospitals are limited in terms of treatments
available (Deressa et al., 2019, Frie K. et al., 2018)
and record chronic stock-outs of essential drugs
against cancer (Agorye et al., 2019, Du Plessis &
Apffelstaedt, 2015), not to mention repeated
breakdowns affecting diagnostic equipment and
materials (Sanuade et al., 2018). Patients are then
engaged in a care pathway interspersed with long
waiting periods (Agorye et al., 2019, Sanuade et al.,
2018), which leads to difficulties of access to care
(Meacham et al., 2016). All these problems and
others affect the ability of patients to progress in
their treatments (Salisu et al., 2021).
Thus, the coordination between healthcare levels
comes to promote better breast cancer
management (McAlearney et al., 2016). Therefore,
it should be conducted in a proactive approach to
bring together healthcare professionals to meet
the needs of patients by providing integrated and
person-centered care (Mollica et al., 2021). In this
regard, the Moroccan health care system
introduced a breast cancer-screening program in
Morocco in 2010. Thus, trained nurses, midwives
and general practitioners perform clinical breast
examinations (CBE) in hospitals and primary care
health centres (PCHCs). They immediately refer
women who are tested positive to the nearest
reproductive health referral centre (RHRC) for a
more in-depth assessment. In total, there are 39
RHRCs in the country. These centres are dedicated
to diagnosis and equipped with state-of-the-art
facilities, such as digital mammography, breast
ultrasound, core biopsy and fine-needle
cytopuncture (Basu et al. , 2018; Ministry of health
et Fondation Lalla Salma, 2020).
Despite all these efforts of this program, there are
still gaps to overcome in coordination of breast
cancer care. Among these issues described in the
literature, there are (a) the lack of resources, (b)
the funding barriers, (c) the lack of communication
between levels of care, (d) the understaffing and
(e) the high patient volume (McAlearney, et al.,
2016). These and other barriers could then
hamper the patient-centered care management
and coordination process. Their identification
would enable improvement of specific actions to
each care context.
Thus, this study aims to describe the coordination
of care in the management of breast cancer in the
prefecture of Marrakech from the perspectives of
patients and healthcare professionals.
METHODS
Study design
This was a qualitative, multicenter, descriptive
study carried out at the oncology and hematology
centre of the regional university hospital, the
RHRC and three different PCHCs. The choice of
these health settings is justified by the nature of
the subject, the availability of personal health and
breast cancer patients, as well as the geographical
proximity.
Study population
The target population consisted of two different
populations, namely women with breast cancer
(n=8) and healthcare professionals (n=10)
practicing at the three aforementioned sites. The
participants were selected based on reasoned
choice sampling. The inclusion criteria were (1)
for healthcare professionals (a) to be involved in
the breast cancer screening or the program
management and (b) to practice in that capacity
for at least one year, (2) for patients (c) to be
diagnosed with breast cancer and (d) be between
30 and 49 years old. The exclusion criteria were
(a) refusing to participate in the study, (b) refusing
the audio recording and (c) withdrawing from
participation in the study through the channels of
contact made available to participants (Phone,
email).
Data collection
Data collection was carried out by the main
researcher using semi-structured, individual
interviews based on pre-established interview
guides. These were administered in French for
professionals and in dialectal Arabic for patients.
The themes developed were (1) with healthcare
professionals (a) the communication with patients,
(b) the relationship with patients and with
professionals from other levels of care, (c) the
transmission of information between different
healthcare levels and (d) the knowledge
development, (2) with patients (e) the cancer
diagnosis, (f) the pathway of care, (g) the
interaction with healthcare professionals, (h) the
communication with healthcare professionals, (i)
the continuity of information, (j) the satisfaction
with care. All interviews were audio-recorded and
conducted during the period May 28 to June 2,
2021.
16
 Data analysis had 10 years or more of professional experience
All audio recordings of the interviews were fully (Table 2).
transcribed and imported into QSR Nvivo software Table 2: Sociodemographic characteristics of
(Version 10.0). It should be noted that the listening healthcare professionals (n=10).
reports from the interviews with the patients were Absolute
Characteristics Modalities
transcribed into Arabic, then translated into Frequencies
French, while preserving fidelity to the statements Female 7
made. The thematic content analysis was carried Sex
out with the same software and enabled the main Male 3
themes to be identified.
Specialist 2
Ethical considerations General
At the beginning of the interviews, interviewees Occupation 3
practitioner
received information about the aim of the study
and signed the informed consent form for Nurse 5
participation. Confidentiality and voluntary <30 years 2
withdrawal from the study were approved by the
study conductors. Age [30 ;40[ years 2
≥40 years 6
RESULTS
Characteristics of participants Professional <10 years 2
1. Patients experience ≥10 years 8
Most of the patients were illiterate, were 30 years
of age and older (5 out of 8). Six patients were Dimensions of care coordination
married and unemployed. Respectively, four had
In total, the content analysis generated 228 nodes.
the Medical Assistance Regime (RAMED) or
These were then categorized into two main
affiliated with a mutual health insurance. In
categories: patients’ perspective and healthcare
addition, 4 out of 8 were from rural areas (Table
professionals’ perspective.
1).
From the patients’ perspective, apart from the
Table 1: Sociodemographic characteristics of sub-category "diagnosis of breast cancer", the
patients (n=8). analysis revealed that the dimensions of care
Absolute coordination were orientation, empathy,
Characteristics Modalities
Frequencies communication and satisfaction.
<30 years 3 From the healthcare professionals’ perspective, the
Age analysis revealed that the coordination of care was
≥30 years 5 communication, relationship, information and
Educational Illiterate 5 training.
level Educated 3 1. Patients’ perspective
Health Mutual 4 – Diagnosis of breast cancer
insurance RAMED 4 Diagnosis of breast cancer is the first step in the
process of caring for patients. Thus, these affirmed
Single 2 that the diagnosis was after the appearance of
Marital status
Married 6 clinical signs and not within the framework of a
systematic screening.
Employment Active 2
“At first, I felt a very severe pain in the breast, I did not
status Unemployed 6 know what it was, and I did not consult until the pain
4 became excruciating and spread to the level of the
Residence Urban
breast of my armpit and my arm” (Patient#2).
area Rural 4 “Breast cancer was suspected when I felt a lump in my
breast” (Patient#1).
2. Healthcare Professionals “In my case, the onset of pain was the first suggestive
Seven on 10 of participants were women, sign I felt, then I started palpating for axillary
including 5 nurses. The other 5 were general lymphadenopathy, so that's where I went to see the
practitioners and specialists. Eight participants doctor” (Patient#4).

17
 – Orientation
According to the verbatim, most of the patients
followed the same pathway of care, namely (1) a
consultation at the nearby PCHC following the
appearance of clinical signs suggestive of the
disease by a clinical examination of the breast, (2)
a referral to the RHRC to perform an ultrasound or
a mammography in case of detection of an
abnormality on palpation and finally (3) a
follow-up of treatment at the level of the oncology
and hematology centre within the university
hospital.
The participants mentioned that the quality of the
care pathway is generally unsatisfactory. They
described it as difficult, given factors such as low
socioeconomic status, the circumstances of the
COVID-19 pandemic and the dates that last for a
very long time.
“It's been almost two years since I had excruciating
pain in my breast, armpit and arm. At first, I didn't
know it was cancer, I didn't do self-examination. I
didn't even know what breast cancer was, and I never
went to the primary health centre for all of my life.
When I went there, the doctor asked me to do a
mammogram after palpating the breast. [...], I did not
start the treatment immediately, given the number of
staff, the very long appointment times and COVID-19.
So I stayed at home and no longer consulted. My
condition became very complicated afterwards. The
pains became very severe” (Patiente#8).
– Empathy
The feeling of empathy on the part of the
healthcare professionals makes the coordination
of care between the three levels easier and
smoother. Thus, some patients appreciated the
interaction with healthcare professionals,
especially when the diagnosis was announced.
“They treated me gently, without breaking my feelings,
with empathy and a lot of awareness” (Patient#5).
Other patients spoke of a lack of empathy on the
part of healthcare professionals.
“I'm not going to lie to you, but there is no empathy in
caregivers. They ignored me so much, no guidance. I
was in pain and they didn't even explain my condition
to me” (Patient#7).
– Communication
Patients admit to having forged communication
links with healthcare professionals at different
levels of breast cancer care.
“I can describe communication with healthcare
professionals as good one. They explained everything
to me about my state of health, namely the disease
itself, the side effects, the duration of treatment, the
time of medication, the possible complications [...].
They gave me all the information I needed. They
communicated with me in a simple and understanding
way according to my level, so I could understand a lot
of things” (Patient#6).
Some participants found it unsatisfactory or
ineffective.
“There is no communication. I remember well when I
asked questions to anyone, nurses or doctors, they told
me that everything is written on my file” (Patient#4).
“There is no notion of communication. The caregivers
always put barriers between us” (Patient#1).
– Satisfaction
Half of the study participants showed
dissatisfaction with care.
“Once I felt a lump in my breast […], I went straight to
the primary health centre. Although it was very far
away, I reported my symptoms to the nurse, who told
me that these are delusions, and that was normal since
I am breastfeeding. I was not convinced! […]”
(Patient#2).
“I am not satisfied with all the care I received during
all levels of care. I wasted a lot of time. It's been almost
2 years now and my condition is still getting worse”
(Patient # 4).
However, the other half noted their satisfaction
with the care given during their care at all three
levels.
“I am satisfied with the care provided by staff in health
structures at different levels, from the primary care
health centre to the oncology centre” (Patient#1).
“I can say that I am satisfied with the care I received”
(Patient#6).
2. Healthcare professionals’
perspective
– Communication
Regarding this dimension, some professionals said
that their communication with patients was rather
satisfactory. It is usually based on the explanation
of the care pathway and its effectiveness.
“I can describe the communication with these patients
as being perfect. When the woman comes to the
referral centre, she is generally shocked, anxious
therefore the health professional must have the
knowledge and of knowing how to communicate”
(Nurse#2).
“Communication between the healthcare professionals
and the patient with breast cancer should ensure
confidence and hope for the woman, explaining that
nowadays the treatment of cancer is easier, more
accessible and less expensive and that the breast
cancer program is more developed” (Nurse#5).
In addition, some professionals indicated that
being a female healthcare professional further
facilitates communication with women, especially
18
those with a low socio-cultural level.
“As nurses, we need to communicate with patients.
Actually, the fact that we are women makes
communication easier and smoother […]” (Nurse#5).
“Communication generally goes smoothly. The fact
that I am a woman makes it easier for women to gain
confidence, given the question of modesty that exists in
Moroccan society; women are more at ease in
revealing themselves, revealing their bodies, their
illness, and all that is related to their intimate details”
(Physician#5).
However, other healthcare professionals have
indicated several difficulties leading to less
communication with patients.
“Communication with patients generally takes place in
good conditions. […]. However, there are several
constraints that make this communication difficult,
such as the lack of materials, of time, of hospital beds,
the number of patients” (Physician#1).
“Sometimes communication is very difficult, given the
unconsciousness of the patients, their very low
socio-cultural and educational level and the neglect of
the disease […]” (Physician#2).
The relationship is a fundamental dimension. It
makes the coordination of care between the
healthcare levels more fluid. On a one hand, all
healthcare professionals agreed that the
relationship with patients is excellent, based
mainly on trust, empathy and mutual respect.
“The nurse-patient relationship must be relevant; it
starts during their cures like a family relationship.
They become attached to us, and vice versa”
(Nurse#1).
“The relationship between the health professional,
whether nurse or doctor, and the patients must be
based on respect, empathy and above all trust. When
the woman learns that the caregiver is experienced
and he is a good practitioner, this is where confidence
is realized, and it helps much more to reassure the
woman and to orient her well to follow her care”
(Nurse#5).
On the other hand, concerning the relationship
between healthcare professionals at different
levels of care, it seems that they do not recognize
any information system linking them apart from
the reference sheet.
“No contact with other levels of breast cancer care.
Since a patient is referred to us at the oncology centre,
she becomes our responsibility” (Physician#1).
“There is no relationship between the first health level
and the second level (RHRC). There is only one referral
sheet circulating between us” (Nurse#4).
– Information
In this sense, the participants stood in two groups.
Those who described the existence of tools
facilitating the transmission of information
between the PCHC and the oncology centre via the
RHRC.
“The information is transmitted after the clinical
examination. This is where the doctor establishes a
prescription or a referral form that contains the
patient's identification and clinical signs. We give the
form to the patient, direct her to the RHRC and always
ask the patient to come back afterwards to inform us
of her prognosis” (Physician#5).
“The transmission of information between the PCHC
and the RHRC is done by telephone or referral form,
which contains the identification, the reason for the
consultation and the associated signs” (Nurse#5).
The second category of participants denies the
existence of this communication tool between
healthcare levels. They mentioned that they do not
regularly receive the counter-referral forms.
“Sometimes we receive a counter-referral from the
second level of care. Otherwise, information is
acquired through patients who return to us to inform
us of their prognosis. Regarding the oncology centre,
there is no transmission between us” (Nurse#4).
“There is no transmission of information between
healthcare levels. I think primary level doctors are
generally scared of patients with cancer, so when they
receive such cases, they always refer them to the
oncology centre” (Physician#1).
– Training
On this aspect, the interviewees were divided
equally between three categories. Most described
that there is a lack of training and that they have
developed their knowledge through their own
efforts.
“It is through personal self-training. For me, I never
had a training regarding the management of breast
cancer. It is a personal effort. I try to find new things
and news to improve my knowledge and to be up to
date” (Physician #5).
“The updating of knowledge is done based on
experience. We have never benefited from training or
a conference since I came to the RHRC in 2014. The
only way to update knowledge is through self-study,
research on the internet” (Nurse#2).
In the second category, the lack of training is
linked to the circumstances of the COVID-19
pandemic.
“Before COVID-19, there was always training
dedicated to staff on the different types of cancer. But
since this pandemic, there is nothing left!” (Nurse#1).
Other participants cited the different ways
available to develop and update knowledge.
“There are several methods, starting with staff and
multidisciplinary meetings that are organized on a
weekly basis, journal consultations, and seminars that
are numerous […]. I can assure you that we are always
up to date with the news in the different regions of
19
Morocco” (Physician#1).
DISCUSSION
In our study, many women reported that their
cancer was diagnosed after the onset of
symptoms. However, the breast cancer diagnosis
should normally be done as part of the screening
that is performed in women without any
symptoms so that the disease can be detected as
early as possible (Bevers et al., 2009).
The interviewees mentioned in their verbatim
dimensions that determine, according to their
perspectives, the breast cancer care coordination.
– Communication
Analysis of the data highlighted the importance of
communication in establishing effective
coordination between the healthcare levels of
breast cancer. It was developed from two
perspectives: the healthcare professionals and the
breast cancer’s patients.
The first category contains three groups. Those
who jointly affirmed effective levels of
caregiver-patient communication. This
communication aims to ensure confidence, hope
and satisfaction for patients. This is confirmed,
since according to Araújo et al. (2010), healthcare
professionals showed that they always seek
quality in the caregiver-patient relationship,
demonstrating efficiency, involvement and
management of various obstacles in
communication.
In the same sense, the analysis of the verbatim
shows that the communication takes place in a
better manner with nurses, which facilitates the
establishment of a relationship of trust, because
the patient is more comfortable to reveal several
things. These results are confirmed, since a
systematic review pointed out that cancer patients
often associated the quality of care with their
relationship with nurses, which is perceived to be
important for the feeling of confidence and
well-being (Rchaidia et al. al., 2009).
On the other hand, healthcare professionals
mentioned barriers preventing the relevant
progress of the communication, such as the lack of
time, the lack of beds, the number of patients and
their socio-cultural and educational levels.
According to Araújo et al. (2010), healthcare
professionals face several challenges that hamper
effective communication with patients, namely the
high number of patients, each reacting in its own
way, variations in location, time, space, feelings
and the social and religious level which differs
from one patient to another.
Moreover, for the patients, two equal groups arose.
The patients of the first group said that they
benefited from explanatory, simple and
comprehensive communication, based on the
explanation of the disease, the treatment, the side
effects and the complications. Indeed, Thi et al.
(2002) state that three out of four patients are
satisfied with the communication established with
their care providers.
However, patients in the second group stated that
their dissatisfaction was due to the lack of
communication on the part of the healthcare
professionals; referring to the lack of information
and active listening. Indeed, patients expect the
healthcare team to listen to them and understand
their needs, believing that trust is the cornerstone
of therapeutic communication to improve the
quality of care (Khoshnazar et al., 2016).
– Relationship
The relationship constitutes an obvious factor for
the success of the coordination between the
different levels of care for breast cancer. The data
analysis revealed two different kinds of
relationship. Firstly, the relationship that unites
professionals from different healthcare levels with
each other. What seems unclear in the verbatim of
the participants is that they declared a complete
absence of this relationship, indicating that no
direct contact between them has ever been
evidenced, apart from the referral form.
This is mentioned by Dossett et al. (2017), who
indicate that the frequency, timing and content of
communication are insufficient, healthcare
professionals at each respective level thus express
their skepticism about the ability of the other level
to play its role. However, they still wish to be more
closely informed by any means of communication
on the experiences of patients. In the same study,
44% of first-level professionals indicated that they
have "sometimes", "rarely" or "never" been
informed of the diagnosis or the results of their
patients in the post-treatment period, and they
have describes a large gap in communication
between diagnosis and the end of treatment.
Secondly, the participants spoke of an excellent
patient-caregiver relationship, based mainly on
three fundamental principles, namely trust,
empathy and mutual respect. They even compare
it to a family relationship. This does not
corroborate with the results of the study by
Friderich (2009) which showed that the quality of
the relationship between caregivers and patients
is characterized by a certain misunderstanding. A
real mistrust on the part of patients regarding the
behavior of healthcare professionals, and a lack of
confidence, which have a negative influence on the
patient-caregiver relationship.
– Information
20
The results inherent in this dimension fell into two
main categories. The first brings together
healthcare professionals who have affirmed the
existence of tools that facilitate the transmission of
information between levels of care, namely, the
referral form.
Indeed, care coordination depends on an efficient
transfer of information between caregivers at the
healthcare levels. However, primary care
professionals tend to describe these informations’
transmissions, when they exist, as slow and
insufficient (Van der Kam, Branger, Van Bemmel, &
Meyboom-de Jong, 1998).
The second category has ruled out the existence of
any means of communication between the
healthcare levels. In fact, according to the
healthcare professionals, there is an absence of
information’s transmission. In addition, according
to them, professionals at the first level are
generally afraid of cancer cases, and therefore
prefer to refer patients systematically to the next
level. Indeed, the lack of targeting, the omissions of
information, the erroneous information, the very
long duration, the noise in the event of oral
transmission and the inattention are all difficulties
cited varying between 20% and 60% by healthcare
professionals as barriers limiting the flow of
information between professionals at different
levels of care (Gandy, 2018). In the same study,
professionals claim that they have never received
training in carrying out transmissions during their
studies, whether in their professional career or at
the university.
In this regard, the present study showed a sharing
of the same point of view by all the participants
concerning the total absence of the
counter-referral forms mentioning that in most
cases, they receive the information on the
prognosis directly by the patient.
– Orientation
The referral of breast cancer patients is done
without any validated regulatory tool. However,
regulating the pathway of patients is an essential
organizational means. It comes highly
recommended and aims to ensure quality while
reducing the cost of care. Through the analysis of
the data, it turned out that the patients were
treated according to the same process, passing
through all three healthcare levels. First, the
consultation at the PCHC for a CBE. Then, the
referral to the RHCH to detect any suggestive
clinical sign of breast cancer. Finally, the
monitoring of treatment at the oncology and
hematology centre.
In this regard, all the participants shared the same
point of view, expressing the difficulties they
faced, namely (a) the disorientation which is the
main drawback, especially in the circumstances of
the COVID-19 pandemic and its socioeconomic
consequences, (b) the lack of transport, (c) the
long duration of the appointments and (e) the
high cost of the treatment.
However, it is recommended (1) to streamline the
patient's care pathway, (2) to facilitate their
knowledge of the healthcare offer, (3) to help them
make decisions, (4) to improve their care and their
support and (5) to optimize the organization of
functions and activities by healthcare teams (INC,
2022).
– Training
According to the results, 1 out of 5 healthcare
professionals working in the three levels of breast
cancer management affirmed benefiting from
training, which aims to provide various medical
news, develop theoretical and practical skills and
up-to-date knowledge.
This proportion remains below that indicated in
the study by OIIQ (2010). With the exception of
one in five caregivers (21.7%) who have not
benefited from continuing training, 20.1% reserve
more than 30 hours/week for this strategy, 29.2%
devote between 15 and 30 hours, while 29% give
it less than 15 hours. Due to the COVID-19
pandemic, courses, conferences and training have
been canceled. This is what was mentioned by
20% of professionals who believe that the lack of
training is strongly linked to COVID-19, by
affirming the periodic existence of these before
the pandemic.
The impact on training following the pandemic
seems obvious. It has delayed the achievement of
the objectives. Thus, in all countries, distance
education means have been used (Gosset et al.,
2021).
– Satisfaction
The measurement of satisfaction helps guide the
strategy of optimal care for patients. The latter do
not share the same point of view. In other words, 4
out of 8 patients declare their satisfaction with
care in the three levels of care whether primary,
secondary or tertiary.
This is confirming the results of a survey whose
objective is to assess the degree of satisfaction of
cancer patients at the Fez University Hospital. It
showed that 87.1% of patients declared to be very
satisfied with care, 12.9% considered it
acceptable, 98.4% will recommend this care to
their relatives, while 67.2% of the patients
considered the cost too expensive. Several items
were deemed very satisfactory by the patients.
This concerned both hotel comfort and the human
aspect in the behavior of medical and nursing
staff, the relationship of patient-caregiver trust
21
and respect (Amazian et al., 2013).
Due to the quality of care provided, the time
wasted, misdiagnosis and disorientation, the cost
deemed too expensive by patients who are mostly
low socioeconomic, poor advice given to patients
regarding their diet and more overall the rules of
hygiene to be followed, 50% of the participants in
the present study however expressed their
dissatisfaction with the care they benefited
(Amazian et al., 2013).
In another study which assessed the satisfaction
with the care for breast cancer patients, the
average satisfaction score was 73 on a scale of 0 to
100. This satisfaction is nevertheless influenced by
several factors like good communication with the
nursing staff, extra-professional activities,
technical skills. Conversely, patient satisfaction was
negatively influenced by poor relationship quality,
the quality of information provided and long wait
times (Vardanega et al., 2019).
– Empathy
In a professional relationship, empathy is listening,
observing to build objective thinking that
facilitates care in the best conditions. It promotes
the quality of the patient-caregiver relationship.
Patients who appreciated the interaction with
caregivers reported that they were treated with
empathy, especially when the diagnosis was
announced.
Other participants pointed to the lack of empathy
and lack of interaction explained by the perceived
ignorance, disorientation, and poor explanation of
their state of health.
Indeed, the empathy of caregivers towards patients
is negatively influenced by several factors,
including the psychological distress of caregivers,
the focus of the task rather than the patient and
the imbalance between the patient-caregiver sexes
(Gleichgerrcht, & Decety, 2013).
Therefore, it would be very interesting to set up
education directly targeted at sensitizing
healthcare professionals at all three levels of care
to empathy and interacting with the different
situations of patients. This would improve the
quality of the patient-caregiver relationship. It
would also help foster relationships of trust and an
optimal understanding of others.
LIMITS OF THE STUDY
This study described the dimensions of
coordination for breast cancer care from tow
perspectives: patients and healthcare
professionals. Given the qualitative design of the
study, limitations remain such as the declarative
nature of the responses, the selection bias of the
participants and the social desirability reduction
during the conduct of the interviews.
RECOMMENDATIONS
Based on the results of the study, several
recommendations are suggested for improving the
coordination of care for breast cancer. It is a
question of (a) developing systematic orientation
tools and encouraging healthcare professionals to
respect them, (b) promoting patient satisfaction
by improving reception conditions, and the
reduction of waiting times, (c) reinforcing the
values of respect, trust, empathy and tolerance, in
order to consolidate the caregiver-patient
relationship, (d) developing a digital information
system linking the three healthcare levels and (e)
making an annual regional training plan taking
into consideration the training needs of healthcare
professionals in order to improve their theoretical
and practical skills in breast cancer diagnosis and
care.
CONCLUSION
Based on the experiences of patients and
healthcare professionals, it appears that the
coordination of care for the management of breast
cancer is not optimal. Its improvement requires
the strengthening of dimensions reported by
participants namely communication, relationship,
information, orientation, empathy, satisfaction and
training.
This study aims to draw up an inventory of the
breast cancer care coordination in Marrakech. It
tried to combine the patients’ and healthcare
professionals’ perspectives. However, it does not
claim to be able to answer to all the questions.
Thus, this subject deserves to be deepened by
further research in perspective, on larger samples
and more robust design. Finally, the discussion
opens up several research perspectives that
should be conducted in the future, to better
understand this situation with a view to
promoting the prevention, diagnosis and
management of breast cancer according to the
required standards.
CONFLICT OF INTEREST
The authors have no conflict of interest.
ETHICAL APPROVAL
This article is part of the main author's doctoral
thesis. The research protocol for this was
approved by the Hospital-University Ethics
Committee of the Faculty of Medicine of
Marrakech, Cadi Ayyad University, Morocco
22
(Reference: 021/20).
FUNDING
This study was conducted with self-funding.
ACKNOWLEDGEMENTS
The authors thank the participants in this study.
Special mention goes to Mrs. Sarah Michaud for
providing a final revision of the English translation
of this paper.
REFERENCES
Agorye, I.-J.-, Chiamaka, I., Chidinma-Egbichi, I.,
Ijeoma, M., & Obiagele, M. (2019). Factors
influencing non–adherence to chemotherapy?:
Perspective of Nigerian breast cancer survivors.
Nursing Practice Today, 6(1), 453-460.
https://doi.org/10.18502/npt.v6i1.392.
Amazian, K., Toughrai, I., Ben, N., Laalim, S. A.,
Alami, M. E. A. E., & Mazaz, K. (2013). Enquête de
satisfaction des patients atteints de cancer dans
un hôpital universitaire au Maroc. Santé
Publique, 25(5), 627‑632.
Ambroggi, M., Biasini, C., Del Giovane, C., Fornari, F.,
& Cavanna, L. (2015). Distance as a Barrier to
Cancer Diagnosis and Treatment?: Review of the
Literature. The Oncologist, 20(12), 1378-1385.
https://doi.org/10.1634/theoncologist.2015-01
10.
Araújo, I. M. de A., Silva, R. M. da, Bonfim, I. M., &
Fernandes, A. F. C. (2010). Nursing
communication in nursing care to
mastectomized women?: A grounded theory
study. Revista Latino-Americana de Enfermagem,
18, 54-60.
https://doi.org/10.1590/S0104-116920100001
00009.
Basu, P., Selmouni, F., Belakhel, L., Sauvaget, C.,
Abousselham, L., Lucas, E., Muwonge, R.,
Sankaranarayanan, R., & Khazraji, Y. C. (2018).
Breast Cancer Screening Program in Morocco?:
Status of implementation, organization and
performance. International Journal of Cancer,
143(12), 3273-3280.
https://doi.org/10.1002/ijc.31749.
Bevers, T. B., Anderson, B. O., Bonaccio, E., Buys, S.,
Daly, M. B., Dempsey, P. J., Farrar, W. B., Fleming,
I., Garber, J. E., Harris, R. E., Heerdt, A. S., Helvie,
M., Huff, J. G., Khakpour, N., Khan, S. A., Krontiras,
H., Lyman, G., Rafferty, E., Shaw, S., Smith, M. L.,
Tsangaris, T. N., Williams, C., & Yankeelov, T.
(2009). Breast Cancer Screening and Diagnosis.
Journal of the National Comprehensive Cancer
Network, 7(10), 1060-1096.
https://doi.org/10.6004/jnccn.2009.0070.
Boutayeb, A., Boutayeb, S., & Boutayeb, W. (2013).
Multi-morbidity of non communicable diseases
and equity in WHO Eastern Mediterranean
countries. International Journal for Equity in
Health, 12(1), 60.
https://doi.org/10.1186/1475-9276-12-60.
Bray, F., Ferlay, J., Soerjomataram, I., Siegel, R. L.,
Torre, L. A., & Jemal, A. (2018). Global cancer
statistics 2018 : GLOBOCAN estimates of
incidence and mortality worldwide for 36
cancers in 185 countries. CA: A Cancer Journal
for Clinicians, 68(6), 394-424.
https://doi.org/10.3322/caac.21492.
Cazap, E., Magrath, I., Kingham, T. P., & Elzawawy,
A. (2016). Structural Barriers to Diagnosis and
Treatment of Cancer in Low- and
Middle-Income Countries?: The Urgent Need for
Scaling Up. Journal of Clinical Oncology, 34(1),
14-19.
https://doi.org/10.1200/JCO.2015.61.9189.
Dedey, F., Wu, L., Ayettey, H., Sanuade, O. A.,
Akingbola, T. S., Hewlett, S. A., Tayo, B. O., Cole,
H. V., de-Graft Aikins, A., Ogedegbe, G., & Adanu,
R. (2016). Factors Associated With Waiting
Time for Breast Cancer Treatment in a Teaching
Hospital in Ghana. Health Education & Behavior,
43(4), 420-427.
https://doi.org/10.1177/1090198115620417.
Deressa, B. T., Cihoric, N., Badra, E. V., Tsikkinis, A.,
& Rauch, D. (2019). Breast cancer care in
northern Ethiopia – cross-sectional analysis.
BMC Cancer, 19(1), 393.
https://doi.org/10.1186/s12885-019-5612-6.
Dossett, L. A., Hudson, J. N., Morris, A. M., Lee, M.
C., Roetzheim, R. G., Fetters, M. D., & Quinn, G. P.
(2017). The primary care provider (PCP)-cancer
specialist relationship?: A systematic review and
mixed-methods meta-synthesis: The
PCP-Cancer Specialist Relationship. CA: A
Cancer Journal for Clinicians, 67(2), 156-169.
https://doi.org/10.3322/caac.21385.
Du Plessis, M., & Apffelstaedt, J. P. (2015).
Treatment outcomes of breast carcinoma in a
resource-limited environment. South African
Journal of Surgery, 53(2), 43.
https://doi.org/10.7196/sajsnew.7842.
Friderich, M. L. (2009). La relation
soignant-soigné au Burkina Faso en 2008?:
Comment les professionnels et les patients la
perçoivent-elles? (hal-01732665) [Thèse de
Médecine Générale, Université Henri Poincaré,
Nancy 1].
https://hal.univ-lorraine.fr/hal-01732665.
Frie K, G., Samoura, H., Diop, S., kamate, B., traore,
C. B., Mall, B., & kantelhardt, E. J. (2018). Why Do
Women with Breast Cancer Get Diagnosed and
Treated Late in Sub-Saharan Africa Perspectives
23
 from Women and Patients in Bamako, Mali.
Breast Care (Basel), 13(1):39-43.
https://doi.org/10.1159/000481087.
Gandy, D. (2018). La place des transmissions
orales dans la continuité des soins.
(hal-01847511) [Mémoire de fin d'études,
Université de Lorraine].
https://hal.univ-lorraine.fr/hal-01847511.
Gleichgerrcht, E., & Decety, J. (2013). Empathy
in Clinical Practice : How Individual
Dispositions, Gender, and Experience
Moderate Empathic Concern, Burnout, and
Emotional Distress in Physicians. PLoS ONE,
8(4), e61526.
https://doi.org/10.1371/journal.pone.00615
26
Gosset, M., Gal, J., Schiappa, R., Dejode, M.,
Fouché, Y., Alazet, F., ... & Barranger, E. (2021).
Impact de la pandémie de COVID-19 sur les
prises en charge pour cancer du sein et
gynécologique. Bulletin du Cancer, 108(1), 3.
Iddrisu, M., Aziato, L., & Dedey, F. (2020).
Psychological and physical effects of breast
cancer diagnosis and treatment on young
Ghanaian women : A qualitative study. BMC
Psychiatry, 20(1), 353.
https://doi.org/10.1186/s12888-020-02760-4.
INC (Éd.). (2022). Contrat d’objectifs et de
performance 2021-2025 entre l’Etat et l’Institut
National du Cancer. Institut National de Cancer.
Available from:
https://www.e-cancer.fr/content/download/42
9128/6465450/file/COP_web_VF_200422_nove
mbre.pdf
Khoshnazar, T. A., Rassouli, M., Akbari, M. E.,
Lotfi-Kashani, F., Momenzadeh, S., Rejeh, N., &
Mohseny, M. (2016). Communication Needs of
Patients with Breast Cancer: A Qualitative Study.
Indian J Palliat Care, 22(4), 402-409.
https://doi.org/10.4103/0973-1075.191763.
McAlearney, A. S., Murray, K., Sieck, C., Lin, J. J.,
Bellacera, B., & Bickell, N. A. (2016). The
Challenge of Improving Breast Cancer Care
Coordination in Safety-net Hospitals. Medical
Care, 54(2), 147–154.
https://doi.org/10.1097/mlr.00000000000004
58.
Meacham, E., Orem, J., Nakigudde, G., Zujewski, J.
A., & Rao, D. (2016). Exploring stigma as a
barrier to cancer service engagement with
breast cancer survivors in Kampala, Uganda?:
Exploring stigma and cancer service
engagement in Kampala, Uganda.
Psycho-Oncology, 25(10), 1206-1211.
https://doi.org/10.1002/pon.4215.
Ministry of Health & Fondation Lalla Salma.
(2020). National Cancer Prevention and Control
Plan 2020-2029. Rabat, Morocco. Available
from:
https://www.sante.gov.ma/Documents/2021/0
3/Plan_National_de_Prevention_et_de_Controle_
du_Cancer_2020-2029_VF.pdf
Mollica, M. A., Buckenmaier, S. S., Halpern, M. T.,
McNeel, T. S., Weaver, S. J., Doose, M., & Kent, E.
E. (2021). Perceptions of care coordination
among older adult cancer survivors?: A
SEER-CAHPS study. Journal of Geriatric
Oncology, 12(3), 446-452.
https://doi.org/10.1016/j.jgo.2020.09.003.
Mutebi, M., Olasehinde, O., Kingham, P.,
Boutin-Foster, C., & Pusic, A. (2018).
Understanding the Breast Cancer Experience of
Women in East Africa?: A Qualitative Study.
Journal of Global Oncology, 4(Supplement 2),
120s-120s.
https://doi.org/10.1200/jgo.18.44100.
Obrist, M., Osei-Bonsu, E., Awuah, B.,
Watanabe-Galloway, S., Merajver, S. D., Schmid,
K., & Soliman, A. S. (2014). Factors related to
incomplete treatment of breast cancer in
Kumasi, Ghana. The Breast, 23(6), 821-828.
https://doi.org/10.1016/j.breast.2014.08.014.
OIIQ. (2010). Rapport statistique sur l'effectif
infirmier 2009-2010: Le Québec et ses régions.
Québec: Ordre des infirmières et infirmiers du
Québec.
Rchaidia, L., Dierckx de Casterlé, B., De Blaeser, L.,
& Gastmans, C. (2009). Cancer patients’
perceptions of the good nurse: a literature
review. Nurs Ethics, 16(5), 528–542.
Salisu, W. J., Mirlashari, J., Varaei, S., & Seylani, K.
(2021). Limited access to care for persons with
breast cancer in Africa?: A systematic review.
European Journal of Oncology Nursing, 50,
101867.
https://doi.org/10.1016/j.ejon.2020.101867.
Sanuade, O. A., Ayettey, H., Hewlett, S., Dedey, F.,
Wu, L., Akingbola, T., Ogedegbe, G., & Aikins, A.
de-Graft. (2018). Understanding the causes of
breast cancer treatment delays at a teaching
hospital in Ghana. Journal of Health Psychology,
26(3), 357-366.
https://doi.org/10.1177/1359105318814152.
Schleimer, L. E., Vianney Dusengimana, J.-M.,
Butonzi, J., Kigonya, C., Natarajan, A.,
Umwizerwa, A., O’Neil, D. S., Costas-Chavarri, A.,
Majyambere, J.-P., Shulman, L. N., Keating, N. L.,
Shyirambere, C., Mpunga, T., & Pace, L. E. (2019).
Barriers to timely surgery for breast cancer in
Rwanda. Surgery, 166(6), 1188-1195.
https://doi.org/10.1016/j.surg.2019.06.021.
Slaoui, M., Razine, R., Ibrahimi, A., Attaleb, M., El
Mzibri, M., & Amrani, M. (2014). Breast Cancer
24
 in Morocco?: A Literature Review. Asian Pacific
Journal of Cancer Prevention, 15(3), 1067-1074.
https://doi.org/10.7314/APJCP.2014.15.3.1067.
Thi, P. L. N., Lê, T. G., Empereur, F., & Briançon, S.
(2002). Satisfaction des patients hospitalisés à
Hô Chi Minh-Ville, Viet Nam. Santé Publique,
14(4), 345-360.
Van der Kam, W. J., Branger, P. J., Van Bemmel, J. H.,
& Meyboom-de Jong, B. (1998). Communication
between physicians and with patients suffering
from breast cancer. Family practice, 15(5),
415-419. https://doi.org/
10.1093/fampra/15.5.415.
Vardanega, J., Henriques, J., Pourcelot, C., Dirand,
C., Nallet, G., Brédart, A., Anota, A., Chaigneau, L.,
Curtit, E., Limat, S., Paget-Bailly, S., & Nerich, V.
(2019). Évaluation de la satisfaction de
patientes atteintes d’un cancer du sein précoce
traitées en adjuvant par hormonothérapie.
Bulletin du Cancer, 106(12), 1104‑1114.
https://doi.org/10.1016/j.bulcan.2019.08.019.
WHO. (2014). Morocco: World Health
Organization - country profiles for cancer, 2014.
Available from:
https://www.who.int/cancer/country-profiles/
mar_fr.pdf
.

25
CONFERENCE PAPER Abir EL-Araby et al., 2022, 26-32

Chitosan Effect on Fungal Strains Responsible for Strawberries Post-harvest

Losses
Abir El-Araby * , Lahsen El Ghadraoui , and Faouzi Errachidi
Functional Ecology and Environment Engineering Laboratory, Faculty of Science and Technology,
Sidi Mohamed Ben Abdellah University, Fez, Morocco

Fungal pathogens are one of the main causes of strawberry’s

post-harvest losses. This is currently prompting producers to overcome
this problem by using natural ingredients that meet food safety standards.
The present study highlights the chitosan effect on fungal strain’s growth
responsible for strawberry fruit post-harvest losses. Chitosan was
extracted from shrimp shells (Parapenaeus longirostris) by using citric
acid during the demineralization step. Produced chitosan was
characterized by Fourier Transform Infrared spectroscopy (FTIR) and
Scanning Electron Microscopy (SEM). FTIR confirms the presence of the
amino group (-NH2) in the obtained chitosan. A deacetylation degree (DD)
of 83% was calculated from the FTIR spectra. The smooth surface and
fibrous structures of chitosan were determined by using SEM. Chitosan
effect was evaluated on the mycelial growth of Botrytis cinerea, Aspergillus
niger, Rhizopus stolonifer, and Penicillium spp. strains isolated from spoiled
strawberry fruits. Growth rates (µ on day-1) was determined from kinetics
curves. Chitosan revealed a strong antifungal activity, dose-dependent
(from 0 to 3%), on all fungal strain’s growth. Three per cent (3%) of
chitosan concentration gives a total inhibition of mycelial growth.
* Cosme et al., 2022). However, these fruits are
Corresponding Author
highly perishable and decay rapidly after harvest
Name: Abir El-Araby
and therefore have a minimal shelf life, typically
Email: abir.elaraby@usmba.ac.ma
five days at 0-4 °C. Strawberries are susceptible to
Phone: +212 634889405 mechanical injury, physiological disorders, and
fungal deterioration during postharvest storage
ISBN: 9788367405256
(Trinetta et al., 2020).
DOI: 10.2478/9788367405256-004
In recent decades, many efforts have been realized
Published by Sciendo. © 2022 Abir EL-Araby et al. This is an to improve strawberries’ shelf life and satisfy
open access article licensed under the Attribution 4.0 consumers by using natural ingredients that meet
International food safety standards. Recently, the edible coating
(https://creativecommons.org/licenses/by/4.0/)
has attracted a lot of interest as an effective way to
extend fruits’ shelf life considered susceptible to
post-harvest losses. The use of bioactive
INTRODUCTION
compounds such as chitosan is a typical example
of edible coatings to control food postharvest
Strawberry’s physiological characteristics provide losses (Qamar et al., 2018 ; Betchem et al., 2019 ;
an ideal environment for fungal infection and Cazón and Vázquez, 2019 ; Quintana et al., 2021).
growth, resulting in substantial economic losses Marine waste extracted chitosan is a versatile and
for the industry. Strawberries are highly nutritious environmentally friendly polymer with a large
fruits and a good source of many bioactive spectrum of antimicrobial and antioxidant
ingredients, including vitamins, minerals, dietary applications. A study done by Abdipour et al.
fibre, and antioxidants (Mohammadi et al., 2021; (2020) reported that the application of chitosan
26
(1%) edible coating and UV irradiation
maintained the postharvest quality and reduced
the weight loss during storage of treated sweet
cherry fruit. The chitosan coating can be used to
maintain the quality of strawberries by reducing
mold growth and consequently enhancing the
shelf life during cold storage compared to the
uncoated treatment (Jiang et al., 2020).
The current study highlights chitosan’s negative
effect on fungal strains’ growth responsible for
strawberry fruit postharvest losses. This work
aims to solve one of the principal causes of
strawberry fruits deterioration which are
sensitive during storage. Chitosan was extracted
from shrimp shells (Parapenaeus longirostris)
marketed in Morocco. It was characterized by
Fourier Transform Infrared spectrometry (FTIR)
and Scanning Electron Microscopy (SEM).
Chitosan effect was evaluated on the mycelial
growth of Botrytis cinerea, Aspergillus niger,
Rhizopus stolonifer, and Penicillium spp. strains
isolated from spoiled strawberry fruits.
MATERIALS AND METHODS
1 Biological materials
Chitosan extraction was carried out from shrimp
shells (Parapenaeus longirostris) marketed in
Morocco. Tested molds are strains isolated from
spoiled strawberries: Botrytis cinerea, Aspergillus
niger, Penicillium spp., and Rhizopus stolonifer.
2 Chitosan extraction
Shrimp shells were washed, dried, and ground.
The deproteinization process was carried out with
30g of powdered shells using NaOH at 5% (1.25
N) with a ratio of 1 g: 12 ml (w/v). The whole was
refluxed at 70˚C for 3 hours. The product was
collected, washed, and then dried. The
discoloration was done with pure acetone for 24 h
(Kandile et al., 2018). The product was recovered
and washed until neutral, then dried. Decolorized
product was demineralized using 1% citric acid
with a ratio of 1 g: 10ml (w/v) for 24 h at room
temperature.
Obtained chitin was collected and washed.
Purified chitin deacetylation was performed by
using NaOH at 55% (12.5 N) with a ratio of 1 g: 5
ml (w/v) at 100˚C for 12 h (Kandile et al., 2018).
Obtained chitosan was washed and wholly dried
until constant weight.
3 Extracted chitosan characterization
Prepared chitosan was characterized by Fourier
Transform Infrared Spectroscopy (FTIR) and
Scanning Electron Microscopy (SEM). These
analyzes were carried out at the Regional
University Center of Interface (CURI) attached to
Sidi Mohamed Ben Abdellah University (USMBA)
in Fez.
Fourier Transform Infrared Spectroscopy
Chitosan was characterized by FTIR spectroscopy
using KBr pellets in the scanning range of 400 to
4000 cm-1. Infrared spectra were measured on a
spectrometer (Bruker Vertex 70) and obtained
with a resolution of 4 cm-1.
Scanning Electron Microscopy
Extracted chitosan morphology was studied by
SEM using a scanning electron microscope (JEOL
JSM-IT500HR) with a magnification range of
×600.000, an accelerating voltage of 0.5 kV-30.0
kV, and a maximum sample size of 200 mm in
diameter and 75 mm in height. The chitosan
sample for SEM was proceeded by depositing onto
a carbon tape.
4 Chitosan effect on strawberries spoilage fungi
growth
Chitosan antifungal activity on Botrytis cinerea,
Aspergillus niger, Rhizopus stolonifer, and
Penicillium spp. mycelial growth was evaluated on
PDA solid medium. Fungi inoculation was done by
deposing aseptically (5 mm) of the previous
culture on a culture medium containing increasing
chitosan concentrations (from 0 to 3%). Growth
kinetics were determined by measuring mycelial
growth diameters. Fungi growth rates (µ on day-1)
were calculated by the following formula (Eq.1):
µ = ( 𝑙𝑛𝐷2−𝑙𝑛𝐷1
∆𝑡 )
(1)
µ: Fungi growth rate (µ on day-1)
D2 and D1: Mycelial growth diameters
∆t: Time Unit (t2-t1)
5 Statistical analysis
The statistical analyses were conducted by
analysis of variance (ANOVA), and the means ±
were compared using Duncan’s multiple rang tests
at P ≤ 0.05 in the SPSS statistical software. The
mathematical models obtained were also
developed with SPSS software.
RESULTS AND DISCUSSION
1 Chitosan extraction
In the present study, chitosan extraction involved
an organic acid, citric acid, for shrimp shells
demineralization. The use of an organic acid is
more environmentally adequate and significantly
reduces production costs, unlike mineral acid
application.
2 Fourier Transform Infrared Spectroscopy
27
The final extracted product was characterized by 1). Assignment of absorption bands is based on
Fourier Transform Infrared Spectroscopy (Figure
literature data (Shigemasa et al., 1996 ; Duarte et bridge) and 1022-1011 cm-1 (C-O stretching
al., 2002). A deacetylation degree (DD) of 83% vibration of alcohol groups) are characteristic of
was calculated from the FTIR spectra. chitosan saccharide structure (Povea et al., 2011).
IR spectral data of chitosan extracted by citric acid 3 Scanning Electron Microscopy
exhibits characteristic absorption bands at 3264
Morphological characteristics of extracted
cm-1 (O-H stretching vibrations), 2919-2853 cm-1
chitosan were studied by Scanning Electron
(CH-stretching aliphatic vibration in the -CH and
Microscopy (SEM). Figure 2 reveals the surface
-CH2 groups), 1653 cm-1 (N-H bond stretching of
morphology of chitosan extracted by citric acid at
amide I), 1580 cm-1 (N-H straining vibrations of
10µm and 5µm. Chitosan micrographs showed
-NH2 groups), 1379 cm-1 (symmetric deformation
very smooth surface and fibrous structures. SEM
of C-H in the -CH3 group), and 1318 cm-1 (vibration
images (figure 2) reveals the fibrous nature of
modes of amide III). Absorption bands at
obtained chitosan (Antonino et al., 2017).
1149cm-1 (anti-symmetric stretching of C-O-C

Figure 1: Infrared (IR) spectral data of extracted chitosan identified by Fourier Transform
Infrared Spectroscopy.

Figure 2: Extracted chitosan morphology, at 10 µm and 5 µm, visualized by Scanning Electron

Microscopy.
Chitosan antifungal activity on Botrytis cinerea,
4 Chitosan effect on strawberries spoilage fungi Aspergillus niger, Rhizopus stolonifer, and
growth
28
Penicillium spp. mycelial growth was evaluated on
PDA solid medium. The following figure
represents the growth rates (µ on day-1) related to
chitosan concentrations (Figure 3). To begin this
study, we developed growth kinetics of molds
isolated from spoiled strawberries. Then we
calculated the growth rates (µ on day-1) evaluated
in the presence and absence of chitosan. The first
observation shows that growth on the culture
medium of molds is very important compared to
that obtained on damaged fruits. This is explained
by the positive contribution of fruit phenolic
compounds, which must be taken into
Aspergillus niger
Rhizopus stolonifer
Figure 3: Growth rates (µ) of fungi according to chitosan concentrations.
Chitosan, a deacetylated derivative of chitin, is a
potential antimicrobial substance due to its good
biocompatibility, biodegradability, non-toxicity,
and abundant availability (Chuan et al., 2019 ;
Shariatinia, 2019 ; Qin et al., 2020). This
biopolymer has been shown to have a broad
spectrum of antifungal activity against a large
variety of foodborne pathogens (Duan et al., 2019
; Abd El-Hack et al., 2020).
Many studies have clearly indicated that chitosan
(positively charged) can bind to fungi or bacteria
(negatively charged) cell surface, producing
plasma membrane permeabilization thereby
causing leakage of intracellular materials (Meng et
consideration to better develop an excellent
antimicrobial agent. At first, we considered it
helpful to work on the microcosms (Petri dishes)
before returning to growth evaluation on fruits.
Chitosan revealed a strong antifungal activity,
dose-dependent (from 0 to 3%), on all fungal
strains growth. Three per cent (3%) of chitosan
concentration gives a total inhibition of mycelial
growth. Chitosan extracted showed a negative
effect on fungal strains growth isolated from
spoiled strawberries. This biopolymer can
significantly reduce the microbial load of
strawberry fruit deterioration.
Botrytis cinerea
Penicillium spp.
al., 2020 ; Verlee et al., 2017 ; Xing et al., 2017).
Results obtained by (Hua et al., 2019) indicated
that chitosan (3.4-342 kDa) significantly inhibited
spore germination and mycelial growth of gray
mold Botrytis cinerea. Dewi and Nur (2018)
reported that chitosan had an inhibitory effect on
Aspergillus spp. mycelial colonies growth.
A study performed by (Arceo-Martínez et al.,
2019) showed the antifungal effects of chitosan (5,
7.5, 10, and 15 mg mL-1), In vitro bioassays,
against all strawberry pathogens tested such as
Colletotrichum gloeosporioides, Colletotrichum
acutatum, Phytophthora spp., and Fusarium
oxysporum. Due to its unique biological properties,
29
chitosan has gained considerable attention in
recent decades as a reference polymer for the
development of active food packaging. The
application of this biopolymer for fruits
post-harvest treatment extends their shelf life by
improving visual appearance and preserving fruits
post-harvest quality considered highly perishable.
Chitosan effect on growth rate is translated
according to 2 models: a linear model reflecting a
specific resistance of Aspergillus niger and
Rhizopus stolonifer and a polynomial model
representing a sensitivity of Penicillium spp. and
Botrytis cinerea strains. This difference reflects a
structural difference in tested strains’ walls.
5 Growth rates mathematical modelling of
tested strains in response to chitosan
Molds strains with an excellent potential for
spoilage of the fruits studied were the subject of
mathematical modelling of their responses to
chitosan as an antimicrobial agent and strawberry
fruits as the matrix used. This information will
provide some answers to make predictive
mathematical modelling later. The following table
(Table 1) illustrates growth rates mathematical
modelling of tested strains, which are isolated
from spoiled strawberries.
From this illustration, it can be concluded that the
growth rate mathematical model depends on
taxonomic position tested strain. Studies are in
progress to further detail these mathematical
models. In the second part of this task, we will
study the Monod growth rate model (Luong,
1987) according to the substrate in culture media
and fruits (Eq. 2).

Table 1: Growth rates mathematical modelling of tested strains in response to chitosan.

Strains Equation Mathematical model R²*
Aspergillus niger µ = -0,4737C + 1,4454 Linear 0,9948
Rhizopus stolonifer µ = -0,5721C + 1,6382 Linear 0,9908
2
Botritis cineria µ = 0,1564C - 0,9595C + 1,511 Polynomial 0,9846
2
Penicillium spp. µ = 0,0426C - 0,5523C + 1,4138 Polynomial 0,9998
µ: Growth rate mathematical model; C: Chitosan concentrations; R²: Weighting coefficient.

𝑑𝑥 𝑆 antifungal activity on the mycelial growth of

𝑑𝑡
= 𝑥µ𝑚 𝐾𝑠+𝑆
(2)
Also according to the presence of inhibitors gives
other models like the Haldane growth kinetics
model (Eq. 3) (Lokshina et al., 2001).
𝑑𝑥 𝑆
𝑑𝑡
= 𝑥µ𝑚 2
𝑆
(3)
𝐾𝑠+𝑆+ 𝐾
𝑖 coating solution to prolong the conservation of
The nature of the growth models of tested strains
can be explained by the Monod model in the
absence of chitosan and the Haldane model in the
presence of chitosan.
CONCLUSION
The development of rots caused by fungal
pathogens is one of the major causes of
post-harvest losses in strawberries. Chitosan as a
biological polysaccharide has received much
attention due to the impending problems
associated with chemical antimicrobial agents. In
this study, FTIR confirms the presence of the
amino group (-NH2) in the obtained chitosan with
a deacetylation degree (DD) of 83%. SEM of
chitosan, extracted by citric acid, reveals its
smooth surface and fibrous structures.
Chitosan extracted showed a negative effect on
fungal strain's growth isolated from spoiled
strawberries. Chitosan revealed significant
Aspergillus niger, Rhizopus stolonifer, Penicillium
spp., and Botrytis cinerea strains known as food
pathogens and post-harvest spoilage
microorganisms of strawberries. As a perspective,
we plan to extend our study by a post-harvest
treatment of strawberries with chitosan as a
these fruits considered sensitive during storage.
AUTHOR CONTRIBUTIONS
All authors listed respectively; Abir EL-ARABY,
Lahsen El GHADRAOUI, and Faouzi ERRACHIDI,
have made a substantial contribution to the work
and approved it for publication.
ACKNOWLEDGEMENTS
This work was supported by the PRIMA project
(Enhancing Mediterranean Fresh Produce
Shelf-Life using Sustainable Preservative
Technologies and Communicating Knowledge on
Dynamic Shelf-Life using Food Cloud Services and
Predictive Modeling “BIOFRESHCLOUD”).
REFERENCES
Abd El-Hack, M. E., El-Saadony, M. T., Shafi, M. E.,
Zabermawi, N. M., Arif, M., Batiha, G. E., Khafaga,
A. F., Abd El-Hakim, Y. M., & Al-Sagheer, A. A.
(2020). Antimicrobial and antioxidant properties

30
 of chitosan and its derivatives and their
applications : A review. International Journal of
Biological Macromolecules, 164, 2726‑2744.
https://doi.org/10.1016/j.ijbiomac.2020.08.153
Abdipour, M., Sadat Malekhossini, P., Hosseinifarahi,
M., & Radi, M. (2020). Integration of UV
irradiation and chitosan coating : A powerful
treatment for maintaining the postharvest quality
of sweet cherry fruit. Scientia Horticulturae, 264,
109197.
https://doi.org/10.1016/j.scienta.2020.109197
Antonino, R. S. C. M. D. Q., Lia Fook, B. R. P., De
Oliveira Lima, V. A., De Farias Rached, R. Í., Lima,
E. P. N., Da Silva Lima, R. J., Peniche Covas, C. A., &
Lia Fook, M. V. (2017). Preparation and
Characterization of Chitosan Obtained from
Shells of Shrimp (Litopenaeus vannamei Boone).
Marine Drugs, 15(5), Article 5.
https://doi.org/10.3390/md15050141
Arceo-Martínez, M. T., Jiménez-Mejía, R.,
Salgado-Garciglia, R., Santoyo, G., López-Meza, J.
E., & Loeza-Lara, P. D. (2019). In vitro and in vivo
anti-fungal effect of chitosan on post-harvest
strawberry pathogens. Agrociencia (Montecillo),
53(8), 1297‑1311.
Betchem, G., Johnson, N. A. N., & Wang, Y. (2019).
The application of chitosan in the control of
post-harvest diseases : A review. Journal of Plant
Diseases and Protection, 126(6), 495‑507.
https://doi.org/10.1007/s41348-019-00248-2
Cazón, P., & Vázquez, M. (2019). Applications of
Chitosan as Food Packaging Materials. In G. Crini
& E. Lichtfouse (Éds.), Sustainable Agriculture
Reviews 36 : Chitin and Chitosan : Applications in
Food, Agriculture, Pharmacy, Medicine and
Wastewater Treatment (p. 81‑123). Springer
International Publishing.
https://doi.org/10.1007/978-3-030-16581-9_3
Chuan, D., Jin, T., Fan, R., Zhou, L., & Guo, G. (2019).
Chitosan for gene delivery : Methods for
improvement and applications. Advances in
Colloid and Interface Science, 268, 25‑38.
https://doi.org/10.1016/j.cis.2019.03.007
Cosme, F., Pinto, T., Aires, A., Morais, M. C., Bacelar,
E., Anjos, R., Ferreira-Cardoso, J., Oliveira, I.,
Vilela, A., & Gonçalves, qu. (2022). Red Fruits
Composition and Their Health Benefits—A
Review. Foods, 11(5), Article 5.
https://doi.org/10.3390/foods11050644
Dewi, R., and Muhammad Nur, R. (2018). Antifungal
activity of chitosan on Aspergillus spp. Int. J.
Bioeng. Biotechnol. 2, 24–30.
Duan, C., Meng, X., Meng, J., Khan, Md. I. H., Dai, L.,
Khan, A., An, X., Zhang, J., Huq, T., & Ni, Y. (2019).
Chitosan as A Preservative for Fruits and
Vegetables : A Review on Chemistry and
Antimicrobial Properties. Journal of Bioresources
and Bioproducts, 4(1), 11‑21.
https://doi.org/wanf
Duarte, M. L., Ferreira, M. C., Marvão, M. R., &
Rocha, J. (2002). An optimised method to
determine the degree of acetylation of chitin and
chitosan by FTIR spectroscopy. International
Journal of Biological Macromolecules, 31(1), 1‑8.
https://doi.org/10.1016/S0141-8130(02)0039-9
Hua, C., Li, Y., Wang, X., Kai, K., Su, M., shi, W., Zhang,
D., & DEX, Y. (2019). The effect of low and high
molecular weight chitosan on the control of gray
mold (Botrytis cinerea) on kiwifruit and host
response. Scientia Horticulturae, 246, 700‑709.
https://doi.org/10.1016/j.scienta.2018.11.038
Jiang, Y., Yu, L., Hu, Y., Zhu, Z., Zhuang, C., Zhao, Y., &
Zhong, an. (2020). The preservation performance
of chitosan coating with different molecular
weight on strawberry using electrostatic spraying
technique. International Journal of Biological
Macromolecules, 151, 278‑285.
https://doi.org/10.1016/j.ijbiomac.2020.02.169
Kandile, N. G., Zaky, H. T., Mohamed, M. I., Nasr, A. S.,
& Ali, Y. G. (2018). Extraction and
Characterization of Chitosan from Shrimp Shells.
Open Journal of Organic Polymer Materials, 8(3),
Article 3.
https://doi.org/10.4236/ojopm.2018.83003
Lokshina, L. Ya., Vavilin, V. A., Kettunen, R. H.,
Rintala, J. A., Holliger, C., & Nozhevnikova, A. N.
(2001). Evaluation of kinetic coefficients using
integrated monod and haldane models for
low-temperature acetoclastic methanogenesis.
Water Research, 35(12), 2913‑2922.
https://doi.org/10.1016/043-1354(00)00595-9
Luong, J. H. T. (1987). Generalization of monod
kinetics for analysis of growth data with
substrate inhibition. Biotechnology and
Bioengineering, 29(2), 242‑248.
https://doi.org/10.1002/bit.260290215
Meng, D., Garba, B., Ren, Y., Yao, M., Xia, X., Li, M., &
Wang, Y. (2020). Antifungal activity of chitosan
against Aspergillus ochraceus and its possible
mechanisms of action. International Journal of
Biological Macromolecules, 158, 1063‑1070.
https://doi.org/10.1016/j.ijbiomac.2020.04.213
Mohammadi, L., Ramezanian, A., Tanaka, F., &
Tanaka, F. (2021). Impact of Aloe vera gel coating
enriched with basil (Ocimum basilicum L.)
essential oil on postharvest quality of strawberry
fruit. Journal of Food Measurement and
Characterization, 15(1), 353‑362.
https://doi.org/10.1007/s11694-020-00634-7
Povea, M. B., Monal, W. A., Cauich-Rodríguez, J. V.,
Pat, A. M., Rivero, N. B., & Covas, C. P. (2011).
Interpenetrated Chitosan-Poly(Acrylic
31
 Acid-Co-Acrylamide) Hydrogels. Synthesis,
Characterization and Sustained Protein Release
Studies. Materials Sciences and Applications,
02(06), Article 06.
https://doi.org/10.4236/msa.2011.26069
Qamar, J., Ejaz, S., Anjum, M. A., Nawaz, A., Hussain,
S., Ali, S., & Saleem, S. (2018). Effect of Aloe vera
Gel, Chitosan and Sodium Alginate Based Edible
Coatings on Postharvest Quality of Refrigerated
Strawberry Fruits of cv. Chandler. Journal of
Horticultural Science & Technology, 8‑16.
https://doi.org/10.46653/jhst180101008
Qin, Y., Li, P., & Guo, Z. (2020). Cationic chitosan
derivatives as potential antifungals : A review of
structural optimization and applications.
Carbohydrate Polymers, 236, 116002.
https://doi.org/10.1016/j.carbpol.2020.116002
Quintana, S. E., Llalla, O., García-Risco, M. R., &
Fornari, T. (2021). Comparison between essential
oils and supercritical extracts into chitosan-based
edible coatings on strawberry quality during cold
storage. The Journal of Supercritical Fluids, 171,
105198.
https://doi.org/10.1016/j.supflu.2021.105198
Shariatinia, Z. (2019). Pharmaceutical applications
of chitosan. Advances in Colloid and Interface
Science, 263, 131‑194.
https://doi.org/10.1016/j.cis.2018.11.008
Shigemasa, Y., Matsuura, H., Sashiwa, H., & Saimoto,
H. (1996). Evaluation of different absorbance
ratios from infrared spectroscopy for analyzing
the degree of deacetylation in chitin.
International Journal of Biological
Macromolecules, 18(3), 237‑242.
https://doi.org/10.116/0141-8130(95)01079-3
Trinetta, V., McDaniel, A., G. Batziakas, K., Yucel, U.,
Nwadike, L., & Pliakoni, E. (2020). Antifungal
Packaging Film to Maintain Quality and Control
Postharvest Diseases in Strawberries. Antibiotics,
9(9), Article 9.
https://doi.org/10.3390/antibiotics9090618
Verlee, A., Mincke, S., & Stevens, C. V. (2017). Recent
developments in antibacterial and antifungal
chitosan and its derivatives. Carbohydrate
Polymers, 164, 268‑283.
https://doi.org/10.1016/j.carbpol.2017.02.001
Xing, K., Liu, Y., Shen, X., Zhu, X., Li, X., Miao, X., Feng,
Z., Peng, X., & Qin, S. (2017). Effect of O-chitosan
nanoparticles on the development and
membrane permeability of Verticillium dahliae.
Carbohydrate Polymers, 165, 334‑343.
https://doi.org/10.1016/j.carbpol.2017.02.063
32
CONFERENCE PAPER Walid Janati et al., 2022, 33-38

Phosphate Solubilizing Bacteria: Value and Response in Two Species of

Papilionaceae Seeds
Walid Janati * , Karima Mikou, Lahsen EL Ghadraoui, and Faouzi Errachidi
Functional Ecology and Environment Engineering Laboratory, Faculty of Science and Technology, Sidi
Mohamed Ben Abdellah University, Fez, Morocco

Low-cost and environmentally friendly agricultural practices have received

increasing attention in recent years. Indeed, microbial inoculants based on
phosphate (P) solubilizing bacteria (PSB) appear to be an emerging
biological solution to increase P availability in the plant rhizosphere. The
present study was conducted on PSBs isolated from soils located at different
bioclimatic stages in Morocco, from various rhizospheres of leguminous
cultures to assess PSBs response on two Papilionaceae species (Pea and
Broad Bean). Phosphate solubilization potency was assessed by the
vanadate-molybdate method. Of all tested strains (64), the AS61 isolate
performed best in NBRIP liquid medium, with 0.148 g.L-1 of soluble P.
Sterilized Pea and Broad Bean seeds were inoculated with six strains (109
CFU mL-1), individually and incubated at 28 ± 2˚C in Petri dishes containing
0.25% agar water. Seedlings treated with PSBs promoted germination and
seedling vigour between 7% and 15%, compared to non-inoculated control.
Therefore, this study reveals the potential effect of PSBs to support
rhizobacterium growth-promoting traits.
* (Graham and Vance, 2003; Bessada et al., 2019).
Corresponding Author
Today's inorganic fertilizers have been widely
Name: Walid Janati
used to meet agriculture, depending mainly on P
Email: walid.janati@usmba.ac.ma
supplied due to their importance in N2 fixation
Phone: +212 620847003 and legume growth.
ISBN: 9788367405256 Natural fertilization represents a promising way to
DOI: 10.2478/9788367405256-005 overcome such problems through sustainable
improvement in plant growth (Pachapur et al.,
Published by Sciendo. © 2022 Walid Janati et al. This is an 2020). Plant Growth Promoting Rhizobacteria
open access article licensed under the Attribution 4.0 (PGPR) may account for most of the world's
International microbial biodiversity and play a mediating role in
(https://creativecommons.org/licenses/by/4.0/)
critical ecosystem processes such as nutrient
dynamics (Khan et al., 2010; Singh et al., 2014).
INTRODUCTION However, Anand et al. (2016) pointed out that
PSBs are also part of this group due to their ability
As the world's population increases, so does the to efficiently solubilize inorganic P forms, as well
demand for food, causing severe food insecurity as the production of siderophores or
coupled with rising food prices, declining food phytohormones and their ability to fix N2.
supply, and reduced land and water resources for In addition, Malhotra et al. (2018) reported that P
food production. Legumes rank second after enhances seed germination, root development,
cereals in terms of human nutritional stem strength, as well as flowers and seed
requirements, providing 33% of dietary protein, formation, together with crop yield and quality.
as well as carbohydrates, fibre, minerals, vitamins, Hence, P is essential for all developmental stages,
and several phytochemicals, mainly offered by from germination to maturity. Moreover, Suleman
Broad Bean, Pea, Chickpea, and Beans grain et al. (2018) indicated that PSB-treated wheat
33
seedlings promoted germination and seedling
vigour (11% increases) compared to
assess the ability of several PSBs to enhance Broad
Bean (Vicia faba) and Pea (Pisum sativum) growth
through increasing germination rate and root
length.
MATERIALS AND METHODS
1 Phosphate solubilizing bacteria isolation
Phosphate solubilizing bacteria were isolated
through serial dilution of the soil suspension. Ten
grams of soil were placed in 100 mL of sterile
phosphate buffer saline (PBS) with a pH of 7.2 and
shaken at 180 rpm for one hour. Then successive
dilutions were done to 10-6. Each serial dilution
was spread evenly on National Botanical Research
in P medium (NBRIP) agar plates (glucose 10 g,
(NH4)2SO4 0.1 g, MgSO4.7H2O 0.25 g, Ca3PO4 5 g,
KCl 0.2 g, and 15 g agar with a pH of 6.8, were
dissolved in sufficient quantity to 1 L distilled
water (DW)) (Nautiyal, 1999) with a 100 µL
aliquot.
After that, the NBRIP medium was supplemented
with cycloheximide to inhibit fungal growth. For
seven days, NBRIP plates were incubated at 28 ±
2°C. PSB colonies showed clear zones formed by P
solubilization were isolated. All morphologically
different colonies were isolated, purified, and
sub-culturing in NBRIP plates to ensure their
solubilization capacity, and finally conserved in
Tryptic Soy Agar (TSA) medium before
cryopreservation in sterile glycerol (20%) at
-80°C.
2 Crops used in the experiment
Rhizobacteria germination ability and root length
enhancement were evaluated on two legume
crops; the Feve-Aguadulce Broad Bean variety
(Vicia faba) and PP-Onward Pea variety (Pisum
sativum). The experiment was carried out under
climate-controlled conditions in Petri dishes.
Seeds were obtained from A.PHY.SEM Company of
agriculture plant section.
3 Phosphate solubilizing activity
A quantitative test was done to measure the PSBs
solubilization capacity of Tricalcium phosphate
(TCP) in a liquid NBRIP medium. After incubation
of a pre-culture for a few days (from 1 to 7 days)
in a shaker incubator (at 28 °C, 150 rpm), a
volume of bacterial suspensions was taken and
centrifuged (12000 rpm for 15 min). Supernatants
were recovered and analyzed for their assimilable
P content on the 7th day, by the ascorbic acid
colourimetric method (Murphy & Riley, 1962; Joe
et al., 2018), and then the pH medium was
non-inoculated seeds. Thus, the present study
aims to
measured.
One mL of supernatant was mixed with 160 µL of
the reaction solution. The Optical Density (OD)
was measured at 880 nm after a few minutes of
incubation at room temperature, with three
replications per isolate. Soluble P content was
calculated based on a concentration range
prepared with standard solutions of KH2PO4 with
P concentrations ranging from 0.1 to 1 mg.l-1.
4 Germinating seed bioassay
For seedling bioassay, six bacterial isolates were
grown in TSA agar medium at 28 °C for 24 h.
Broad Bean (Vicia faba) and Pea (Pisum sativum)
seeds were surface sterilized with sodium
hypochlorite (2%) and washed five times with
distilled sterile (DW) (Suleman et al., 2018; Valetti
et al., 2018). Sterilized seeds were inoculated with
bacteria using a suspension containing 109
bacteria/mL, separately.
Five pre-germinated seeds were used in each Petri
Dish. All treatments were performed in three
replicates and incubated at 28 ± 2 °C. After seven
days post-inoculation, radicals were more than
seed size and germination percentage was
registered (Figure 1). The root length, expressed
in mm, of each seedling, was measured seven days
after incubation and compared to the
non-inoculated seeds. All used isolates were found
to enhance germination and root length
significantly. For this purpose, Bergey's manual
(Systematic Bacteriology, 2nd ed., vol. 5, eds., 2012)
was used to identify isolates based on their
morphological, physiological and biochemical
characteristics.
5 Statistical analysis
SPSS software was used to analyze the data, and
results were presented as means ± standard
deviation of three replications. Furthermore, the
findings were examined by ANOVA, while the
comparison of means was carried out by Duncan's
multiple range test at p ≤ 0.05.
RESULTS
Several strains were selected based on colony
morphology and significant P solubilization halo
as a qualitative indication of PSB. Bacterial
colonies that grew without forming a halo were
also isolated to assess solubilization capacity
(Figure 1). As a result, 134 isolates were selected,
and transplanted onto NBRIP medium five times
to be purified. Of 134 isolates, 64 were designated
as PSB from their P solubilization on NBRIP liquid
34
medium.

Figure 1: A) Solubilization halo of PSBs isolated on NBRIP medium containing TCP. B) Broad Bean
germination. C) Pea germination.
Quantitative measurement of P release by the with a parallel decrease of pH culture medium (pH
isolates in NBRIP broth medium showed a higher 4.3). Compared to the commercial control, 0.036
P solubilization capacity associated with a g.L-1 of P was solubilized in the culture medium
decrease in pH medium when compared to control with (pH 5.74). The amounts of Soluble-P and pH
in TCP-containing medium. Isolate AS61 is the variation are presented in (Figure 2), with
most effective in solubilizing P with a P significant variations among different isolates (p ≤
concentration of 0.036 g.L-1, and this coincides 0.05).

Figure 2: Solubilization capacity of TCP during seven days in liquid medium by the isolated
bacteria. Data represented by the same letter are not significantly different according to the
Duncan test performed (P < 0.05).
A plate assay was carried out to study the P Broad Bean inoculated seeds better than Pea
solubilizing bacteria effect at the early seedling inoculated seeds (Figure 3). Our results showed a
stage of Pea and Broad Bean. All strains show a significant relationship between root length, root
significantly positive impact by promoting Pea and number, and germination percentage of inoculated
Broad Bean seedling growth. The germination seeds. Root length and root number increased
percentage and root length compared to both considerably with increasing germination
positive (commercial-isolate) and negative percentage, which explains a strong positive
(non-inoculated) control were increased. The correlation (R² = 0.9239) (Figure 4). The classical
maximum hypocotyl and radical length results isolates identified were carried out following
were observed in seeds inoculated, with the AS53 morphological, physiological, and biochemical
strain followed by the AS61 strain. Besides that, characteristics according to Bergey’s Manual,
the present results showed a significant response which show that our isolates belong to Bacillus,
regarding germination rate and root length of Pseudomonas, and Rhizobium genus.

35
Figure 3: Roots length of both Pea and Broad Bean inoculated seeds. Data represented with the
same letter are not significantly different according to the Duncan test performed (P < 0.05).

Figure 4: Germination percentage and rooting number of Pea and Broad Bean seeds inoculated
with different PSB isolates. Data represented with the same letter are not significantly different
according to the Duncan test performed (P < 0.05).
DISCUSSION the P concentration was 0.148 g.L-1. These results
agree with Khan et al. (2010) and Anand et al.,
In the present investigation, studies were focused
2016 studies which report that PSBs can solubilize
on evaluating the effect of several PSBs to promote
insoluble inorganic P compounds, such as TCP,
legume growth by increasing germination
dicalcium P (DCP), hydroxyapatite, and RP.
percentage. PSB isolates used in this work were
Phosphate solubilization is accompanied by a pH
able to increase P availability in an NBRIP liquid
medium decrease compared to the control. In
medium containing TCP as the only source of P. Our
addition, we note that as the pH decreases, P
findings support the idea that PSBs are
content increases, which is emphasized by Batool &
characterized by their ability to readily and
Iqbal. (2019), which indicates a strong correlation
efficiently solubilize inorganic P forms (Elkoca et
between P solubilization and pH decrease.
al., 2008; Anand et al., 2016; M. A. Khan et al.,
Likewise, Alori et al. (2017) observed a negative
2020).
correlation between the amount of P solubilized by
Maximum P solubilization was observed in a B. cepacia (SCAUK0330) and pH drop, which leads
TCP-containing medium with AS61 isolate, where to an increase in P solubilization. In the seed
36
germination experiment, all tested isolates showed
increasing results of hypocotyl and radical length,
especially seeds inoculated with AS53 followed by
AS61.
Our results correlate with Song et al. (2022), which
reported that after treatment with PSB strains, the
germination rate of maize seeds increased by
23.4%, 20.7% and 20.0%, respectively. In addition,
Wahyudi et al. (2011) signalled that the ability of
the isolates to increase plant growth in germinating
seed bioassay is highly related to the Indole Acetic
Acid (IAA) production, which was produced by
Bacillus sp. isolates. However, PSM biotechnology
offers an excellent opportunity to develop
environmentally friendly P biofertilizers that will
be used as supplements and, or alternatives to
chemical fertilizers to overcome agricultural
challenges imposed by continuously increasing
food demand (Sharma et al., 2013).
CONCLUSION
This study showed that P-solubilizing bacteria have
massive potential as bio-fertilizers. The
experiments on seed germination reveal that PSBs
could promote plant growth much faster than
untreated seeds, although those treated with PSBs
showed a higher germination rate and rooting
length. PSBs as biological fertilization and
sustainable improvement of agriculture is,
therefore, a promising way to overcome inorganic
fertilizers that have been widely used to meet
world food demand.
AUTHOR CONTRIBUTIONS
All authors listed have made a substantial, direct
and intellectual contribution to this work and
approved it for publication.
ACKNOWLEDGEMENT
The authors thank and appreciate all the support
provided to carry out this research.
REFERENCES
Alori, E. T., Glick, B. R., & Babalola, O. O. (2017).
Microbial Phosphorus Solubilization and Its
Potential for Use in Sustainable Agriculture.
Frontiers in Microbiology, 8.
https://www.frontiersin.org/article/10.3389/fm
icb.2017.00971
Anand, K., Kumari, B., & Mallick, M. A. (2016).
PHOSPHATE SOLUBILIZING MICROBES: AN
EFFECTIVE AND ALTERNATIVE APPROACH AS
BIOFERTILIZERS. 8(2), 5.
Batool, S., & Iqbal, A. (2019). Phosphate solubilizing
rhizobacteria as alternative of chemical fertilizer
for growth and yield of Triticum aestivum (Var.
Galaxy 2013). Saudi Journal of Biological
Sciences, 26(7), 1400–1410.
https://doi.org/10.1016/j.sjbs.2018.05.024
Bessada, S. M. F., Barreira, J. C. M., & Oliveira, M. B. P.
P. (2019). Pulses and food security: Dietary
protein, digestibility, bioactive and functional
properties. Trends in Food Science & Technology,
93, 53–68.
https://doi.org/10.1016/j.tifs.2019.08.022
Elkoca, E., Kantar, F., & Sahin, F. (2008). Influence of
Nitrogen Fixing and Phosphorus Solubilizing
Bacteria on the Nodulation, Plant Growth, and
Yield of Chickpea. Journal of Plant Nutrition, 31,
157–171.
https://doi.org/10.1080/01904160701742097
Joe, M. M., Deivaraj, S., Benson, A., Henry, A. J., &
Narendrakumar, G. (2018). Soil extract calcium
phosphate media for screening of
phosphate-solubilizing bacteria. Agriculture and
Natural Resources, 52(3), 305–308.
https://doi.org/10.1016/j.anres.2018.09.014
Khan, M. A., Asaf, S., Khan, A. L., Jan, R., Kang, S.-M.,
Kim, K.-M., & Lee, I.-J. (2020). Thermotolerance
effect of plant growth-promoting Bacillus cereus
SA1 on soybean during heat stress. BMC
Microbiology, 20(1), 175.
https://doi.org/10.1186/s12866-020-01822-7
Khan, M. S., Musarrat, J., & Zaidi, A. (Eds.). (2010).
Microbes for Legume Improvement. Springer
Vienna.
https://doi.org/10.1007/978-3-211-99753-6
Malhotra, H., Vandana, Sharma, S., & Pandey, R.
(2018). Phosphorus Nutrition: Plant Growth in
Response to Deficiency and Excess. In Plant
Nutrients and Abiotic Stress Tolerance (pp.
171–190).
https://doi.org/10.1007/978-981-10-9044-8_7
Murphy, J., & Riley, J. P. (1962). A modified single
solution method for the determination of
phosphate in natural waters. Analytica Chimica
Acta, 27, 31–36.
https://doi.org/10.1016/S0003-2670(00)88444-
5
Nautiyal, C. S. (1999). An e¤cient microbiological
growth medium for screening phosphate
solubilizing microorganisms. FEMS Microbiology
Letters, 6.
Pachapur, P. K., Pachapur, V. L., Brar, S. K., Galvez, R.,
Le Bihan, Y., & Surampalli, R. Y. (2020). Food
Security and Sustainability. In R. Surampalli, T.
Zhang, M. K. Goyal, S. Brar, & R. Tyagi (Eds.),
Sustainability (1st ed., pp. 357–374). Wiley.
https://doi.org/10.1002/9781119434016.ch17
Sharma, S. B., Sayyed, R. Z., Trivedi, M. H., & Gobi, T.
A. (2013). Phosphate solubilizing microbes:
37
 Sustainable approach for managing phosphorus
deficiency in agricultural soils. SpringerPlus, 2(1),
587. https://doi.org/10.1186/2193-1801-2-587
Singh, O., Gupta, M., Mittal, V., Kiran, S., Nayyar, H.,
Gulati, A., & Tewari, R. (2014). Novel phosphate
solubilizing bacteria ‘Pantoea cypripedii PS1’
along with Enterobacter aerogenes PS16 and
Rhizobium ciceri enhance the growth of chickpea
(Cicer arietinum L.). Plant Growth Regulation,
73(1), 79–89.
https://doi.org/10.1007/s10725-013-9869-5
Song, C., Wang, W., Gan, Y., Wang, L., Chang, X.,
Wang, Y., & Yang, W. (2022). Growth promotion
ability of phosphate‐solubilizing bacteria from
the soybean rhizosphere under maize–soybean
intercropping systems. Journal of the Science of
Food and Agriculture, 102(4), 1430–1442.
https://doi.org/10.1002/jsfa.11477
Suleman, M., Yasmin, S., Rasul, M., Yahya, M., Atta, B.
M., & Mirza, M. S. (2018). Phosphate solubilizing
bacteria with glucose dehydrogenase gene for
phosphorus uptake and beneficial effects on
wheat. PLOS ONE, 13(9), e0204408.
https://doi.org/10.1371/journal.pone.0204408
Valetti, L., Iriarte, L., & Fabra, A. (2018). Growth
promotion of rapeseed (Brassica napus)
associated with the inoculation of phosphate
solubilizing bacteria. Applied Soil Ecology, 132,
1–10.
https://doi.org/10.1016/j.apsoil.2018.08.017
Wahyudi, A. T., Astuti, R. P., Widyawati, A.,
Meryandini, A., & Nawangsih, A. A. (2011).
Characterization of Bacillus sp. Strains isolated
from rhizosphere of soybean plants for their use
as potential plant growth for promoting
Rhizobacteria. 7.
38
CONFERENCE PAPER Amr Farhan et al.2022, 39-44

Comparison of automatic and manual methods for analyzing heart rate

variability parameters
Amr Farhan 1,2 * , Mourad Rattal 1,2 , Badreddine Labakoum 1,2 , Elmaati Essoukaki 1,2 , Azeddine
Mouhsen 2 , and Aissam Lyazidi 1,2
1
Hassan First University of Settat, Radiation-Matter Instrumentation Laboratory (RMI), Settat,
Morocco.
2
Hassan First University of Settat, Higher Institute of Health Sciences (ISSS), Settat, Morocco.

Heart rate variability (HRV) is a simple, non-invasive measure to explore

the influence of the autonomic nervous system on the cardiovascular
system. The current study aims to validate the results obtained by BIOPAC
through the extraction of the ECG signal and measure the HRV in two
ways, the first by Acqknowledge software program and the second
manually. The levels of agreement and the relationship between the
time-domain parameters derived by BIOPAC and the manual calculation
were examined using a Pearson product-moment correlation and a
Bland-Altman plot. Materials and methods: The ECG signal of lead II was
recorded, and then the HRV time-domain was calculated using software
named Acqknowledge. To verify the validity of these results, we conducted
a comparative study by manually calculating the HRV time-domain, and
applied the Bland-Atman plot analysis as this method allows us to be sure
of your result in a reliable way. Results: The correlation coefficient r was
calculated for each parameter RMSSD (r=0.98), SDNN(r=0.98),
PNN50%(r=0.99) and had a positive coefficient between HRV measured.
The Bland-Altman plot reflects a good agreement between the
measurement’s methods used. Conclusion: The present comparative study
shows that the values of the two methods are similar and can be used
BIOPAC in wider research, even for diagnosis.

* decompress blood in a similar cardiac cycle.

Corresponding Author
cardiac cycle(Alberdi et al., 2016) . The ECG signal
Name: Amr Farhan
has a quasi-periodic pattern with a varying
Email: a.farhan@uhp.ac.ma
frequency with time, making it a useful tool for
Phone: +212633280045
various non-invasive biomedical applications such
ISBN: 9788367405256 heart rate estimate, heart rate monitoring,
emotion detection, biometric identification, and
DOI: 10.2478/9788367405256-006 cardiac abnormality diagnostics (Kumar et al.,
Published by Sciendo. © 2022 Amr Farhan et al. This is an 2020). Disposable Ag/AgCl electrodes are placed
open access article licensed under the Attribution 4.0 on the skin surfaces of the legs, arms, and chest to
International gather ECG data. Electrical impulses from various
(https://creativecommons.org/licenses/by/4.0/) areas of the heart can be detected using these
electrodes (Olkkonen, 2011). These electrodes can
detect electrical impulses from different parts of
INTRODUCTION the heart. The ECG provides heart rates and their
rhythms, whether the heartbeat is irregular or
The heart generates an electrical signal called an regular (Hammad et al., 2018). The time interval
electrocardiogram (ECG). The electrical impulse between successive heartbeats is defined as heart
drives the human heart's muscles to compress and
39
rate variability (HRV), and the Taskforce of the
European Society of Cardiology and the North
American Society of Pacing and Electrophysiology
set the criteria two decades ago (Guidelines et al.,
1996), (Kiran kumar et al., 2021). HRV analysis is
a technique for estimating cardiovascular
mechanisms in close contact with the
parasympathetic system, whose activity it
observes non-invasively since it is associated with
the heart rate and muscular activity of the body.
This fluctuation can offer an assessment of a
person's autonomic nervous system activity by
indicating the difference between subsequent
heartbeats, and the time difference between them
(Farhan et al., 2023; Malik et al., 1996; Mejía-Mejía
et al., 2020). It is one of the most simple,
non-invasive, and sensitive tests for assessing
autonomic changes in various functional and
clinical conditions. An increase in sympathetic
activity or a decrease in parasympathetic activity,
or both, has been interpreted as a decrease in HRV
(Maheshkumar et al., 2016). Linear and nonlinear
methods have been used for HRV analysis. There
are several methods for measuring HRV: time
domain, Spectral methods, Geometric methods. In
our study, we focused on the time domain method
as it is considered. The variation of RR intervals is
related to the standard HRV measurements stated
in the recommendations. The period from the ith
to the i+1st R peak is denoted by RRi. The average
interval is RR, and there are n intervals in
total(Hartikainen et al., 1996).
• RR - Mean NN interval (msec).
• SDNN - Standard deviation of NN interval
(msec).
• RMSSD - Root mean square of the successive
difference of NN interval (msec).
• pNN50 - The percentage of numbers of pairs of
NN that is longer than 50(Vega-Martínez et al.,
2014).
The average heart rate and heart rate variations
per second affect each time measurement based
on absolute differences of RR intervals. Absolute
differences must be steady in order to get the
same HRV. Even if your average heart rate rises.
The main aim of this study is the validation of
automatic analysis by comparison with the
manual analysis of HRV, which in the future will
allow working on many patients for long, reliable,
and reproducible recording periods.
METHODS
Participants
The study included ten volunteers (5 men and 5
women) within the laboratory located at the
university from the doctoral students between 24
- 35. The investigation began after obtaining the
consent of the volunteers. The medical record of
each volunteer, medicines, and routine activities
in daily life was taken into consideration. Since
they are researchers, it was easy to deal with
them and assess their condition and explain the
necessary information of the nature of the study
and its purpose with guidelines of the Clinical
Research, as they were instructed with some
instructions such as not drinking alcohol and
medication the day before obtaining the data, as
well as not smoking before getting the data
directly, as well as muscle effort and exercising the
day before Data acquisition (Table1).
Table 1. Demographic parameters of the volunteers
Variables Mean-+SD Median 95%CI
Age (YRS) 30 ± 4.12 31 26.19-33.81
Weight (KGS) 78.86 ± 10.87 78 68.81-88.91
Height (cm) 172.29 ± 4.15 170 168.45- 176.13
BMI (Kg/m2) 26.52 ± 3.15 26.99 23.61-29.44
*Where BMI: Body Mass Index (Kg per square meter)
Data acquisition
ECG data were taken in the sitting position from
the ten volunteers. The sitting position was
chosen to reduce any possible parasympathetic
saturation, often seen in people with low resting
heart rates(Plews et al., 2013). For this study, the
ECG data were obtained using BIOPAC MP160
equipment, a data acquisition system. The signal
was recorded, and lead II was taken because it
gives us a higher R peak than leads I and III. For
obtained lead II, we connect the positive terminal
to the left leg (LL), the negative terminal to the
right hand (RA), and the ground to the right leg
(RL). We recorded the signal in the short-term.
Ten volunteer signals were obtained for two
minutes. These data were used in the HRV
analysis of each volunteer.
Method of analysis
We analyzed HRV with the time domain. A
time-domain parameter was chosen to reflect the
variations in vagal modulation. It was preferred
because of its more excellent reliability in
revealing parasympathetic activity than other
power spectral density indices(Al Haddad et al.,
2011). The static analysis was based on the
difference between the successive R to R peaks as
shown below, and from this, we can calculate the
different parameters (Figure 1).
To measure HRV, we used the Acqknowledge
software, which is attached to the BioPac system
(MP160, USA), versus a manual evaluation of the
parameters (RMMSD-SDNN-PNN50) calculated
every 10 seconds. By using the following
equations(Vollmer, 2015):
40
 𝑛−1
1 2
𝑅𝑀𝑆𝑆𝐷 = 𝑛−1
∑ (𝑅𝑅𝑖 + 1 − 𝑅𝑅𝑖) (1)
𝑛=1
1
𝑛 2
𝑆𝐷𝑁𝑁 = 𝑛−1
∑ (𝑅𝑅𝑖 − 𝑅𝑅) (2)
𝑛=1
𝑝𝑁𝑁50 = 𝑃( |𝑅𝑅𝑖 + 1 − 𝑅𝑅𝑖| > 50𝑚𝑠 ) (3)
Figure: Successive R-R intervals were used to measure HRV
Statistical analysis
The mean and standard deviation of continuous
variables are given. To compare the discrepancies
in measures, a one-sample t-test was performed.
We used Bland-Altman technique and standard
linear regression to test method agreement, and
Pearson product-moment correlation analysis to
calculate the coefficient of correlation (CC). A
correlation coefficient was chosen to indicate an
acceptable level of relationship(Bunce, 2009;
Errotta et al., 2017) . The statistical analysis was PNN50). The correlation findings for the
performed using Microsoft Office Excel 2019. The
Bland-Altman (B&A) plot was developed by Bland
and Altman to depict the agreement between two
quantitative data by creating limits of agreement
(Doğan, 2018). The mean and standard deviation
(SD) of the discrepancies between two
measurements are used to compute these
statistical limits. They employed a graphical
technique to test their assumptions about the
normalcy of differences and other distributions.
properties(Altman & Bland, 2017). The resultant
graph is a scatter plot XY, with the Y-axis
representing the difference between the two
paired measurements (A-B), and the X-axis
representing the average of these data ((A+B)/2).
In other words, the difference between the two
paired measurements is plotted against the mean
of the two measurements(Giavarina, 2015). B&A
suggested that 95% of the data points be within ±
2s standard deviations of the mean difference.
This is the most typical way to plot the B&A
method, however the differences can alternatively
For each parameter, we utilized Bland-Altman plot
analysis, which allowed us to compare the
findings collected and plotted.
be shown as percentages or ratios, and the first or
second technique can be used instead of the mean
of both ways (Shaffer & Ginsberg, 2017). The bias
is calculated by subtracting the value determined
by one technique from the value determined by
the other.
RESULTS
We can see from the findings that all variables had
a p-value of less than 0.05. (RMSSD, SDNN,
characteristics obtained from the subjects'
automated and manual ECG readings are shown in
table.2. The time-domain parameters (RMSSD,
SDNN, and pNN50) show a strong correlation (r >
0.99) between the Automatic and manual
measurement. When comparing the two
approaches for calculating the same index for a
specific person, we found that the mean index of
the HRV parameters had statistically comparable
Table 2. Results of time-domain parameters
Variable time Correlation 95%CI
domaine coefficient (r)
RMSSD 0.99 1.94 - 4.2
SDNN 0.99 2.11 - 4.05
PNN50% 1.0 0.01- 0.58
Bland-Altman analysis of all features calculated
from the ECG shows close agreement for all
parameters SDNN, RMSSD, and pNN50,
respectively (Figure 2).
41
DISCUSSION measuring vagal activity reflected in HRV. PNN50
is strongly connected to both PNS and RMSSD
The main conclusion of this study is that the
observations(Shaffer & Ginsberg, 2017). The
parameters evaluated automatically using BIOPAC
correlation coefficient we observed (r=0.99,
and calculated manually have acceptable levels of
CI95%=2.11 - 4.05, p<0.05; r=0.99, CI95%=1.94 -
agreement. SDNN reflects sympathetic nervous
4.2, p<0.05; r=1.0, CI95%==0.01- 0.58, p<0.05) for
system (SNS) and parasympathetic nervous
SDNN, RMSSD, PNN50% respectively.
system activity. However, respiratory sinus
arrhythmia is regarded to be the predominant
source of variance in short-term recordings, and
RMSSD is the primary time-domain measure for

42
 Figure 2: Bland–Altman plots of time-domain indexes, i.e. (a1) SDNN, (b1) RMSSD, and (c1)
pNN50% showing mean values indexes against their difference and linear regression, (a2) SDNN,
(b2) RMSSD, and (c2) PNN50% showing automatic measurement against manual measurement.
MATLAB and are validated by Kubios(Kumar et
Demonstration of an extremely significant
al., 2020). also, other software is specialized for
relationship between BioPac and manual
HRV analysis like Kubios, which is used to validate
measurement. The results of the Bland Altman
several studies like the elite HRV smartphone
analyses indicate a positive bias (3.28), standard
application with Kubios(Errotta et al., 2017).
deviation (5.54), and limit of agreement (-7.57 to
14.14) for SDNN, a bias (3.07), standard deviation
CONCLUSIONS
(5.33), and limit of agreement (-7.37 to 13.52) for
RMSSD, and a bias (0.285), standard deviation
(5.54), and limit of agreement (-7.57 to 14.14) for Time-domain parameters and other linear HRV
SDNN, a bias (3.07), standard deviation (5.33), analysis parameters give critical information
and limit of agreement (-7.37 to 13.52) for regarding the heart's responses. Heart rate
RMSSD, and a bias (0.285), standard deviation changes are linked to variations in HRV
(1.398), and limit of agreement (-2.45 to 3.02) for parameters in ECG data. This paper presents the
PNN50%. linear HRV parameters evaluated in two ways
automatically and manually. We observe that the
As shown in Fig.2, we can observe a good results obtained from the software and the
agreement because, firstly, the points lie along the manual results are comparable, so we can use it to
line of equality. secondly, 95% of the points lie investigate in a large number of patients and for
between the limits of agreement. Therefore, we long recordings. This model can be improvised to
have a good agreement between the two make predictions for wider amplitude and
measures. frequency ranges describing cardiac disorders
In this study, we compared HRV assessment and arrhythmia. The current system uses
parameters of 10 healthy volunteers obtained temporal features of HRV that can be used to
automatically by the Acqknowledge software examine the ANS, and classify and predict ECG
attached to the BioPac MP160 and the manual arrhythmia signals.
calculation of these same parameters. To perform
clinical studies of HRV for a more significant ACKNOWLEDGEMENTS
number of patients and to help in the diagnosis,
the automatic analysis using the BioPac system REFERENCES
allows evaluating longer recordings, to have Al Haddad, H., Laursen, P. B., Chollet, D., Ahmaidi,
reproducible results, and to save time. Moreover, S., & Buchheit, M. (2011). Reliability of resting
we found that it can be used very judiciously. and postexercise heart rate measures.
Other studies use the BioPac MP36 to measure International Journal of Sports Medicine, 32(8),
HRV, and they found it capable of making the 598–605.
diagnosis. They validated the simple analogue https://doi.org/10.1055/s-0031-1275356
amplifier with a PC-based sound card by the Alberdi, A., Aztiria, A., & Basarab, A. (2016).
BioPac MP36(Maheshkumar et al., 2016). Towards an automatic early stress recognition
Another study uses the BioPac MP45 to extract system for office environments based on
ECG signal, and they have analyzed the HRV by multimodal measurements: A review. Journal of

43
 Biomedical Informatics, 59, 49–75.
https://doi.org/10.1016/j.jbi.2015.11.007
Altman, D. G., & Bland, J. M. (2017). Assessing
agreement between methods of measurement.
Clinical Chemistry, 63(10), 1653–1654.
https://doi.org/10.1373/clinchem.2016.26887
0 Kumar, P., Das, A. K., Prachita, & Halder, S. (2020).
Bunce, C. (2009). Correlation, Agreement, and
Bland-Altman Analysis: Statistical Analysis of
Method Comparison Studies. American Journal
of Ophthalmology, 148(1), 4–6.
https://doi.org/10.1016/j.ajo.2008.09.032
Doğan, N. Ö. (2018). Bland-Altman analysis: A
paradigm to understand correlation and
agreement. Turkish Journal of Emergency
Medicine, 18(4), 139–141.
https://doi.org/10.1016/j.tjem.2018.09.001
Errotta, A. N. S. P., Eklin, A. N. T. J., Ives, B. E. N. A.
H., Eanwell, L. E. A. H. E. M., & Arburton, D. A. E.
R. W. (2017). V e hrv s a e h r v f -b s.
2296–2302.
Farhan, A., Lyazidi, A., Elkettani, S., Labakoum, B.,
Rattal, M., & Mouhsen, A. (2023). Linear
Analysis of ECG Data Variability to Assess The
Autonomic Nervous System in Two Different
Body Positions. The Egyptian Journal of Hospital
Medicine, 90(1), 459–464.
https://doi.org/10.21608/ejhm.2023.279661
Giavarina, D. (2015). Understanding Bland Altman
analysis Lessons in biostatistics. Biochemia
Medica, 25(2), 141–151.
https://doi.org/10.11613/BM.2015.015
Guidelines, American, T. N., & Guidelines. (1996).
Guidelines Heart rate variability. European
Heart Journal, 17, 354–381.
https://doi.org/10.1161/01.CIR.93.5.1043
Hammad, M., Maher, A., Wang, K., Jiang, F., &
Amrani, M. (2018). Detection of abnormal heart
conditions based on characteristics of ECG
signals. Measurement: Journal of the
International Measurement Confederation,
125(May), 634–644.
https://doi.org/10.1016/j.measurement.2018.0
5.033
Hartikainen, J. E. K., Malik, M., Staunton, A.,
Poloniecki, J., & Camm, A. J. (1996). Distinction
between arrhythmic and nonarrhythmic death
after acute myocardial infarction based on heart
rate variability, signal-averaged
electrocardiogram, ventricular arrhythmias and
left ventricular ejection fraction. Journal of the
American College of Cardiology, 28(2), 296–304.
https://doi.org/10.1016/0735-1097(96)00169
-6
Kiran kumar, C., Manaswini, M., Maruthy, K. N.,
Siva Kumar, A. V., & Mahesh kumar, K. (2021).
Association of Heart rate variability measured
by RR interval from ECG and pulse to pulse
interval from Photoplethysmography. Clinical
Epidemiology and Global Health, 10(December
2020), 100698.
https://doi.org/10.1016/j.cegh.2021.100698
Time-domain HRV Analysis of ECG Signal under
Different Body Postures. Procedia Computer
Science, 167, 1705–1710.
https://doi.org/10.1016/j.procs.2020.03.435
Maheshkumar, K., Dilara, K., Maruthy, K. N., &
Sundareswaren, L. (2016). Validation of
PC–based sound card with biopac for
digitalization of ECG recording in short–term
HRV analysis. North American Journal of
Medical Sciences, 8(7), 307–311.
https://doi.org/10.4103/1947-2714.187150
Malik, M., Bigger, J. T., Camm, A. J., Kleiger, R. E.,
Malliani, A., Moss, A. J., & Schwartz, P. J. (1996).
Heart rate variability: Standards of
Measurement, Physiological. European Heart
Journal, 17, 354–381.
Mejía-Mejía, E., Budidha, K., Abay, T. Y., May, J. M., &
Kyriacou, P. A. (2020). Heart Rate Variability
(HRV) and Pulse Rate Variability (PRV) for the
Assessment of Autonomic Responses. Frontiers
in Physiology, 11(July), 1–17.
https://doi.org/10.3389/fphys.2020.00779
Olkkonen, J. (2011). DISCRETE WAVELET
TRANSFORMS - THEORY AND APPLICATIONS
Edited by Juuso Olkkonen. In Book.
Plews, D. J., Laursen, P. B., Stanley, J., Kilding, A. E.,
& Buchheit, M. (2013). Training adaptation and
heart rate variability in elite endurance athletes:
Opening the door to effective monitoring. Sports
Medicine, 43(9), 773–781.
https://doi.org/10.1007/s40279-013-0071-8
Shaffer, F., & Ginsberg, J. P. (2017). An Overview of
Heart Rate Variability Metrics and Norms.
Frontiers in Public Health, 5(September), 1–17.
https://doi.org/10.3389/fpubh.2017.00258
Vega-Martínez, G., Toledo-Peral, C.,
Alvarado-Serrano, C., Leija-Salas, L.,
Aztati-Aguilar, O. G., & De Vizcaya-Ruiz, A.
(2014). SDNN index of heart rate variability as
an indicator of change in rats exposed to fine
particles: Study of the impact of air pollution in
Mexico City. 2014 11th International Conference
on Electrical Engineering, Computing Science
and Automatic Control, CCE 2014, 124–127.
https://doi.org/10.1109/ICEEE.2014.6978319
Vollmer, M. (2015). A robust, simple and reliable
measure of heart rate variability using relative
RR intervals. Computing in Cardiology, 42(6),
609–612.
https://doi.org/10.1109/CIC.2015.7410984
44
 CONFERENCE PAPER Hamza Elbaylek and Soumia Ammor, 2022, 45-51

Dietary Transition and Colorectal Cancer: A Case-Control Study of Dietary

Factors in the Region Marrakech-Safi, Morocco
Hamza Elbaylek* and Soumia Ammor
Faculty of Science Semlalia, Department of biology, University Cadi Ayyad, Marrakech, Morocco.

In Morocco, the dietary transition impacted on the incidence of colorectal

cancer or CRC, currently it’s the first digestive cancer. Thus, the aims of our
work are to study the dietary risk factors in the Moroccan population. It is a
retrospective case-control study carried out in the Mohammed VI University
Hospital of Marrakech. During 7 months, the study included 160 cases and
160 controls. The results showed an association of the disease with the
consumption of commercial bread (OR=3.67 : IC95%= 1.55-8.67), Msseman
or Moroccan pancake (OR=4.304 : IC95%=1.32-16.37), Khlii (dried and
salted meat) (OR=4.167 : CI95%=1.601-10.846), Smen (rancid butter having
undergone butyric fermentation) (OR=4.832 : CI95%=1.866-12.514) with
an increased risk of CRC , while daily consumption of vegetables (OR=0.192 :
CI95%=0.066-0.561) , milk (OR=0. 176: IC95%=0.078-0.369) and white
meat (OR=0.14: IC95%=0.052-0.379) is associated with a reducing risk of
CRC. A balanced diet rich in fiber, vitamins and other micronutrients while
avoiding red meats and processed foods may prevent CRC.

*
Corresponding Author
INTRODUCTION
Name: Hamza Elbaylek
Email: During the last few years, Morocco has
Hamza.elbaylek@ced.uca.ma experienced a food transition. This transition is
Phone: +212 695 07 35 17 due to changes on several levels, such as
globalization and urbanization. The Moroccan diet
ISBN: 9788367405256 which is of Mediterranean type based on a large
DOI: 10.2478/9788367405256-007 consumption of fruits, vegetables and cereals, but
following the transition and the change of the way
Published by Sciendo. © 2022 Hamza Elbaylek and Soumia of life of the Moroccan population, the diet is
Ammor. This is an open access article licensed under the
directed more and more to the consumption of
Attribution 4.0 International
(https://creativecommons.org/licenses/by/4.0/) food outside the home. Who is characterize by
their richness in sugars and fat with the reduction
of the consumption of food of vegetable origin and

45
rich in micronutrients.
This dietary transition has had several
repercussions on the health of the Moroccan
population, with the increase of certain types of
disease or cancer, among them colorectal cancer
(CRC), According to a study conducted at the INO
(National Institute of Oncology) in Rabat, CRC
ranks first among digestive cancers with a
percentage of 40.8%. The incidence of CRC is
increasing, as evidenced by the evolution of the
incidence of this cancer in the Greater Casablanca
region, which increased from 7.3 cases/100000
inhabitants in 2004-2007 to 9.6 cases/10000
inhabitants in 2008-2012 (Cancer Registry of the
Greater Casablanca Region ,2016).
The cohabitation of several cultures within the
same country, gives Morocco a specificity, that
from one region of the country to another we can
notice different customs or cultures. These
differences can have an influence either in a direct
or indirect way on the diet of the population, the
study’s objectives were to describe and analyze
dietary behavior and its relationship with CRC,
determine the most frequent dietary risk factors
and finally shed light on the impact of dietary
transition on the occurrence of colorectal cancer.
Compare the results of the Marrakech-Safi region
to the results of other regions and highlight the
most frequent risk factors in the Moroccan
population.
METHODS
The study was in the form of a retrospective
epidemiological survey: case-controls in the
Mohamed VI University Hospital Marrakech, more
specifically at the level of the day hospital. The
controls were recruited in the same hospital
center but in different services. The recruitment of
cases and controls was according to eligibility
criteria for cases: diagnosis of colorectal cancer,
being at least 18 years old at the time of diagnosis,
absence of another tumor, lack of a pathology that
requires a specific diet, lack of dietary and
cognitive disorders, patient not bedridden and not
confined to his chair or bed; for controls: lack of
cancer, being 18 years old, lack of a pathology that
requires a specific diet, and absence of eating and
cognitive disorders. The was collect of information
was through a questionnaire based on the Food
frequency questionnaire , detailing 31 food groups
according to the frequency of consumption of the
food(divided into seven categories of
consumption: F1: never/rarely, F2: 1 time per
month, F3: 1 time per week, F4: 2-5 times per
week, F5: 1 time per day, F6: 2-3 times per day
and F7: more than four times per day)(
Appendix1) . The data entry and analysis were by
SPSS V23.0 software.
RESULTS
During the study period, a total of 160 cases and
160 controls. Were included among them 80 males
and 80 females in each group, with a sex ratio of 1;
the location of the tumor in the patients was 56.1%
in the colon, 28.1% in the rectum and 15.8% in the
colorectal. The mean age of the cases was 51.74 ±
8.366 and 50.67 ± 4.20 for the controls with no
significant difference (p=0.393).
Cereals :
According to the data collected for the cereal
category: 80% of the cases never or rarely
consumed wholemeal bread against 94.7% for the
controls without significant difference (p=0.32), for
commercial bread , a high intake (F7) of
consumption in 45.6% of the cases against 17.5%
for the controls, we notice a significant difference
(p=0.003) between the two groups, the risk of CRC
for daily consumption of homemade bread (F5) is
OR=0.095 (IC95%=0.012-0.779) in 1.7% of the
cases against 15.7% in the controls with a
significant difference (p=0.028). Finally, for
"Msemmen" or Moroccan pancake, the risk of
weekly consumption (F3) is OR=4.304
(CI95%=1.32-16.37) with a significant difference
(p=0.032). Moreover it we notice that for a rare
consumption (F1), the risk is OR=0.3
(CI95%=0.136-0.658) with a significant difference
(p=0.03) .
Vegetables and fruits:
For the vegetable category, only 8.7% of the cases
consumed vegetables daily (F5) against 33.3% of
the controls, the risk of CRC was OR=0.192
(IC95%=0.066-0.561) with a significant difference
(p=0.003), for a consumption of 2 to 3 times a week
(F4) the risk was OR=0.211 (IC95%=0.095-0.466)
and a significant difference (p=0.008). Weekly
consumption (F3) of pulses was associated with a
decreased risk OR=0.321 (CI95%=0.146-0.705)
with a significant difference (p=0.005).
In the fruit category, consumption of fruit two to
five times a week (F4) was associated with a
decreased risk of CRC OR=0.156 (CI95%=0.068-
0.358) with a significant difference (p=0.001).
Similarly, weekly consumption (F3) of bananas is
associated with a reduced risk of CRC OR=0.315
(IC95%=0.147-0.678) with a significant difference
(p=0.003), For watermelons, a weekly intake (F3) is
associated with a reduced risk of CRC OR=0.06
(CI95%=0.008-0.48) with a significant difference
(p=0.008).
Meat:
Weekly consumption (F3) of red meat was
associated with an increased risk of CRC OR=2.773
(CI95%=1.908-8.474) with a significant difference
(p=0.007). In contrast, weekly consumption (F4) of
white meat, specifically turkey, was associated with
a decreased risk of CRC OR=0.14 (CI95%=0.052-
0.379) with a significant difference (p=0.029).
46
A rare or low consumption (F1) of "Traked" or
brines (preserved by salt or vinegar) is associated DISCUSSION
with a low risk of CRC OR=0.181 (IC95%=0.075-
0.437) with a significant difference (p=0.002), the Our results highlight the large consumption of
more the frequency of consumption increases, the cereals in the Moroccan population even with the
more the risk of CRC increases (F2 OR: 3.915, dietary transition. Moreover, we note the beneficial
IC95% =1.191-12.866;(P=0.025)). effect of certain cereal-based foods such as
Others: homemade bread and wholemeal bread; the results
For clarified butter or "Smen" a weekly of our study are consistent with the literature,
consumption is associated with increased risk of according to a meta-analysis carried out by
CRC OR=4.832 (CI95%=1.866-12.514) with a WCRF/AICR, a consumption of 10g/d of the fiber
significant difference (p=0.001), similarly for reduces the risk of CRC by 10% (WCRF,2011).
"Khlii" (dried and salted meat) increase the risk of The relationship between cereals or cereal-based
CRC OR=4.167 (CI95%=1.601-10.846) with a foods and the reduction of the risk of CRC is due to
significant difference (p= 0.003). The regularity of the richness of the cereals in fiber, more specifically
the food rhythm is associated with reducing the the fibers having laxative effects, by the ticket of
risk of CRC OR=0,195 (IC95%=0,088-0,433) with two mechanisms: the first one which is by an
only 33,3% of the cases with a regular rhythm increase of the volume of the stools which will lead
against 71,9% of the controls with a significant to a dilution of the carcinogenic elements. The
difference (p=0,004). second mechanism is the acceleration of the transit
Table 1. Food consumption frequency. time, which will cause a reduction in contact
between the carcinogenic substances and the
Food Case Control % colorectal wall (Lipkin M et al. 1999).
% Some foods such as "Mseman" and "commercial
Commercial bread (F7) 45.6 17.5 bread", even if they are based on cereals, the study
Homemade bread (F5) 1.7 15.7 shows that they are risk factors: this may be related
Mseman (F1) 45.6 73.6 to the way of preparation, such as a large amount of
Mseman (F3) 19.2 5.2
oil used during the preparation, as well as salt and
sugar in commercial bread. Bread or even
Vegetables (F4) 28 64.9
"Mseman" was always prepared at home, but the
Vegetables (F5) 8.7 33.4 lifestyle change induces people to buy or consume
Fruit (F4) 36.8 78.9 them outside. A similar study in the region of
Banana (F3) 36.8 64.9 Casablanca also reported high consumption of
Watermelon (F3) 1.7 22.8 Mseman among patients but without a significant
Red meat (F3) 21 8.7 difference (p=0.08) ( Imad F et al. 2020).
Furthermore, the results of our study underline
White meat (F4) 10.5 45.6
once again the protective effect of vegetables and
Traked (F1) 49.1 84.2 fruits consumed at least 2 to 5 times per week
Traked (F2) 22.8 0.7 against CRC; a meta-analysis showed a 9% reduced
Smen (F4) 40.3 12.2 risk of colorectal cancer between the high and low
Khlii (F4) 36.8 12.2 consumption of vegetables (Slavin J. 2000);
whereas for fruits the WCRF/AICR meta-analysis
showed a risk reduction of only 3% for 100 g/d
(Aune D. 2011). paradoxically, in comparison with
the other regions, fruit consumption was higher for
cases in Casablanca (32% vs 16.9) and Fez (59.9 vs
23.8). For vegetables, the region of Casablanca also
highlights a higher consumption among controls
(10.2% vs. 23.6%) but no difference in the Fez
region (40.5% vs. 45.3%)( Slavin J. 2000 ; Touyar A.
2011).
The effect of fruits and vegetables could be due to
their content in several nutrients and components
that are beneficial effects on CRC risk such as
antioxidants, vitamins and minerals for fruits that
are likely to promote the activity of detoxifying
enzymes and the reduction of oxidative stress and
inflammation. While for vegetable, elements such
as flavonoids, carotenoids, selenium will interact
with free radicals to protect against oxidative
47
damage (Kelsay J. et al. 1978).
In addition, in our study, we noted the association
between red meat consumption and increased risk
of CRC. Those results are consistent with those of
the region Casablanca where daily consumption
was significantly high among cases (23.1% vs 9.8),
while for the region of Fez there was no significant
difference ( 9.5% vs 4.8%)( Slavin J. 2000 ; Touyar
A. 2011).The relationship between red meat and
colorectal cancer can be explained by the heme in
red meat , which will lead to the formation of
carcinogenic N-nitro compounds by the colon
bacteria as well as the production of
pro-inflammatory cytokines, free radicals (Carr, P.
R. et al. 2018); some studies highlight that the
nature of the meat can affect the risk of CRC, this is
consistent with our study where the consumption
of turkey (white meat) is associated with a
decreased risk of CRC, this difference could be
explained by their composition: fat and fatty acid
content with a ratio (polyunsaturated fatty
acids/saturated fatty acids that is 0. 1 for beef and
0.7 for chicken) (Sinha R. et al. 2001).
Thus, our study suggests a possible relationship
between the consumption of "Traked", "Smen",
"Khlii" and the increased risk of CRC, the common
point between most of these foods is their high
fatty acid content, the relationship between CRC
and fatty acids base on two aspects: a first
functional aspect where fatty acids are precursors
to carcinogenic molecules; a second structural
aspect for the formation of the cell membrane
during cell growth (Nkondjock A. et al. 2003). The
second common point is the traditional
preservation methods that could induce the
production of specific carcinogenic molecules by
the bacteria.
Several studies confirm that a disorganized dietary
pattern is associated with an increased risk of CRC
(Gerhardsson M. et al. 1992 ;Shoff SM. et al. 1997),
which is entirely consistent with the results of our
study; it is clear that a disorganized dietary pattern
impedes proper digestion by influencing primary
bile acid flow (Shoff SM. et al. 1997).
The comparison of the results of our study with the
two studies carried out in Casablanca and Fez,
shows certain similarities with the Casablanca
region. With a low consumption of meats and
especially red meats, contrary to the region of Fez
where the consumption of meats was high in the
cases as well as in the controls respectively 85.7
and 88.2. A higher consumption of fruits in
Marrakech than in the other two regions was noted
(Appendix 2).
CONCLUSION
The transition that Morocco has recognized has had
a significant influence on the lifestyle and, more
specifically , on an vital component which is the
diet. The Moroccan food profile is increasingly
moving towards a poor consumption of cereals,
fruits and vegetables which results in deficiencies
in several essential micronutrients, for the proper
functioning of our body. In parallel, the high
consumption of certain foods, such as red meats
and processed products, provides our body with
certain carcinogenic elements or molecular control
defects, which can lead to the development of CRC.
The comparison of the three regions shows a
certain variability in the regions and even in the
frequency of consumption. The results of the study
are still preliminary, but studies on a larger sample
and on several regions will allow to better
understand the dietary behavior of the Moroccan
population. This may allow the implementation of
an effective preventive strategy against CRC.
ACKNOWLEDGEMENTS
we want to thank the contributors of this work, from
CHU Mohamed VI, Marrakech and Laboratory of
Pharmacology, Neurobiology, Anthropobiology and
Environment, Faculty of Sciences Semlalia, Cadi Ayyad
University, Marrakech .
REFERENCES
Aune D, Lau R, Chan D, Vieira R, Greenwood D,
Kampman F, et al. 2011 . Nonlinear reduction in
risk for colorectal cancer by fruit and vegetable
intake based on meta-analysis of prospective
studies. Gastroenterology. 106—18. 141(1).
Cancer Registry of the Greater Casablanca Region,
2008-2012 data. 2016 Edition. Available at:
https://www.contrelecancer.ma/site_media/uploa
ded_files/RCRGC.pdf
Carr, P. R., Weigl, K., Jansen, L., Walter, V., Erben, V.,
Chang-Claude, J., Brenner, H., & Hoffmeister, M.
2018. Healthy Lifestyle Factors Associated With
Lower Risk of Colorectal Cancer Irrespective of
Genetic Risk. Gastroenterology. 1805–1815.e5.
155(6).
Gerhardsson M, Longnecker MP. 1992. Eating
frequency: a neglected risk factor for colon cancer?
Cancer Causes Control. 77-81. 3(1).
Imad F .et al. 2020 . Facteurs de risque alimentaires
du cancer colorectal au Maroc: étude cas-témoin.
Pan Afr. med. j.35:59.
Kelsay J, Kehall KM, Prather ES. 1978. Effect of fiber
from fruits and vegetables on metabolic responses
of human subjects in bowel transit time, number of
defecations, fecal weight, urinary excretions of
energy and nitrogen and apparent digestibilities of
energy, nitrogen, and fat. Am j Clin Nutr. 1149—53.
31(7).
48
 des cancers colorectaux dans la région de Fes ,
Lipkin M, Reddy B, Newmark H, Lamprecht S. 1999 étude cas-témoins . Faculty of medicine and
. Dietary factors in human colorectal cancer. Annu . pharmacy of fez ( Phd thesis).
Rev . Nutr.545—86,19.
World Cancer Research Fund, American institute
Nkondjock A, Shatenstein B, Maisonneuve P, for cancer research . 2011. continuous update
Ghadirian P. 2003 . Assessment of risk associated project report. Food, nutrition, physical activity,
with specific fatty acids and colorectal cancer and the prevention of colorectal cancer. Colorectal
among french-Canadians in Montreal : a cancer 2011 report. Aicr.
case-control study. Internat j Epidemiol .
200-209.32.

Shoff SM, Nawcomb PA, Longnecker MP. 1997.

Frequency of eating and risk of colorectal cancer in
women. Nutr cancer. 22- 25. 27(1).

Sinha R, Kulldorff M, Chow WH, et al. 2001. Dietary

intake of heterocyclic amines, meat- derived
mutagenic activity, and risk of colorectal adenomas.
Cancer Epidemiol Biomarkers Prev. 559-562. 10.

Slavin J. 2000. Mechanisms for the impact of

whole-grain foods on cancer risk. J. Am. Diet.
Assoc.300s—7s.19(3suppl).

Touyar A . 2011 . Facteurs de risuqe nutritionnels

49
Appendix 1

Food F1 F2 F3 F4 F5 F6 F7
Commercial bread
Wholemeal bread
Homemade bread
Mseman
White sugar
honey
Olive oil
Sunflower oil
Beans
Lentil
Chickpea
Green vegetables
Fruits
Couscous with vegetables
Couscous with raisin and
onion
Beef
sheep
lamb
chicken
turkey
Fatty fish
White fish
Seafood
Full cream milk
Fermented milk
Soy milk
Processed cheese
Eggs
Traked
Smen
Khlii

50
Appendix 2

Food Marrakech Casablanca (Imad F. et al . Fez (Touyar A. 2011)

2020)
Read meat
Case (%) 21 23.1 85.7
Control (%) 8.7 9.8 88.2
OR(Cl95%) 2.77(1.9-8.4) 4.4(1.6-11.9) _____
White meat
Case (%) 10.5 36 92.9
Control (%) 45.6 41.8 88.1
OR(Cl95%) 0.14(0.05-0.37) 1.7(0.6-4.4) _____
Fruits
Case (%) 36.8 16.4 9.5
Control (%) 78.9 33.8 21.5
OR(Cl95%) 0.15(0.06-0.35) 0.42(0.1-1.3) _____
Vegetebles
Case (%) 28 20 57.2
Control (%) 64.9 42.2 52.4
OR(Cl95%) 0.21(0.09-0.46) 0.13(0.07-0.2) _____
Bread
Case (%) 1.7 _____ 78.5
Control (%) 15.7 _____ 92.8
OR(Cl95%) 0.09(0.01-0.77) _____ _____
Full cream milk
Case (%) 35 9.8 38.1
Control (%) 75.4 14.7 64.3
OR(Cl95%) 0.17(0.07-0.36) 0.7(0.4-1.3) _____
Fish
Case (%) 20 24.9 28.6
Control (%) 42 43.1 42.9
OR(Cl95%) 0.02(0.006-0.12) 0.35(0.15-0.7) _____

51
CONFERENCE PAPER Hicham Gougueni et al., 2022, 52-58

Potability of drinking water in rural areas of Tiznit in southern Morocco

Hicham Gougueni1,2 , Abdelkrim Ezaidi1, Latifa Ez-Zaher3, Youssef Bouchriti1,2, Mohamed Ait Haddou1,
Rachid Amiha1 And Mohamed En-Nasiry1
1
Geosciences, Environment and Geomatics Laboratory; Department of Geology; Faculty of Science;
University Ibn Zohr Agadir. Morocco
2
Higher institute of nursing professions and health techniques, Agadir. Morocco
3
Team of Cellular Biology and Molecular Genetics; Department of Biology; Faculty of Science; U.I.Z
Agadir. Morocco

Water quality remains one of the significant environmental problems for its
health and economic consequences. The present work aims to assess the
bacteriological quality of water consumed by the rural population of the
province of Tiznit. A total of 133 water samples were studied at the level of
62
wells, 49 cisterns (matfias) and 7 sources. These samples were subject to
bacteriological analysis and some Physico-chemical parameters. The results
show that the rate of non-compliance of samples taken in rural areas
reaches 80% due to bacterial contamination mainly by Escherichia coli and
total coliforms (69.1%; 68.42%), fecal coliforms (55.64%) and Anaerobic
Sulphito-reducing Spores (27.82%). The nature of water that recorded a
high rate of non-compliance is that of untreated water with a percentage of
(98%). Non-compliance is particularly high for the following types of water:
untreated networks, untreated standpipes, wells and cisterns. These results
could explain the large number of cases of diarrhea reached 2437 cases
in2019 in the rural areas of Tiznit.
* the problems of availability and accessibility of
Corresponding Author
drinking water in peri-urban and rural areas. The
Name: Hicham Gougueni
quantity and quality of available water resources
Email:
are currently posing increasingly complex and
hicham.gougueni@edu.uiz.ac.ma challenging problems, especially, in
Phone :00212663439857 underdeveloped countries (Gbohaida et al., 2016).
Such quality is affected by several problems,
ISBN: 9788367405256
including pollution that is related to anthropogenic
DOI: 10.2478/9788367405256-008 activities (Sokegbe et al., 2018). The
bacteriological and physico-chemical quality of the
Published by Sciendo. © 2023 Hicham Gougueni et al. This
is an open access article licensed under the Attribution 4.0
drinking water served to the population has been
International the subject of numerous studies that have shown
(https://creativecommons.org/licenses/by/4.0/) the sanitary risks and the impact of drinking water
of questionable quality on human health (Abegaz
& Midekssa, 2021; Bain, Cronk, Wright, et al., 2014;
INTRODUCTION Bain et al., 2020; Cairncross et al., 2010b; Lalami,
2014; Tousi et al., 2021).
Water is an indispensable substance for life. Consumption of water that does not meet the
Currently, people worldwide are confronted with quality standards required by the World Health

52
Organization or national standards of drinking Maximum
water quality can affect human health (Abegaz & permissible
Parameters Commentary
values
Midekssa, 2021; Gougueni et al., 2021 Kapembo et
Residual chlorine levels
al., 2022;). However, water intended for human E.coli 0/100
ml must be between: 0.1 and
consumption should not contain dangerous
1 mg/l at distribution 0.5
chemical substances or germs harmful to 0/100 to 1.0 mg/l at production
health(WHO,2017). I.E
ml
Several efforts are being made to increase safe - No coliforms in
water and sanitation services. However, billions of 95% of the samples taken
people in rural areas lack access to these essential over 12 month
0/100 - No positive
services. According to World Health Organization Coliforms ml
2.2 billion people lack drinking water services results in two consecutive
samples
managed safely (WHO,2019).
This parameter must be
Up to now, no study has been realized in the rural measured when the water
region of Tiznit on the bacteriological quality of 0/100 is of the superficial origin
S.A.S.R ml
the water consumed by the local population. It is or influenced by the water
therefore essential to assess the quality of water of superficial origin.
from different drinking water sources in such a Revivable 20/100 ml at Variation in a ratio of 10
rural township to determine and characterize its microorganism 37°C concerning the usual Value
quality concerning national and WHO standards. at 22°C and 100/100 ml
37°C at 22°C
● The national epidemiological situation in
IE : intestinal enterococci ; S.A.S.R : Spores of
Morocco
sulfite-reducing anaerobic micro-organisms
In 2017, the Epidemiology and Environmental
WHO published the fourth edition of the
Health Laboratories performed 70526
Guidelines for Drinking Water Quality in 2017.
bacteriological and physico-chemical analyses for
These guidelines provide guide values for many
16798 water and food samples. This is part of the
more chemical contaminants than those that will
food hygiene and water sanitary control programs
actually affect a particular water supply.
(DELM,2018). The non-compliance rates recorded
Assessment of the chemical quality of drinking
vary between 20% and 24%. The highest
water is based on the comparison with guide
non-compliances on drinking water during the
values (Table 2).
year 2017, were recorded at the level of the
regions of Tangier-Tetouan-Al- Hoceima (39%), In addition, the WHO guideline values are
Marrakech-Safi (37%) and Beni-Mellal-khénifra considered scientifically sound starting points for
(36%). However, the category that presents the the establishment of national or regional
highest rate of non-compliance is that of untreated quantitative drinking water quality standards.
water (88%). Types of germs detected in drinking There is no single universally applicable strategy.
water are mainly fecal streptococci (77%) and E. Drinking water standards may therefore vary
coli (68%)(DELM,2018). between countries or regions.
● Quality standards Table 2. Maximum permissible values for some
physico- chemical parameters and toxic minerals
According to WHO (2017), microbial water quality
according to the Moroccan standard NM 03.7.001.
is based primarily on analysing microorganisms Maximum
indicator of fecal contamination, including Expression
Parameters permissible
Escherichia coli and thermotolerant coliform of results
values
bacteria. The presence of Escherichia coli brings Hydrogen
6,5< pH< 8,5 units pH
incontestable proof of recent fecal contamination. Potential (pH)
This micro-organism must be completely absent Conductivity 2700 µS/cm à 20°C
from the drinking water. Additional indicators,
such as bacteriophages or bacterial spores, may be Chlorides 750 Cl:mg/l
used in some situations.
Sulfates 400 SO4:mg/l
In Morocco, the standard NM 03.7.001 sets the
requirements for the quality of human food water Dissolved
5 ≤ O2 ≤ 8 O2:mg O2/l
(Table 1). oxygen
Ammonium 0,5 NH4: mg/l
Table 1. Maximum permissible values for
bacteriological parameters according to the
Nitrites 0,5 NO2:mg/l
Moroccan standard: NM 03.7.001.
53
 Nitrates 50 NO3:mg/l Lead 10 Pb : µg /1

Copper 2 Cu : mg/1

Fluorides 1,5 F : mg/1

METHODS
● Data collection
isothermal boxes to maintain the temperature
This retrospective study (2015-2019) aims to
below 4°C. The pH and temperature are measured
describe the bacteriological quality of rural
in situ.
drinking water in the Tiznit province. The water
samples were taken by health and environmental ● Data analysis
technicians according to standard methods of the
The data were analyzed using Microsoft Excel
WHO guidelines and the national standard.
software. Our bacterial analysis results were
Water sampling for bacteriological analysis was compared to the Moroccan standard NM 03.7.001
performed aseptically, using sterile glass vials to for drinking water quality and interpreted as
prevent external contamination. The transport to compliant or non-compliant.
the provincial laboratory of health and
environment is done the same day using
RESULTS
The water points studied are 118 in number, divided into 62 wells, 49 cisterns and springs (Figure 1).

Figure 1: Distribution of the studied water points according to the type

In the samples studied, untreated water represents 98%, while treated water represents only 2% (Figure 2).

54
The water temperature does not exceed 22 °C as a maximum value and 15 °C as a minimum value (Figure 3).

Figure 3: temperature of the water samples taken in the different points

The Hydrogen Potential (pH) values of the samples are between 6,7 and 7,8 and the pH values of the samples taken are
within the standards (Figure 4).

Figure 4: Distribution of the samples according to the pH values.

E. Coli and total coliforms(CT) are detected in most of the samples studied (69% and 68%). Faecal coliforms (CF) are
identified in half of the samples (55.64%), (Figure 5)

55
The rate of non-conformity of the studied samples is 80% compared to a rate of conformity that does not exceed 20%
(Figure 6).
DISCUSSION
According to the study results, the nature of the
water taken from these points is, in almost all
cases, untreated (98%). Given that the treatment
operation in rural areas is based on disinfection
by chlorination, this shows that this activity is not
carried out regularly.
It also appears that the temperature of the water
samples studied varies between 15°C as a
minimum value and 22°C as a maximum value.
Indeed, the study conducted on the waters of the
aquatic fauna of The Tiznit province, shows that
the temperature varies between 20.9 °C and 22.4
°C. This relative thermal constancy of the water is
a general character and should not be surprising
since it has already been observed and highlighted
many times, both in Europe and Morocco (Boulal
et al., 2017).
Another essential parameter is the pH (hydrogen
potential), which measures the concentration of
H⁺ ions in water and thus translates the balance
between acids and bases on a logarithmic scale
from 0 to 14. This parameter conditions a large
number of physicochemical equilibria. Its values
are between 6 and 8.5 in natural waters (Akil et al.,
2014). As for the present study results, the pH
values of the samples are between 6.7 and 7.8. So
these values are within the required standards for
drinking water.
It also emerges from the results of this study that
the bacterial contamination by E. COLI and total
coliform bacteria (TC) interests the majority of the
samples taken (69.1%;68.42%), followed by fecal
coliforms (CF) (55.64%). The presence of E. coli is
an indicator of recent environmental
contamination by human or warm-blooded
animal fecal material. This means that there
would be a probable risk that the pathogens are
present in these waters (Sokegbe et al., 2018). The
WHO describes coliform bacteria as a good test for
evaluating disinfection practices, but not
necessarily an indicator of health risk. E. coli is
considered the best indicator of fecal
contamination (Rubino et al., 2018).
Contaminated water poses a serious threat to
human health and requires special efforts to
control its quality (Slavik et al., 2020).
Different factors can contribute to water
contamination. The microbial quality of drinking
water is intrinsically linked to poor sanitation
practices. In rural areas where hygiene and
sanitation standards are inadequate, faecal
pathogens are the most common source of
drinking water contamination (Yongsi, 2010).
100% of unmanaged/undeveloped wells surveyed
are heavily contaminated with fecal matter
(Kapembo et al., 2022). Also, the lack of regular
treatment or degradation of the disinfection agent
can be a factor. Degradation of the disinfection
agent and formation of by-products are the most
common chemical problems that can lead to
microbiological processes resulting in water
quality deterioration (Slavik et al., 2020). Microbial
regrowth in biofilms at water points results from
the lack of secondary and regular disinfection.
This would mean a vulnerability of the drinking
water management system in rural areas (Daley et
al., 2018).
56
CONCLUSIONS Boulal, M., Boulanouar, M., Ghamizi, M., & Boutin, C.
2017. Qualité de l’eau et faune aquatique des puits
Most well, spring and cistern water samples from the
dans la région de Tiznit (Anti-Atlas occidental,
rural region of Tiznit were contaminated with germs
Maroc). Bulletin de la Société d'histoire naturelle de
indicative of faucal contamination. E. coli and total
Toulouse,153 :25-41
coliforms were detected respectively in (69.1%;
68.42%) of the samples analyzed. Although these Cairncross, S., Hunt, C., Boisson, S., Bostoen, K., Curtis,
species are non-pathogenic and do not represent a V., Fung, I. C., & Schmidt, W.-P. 2010b. Water,
direct health risk. These waters' quality is considered sanitation and hygiene for the prevention of
non-compliant with WHO recommendations and the diarrhoea. International Journal of Epidemiology,
Moroccan standard for drinking water quality. 39(suppl_1), 193-205.
To consume water safely, it must therefore undergo
Daley, K., Truelstrup Hansen, L., Jamieson, R. C.,
various bacteriological and physicochemical analyses
Hayward, J. L., Piorkowski, G. S., Krkosek, W.,
that will define its quality for human consumption to
Gagnon, G. A., Castleden, H., MacNeil, K.,
avoid the risks of waterborne diseases for
Poltarowicz, J., Corriveau, E., Jackson, A., Lywood, J.,
consumers. In the same way, respect for the hygiene
& Huang, Y. 2018. Chemical and microbial
rules and the regular treatment of water by the
characteristics of municipal drinking water supply
populations are essential measures to avoid the
systems in the Canadian Arctic. Environmental
appearance of waterborne diseases.
Science and Pollution Research, 25(33),
Further studies are needed to investigate the 32926‑32937.
physicochemical quality of the water consumed by
the rural population and its impact on health. Direction de l’épidémiologie et lutte contre les
Environmental scientists will need to work together maladies (DELM).2018. Bulletin d’épidémiologie et
with health authorities and the local population to de santé publique N° 76. Ministère de la santé,
manage and improve the quality of the water Maroc.
consumed. Gbohaida, V., Agbangnan, D. C. P., Ngossanga, M. B.,
Medoatinsa, S. E., Dovonon, L. F. C., Wotto, D. V.,
ACKNOWLEDGEMENTS Avlessi, F., & Sohounhloue, D. C. K. 2016. Etude de la
The author thanks the technicians of health and qualité physico-chimique de l’eau de boisson dans
environment and the staff of the Laboratory of deux localités du Bénin : Cotonou et Dassa-Zoumè.
Health and Environment of the Tiznit province. He International Journal of Biological and Chemical
also thanks K.Iziki; S. Garmoum and I. Fadladi for Sciences, 10(1), 422.
their contribution in data collection. Gougueni, H., Ez-Zaher, L., Bouchriti, Y., Ait Haddou,
M., & Ezaidi, A. 2021. Health effects of the
REFERENCES consumed water from the traditional reservoirs on
the population of Tiznit in Morocco. E3S Web of
Abegaz, M. T., & Midekssa, M. J. 2021. Quality and
Conferences, 234, 00073.
Safety of Rural Community Drinking Water Sources
in Guto Gida District, Oromia, Ethiopia. Journal of Kapembo, M. L., Mukeba, F. B., Sivalingam, P., Mukoko,
Environmental and Public Health. 1‑13. J. B., Bokolo, M. K., Mulaji, C. K., Mpiana, P. T., & Poté,
J. W. 2022. Survey of water supply and assessment
Akil, A., Hassan, T., Fatima, E. H., Lahcen, B., &
of groundwater quality in the suburban communes
Abderrahim, L. 2014. Étude de la qualité
of Selembao and Kimbanseke, Kinshasa in
physico-chimique et contamination métallique des
Democratic Republic of the Congo. Sustainable
eaux de surface du bassin versant de guigou, maroc.
Water Resources Management, 8(1), 3.
European Scientific Journal(ESJ), 10(23), 11.
Lalami, A. E. O. 2014. Risque Sanitaire Liés à la
Bain, R., Cronk, R., Wright, J., Yang, H., Slaymaker, T., &
Consommation des eaux de Boisson Conditionnées
Bartram, J. 2014. Fecal contamination of
drinking-water in low- and middle-income et Commercialisées dans la Ville de Fès (Maroc)
countries: A systematic review and meta-analysis. european journal of scientific research. 125(2), 15.
PLoS Medicine, 11(5), Rubino, F., Corona, Y., Pérez, J. G. J., & Smith, C. 2018.
Bain, R., Johnston, R., & Slaymaker, T. 2020. Drinking Bacterial Contamination of Drinking Water in
water quality and the SDGs. Npj Clean Water, Guadalajara, Mexico. International Journal of
3(1),37. Environmental Research and Public Health, 16(1),
67.

57
Slavik, I., Oliveira, K. R., Cheung, P. B., & Uhl, W. 2020.
Water quality aspects related to domestic drinking
water storage tanks and consideration in current
standards and guidelines throughout the world—A
review. Journal of Water and Health, 18(4),
439‑463.
Sokegbe, O. Y., Djeri, B., Kogno, E., Kangnidossou, M.,
Mensah, R. T., Soncy, K., & Ameyapoh, Y. 2018. Les
risques sanitaires liés aux sources d’eau de boisson
dans le district n°2 de Lomé-commune : Cas du
quartier d’Adakpamé. International Journal of
Biological and Chemical Sciences, 11(5), 2341.
Tousi, E. G., Duan, J. G., Gundy, P. M., Bright, K. R., &
Gerba, C. P. 2021. Evaluation of E. coli in sediment
for assessing irrigation water quality using machine
learning. Science of The Total Environment, 799,
149286.
WHO.2017. Guidelines for drinking-water quality,
4th edition, incorporating the 1st
addendum.https://www.who.int/publications-deta
il-redirect/9789241549950
WHO.2019. 1 personne sur 3 dans le monde n’a pas
accès à de l’eau salubre – UNICEF, OMS. Consulted
on 21 avril 2022, at
https://www.who.int/fr/news/item/18-06-2019-1
-in-3-people-globally-do-not-have-access-to-safe-dr
inking-water-–-unicef-who
Yongsi, H. B. N. 2010. Suffering for Water, suffering
from Water: Access to Drinking-water and
Associated Health Risks in Cameroon. Journal of
Health, Population and Nutrition, 28(5), 424‑435.

58
 CONFERENCE PAPER Yassine Zine-eddine et al., 2022, 59-67

The effect of physicochemical parameters and enzyme concentration on the

clotting and cutting times properties of Alpine breed goat's milk
Yassine Zine-eddine*1, Hassan Charii2, Fouzia Kzaiber1, Abdelkhalek Oussama1, Amine Moubarik2 and
Khalid Boutoial1
1
Laboratory of the Engineering and Applied Technologies, Higher School of Technology, Sultan
Moulay Slimane University, Beni Mellal, Morocco
2
Laboratory of Chemical Processes and Applied Materials, Polydisciplinary Faculty, Sultan Moulay
Slimane University, Béni-Mellal, Morocco

The coagulation process significantly impacts cheese yield and the

product's rheological properties. For example, cutting the curd after a
predetermined time influences its firmness, resulting in a variance in the
optimal cutting time and a delay in syneresis, resulting in a high moisture
cheese. Conversely, cutting the curd too soft will reduce the cheese yield
due to increased fat and curd loss in the whey. So, it is critical to
understand the process and measuring tools to achieve the best
transformation performance and processing efficiency. For this, by using
the Berridge and viscometric methods, uncoded unit regression equations
of clotting and cutting time as a function of physicochemical factors (pH,
CaCl2, temperature, enzyme concentration) were developed. These models
are more significant and have a superior forecasting ability for novel
observations. The characteristics that most influenced clotting time were
pH, temperature, and coagulating enzyme concentration, in that order.
Specifically, increasing these parameters reduces the clotting time. While
cutting time was influenced successively by pH, enzyme concentration,
and temperature. Calcium chloride reinforcement seems to be negative at
some pH values, resulting in protracted clotting and cutting time. In
addition, increasing the temperature to a certain extent can affect enzyme
activity.

* INTRODUCTION
Corresponding Author
Name: Yassine Zine-eddine
Email:
Milk coagulation is described as the destabilization
zineeddine.yassine95@gmail.com of casein micelles, which flocculate and combine to
ISBN : 9788367405256 create a gel containing the soluble milk components
(Lapointe-Vignola 2002). It is a critical stage that is
DOI: 10.2478/9788367405256-009 heavily influenced by the protein content of milk and
Published by Sciendo. © 2022 Yassine Zine-eddine et al.
has a significant impact on the efficiency of cheese
This is an open access article licensed under the Attribution manufacturing (Troch et al. 2017). It is a critical
4.0 International phase in the cheese-making process since it dictates
(https://creativecommons.org/licenses/by/4.0/) the final cheese properties (García et al. 2012). Milk
coagulation is crucial because it impacts
cheese-making ability, cheese production, and
cheese quality (Joudu et al. 2008). Because it is such
a crucial phase in cheese production, it has been
59
extensively researched (García et al. 2015; Pretto et
al. 2011). It is vital to understand the process and
the measurement tools to obtain the optimum
transformation performance and maximum
processing efficiency.
Milk coagulation can be caused by acidification,
enzyme activity, or a combination of the two (Troch
et al. 2017). Coagulation is often separated into two
steps: the enzymatic phase, in which chymosin
degrades the κ-casein, and the non-enzymatic phase,
which corresponds to the development of a gel by
the aggregation of destroyed micelles. These two
phases are distinct in character but are not
completely separated in time (Frederiksen et al.
2011).
There are considerable interspecies variances in
milk content, which impact milk coagulation and are
reflected in the properties of the cheeses made from
it. There are also considerable variances in milk
composition across species and breeds, which
impact cheese quality, as do seasonal, lactational,
and nutritional variables, the health of the producing
animal, and, of course, milk production, storage, and
collecting procedures (Fox et al. 2017).
A variety of factors influence the cheese-making
qualities of milk, including temperature, milk pH,
and Ca2+ content (Daviau et al. 2000; Gunasekaran
and Ay 1996; Payne et al. 1993; Picon et al. 1995);
these factors have to be controlled (Balcones et al.
1996).
The rate of rennet coagulation's enzymatic phase is
proportional to the amount of rennet employed;
there is an excellent linear link between enzyme
concentration and milk-clotting activity (Fox et al.
2017; Nájera et al. 2003). Clotting time lowers when
enzyme concentration is raised because of a higher
level of proteolysis of k-casein (Nájera et al. 2003).
Increased rennet concentration results in a shorter
flocculation time, earlier onset of gel hardness, and a
faster rate of rising (Law and Tamime 2011).
The pH has a significant impact on clotting time.
Because of the influence of pH on enzyme activity,
the rennet coagulation time increases with
increasing pH, while decreasing milk pH produces a
decrease in clotting time (Fox et al. 2017). As a
result, the most critical impacts of reducing milk pH
are the solubilization of micellar calcium phosphate
(Le Graet and Brulé 1993), casein dissociation from
micelles and a reduction in the net charge of the
casein molecule (Gastaldi et al. 1996). It has also
been noted that decreasing the pH produces an
increase in the firming rate of the curd (Daviau et al.
2000). However, at pH levels below 5.0, renneted
milk coagulation is ineffective (Nájera et al. 2003).
Lowering the pH of milk while the rennet content
results in a faster initial increase in gel firmness and
a quicker coagulation time (Law and Tamime 2011).
The negative charge of caseins decreases when pH
decreases, facilitating aggregation and link
formation. However, it also causes calcium
phosphate to dissolve from casein micelles,
increasing the negative charge in the opposite way
(Law and Tamime 2011).
The temperature has a significant impact on milk
coagulation (Gunasekaran and Ay 1996;
Lapointe-Vignola 2002). Temperature is a significant
factor influencing enzyme activity (Siboukeur et al.
2005). Temperature changes in the milk before the
addition of rennet also affect its coagulation
characteristics (Troch et al. 2017). The enzyme is
still active at temperatures below 10 °C; however,
gelation does not occur. Coagulation is sluggish
between 10 and 20 °C. Clotting occurs gradually
between 30 and 42 °C. Coagulation begins to slow
above 42 °C and ceases altogether at 55 °C (Troch et
al. 2017).
The rate of coagulation, gel firming, and syneresis
will increase as the cheese milk temperature rises.
Changing the gel formation temperature impacts the
rate of enzymatic and aggregation processes, as well
as the initial rate of gel firming (Law and Tamime
2011). (Zoon et al. 1988) investigated the effect of
temperature thoroughly, and found that the gel
becomes more viscous-like and less firm with
increasing temperature of formation, seen by an
increasing phase angle and decreasing maximum
firmness with increasing temperature. A reduction
in temperature causes an increase in firmness and
vice versa (Law and Tamime 2011)
The gel strength and the calcium content have been
found to have a very significant relationship (Remeuf
et al. 1991). The concentration of Ca2+ ions impacts
rennet activity (Troch et al. 2017). The gelation
period is reduced when calcium chloride is added to
the milk (Law and Tamime 2011; Nájera et al. 2003).
It has also been found that raising the concentration
of Ca2+ affects curd firmness (Solorza and Bell 1998;
Balcones et al. 1996). The coagulation velocity
increases as the calcium/(phosphate+citrate) ratio
in the aqueous phase rises (Troch et al. 2017).
Furthermore, the addition of CaCl2 lowers the pH of
milk, resulting in a higher rate of protein aggregation
(Van Hooydonk and Berg 1988).
This work aimed to study the effect of the
physicochemical parameters (temperature, pH,
calcium chloride, and enzyme concentration) on the
coagulation properties of the breed alpine goat milk.
And develop a mathematical equation for optimizing
the clotting and cutting time; based on the Berridge
method (for the clotting time) and the viscometric
method via a rotary rheometer for their use in
industrial cheese production to attain the highest
transformation and processing efficiency possible.
Materials and methods
Milk simples
Fresh goat milk of the alpine breed was studied from
60
a herd of 24 goats on a farm located in Béni Mellal coagulation enzyme solution is added.
city Morocco. The goats are fed with a mixture of Simultaneously, a stopwatch is activated, and when
fodder and pasture. the first flocculation is observed on the tube wall, the
Milk samples are stored at 4 ° C until laboratory clotting time is noted on the stopwatch (LÓOPEZ et
analysis; these analyzes are carried out on the same al. 1999).
day of milking; all analyzes are done in triplicate. A cheesemaker trainer did the cutting time
determination. The cutting time was when the curd
Physicochemical analyses could be removed off the vessel glass's surface with a
spatula without tearing. A sharp knife was used to
Physicochemical parameters are analyzed by Milko make at least three strokes in separate areas. The
Skan in a combi Foss 5000, Foss Electric, Hillerod, strokes were performed at intervals of at least 1
Denmark. minute. The cutting time referred to the period when
The acidity of the milk is measured by acid-base a cut through the gel resulted in a smooth, readily
titration with sodium hydroxide (NaOH) as separated surface. For each experiment, this test was
described by (Guiraud 1998) and phenolphthalein as carried out three times (Lopez et al. 1998).
a color indicator. The results are expressed in Dornic Viscosity was measured by Anton Paar RheolabQC
degrees (°D). rotary rheometer (27 cc cylinder) with a
temperature-controlled device. A known volume of
Factorial design the calcium chloride solution is mixed with 20 ml of
the milk. The mixture is allowed to take the desired
To determine the effect of physicochemical temperature by the viscotherm, and a known volume
parameters in the clotting and cutting time and their of the milk coagulation enzyme solution is added.
optimization in cheese making, a factorial design is The viscosity was determined every 6 seconds from
used by Minitab 18 software. It was a four-factor the addition of the enzyme until the viscosity
design with three replications. It is a type of remained constant. The time corresponds to the
experiments design that allows studying several maximum value of the viscosity is noted, in order to
factors' effects on a response. The pH is studied in a know the relationship between this value and
range of 5.2 to 6.6, likewise the temperature cutting time. The measuring instruments, rotor and
between 30 and 46 ° C, calcium chloride from 0.5 cylinder were kept in a detergent solution between
mM to 3 mM (García et al. 2015; Fagan et al. 2007) measurements and rinsed with distilled water. All
and the coagulating enzyme concentration of 1 IMCU measurements were repeated three times.
to 2 IMCU for 20 ml volume of milk.
Data analysis
pH adjustment
The factorial design, their analysis, the normal plot
In this study, the pH is studied in 5.2 to 6.6. 88% of the standardized effects, the Pareto and the
extra pure lactic acid (LOBA CHEMIE PVT.LTD.) is descriptive statistics of goat milk composition are
used to adjust its values. determined using the Minitab 18 software (version
18, Minitab Inc., in the United States and other
Calcium chloride countries).

Calcium chloride dihydrate (SIGMA-ALDRICH

GERMANY) is used to prepare a solution of calcium Results and discussion
chloride with a concentration of 510g / l.
Goat milk composition
Rennet coagulation
The mean values and standard deviations for the
This study used HANNILASE XP 200 CHR-HANSEN, physicochemical compositions of 24 bulk milk
DENMARK (Activity: ~ 200 IMCU/ml, Dosage: 33-66 samples are summarized in Table 1. According to
IMCU / L of milk) originates from Rhizomucor these results, it is concluded that the goat milk
miehei as coagulant. For clotting time determination, samples used in this study were homogeneous and
the only internationally accepted one is the Berridge that the samples could be prepared as a mixture,
method, as described by the International Dairy which was the case in this research.
Federation (LÓOPEZ et al. 1999; García et al. 2015).
For microbial enzymes and other coagulants, similar
approaches have been devised (Tabayehnejad et al.
2012). A known volume of the calcium chloride
solution is mixed with a known amount of the milk,
the mixture is kept in a water bath at a given
temperature, and the known volume of the milk
61
Table 1. The mean values and standard 8 5.2 30 3 2 20 74 210 2.84
9 6.6 30 0.5 2 30 384 1020 2.66
deviations of physicochemical compositions of 10 5.2 46 0.5 2 13 86 228 2.65
milk samples. 11 5.2 30 0.5 1 21 66 184 2.79
12 6.6 30 3 1 32 790 1990 2.52
Standard 13 5.2 46 3 1 18.5 108 270 2.50
Variable Mean
deviation 14 5.2 30 0.5 1 20 70 193 2.76
Fat % 3.55 0.60 15 6.6 30 3 1 32.5 786 1970 2.51
Solid-non-fat % 8.42 0.29 16 5.2 30 3 2 19 82 226 2.76
Density 1.03 0.00 17 6.6 46 0.5 1 29 432 1050 2.43
18 5.2 46 0.5 2 13.5 90 240 2.67
Freezing point °C 0.53 0.02
19 5.2 30 3 2 19.5 78 218 2.79
Protein % 3.07 0.10 20 6.6 46 0.5 1 31 428 1044 2.44
Lactose % 4.61 0.15 21 6.6 46 3 2 13.5 308 781 2.53
Salts % 0.69 0.02 22 5.2 30 0.5 1 20.5 62 175 2.82
Acidity °D 17.25 1.33 23 5.2 46 3 1 19 112 274 2.45
24 6.6 30 0.5 2 31 380 1008 2.65
pH 6.77 0.07
T: Temperature; E: enzyme concentration; Cl.t: Clotting time; Cu.t:
Cutting time; tvm: The time corresponding to the maximum viscosity
The fat contents were above the previous values
described in this breed by (Voutsinas et al. 1990;
Zeng et al. 1997) and below that described by The relation between the cutting time and the time
(Pereira et al. 2010). The protein content was lower corresponding to the maximum viscosity
than indicated by (Voutsinas et al. 1990; Pereira et
al. 2010) and higher than described by (Zeng et al. Table 3 presents the factor's mean value and
1997). For the lactose content was the same value standard deviation (cutting time/ the time
found by (Pereira et al. 2010); it was greater than the corresponding to the maximum viscosity). The
percentages given by (Voutsinas et al. 1990; Zeng et results show that the mean of this factor equal to β=
al. 1997). However, the pH, density and solid-non-fat 2.61. The standard deviation equals 0.13, which
were higher than the values described by (Voutsinas means that the values are less dispersed around the
et al. 1990; Zeng et al. 1997; Pereira et al. 2010). mean, indicating that the average better describes
These differences reflect the lactation period the sample studied. The standard error of the mean
(Wanjekeche et al. 2016), genetic selection, climatic equals 0.03, demonstrating that the estimate of the
and dietary conditions (LÓOPEZ et al. 1999). sample average is more accurate. We can conclude
Factorial design that Cu.t= β *tvm.
Mean
N SE Mean StDev
(β)
(Cu.t/ tvm) 24 2.61 0.03 0.13 Table 3. Descriptive statistics of the factor
Cu.t: Cutting time; tvm: The time corresponding to the (Cu.t/tvm).
maximum viscosity; StDev: Standard deviation; SE
Mean: Standard error of the mean
Clotting time prediction
The design table (Table 2) shows the experimental
From the results of table 4, an uncoded unit
conditions or settings for each factor for the design
regression equation of the clotting time as a function
points. A total of 24 tests are performed with an
of the four physicochemical parameters (pH, CaCl2,
order to be respected, and these results (clotting,
cutting time and the time corresponding to thetemperature, enzyme concentration) is determined
maximum viscosity) are described in table 2. using the experimental procedure. The goodness of
the model's fit was checked by the determination of
R squared (Khan et al. 2006). In this case, the R
Table 2. The mean values and standard squared was 99.42% indicated that the model did
deviations of physicochemical compositions of not explain only 0.58% of the total variations, which
milk samples. shows a better adjustment of this model to the data.
C Furthermore, the value of the adjusted R squared
R
u
a E
C C (R-sq (adj)= 99.17%) was very high, indicating that
T C ( tv
n
( l I Cl. m
u. u. an increased significance of the model. In addition,
O p t t
r H
° 2 M t (
( /
the predicted R squared is high (98.70%), which
C ( C (s) s makes this model have a better forecasting capacity
d s tv
) m U )
e ) m for new observations.
M )
r
)
1 6.6 30 3 1 33 782 1950 2.49
2 6.6 46 3 2 14 312 798 2.56 Table 4. The uncoded unit regression equation of
3 6.6 30 0.5 2 29 388 1032 2.66
the clotting time.
4 6.6 46 3 2 13 316 815 2.58
5 6.6 46 0.5 1 30 436 1051 2.41
6 5.2 46 3 1 18 104 266 2.56
7 5.2 46 0.5 2 14 94 252 2.68

62
 R Table 5. The coded coefficients for the analysis of
- the variability of the regression equation.
R-
R s
sq SE P-Valu
Regression equation in - q Term Effect Coefficient VIF
(p Coef e
uncoded units s (
re Constant 22.25 0.13 0.00
q a
d) pH 8.50 4.25 0.13 0.00 1.00
d
T -6.75 -3.37 0.13 0.00 1.00
j)
CaCl2 -2.50 -1.25 0.13 0.00 1.00
Cl.t= -109.10 + 26.87 A + 1.027 B
99.4 99.1 98.70 E -6.25 -3.12 0.13 0.00 1.00
+ 14.171 C + 21.14 D – 0.2455 A*B
2% 7% % pH*T -2.75 -1.37 0.13 0.00 1.00
– 2.571 A*C – 4.643 A*D
pH*CaCl2 -4.50 -2.25 0.13 0.00 1.00
Cl.t: Clotting time (s); A: pH; B: Temperature (°C);
pH*E -3.25 -1.62 0.13 0.00 1.00
C: sodium chloride concentration (mM); D: SE Coef: coefficient standard error ; VIF: the variance inflation
enzyme concentration (IMCU); R-sq : R squared; factor; T: Temperature; E: Enzyme concentration
R-sq (adj): adjusted R squared; R-sq (pred):
predicted R squared The Pareto chart is used to determine the size and
importance of the terms of the effects (Fig. 1). It
Table 5 represents the coded coefficients for the shows the absolute values of the normalized effects,
analysis of the variability of the regression equation. ranking them from highest to lowest. The terms
The effect of a factor represents the expected change indicated by the diagram were statistically
in the mean response as the element moves from the significant, where they exceeded the reference line at
lower level to the higher. The pH presents the major a 95% confidence interval. The pH, temperature,
positive effect; therefore, this parameter influences concentration of coagulating enzyme were the most
the direction of increase of the response (clotting parameters that affect the clotting time of goat milk
time) going from a pH of 5.2 to 6.6; the same results studied in these experiences.
are described by (Fox et al. 2017; Nájera et al. 2003).
Calcium gradually dissociates from the casein
micelle at low pH. Furthermore, the neutralizing of
the casein's negative charges promotes widespread
aggregation and fusing amongst the micelles, which
tend to form a gel. At pH 4.6, a casein network forms,
encasing the other components (Park 2007).
However, the other terms had negative signs, namely
temperature, enzyme concentration, term pH * CaCl2
concentration, pH * enzyme concentration, pH *
temperature, and calcium chloride successively
influence in reducing the response, i.e., increasing
these terms leads to reducing clotting time. In the
same way (Fox et al. 2017) noted that the reciprocal
enzyme concentration has an excellent linear Fig. 1. The Pareto chart of the absolute values of
connection with clotting time. Also, (Troch et al. the normalized effects.
2017) deduced that increasing the temperature and
increasing the enzyme concentration reduces this To examine the effect of the terms on the response
time. It has been discovered that the temperature of (clotting time), the main effects plot for clotting time
the milk has a significant impact on the pace of is used (Fig. 2). Going from a pH of 6.6 to 5.2, the
protein aggregation and that a higher temperature clotting time decreases, then the decrease in the pH
enhances the rate of gel firming (Nájera et al. 2003). value leads to a reduction of the clotting time. Also,
All terms had a coefficient standard error (The increasing the temperature and increasing the
magnitude of the coefficient is half that of the effect) enzyme concentration reduces this time. These
of 0.13, which is small, which means the estimate results confirmed the previous ones illustrated.
was accurate. Adding that the p-value was less than Although the reinforcement of calcium chloride
the significance level α = 0.05 (0.05 the generally decreases this time, but not in a weak way when
accepted margin of error in the humanities and compared with the other parameters.
social sciences) (Zine‐eddine et al. 2021a;
Zine-eddine et al. 2021b); then, there is a statistically
significant association between the response
variable (clotting time) and the terms. Likewise, the
variance inflation factor (VIF) equals to 1, indicating
no multicollinearity between the predictors (the
absence of multicollinearity simplifies the
determination of statistical significance).

63

Fig. 2. The main effects graph for clotting time.
To show the relationship between the factors
(physicochemical parameters) and the response (the
clotting time), the cube plot (fitted means) was applied.
The results are illustrated in Fig. 3a and Fig. 3b. They
show the clotting time values calculated from the
multiple linear regression equation reported in Table 4
by considering all the possible combinations of the
factors pH, coagulation temperature (T), the
concentration of CaCl2, and concentration of
coagulating enzyme (E).
Fig. 3a. The cube plot (fitted means) for clotting
time ([Enzyme]=1 IMCU).
Fig. 3b. The cube plot (fitted means) for clotting
time ([Enzyme]=2 IMCU).
Fig. 3a indicates the response means when the
enzyme concentration is 1 IMCU, and Fig. 3b shows
the response means when the enzyme concentration
is 2 IMCU.
The low clotting times are presented by the values
pH = 5.2 and temperature = 46 in the two
concentrations of enzymes. It is mentioned that the
high concentration shows the lowest clotting times.
So, lowering the pH makes the clotting time shorter;
likewise, increasing the temperature and the enzyme
concentration makes this time quicker. Adding that
the increase in the level of CaCl2 decreases the
coagulation time and promotes syneresis; but for the
values of pH = 5.2, despite increasing the rate of the
latter, the effect seems to be negative at specific pH
values and at high concentrations of calcium, there
will not be a decrease in the coagulation time but the
reverse. The adverse effect of an elevated calcium
concentration has been attributed to the interaction
of Ca2+ with the aspartate and glutamate groups of
proteins, increasing the net positive charge and
suppression of syneresis (Fox et al. 2017).
Cutting time prediction
The results of table 6 are used to calculate an
uncoded unit regression equation of the time
corresponding to the cutting time as a function of
four physicochemical parameters (pH, CaCl2,
temperature, and enzyme concentration). This
model's R squared was 99.98%, which means that
the model explains 99.98% of the variation in the
response, shows that this model was adjusted to the
data. Furthermore, the predicted R squared is high
(99.96%), indicating that this model has a higher
capacity for forecasting new observations.
Additionally, the adjusted R squared value (R-sq
adj)= 99.97%) was quite high, indicating that the
model was more significant.
Table 6. The uncoded unit regression equation of
the cutting time.
R-
R-s
sq
Regression equation in R-s q
(p
uncoded units q (a
re
dj)
d)
Cu.t= 2.61*(-4671.3 + 901.29 A
+ 57.402 B – 181.20 C 99.98 99.97 99.96
+ 955.3 D – 10.714 A*B % % %
+ 36.00 A*C – 184.29 A*D)
Cu.t: Cutting time (s); A: pH; B: Temperature (°C); C:
sodium chloride concentration (mM); D: enzyme
concentration (IMCU); R-sq : R squared; R-sq (adj):
Adjusted R squared ; R-sq (pred): predicted R squared
Table 7 represents the coded coefficients for the
analysis of the variability of the cutting time
regression equation. The effect of a factor represents
the expected change in the mean response as the
item moves from the lower level to the higher level.
All terms had a low coefficient standard error (the
magnitude of the coefficient is half that of the effect)
of 0.816, which means the estimate was accurate.
Adding that the p-value was below the significance
level α = 0.05; then, there is a statistically significant
association between the response variable (cutting
time) and the terms. Likewise, the variance inflation
factor (VIF) equals 1, indicating the absence of
64
multicollinearity between the predictors (the lack of
multicollinearity simplifies the determination of
statistical significance).
Table 7. The coded coefficients for the analysis of
the variability of the cutting time regression
equation.
Term Coefficient SE Coef P-Value VIF
Constant 282.00 0.82 0.00
pH 196.50 0.82 0.00 1.00
T -46.50 0.82 0.00 1.00
CaCl2 39.00 0.82 0.00 1.00
E -66.00 0.82 0.00 1.00
pH*T -60.00 0.82 0.00 1.00 Fig. 4. The normal probability plot of the
pH*CaCl2 31.50 0.82 0.00 1.00 standardized effects.
pH*E -64.50 0.82 0.00 1.00
Fig. 5 illustrates the main effects graph for cutting
SE Coef: coefficient standard error ; VIF: the variance
inflation factor time. According to this Fig., the pH is the factor that
T: Temperature; E: Enzyme concentration had the greatest effect on the response (cutting
time), where going from a pH of 6.6 to 5.2, the
To determine the magnitude, the direction, and the cutting time decreased. The increase in the enzyme
importance of the effects, the normal probability plot concentration also resulted in a decrease in the
of the effects (Fig. 4) is used. It shows the cutting time but with less effect than the pH. Adding
standardized effects relative to a distribution fit line that the temperature rise still decreased the
when all the effects are 0. On this plot, effects that response but less than the two previous factors. On
are further from 0 are statistically significant. The the contrary, strengthening the calcium chloride
factors presented by the Normal Probability of increases the cutting time but weakly compared to
Effects graph were statistically significant at the 0.05 the other parameters. These results confirm the
level. The positive effects are shown to the right of earlier results of the normal plot of the standardized
Henry's line, and the negative ones are displayed to effects.
the left. The pH has the major positive effect;
consequently, this parameter influences the
direction of increase of the response (cutting time),
going from a pH of 5.2 to 6.6; (Lopez et al. 1998) are
mentioned that a decrease in pH results in a faster
rate of micellar aggregation and bonding, then
shorter cutting time. However, the other terms had
negative signs, namely the concentration of
coagulating enzyme, the term pH * enzyme
concentration, pH * temperature, and temperature
successively influence the reduction of the response, Fig. 5. The main effects graph for cutting time.
and they had negative signs i.e., increasing these
terms results in a decrease in cutting time; (Castillo
et al. 2000) also noted that clotting time and Fig. 6a and Fig. 6b represent the cube plot (fitted
observed cutting time decreased as enzyme means) for cutting time. They show the values of the
concentration and temperature increased and pH cutting time calculated from the multiple linear
decreased. Adding that the calcium chloride and the regression equation reported in table 6, considering
term (CaCl2 * pH) had an effect increasing the all the possible combinations of the physicochemical
response but less than the previous effects (the parameters studied.
effect of the pH shows approximately five times the
effect of these two positive terms) (Fig. 4). At
addition levels greater than 0.02 g CaCl2 / l (i.e., ~ 2
mM Ca), the rate of curd firming and curd firmness
stabilize and decline again to levels greater than or
equal to 0.1 g / l (i.e.,≥ 9 mM Ca). The decrease in
curd tension at higher Ca2+ levels may be due to the
effect of the interaction of excess Ca2+ with negatively
charged carboxyl groups on paracasein; this would
result in increasing the positive charge on the casein,
making it less prone to aggregation (Fox et al. 2017).

65
Fig. 6a. The cube plot (fitted means) for cutting successively the parameters most influencing cutting
time ([Enzyme]=1 IMCU). time. It should be noted that calcium chloride
reinforcement appears to be negative at specific pH
values, making clotting and cutting time long,
confirming the results of previous research. It is
adding that increasing the temperature to a certain
degree can affect the enzyme's activity. These results
could contribute to optimizing the cheese
manufacturing process on an industrial scale. To
know the effect of the parameters studied on the
coagulation process and how to exploit them to
achieve the best transformations.

REFERENCES

E Balcones, A Olano and M M Calvo (1996). Factors

Fig. 6b. The cube plot (fitted means) for cutting
time ([Enzyme]=2 IMCU).
Fig. 6a indicates the response means when the
enzyme concentration is 1 IMCU, and Fig. 6b shows
the response means when the enzyme concentration
is 2 IMCU. The values pH=5.2 and temperature = 46
presented the low values of the cutting time in the
two concentrations of coagulating enzyme, namely
that the high concentration had the less weak. These
results confirmed those previously described
mentioning that the cutting time decreases by
increasing the enzyme concentration and
temperature. It is to highlight that the decrease in pH
does not affect the enzyme activity. In the value of
pH = 5.2 and the two concentrations of enzymes and
calcium chloride, the increase in temperature led to
an increase in the cutting time; despite this increase,
this time remains still short. This slight increase in
this time may be due to the thermal sensitivity of the
coagulating enzyme (Rhizomucor miehei), where the
latter loses its activity at 47 ° C (Fox et al. 2017).
Going from a CaCl2 concentration of 0.5 mM to 3 mM,
an increase in cutting time is noticed. As previously
described and at high concentrations of calcium
chloride, the latter resulted in an undesirable effect
leading to increased cutting time.
CONCLUSIONS
Uncoded unit regression equations of the clotting
and cutting time as a function of the physicochemical
parameters (pH, CaCl2, temperature, enzyme
concentration) were elaborated, which show an
increased significance of these models and a better
forecasting capacity for new observations. The pH,
the temperature and the concentration of
coagulating enzyme were successively the
parameters most influencing clotting time. Namely
that the increase in these parameters makes the
clotting time shorter. However, the increase in the
level of calcium chloride makes the clotting time
shorter but not of the same effect as the previous
parameters. While for the cutting time, the pH, the
enzyme concentration and the temperature were
affecting the rennet clotting properties of ewe's
milk. Journal of Agricultural and Food Chemistry 44
1993-1996.
M Castillo, F Payne, C Hicks and M Lopez (2000).
Predicting cutting and clotting time of coagulating
goat's milk using diffuse reflectance: effect of pH,
temperature and enzyme concentration.
International Dairy Journal 10 551-562.
C Daviau, M-H Famelart, A Pierre, H Goudédranche
and J-L Maubois (2000). Rennet coagulation of
skim milk and curd drainage: effect of pH, casein
concentration, ionic strength and heat treatment.
Le Lait 80 397-415.
P F Fox, T P Guinee, T M Cogan and P L Mcsweeney
(2017). Fundamentals of cheese science. Springer.
P Frederiksen, K Andersen, M Hammershøj, et al.
(2011). Composition and effect of blending of
noncoagulating, poorly coagulating, and
well-coagulating bovine milk from individual
Danish Holstein cows. Journal of Dairy Science 94
4787-4799.
V García, S Rovira, K Boutoial, D Álvarez and M
López (2015). A comparison of the use of thistle
(Cynara cardunculus L.) and artichoke (Cynara
scolymus L.) aqueous extracts for milk coagulation.
Dairy science & technology 95 197-208.
V García, S Rovira, R Teruel, et al. (2012). Effect of
vegetable coagulant, microbial coagulant and calf
rennet on physicochemical, proteolysis, sensory
and texture profiles of fresh goats cheese. Dairy
science & technology 92 691-707.
E Gastaldi, A Lagaude and B T De La Fuente (1996).
Micellar transition state in casein between pH 5.5
and 5.0. Journal of Food Science 61 59-64.
J-P Guiraud (1998). La microbiologie alimentaire.
S Gunasekaran and C Ay (1996). Milk coagulation
cut‐time determination using ultrasonics. Journal
of Food Process Engineering 19 63-73.
I Joudu, M Henno, T Kaart, T Püssa and O Kärt
(2008). The effect of milk protein contents on the
rennet coagulation properties of milk from
individual dairy cows. International Dairy Journal
18 964-967.
S Khan, A Misra, C Tripathi, B Mishra and V Bihari
66
(2006). Response surface optimization of effective
medium constituents for the production of alkaline
protease from a newly isolated strain of
Pseudomonas aeruginosa.
C Lapointe-Vignola (2002). Science et technologie
du lait: transformation du lait. Presses inter
Polytechnique.
B A Law and A Y Tamime (2011). Technology of
cheesemaking. John Wiley & Sons.
Y Le Graet and G Brulé (1993). Les équilibres
minéraux du lait: influence du pH et de la force
ionique. Le Lait 73 51-60.
M Lóopez, M Jordán, M Granados, J Fernández, M
Castillo and J Laencina (1999). Viscosity changes
during rennet coagulation of Murciano‐Granadina
goat milk. International journal of dairy technology
52 102-106.
M Lopez, S Lomholt and K Qvist (1998).
Rheological properties and cutting time of rennet
gels. Effect of pH and enzyme concentration.
International Dairy Journal 8 289-293.
A Nájera, M De Renobales and L Barron (2003).
Effects of pH, temperature, CaCl2 and enzyme
concentrations on the rennet-clotting properties of
milk: a multifactorial study. Food Chemistry 80
345-352.
Y Park (2007). Rheological characteristics of goat
and sheep milk. Small Ruminant Research 68
73-87.
F Payne, C Hicks, S Madangopal and S Shearer
(1993). Fiber optic sensor for predicting the cutting
time of coagulating milk for cheese production.
Transactions of the ASAE 36 841-847.
R Pereira, C Oliveira, A Medeiros, R Costa, M
Bomfim and R Queiroga (2010). Physicochemical
and sensory characteristics of milk from goats
supplemented with castor or licuri oil. Journal of
Dairy Science 93 456-462.
A Picon, P Gaya, M Medina and M Nunez
(1995).Kinetics of milk coagulation by mixtures of
cyprosin and chymosin. Milchwissenschaft
(Germany).
D Pretto, T Kaart, M Vallas, et al. (2011).
Relationships between milk coagulation property
traits analyzed with different methodologies.
Journal of Dairy Science 94 4336-4346.
F Remeuf, V Cossin, C Dervin, J Lenoir and R
Tomassone (1991). Relations entre les caractères
physico-chimiques des laits et leur aptitude
fromagère. Le Lait 71 397-421.
O Siboukeur, A Mati and B Hessas (2005).
Amélioration de l'aptitude à la coagulation du lait
cameline (Camelus dromedarius): utilisation
d'extraits enzymatiques coagulants gastriques de
dromadaires. Cahiers agricultures 14 473-478
(471).
Fagan CC, Castillo M, Payne F, O’Donnell C,
O’Callaghan D (2007). Effect of cutting time,
temperature, and calcium on curd moisture, whey
fat losses, and curd yield by response surface
methodology. Journal of dairy science 90
4499-4512.
F Solorza and A Bell (1998). The effect of calcium
addition on the rheological properties of a soft
cheese at various stages of manufacture.
International journal of dairy technology 51 23-29.
N Tabayehnejad, M Castillo and F Payne (2012).
Comparison of total milk-clotting activity
measurement precision using the Berridge clotting
time method and a proposed optical method.
Journal of Food Engineering 108 549-556.
T Troch, É Lefébure, V Baeten, F Colinet, N Gengler
and M Sindic (2017). Cow milk coagulation:
process description, variation factors and
evaluation methodologies. A review.
Biotechnologie, Agronomie, Société et
Environnement 21.
A Van Hooydonk and G Berg (1988). Control and
determination of the curd-setting during
cheesemaking. Federaion Internationale de
Laiterie.
L Voutsinas, C Pappas and M Katsiari (1990). The
composition of Alpine goats' milk during lactation
in Greece. Journal of Dairy Research 57 41-51.
E Wanjekeche, Z Macosore, A Kiptanui and T Lobeta
(2016). Quality and consumer acceptability of goat
milk with respect to goat breed and lactation stage.
African Crop Science Journal 24 95-99.
S Zeng, E Escobar and T Popham (1997). Daily
variations in somatic cell count, composition, and
production of Alpine goat milk. Small Ruminant
Research 26 253-260.
Y Zine-Eddine, L H Zinelabidine, F Kzaiber, A
Oussama and K Boutoial (2021a). Analysis of
acceptance and factors affecting the consumption
of goat's milk in Morocco. Small Ruminant Research
197 106338.
Y Zine‐Eddine, L H Zinelabidine, F Kzaiber, A
Oussama and K Boutoial (2021b). The use of paired
comparison test on the goat's milk adulteration
detection (whole and semiskimmed). Journal of
Sensory Studies e12690.
P Zoon, T Van Vliet and P Walstra (1988).
Rheological properties of rennet-induced skim milk
gels. 2. The effect of temperature. Netherlands Milk
and Dairy Journal 42 271-294.
67
CONFERENCE PAPER Hajri Zakariae et al., 2022, 68-72

Effect of Cooking and Washing on Sulfites (SO2) Content of Dried Fruits

Zakariae Hajri1* , Abdelkhalek Oussama1 , Mohammed Chigr2 , Fouzia Kzaiber1 , Khalid Boutoial
1
Laboratory of the Engineering and Applied Technologies, Higher School of Technology, Sultan
Moulay Slimane University, Beni Mellal, Morocco
2
Laboratory of Bio-Organic and Analytical Chemistry, Faculty of Science and Technology, Sultan
Moulay Slimane University, Beni Mellal, Morocco

Sulfites are a group of chemical food additives mainly used in the food
industry as preservatives. This study was conducted to determine the
impact of three cooking methods and washing on the sulfites content of
dried apricots and raisins, which allows us to estimate the potential average
intake of sulfites through these dried fruits in the actual conditions of
consumption. Sulfites concentrations of raw and cooked dried fruit samples
were analyzed according to the optimized Monier-Williams method. As a
result, the average concentration of sulfites in raw dried apricots was 2008
mg/kg, while the raisins contain sulfites with a mean concentration of 374
mg/kg. Washing allowed the removal of 16% of the initial sulfite content for
dried apricots and about 30% for raisins. Steam cooking reduced the
sulfites content by 16% in dried apricots and 20% in raisins. Boiling
reduces sulfites by 44% in dried apricots and 41% in raisins.
Caramelization eliminated 41% of sulfites for dried apricots and 34% for
raisins. This study shows the considerable effect of different cooking
methods and washing on eliminating part of sulfites from dried fruits.
Therefore, appropriate washing and cooking of sulfites-containing dried
fruits significantly reduce consumer exposure to sulfites, thus minimizing
possible adverse health effects.

* 2008), the family of sulfites includes eight food

Corresponding Author
additives, namely, sulfur dioxide (E220), sodium
Name: Hajri Zakariae sulfite (E221), sodium acid sulfite (E222), sodium
Email: zakariae.hajri@gmail.com disulfite (E223), potassium disulfite (E224),
Phone: +212632646327 calcium sulfite (E226), calcium acid sulfite (E227),
and potassium acid sulfite (E228). These additives
ISBN: 9788367405256 can be used in the food sector as antioxidant
DOI: 10.2478/9788367405256-010 agents, bleaching agents, flour treatment agents, or
preservatives (Codex Alimentarius, 2019).
Published by Sciendo. © 2022 Hajri Zakariae et al. This is an
open access article licensed under the Attribution 4.0 Since the 1970s, questions and observations have
International been raised about the safety of sulfites in foods
(https://creativecommons.org/licenses/by/4.0/)
(Lester, 1995; Babgaleh et al., 2004; Taylor et al.,
2014). Multiple studies have confirmed that the
significant risk of sulfites concerns only a part of
INTRODUCTION the asthmatic population (Armentia-Alvarez et al.,
1993; Vally et al., 2009). However, a range of
The term sulfites refer to sulfur dioxide (SO2) and adverse effects has been attributed to sulfites,
several other forms of inorganic sulfites that including asthma, anaphylaxis, hives, diarrhea,
release SO2 (Taylor et al., 1986). According to the abdominal pain and cramps, nausea and vomiting,
European Commission (European Commission, pruritus, fainting, headaches, chest pain, loss of
68
consciousness, skin rashes (Wilson et Bahna, 2005;
Taylor et al., 2014). Nevertheless, the appearance
or not of adverse effects depends on the sensitivity
degree of the individual, the type of food involved,
and the residual levels of SO2 in foods 2 (Taylor et
al., 1986; Bush et al., 1990). The Joint FAO/WHO
Expert Committee on Food Additives (JECFA) has
established an Acceptable Daily Intake (ADI) for
sulfites of 0.7 mg/kg body weight expressed as
sulfur dioxide (WHO/FAO, 1974).
Sulfites are permitted as food additives in more
than 100 foods with Maximum Permitted Levels
(MPLs) by the European regulation (European
Commission, 2008), Moroccan regulation (Ministry
of Agriculture/Ministry of Health, 2014), and by
the Codex Alimentarius (Codex Alimentarius,
2019), including fruits and vegetables (fresh,
frozen, dried, canned), confectionery, shellfish,
mustard, fruit juice and processed potato products.
Sulfites are also subject to restrictions where their
use in olives or processed meats, for example, is
prohibited. Among all these food products, dried
fruits, particularly apricots, peaches, grapes, plums,
and figs, are allowed to contain up to 2000 mg/kg,
the highest permitted level compared with all other
foods. Sulfites are also subject to restrictions where
their use in olives or processed meats, for example,
is prohibited.
Different studies have confirmed the reducing
effect of cooking on the concentration of sulfites in
some foods. A study in Spain (Pena-Egido et al.,
2005) found that cooking on the grill of meat
burgers samples (beef and chicken) reduced the
initial content of sulfites by 28 % for the total SO2,
29 % for the free SO2, and 31 % for the
S-sulphonates. Another study conducted in Spain
(Armentia-Alvarez et al., 1993) on the influence of
frying on sulfites content in beef, pork, and chicken
burgers, concluded that the process of frying with
vegetable oil leads to a reduction of the initial range
of total SO2 by 41% (± 12.6%) for all the analyzed
samples. Cooking is not the only process that
allows the reduction of the initial concentration of
sulfites. Storage conditions also have a reducing
effect (Vandevijvere et al., 2010).
This study aims to evaluate the effect of cooking
and washing processes on the residual sulfite
content of some potentially containing-sulfites
dried fruits. Indeed, this study consists, firstly, in
the analytic measurement of sulfites concentration
in three raw dried fruits (apricots, grapes and
plums) highly coveted by Moroccan consumers. In a
second step, the same batches of dried fruits
underwent different cooking methods (cooking in
boiling water, steaming and caramelization) as well
as a simple washing in running water, then were
analyzed a second time to determine the effect of
each method of cooking and washing on the
reduction of the final residual sulfite content. The
results would give an idea of the exposure level to
sulfites in real consumption conditions.
MATERIAL AND METHODS
Samples
In Morocco, the main dried fruits consumed raw or
cooked in traditional dishes are apricots, plums and
grapes. For that reason, these three foods are
chosen for this study. Two batches of 500g of each
dried fruit were collected from local shops in Beni
Mellal city (north-central Morocco). The first batch
was collected from local grocery stores for each
dried fruit and the second from supermarkets. All
samples collected were put in clean plastic bags
with a zip closure, identified and stored in a
protected place.
Analytical method
The determination of sulfites concentrations in the
collected samples of dried fruits was performed
according to the optimized MonierWilliams method
(AOAC, 2000).
With a limit of quantification of 10 mg/kg, this
method allows the measurement of free sulfites
and the reproducible portion of bound sulfites such
as carbonyl addition products present in foods
(AOAC, 2000). A 50g portion of the sample is
ground in a blender with an ethanol/water mixture
(5%/95%). The sample contained in the
ethanol/water solution is transferred to a
round-bottom flask where it is heated under reflux
with a hydrochloric acid solution (90ml) for 105
minutes to transform the sulfites into sulfur dioxide
(SO2). A stream of nitrogen injected into the
refluxing solution allows the sulfur dioxide to be
carried away to the neutral solution of hydrogen
peroxide (3%) contained in a bubbler. On arrival in
this solution, the sulfur dioxide is oxidized and
transformed into sulfuric acid, which is titrated by
a standard sodium hydroxide solution.
After titration, each milliliter of sodium hydroxide
obtained equals 0.32 mg of sulfur dioxide. The
sulfur dioxide content, expressed in mg/kg of the
sample analyzed, is then calculated by the following
formula:
SO2 = 32.03 x V x M x 1000 / m
Where 32.03 is the milliequivalent mass of SO2, V is
the volume (ml) of 0.01M sodium hydroxide
obtained in the titration, M is the molarity of the
sodium hydroxide solution, 1000 is the conversion
factor from milliequivalents to micro equivalents,
and m is the weight (g) of the test sample. The
results obtained are rounded without decimal
places.
Any sample that presents a result lower or equal to
69
the limit of quantification (10 mg/kg) is considered
sulfites-free (negative sample).
Determination of sulfites levels in raw samples
The first step was to determine the sulfite
concentrations of all raw dried fruits without any
treatment or washing (as collected). The dried
fruits were collected and analyzed separately to
avoid any contamination.
Determination of sulfites levels after cooking
and washing
In this step, the samples with positive results
(presence of sulfites) are analyzed a second time,
using the same method, just after being washed
with clean water and then applying three cooking
methods. For washing, dried fruits samples
containing sulfites are washed with tap water in a
stainless steel sieve for 2 minutes. The three
cooking methods applied are: cooking in boiling
water, steam cooking and caramelization. The first
cooking method consists in putting the raw dried
fruits in a stainless steel pan filled with boiling
water for 15 minutes. The second method is to
steam them in a stainless steel steamer for 15
minutes, and the last process consists in
caramelizing them in a stainless steel pan by
adding water and granulated sugar.
The choice of these cooking methods is justified by
how these dried fruits are used in Moroccan
culinary. Indeed, the dried fruits studied are used in
traditional dishes (beef or chicken tagine,
couscous...) where they undergo cooking processes
similar to those chosen for this study.
All samples were analyzed twice (two tests per
analysis) for both phases. The analysis results are
reported in an individual analysis report with all
the necessary information (such as place and
number of sampling, food concerned, name and
address of the manufacturer, the formula of
calculation of the results).
Statistical analysis
The statistical analysis was carried out using
Microsoft Office Excel (version 2010, Microsoft,
USA) and Minitab Statistical Software (version 16,
Minitab Inc. USA). The results obtained are
expressed as "mean ± standard deviation."
Significant differences between means were
determined using a one-way analysis of variance
(ANOVA test) combined with a Tukey's test with a
significance level of ≤0.05.
RESULTS AND DISCUSSION
Sulfites level in raw dried fruits
The concentrations of sulfites in raw dried fruits
(without washing or cooking) are given in Table 1.
Results showed that all dried apricots samples
analyzed contain sulfites with an average
concentration of 2008 mg/kg. Such these found
average concentrations are similar to those
determined in Australia (2097 mg/kg; FSANZ,
2005) but well below those found in France (661
mg/kg; Mareschi et al., 1992) and in Belgium (533
mg/kg; Vandevijvere et al., 2010). Both batches of
dried apricots had sulfite levels close to the
maximum permitted level of 2000 mg/kg required
by Moroccan regulations for the dried fruits
analyzed (Ministry of Agriculture/Ministry of
Health, 2014).
All samples of raisins also contain sulfites with an
average residual level of 374 mg/kg lower than the
concentration found in a study from France that
reported a mean content of 173 mg/kg (Mareschi
et al., 1992); these values explain that the sulfites
content complies with the Moroccan regulations.
Dried plums show a concentration below the limit
of quantification (<10 mg/kg) and are therefore
considered untreated by sulfites.
Table 1: Sulfites levels (SO2) in raw dried fruits.
Dried fruits Mean level of MPL (mg/kg)
SO2 (mg/kg)
Dried apricots 2008±8
Raisins 374±77 2000
Dried plums <10
*MPL: Maximum Permitted Level according to Moroccan
regulation.
Sulfites level in dried fruits after washing and
cooking
Tables 2 and 3 present the sulfites concentrations
in dried apricots and raisins after washing and
cooking, respectively. The applied washing allowed
an average removal of 320 mg/kg of sulfites for
dried apricots (16%) and 111 mg/kg of sulfites for
raisins (29.5%). The first cooking method, boiling,
resulted in an average sulfites reduction of 892
mg/kg for dried apricots (44.4%) and 154 mg/kg
for raisins (41%). The steam cooking removed 315
mg/kg of sulfites for dried apricots (15.7%) and 76
mg/kg of sulfites for raisins (20%). The last
cooking method applied, caramelization, resulted
in an average sulfites reduction of 820 mg/kg for
dried apricots (almost 49%) and 127 mg/kg for
dried raisins (34%).
Therefore it is apparent that cooking in boiling
water leads to the reduction of a higher percentage
of sulfites content for both dried fruits, followed by
caramelization. While the treatment that removes a
small amount of sulfite is steam cooking. These
results show that washing the studied dried fruits
70
combined with boiling water cooking allows a doses with which these dried fruits are treated by
reduction of up to 60.4% of the sulfite content for manufacturers, given that the majority of dried
dried apricots and 70.5% for raisins. Nevertheless, apricots and raisins present in the Moroccan
if these foods are consumed without any treatment, market are imported. REFERENCES
that can lead to possible health risks for the
consumer. AOAC. (2000). Official Method 990.28. Sulfites in
Foods, Optimized Monier-Williams Method. 17th
Table 2: Mean levels of sulfites (mg/kg) in dried
apricots after washing and cooking. Edition.
Armentia‐Alvarez, A., Fernandez‐Casero, A.,
Treatment Mean Reduced % SO2
level mean level eliminated Garcia‐Moreno, C., Pena‐Egido, M. J. (1993).
Residual levels of free and total sulphite in fresh
Without 2008±8a - - and cooked burgers, Food Addit. Contam., 10(2),
treatment
157-165.
Washing 1688±8b 320 16%
Babgaleh, T., Koehler, M. K., Wolyniak, C., Klontz, K.
d
Boiling water 1116±3 892 44.4% C. (2004). Sulfites - A Food and Drug
Steam cooking 1693±10b 315 15.7% Administration review of recalls and reported
adverse events. J. Food Prot., 67(8), 1806–1811.
Caramelization 1180±20c 820 40.8%
Bush, R. K., Zoratti, E., Taylor, S. L. (1990). Diagnosis
of sulfite and aspirin sensitivity. Clin. Rev. Allergy,
The comparison of the effect of washing on the
8(2-3), 159-78.
reduction of sulfite content of dried apricots and
raisins shows a significant difference (16% vs. Codex Alimentarius, World Health Organization
29.5%). This can be explained by the volume of the (WHO) and Food and Agriculture Organization of
two dried fruits (dried apricots being more the United Nations (FAO), (2019). General
voluminous than raisins) and by the initial sulfites standard for food additives. Adopted in 1995, last
content (2008 mg/kg for dried apricots vs. 374 revision in 2019.
mg/kg for raisins). The three cooking methods European Commission (EC). (2008). Regulation No
tested have a more or less similar effect in sulfite 1333/2008 of the European parliament and of
reduction for both dried fruits. the council of 16 December 2008 on food
Table 3: Mean levels of sulfites (mg/kg) in raisins additives. OJ L 354.
after washing and cooking. Food Standards Australia New Zealand (FSANZ).
Mean Reduced % SO2 (2005). The 21st Australian Total Diet Study. A
Treatment
level mean level eliminated total diet study of sulphites, benzoates and
sorbates. FSANZ, Canberra, Australia.
Without 374±77a - -
treatment Lester, R. M. (1995). Sulfite sensitivity: significance
Washing 263±63ab 111 29.5%
in human health. J. Am. Coll. Nutr., 14(3), 229-
232.
Boiling water 220±50b 154 41%
Mareschi, J. P., François‐Collange, M., Suschetet, M.
ab
Steam cooking 298±67 76 20% (1992). Estimation of sulphite in food in France.
Caramelization 247±52 ab
127 34% Food Addit. Contam., 9(5), 541-549.
Ministry of Agriculture/Ministry of Health,
CONCLUSION Morocco (2014). Joint Order No. 1795-14 of May
14, 2014, establishing the list and limits of food
The reducing effect of conventional cooking additives authorized for use in primary products
methods and washing on the residual sulfite and foodstuffs, as well as the indications that
content of dried fruits has been demonstrated their packaging must bear (in French).
through this study. Washing dried fruits abundantly Peña-Egido, M. J., García-Alonso, B., GarcíaMoreno,
before consumption is an important and desirable C. (2005). S-sulfonate contents in raw and cooked
action to reduce the high levels of sulfites that the meat products. J. Agric. Food Chem., 53(10),
consumer can ingest through these foods. Thus, the 4198–4201.
cooking methods of dried fruits used in Moroccan
culinary eliminate a significant part of the initial Taylor, L. S., Higley, N. A., Bush, R. K. (1986). Sulfites
sulfite content. Nevertheless, if these dried fruits in foods: Uses, analytical methods, residues, fate,
are consumed without any washing or cooking, the exposure assessment, metabolism, toxicity, and
consumer would be exposed to relatively high hypersensitivity. Adv. Food Res., 30, 1-76.
concentrations of sulfites. For this reason, a Taylor, L.S., Bush, R. K., Nordlee, A. J. (2014). Sulfites
downward revision of the maximum permitted in Food Allergy: Adverse Reactions to Foods and
level (2000 mg/kg) may be necessary to reduce the Food Additives, Fifth Edition, 361- 374. Edited by
71
 Metcalfe D. D., Sampson A. H., Simon A. R., Lack G.
John Wiley & Sons, Ltd.
Vally, H., Misso, N. L., Madan, V. (2009). Clinical
effects of sulphite additives. Clin. Exp. Allergy, 39,
1643–1651.
Vandevijvere, S., Temme, E., Andjelkovic, M., De Wil,
M., Vinkx, C., Goeyens, L., Van Loco, J. (2010).
Estimate of intake of sulfites in the Belgian adult
population, Food Addit. Contam., 27(8),
1072-1083.
Wilson, G. B., Bahna, L. S. (2005). Adverse reactions
to food additives. Ann. Allergy Asthma Immunol.,
95(6), 499–507.
World Health Organization (WHO) and Food and
Agriculture Organization of the United Nations
(FAO).
Joint FAO/WHO Expert Committee on Food
Additives (JECFA), (1974). Toxicological
evaluation of certain food additives with a review
of general principles and of specifications:
seventeenth report of the JECFA, Geneva.

72
CONFERENCE PAPER Najem Ayoub et al., 2022, 73-77

Citrus Polymethoxyflavones as Corrosion Inhibitor for Mild Steel in HCl medium

Najem Ayoub* , Sabiha Malika , Echihi Siham , Bellaouchou Abdelkbir , and Boudalia Maria
Laboratory of Nanotechnology, Materials and Environment, Chemistry department, Faculty of
Sciences, Av. Ibn Battouta, BP 1014 RP, M-10000, Rabat, Morocco

Citrus polymethoxyflavones CPMFs are natural organic compounds,

they are mainly found in different varieties of citrus and with high
percentage. They exhibit several pharmacological activities, such as
antioxidant (Lou et al., 2014), anti-inflammatory (Wang et al., 2018),
antibacterial (Wu et al., 2013). However, the main aim of this study is to
perform anti-corrosion test on mild steel in acidic medium HCl 1M.
Indeed, Weight Loss measurement (WL) and Electrochemical Impedance
Spectroscopy (EIS) were carried out through this work. The results
depicted significant efficiency around 95.78% using only 100ppm and
high inhibition activity was recorded.

* the exorbitant service (Ashassi-Sorkhabi et al.,

Corresponding Author
2009). Several studies have shown the use of
Name: Najem Ayoub green inhibitors to combat the degradation of a
Email: Najem-ayoub @hotmail.com wide range of metals: steel, copper, aluminum…
Phone: +212 673297310 in acidic media (Kamal et al., 2012), (Chaubey et
al., 2021). Our main target is to experiment citrus
ISBN : 9788367405256 polymethoxyflavones (PMFs) elements as
DOI: 10.2478/9788367405256-011 organic natural inhibitor agent to preserve mild
steel from corrosion phenomenon. Nowadays,
Published by Sciendo. © 2022 Alfonso Infante-Moro et al. corrosion is still a significant challenge faced by
This is an open access article licensed under the Attribution
4.0 International
industries and infrastructure worldwide, leading
(https://creativecommons.org/licenses/by/4.0/) to extensive financial losses and safety concerns.
Specifically, the mild steel, which is considered as
a commonly used material in various
INTRODUCTION applications, is particularly susceptible to
corrosion, especially in acidic environments. For
Corrosion is a group of actions that occur due to this reason, the economy is looking for agents to
the interaction of the environment with a certain mitigate this problem. Researchers have been
material, resulting in the deterioration and loss exploring the potential of natural compounds as
of its properties (Afolalu et al., 2017). Mild steel corrosion inhibitors, aiming to develop
is known as the most popular alloy used in the eco-friendly and cost-effective alternatives to
manufacturing industry due to its properties and synthetic inhibitors. One of the promising natural
attributes such as strength, toughness, buckling, compounds of interest is citrus peels, which
toughness, etc. Accordingly, tremendous contain organic acids and polyphenols that have
destructions and damages happens by piloting shown promise as corrosion inhibitors. This
work involves investigating the effectiveness of
73
 citrus peels and particularly, a specific class of
compound, named PMFs obtained from
Moroccan citrus of Beni Mellal region to make a
barrier for corrosion inhibitor that happens to
mild steel in acidic medium, specifically
hydrochloric acid (HCl).
METHODS
Weight Loss assay
The classical way is still quite used as guide
during the first evaluation of corrosion inhibition
to get approximative pattern, however the
selected material could have a potential
inhibition. The weight loss testing was primarily
performed. A required immersion time is
accounted. The material is first measured and
then soaked into 100 mL with the inhibitor, as
well as without the presence of inhibitor. A series
of different concentrations from 25 ppm to 100
ppm was developed. Then the specimen was
carefully removed from the solution with
tweezers, rinsed with distilled water, then
followed by acetone, and eventually dried using
hot air. At the time of this assay, the basic
characteristic is the calculation of ∆W using the
unit of mg. The investigations were performed
under an aggressively aerated environment. The
effectiveness of inhibition of citrus PMFs was
derived using the following equations (1 and 2):
(1)
(2)
Where Wcorr° and Wcorrinh are depicting the
corrosion rate, both in the absence of inhibitor
and when citrus PMFs inhibitor is present. Δm, S
and T are respectively, describing the average
weight loss before submersion as well as after
submersion (mg), the overall surface area (cm2)
and the time of submersion (h).
Electrochemical Impedance Spectroscopy
Results obtained in the previous method; the
weight loss should be confirmed along with the
EIS method. Electrochemical impedance
spectroscopy is more sophisticated method and
considered as the best way to evaluate the quality
of the results. This analytical technique was
applied to all concentrations. As parameters we
set up 10 mV peak as the amplitude of the
sinusoid imposed on the free potential. The range
of frequency was set between 100 kHz to 10 MHz
Measuring electrochemical impedance and
achieving efficiency scores was done with the PGZ
100 potentiostat apparatus driven by the
Voltamaster 4 software. For this purpose, three
electrodes designated; saturated calomel, short
form SCE, auxiliary, and subsequently our working
electrode, short form MS, were employed in the
sense of mild steel. A volume of 500 mL has been
distributed to the double-walled thermostat
volume. The EIS measurement extracted loop is
attained after a necessary stabilization of the work
electrode into the aggressive medium of HCl. A
period of 30 minutes was the criteria required in
open circuit potential, before carrying out the
tests. The Nyquist curve model was the one
followed to obtain a shape of loop and to get also
the Rct and Cdl named consecutively as
polarization resistance and double layer capacity.
Afterwards, the effectiveness of our organic
natural reducer has been calculated with the
sequence (3):
(3)
Where, Rctinh defining the resistance of charge
transfer in case of an inhibitor and Rct° defining the
resistance of charge transfer in case of no inhibitor.
RESULTS
Weight Loss assay
In this primary focus, weight loss will be explored
by submerging the preselected material in the
aggressive medium of HCl. Such environment is
known as a damaging agent, since it causes pitting
and cracking on the mild steel surface. We will
investigate the overall effect of CPMFs and
determine how long this material can protect our
metal. The measure of weight loss could provide us
thoughtful results of the corrosion procedure with
the presence of CPMFs and without it (Zhang et al.,
2018).
Table 1: Mild steel weight loss data in 1 M HCl
solution in the absence and in the presence of
PMFs at different
C Wcorr IEWL
(ppm) (mg cm-2h-1) (%)
Blank 1.4271 -
100 0.1946 90.50
75 0.2852 88.49
50 0.4061 71.61
25 0.7314 64.05
74
 Based on the findings results in the above table,
we perceive that there is a huge difference of
weight loss between the intervention of CPMFs
agent and without it. Hence, by using a moderate
concentration of CPMFs, the weight loss of the
mild steel is greatly reduced. We further observe
that the corrosion rate as well as the efficiency
shift inversely from the following figure.
frequency characterizing the charge transfer process
(Biswas et al., 2015). The needed corrosion settings
with respect to the impedance mode have been
summarized in Table 2 below.
Figure 2 : Nyquist curves of MS with and without the
presence of various concentrations of PMFs

Figure 1 : The change of the rate of corrosion (Wcorr) In all released measurements for the different
and the efficiency of PMFs with various concentrations, we can observe only one depressed
concentrations capacitive loop. As a consequence, there is just one
phenomenon obtained. We could deeply interpret
According to the above figure, we find that at the the obtained capacitive loops through two main
concentration of 100 ppm, the rate of corrosion gets events process; the charge transfer and the
slower and the effectiveness achieves a great construction of the double layer. As a matter of fact,
performance. Consequently, our investigated sample by adding the amount of PMFs to hydrochloric acid,
(CPMFs) proved to play an outstanding role in there is an increase in the inhibitor concentration,
mitigating the weight of mild steel and in saving it and the Nyquist plots grow larger with respect to
from the phenomenon of corrosion. PMFs appear to hydrochloric acid alone without inhibitor.
be an excellent corrosion attenuator agent. The Concurrently, the size of the loops becomes larger.
approach of inhibition could be due to the existence The pattern followed is an equivalent simple Randle
of methoxy groups (Verma et al., 2018). As it was circuit.
mentioned by (M’hiri et al., 2016) a good inhibitory
attribute was related to the presence of compounds
such as flavonoids and in particular PMFs. The
subsequent trial will be conducted with
electrochemical impedance spectroscopy to validate Figure 3 : Equivalent circuit model tried during this
the results of the present test. study to fit the metal/solution interface

Electrochemical Impedance Spectroscopy
Figure 2 below, plots the EIS results in varying
concentrations of CPMFs, and Table 5 below displays
the measurement parameters. To understand better
and acquire information of other angle about the
mechanism that took place between the CPMFs and
the chosen mild steel electrode surface, the
electrochemical impedance
spectroscopy experiments was undertaken. The
measurements have been thoroughly guided under
the potentiostatic settings with the intervention of
various concentrations of CPMFs and in the absence
of this material. We have operated the more used
model of Nyquist to provide more important
informations about the test done. The weakest
With Rs being the resistance of the solution, CPE
being the constant phase element, Rct being the
charge transfer resistance. Figure 3 plots the
equivalent circuit taken during the course of our
electrochemical test. All upcoming published EIS
plots are to be interpreted in accordance with this
modified Randles circuit. Subsequently, the
parameters that were obtained were extracted
using the Z-view software. The equivalent circuit
modeling was used as in approach to study
corrosion inhibition. It allows us to depict the
electrochemical behavior of the corroding material
used during this work (MS). By the use of an
electrical circuit analogy, we were able to provide

75
insights into the electrochemical processes
occurring at the metal-electrolyte interface and
helps analyze the efficiency of corrosion inhibitors.
Table 1: Parameters of Electrochemical Impedance
Spectroscopy method
enhanced through modifications in their structure
or by combining them with other inhibitors or
additives. Additionally, the environmentally
friendly nature of PMFs makes them an attractive
alternative to synthetic corrosion inhibitors.

C Rs R ct Q
(ppm) (Ω.cm2) (Ω.cm2) Further research is needed to fully understand the
HCl 0.56313 20.77 0.0047848 mechanism of action of PMFs as corrosion
6
inhibitors and optimize their performance.
100 3.406 492.7 0.000780
Parameters such as long-term stability,
10-6
compatibility with different corrosive
75 3.243 384.2 0.008315
environments, and the influence of PMF
10-6
concentration on inhibition efficiency require
50 2.577 224.5 0.001081 further investigation to establish the practical
10-6 application of PMFs in corrosion protection.
Polymethoxyflavones derived from citrus fruits
25 2.417 82.76 0.002430
offer a potential solution for corrosion inhibition
10-6
in various industries. Their natural origin,
inhibitory properties, and compatibility with
metals make them an intriguing area of research.
The outcome plots indicate that the Rct values are Continued exploration of PMFs as corrosion
increased when the PMFs dose is increased, thus inhibitors could lead to the development of
leading to a great efficiency as well as a good sustainable and effective solutions for mitigating
surface treatment. And presumably the double corrosion-related challenges in different sectors.
layer capacity (Cdl) will get lowered in the existing
of our reducer agent (CPMFs). Accordingly, we will CONCLUSIONS
experience a reduction in the features afforded in
the inhibitor through the adsorption of the The CPMFs have been found to play a pivoted part
metal-acid interface (el Bribri et al., 2013). Similar in preventing and mitigating corrosion failure. It
efficiency surrounded 95% were obtained also by has been shown to act as a mixed inhibitor against
(Asadi et al., 2019) using Lemon Balm extract, cathodic as well as anodic reactions with an
while our work remains more friendly in terms of effectiveness of 95.78% at a small concentration of
using citrus waste rather than destroying nature by 100 ppm. As a perspective, PMFs could be
cutting medicinal plants. investigated through potentiodynamic polarization
tools (PDP) and also scanning electron microscope
DISCUSSION (SEM) to get information on the surface
morphology.
These results depict the potential of citrus peel
extract as corrosion inhibitors. The inhibitory
effect of citrus can be attributed mainly to the REFERENCES
presence of polymethoxyflavones. This class of
natural compounds can gain a big attention in the Afolalu, S. A., Adetunji, O. R., Salawu, E. Y.,
coming years. They can show promise in Okokpujie, I. P., Abioye, A. A., & Abioye, O. P.
protecting metals from degradation, specifically (2017). Cost of Corrosion of Metallic Products in
mild steel. The inhibitory mechanism of PMFs Federal. In International Journal of Applied
involves their adsorption onto the metal surface, Engineering Research (Vol. 12). Retrieved from
which is influenced by factors such as http://www.ripublication.com
concentration, temperature, and pH of the Asadi, N., Ramezanzadeh, M., Bahlakeh, G., &
corrosive medium. The presence of methoxy Ramezanzadeh, B. (2019). Utilizing Lemon
groups enhances the adsorption of PMFs onto the Balm extract as an effective green corrosion
metal surface, forming a barrier that prevents the inhibitor for mild steel in 1M HCl solution: A
corrosive agents from reaching the metal and detailed experimental, molecular dynamics,
inhibiting the electrochemical reactions that Monte Carlo and quantum mechanics study.
initiate corrosion. Journal of the Taiwan Institute of Chemical
Engineers, 95, 252–272. doi:
The inhibition efficiency of PMFs can be further 10.1016/j.jtice.2018.07.011
76
Ashassi-Sorkhabi, H., Masoumi, B., Ejbari, P., &
Asghari, E. (2009). Corrosion inhibition of mild
steel in acidic media by Basic yellow 13 dye.
Journal of Applied Electrochemistry, 39(9),
1497–1501. doi: 10.1007/s10800-009-9829-y
Biswas, A., Pal, S., & Udayabhanu, G. (2015).
Experimental and theoretical studies of xanthan
gum and its graft co-polymer as corrosion
inhibitor for mild steel in 15% HCl. Applied
Surface Science, 353, 173–183. doi:
10.1016/j.apsusc.2015.06.128
Chaubey, N., Savita, Qurashi, A., Chauhan, D. S., &
Quraishi, M. A. (2021). Frontiers and advances in
green and sustainable inhibitors for corrosion
applications: A critical review. In Journal of
Molecular Liquids (Vol. 321). Elsevier B.V. doi:
10.1016/j.molliq.2020.114385
el Bribri, A., Tabyaoui, M., Tabyaoui, B., el Attari, H.,
& Bentiss, F. (2013). The use of Euphorbia falcata
extract as eco-friendly corrosion inhibitor of
carbon steel in hydrochloric acid solution.
Materials Chemistry and Physics, 141(1), 240–247.
doi: 10.1016/j.matchemphys.2013.05.006
Kamal, C., & Sethuraman, M. G. (2012). Spirulina
platensis - A novel green inhibitor for acid
corrosion of mild steel. Arabian Journal of
Chemistry, 5(2), 155–161. doi:
10.1016/j.arabjc.2010.08.006
Lou, S. N., Hsu, Y. S., & Ho, C. T. (2014). Flavonoid
compositions and antioxidant activity of
calamondin extracts prepared using different
solvents. Journal of Food and Drug Analysis, 22(3),
290–295. doi: 10.1016/j.jfda.2014.01.020
M’hiri, N., Veys-Renaux, D., Rocca, E., Ioannou, I.,
Boudhrioua, N. M., & Ghoul, M. (2016). Corrosion
inhibition of carbon steel in acidic medium by
orange peel extract and its main antioxidant
compounds. Corrosion Science, 102, 55–62. doi:
10.1016/j.corsci.2015.09.017
Verma, C., Olasunkanmi, L. O., Ebenso, E. E., &
Quraishi, M. A. (2018). Substituents effect on
corrosion inhibition performance of organic
compounds in aggressive ionic solutions: A
review. In Journal of Molecular Liquids (Vol. 251,
pp. 100–118). Elsevier B.V. doi:
10.1016/j.molliq.2017.12.055
Wang, X., Li, S., Wei, C.-C., Huang, J., Pan, M.-H.,
Shahidi, F., & Ho, C.-T. (2018). Anti-inflammatory
effects of polymethoxyflavones from citrus peels:
a review. Journal of Food Bioactives, 3, 76–86. doi:
10.31665/jfb.2018.3150
Wu, T., He, M., Zang, X., Zhou, Y., Qiu, T., Pan, S., & Xu,
X. (2013). A structure-activity relationship study
of flavonoids as inhibitors of E. coli by membrane
interaction effect. Biochimica et Biophysica Acta -
Biomembranes, 1828(11), 2751–2756. doi:
10.1016/j.bbamem.2013.07.029
Zhang, K., Yang, W., Yin, X., Chen, Y., Liu, Y., Le, J., &
Xu, B. (2018). Amino acids modified konjac
glucomannan as green corrosion inhibitors for
mild steel in HCl solution. Carbohydrate Polymers,
181, 191–199. doi:
10.1016/j.carbpol.2017.10.069
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 CONFERENCE PAPER Sanae Berrada et al., 2022, 78-86

Screening and risk factors for nasal carriage of Staphylococcus aureus in a sample
of Moroccan haemodialysis patients

Sanae Berrada 1 , Ghita Benjelloun Touimi 2, Tarik Sqalli Houssaini 3, Mohammed Cherigui 4,
Abdellatif Alami 4, Fatima Zahra Squali Houssaini 1 and Laila Bennani 5

1: Biotechnology, environment, agro-food and health Laboratory, Faculty of Science Dhar El Mahraz, Sidi
Mohamed Ben Abdellah University, Fez, Morocco
2: Laboratory of Human Pathology Biomedicine and Environment, Faculty of Medicine and Pharmacy,| Sidi
Mohammed Ben Abdellah University, Fez, Morocco
3: Nephrology Department, Hassan II University Hospital, Fez, Morocco.
4: Regional Laboratory for Epidemiological Diagnosis and Environmental Hygiene, Fez-Morocco.
5: The Superior Institute of Nursing Professions and Health Technology of Fez (ISPITS), Fez, Morocco

Patients with end-stage renal disease are in particular risk for S.aureus
infections, primarily via nasal colonization. We aimed to investigate nasal
carriage categories, risk factors associated to colonization, and antimicrobial
susceptibility of S. aureus isolates among patients undergoing haemodialysis.
Over three months (January-March 2015), we have achieved a prospective
transversal study in a public haemodialysis center, we collected clinical data and
performed screening cultures of S. aureus from the anterior nares of 64 patients
on long-term dialysis in the hospitals. In order to determinate carriers nasal
types of S. aureus among patients, we performed two nasal samples for each
participant among a mean period of fifteen days (D0 and D15). Thirty seven (37)
patients have had a coagulase-positive Staphylococcus. BMI was the only risk
factor associated to nasal carriage of S. aureus. Among participants, 17% were
persistent carriers. Out of all isolates, only two (5.4%) were methicillin-resistant.
We have also noticed a multi-resistance of MRSA in more than three antibiotics.
To prevent SARM infections, each haemodialysis center must make a periodic
assessment of S. aureus antibiotics sensitivity currently used and improve
hygiene measures and the conditions of hospitalisation.

*
Corresponding Author
INTRODUCTION
Name: Sanae Berrada
Email: sanae.berrada@usmba.ac.ma Infection is one of the most common causes of
Phone: (+212)661179530 hospitalization, morbidity, and mortality among
● https://orcid.org/0000-0003-0108-910X patients with End-Stage Renal Disease(ESRD)
ISBN: 9788367405256 undergoing haemodialysis(Fan & Li, 2015). Due to
DOI: 10.2478/9788367405256-012 the immunodeficiency of the patients and to the
haemodialysis process, Staphylococcal translocation
Published by Sciendo. © 2022 Sanae Berrada et al. This is from the colonized nasal mucosa to the
an open access article licensed under the Attribution 4.0 bloodstream has been considered the potential
International
(https://creativecommons.org/licenses/by/4.0/)
source of bacterial invasion for these patients
(Oumokhtar et al., 2013). The carriage of these
microorganisms, especially Staphylococcus aureus
(S.aureus) in the Anterior nares, appears to play a
78
crucial role in the epidemiology and pathogenesis
of Healthcare-associated infections ((HAIs)
(Kozioł-Montewka et al., 2006). Moreover,
worldwide concern in public health is the
remarkable aptitude of these microorganisms to
develop resistance against a broad spectrum of
antibiotics and the increasing incidence of illnesses
caused by coagulase-negative Staphylococcus (CNS)
(Bettin et al., 2012; Kozioł-Montewka et al., 2006).
Strategies for controlling Methicillin-resistant
Staphylococcus aureus (MRSA) transmission in
hospitals require baseline information on the
prevalence and characteristics of circulating MRSA
strains. Hence, the fine establishment of
Staphylococcal nasal carrier status accompanied by
complete and accurate characterization of cultured
isolates and antibiotic susceptibility testing is
crucial for haemodialysis patients (Wang et al.,
2012).
The present study aims to investigate nasal carriage
of Staphylococcus, principally S.aureus, carriage
categories, risk factors associated with colonization,
and antimicrobial susceptibility of S.aureus isolates
among patients undergoing haemodialysis.
MATERIALS AND METHODS
Design and study population
We have conducted this study at the haemodialysis
center of the Al Ghassani Hospital in Fez, Morocco,
from January to March 2015. In the beginning,
participants were informed about the study’s
objectives and have delivered oral consent before
specimen collection. We asked patients who agreed
to participate to complete a written survey based on
their demographics and medical history. The
questionnaire included data that is related to
patients such as age, gender, illness-associated
pathologies, the number of haemodialysis sessions
per week as well as antibiotic usage in the previous
three months, and smoking habits. We approved the
study by the Local Ethics Committee, and considered
ethical considerations, including the privacy of
personal data during all research steps.
Sampling mode
Among all participants, we performed samples using
single-use sterile dry or moist cotton swabs, inserted
into the patient’s anterior nostril (1-2cm). We
collected nasal secretions by performing five
complete rotations of the swab, the same swab being
used for both nostrils. We rapidly transported the
swabs specimens in a cooler kept at four degree
Celsius, to the bacteriology laboratory.
Bacteriological Analysis
After nonselective enrichment in Brain heart
infusion (24 hours, 36°C), we have streaked
swabs onto Mannitol Chapman Agar (MCA) and
incubated them at 37 ± 1◦C for 48 hours. The
bacterial load was determined and expressed as
colony-forming units (CFU)/swabs. We performed
preliminary identification of Staphylococcal
isolates based onn colonial morphology, cultural
characteristics on MCA media, Gram-reaction,
oxidase, catalase, DNase, and coagulase tests.
Susceptibility to Antibiotics
Antimicrobial susceptibility was performed on the
isolates using the disk diffusion method on
Mueller Hinton agar (Microbiology Systems, USA)
according to the Committee of Antimicrobial
Testing of the French Society of Microbiology
(Dubreuil et al., 2020). The tested antibiotics were
penicillin G (PG-10UI), ampicillin (AMP-10µg),
amoxicillin (AML-25 µg), amoxicillin + clavulanic
acid (AMC- 20/10µg), oxacillin (Ox 5µg), cefoxitin
(FOX-30 µg), trimethoprim + sulfamethoxazole
(SXT1.25/23.75 µg), tobramycin (TOB-10µg),
Ciprofloxacin (CIP-5µg), tetracycline (TET-30µg),
fusidic acid (FA-10 µg), ofloxacin (OFX-5 µg),
fosfomycin (FOS-5 µg) and vancomycin (VA-5 µg).
Isolates were classified as methicillin-resistant
(MRSA) if they demonstrated resistance to
Oxacillin/Cefoxitin, and methicillin-susceptible
S.aureus (MSSA) if they were susceptible. We
prepared all culture media according to standard
(MN 08.0.155, 2006), and checked before use
their pH and their sterility. Moreover, we carried
out daily quality control of the distilled water,
materials, and equipment (incubator, autoclave,
etc.), as well as ambient conditions of the analysis
environment, according to the internal laboratory
procedure and written according to Canadian
guidelines (DR-12-SCA-02, 2014).
Types of S. aureus carriage
To define carriers nasal types of S.aureus among
patients, we performed two sampling nasal for
each participant on a mean period of fifteen days
(D0 and D15). Based on the results related to the
loading of contamination and according to the
definitions proposed by Verhoeven and al
(Verhoeven et al., 2012), we classified patients into
three categories: persistent carriers, non-carriers,
and intermittent carriers. Participants were
classified as persistent if the bacterial load was
>103CFU for the first swab or load bacterial >
79
 102UFC for the two swabs, and as non-carriers if
the first nasal swab was negative. In other cases,
the subjects were classified as non-persistent
carriers.
Statistical analysis
We expressed results as the mean, standard
deviation. We have made the comparison by the
Chi-square test. The tests that are considered to be
significant concern the values of p < 0.05. Data
entry and processing were done using Epi 3.4
version 2007 software.
RESULTS
Characteristics of participants
Among eighty-four (84) haemodialysis patients, we
swabbed only participants who accepted to
participate at the present study (64). The age of
the participants ranged between 22 and 82 years.
The mean age of 51.38 years (± 14.708 years) and
sex ratio of 1.20 has been registered. 54.7% of
patients perform haemodialysis three times a
week. 34.4% of patients have high blood pressure
(HBP), 10.9% are diabetic, 56.3% are anaemic,
10.9% have hepatitis B, 25% hepatitis C, and
28.1% contract other infection during the study
period. The results also show that 67.2% of
patients have inflammation (C Reactive Protein
(CRP) > 6 mg/l) and 28.1% have an albumin level <
35 mg/l (Table 1).
Associations with possible risk factors for nasal
S. aureus colonization
Out of sixty-four (64) participants, 87.5% and 12.5%
(n=8) of patients have had respectively positive and
negative nasal cultures. For the patients that have
been revealed positive, 37 have had a
coagulase-positive Staphylococcus, and 19 a
coagulase-negative Staphylococcus. Results have
shown that the nasal carriage rate of S.aureus was
57.8%. The univariate analysis does not show any
relationship between the carriage of positive
Staphylococcus coagulase and the variables studied,
such as age group, gender, associated pathologies,
and duration of haemodialysis and smoking (Table
2). We have notified a statistically significant
difference in the colonization of Staphylococcus
aureus, only in the case of Body mass index (p
<0.05).
S. aureus antibiotics susceptibility
We have subjected the 37 S.aureus strains
obtained at the first nasal swab survey to
antibiotic susceptibility testing. The results
revealed high resistance to penicillin G and
ampicillin. Resistances to amoxicillin,
trimethoprim-sulfamethoxazole, and
amoxicillin-clavulanic acid were also remarkable,
respectively 64.8%, 59.45%, and 54.05 %.
Resistance to tetracycline was around 16.8%.
Oxacillin, cefoxitin, fusidic acid (FA), ofloxacin,
ciprofloxacin, fosfomycin, and tobramycin
showed good activity on the strains tested.
Vancomycin revealed maximal activity (Table 3).
S. aureus persistence of nasal colonization
Concerning persistence of nasal colonization,
results showed that 17% (n = 11) were persistent
carriers, 40% (n = 26) were intermittent carriers,
and 43% (n = 28) were non-carriers.
Furthermore, no persistent carriers were
colonized with MRSA strains (Figure 1).
DISCUSSION
S. aureus is a significant cause of
infectious-disease morbidity and mortality,
mainly due to increasing antibiotic resistance
among strains (Alexander et al., 2011). Its
carriage is considered a major risk factor for the
subsequent development of hospital-acquired
infections (Al-Tamimi et al., 2018; Bettin et al.,
2012). We recognized the anterior nares as the
primary site for colonization with an increased
risk of invasive infection (Claassen-Weitz et al.,
2016). Moreover, patients on chronic
haemodialysis have been reported to have over
twice the rate of S. aureus nasal colonization as
healthy controls. Thus, particular attention
should be paid not only to colonization with
MRSA isolates, but also to MSSA colonization
since both groups of microorganisms may be
previous to the development of invasive
infections (Alexander et al., 2011).
In the present study, we recruited sixty-four (64)
haemodialysis patients. Males constituted
54.69% of the participants, and 45.31% were
females. The mean age of our participants was
51.37±14.70 years, and 68.75% of them belonged
to the age group [45-82] years. This result could
be linked to the increase with age in the
prevalence of associated pathologies such as high
blood pressure, the most potent factor in the
progression of renal failure (Birkui et al., 2017).
80
In Morocco, the prevalence of hypertension is
53.8% among people over 40 years and 72.2%
among those over 65 years (ALD 14, 2012).
Moreover, 56.25% of participants have anaemia,
which could be due to a decrease in the renal
production of erythropoietin (EPO), the
resistance of the action of EPO resulting from iron
deficiency, and erythropoiesis inhibitors, the
main ones being pro-inflammatory cytokines
(Novo, 2020).
Based on nasal swabs taken from 64
haemodialysis patients, the results of our study
indicate that carriage rates were 29.7% for
coagulase-negative Staphylococcus and 57.8% for
S.aureus. This rate is frequent in haemodialysis
patients and varies from one study to another
(Mezghani M. S., Toumi, 2021). Our S.aureus rate
carriage is similar to the ones reported in related
studies of some countries such as Poland
(Kozioł-Montewka et al., 2006) and Spain (Peña
et al., 2004), but higher than those reported in
other countries (Al-Tamimi et al., 2018; Halablab
et al., 2010; Nakamura et al., 2017; Tsige et al.,
2020; Weterings et al., 2019) and other studies
carried out in Morocco (Diawara et al., 2014;
Flouchi et al., 2021; Oumokhtar et al., 2013; Souly
et al., 2011). The difference may be related to
reasons linked to patient-related such as race,
sex, age, hormonal factors, nasal anatomical
alterations, the bactericidal activity of nasal
secretions, the existence of receptors on
epithelial cells, and local immunity linked to Ig A
(Sollid et al., 2014). The variance could also be
due to the sampling size and to the culture
techniques, procedure-related factors including
hospital infrastructure, poor hygiene, failure to
follow-up on the patient’s treatment,
overwhelmed healthcare services due to
population burden, and contamination from the
external environment essentially resulting from
the length of hospitalization and the nature of the
service (Ali & Al-Jaff, 2021; Kluytmans et al.,
1997; Sakr et al., 2018; Sollid et al., 2014).
Furthermore, the increase in the rate of nasal
carriage to the one found in the previous study
carried out in the exact center (Oumokhtar et al.,
2013), could result from the lack of hygiene
measures and compliance with standard
precautions, which we raised during our
preliminary study (Berrada et al., 2015), close
contact with nasal wearers favoured particularly
in a hospital setting (Sherertz et al., 2001).
By univariate analysis, the results showed that
the frequency of S.aureus nasal carriage was high
both in patients treated with haemodialysis three
sessions a week and in those with associated
pathologies such as diabetes, arterial
hypertension, anemia, and overweight. However,
the difference between the two groups of patients
(carriers and non-carriers of S.aureus) was
insignificant for almost all studied parameters.
Comparable results have been found in other
studies carried out on populations of
haemodialysis patients (Kang et al., 2012;
Mezghani M. S., Toumi, 2021). However, studies in
broader populations have shown that the male
sex, the young age, diabetes, immunosuppression,
and the duration of dialysis were risk factors for
significant carriage of S. aureus (Askarian et al.,
2009; Oumokhtar et al., 2013; Souly et al., 2011).
Other studies show that S.aureus and MRSA
colonization is more related to patient habits,
such as hygiene, bathing frequency, and
antibacterial soap use (Ahmadi et al., 2019; Wang
et al., 2012). Furthermore, associated diseases
such as diabetes, cardiovascular disease, and
obesity have been identified as risk factors for
nasal carriage of S.aureus (Olsen et al., 2013;
Sollid et al., 2014). In our study, the difference
was only statistically significant in the case of an
increase in Body Mass Index (BMI). Obesity leads
to a disturbance of the ratio between pro-and
anti-inflammatory substances by an intense
secretion of cytokines and adipokines with
predominance pro-inflammatory, as well as an
imbalance of the leptin-adiponectin balance,
resulting in chronic low-grade inflammation and
perturbation of immune function, including
cutaneous antimicrobial defence, which could
promote nasal carriage of S.aureus (Meckenstock
& Therby, 2015; Olsen et al., 2013).
As for the anaemia, it could be a consequence of
the S.aureus nasal carriage. S.aureus is thought to
produce toxins that lyse erythrocytes, releasing
haemoglobin, the most abundant iron source in
mammals, promoting, thus, its survival in the
host (Spaan et al., 2015).
As haemodialysis patients are more exposed to
develop an endogenous S.aureus infection,
persistent carriage proportion for these patients
is higher than in the general population
(Kozioł-Montewka et al., 2006). In our study,
eleven (11) patients among the 64-haemodialysis
patients were classified as persistent carriers
81
(17%), 26 carriers intermittent (40%) with a
bacterial load higher in persistent carriers, and
28 were non-carriers (43%). Our rate of
persistent carriage is lower than the ones
reported in other studies (Alexander et al., 2011;
Mezghani M. S., Toumi, 2021; Verhoeven et al.,
n.d., 2012). The variability of the carrying
proportion is essentially related to the absence of
a consensual definition of nasal carriage states of
S.aureus, and assuming that a persistent carrier
could be defined by using seven samples taken
during 5-weeks, as well as the number (Diawara
et al., 2014) of nasal swabs to be taken, and the
duration of monitoring (Verhoeven et al., 2012).
Although the rate found is lower than the one
reported in other studies, screening and
decontamination of persistent carriers, as well as
verification of mupirocin activity are currently
considered decisive and essential measures in
reducing the risk of developing an endogenous
S.aureus infection in haemodialysis (Mezghani M.
S., Toumi, 2021).
Antimicrobial susceptibility testing provides
essential data on antimicrobial resistance. We
notified a very high resistance for S.aureus
isolates to penicillin G and ampicillin (91.9 %), a
high resistance to amoxicillin (64.8%),
trimethoprim-sulfamethoxazole (59.45%), and
amoxicillin-clavulanic acid (54.05 %), which
means multidrug resistance. Nevertheless, strains
of S.aureus resistant to β lactams showed good
susceptibility to antibiotics recognized as active
both in Morocco and other countries, such as
fusidic acid, ofloxacin, ciprofloxacin, fosfomycin,
and vancomycin. Other authors reported similar
results (Askarian et al., 2009; Diawara et al.,
2014). Antibiotic resistance profile varies with
geographical region and changes over time
(Elhamzaoui et al., 2009). Regarding the
prevalence of MRSA, our prevalence (5.4%) is
consistent with other studies (Askarian et al.,
2009; Bogut et al., 2007; Kozioł-Montewka et al.,
2006; Oumokhtar et al., 2013), but different from
those found by other authors (Diawara et al.,
2014; Doh et al., 2003; Flouchi et al., 2021).
Multidrug resistance results could be explained
by antibiotics overuse and by patient’s
self-medication on one hand (Oumokhtar et al.,
2013), and by the cross-transmission of strains,
favored by the lack of hygiene, the promiscuity of
patients, the sharing of possibly contaminated
objects, the immune status of people in contact
and the characteristics of the strains on the other
hand (Carlet et al., 2012; HCSP, 2014).
To reduce the emergence and spread of antibiotic
resistance worldwide, many actions could be
taken. Main of them consist of rational use of
antibiotics in all settings, implementation of
infection control measures in healthcare settings,
development of approaches to diminish the risks
of environmental exposure, promotion of
antibacterial resistance prevention and
monitoring, research and development of novel
antimicrobial strategies and antibacterial agents
(Carlet et al., 2012; Cookson, 2005; D’Agata, 2002;
Roca et al., 2015).
This study is not without limitations. These
include the short period of time, and small size of
our population. Therefore, the results obtained
are not completely representative of the nasal
carriage rates of the haemodialysis patients.
Important risk factors associated with MRSA
acquisition were not recorded. In addition, due
to the limitation of resources in the
microbiological laboratory, we didn’t perform
molecular biology for identifying the resistance
genes of S. aureus.
CONCLUSIONS
Our study showed that S. aureus, and MRSA nasal
carriage rates among haemodialysis patients are
not negligible. There were no gender differences
between the isolate carriers and non-carriers or
comorbidity factors such as infections, diabetes,
chronic smoking, recent hospitalization or
antibiotic therapy. Body Mass Index was the only
risk factor associated with nasal carriage of
S.aureus. Among participants, 17% were
persistent carriers. Out of all isolates, only two
(5.4%) were methicillin-resistant. Concerning
antibiotic sensitivity, we found a multi-resistance
of MRSA to more than three antibiotics. These
results provide the importance of screening
patients for nasal colonization with S.aureus and
monitoring susceptibility in treating of infections
generated by this specie.
To reduce the risk of Healthcare-associated
infections, a bundle of activities must be
implemented, including clinical risk assessment,
hand hygiene, evidence-based environmental
cleaning and disinfection procedures, structured
training for environmental services staff, and
timely feedback from cleaning and cleanliness
monitoring. Furthermore, so that SARM
82
 infections will be prevented from, each
haemodialysis center must make a periodic
assessment of S.aureus antibiotics sensitivity
currently used and improve hygiene measures
and the hospitalization conditions.
ACKNOWLEDGEMENTS
Authors would like to thank the Fez-Meknes
regional health directorate. They also thank the
team and health professionals of the
haemodialysis center of Al Ghassani Hospital and
the public health laboratory of the delegation of
the Ministry of Health in Fez for their
collaboration, and their precious help.
DATA AVAILABILITY
All data are available from the corresponding
author upon kind request.
CONFLICTS OF INTEREST
The authors declare no conflicts of interest.
REFERENCES
Ahmadi, E., Khojasteh, M., Mortazavi, S. M.,
Khan-Mohammadi, F., Kazemnia, A.,
Beheshtipour, J., & Raeeszadeh, M. (2019).
Prevalence of and risk factors for
methicillin-resistant Staphylococcus aureus
nasal carriage in the West of Iran: A
population-based cross-sectional study. BMC
Infectious Diseases, 19(1), 1–8.
https://doi.org/10.1186/s12879-019-4567-1
Al-Tamimi, M., Himsawi, N., Abu-Raideh, J., Jazar, D. A.,
Al-Jawaldeh, H., Mahmoud, S. A. H., Hijjawi, N., &
Hawamdeh, H. (2018). Nasal colonization by
methicillin-sensitive and methicillin-resistant
staphylococcus aureus among medical students.
Journal of Infection in Developing Countries,
12(5), 326–335.
https://doi.org/10.3855/jidc.9908
ALD 14, 2012. (2012). Recommandations de Bonnes
Pratiques Médicales L’hypertension artérielle de
l’adulte. 1–85.
Alexander, E. L., Morgan, D. J., Kesh, S., Weisenberg, S.
A., Zaleskas, J. M., Kaltsas, A., Chevalier, J. M.,
Silberzweig, J., Barrón, Y., Mediavilla, J. R.,
Kreiswirth, B. N., & Rhee, K. Y. (2011).
Prevalence, persistence, and microbiology of
Staphylococcus aureus nasal carriage among
hemodialysis outpatients at a major New York
Hospital. Diagnostic Microbiology and Infectious
Disease, 70(1), 37–44.
https://doi.org/10.1016/j.diagmicrobio.2010.1
2.005
Ali, K. M., & Al-Jaff, B. M. A. (2021). A Study on the
Prevalence and Source of Staphylococci and
Methicillin-resistant Staphylococcus aureus
Causing Superficial Incisional Surgical Site
Infection. Aro-the Scientific Journal of Koya
University, 9(1), 114–121.
https://doi.org/10.14500/aro.10752
Askarian, M., Zeinalzadeh, A., Japoni, A., Alborzi, A., &
Memish, Z. A. (2009). Prevalence of nasal
carriage of methicillin-resistant Staphylococcus
aureus and its antibiotic susceptibility pattern in
healthcare workers at Namazi Hospital, Shiraz,
Iran. International Journal of Infectious Diseases,
13(5).
https://doi.org/10.1016/j.ijid.2008.11.026
Berrada, S., Houssaini Sqalli, T., Oumokhtar, B.,
Bennani, L., El Ouali Lalami, A., Hanin, H., El
Fakir, S., Benaich, N., & Houssaini Squali, F. Z.
(2015). Évaluation de la conception et des
pratiques standard d’un centre d’hémodialyse
au Maroc. Revue Francophone Internationale de
Recherche Infirmiere, 1(4), 225–232.
https://doi.org/10.1016/j.refiri.2015.10.009
Bettin, A., Causil, C., & Reyes, N. (2012). Molecular
identification and antimicrobial susceptibility of
Staphylococcus aureus nasal isolates from
medical students in Cartagena, Colombia.
Brazilian Journal of Infectious Diseases, 16(4),
329–334.
https://doi.org/10.1016/j.bjid.2012.06.017
Birkui, P. J., Janiaud, P., Carteron, H., & Chabanel, A.
(2017). To cite this version : Insuffisance rénale
chronique Etiologies , moyens.
Bogut, A., Kozioł-Montewka, M., Baranowicz, I.,
Jóźwiak, L., Ksia̧zek, A., Al-Doori, Z., Morrison, D.,
Kaczor, D., & Paluch-Oleś, J. (2007).
Characterisation of Staphylococcus aureus nasal
and skin carriage among patients undergoing
haemodialysis treatment. New Microbiologica,
30(2), 149–154.
Carlet, J., Rambaud, C., & Pulcini, C. (2012). Alliance
contre les bactéries multirésistantes : Sauvons
les antibiotiques ! Annales Francaises
d’Anesthesie et de Reanimation, 31(9), 704–708.
https://doi.org/10.1016/j.annfar.2012.07.003
Claassen-Weitz, S., Shittu, A. O., Ngwarai, M. R.,
Thabane, L., Nicol, M. P., & Kaba, M. (2016). Fecal
carriage of Staphylococcus aureus in the hospital
and community setting: A systematic review.
Frontiers in Microbiology, 7(MAY).
https://doi.org/10.3389/fmicb.2016.00449
Cookson, B. (2005). A review: Clinical significance of
emergence of bacterial antimicrobial resistance
in the hospital environment. Journal of Applied
Microbiology, 99(5), 989–996.
https://doi.org/10.1111/j.1365-2672.2005.026
93.x
D’Agata, E. M. C. (2002). Antimicrobial-resistant,
gram-positive bacteria among patients
undergoing chronic hemodialysis. Clinical
Infectious Diseases, 35(10), 1212–1218.
https://doi.org/10.1086/344282
Diawara, I., Bekhti, K., Elhabchi, D., Saile, R.,
Elmdaghri, N., Timinouni, M., & Elazhari, M.
(2014). Staphylococcus aureus nasal carriage in
hemodialysis centers of Fez, Morocco. Iranian
83
 Journal of Microbiology, 6(3), 175–183.
Doh, V. E., Adou, D. G., Ia, H. T., & Nonsahe, D. G.
(2003). Epidemiologie Et Prevention Du Portage
Nasal De Staphylococcus aureus chez les
malades et le personnel du centre d’hémodialyse
de Cocody-Abidjan. Med Trop, 63:(225),
590–592.
DR-12-SCA-02, 2014. (2014). Guidelines for analytical
work in microbiology.
Dubreuil, L., Goldstein, F., Jarlier, V., Morel, C.,
Philippon, A., Rouveix, B., Sirot, J., Soussy, C., &
Thabaut, A. (2020). Comité de l’antibiogramme
de la Société Française de Microbiologie. Société
Française de Microbiologie, 1.1, 181.
Elhamzaoui, S., Benouda, A., Allali, F., Abouqual, R., &
Elouennass, M. (2009). Sensibilité aux
antibiotiques des souches de Staphylocoques
aureus isolées dans deux hôpitaux universitaires
à Rabat, Maroc. Medecine et Maladies
Infectieuses, 39(12), 891–895.
https://doi.org/10.1016/j.medmal.2009.01.004
Fan, Z., & Li, B. (2015). Nasal hygiene in patients with
end-stage renal disease. Integrative Molecular
Medicine, 2(6), 428–431.
https://doi.org/10.15761/imm.1000181
Flouchi, R., Elmniai, A., Hibatallah, A., Fahsi, K.,
Touzani, I., & Fikri-Benbrahim, K. (2021). The
Relationship between Nasal Carriage of
Staphylococcus aureus and Surgical Site
Infections in a Hospital Center in Morocco.
Hindawi International Journal of Microbiology,
2021. https://doi.org/10.1155/2021/5585588
Halablab, M. A., Hijazi, S. M., Fawzi, M. A., & Araj, G. F.
(2010). Staphylococcus aureus nasal carriage
rate and associated risk factors in individuals in
the community. Epidemiology and Infection,
138(5), 702–706.
https://doi.org/10.1017/S0950268809991233
HCSP. (2014). Conduite à tenir lors d’épisodes de cas
groupés d’infections cutanées suppuratives liées
aux souches de SARM Co.
Kang, Y. C., Tai, W. C., Yu, C. C., Kang, J. H., & Huang, Y. C.
(2012). Methicillin-resistant Staphylococcus
aureus nasal carriage among patients receiving
hemodialysis in Taiwan: Prevalence rate,
molecular characterization and de-colonization.
BMC Infectious Diseases, 12.
https://doi.org/10.1186/1471-2334-12-284
Kluytmans, J., Van Belkum, A., & Verbrugh, H. (1997).
Nasal carriage of Staphylococcus aureus:
Epidemiology, underlying mechanisms, and
associated risks. Clinical Microbiology Reviews,
10(3), 505–520.
https://doi.org/10.1128/cmr.10.3.505-520.199
7 6(February
Kozioł-Montewka, M., Szczepanik, A., Baranowicz, I.,
Jóźwiak, L., Ksiazek, A., & Kaczor, D. (2006). The
investigation of Staphylococcus aureus and
coagulase-negative staphylococci nasal carriage
among patients undergoing haemodialysis.
Microbiological Research, 161(4), 281–287.
https://doi.org/10.1016/j.micres.2005.10.002
Meckenstock, R., & Therby, A. (2015). Modifications of
immunity in obesity: The impact on the risk of
infection. Revue de Medecine Interne, 36(11),
760–768.
https://doi.org/10.1016/j.revmed.2015.07.013
Mezghani M. S., Toumi, ; S. Chakroun; A et al. (2021).
Screening for Staphylococcus aureus nasal
carriage in chronic hemodialysis patients. J.I. M.
Sfax, 37, 24–30.
MN 08.0.155. (2006). Guide for the preparation and
production of culture media in the laboratory.
Nakamura, M., Shimakawa, T., Nakano, S., Chikawa, T.,
Yoshioka, S., Kashima, M., Toki, S., & Sairyo, K.
(2017). Screening for nasal carriage of
Staphylococcus aureus among patients
scheduled to undergo orthopedic surgery:
Incidence of surgical site infection by nasal
carriage. Journal of Orthopaedic Science, 22(4),
778–782.
https://doi.org/10.1016/j.jos.2017.03.005
Novo, R. (2020). Chapitre 81 - Anémie de
l’insuffisance rénale chronique. In
N&#233;phrologie de l’enfant. Elsevier Masson.
https://doi.org/10.1016/B978-2-294-76353-3.
00081-9
Olsen, K., Danielsen, K., Wilsgaard, T., Sangvik, M.,
Sollid, J. U. E., Thune, I., Eggen, A. E., Simonsen, G.
S., & Furberg, A. S. (2013). Obesity and
Staphylococcus aureus Nasal Colonization
among Women and Men in a General Population.
PLoS ONE, 8(5), 19–25.
https://doi.org/10.1371/journal.pone.0063716
Oumokhtar, B., Elazhari, M., Timinouni, M.,
Bendahhou, K., Bennani, B., Mahmoud, M., El
Ouali Lalami, A., Berrada, S., Arrayhani, M., &
Squalli Houssaini, T. (2013). Staphylococcus
aureus nasal carriage in a moroccan dialysis
center and isolates characterization.
Hemodialysis International, 17(4), 542–547.
https://doi.org/10.1111/j.1542-4758.2012.007
59.x
Peña, C., Fernández-Sabe, N., & Domínguez, M. A. et Al.
(2004). Staphylococcus aureus nasal carriage in
patients on haemodialysis: Role of cutaneous
colonization. Journal of Hospital Infection, 58(1),
20–27.
https://doi.org/10.1016/j.jhin.2004.04.018
Roca, I., Akova, M., Baquero, F., Carlet, J., Cavaleri, M.,
Coenen, S., Cohen, J., Findlay, D., Gyssens, I.,
Heure, O. E., Kahlmeter, G., Kruse, H.,
Laxminarayan, R., Liébana, E., López-Cerero, L.,
MacGowan, A., Martins, M., Rodríguez-Baño, J.,
Rolain, J. M., … Vila, J. (2015). The global threat
of antimicrobial resistance: Science for
intervention. New Microbes and New Infections,
2015), 22–29.
https://doi.org/10.1016/j.nmni.2015.02.007
Sakr, A., Brégeon, F., Mège, J. L., Rolain, J. M., & Blin, O.
(2018). Staphylococcus aureus nasal
colonization: An update on mechanisms,
epidemiology, risk factors, and subsequent
infections. Frontiers in Microbiology, 9(OCT),
1–15.
https://doi.org/10.3389/fmicb.2018.02419
84
Sherertz, R. J., Bassetti, S., & Bassetti-Wyss, B. (2001). Kluytmans, J. (2019). Prevalence of nasal
“Cloud” health-care workers. Emerging Infectious carriage of methicillin-resistant Staphylococcus
Diseases, 7(2), 241–244. aureus in patients at hospital admission in The
https://doi.org/10.3201/eid0702.010218 Netherlands, 2010–2017: an observational
Sollid, J. U. E., Furberg, A. S., Hanssen, A. M., & study. Clinical Microbiology and Infection, 25(11),
Johannessen, M. (2014). Staphylococcus aureus: 1428.e1-1428.e5.
Determinants of human carriage. Infection, https://doi.org/10.1016/j.cmi.2019.03.012
Genetics and Evolution, 21, 531–541.
https://doi.org/10.1016/j.meegid.2013.03.020
Souly, K., Ait El kadi, M., Lahmadi, K., Biougnach, H.,
Boughaidi, A., Zouhdi, M., Benasila, S., Elyoussefi,
Z., Bouattar, T., Zbiti, N., Skalli, Z., Rhou, H.,
Ouzeddoun, N., Bayahia, R., & Benamar, L.
(2011). Epidemiology and prevention of
Staphylococcus aureus nasal carriage in
hemodialyzed patients. Medecine et Maladies
Infectieuses, 41(9), 469–474.
https://doi.org/10.1016/j.medmal.2011.05.005
Spaan, A. N., Reyes-Robles, T., Badiou, C., Cochet, S.,
Boguslawski, K. M., Yoong, P., Day, C. J., De Haas,
C. J. C., Van Kessel, K. P. M., Vandenesch, F.,
Jennings, M. P., Le Van Kim, C., Colin, Y., Van
Strijp, J. A. G., Henry, T., & Torres, V. J. (2015).
Staphylococcus aureus Targets the Duffy Antigen
Receptor for Chemokines (DARC) to Lyse
Erythrocytes. Cell Host and Microbe, 18(3),
363–370. Figure 1: S. aureus carrier types
https://doi.org/10.1016/j.chom.2015.08.001
Tsige, Y., Tadesse, S., G/Eyesus, T., Tefera, M. M., Table 1: Study participant’s information.
Amsalu, A., Menberu, M. A., & Gelaw, B. (2020). Demographic, social, and clinical
Prevalence of Methicillin-Resistant characteristics of study participants
Staphylococcus aureus and Associated Risk
Factors among Patients with Wound Infection at Demographic Male Female
Referral Hospital, Northeast Ethiopia. Journal of and clinical N=35 N=29
Pathogens, 1–7. characteristics N (%) N (%)
https://doi.org/10.1155/2020/3168325 Age (years) Max 82 82
Min 22 26
Verhoeven, P. O., Gagnaire, J., Haddar, C. H., Grattard, F., Mean 49.26 53.93
Thibaudin, D., Afiani, A., Cazorla, C., Carricajo, A., Ecartype 16.028 12.745
Mariat, C., Alamartine, E., Lucht, F., Garraud, O., Age range [22 - 30] 4 (11.42%) 2 (6.9%)
Pozzetto, B., Botelho-Nevers, E., & Berthelot, P. [31 - 44] 11(31.42%) 3(10.34%)
[45 - 82] 20(57.16%) 24(82.76%)
(n.d.). Identifying Hemodialysis Patients With the Seniority of >10years 11(17.2%) 9(14.1%)
Highest Risk of Staphylococcus aureus haemodialysis ≤10years 24(37.5%) 20(31.2%)
Endogenous Infection Through a Simple Nasal Number of 2 15(23.4%) 14(21.9%)
Sampling Algorithm. haemodialysis
session/week 3 20(31.2%) 15(23.4%)
https://doi.org/10.1097/MD.00000000000032
Number of [8-10] 15(23.4%) 14(21.9%)
31 haemodialysis [11-12]
Verhoeven, P. O., Grattard, F., Carricajo, A., Lucht, F., hour/week 20(31.2%) 15(23.4%)
Cazorla, C., Garraud, O., Pozzetto, B., & Berthelot, Associated HBP 11(17.18%) 11(17.18%)
P. (2012). An algorithm based on one or two pathology Diabetes 5(7.8%) 2(3.1%)
Anaemia 22(34.4%) 14(21.9%)
nasal samples is accurate to identify persistent Hepatitis B 6(9.37% 1(1.56%)
nasal carriers of Staphylococcus aureus. Clinical Hepatitis C 8(12.5%) 8(12.5%)
Microbiology and Infection, 18(6), 551–557. BMI (Kg/m2 [13-18.5[ 8(12.5%) 4(6.2%)
https://doi.org/10.1111/j.1469-0691.2011.036 [18.5-22.5[ 21(32.8%) 17(26.6%)
[22.5-25[ 1(1.6%) 1(1.6%)
11.x [25-30[ 5(7.8%) 5(7.8%)
Wang, C. Y., Wu, V. C., Wang, W. J., Lin, Y. F., Lin, Y. H., [30-32,5[ 0 2(3.1%)
Chen, Y. M., Su, C. T., Wang, J. Y., Wu, K. D., & CRP (mg/l) [1-6] 8(12.5%) 13(20.3%)
Hsueh, P. R. (2012). Risk factors for nasal [7-200] 27(42.2%) 16(25.0%)
Albumin (mg/l) < 35 13(20.3%) 5(7.8%)
carriage of methicillin-resistant Staphylococcus ≥ 35 22 (30.4%) 24(37.5%)
aureus among patients with end-stage renal
disease in Taiwan. Journal of the Formosan
Medical Association, 111(1), 14–18. Undercurrent
infection : Yes 25(39.1%) 21(32.8%)
https://doi.org/10.1016/j.jfma.2012.01.001 No 10(15.6%) 8(12.5%)
Weterings, V., Veenemans, J., van Rijen, M., &
85
Table 2: Risk factors for nasal S. aureus Table 3: Antibiotics susceptibility of S. aureus
colonization isolates (N= 37).

Variables Carriers Non-carr p-value Antibiotics S. aureus

patients iers Resistant Sensitive
patients
N (%) N (%) Penicillin 91.9% 8.10%
Gender Mal 20(54.1%) 15(55.6%) Ampicillin 91.9% 8.10%
Female 17(45.9%) 12(44.4%) 1.000 Amoxicillin 64.8% 35.2%
Age range [22 - 30] 4(10.81%) 2(7.40%)
Amoxicillin-clavul 54.1% 45.9 %
[31 - 44] 8(21.62%) 6(22.22%) 0.89
[45 - 82] 25(67.57%) 19(70.38%)
anic acid
Seniority of >10years 16(59.1%) 13(40.9%) 0.759 Oxacillin 5.4% 94.6%
haemodialysis ≤10years 11(54.0%) 14(45%) Cefoxitin 5.4% 94.6%
Number of 2 16(55.2%) 13(44.8%) 0.697 Fusidic acid 5.4% 94.6%
haemodialysis
session/week 3 21(60%) 14(40%) Trimethoprim-sulf 59.45% 40.55%
amethoxazole
Number of [8-10] 21(60%) 14(40%) 0.697
haemodialysis Fosfomycin 13.5% 86.5%
hour/week [11-12] 16(55.2%) 13(44.8%) Ofloxacin 24.3% 75.7%
HBP 13(59.1%) 9(40.9%) 0.548
Diabetes 5 (71.42%) 2(28.58%) 0.364 Tobramycin 24.3% 75.7%
Anaemia 20(54.05%) 17(45.95%) 0.14 Ciprofloxacin 21.6% 78.4%
BMI (Kg/m2 [13-18.5[ 4(33.3%) 8(66.7%) 0.047 Tetracyclin 16.8% 84.2%
[18.5-22.5[ 24(63.2%) 14(36.8%)
[22.5-25[ 2(100%) 0(0.0%)
Vancomycin 0% 100%
[25-30[ 7(70%) 3(30%)
[30-32.5[ 0(0.0%) 2(100%)
CRP (mg/l) [1-6] 11(52.4%) 10(47.6%) 0.539
[7-200] 26(60.5%) 17(39.5%)
Albumin (mg/l) < 35 11(61.1%) 7(38.9%) 0.738
≥ 35 226(56.5%) 20(43.5%)

Undercurrent
infection : Yes 8(44.4%) 10(55.6%) 0.176
No 29(63.0%) 17(37.0%)

Taking
treatment: Yes 7(55.6%) 24(44.4%) 0.498
No 30(70%) 3(30%)
Smoking: Yes 5(71.4%) 2(28.6%) 0.689
No 32(56.1%) 25(43.9%)

86
CONFERENCE PAPER Fatima-Ezzahra Azzouzi et al., 2022, 87-96

Hormonal Contraception and Breast Cancer Among Women in the Region

Marrakech-Safi, Morocco
Fatima-Ezzahra Azzouzi 1 2, Mohamed-Amine Lyaazale³, Saloua Lamtali4 5, and Ibtissam Youlyouz1
1
Faculty of Medicine and Pharmacy, Cadi Ayyad University, Biosciences and Health
Laboratory-Marrakech-Morocco
2
Higher Institute of Health Sciences, University Hassan I -Settat-Morocco
³ Reproductive health reference center for early detection of breast and cervical cancers -Marrakech-Morocco
4
Faculty of Sciences Semlalia, Cadi Ayaad University, Laboratory of Pharmacology, Neurobiology,
Anthropobiology and Environment-Marrakech-Morocco.
5
Higher institute of nursing professions and health techniques-Marrakech-Morocco

Breast cancer is the most common cancer in women worldwide. In Morocco,

its incidence continues to increase, making it the most common cancer in
women. Every year, 1.4 million new cases are diagnosed and more than 450,000
women die of the disease, Hormonal contraception is likely a risk factor whether
pill, patch or IUD. The survey on women's hormonal contraception is likely to
help identify women at risk, and provide guidance on the development of
effective health promotion interventions. To synthesize the links between
hormonal contraception and breast cancer among women in the Marrakech-Safi
region. A case-control study was conducted with an exhaustive record of all
cases of women with histologically confirmed breast cancer in all public health
facilities involved in screening of cancer in all the provinces of the
Marrakech-Safi region "Rhamna-Youssoufia-Kalâa of sraghna-Marrakech-
Essaouira-Chichaoua-Safi-Elhaouz". The total sample included 1200 women
with 600 confirmed cases of cancer and 600 female controls (Confirmed healthy
by normal radio-clinical examination). Data processing and statistical analysis
were done using software SPSS and Excel, the level of significance was set at
3.6%. Results: The univariate analysis showed that among women in the
Marrakech-Safi region, there is a statistically significant association with the use
of oral contraceptives and the occurrence of the disease OR = 1.44.
*Corresponding Author
Name: Fatima-Ezzahra Azzouzi Less developed countries account for about 55.6%
Email: of all breast cancer cases and 62.1% of deaths
fatimaezzahraazzouzi@gmail.com (Global Cancer Statistics,2018). In Morocco, The
incidence rate of this disease has risen in the past
ISBN : 9788367405256 years, likely due to changes in the pattern of breast
DOI: 10.2478/9788367405256-013 cancer risk factors caused by adoption of a Western
lifestyle. According to the cancer registry of the
Published by Sciendo. © 2022 Fatima-Ezzahra Azzouzi et
al. This is an open access article licensed under the greater Casablanca region (RCGC), its incidence is
Attribution 4.0 International 36.4 new cases / 100,000 inhabitants per year
(https://creativecommons.org/licenses/by/4.0/) (Cancer Registry of Greater Casablanca, 2012).
Every year, 1.4 million new cases are diagnosed,
and more than 450,000 women die due to this
INTRODUCTION disease.
Breast cancer is the most frequently diagnosed
cancer and the leading cause of cancer death among
women worldwide (OMS,2020).
87
The median age is 50, and young women under 35 METHODS
account for 6.7% of the cases. The incidence is
higher in the age groups 45-54 (144 per 100,000) Design
and 55-64 (131 per 100,000) and remains It’s a case control study conducted, in 2019,
relatively low in the 25-34 age group (16 per at all the public health establishments
1,000). 100,000) (Alami. Z et Al, 2012). concerned by cancer screening in the
Marrakech-Safi region: "Rhamna, Youssoufia,
Indeed, breast cancer is a multifactorial disease. Kalâa des sraghna, Marrakech, Essaouira,
This means that several factors influence the risk of Chichaoua, Safi, Elhaouz.
its occurrence.
We talk about risk factors Participants
(André.N&Parviz.G,2005). Hormonal contraception Data were collected from women record.
is among breast cancer’s risk factors. Indeed, 1200 women’s file were examined:
Hormonal contraception, such as pill, patch or IUD, - 600 cases: who have histologically
is linked to an increased risk of breast cancer confirmed breast cancer
(Mørch.L, 2017). - 600 controls : who were confirmed
Association between hormonal contraception and healthy by normal radio-clinical examination
breast cancer have been known for more than 30
years, and are a source of frequent questions and We excluded uncompleted records and those
concerns for patients attending contraceptive with an inconclusive pathological
consultations (Polly A Marchbanks et Al , 2002). examination. Also, institutions for wich we
Studies showed that the risk of developing breast couldn’t have authorization to explore the
hormonal contraceptives and the risk increase records were excluded.
when the duration of use is longer. Beyond 10 years,
it is evaluated at 38% (Mørch.L, 2017). Ethical considerations
According to the National Cancer Institute, "a
woman on a combined pill seems to be slightly We conducted our study in an ethical way,
increasing her risk of breast cancer, especially respecting anonymity of all files.
young women who are taking it recently and those The data collection has been authorized by
who have been using it for 10 years or more and the local authorities of the ministry of health
this risk is the normal 10 years after you stop .
taking it. "(National Cancer Institute of
France,2018)
Exploitation sheet
However the risk decrease, 10 years after stopping
hormonal contraception to become
Data were collected throughout the year
normal.(Martin.P, 1999)
2019, from women record. So, we have
In Morocco, 67% of women aged 15 to 49 use
developed an exploitation file for that
contraception in the form of estrogen / progestin
including : (a) Socio- demographic
pills (Morocco National Survey on Population and
informations (b) Reproductive
Family Health,2011). Also, nurses who work at
characteristics of women (c) Family history
Marrakesh Reference Center (CR) report that use of
of breast cancer (d) Hormonal contraception.
Hormonal contraception is hight among women
with
histologically confirmed breast cancer and often
exceeds 10 years of use.
Therefore, it seems important to study association
between hormonal contraception and the risk of
breast cancer occurring in women in Morocco. This
study focuses on women in the Marrakech-Safi
region.

88
Data analysis
The third group has an age between 40 and
Data was analyzed using the Statistical Package for 50 years and that present 28% and finally
Social Sciences (SPSS, version 21). 41% of women have the age that exceeds 50
Descriptive statistics (percentages and frequencies) years.
were used to describe the population (case and For the record, our sample includes women
control) and univariate analysis was carried out between 20 and 88 years old.
using Chi-square test to compare percentages.
The confidence Interval was 95% and P value of Table2: Sociodemographic characteristics
<0.05 was taken as significant. “Province”
Case Control

Proportionality hypothesis of logistic regression of Provinces

proportional probabilities has been evaluated from Effective % Effective %
the bivariate models of predictor result (Bivariate
analysis of hormonal contraception as a risk factor 480 80 498 83
associated with breast cancer), and the results of Marrakech
each combination rib ratios. 12 2 12 2
El Haouz
RESULTS
12 2 12 2
Sociodemographic characteristics Youssoufia
36 6 18 3
Table1: Sociodemographic characteristics “Age” Safi
Case Control 6 1 6 1
Chichaoua
Variabl Categorie Effectiv e % Effectiv e %
e s 18 3 6 1
N=18 3 N=12 2 Essaouira
20-30
years 30 5 48 8
N=150 25 N=174 29 Rhamna
Age
30-40
years 6 1 6 1
N=168 28 N=168 28 Kalâa
40-50 Sraghna
years
N=264 44 N=264 41
+50 years

The above table shows the distribution of women

with histologically confirmed breast cancer by age.
It shows that 3% of women are between 20-30
years old, 25% are between 30-40 years old.
The third group has an age between 40 and 50
years which present 28% and finally 44% of
women have the age that exceeds 50 years.
Note: It should be noted that our sample includes
women between 20 and 88 years.
In the same context, the distribution of control
women (women with positive breast screening and
negative radiological examination by age).
It emerges that 2% of women have an age of 20 to
30 years, 29% have the age between 30 and 40
years.
The table above presents the distribution of
women with histologically confirmed breast
cancer according to province. It emerges that
the provinces of Chichaoua and Kalâa
sraghna, represent 1% of women with
histologically proven breast cancer, 2% in
Haouz and Youssoufia, 3% in Essaouira, 5%
in Rhamna, 6% in Safi and finally the
majority of cases with a percentage of 80%
in Marrakech. "For the cases .
Concerning the female witnesses, the
majority of cases with a percentage of 83%
in Marrakech.
The province of Rhamna represents 8%, a
percentage of 3% in Safi, 2% in Haouz and
Youssoufia, 1% in Essaouira, Chichaoua and
Kalâa sraghna.

89
 Table3: Sociodemographic characteristics
“Family situation”
Case Control
followed by women who had their menstrual
period after the age of 12, which represents
Variable Categories Effective % Effective % 68%.
And in last rank 10% of women whose age of
378 63 390 65 first menstruation was not mentioned on the
Married consulted files.
Family For the distribution of "Control" women
48 8 72 12
situation
Divorced
according to the age of their first menstrual
period.
96 16 84 14
widow It emerges that the population studied is
78 13 54 9 constituted in first place by women who had
single their first menstrual period early (before the
age of 12) and who represent 30%, followed
by women who had their period after the age
of 12 who represent 62%, and lastly 8% of
women whose age of first menstrual period
The table above shows the distribution of the family was not mentioned on the files consulted.
status of women with histologically confirmed
breast cancer. Table5: Reproductive characteristics of
It shows a minority of 8% divorced, 13% with a women “Menopause
single status, 16% of women widowed and finally
63% of cases with a married status. Case Control
The distribution of the family situation of women
exposed to hormonal contraception and not sick
Variable Categorie Effective % Effecti ve %
"Witnesses" according to family status.
This shows a minority of 9% divorced, 12% with a Yes 264 44 264 44
single status, 14% of widowed women and finally
65% of cases with a married status. Menopa No 330 55 336 56
u se

Reproductive characteristics of women: N.M 6 1 0

0
Table 4: Reproductive characteristics of NM: Non mentioned
women “Age of first menstruation”
Case Control The table above presents the distribution of
Variable Categor ies Effecti ve % Effectiv e % women with histologically confirmed breast
cancer according to menopause, showing
<12 13 2 22 180 30 that the study population is made up of 44%
Age of years of postmenopausal women, and 55% of
first >12 68 372 62
menstrua years 40 8
non-menopausal women.
tion
For the distribution of the " Witnesses "
The table above presents the distribution of women women according to menopause. It emerges
with histologically confirmed breast cancer that the population studied is constituted in
according to the age of their first menstrual period. first rank of 44% of the menopaused women,
It shows that the study population is made up first and in second rank we have 56% of the non
of women who had their first menstrual period menopaused women.
early; before the age of 12, which represents 22%,

90
 Hormonal contraception
Family history of breast cancer Table 7: Taking hormonal contraception by
women with breast cancer–Case-Control
This section of the results will focus on the
Case Control
distribution of "case" and "control" women with
histologically confirmed breast cancer according Variable Categorie Effective % Effetive %
to their family history.

Table 6: Family history of breast cancer Yes 507 84, 69 11.

Case Control 4 5
Hormonal
contracepti on No 93 15. 531
6 88.
Variable Categories Effective % Effective % 5

The table above shows the use of hormonal

Yes 87 14.5 69 11.5 contraception by women with breast cancer
according to the type of contraception.
Family This distribution shows that 84.4% of
history No 513 85.5 531 88.5
women take hormonal contraception and
only 15.6% do not.
of On the other hand, the use of hormonal
breast Mère 41 4 69 252 42 contraception by "Control" women according
cancer to the type of contraception showed 88.5%
of women who take hormonal contraception
84 14 31 and only 11.5% who do not take it.
Soeur 186 Case Control

Cousine 60 10 72 12 Categories Effective % Effective %

COC 483 95.2 66 95.8

Autres 42 7 90 15 Injectables 7 1.38 1 1.69

Other method 7 1.38 0 0.37

N.M 10 2.04 2 2.07

Table8: Taking hormonal

contraception by women with breast
The table above shows the distribution of women
with histologically confirmed breast cancer cancer by type –case-Control
according to family history. It appears that 85.50% N.M: Non mentioned
have no family history and 14.50% of the study
population have a family history which is Regarding the type of contraception used by
constituted in the first place by 69% of the women women with breast cancer, the majority of
who have as antecedent the mother, 14% sister, women used combined oral contraceptives
10% cousin and 7% who represents another family (COC) with a percentage of 95.2%, including
member. Concerning the distribution of women 1.38% of women who used injectables as a
witnesses according to family history. method of contraception and 1.38% of
It emerges that 88.50% have no family history and women used other methods of
11.50% of the study population have a family contraception.
history which is constituted in the first place by On the other hand, the type of contraceptive
42% mother, 31% sister, 12% cousin and 15% who used by the control women; the majority of
represents another family member. women used combined oral contraceptives
(COC) with a percentage of 95.8%, including
1.69% of women who used injectables as a
method of contraception, and 0.37% of
women used other methods of
contraception..

91
Table 9: Taking hormonal contraception by
women case-control with breast cancer by
type/duration

Case Control
The table above presents the type of
Durat hormonal contraception taken by women
ion Categ Effe Effe % with breast cancer according to duration
ories ctiv e % ctive This breakdown shows that 62% of women
Type take COC for less than 10 years, 36% for
more than 10 years, and 1.86% for which the
<2 73 15.14
68
duration was not mentioned in the woman's
13.35
file.
2-5 17.84 91
Regarding all other types of contraception
COC 17.87
86 used by women with breast cancer,
5-10
"Injectables and others", 57.13% take them
29.04 160 31.43
140 for less than 10 years and 42.85% take them
>10
for more than 10 years.
151 157 30.84
31.32 On the other hand, this table presents the
>20
type of hormonal contraception taken by the
28 5.50
23 4.77 control women according to the duration.
N.M 1.86 5 0.98
9 This breakdown shows that 62.7% of women
take COC for less than 10 years, 36.35% take
them for
more than 10 years, and 0.98% whose
<2 0 0 0 0 duration was not mentioned in the woman's
Inject file.
able 2-5 14.28 4 44.44 Regarding any other type of contraception
1
used by women with breast cancer
5-10 42.85 5 55.55 "Injectables, and others"; it shows 0% for a
3
duration of less than 10 years and more than
>10 42.85 0 0 10 years.
3
>20 0 0 0 0

N.M 0 0 0 0

<2
0 0 0 0

Other 2-5 0 0 0 0
metho d
5-10 0 0 0 0

>10 0 0 0 0

>20 0 0 0 0

N.M 2 100 2 100

92
 Table 10: Association between hormonal
contraception as a risk factor and breast
cancer among women in the Marrakech-Safi
region
The table shows that there is a statistically
significant association between :
Contr
characteri Total Case ol OR Ch P contraceptive use and breast cancer (OR=
stics (1 200) N N [CI] i val 1.44, 95% CI= [1.024.1, 996]), women who
= = ue take hormonal contraception had a higher
600 à squ
600
95% are risk of developing breast cancer than those
who do not take it.
Taking
contrace
ption We also found a statistically significant
association between the type of hormonal
contraception and the occurrence of the
Yes 1037 506 531 1.44 4.452 0.036 disease (OR= 1.37, 95% CI= [1.014.1, 849]),
= = =
women taking oral contraceptives; COC, had
[1.024.
1,996] a higher risk of developing breast cancer
86,41 84,3 88,5 than women taking other methods of
%
% contraception or those who were not taking
%
any oral contraceptives.

No 163 94 69 In addition, a statistically significant

= association was found between the use of
= =
13,5
oral contraceptives and the occurrence of the
%) 15,6 11 ,5 disease, that is, the use of oral contraceptives
%) %) for more than 10 years the risk of developing
Types breast cancer is so frequent (OR=2.37, 95%
COC 991 509 482 CI [1.74-3.22]).
= = =
DISCUSSION
82,6% 84,8 80% 1.37 4,224 0,040
%
In this study, the same socio-demographic
[1.014.
1,849]
characteristics were observed in our sample
control cases.
In this case-control study, we found that
209 91(1 118
AUTRE 5,2)
hormonal contraception is a risk factor
(17,4 20% associated with the development of breast
) cancer among women in the Marrakech-Safi
%) region. Indeed, the results showed that
Duratio among women in the Marrakech Safi region,
n taking oral contraceptives would increase
387 80(1 307 2,37 30,93 0,00
<10ans 3,3 the risk of breast cancer, this has been found
(32,25 %) ( [1,74-
in many studies (White E, and Al, 1994) and
%)
51,2 3,22] (Ursin G, and Al, 1998) Our study also
%) showed that women who take hormonal
474 181 293 contraception, especially oral contraceptives
>10ans for more than 10 years, are more likely to
(39,5 (30, (48,8 develop breast cancer than women who take
%) 2%) %) them for a period of less than 10 years.

93
It has been found in other studies as well as
in (Mørch et al., 2017) that dusein cancer
increases by 20% in women taking these
hormonal contraceptives. Another element,
the more sustainable the use, the higher the
risks. Beyond 10 years, they are evaluated at
38%.
In the same vein, (Hunter et al., 2010), and
(Kumle et al., 2002) in their studies indicate
that there is an over-risk of breast cancer
while taking estroprogestative
contraception, with a risk relative between
1.2 and 1.6.
To our knowledge, our study is the first case-
control study in women from the
Marrakech-Safi region to study hormonal
contraception as a risk factor for breast
cancer already known in the literature.
The results found were similar to those seen
in other studies. On the other hand, the time
constraint, the non-exploitable state of
certain files and the lack of access to files in
some institutions like the CHU, all these
constraints have limited our study.
Moreover, the choice of cases that had the
same socio-demographic characteristics as
the witnesses; of the same age and
geographical area were a strong point for the
credibility of the data collection and
therefore the analysis of the results of our
study.And although the results can not be
generalized, they will remain the only
reference that was interested in hormonal
contraception as a risk factor associated with
breast cancer among women in the
Marrakech- Safi region of our country.
Other larger studies, including private and
semi- public sector institutions and UHCs in
all provinces of the Marrakech-Safi region,
would be an addition to the progress of our
study.
CONCLUSION
Early detection activities are very important
in reducing the incidence of some cancers.
Through efficient information and early
detection programs and therapeutic
management at the beginning of their onset,
morbidity and mortality related to some
cancers can be reduced by
one third.
The National Cancer Prevention and Control
Plan (PNPCC) has identified the detection of
breast
cancer as one of its priorities.
But on the other hand, it is necessary to
highlight the various major factors
associated with the occurrence of breast
cancer, just like hormonal contraception, in
order to emit courses of action that will be
taken into consideration for the main and
ultimate goal that is progress in improving
the management of women with breast
cancer by setting up an organized system of
screening, early diagnosis and treatment of
these cancers, which is the first cancer in
women in Morocco and in the world whole.
On the occasion of this study, the suggestions
are, on the one hand, derived from the
synthesis of the perspectives given by the
nurses, and on the other hand, the
consequence of the observations that we
have made.
These suggestions and recommendations are
grouped in three axes:(a) Recommendations
for breast cancer screening (b) Age-related
recommendations for women screened for
(c) Recommendations for hormonal
contraception.
Recommendations for breast cancer
screening:
-To identify participating women referred by
CSU health professionals and CSC for a
clinical abnormality of the breast or having a
family history (mother, sister, cousin or
other), or breast cancer staff.
-Simplify the procedures for diagnosis and
treatment of breast cancer;
-Equip screening and early diagnosis
services for breast cancer
-Strengthen the technical platform at all
levels of support.
-Develop the skills of health professionals as
part of the training basic and especially
continuing education
Recommendations related to the age
group of women concerned by the
screening:
-Extend the age range of screening and early
detection of breast cancer including women
from age 35 in the program or having family
history of breast cancer.
94

Recommendations related to hormonal
contraception:
-To prohibit any use of combination contraceptives
from age 35 instead of 40 years.
-Promote locally-based progesterone-based
contraceptive methods (Hormonal IUD)
-Promote first-line non-hormonal contraceptive
methods in the care package of basic health care
facilities
-Expand awareness on the copper IUD
-Microprogestant use especially long-term
(contraceptive implant)
-Training all qualified health professionals on copper
IUD insertion as part of the family planning program.
ACKNOWLEDGEMENTS
The authors thank all the participants women in this
study and research assistants for the data collection
in the provinces of the region Mrrakech-Safi
REFERENCES
Bray F., Ferlay J., Soerjomataram I. Siegel R. and Torre
L.A. Global Cancer Statistics 2018: GLOBOCAN
Estimates of Incidence and Mortality Worldwide for
36 Cancers in 185 Countries CANCER J CLIN 2018;
68:394–424
Registre du cancer de la région du grand Casablanca
(2012) (Cancer Registry of Greater Casablanca
(2012)).Site http://www.contrelecancer.ma/site_m
edia/uploaded_files/RCRC
Accessed December 20, 2019.
-Alami, Z. (2012). Chimiothérapie neoadjuvante dans
le cancer du sein inflammatoire (A propos de 41 cas).
Sidi mohamed Ben Abdellah, Fès.
Nkondjock, A., & Ghadirian, P. (2005). Facteurs
de risque du cancer du sein. médecine/sciences,
21(2), 175‑180.
https://doi.org/10.1051/medsci/2005212175
-Qu’est-ce que le cancer du sein ? | Biron. (s. d.).
Consulté 8 juin 2019, à l’adresse
https://www.biron.com/fr/centre-du-savoir/petit-
guide-biron/cancer-du-sein/
- Mørch, L. S., Skovlund, C. W., Hannaford, P. C.,
Iversen, L., Fielding, S., & Lidegaard, Ø. (2017).
Contemporary Hormonal Contraception and the Risk
of Breast Cancer. New England Journal of Medicine,
377(23), 2228‑2239.
https://doi.org/10.1056/NEJMoa1700732
- Marchbanks, P. A., McDonald, J. A., Wilson, H.
G., Folger, S. G., Mandel, M. G., Daling, J. R., … Weiss,
L. K. (2002). Oral contraceptives and the risk
of breast cancer. The New England Journal of
Medicine, 346(26), 2025‑2032.
https://doi.org/10.1056/NEJMoa01320
-Collaborative group on hormonal factors in
breast cancer. Breast cancer and hormonal
contraceptives : collaborative reanalysis of
individual data on 53,297 women with
breast cancer and 100,239 women without
breast cancer from 54 epidemiological
studies. Lancet 1996 ; 347 : 1713-27.
-National Population and Family Health
Survey,2011
Anne, T. (2014). Imagerie de la femme :
sénologie (Coll. Imagerie médicale).
Lavoisier.
Chopier, J., Jalaguier-Coudray, A., &
Thomassin- Naggara, I. (2011). Variations du
sein normal.
Aspects mammographiques et
échographiques.
/data/traites/r5/34-56763/. Consulté à
l’adresse https://www.em-
consulte.com/en/article/284389#N104CC
Gavenonis, S. C. (2011). Breast MR imaging:
normal anatomy. Magnetic Resonance
Imaging Clinics of North America, 19(3),
507‑519; viii.
https://doi.org/10.1016/j.mric.2011.05.
Hannaford, P. C., Selvaraj, S., Elliott, A. M.,
Angus, V., Iversen, L., & Lee, A. J. (2007).
Cancer risk among users of oral
contraceptives: cohort data from the Royal
College of General Practitioner’s oral
contraception study. BMJ (Clinical Research
Ed.), 335(7621), 651.
https://doi.org/10.1136/bmj.39289.649410
.55
Hunter, D. J., Colditz, G. A., Hankinson, S. E.,
Malspeis, S., Spiegelman, D., Chen,
W., … Willett, W. C. (2010). Oral
contraceptive use and breast cancer: a
prospective study of young women. Cancer
Epidemiology, Biomarkers & Prevention: A
Publication of the American Association for
Cancer Research, Cosponsored by the
American Society of Preventive Oncology,
19(10), 2496‑2502.
https://doi.org/10.1158/1055-
9965.EPI-10-0747
Kumle, M., Weiderpass, E., Braaten, T.,
Persson, I., Adami, H.-O., & Lund, E. (2002).
Use of oral contraceptives and breast cancer
95
risk: The Norwegian-Swedish Women’s
Lifestyle and Health Cohort Study. Cancer
Epidemiology, Biomarkers & Prevention: A
Publication of the American Association for
Cancer Research, Cosponsored by the
American Society of Preventive Oncology,
11(11), 1375‑1381.
Lansac, J. (2006). COLLÈGE NATIONAL DES
GYNÉCOLOGUES ET OBSTÉTRICIENS
FRANÇAIS, Extrait des mises à jour en
Gynécologie Médicale.
Latest world cancer statistics – GLOBOCAN
2012: Estimated Cancer Incidence, Mortality
and Prevalence Worldwide in 2012 – IARC.
(s. d.). Consulté 8 juin 2019, à l’adresse
https://www.iarc.fr/news-events/latest-wor
ld-cancer-
statistics-globocan-2012-estimated-cancer-
incidence-mortality-and-prevalence-worldwi
de-in- 2012/
le registre des cancers de la région de Rabat.
(2005). RCRGC le registre des cancers de la
région du gra.pdf. (s. d.).
Tazi, M. A., Er-Raki, A., & Benjaafar, N. (2013).
Cancer incidence in Rabat, Morocco:
2006-2008. Ecancermedicalscience, 7, 338.
https://doi.org/10.3332/ecancer.2013.338
Winters, S., Martin, C., Murphy, D., & Shokar,
N.
K. (2017). Breast Cancer Epidemiology,
Prevention, and Screening. Progress in
Molecular Biology and Translational Science,
151, 1‑32.
https://doi.org/10.1016/bs.pmbts.2017.07.
002
White E, Malone KE, Weiss NS, Daling JR.
Breast cancer among young United States
women in relation to oral contraceptive use. J
Natl Cancer Inst 1994; 86:505 – 14
Ursin G, Ross RK, Sullivan-Halley J, Hanisch
R,
Henderson B, Bernstein L: Use of
oral contraceptives and risk of breast cancer
in young women. Breast Cancer Res Treat
,1998, 50: 175-184
Najmeh Tehranian, Fatemeh Shobeiri,
Farahnaz Hafezi Pour, Ebrahim Hagizadeh.
Risk Factors for Breast Cancer in Iranian
Women Aged Less than 40 Years. Asian
Pacific Journal of Cancer Prevention, Vol 11,
2010, 1723-1725.
Mirra AP, Cole P, Macmahon B. Breast cancer
in an area of high parity: Sao Paulo,
Brazil. Cancer Res ,1971;31:77- 83.
Mandana Ebrahimi, Mariam Vahdaninia and
Ali Montazeri. Risk factors for breast
cancer in Iran: a case–control study. Breast
Cancer Research, 2002, Vol 4 No 5.
96
 CONFERENCE PAPER Youssef S’hih et al. 2022,97_105

Contribution to the study of the acute and subacute toxicity of aqueous extract
of Lawsonia Inermis L. leaves in Wistar rats
Youssef S’hih1*, Ibrahim HINAD1, Radia ELGUI2, Aboubaker ELHESSNI1, Abdelhalim MESFIOUI1,
Abdechahid LOUKILI3 and Moulay Laarbi OUAHIDI1
1
Laboratory of Biology and Health, Department of Life Sciences, Faculty of Sciences, Kenitra, Ibn Tofail
University, Morocco
2
Laboratory of Pathological Anatomy and Cytology, El Idrissi Public Hospital, Kenitra, Morocco
3
Laboratory of scientific research and pedagogical innovation, Regional Centre for Education and
Training, Rabat-Sale-Kenitra, Morocco

Lawsonia inermis (LI) leaves are widely used in traditional phytotherapy

without any control, and research on its toxicity is limited. For this, this
study aims to investigate the acute and subacute toxicity of aqueous extract
of LI leaves (AELIL) in the Wistar rat. For acute toxicity, five dose levels (500,
1000, 2000, 3000 and 5000 mg.kg-1bw) were tested by single oral
administration. Subacute toxicity for 28 days was obtained by daily oral
administration of AELIL at doses of 1000, 2000 and 3000 mg.kg-1bw. No
deaths were recorded, and clinical signs were observed in the few rats at the
highest doses compared to controls. The subacute toxicity results showed in
rats of group 4 (3000mgkg-1bw) a significant difference in body weight, hair
straightening, drowsiness, hypoactivity, isolation and weakness from the
second week of treatment, while the histopathological examination showed
that the kidneys remained intact, as well as degeneration and steatosis of
hepatocytes, while the haematological profile has shown no significant
modification of blood parameters. AELIL should be used with caution as it
has caused signs of toxicity and may affect the liver at repeated doses above
2000 mg.kg-1bw. However, further studies are needed to verify and clarify the
toxic aspect of AELIL.
INTRODUCTION
*
Corresponding Author Phytotherapy is widely practiced in most
Name: Youssef S’hih countries of the world, including Morocco,
Email: youssef.shih@uit.ac.ma whose population still depends exclusively on
Phone:00212665805984 plants as a source of therapy through different
traditional methods (Elachouri et al., 2021).
The World Health Organization (WHO)
ISBN: 9788367405256
estimates that around 80% of the population in
DOI: 10.2478/9788367405256-014 developing countries uses traditional medicine
for essential medical care. Nevertheless,
Published by Sciendo. © 2022 Youssef S’hih et al.
scientific research is needed to support these
This is an open access article licensed under the practices, and this research must respect the
Attribution 4.0 International
(https://creativecommons.org/licenses/by/4.0
knowledge and experience gained through
/) long-established practices (WHO, 2000).
However, there is a major misconception that
herbal medicines are harmless since they are
natural, and these plants are mistakenly
considered to be safe (WHO, 2004), but they
can contain powerful chemical compounds
97
which are responsible for adverse effects and
significant toxicity.
Among the medicinal plants most used in
herbal medicine is Lawsonia inermis (LI),
which has been traditionally used for centuries
for medicinal purposes as an anti-diabetic and
hypo-lipidemic (Arayne et al., 2007; Badoni
Semwal et al., 2014; Singh et al., 2015), diuretic
(Chandra Kalyan Reddy et al., 2011),
antimicrobial (Sharma and Goel, 2018),
hepatoprotective (Anand et al., 1992),
anti-inflammatory, analgesic, antipyretic and
anti-arthritic (Al Saif, 2016), cytotoxic (Li et al.,
2014), antioxidant (Hsouna et al., 2011) and
anticancer (Priya et al., 2011; Raja and Pandey,
2020; Singh and Luqman, 2014), and has been
considered harmless from prehistoric times to
the present day. Therefore, further study of oral
toxicity is essential to identify the range of
doses that could be used in the future (Jothy et
al., 2011).
The toxicological study in vivo of the aqueous
extract of LI leaves (AELIL) has not been
considered yet. A preclinical toxicity study is
fundamental to approving its medicinal use.
The present study determines the possible
acute and subacute toxicity depending on the
dose administered by single and repeated oral
gavage (28 days) of AELIL to Wistar rats using
the guidelines of the Organization for Economic
Cooperation and Development (OECD 420 and
407).
MATERIALS AND METHODS
Material
Plant material
- The harvest of the plant: LI leaves were
collected from the region of Errachidia
(South-East of Morocco) between April and May
2021, identified and authenticated by
professors from the life sciences department,
and a good specimen of the reference has been
deposited from the plant herbarium of Ibn Tofail
University’s Faculty of Sciences in Kenitra under
the number HFSKLI06/21.
- Sample preparation: The plant leaves were
thoroughly cleaned with water, allowed to air
dry at room temperature, and then ground into
a coarse powder using an electric grinder. They
were then kept at room temperature in an
airtight jar.
Animal material
Wistar rats were received from the animal
facility at the Faculty of Sciences of Ibn Tofail
University in Kenitra, Morocco. Before the
experiment started, the rats were weighed,
divided into groups, and given seven days to
become acclimated to the lab environment with
total access to water and food (Hinad et al.,
2021).
For testing acute and subacute toxicity, we used
sixty female Wistar rats weighing 160–200g,
divided into ten groups of six animals each. The
NC3Rs (ARRIVE) recommendations for in vivo
studies, the UK's Scientific Procedures Act of
1986, and EU Directive 2010/63/EU were all
followed while testing animals. The practices
used have complied with the guidelines outlined
in the eighth edition on the care and use of
laboratory animals (National Academy of
Sciences, 2010).
Methods
Preparation of the extract
The leaves of LI were used to prepare an
aqueous extract. One hundred grams of powder
was placed in one thousand milliliters of
distilled water, which was boiled for thirty
minutes. The resultant extract was centrifuged,
filtered, and concentrated to dryness using a
rotary evaporator under a vacuum at 40°C.
Then frozen at -20°C and lyophilized (Free
Zone® Dry 4.5) to give a yield of 14% (28g)
(w/w) dry extract. The extract was collected in
a clean flask and stored in the -4°C freezer.
Acute toxicity assessment
The recommendations of OECD420 are
followed in the conduct of the acute oral
toxicity study (OCDE 420, 2001). Before the
treatment, the animals fasted for twelve hours,
then those in group 1 (C) were given distilled
water, and the remaining groups orally received
500, 1000, 2000, 3000 and 5000 mg.kg-1bw of
AELIL. The doses were prepared using distilled
water. Animals were carefully monitored for the
first six hours, the following day, and once per
day for the following fourteen days of the
experiment to record their behaviors and
several toxicological indicators.
Subacute Toxicity Assessment
The subacute oral toxicity study was carried out
according to OCDE 407 (2008). After fasting for
twelve hours, the AELIL were administered to
rats at different doses by gavage every morning,
seven days a week for four weeks. Group 1
functioned as the control and obtained distilled
water, while the other groups (2, 3, and 4)
received orally 1000, 2000, and 3000 mgkg-1bw
of AELIL respectively every day for
twenty-eight days successive. The animals were
observed after extract administration every 30
minutes for 8 hours on the first day and at the
end of each day of therapy. During this period,
the number of deaths and signs of toxicity and
98
 symptomatology disorders were noted. organs was performed before fixation to note
Throughout the study period, food intake was any changes in shape or color, and then fixed in
recorded every day, and body weight was 10% paraformaldehyde for histology and
recorded once a week to search for any changes treated with paraffin according to the standard
induced by the extract treatment (Thounaojam protocol. Seven µm thick sections of each tissue
et al., 2011). were stained with hematoxylin and eosin and
observed for histopathological damage.
Blood Sampling and hematological analysis
Histological sections were made, including
Group1 Group2 Group3 Group4
Day (Control) 1000mgkg-1 2000mgkg-1 3000mgkg-1 dehydration, inclusions, and sections (S’hih et
1 N N N N al., 2022).
2 N N N N
3 N N N N RESULTS
4 N N N N
5 N N N N Acute toxicity
6 N N N N Acute oral toxicity studies in Wistar rats after a
7 N N N N single administration of five dose levels of
8 N N N N AELIL, revealed some toxicity symptoms in rats
9 N N N N of group 5 (3000 mg.kg-1bw) and 6 (5000
10 N N N N mg.kg-1bw) such as sedation, dyspnoea,
11 N N N N convulsions and hypo-activity while the control
12 N N N N group of rats given distilled water showed
13 N N N N normal behavior, and no mortality was
14 N N N HS recorded after the seventy-two hours and
15 N N N HS during the fourteen days of monitoring. It
16 N N N HS shows that no mortality were recorded in the
17 N N N HS groups receiving the different dose levels. The
18 N N N HS DL50 is greater than 5000mg.kg-1bw, and the
19 N N N HS
AELIL is non-toxic at this dose (class V, WHO).
20 N N N HS
Subacute toxicity
21 N N N Dr
-Daily monitoring and observation for signs of
22 N N N Dr
toxicity and symptomatic disturbances:
23 N N N HA
24 N N N HA The rats in group 1 (C), 2 and 3 remained
25 N N N HA normal (N) and showed no signs of intoxication.
26 N N N ISO, W in contrast, rats in group 4 showed abnormal
27 N N N ISO, W symptoms such as a hair straightening (HS)
28 N N N ISO, W from the 14th day and drowsiness (Dr),
After twenty-eight days of treatment with hypoactivity (HA), isolation (ISO) and weakness
AELIL, the rats were anaesthetized with 10% (W) from the 21st day of the study. (Table 1).
chloral (3ml.kg-1bw) and blood was collected Evolution of body weight of rats
from the fasting animals overnight by
puncturing the lateral vein of the tail and During this study, we recorded in group 4
collected in tubes containing EDTA (Li et al., (3000 mg.kg-1pc) a significant difference α <
2014). It was collected for the measurement of 0.05) in body weight with slow growth in
blood parameters, including red blood cells group 2 and 3 of rats (Table 2).
(RBC), white blood cells (WBC), mean platelet Table 1: signs of toxicity and symptomatology
volume (MPV), haemoglobin level (HB), disorders.
haematocrit (HT), mean blood cell volume
Haematological parameters
(MCV), mean corpuscular haemoglobin
After administration of AELIL for four weeks,
(CMHC), neutrophil count (NPC), eosinophil
there were no significant changes (ns at
count (EPC), basophil count (BPC), lymphocyte
α<0.05) in haematological parameters in rats of
count (LC) and the number of monocytes (MC).
groups 2, 3, and 4 compared to group 1 (C)
Histopathology
(Table 3).
The animals were then sacrificed by cervical
Macroscopic and histopathological analysis of
dislocation under 10% chloral anesthesia
rat organs
(3ml.kg-1bw) for autopsy; the liver and kidneys
Macroscopic examination of the kidneys and
were excised, and rinsed with physiological
liver of rats treated with the different doses of
water, a macroscopic examination of these vital
AELIL showed no change in the appearance of

99
 these organs compared to the control group. 1B). Histopathological examination of kidney
slides in groups 2 (1000 mg.kg-1bw), 3 (2000
The histopathological examination of liver
mg.kg-1bw) and 4 (3000 mg.kg-1bw) of Wistar
slides from groups 2 (1000 mg.kg-1bw) and 3
rats showed that the glomerulus has a normal
(2000 mg.kg-1bw) showed regular portal triad
size and cellularity, no increase in mesangial
areas without abnormalities and are like those
matrix or thickening of glomerular basement
of group 1 (C) (Figure 1A). While in group 4
membrane (Figure 1D) compared to group 1
(3000 mg.kg-1bw), pathological changes are
(control) with normal appearance of renal
observed with an occasional region of
capsules and tubules (Figure 1C).
hepatocyte degeneration and steatosis with
lymphatic infiltration in the portal area (Figure
Table 1: Evolution of the body weight of rats.
Groups Initial weight Week 1 Week 2 Week 3 Week 4 Variation (g)
Group 1© 186,00 ± 16 192,00 ± 15 203,00 ± 15 215,00 ± 15 226,00 ± 13 +40,00 ± 2,51 ns
Group 2 (1000 mg.kg-1) 172,00 ± 20 178,00 ± 19 185,00 ± 15 191,00 ± 15 196,00 ± 16 +24,00 ± 5,31 ns
Group 3 (2000 mg.kg-1) 161,00 ± 08 168,00 ± 08 177,00 ± 07 184,00 ± 09 190,00 ± 10 +29,00 ± 1,68 ns
Group 4 (3000 mg.kg-1) 164,00 ± 08 164,50 ± 10 166,00 ± 05 168,00 ± 15 171,50 ± 17 07,50 ± 1,24*
ns (Not significant), * (p ⩽ 0.05)
Table 2: The hematological parameters of the different groups studied.
Group 1 © Group 3 Group 4
Group 2
Parameters (0 -1 (2000mg (3000mg
(1000mg kg bw)
mg.kg-1bw) kg-1bw) kg-1bw)
RBC
05,34±0,17 04,96±0,24 ns 05,84±0,16 ns 06,38±0,33 ns
(106/mm3)
WBC
12,30±0,45 12,55±0,61 ns 12,83±0,74 ns 13,43±1,08 ns
(103//mm3)
MPV 251,00±12, 249,00±10,3 252,00±14,73 254,00±16,08
(103//mm3) 72 8 ns ns ns

ns
Hb (g/dL) 10,30±0,88 10,56±0,29 10,91±0,46 ns 11,15±0,18 ns
ns
Ht (%) 29,40 29,40 29,40 ns 31,80 ns
MGV ( µ3) 52,00±3,93 52,84±3,27 ns 53,42±2,98 ns 54,00±3,05 ns
ns
MCH (pg) 17,55±1,13 17,46±2,12 17,89±2,24 ns 18,00±1,57 ns
ns
MCHC (%) 33,45 32,97 33,65 ns 34,00 ns
PNN 01,93 ±0,02
01,77 ±0,08 01,85 ±0,11 ns 01,95± 0,07 ns
(103//mm3) ns

PNE 00,48 ±0,01

00,44 ±0,03 00,55 ±0,09 ns 00,59 ±0,04 ns
(103//mm3) ns

PNB 00,08 ±0,02

00,09 ±0,01 00,09 ±0,03 ns 00,10±0,05 ns
(103//mm3) ns

08,26 ±0,38
L (103//mm3) 08,58 ±0,49 ns 09,07 ±0,19 ns 09,24 ±0,56 ns

ns (Not significant), * (p ⩽ 0.05)

100
 (A) (B)

(C) (D)
Figure1: Histological section of (A) Normal liver of control group rats given distilled water and fed
a normal diet for 28 days; (B) Liver of rats having received a dose of 3000 mg.kg-1bw of AELIL for 28
days showing steatosis, degeneration of hepatocytes; (C) Normal kidney of control group rats were
given distilled water and fed a normal diet for 28 days. (D) Healthy kidney shows in rats having
received 3000 mg.kg-1bw of AELIL for 28 days.

DISCUSSION and use of the extract.

During the acute toxicity experiment on Wistar
rats that received the AELIL, no mortality is
recorded over the entire follow-up period of the
five groups of rats after the single administration
of the five doses (500, 1000, 2000, 3000 and 5000
mg.kg-1bw), it is observed that only the high doses
(3000 and 5000 mg.kg-1bw) cause some signs of
abnormal behavior in certain rats such as sedation,
dyspnoea, convulsions and hypoactivity during the
seventy-two hours of follow-up. The DL50 is
greater than 5000 mg.kg-1bw, and AELIL can be
classified as non-dangerous up to this dose level
(class V, WHO). While other acute oral toxicity
studies indicated dose-dependent toxicity
symptoms ranging from loss of appetite, lethargy,
inactivity, and respiratory failure to death, and the
DL50 of AELIL was 750 mg.kg-1bw (Olawuyi et al.,
2019), while the work of Kaur et al. (2014)
showed at a dose of 2000 mg.kg-1bw 50% mortality
after 48 hours and 100% after seventy-two hours
of rats died. But those of Shastry et al. (2012)
showed that AELIL was found to be safe up to 2000
mg.kg-1bw, but after twenty-four hours, the animals
were well tolerated, and there were no death and
no intoxication symptoms. These differences in
results are due to a difference in the preparation
The subacute oral toxicity study was carried out
with daily oral gavage of three dose levels (1000,
2000 and 3000 mg.kg-1bw) of AELIL to different
groups of rats for twenty-eight days. The
monitoring of rats during this period showed that
this extract is not very toxic, only produces late
toxicity (without mortality), which is associated
with clinical manifestations at a dose of 3000
mg.kg-1bw, since after two weeks of treatment, it
was observed in rats of group 4 a hair
straightening (HS), while drowsiness (Dr),
hypoactivity (HA), isolation (ISO) and weakness
(W) from the 21st-day (Table 1). However, the
study by Abdallah and Aldameg (2012) on rats
treated with the methanolic extract of LI leaves did
not show any sign of intoxication. We also reported
in our study a decrease in daily food and water
intake in groups 3 and 4 compared to the control
group, which was supposed to be responsible for
the considerable decrease in the body weight of
the rats compared to the control group (Table 2),
because a reduction of appetite is frequently
associated with bodyweight decrease and
significant variations in body weight are
recognized as predictors of unfavorable chemical
and pharmacological effects (Raza et al., 2002),
101
this is due to disturbances in the metabolism of
carbohydrates, proteins or fats (Saganuwan, 2017).
While the study of Abdallah and Aldameg (2012)
on rats administered a methanolic extract of LI
leaves revealed no significant variations in body
weight.
The hematopoietic system is one of the most
vulnerable targets for toxic substances. Therefore,
it is necessary to document any potential change
caused by a chemical substance (Daas et al., 2019).
Then, changes in hematological parameters have a
higher predictive value when information on
chemical toxicity from animal research is
transformed for use in the medical field (Olson et
al., 2000). Then, the typical hematological profile
of the three groups (2, 3 and 4) treated with AELIL
compared to the control group (group 1) can be
explained by the non-toxic nature of the extract at
these doses (1000, 2000, 3000 mg.kg-1bw), because
there was a non-significant variation in
hematological parameters (RBC, WBC, MPV, Hb, Ht,
MGV, MCH, MCHC, etc.). These results allow us to
affirm that the repeated administration of AELIL at
doses of 1000, 2000 and 3000 mg.kg-1bw does not
cause any alteration of these parameters in the
Wistar rat, and confirms the results obtained by
Abdallah and Aldameg (2012), which does not
show any clinical change in rat blood parameters.
But the lawsone (2-hydroxy-1,4 naphthoquinone)
is believed to be the active component of LI
(Munday et al., 1991), responsible for its
haemato-toxicity (Sauriasari et al., 2007), the
Lawsone molecule has also been reported to be
able to induce oxidative injury to red blood normal
and hemolytic anemia in people with
glucose-6-phosphate (G6PD) deficiency (Kök et al.,
2004).
After the twenty-eight days of daily treatment by
AELIL, the Wistar rats were sacrificed, and the
organs are removed, whereas a meticulous
macroscopic observation of these organs showed
no adverse effect on the morphology of the kidneys
and the liver, which could be a good indicator of
the no-toxicity of this extract.
The liver is the body's primary detoxification site
for any drugs or toxins. It is important to all the
vital processes, including the metabolism of
substances which enter the body from some
sources (Latha, 2004), as well as many medicinal
plants that are toxic to the liver, so it is an essential
organ in any toxicological study (Treadway, 1998).
The observations of histological sections of the
liver after the twenty-eight days of treatment with
AELIL at the doses of 1000 and 2000 mg.kg-1bw
appeared normal and like the control group
(Figure 1A). They showed regular portal triad
areas without abnormalities, while severe
abnormalities were observed in sections from rats
treated at the dose of 3000 mg.kg-1bw with an area
of liver degeneration and steatosis (Figure 1B),
suggesting a possible toxic effect of LI leaves on
rats. Liver abnormalities were also observed by
Alferah (2012) at a dose of 1000 mg.kg-1bw of
AELIL, just as hyperbilirubinemia is always
associated with periportal liver lesions previously
described in goats by (Ali and Adam, 1978).
Also, the kidneys are the main organs of the body,
sensitive to the toxic effects of drugs (Adekomi et
al., 2011), which can trigger tubular, interstitial,
tubule-interstitial and glomerular nephritis (Pletz
et al., 2018). Histological sections of the kidneys of
the groups of rats treated with doses of 1000, 2000
and 3000 mg.kg-1bw did not show any difference
compared to the sections of the control group
(Figure 1D), the tubules were within the standard
limits, and the glomerulus were of normal size and
cellularity, with no increase in the mesangial
matrix (Figure 1C). Our results disagree with the
conclusions of Alferah (2012), who detected
degeneration and desquamation of the renal
tubular mucosa, and those of Kaur et al. (2014),
who noticed degenerative changes in the tubular
epithelium, an area of necrosis tubular and
capillary congestion of some glomeruli in animals
treated with the dose of 1000 mg.kg-1bw. In
addition, a case of acute renal failure was observed
due to the nephrotoxic effect of henna in a
34-year-old man with G6PD deficiency from
Yangon, Myanmar after ingesting a herbal remedy
based on boiled henna leaves (Khine, 2017).
CONCLUSIONS
Many researchers are interested in biologically
active compounds isolated from plant extracts
which are veritable chemical factories, hence,
maximum benefit should be obtained. The leaves
of Lawsonia inermis are highly valued and widely
used in traditional medicine. The results of our
acute toxicity study show that the single oral
administration of aqueous extract of these plant
leaves at a high dose (5000 mg.kg-1bw) did not
cause the death of the rats. While those of subacute
toxicity during repeated consumption for
twenty-eight days at a dose of 3000 mg.kg-1bw
modified the behavior of rats and caused changes
in liver tissue.
This study assesses the toxicity of this extract and
defines it as non-toxic at a lower dose of 3000
mg.kg-1bw in repeated consumption. So, its use
requires more caution in the use of these leaves in
the medium and long term at high doses.
Hopefully, these results of toxicity on this plant
102
leaves will be beneficial in generating more
interest in this herb and its healthy use, and
further studies are needed to screen for the
phytochemicals responsible for these effects and
to better assess their toxicity by chronic toxicity
tests, defining new pharmacological and clinical
applications that may be useful in the
development of new formulations in the future
with caution.
HUMAN AND ANIMAL RIGHTS
No human is used in the study.
The animals were used with the greatest
minimization of numbers and suffering following
the ethics report for research involving animals.
ACKNOWLEDGEMENTS
Thanks to Dr Hajar Hechlaf; Specialist in Histology
at the El-Idrissi Hospital, Kenitra. Morocco for
reading the histological sections of the study.
REFERENCES
Abdallah, E., Aldameg, M., 2011. Antibacterial and
toxicological assessment of Lawsonia innermis
linn. (Henna) leaves on rats. Res. J. Biol. Sci. 6,
275–280. https://doi.org/10.3923/rjbsci.2011.
275.280
Adekomi, D., Tijani, A., Adeniy, T., Olajide, J., 2011.
Some of the Effects of Aqueous Leaf Extract of
Cnidoscolous aconitifolius (Euphorbiaceae) on
the Morphology and Histology of the Kidney and
Liver of Sprague Dawley Rat. Trop. J. Health Sci.
18. https://doi.org/10.4314/tjhc.v18i1.64478
Al Saif, F., 2016. Henna beyond skin arts:
Literatures review. J. Pak. Assoc. Dermatol. 26,
58-65.
Alferah, M., 2012. Toxicity Induced Histological
Changes in Selected Organs of Male (Wistar) Rats
by Lawsonia inermis Leaf Extract. Eur. J. Med.
Plants 2, 151–158. https://doi.org/10.9734/
EJMP/2012/1061
Alferah, M.A.Z., 2012. Toxicity Induced Histological
Changes in Selected Organs of Male (Wistar) Rats
by Lawsonia inermis Leaf Extract. Eur. J. Med.
Plants 151–158. https://doi.org/10.9734/EJMP
/2012/1061
Ali, B., Adam, S.E.I., 1978. Effects of
Acanthospermum hispidum on goats. J. Comp.
Pathol. 88, 533–544. https://doi.org/10.1016/
0021-9975(78)90007-5
Anand, K., Singh, B., Chand, D., Chandan, B., 1992.
An evaluation of Lawsonia alba extract as
hepatoprotective agent. Planta Med. 58, 22–25.
https://doi.org/10.1055/s-2006-961382
Arayne, M.S., Sultana, N., Mirza, A.Z., Zuberi, M.H.,
Siddiqui, F.A., 2007. In vitro hypoglycemic
activity of methanolic extract of some indigenous
plants. Pak J Pharm Sci 7.
Badoni Semwal, R., Semwal, D.K., Combrinck, S.,
Cartwright-Jones, C., Viljoen, A., 2014. Lawsonia
inermis L. (henna): Ethnobotanical,
phytochemical and pharmacological aspects. J.
Ethnopharmacol. 155, 80–103. https://doi.org
/10.1016/j.jep.2014.05.042
Chandra Kalyan Reddy, Y., Sandya, L., Sandeep, D.,
Ruth Salomi, K., Nagarjuna, S., Padmanabha
Reddy, Y., 2011. Evaluation of diuretic activity of
aqueous and ethanolic extracts of Lawsonia
inermis leaves in rats. Asian J. Plant Sci. Res.
28–33.
Daas, S.I., Rizeq, B.R., Nasrallah, G.K., 2019. Adipose
tissue dysfunction in cancer cachexia: DAAS et al.
J. Cell. Physiol. 234, 13–22.
https://doi.org/10.1002/jcp.26811
Elachouri, M., Kharchoufa, L., Fakchich, J.,
Lorigooini, Z., Subhasis, P., Subhash, M., 2021.
Ancestral phytotherapeutic practices in Morocco:
regards on history, current state, regulatory and
safety of commonly used herbal medicine 17.
Hinad, I., S’hih, Y., El Gui, R., Elhessni, A., Mesfioui,
A., Ouahidi, M. laarbi, 2021. Acute and subacute
toxicity study of the methanolic extract of olea
europea.L leaves in Wistar rat. E3S Web Conf.
319,01094.https://doi.org/10.1051/e3sconf/20
2131901094
Hsouna, A.B., Trigui, M., Culioli, G., Blache, Y., Jaoua,
S., 2011. Antioxidant constituents from Lawsonia
inermis leaves: Isolation, structure elucidation
and antioxidative capacity. Food Chem. 125,
193–200.https://doi.org/10.1016/j.foodchem.20
10.08.060
Jothy, S.L., Zakaria, Z., Chen, Y., Lau, Y.L., Latha, L.Y.,
Sasidharan, S., 2011. Acute Oral Toxicity of
Methanolic Seed Extract of Cassia fistula in Mice.
Molecules 16, 5268–5282.
https://doi.org/10.3390/molecules16065268
Kaur, M., Dangi, C., Singhai, A., Singh, M., Kosta, S.,
Singh, H., Peter, J., Jain, S., 2014. Toxicity profile of
ethanolic extract of Lawsonia inermis leaves in
albino wistar rats. World J. Pharm. Pharm. Sci. 3,
14.
Khine, Y.Y., 2017. Acute Kidney Injury following
Ingestion of Henna Leaf Extract: A Case Report
from Myanmar. Blood Purif. 44, 41–45.
https://doi.org/10.1159/000479618
Kök, A.N., Ertekin, M.V., Ertekin, V., Avci, B., 2004.
Henna (Lawsonia inermis Linn.) induced
haemolytic anaemia in siblings: Henna
(Lawsonia Inermis Linn.) Induced Haemolytic
Anaemia. Int. J. Clin. Pract. 58,530–532.
103
 https://doi.org/10.11
11/j.13685031.2004.00048.x
Latha, M., 2004. Protective activity of Glycyrrhiza
glabra Linn. on carbon tetrachloride-induced
peroxidative damage. Indian J. Pharmacol. 36,
284.
Li, Q., Gao, W., Cao, J., Bi, X., Chen, G., Zhang, X., Xia,
X., Zhao, Y., 2014. New cytotoxic compounds from
flowers of Lawsonia inermis L. Fitoterapia 94,
148–154.
https://doi.org/10.1016/j.fitote.2014.02.007
Munday, R., Smith, B.L., Fowke, E.A., 1991.
Haemolytic activity and nephrotoxicity of
2-hydroxy-1,4-naphthoquinone in rats. J. Appl.
Toxicol. 11, 85–90. https://doi.org/10.1002/jat.
2550110203
National Academy of Sciences, T.N.A.P.,
Washington, DC, 2010. Législation pour la
protection des animaux utilisés à des fins
scientifiques - Environnement - Commission
européenne. URL https://ec.europa.eu/
environment/chemicals/lab_animals/legislation
_en.htm (accessed 9.23.21).
OCDE 407, 2008. Toxicité orale à doses répétées -
pendant 28 jours sur les rongeurs (Text No. Essai
n° 407).
OCDE 420, 2001. Toxicité orale aiguë - Méthode de
la dose prédéterminée (Text No. Essai n° 420).
Olawuyi, T.S., Akinola, K.B., Adelakun, S.A.,
Ogunlade, B.S., Akingbade, G.T., 2019. Effects of
Aqueous Leaf Extract of Lawsonia inermis on
Aluminum-induced Oxidative Stress and Adult
Wistar Rat Pituitary Gland Histology. JBRA Assist.
Reprod. 23, 117–122. https://doi.org/10.5935/
1518-0557.20190024
Olson, H., Betton, G., Robinson, D., Thomas, K.,
Monro, A., Kolaja, G., Lilly, P., Sanders, J., Sipes, G.,
Bracken, W., Dorato, M., Van Deun, K., Smith, P.,
Berger, B., Heller, A., 2000. Concordance of the
Toxicity of Pharmaceuticals in Humans and in
Animals. Regul. Toxicol. Pharmacol. 32, 56–67.
https://doi.org/10.1006/rtph.2000.1399
Pletz, J., Enoch, S.J., Jais, D.M., Mellor, C.L., Pawar, G.,
Firman, J.W., Madden, J.C., Webb, S.D., Tagliati,
C.A., Cronin, M.T.D., 2018. A critical review of
adverse effects to the kidney: mechanisms, data
sources, and in silico tools to assist prediction.
Expert Opin. Drug Metab. Toxicol. 14,
1225–1253.
https://doi.org/10.1080/17425255.2018.15390
76
Priya, R., Ilavenil, S., Kaleeswaran, B., Srigopalram,
S., Ravikumar, S., 2011. Effect of Lawsonia
inermis on tumor expression induced by Dalton’s
lymphoma ascites in Swiss albino mice. Saudi J.
Biol.Sci.18,353–359.https://doi.org/10.1016/j.
sjbs.2011.04.001
Raja, W., Pandey, S., 2020. Antitumoral effect of
lawsonia inermis extract on melanoma
tumor-bearing C57BL/6 mice. Pharmacogn. Mag.
16, 435.
https://doi.org/10.4103/pm.pm_219_19
Raza, M., Oa, A.-S., Tm, E.-H., Aa, A.-M., 2002. Effect
of prolonged vigabatrin treatment on
hematological and biochemical parameters in
plasma, liver and kidney of Swiss albino mice.
Sci. Pharm. 70, 135–145. https://doi.org/
10.3797/scipharm.aut-02-16
Saganuwan, S., 2017. Toxicity studies of drugs and
chemicals in animals: an overview. Bulg. J. Vet.
Med. 20, 291–318. https://doi.org/10.15547/
bjvm.983
Sauriasari, R., Wang, D., Takemura, Y., Tsutsui, K.,
Masuoka, N., Sano, K., Horita, M., Wang, B., Ogino,
K., 2007. Cytotoxicity of lawsone and
cytoprotective activity of antioxidants in catalase
mutant Escherichia coli. Toxicology 235,
103–111. https://doi.org/10.1016/j.tox.2007.
03.019
Sharma, R.K., Goel, A., 2018. Identification of
Phytoconstituents in Lawsonia inermis Linn.
Leaves Extract by GC-MS and their Antibacterial
Potential. Pharmacogn. J. 10, 1101–1108.
https://doi.org/10.5530/pj.2018.6.187
Shastry, C., Kiran, U., Aswathanarayana, B., 2012.
Effect of Acute and Chronic Administration of the
Aqueous Extract of Lawsonia Inermis Leaves on
Haloperidol Induced Catalepsy in Albino Mice.
Res. J. Pharm. Biol. Chem. Sci. 10.
S’hih, Y., Hinad, I., Gui, R.E., Elhessni, A., Mesfioui,
A., Loukili, A., Ouahidi, M.L., 2022. Evaluation of
the Acute and Subacute Toxicity of Aqueous
Extract of Coriandrum sativum L. Seeds in Wistar
Rats. Curr. Drug Saf. 18, 504–510.
https://doi.org/10.2174/157488631766622060
6153524.
Singh, D.K., Luqman, S., 2014. Lawsonia inermis
(L.): A perspective on anticancer potential of
Mehndi/Henna. Biomed. Res. Ther. 1, 18.
https://doi.org/10.7603/s40730-014-0018-1
Singh, D.K., Luqman, S., Mathur, A.K., 2015.
Lawsonia inermis L. – A commercially important
primaeval dying and medicinal plant with
diverse pharmacological activity: A review. Ind.
Crops Prod. 65, 269–286. https://doi.org/
10.1016/j.indcrop.2014.11.025
Thounaojam, M.C., Jadeja, R.N., Valodkar, M., Nagar,
P.S., Devkar, R.V., Thakore, S., 2011. Oxidative
stress induced apoptosis of human lung
carcinoma (A549) cells by a novel copper
nanorod formulation. Food Chem. Toxicol. 49,
2990–2996. https://doi.org/10.1016/j.fct.2011.
104
 07.055
Treadway, S., 1998. An Ayurvedic Herbal Approach
To A Healthy Liver 3.
WHO, 2004. Rapport sur la santé dans le monde
2004: changer le cours de l’histoire. Organisation
mondiale de la santé, Genève.
WHO (Ed.), 2000. Pour un système de santé plus
performant, Rapport sur la santé dans le monde.
OMS, Genève.

105
CONFERENCE PAPER Myriam Chems et al. 2022, 106-112

Photocatalytic degradation of dye using ZnO and 1%Al-Doped ZnO: a

comparative investigation of nanocatalysts properties and operating conditions

Myriam Chems* ,Kaoutar El Hassani , Hajar Jabkhiro And Abdellah Anouar

Hassan First University of Settat, Faculty of Sciences and Techniques, Laboratory of Applied
Chemistry and Environment,26000 Settat, Morocco

Zinc oxide and 1% aluminum-doped ZnO were successfully synthesized

through a one-step and low-cost chemical route. The structural, functional
groups, and morphological properties were investigated by employing X-ray
Diffraction (XRD), Scanning Electron Microscopy (SEM), and Fourier
Transform Infrared Spectroscopy (ART-FTIR) respectively. The obtained
nanoparticles were subsequently used for the photocatalytic degradation of
Methyl Orange (MO) dye from its aqueous solution under UV-light
irradiation and measured using a spectrophotometric method. Various such
as dye solution pH, dye concentration, contact time, and parameters catalyst
dose were examined. 1%Al-ZnO showed an excellent (MO) removal
efficiency of 98-80% at a large pH range compared to the photocatalytic
activity of pure ZnO nanocatalyst under the same conditions.

* UV and solar irradiation (Ebrahimpour et al.,

Corresponding Author
2021; Lee et al., 2016; Ong et al., 2018).
Name: Myriam Chems
Email: m.chems@uhp.ac.ma Thus, photocatalysis is one of the Advanced
Phone : Oxidation Processes (AOPs) and is known to be
the most environmentally friendly treatment so
ISBN : 9788367405256 far due to the ability to reach complete
DOI: 10.2478/9788367405256-015 mineralization and nearly zero waste (Sundar &
Kanmani, 2020). Several types of catalysts are
Published by Sciendo. © 2022. Myriam Chems et al. promising to enhance the photocatalytic
This is an open access article licensed under the Attribution degradation of various organic contaminants like
4.0 International metal oxides (TiO2, ZnO, ZrO2….) (Balaprakash et
(https://creativecommons.org/licenses/by/4.0/) al., 2018; Trandafilović et al., 2017; Ullah et al.,
2017).

ZnO has gained a spotlight in the scientific

INTRODUCTION
Nowadays, the water crisis is becoming an
increasingly serious issue, in an ever-expanding
global economy and population, due to the limited
access to clean water resources and excessive
demand for it. One of the most appealing
responses to this worldwide problem is to
implement sustainable treatment of various
industrial effluents from processes such as food,
cosmetics, textile, pharmaceutical, etc. The most
encouraging process is the treatment process
coupled with unconventional energy-source as
community, thanks to its semiconducting
properties, a large exciton binding energy of ∼60
meV at room temperature, and a wide bandgap of
3.4 eV (Ahammed et al., 2018), as well as its doped
derivatives. Besides Al is an abundant, low cost
and non-toxic metal and its combination with ZnO
has succeeded in other applications.
This work presents the synthesis of ZnO and 1%Al
doped ZnO nanocatalysts and the investigation of
their photocatalytic degradation of Methyl Orange
under UV light with a comparative approach
106
MATERIALS AND METHODS
Chemicals
In this study, all the chemicals were analytic grade
and used without any further purification. Zinc
nitrate hexahydrate (Zn (NO3).6H2O) 99%, and
Aluminum nitrate nonahydrate (Al (NO3)3.9H2O)
99% were used as precursors, sodium hydroxide
(NaOH) 97% was used as a precepting agent and
MO (C14H14N3NaO3S) dye was used as a
pollutant, Distilled water was used for all
preparation solutions.
Catalyst synthesis
Through a simple precipitation method, ZnO and
1%Al-ZnO nanoparticles were synthesized. 0.3M of
zinc nitrate hexahydrate and 0.03M of aluminum
nitrate nonahydrate were dissolved in distilled
water to prepare 2 precursors solutions with a
different molar ratio of Al/ZnO (0% and 1%). Each
precursors solution was mixed and heated at 70°C,
and then 1M of NaOH solution was added drop
ways until pH≈10. The remaining white precipitate
was filtered, washed, and dried at 70°C/24h. The
dried samples were grinded, and calcinated at
600°C/4h.
Characterization
The photocatalysts were prepared and
characterized by X-ray Diffraction (XRD), Bruker
diffractometer (D2 Phaser, monochromatized
radiation Cu Kα λ=1.541) were used to collect the
patterns, and LYNXEYE detector at 2θ ranging from
10° to 80°. Fourier Transform Infrared
spectroscopy was used to determine surface
functional groups (FTIR-ATR Spectrum two
PerkinElmer), 400-4000 cm-1 were the recorded
range of the Spectra, and the morphological
features of the samples were examined by
Scanning Electron Microscopy (SEM).
Furthermore, DR 6000 UV-Vis spectrophotometer
(Hach, Germany) was used to examine the light
absorption and the photocatalytic activity.
Photocatalytic degradation experiment
The photocatalytic activity of the nanocatalysts
was investigated by testing the degradation of a
synthetic solution of MO under UV-light irradiation
and continuous stirring. This experiment was
executed in a photoreactor at 25°C with different
initial conditions (initial pH, dye concentration,
contact time, and catalyst dose).
UV-Vis spectrophotometer was used to measure
the absorbance of MO dye at its maximum
wavelength (465 nm). The degradation yield (%)
was calculated by Equation 1.
(𝐶0−𝐶𝑡)
% 𝐷𝑒𝑔𝑟𝑎𝑑𝑎𝑡𝑖𝑜𝑛 = 𝐶0
* 100 (1)
C0 and Ct are dye concentrations at t=0 and t time
during the degradation process.
RESULTS AND DISCUSSION
Characterization
X-ray diffraction analysis
The XRD patterns of ZnO and 1%Al-ZnO
nanocatalysts were shown in (Figure 1). Eleven
peaks of the XRD spectrum have been detected at
2θ = 31.96, 34.45, 36.45, 47.74, 56.75, 63.06, 66.49,
68.15, 69.26, 72.69, 77.17 and 2θ = 31.78, 34.42,
36.26, 47.56, 56.57, 62.86, 66.53, 67.97, 69.02,
72.55, 76.89 position for ZnO and 1%Al-ZnO
respectively. The diffraction peaks (100), (002),
(101), (102), (110), (103), (200), (112), (201),
(004) and (202) correlate to the hexagonal
crystalline planes of Zinc oxide. The good
crystalline quality of ZnO and 1%Al-ZnO
nanoparticles was confirmed by the well-defined
and sharp peaks. Furthermore, all the XRD spectra
of the samples revealed the characteristic peaks of
hexagonal wurtzite structure according to JCPDS
card No. (36-1451)(El Hassani et al., 2020).
The Scherer formula as appeared in Equation 2
was used to calculate the average crystalline size of
the synthesized samples.
𝑘λ
𝐷 =
β𝑐𝑜𝑠⁡(θ)
(2)
D: the average crystalline size, λ: the wavelength
(Å), β: the FWHM (rad), and θ: the diffraction angle
(°).
ZnO and 1%Al-ZnO nanoparticles average
crystalline size was found at 38.76 nm and 42.37
nm, respectively.
FTIR Analysis
In (Figure 2), the FTIR spectra of the ZnO and
1%Al-ZnO are displayed, respectively. The broad
low transmittance peaks at 3431 cm-1 and 3416
cm-1 results from -OH group stretching vibration
mode in the adsorbed water molecules. Zn-OH
bonding was confirmed by the peaks at 889 cm-1
and 872 cm-1 (Zhu et al., 2010). Finally, the peaks
at 461-419 cm-1 correspond to Zn-O symmetric
and asymmetric vibration (Ahammed et al., 2018).
107
 Figure 1: XRD spectrum of ZnO and 1%Al-ZnO.

Figure 2: FTIR Spectra pattern of ZnO and 1% Al-ZnO.

SEM analysis average size of 97.22 nm and 55.56 nm for ZnO

The morphological properties of the ZnO and
1%Al-ZnO nanoparticles were presented in (Figure
3). The SEM images exhibit compact structures
with highly uniformed spherical particles with an
and 1%Al-ZnO nanocatalysts, respectively.
By the application of XRD, FTIR, and SEM
techniques, the synthesized materials (ZnO and

108
1%Al-ZnO) approved its pure structure as well as
its nano-scale morphology.
Figure 3. SEM images of (a) ZnO and (b) 1%Al-ZnO.
Photocatalytic degradation
Initial pH effect
The pH is a key parameter in photocatalytic
investigation since it regulates reactions during the
degradation of organic compounds and the
formation of hydroxyl radicals (Isai & Shrivastava,
2019; Mohammadzadeh et al., 2015). The effect of
initial pH (2-13) was investigated on the
photocatalytic activity of ZnO and 1%Al-ZnO on
MO degradation as shown in (Figure 4), under the
conditions ([MO] = 20mg/l, catalyst dose = 1g/l for
30 min in the dark and for 180 min in irradiation).
The photocatalytic degradation reached its
maximum of 74.8% and 98.1% at pH 8 and 4 in the
presence of ZnO and 1%Al-ZnO nanomaterials,
respectively.
Figure 4. Initial pH effect on MO photocatalytic degradation in presence of ZnO and 1% Al-ZnO
Dye concentration effect
At the optimized initial pH 4 and 8 in the presence
of 1%Al-ZnO and ZnO nanomaterials, respectively.
The influence of initial MO concentration on
degradation rate was examined by adjusting the
initial concentration of the dye from 10mg/l to 50
mg/l, under the conditions (1g/l as a catalysts
dose, for 30 min in the dark and 180 min of
irradiation time (Figure 5).
The degradation rate attended its highest at 10
mg/l and 40mg/l in the presence of ZnO and
1%Al-ZnO. Afterward, the photocatalytic activity
decreases with the augmentation of MO
concentration.
109
 under conditions: [MO] = 20mg/l, catalyst dose = 1g/l for 30min in the dark and irradiation
time =180 min.

Figure 5. Effect of initial MO concentration on photocatalytic degradation under conditions (pH =

[8 and 4] for ZnO and 1%Al-ZnO, respectively, catalyst dose = 1g/l for 30 min the dark and
irradiation time = 180 min.

Contact time effect

The influence of contact time on photocatalytic
activity was investigated under the optimum
conditions of pH 8, 4 and MO concentration
10mg/l, in the presence of 1g/l of ZnO and
1%Al-ZnO nanoparticles as the dose of the catalyst.
As presented in (Figure 6), the degradation
efficiency increases proportionally with irradiation
time. The degradation rate reached its maximum
after 180 min for ZnO and 50 min for 1%Al-ZnO.
The impact of ZnO and 1%Al-ZnO nanomaterials
dose (Figure 7) was investigated under the
optimum conditions of (pH 8 and 4, [MO] =
10mg/l, and 180 min of irradiation time)
sequentially. The dose of the photocatalyst varied
from 0.2g/l to 2g/l. As presented in (Figure 7), the
degradation rate increases rapidly as the catalyst
dose increase and reached its maximum at 1g/l
and 1.5g/l for 1%Al-ZnO and ZnO, however, it
slightly decreases afterward.

Photocatalyst dosage effect

110
 Figure 6. Contact time effect on MO photocatalytic degradation under conditions: [MO]= 10mg/l
and catalyst dose = 1g/l.

Figure 7. Photocatalyst dosage effect on MO photocatalytic degradation under conditions: pH= [8

and 4] for ZnO and 1%Al-ZnO, respectively, [MO] = 10mg/l, in the dark for 30 min, and irradiation
time of 180 min.

examined ZnO and 1%Al-ZnO with a comparative

CONCLUSION approach. The results of the photocatalytic study
reviled the promising potential of 1%
aluminum-doped ZnO in enhancing the
Via a simple and low-cost precipitation method,
degradation of MO from 72% to 98% under the
ZnO and 1% Al-doped Zinc oxide photocatalysts
optimized conditions.
were successfully prepared. The XRD patterns
showed a hexagonal wurtzite structure for both
ZnO and 1%Al-ZnO nanomaterials with ~38.76 nm REFERENCES
and 42.37 nm as average crystalline sizes,
respectively. the samples were also investigated by
Ahammed, N., Samim Hassan, M. D., & Hassan, M.
SEM and FTIR. The photocatalytic experiment
(2018). Effects of aluminum (Al)
111
 incorporation on structural, optical and
thermal properties of ZnO nanoparticles.
Materials Science- Poland, 36(3), 419–426.
https://doi.org/10.1515/msp-2018-0018
Balaprakash, V., Gowrisankar, P., Sudha, S., &
Rajkumar, R. (2018). Aluminum doped ZnO
transparent conducting thin films prepared
by sol-gel dip coating technique for solar cells
and optoelectronic applications. Materials
Technology, 33(6), 414–420.
https://doi.org/10.1080/10667857.2018.1455384
Ebrahimpour, Z., Pliekhova, O., Cabrera, H.,
Abdelhamid, M., Korte, D., Segbéya
Gadedjisso-Tossou, K., Niemela, J., Lavrencic
Stangar, U., & Franko, M. (2021).
Photodegradation mechanisms of reactive
blue 19 dye under UV and simulated solar
light irradiation. Spectrochimica Acta - Part A:
Molecular and Biomolecular Spectroscopy,
252, 119481.
https://doi.org/10.1016/j.saa.2021.119481
El Hassani, K., Bayram, O., Anouar, A., & Mavi, A.
(2020). Thin films derived from Zn(Al)O
mixed metal oxides nanoparticles dispersed
in polyethylene glycol: Structural,
morphological and optical properties.
Ceramics International, 46(10), 14733–14738.
https://doi.org/10.1016/j.ceramint.2020.02.
277
Isai, K. A., & Shrivastava, V. S. (2019). Photocatalytic
degradation of methylene blue using ZnO and
2%Fe–ZnO semiconductor nanomaterials
synthesized by sol–gel method: a comparative
study. SN Applied Sciences, 1(10), 1–11.
https://doi.org/10.1007/s42452-019-1279-5
Lee, K. M., Lai, C. W., Ngai, K. S., & Juan, J. C. (2016).
Recent developments of zinc oxide based
photocatalyst in water treatment technology:
A review. Water Research, 88, 428–448.
https://doi.org/10.1016/j.watres.2015.09.04
5 Research Bulletin, 45(12), 2024–2030.
Mohammadzadeh, S., Olya, M. E., Arabi, A. M.,
Shariati, A., & Khosravi Nikou, M. R. (2015).
Synthesis, characterization and application of
ZnO-Ag as a nanophotocatalyst for organic
compounds degradation, mechanism and
economic study. Journal of Environmental
Sciences (China), 35, 194–207.
https://doi.org/10.1016/j.jes.2015.03.030
Ong, C. B., Ng, L. Y., & Mohammad, A. W. (2018). A
review of ZnO nanoparticles as solar
photocatalysts: Synthesis, mechanisms and
applications. Renewable and Sustainable
Energy Reviews, 81(July 2016), 536–551.
https://doi.org/10.1016/j.rser.2017.08.020
Sundar, K. P., & Kanmani, S. (2020). Progression of
Photocatalytic reactors and it’s comparison: A
Review. Chemical Engineering Research and
Design, 154, 135–150.
https://doi.org/10.1016/j.cherd.2019.11.035
Trandafilović, L. V., Jovanović, D. J., Zhang, X.,
Ptasińska, S., & Dramićanin, M. D. (2017).
Enhanced photocatalytic degradation of
methylene blue and methyl orange by ZnO:Eu
nanoparticles. Applied Catalysis B:
Environmental, 203, 740–752.
https://doi.org/10.1016/j.apcatb.2016.10.06
3
Ullah, A. K. M. A., Kibria, A. K. M. F., Akter, M., Khan,
M. N. I., Maksud, M. A., Jahan, R. A., & Firoz, S.
H. (2017). Synthesis of Mn3O4 nanoparticles
via a facile gel formation route and study of
their phase and structural transformation
with distinct surface morphology upon heat
treatment. Journal of Saudi Chemical Society,
21(7), 830–836.
https://doi.org/10.1016/j.jscs.2017.03.008
Zhu, Q., Chen, J., Zhu, Q., Cui, Y., Liu, L., Li, B., & Zhou,
X. (2010). Monodispersed hollow
microsphere of ZnO mesoporous nanopieces:
Preparation, growth mechanism and
photocatalytic performance. Materials
https://doi.org/10.1016/j.materresbull.2010.
06.016
112
CONFERENCE PAPER Youssef Nouhi et al., 2022, 113-120

Association of aldosterone synthase gene -344C/T polymorphism with type 2

diabetes in Moroccan population
Youssef Nouhi1*, Bouchra Benazzouz1, Otmane El Brini2, Younes Filali-Zegzouti3, Leila Bikjdaouene1,
Rajae Ramdan1, Omar Akhouayri1
1
Laboratory of Biology and Health, Ibn Tofail University, Kenitra. Morocco
2
Diagnostic Center-Moulay Youssef Hospital. Rabat. Morocco
3
Laboratory of Biology, Environment, and Health, Moulay Ismail University, Meknes. Morocco

Objective: The Aldosterone synthase (CYP11B2) is a key enzyme that plays

a role in the production of aldosterone. The existence of a common
polymorphism in the gene of this enzyme has been hypothesized to
contribute to the pathogenesis of type 2 diabetes. The current study
explored the relationship between -344C/T polymorphism in CYP11B2 gene
and type 2 diabetic Moroccan population. Material and Methods: A
case-control study was conducted with 113 healthy control subjects and 128
type 2 diabetic patients from the Moroccan population. Triglyceride, waist,
HDL, LDL, and total cholesterol were investigated. The Polymerase Chain
Reaction-Restriction Length Polymorphism (PCR/RFLP) technique was used
to genotype the CYP11B2 gene. Results: There is no difference between the
patients with type 2 diabetes and the control group in the distribution of the
genotype CYP11B2 -344C/T polymorphisms (χ2=4.9; P=0.086). The C allele
frequency was significantly higher in type 2 diabetic group than in the
control group (χ2=4.66; P=0.03). Conclusion: These data imply that there is
no meaningful relationship between the CYP11B2 polymorphism (-344C/T)
and type 2 diabetes in the Moroccan population. However, The C allele may
be a genetic factor linked to type 2 diabetes susceptibility.

* Eastern Mediterranean region is particularly

Corresponding Author
Name: Youssef Nouhi
Email: nouhi.ysf@gmail.com
Phone: +212679141811
ISBN: 9788367405256
DOI: 10.2478/9788367405256-016
Published by Sciendo. © 2022 Youssef Nouhi et al..
This is an open access article licensed under the Attribution
4.0 International
(https://creativecommons.org/licenses/by/4.0/)
INTRODUCTION
Diabetes is the most frequent metabolic disease.
According to the International Diabetes Federation
(IDF), 537 million people in the world are diabetic
in 2021 and this number is likely to increase by
46% in 2045 to 783 million (IDF, 2021). The
concerned. Morocco does not escape this global
trend. Based on World Health Organization (WHO),
the prevalence rate of diabetes in the adult
population is 12.4%. This pathology is the cause of
more than 12,000 deaths per year and is at the
origin of 32,000 additional deaths, attributable to
complications due to the high level of blood
glucose (WHO, 2016).
Type 2 Diabetes (T2D) is a multifactorial disease
with a strong genetic component (Alberti and
Zimmet, 1998). The search for polymorphisms or
mutations of candidate genes related to the
pathophysiology of the disease reduces the risk of
developing diabetes by promoting the practice of
physical activity and/or modification of eating
habits in people with predisposition genes.
One of the principal effectors of the renin
angiotensin system (RAS) is aldosterone. This
hormone affects blood pressure and controls
113
intravascular volume and sodium homeostasis
(Bureik et al., 2002; Epstein and White, 1994).
Aldosterone controls potassium flow, which is
linked to insulin's reaction to nutrition. It also
includes the insulin-mediated absorption of
glucose into muscle cells, which is a symptom of
insulin sensitivity resistance (Davies et al., 1999).
The benefic act of aldosterone on insulin
resistance, glucose tolerance, and Metabolic
Syndrome (T2D is one of its factors) has been
previously studied (Bellili et al., 2010; Kim et al.,
2014).
The degree of expression of the aldosterone
synthase, a mitochondrial cytochrome P450
enzyme, regulates aldosterone secretion
(CYP11B2) which is necessary for its biosynthesis
(Curnow et al., 1991). The CYP11B2 gene has nine
exons and eight introns and is located on
chromosome 8 (Brand et al., 1998; Hilgers and
Schmidt, 2005; Kupari et al., 1998). The CYP11B2
gene was identified as a possible candidate for the
development of complications of diabetes (Ma et
al., 2017), and it was found to have an impact on
glucose homeostasis and body mass in humans
(Russo et al., 2007). T2D elevated CYP11B2 mRNA
and protein in the kidney and renal cortical,
according to Xue and Siragy (Xue and Siragy,
2005).
The -344C/T polymorphism (rs1799998) involves
the substitution of cytosine for thymidine at the
steroidogenic transcription factor binding site, in
the promoter region of the CYP11B2 gene (Munshi
et al., 2010). It has been examined extensively, and
researchers have discovered a relationship
between blood pressure and aldosterone
production (Russo et al., 2002), plasma glucose
levels (Barbato et al., 2004; Kumar et al., 2003;
Ranade et al., 2001), and T2D (Purkait et al., 2012)
and its related complications (Yilmaz et al., 2015).
The aim of this study is to investigate, for the first
time in Morocco, the relationship between the
genetic polymorphisms -344C/T of CYP11B2 gene,
with the prevalence of T2D in Moroccan
population.
MATERIAL AND METHODS
Population Study
This case-controlled study was conducted on 113
volunteer Moroccan’s diabetics patients consulted
at Diagnostic Center from Rabat, with blood
glucose level more than 1.10g/l. Compared to 128
supposedly healthful control subjects having
glycemia inferior to 1.10g/l. The patients and
control group are both genders and their ages vary
between 34 and 64 years old.
Measurements of Clinical and Anthropometric
Traits
Glycemia (g/l), waist (cm), total cholesterol (TC in
g/l), high-density lipoproteins (HDL in g/l),
low-density lipoproteins (LDL in g/l), triglyceride
(TG in g/l), systolic blood pressure (SBP in mmHg)
and diastolic blood pressure (DBP in mmHg) were
investigated.
Polymorphism Analysis
DNA used as an amplification matrix for PCR
reactions was extracted by the high ionic protein
recharging method (Montgomery and Sise, 2012).
The blood is initially centrifuged and, after
recovery of the white cloud containing the
leukocytes, the cell pellet is treated with a white
blood cell lysis solution (SLB: EDTA-Na2 10mM;
SDS 0.2%; Tris-HCl 10mM, pH 7.5; NaCl 50mM)
and a K proteinase that digests cell proteins. The
latter will subsequently be relegated through an
ionic force of the NaCl (6 M). Finally, the
precipitation of the genomic DNA is conducted
using a 95% cold ethanol solution (-20°C).
Using forward (5' GTG TCA GGG CAG GGG GTA 3')
and reverse (5' AGG CGT GGG GTC TGG ACT 3')
primers, we identified the polymorphism -344C/T
of CYP11B2 gene using the Polymerase Chain
Reaction Restriction Fragment Length
Polymorphism (PCR-RFLP) method. A 25µL
reaction mixture including 80ng of genomic DNA,
5x reaction buffer, and My TaqTM Polymerase was
used to perform standard PCR amplification. (5mM
dNTP, 15mM MgCl2, stabilizers and enhancers)
and 5U/µL of My Taq polymerase. DNA occurs in
35 cycles with denaturation (94˚C for 30sec),
followed by annealing (60˚C for 30sec) and finally
extension (72˚C for 1min). The 228bp PCR
products were daggered with a restriction
endonuclease, HaeIII at 37˚C for 13h.
The digested samples were electrophoresed in a
2% agarose gel and examined under UV light. The
C allele creates a new restriction site. Cases with
heterozygous (CT) genotype generate four
fragments (175, 106, 69, and 53bp); those with
homozygous (CC) genotype generate three
fragments (106, 69, and 53bp); and homozygous
(TT) patients generate two fragments (175 and
53bp) (Figure 1).
Statistical Analysis
The data was reported as a mean or as a
percentage with standard deviation. The ANOVA
test and Student's T-Test were used to evaluate
biological characteristics. Between T2D and
controls, frequencies of genotype and allele of the
CYP11B2 -344C/T polymorphism are identified
and compared by the χ2 test, and Hardy Weinberg
114
equilibrium was assessed. The odds ratio was used studied and T2D prevalence.
to calculate the relationship between the SNPs

Figure 1: PCR-RFLP digestion for the CYP11B2 (-344C/T) polymorphism on an agarose gel
electrophoresis. Lanes 1, 8, 11, 12, and 14 represent the TT genotype; lanes 7 and 10 represent the
CC genotype; and lanes 2, 3, 4, 5, 6, 9, and 13 represent the CT genotype. M represents the ladder.
and C are 70.5% and 29.5% in patients with T2D,
RESULTS and 82.0% and 18.0% in healthy controls,
respectively. The genotype of the -344C/T
We compared baseline characteristics of subjects polymorphism was not significantly different (p for
between non-diabetic and diabetics patients. trend=0.086) between the diabetics and control
Diabetic patients had significantly higher values groups (Table 2). In detail, heterozygote variant TC
for, glycemia (P<0.001), waist (P<0.001), TC at CYP11B2 -344C/T polymorphism seemed not to
(P=0.002), TG (P<0.001), LDL (P=0.042), DBP be associated with T2D risk [OR=1.38 (95%
(P<0.001) and SBP (P<0.001) than control CI=0.79-2.39); p=0.25] (Table 2), but a higher
subjects, but no difference is observed for HDL prevalence of the homozygous mutant genotype
cholesterol (P=0.148) between the two groups (CC) was observed in patient group compared with
(Table 1). healthy group [OR=3.36 (95% CI=1.00-11.20);
The genotype and allele frequencies of the p=0.04]. Analysis of allelic frequencies showed that
CYP11B2 -344C/T polymorphism of the diabetic the C allele was significantly associated with the
and control groups were compared (Table 2). risk of T2D in Morocco [OR=1.61 (95%
Digestion product size in agarose gel CI=1.04-2.49); p=0.03].
electrophoresis of CYP11B2 gene (-344C/T) Table 3 shows the comparison of clinical
polymorphism is shown in figure 1. In both groups, characteristics of T2D patients depending on the
the distributions were like those predicted by genotype of CYP11B2 gene polymorphism. Control
Hardy–Weinberg equilibrium (diabetic group p= patients with CC genotype were older than control
0.26 and control group p= 0.93). The genotype patients with TT and TC genotypes (p=0.03). The
frequencies are as follows: 56.6% (TT), 8.8% (CC) second significative difference was observed in
and 34.5% (TC), in patients with T2D, and 67.2% waist between T2D patients with TT genotype and
(TT), 3.4% (CC) and 29.7% (TC) in healthy group other genotypes in the same group (p=0.04).
(Table 2). In addition, the frequencies of alleles T

Table 1: Basic characteristics of T2D and control groups.

Clinical characteristics T2D patients Control Patients P-value
Age 53.83±10.48 48.87±14.18 0.003
Blood glucose (g/l) 1.89±0.62 0.89±0.16 < 0.001
Waist (cm) 89.2±8.64 82.70±7.00 < 0.001
TC (g/l) 1.96±0.43 1.80±0.40 0.002
HDL (g/l) 0.52±0.22 0.56±0.19 0.148
LDL (g/l) 1.13±0.41 1.03±0.36 0.042
TG (g/l) 1.52±0.97 0.98±0.56 < 0.001
SBP (mmHg) 137.92±16.64 128.47±11.25 < 0.001
DBP (mmHg) 76.93±13.41 72.51±6.13 0.001

115
The data are presented in Forms means ± SD. T2D, type 2 diabetes; TC, total cholesterol; HDL,
high-density lipoprotein cholesterol; LDL, low-density lipoprotein cholesterol; TG, triglyceride;
SBP, systolic blood pressure; DBP, diastolic blood pressure.

116
 Table 2: The genotype and allele frequencies of the CYP11B2 -344C/T polymorphism in T2D
patients and controls.
Control
CYP11B2 -344C/T T2D (n=113) OR (95% CI) P-value
(n=128)
TT 64 (56.6%) 86 (67.2%) 1.00 (Reference)
TC 39 (34.5%) 38 (29.7%) 1.38 (0.79-2.39) 0.25
CC 10 (8.8%) 4 (3.1%) 3.36 (1.00 - 11.20) 0.04
P of trend 0.08

T 167 (71.0%) 210 (82.0%) 1.00 (Reference)

C 59 (29.0%) 46 (18.0%) 1.61 (1.04-2.49) 0.03

T2D. type 2 diabetes; OR. odd ratio; CI. confidence interval

DISCUSSION subjects (48.87±14.18) which strengthens the

conclusion of Purkait and al., that older age can be
To our knowledge, this is the first study analyzing a susceptibility factor to getting T2D (Purkait et al.,
the correlation between -344C/T polymorphism of 2013).
the aldosterone synthase gene and T2D in
As to other studies (Fabris et al., 2005; Pulakes
Moroccan population.
Purkait et al., 2013), we observed an increase in
The -344C/T polymorphism of the CYP11B2 gene the level of SBP in T2D subjects, suggesting that
did not differ significantly between the two groups, SBP influences the development of diabetes and its
according to our findings (p for trend=0.086), but complication.
the CC genotype is 2.5 times more present in T2D
CYP11B2 -344C/T SNP has been related to
patients than in control group. In addition, we
diabetes and glucose homeostasis in several
found that the frequency of the C allele was
studies. A study of combined American and
statistically different between the two groups, and
Taiwanese populations of Chinese and Japanese
patients carrying this allele were at increased risk
ancestry was performed (Ranade et al., 2001).
for T2D. The age of T2D patients (53.83±10.48)
showed a higher value than the age of control

Table 3: Clinical characteristics of T2D patients and control based on CYP11B2 gene polymorphism
comparison.
Control (N 128) T2D (N 113)
Clinical
TT (n=86) CC (n=4) TC (n=38) P-value TT (n=64) CC (n=10) TC (n=39) P-value
characteristics
Age 47.83±13.75 67.00±24.58 49.31±13.00 0.03 53.18±10.15 57.50±8.48 53.94±11.41 0.48
BG (g/l) 0.87±0.09 0.91±0.12 0.91±0.25 0.56 1.80±0.54 1.86±0.52 2.06±0.74 0.11
Waist (cm) 82.00±7.00 86.00±12.19 84.00±6.66 0.23 87.50±8.75 89.80±8.70 91.84±8.00 0.04
TC (g/l) 1.78±0.39 1.77±0.52 1.80±0.43 1.00 1.97±0.35 2.15±0.74 1.90±0.44 0.23
HDL (g/l) 0.55±0.17 0.62±0.22 0.56±0.21 0.80 0.52±0.18 0.52±0.17 0.53±0.28 0.97
LDL (g/l) 1.03±0.34 0.92±0.38 1.03±0.40 0.83 1.15±0.35 1.35±0.56 1.03±0.44 0.07
TG (g/l) 0.98±0.60 1.13±0.74 0.98±0.46 0.87 1.47±0.84 1.40±1.00 1.63±1.17 0.65
SBP (mmHg) 127.51±11.08 129.25±2.50 130.58±12.03 0.37 134.87±15.08 142.90±19.37 141.66±17.72 0.08
DBP (mmHg) 72.30±6.10 69.75±0.507 73.31±6.46 0.46 74.73±14.26 81.30±12.50 79.43±11.70 0.12
The data are presented in Forms means ± SD deviation. The differences between the three
genotypes were examined by the variance analysis to a factor (ANOVA) with the Tukey test for the
post hoc comparisons of each group.

According to Ranade et al., there is a link between
this SNP and blood glucose levels, as well as
glucose intolerance. They found that this
polymorphism is practically associated with
variations in fasting blood glucose or after a
provoked hyperglycemia test. Diabetes was more
common in the -344C homozygotes, as were
impaired fasting glucose levels (Ranade et al.,
2001). Another study found an association
between the -344C/T polymorphism and the
occurrence of T2D in European men who had the C
allele as the risk variant (Bellili et al., 2010). Li. et

117
al. have found that the -344C variant is associated with b-cell activity and reduced insulin
sensitivity, as well as increased fasting and
postprandial glycemia (Li et al., 2011). Another
study discovered that having the CC genotype was
related to a lower risk of T2D in healthy people
(Al-Safar et al., 2013); unlike in the French study,
the CC genotype was found to be highly associated
with a high incidence of T2D in men (Bellili et al.,
2010). Considering these studies and our data, it
seems there is a positive association between
CYP11B2 -344C/T polymorphism and T2D, and the
presence of the CYP11B2 -344C allele may
contribute to the threat of developing T2D.
In the contrast, a similar study done on the Indian
population showed no relationship between T2D
and -344C/T polymorphism variants (Purkait et
al., 2013). Another study by the same author
investigated the relationship between the
CYP11B2 -344C/T polymorphism and Sex in T2D
patients in the Indian community; no significant
link between the CYP11B2 -344C/T polymorphism
and sex in the studied cohort was shown (Pulakes
et al., 2015).

CONCLUSION

The significantly higher frequency of CYP11B2

-344 C allele in the T2D group compared to the
control group may offer an etiological link between
the presence of CYP11B2 -344C allele and the risk
of T2D. However, although 128 cases (a total of
241) can be considered a good sample size, it
remains relatively small. Therefore, further studies
in larger populations containing other inheritable
factors are needed to achieve a definitive
conclusion. This study adds to the data that people
with the -344C allele are more likely to develop
T2D in our population.

REFERENCES

Al-Safar, H., Pillai, S. S., Abood, L. J., Chandhoke, A.

S., Usman, S. K., Hasoun, A., and Rais, N. 2013.
Interaction between angiotensin converting
enzyme (ACE) insertion/deletion and
aldosterone synthase (CYP11B2) -344C/T
polymorphisms in relation to type 2 diabetes
mellitus risk in Emiratis. International Journal of
Genetics and Molecular Biology, 5(2), 20–27.
Alberti, K. G. M. M., and Zimmet, P. Z. 1998.
Definition, diagnosis and classification of
diabetes mellitus and its complications. Part 1:
diagnosis and classification of diabetes mellitus.
Provisional report of a WHO Consultation.
Diabetic Medicine, 15(7), 539–553.
Barbato, A., Russo, P., Siani, A., Folkerd, E. J., Miller,

118
 M. A., Venezia, A., … Cappuccio, F. P. 2004.
Aldosterone synthase gene (CYP11B2) C-344T
polymorphism, plasma aldosterone, renin
activity and blood pressure in a multi-ethnic
population. Journal of Hypertension, 22(10),
1895–1901.
Bellili, N. M., Foucan, L., Fumeron, F., Mohammedi,
K., Travert, F., Roussel, R., … Marre, M. 2010.
Associations of the 344 T>C and the 3097 G>A
polymorphisms of CYP11B2 gene with
hypertension, type 2 diabetes, and metabolic
syndrome in a French population. American
Journal of Hypertension, 23(6), 660–667.
Brand, E., Chatelain, N., Mulatero, P., Féry, I.,
Curnow, K., Jeunemaitre, X., … Soubrier, F. 1998.
Structural analysis and evaluation of the
aldosterone synthase gene in hypertension.
Hypertension, 32(2), 198–204.
Bureik, M., Lisurek, M., and Bernhardt, R. 2002. The
human steroid hydroxylase CYP11B1 and
CYP11B2. Biological Chemistry, 383(10),
1537–1551.
Curnow, K. M., Tusie-Lunaf, M. T., Pascoe, L.,
Natarajan, R., Gu, J. L., Nadler, J. L., and Whitef, P.
C. 1991. The product of the CYP11B2 gene is
required for aldosterone biosynthesis in the
human adrenal cortex. Molecular Endocrinology,
5(10), 1513–1522.
Davies, E., Holloway, C. D., Ingram, M. C., Inglis, G.
C., Friel, E. C., Morrison, C., … Connell, J. M. C.
1999. Aldosterone excretion rate and blood
pressure in essential hypertension are related to
polymorphic differences in the aldosterone
synthase gene CYP11B2. Hypertension, 33(2),
703–707.
Epstein, F. H., and White, P. C. 1994. Disorders of
Aldosterone Biosynthesis and Action. New
England Journal of Medicine, 331(4), 250–258.
Fabris, B., Bortoletto, M., Candido, R., Barbone, F.,
Cattin, M. R., Calci, M., … Carretta, R. 2005.
Genetic polymorphisms of the
renin-angiotensin-aldosterone system and renal
insufficiency in essential hypertension. Journal of
Hypertension, 23(2), 309–316.
Hilgers, K. F., and Schmidt, B. M. W. 2005. Gene
variants of aldosterone synthase and
hypertension. Journal of Hypertension, 23(11),
1957–1959.
IDF. 2021. Diabetes Atlas 10th edition (10th ed.).
Retrieved from
https://diabetesatlas.org/idfawp/resource-files/
2021/07/IDF_Atlas_10th_Edition_2021.pdf
Kim, Y. R., Kim, S. H., Kang, S. H., Kim, H. J., Kong, M.
H., and Hong, S. H. 2014. Association of CYP11B2
polymorphisms with metabolic syndrome
patients. Biomedical Reports, 2(5), 749–754.
Kumar, N. N., Benjafield, A. V., Lin, R. C. Y., Wang, W.
Y. S., Stowasser, M., and Morris, B. J. 2003.
Haplotype analysis of aldosterone synthase gene
(CYP11B2) polymorphisms shows association
with essential hypertension. Journal of
Hypertension, 21(7), 1331–1337.
Kupari, M., Hautanen, A., Lankinen, L., Koskinen, P.,
Virolainen, J., Nikkila, H., and White, P. C. 1998.
Associations between human aldosterone
synthase (CYP11B2) gene polymorphisms and
left ventricular size, mass, and function.
Circulation, 97(6), 569–575.
Li, X. M., Ling, Y., Lu, D. R., Lu, Z. Q., Yi, Q. L., Liu, Y.,
… Gao, X. 2011. Association of the aldosterone
synthase gene -344T>C polymorphism with
essential hypertension and glucose homeostasis:
A case-control study in a Han Chinese
population. Clinical and Experimental
Pharmacology and Physiology, 38(9), 598–604.
Ma, Y., Ionin, V., Ulitina, A., Pchelina, S., Bazhenova,
E., Zaslavskaya, E., … Baranova, E. 2017. C-344T
polymorphism gene aldosterone synthase
(CYP11B), serum aldosterone and association
with arterial fibrillation in metabolism syndrome
patients. Journal of Hypertension,
35(Supplement 2), e203.
Montgomery, G. W., and Sise, J. A. 2012. Extraction
of DNA from sheep white blood cells. New
Zealand Journal of Agricultural Research, 33(3),
437–441.
Munshi, A., Sharma, V., Kaul, S., Rajeshwar, K., Babu,
M. S., Shafi, G., … Jyothy, A. 2010. Association of
the −344C/T aldosterone synthase (CYP11B2)
gene variant with hypertension and stroke.
Journal of the Neurological Sciences, 296(1),
34–38.
Purkait, P., Sarkar, B., and Naidu, J. 2012. Null
association of aldosterone synthase (cyp11b2)
gene-344 c > t promoter polymorphism in type 2
diabetic patients among Indian population.
Diabetes Metabolism, 3(8).
Purkait, P., Raychodhury, P., Bandhyopadhya, S.,
Naidu, J., and Sarkar, B. 2013. Analysis of
Aldosterone Synthase Gene Promoter (- 344 C
>T) Polymorphism in Indian Diabetic
Nephropathy Patients. J Diabetes Metab Citation,
4, 271.
Purkait, P., Roy, A., Halder, K., Suthar, K.,
Raychaudhuri, P., Bhattacharya, S., … Naidu, J. M.
2015. Sex-based Association of CYP11B2 (-344
C/T) Polymorphism in Indian Type 2 Diabetic
Patients. In Int J Diabetol Vasc Dis Res (Vol. 3).
Ranade, K., Wu, K. D., Risch, N., Olivier, M., Pei, D.,
Hsiao, C. F., … Botstein, D. 2001. Genetic
variation in aldosterone synthase predicts
plasma glucose levels. Proceedings of the
National Academy of Sciences of the United
119
States of America, 98(23), 13219–13224. Russo, P., Lauria, F., Loguercio, M., Barba, G.,
Arnout, J., Cappuccio, F. P., … Siani, A. 2007. -344
C/T Variant in the Promoter of the Aldosterone
Synthase Gene (CYP11B2) Is Associated With
Metabolic Syndrome in Men. American Journal of
Hypertension, 20(2), 218–222.
Russo, P., Siani, A., Venezia, A., Iacone, R., Russo, O.,
Barba, G., … Strazzullo, P. 2002. Interaction
between the C(-344)T polymorphism of
CYP11B2 and age in the regulation of blood
pressure and plasma aldosterone levels:
Cross-sectional and longitudinal findings of the
Olivetti Prospective Heart Study. Journal of
Hypertension, 20(9), 1785–1792.
WHO. 2016. Journée mondiale de la Santé :
ensemble contre le diabète. Retrieved April 26,
2022, from
http://www.emro.who.int/fr/mor/morocco-new
s/journee-mondiale-de-la-sante-ensemble-contr
e-le-diabete.html
Xue, C., and Siragy, H. M. 2005. Local renal
aldosterone system and its regulation by salt,
diabetes, and angiotensin II type 1 receptor.
Hypertension, 46(3), 584–590.
Yilmaz, M., Sari, I., Bagcı, B., Gumus, E., and
Ozdemir, O. 2015. Aldosterone synthase
CYP11B2 gene promoter polymorphism in a
Turkish population with chronic kidney disease.
Iranien Journal of Kidney Diseases, 9(3),
209–214.

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 CONFERENCE PAPER Nour El Yakine Lakhdar et al.,2022, 121-133

Optimizing Fitness in Obesity: A Program Combining Various Exercise Techniques

Nour El Yakine Lakhdar*1, Driss Lamri2, Moulay Laarbi Ouahidi1

1 Biology and Health Laboratory, Faculty of Science, Kenitra, Morocco

2Regional Training and Education Centre, Rabat-Salé-Kenitra, Morocco

BACKGROUND.
Obesity, expressed as a high body mass index (BMI), is associated with a risk of
decrease in functional capacity and muscle strength, in particular in weight-
bearing joints, but so far, no study has been able to show a sufficiently strong
relationship between these factors. two options in conclusion.
AIM. The main objective of this study is to propose a mixed physical activity
exercise for individuals suffering from obesity.
MATERIALS AND METHODS. We collected data from 20 obese subjects (12 females
and 8 males) and 13 control subjects (9 females and 4 males). The "obese" group
included all subjects with a BMI ≥ 25.0 kg/m2, while the "control" group included
subjects with a BMI < 25.0 kg/m2.
Our physical training program consisted of walking, running, and muscle
stretching, with 2 sessions per week. In the initial phase (8 sessions), the subjects
were trained at the fat oxidation threshold, gradually increasing the duration of the
sessions until reaching 60 minutes of continuous exercise. From the ninth session
onwards, the program alternated between sessions at the fat threshold and more
intense activity sessions at the anaerobic threshold. The goal was to achieve a total
of 4 periods of 12 minutes of exercise at the anaerobic threshold.
Anthropometric parameters and various physiological characteristics were
measured before the first and after the last session. These measurements were
subsequently compared and analyzed.
RESULTS. Our physical activity program led to significant changes in body
composition for both obese women and men, but not for the control subjects.
Obese women experienced a weight loss of 1.7 ± 0.6 kg (p=0.006), while obese men
lost 5.8 ± 2.1 kg (p=0.02). The fat mass reduction was 2.6 ± 0.6 kg (p<0.001) for
women and 4.9 ± 1.1 kg (p=0.003) for men.
Furthermore, the obese subjects improved their exercise tolerance as a result of the
training they received. After the training period, they were able to reach
significantly higher fat and anaerobic thresholds during their efforts. The
performance increase was +14.3% (p=0.03) at the fat threshold and +12.1% at the
anaerobic threshold (p=0.02). This performance improvement occurred without a
significant change in heart rate. Specifically, heart rate only increased by 3.9%
(p=0.11) at the fat threshold and 4.3% (p=0.06) at the anaerobic threshold.
DISCUSSION. Two significant findings emerge from this study. Firstly, our exercise
program tailored to the needs of obese patients, without controlled diet,
significantly improves the body composition of our obese subjects. It has led to
significant weight and fat mass losses, surpassing those observed in previous
studies. Secondly, our program enables obese individuals to achieve greater
performance at the fat and anaerobic thresholds, with nearly comparable heart
rates. Consequently, their capacity for fat oxidation becomes enhanced.
.

121

* alarmingly increasing. Childhood obesity is a
Corresponding Author
Name: NOUR EL YAKINE LAKHDAR
Email : Nourelyakine.lakhdar@uit.ac.ma
Phone : 00212660317414
ISBN: 9788367405256
DOI: 10.2478/9788367405256-017
Published by Sciendo. © 2022 Nourelyakine.lakhdar et
al. This is an open access article licensed under the
Attribution 4.0 International
(https://creativecommons.org/licenses/by/4.0/)
Introduction
The phenomenon of obesity is reaching alarming
proportions in Morocco. 25.2% of the Moroccan
population is overweight, and 10.3% suffer from
obesity(Schuch, F. B., Vasconcelos-Moreno, M. P.,
Borowsky, C., Zimmermann, A. B., Rocha, N. S., &
Fleck, M. P, 2015). Nearly one-third of the
population in Morocco, approximately 10.3
million people, experience overweight issues,
which have become a public health concern that
has been increasing over the past ten years, as
highlighted in a study by the High Commission
for Planning (HCP) of Morocco in 2011.
According to this survey conducted by the HCP
among 2426 households, Morocco has made
significant progress in combating undernutrition.
In children under the age of five, underweight
rates decreased from 9.3% in 2004 to 3.1% in
2011, placing the Kingdom in a very favorable
position compared to the global average (16%).
For adults over 20 years old, the prevalence of
underweight is now only 3.3%, compared to
3.9% in 2001. However, Morocco is facing serious
obesity problems. In total, 10.3 million adult
Moroccans, 63.1% of whom are women, are
affected by obesity or pre-obesity, according to
the report. Among these individuals, 3.6 million,
which is nearly one in five Moroccan adults, are
affected by severe and morbid obesity. Women
are the most affected (26.8%), particularly in
urban areas. Severe and morbid obesity has
increased at an average rate of 7.3% per year
over the past 10 years, alarming the HCP, which
notes that inactivity, as well as living standards
and education levels, influence this phenomenon.
Indeed, the frequency of overweight and obesity
is increasing rapidly, especially among children,
making it a major global health problem(Carneiro
LSF, Fonseca AM, Vieira-Coelho MA, Mota MP,
Vasconcelos-Raposo J, 2015). Morocco does not
appear to be spared from the phenomenon of
childhood obesity, the prevalence of which is
major risk factor for cardiovascular diseases and
can lead to joint, respiratory, metabolic,
endocrine, and even orthopedic problems.
Beyond the somatic consequences, childhood
obesity can cause numerous psychosocial
disorders. Furthermore, obesity in children
presents a significant risk of persisting into
adulthood. These multiple complications of
obesity emphasize the importance of effective
preventive approaches that should be
implemented from childhood(Rahim and Baali,
2011). Indeed, there is an association between
excess weight in early childhood and numerous
complications, and this excess weight is also
linked to an increased risk of morbidity and
premature death in adulthood (Rahim and Baali,
2011). These consequences on physical health
add to the psychological and social repercussions
related to the altered body image caused by
obesity in children. Considering its rapid
progression worldwide, childhood obesity is
becoming an increasingly worrying health
phenomenon. It represents one of the
consequences of the nutrition transition and
sedentary lifestyles that have emerged in many
countries, particularly in urban areas (Gupta N,
Goel K, Shah P, Misra A., no date). In 2002, the
World Health Organization (WHO) considered
that 60 to 85% of the global population had a
sedentary lifestyle, including two-thirds of
children, and attributed two million deaths per
year to it. It is, therefore, one of the most serious
public health problems of our time, and although
it is not yet receiving sufficient attention, some
refer to it as an epidemic of physical inactivity
(World Health Organization, 2002).
Regular and moderate physical activity has
beneficial effects on longevity by reducing overall
mortality, particularly cardiovascular mortality.
The initial epidemiological data presented in the
1980s on Harvard alumni (Paffenbarger RS, Hyde
RT, Wing AL, Hsieh CC., no date) have been
extensively confirmed by several studies
conducted in large and diverse populations of
men and women of varying ages (Andersen,
1995). It is well accepted that physical activity
plays a protective role against obesity. However,
the combination of a suitable hypocaloric diet is
necessary (Shephard RJ, Balady GJ., no date).
Additionally, physical activity has a beneficial
effect on fat distribution, particularly visceral fat,
and increases muscle mass.
Study Design and Method
Type and Study Method:
Study site. This is a cross-sectional descriptive
122
 research study that was conducted on a
representative sample of obese individuals in the
Fes-Meknes region. This means that the study
aimed to describe the characteristics of the
sample of obese individuals in the region at a
specific point in time.
Time of the study. The study took place from
September 06 to November 11, 2022.
Study populations:
Inclusion criteria: Our study focused on obese
individuals aged 18 to 40 years.
Exclusion criteria: In our study, participants
with uncontrolled cardiovascular diseases,
endocrine dysfunction, musculoskeletal
impairment preventing them from continuing
their training, as well as those with diabetes
and/or osteoarticular disorders were excluded.
This allowed us to focus on the effect of intense
exercise on functional capacity and muscle power
among obese individuals without these
conditions
Sampling method from the study population
All participants were randomly selected from
different sports clubs and sportive institutions.
Study design Prospective one-sample
comparative.
Description of medical intervention
We collected data from 20 obese subjects (12
women and 8 men) and 12 control subjects (7
women and 5 men). We included in the "obese"
group all subjects with a BMI ≥ 25.0 kg/m2, and
in the "control" group all subjects with a BMI <
25.0 kg/m2. The subjects participated in the
physical activity program either on medical
recommendation or on their own initiative.
Excluded from the study were subjects under the
age of 17 or over 65, subjects who had undergone
or were undergoing weight-loss surgery (such as
gastric bypass or gastric banding), and subjects
who did not participate in the exercise program
for at least 6 weeks. The subjects did not receive
any specific dietary instruction at any time.
Methods
Anthropometric measurements
● Body mass (BM)
The body mass was measured using an electronic
scale (Balance NRBF701-17) (Accuracy: 0.1 kg).
The participants were lightly clothed.
The triangular exercise tests. was used to
determine the fat and anaerobic thresholds for
each subject. We defined the fat threshold as the
point where the Respiratory Quotient (RQ)
ranged from 0.88 to 0.90, and the anaerobic
threshold as the point where the RQ ranged from
0.96 to 1.00.
During the triangular exercise test, the subjects
were accompanied by an experienced sports
monitor to collect data while running on a
variable-speed and incline treadmill (Technogym,
Italy). Heart rate was recorded using a Polar
Tempo system. Measurement of the Respiratory
Quotient (RQ) was conducted using an Altitrainer
200 facial mask, which measured oxygen
consumption, carbon dioxide production, and
ventilation.
After the triangular exercise test, the subjects
began a training program twice a week. The first
eight sessions aimed to develop endurance at the
fat threshold. During the initial sessions, subjects
were encouraged to brisk walk or lightly jog
(according to their abilities) for 10 to 20 minutes,
monitoring their heart rate to not exceed the
level corresponding to an RQ of 0.90 during the
initial triangular exercise test. This exercise
period was followed by 10 minutes of muscle
stretching, then 1 to 2 additional exercise periods
of 10 to 20 minutes, identical to the first one.
Each session concluded with 10 minutes of
muscle stretching. As the sessions progressed,
subjects were encouraged to extend the duration
of their first exercise period, gradually reaching
60 minutes of continuous exercise.
From the fifth week, starting from the ninth
training session, one of the two weekly sessions
focused on resistance training at the anaerobic
threshold. The goal was to achieve a higher heart
rate that corresponded to an RQ ranging from
0.96 to 1.00.
The resistance training sessions were conducted
as follows: the subject began with a period of
brisk walking or light jogging for 10 to 20
minutes, similar to the first 8 sessions. Then, the
subject performed 4 sets of 5-minute runs each,
reaching a heart rate compatible with the
anaerobic threshold. The 4 sets were separated
by 2-minute rest intervals. Afterwards, the
subject engaged in a 5 to 10-minute walking
period to lower the heart rate to a level
compatible with an RQ below 0.90. Finally, the
subject performed 10 minutes of muscle
stretching. The objective after 16 or more
sessions of this type was to complete 4 periods of
12 minutes each at the anaerobic threshold.
Statistical analysis: The analysis of the results
was conducted using three statistical software
programs, SPSS 18.0 (IBM, Inc., Chicago, IL),
Graph Pad Prism (version 6.0, Inc., CA), and WHO
Anthro Plus.
Ethical considerations: The participants in the
survey were informed about the reasons for the
study. Those who were literate were all asked to
fill out a personal identification form. For those
who had no formal education, oral consent was
obtained. They then agreed to have their
parameters measured. Their informed and
voluntary consent was obtained prior to the start
of the research. They were free to withdraw from
123
 the survey at any time without any negative
consequences. The data was collected
anonymously.
Results
Table 1 presents the basic characteristics and
average training duration of the four groups.
On average, participants of all genders have an
age of 42.2 ± 1.9 years. There is no significant
difference in terms of age between the groups of
women and the groups of men.

Table 01. Basic characteristics and average training duration of the 04 groups.
OBESE CONTROLS P (Obese Vs controls)
W M W M W M
Number 12 08 07 05
Age 41,6 ±1,6 40,6 ±2,8 41,8 ±3,5 42,1 ±2,1 0,97 0,82
Weight 88.1 ± 2.2 98 ± 6.6 64.2 ± 1.6 66.3 ± 3.1 < 0.03
0.0001
BMI 31.0 ± 0.8 32.3 ± 2.7 23.3 ± 0.6 24.1 ± 0.6 < 0.02
0.0001
Body fat (%) 29.5 ± 1.8 32.0 ± 1.9 24.4 ± 1.8 < 0.09
42.3 ± 0.9 0.0001

Waist 117.8 ± 4.8 85.7 ± 2.1 82.1 ± 3.5 0.006 0.006

circumference 99.2 ± 2.0 (n=6) (n=6) (n=4)

Hip 114.5 ± 1.9 114.1 ± 3.9 96.3 ± 1.7 91.8 ± 2.1 < 0.008
circumference (n=6) (n=6) (n=4) 0.0001
Waist-to-Hip 0.84 ± 0.01 1.01 ± 0.02 0.84 ± 0.02 0.88 ± 0.998 0.03
ratio (n=6) (n=6) 0.02
(n=4)
Duration 23.9 ± 2.8 30.1 ± 5.3 22.7 ± 5.4 43.7 ± 6.3 0.69 0.19
(weeks)

The results show a significant difference between

the two groups in terms of weight. Obese women
have an average weight of 88.1 ± 2.2 kg, while
control women have an average weight of 64.2 ±
1.6 kg (p<0.0001). For men, obese individuals
have an average weight of 98 ± 6.6 kg, while
controls have an average weight of 66.3 ± 3.1 kg
(p=0.03).
There is a highly significant difference between
obese women and control women in terms of
body fat percentage (42.3 ± 0.9% and 32.0 ±
1.9%, respectively, p<0.0001). However, there is
no significant difference between obese men and
control men in terms of body fat percentage (29.5
± 1.8% and 24.4 ± 1.8%, respectively, p=0.08).
Finally, according to the table, it can be observed
that the average durations of training periods do
not show statistically significant differences
between the different groups. Thus, the average
training duration for obese women is 23.9 ± 2.8
weeks, while it is 22.7 ± 5.4 weeks for control
women (p=0.69). For the two respective groups
of men, the average training duration is 30.1 ± 5.3
and 43.7 ± 6.3 weeks, but there is no statistically
significant difference between them (p=0.19).

124
 Table 02. Comparison of performance and physiological characteristics of obese subjects and control
subjects before the training period.
OBESE CONTROLS P (Obese Vs controls)
W M W M W M
Threshold fat speed 5.2 ± 4.5 ± 0.4 4.8 ± 0.2 6.3 ± 0.8 0.35 0.10
(km/h) 0.5 (n=6) (n=6) (n=4)
(n=10)
Anaerobic threshold 5.5 ± 6.5 ± 0.5 6.9 ± 0.2 8.5 ± 0.4 0.01 0.02
speed (km/h) 0.4 (n=7) (n=5) (n=5)
(n=09)
Heart rate at the fat 118.0 ± 114 ± 119.6 ± 4.9 121 ± 17.2 0.97 0.33
threshold (bpm) 3.5 3.9 (n=6) (n=5)
(n=12) (n=7)
Heart rate at the 139 ± 130.1 ± 145 ± 5.3 156.3 ± 10.5 0.22 0.04
anaerobic threshold 4.1 4.5 (n=6) (n=4)
(bpm) (n=12) (n=7)
Oxygen consumption 16.2 ± 18.4 ± 17.8 ± 0.6 25.6 ± 6.1 0.19 0.19
at the fat threshold 0.5 2.1 (n=6) (n=4)
(ml/kg/min) (n=12) (n=7)
Oxygen consumption 19.8 ± 23.5 ± 24.3 ± 1.3 32.1 ± 4.3 0.004 0.10
at the anaerobic 1.1 2.3 (n=6) (n=4)
threshold (n=12) (n=7)
(ml/kg/min)
Ventilation at the fat 34.8 ± 44.7 ± 24.7 ± 1.4 41.3 ± 13.3 0.001 0.72
threshold (l/min) 1.5 2.9 (n=6) (n=4)
(n=12) (n=7)
Ventilation at the 48.6 ± 61.4 ± 42.3 ± 2.9 62.3 ± 13.1 0.23 0.76
anaerobic threshold 3.1 4.8 (n=6) (n=4)
(l/min) (n=12) (n=7)

The speeds reached at the fat threshold do not
show significant differences between the groups
of women and the groups of men. For obese
women, the average speed at the fat threshold is
5.2 ± 0.5 kilometers per hour (km/h), while
control women have a speed of 4.8 ± 0.2 km/h (p
= 0.35). Men, on the other hand, reach speeds of
4.5 ± 0.4 km/h and 6.3 ± 0.8 km/h (p = 0.10),
respectively.
The speeds reached at the anaerobic threshold
differ significantly between the groups, unlike the
speeds at the fat threshold. Obese women reach a
speed of only 5.5 ± 0.4 km/h, while control
women reach 6.9 ± 0.2 km/h (p=0.01). In men,
the speeds are 6.5 ± 0.5 km/h for the obese and
8.5 ± 0.4 km/h for the controls (p=0.02).
The results show that the heart rates of obese
subjects and control subjects do not differ
significantly, either at the fat threshold or at the
anaerobic threshold. However, it is important to
note that a slight difference approaching the
threshold of significance was observed in men at
the anaerobic threshold. For women, the average
heart rate at the fat threshold is 118.0 ± 3.5 beats
per minute (bpm) for the obese and 119.6 ± 4.9
bpm for the controls (p=0.97). At the anaerobic
threshold, the mean values are 139 ± 4.1 bpm
and 145 ± 5.3 bpm, respectively (p=0.22). For
men, the mean values at the fat threshold are 114
± 3.9 bpm for the obese and 121 ± 17.2 bpm for
the controls (p=0.33). At the anaerobic threshold,
the mean values are 130.1 ± 4.5 bpm and 156.3 ±
10.5 bpm, respectively (p=0.04).
Finally, a comparison of the ventilatory efforts
provided by the different groups was also
conducted. These data are expressed in liters per
minute (l/min). At the fat threshold, obese
women have a ventilation of 48.6 ± 3.1 l/min,
while control women have a ventilation of 24.7 ±
1.4 l/min (p=0.001). Although this difference is
highly significant, it is not significant in men, who
have values of 44.7 ± 2.9 l/min for the obese and
41.3 ± 13.3 l/min for the controls (p=0.72). At the
anaerobic threshold, no significant difference was
observed in both sexes. Obese and control
women have values of 48.6 ± 3.1 l/min and 42.3 ±
2.9 l/min, respectively (p=0.23). In obese and
control men, ventilation was 61.4 ± 4.8 l/min and
62.3 ± 13.1 l/min, respectively (p=0.76).

125
 Impact of the physical exercise program on body composition
Effect of exercise on weight
Table 03. Effect of exercise on ventilation at the fat threshold

Weight (kg) Weight (kg) weight Difference

before after exercise difference between P
exercise Average (kg) per before and
week after

87.5 ± 2.7 85.8 ± 2.7 -0.09 ± 0.16 - 1.9% 0.006

Obese women (12)
Obese men (08) 104.0 ± 7.4 98.2 ± 6.4 -0.26 ± 0.26 - 5.6%

61.6 ± 1.3
Obese women (07) 60.7 ± 1.5 -0.03 ± 0.12 - 1.5% 0.35
Obese men (05) 68.1 ± 3.9
67.4 ± 4.7 -0.02 ± 0.07 - 1.0%

The weight data was available for all subjects in kg/week (p=0.02).
the study. A significant decrease in weight was However, neither the control women nor the
observed in both obese women and obese men. control men showed a significant variation in
Obese women went from 87.5 ± 2.7 kg before their weight. Control women went from 61.6 ±
exercise to 85.8 ± 2.7 kg after exercise, 1.3 kg to 60.7 ± 1.5 kg (-1.5%; -0.03 kg/week;
corresponding to a decrease of -1.9% or a weight p=0.35), while control men went from 68.1 ± 3.9
loss of -0.09 ± 0.16 kg/week (p=0.006). Obese kg to 67.4 ± 4.7 kg (-1.0%; -0.02 kg/week;
men, on the other hand, went from 104.0 ± 7.4 kg p=0.73).
before to 98.2 ± 6.4 kg after exercise,
representing a decrease of -5.6% or -0.26 ± 0.26
Effect of exercise on body fat quantity
Table 05. Effect of exercise on body fat
Body fat before Body fat after difference
exercise exercise (%) P
In % In kg In % In kg between
before
and after
42,3 ± 36.6 37.2 ± 34.6 - 6.6 % < 0.001
0,9 ± 1.8 0.8 ± 1.6
Obese
women (12) 29.5 ± 31,2 - 11.7 % 0,004
1.8 ± 3,8 23.7 ± 24.7
Obese men 1.5 ± 2.9
(08)
32.0 ± 19.4 28.2 ± 18.6 - 7.5 % 0.09
1.9 ± 1.4 2.7 ± 1.9
Obese - 15.1% 0,30
women (07) 24.4 ± 16.3 21.3 ± 14.3
1.8 ± 0.6 1.8 ± 1.7
Obese men
(05)

decrease in body fat, while no significant

Body fat was measured in a group of 12 obese decrease was observed in the control subjects.
women, 8 obese men, 7 control women, and 5 Obese women reduced their body fat percentage
control men. from 42.3 ± 0.9% to 37.2 ± 0.8% after exercise,
Among all obese subjects, there was a significant which corresponds to a decrease of 6.6% or a fat

126
loss of -2.0 ± 0.4 kg (p<0.001). Obese men control women decreased their body fat
decreased their body fat percentage from 29.5 ± percentage from 32.0 ± 1.9% to 28.2 ± 2.7%, and
1.7% to 24.7 ± 2.9%, resulting in a decrease of control men reduced their body fat percentage
11.7% or a fat loss of -6.5 ± 2.4 kg (p=0.004). from 24.4 ± 1.8% to 21.3 ± 1.8%.
The results for the control subjects are as follows:
Effect of exercise on waist circumference
Table 05. Effect of exercise on waist circumference (W)
W (cm) W (cm) after Difference P
before exercise between
exercise before and
after
Obese women 99.2 ± 2.0 95.1 ± 2.0 - 1.9 % 0.001

Obese men 117.8 ± 112.6 ± 5.7 - 4.8 % 0.02

4.8
Obese women ( 85.7 ± 2.1 83.1 ± 2.6 - 3.6 % 0.03

Obese men 82.1 ± 3.5 81.4 ± 1.6 - 0.4 % 0,76

The waist circumference was measured in 12 decreases to 95.1 ± 2.0 cm. For obese men, it
obese women, 08 obese men, 07 control women, changes from 117.8 ± 4.8 cm to 112.6 ± 5.7 cm.
and 05 control men. Significant changes are In control women, a decrease of -3.6% is
observed in this parameter for both obese observed, with a circumference of 85.7 ± 2.1 cm
women and men, as well as control women. Only before exercise and 83.1 ± 2.6 cm after exercise
control men show no significant variation. (p=0.03). Finally, in control men, the
Before exercise, the waist circumference of obese circumference changes from 82.1 ± 3.3 cm before
women is 99.2 ± 2.0 cm, while after exercise, it to 81.4 ± 1.6 cm after exercise.
Effect of the physical exercise program on performance and physiological characteristics at the
fat and anaerobic thresholds
Table 06. Effect of exercise on performance at the fat threshold
Speed Speed Difference p
achieved achieved between
before after before and
exercise exercise after
(km/h) (km/h) exercise
5.2 ± 0.5
Obese Women (n=12) 5.9 ± 0.5 + 11.1% 0.14

Obese Men (n=8) 4.5 ± 0.4 0,13

5.4 ± 0.4 + 18.4%
Women en Men Obese
(n=20) 4.8 ± 0.2 0,03
5.3± 0.2 + 13.8 %
1.8 ± 0.2
Obese Women (n=07) 4.8 ± 0.2 ± 0.0 1.0

Obese Men (n=05) 6.3 ± 0.8 0.40

6.6 ± 0.8 + 10.2%
Women en Men Obese 4.9 ± 0.4
(n=12) 5.2 ± 0.6 +4.8 % 0.49

of 13.8%. When we divide the data by gender

The data were available for 20 obese subjects (12
women; 8 men) and 12 control subjects (7
women; 5 men). It is observed that obese
subjects reach a fat threshold at a speed of 4.8 ±
0.2 km/h before exercise and 5.3 ± 0.2 km/h after
exercise, which represents a significant increase
(not shown), the changes are no longer
significant. Obese women achieve an average
speed of 5.2 ± 0.5 km/h before exercise and 5.9 ±
0.5 km/h after exercise, while obese men go from
4.5 ± 0.4 km/h to 5.4 ± 0.4 km/h. As for the
control subjects, their performance at the fat

127
threshold does not show significant variation. speed of 4.8 ± 0.2 km/h before and after exercise
The speed reached before exercise is 4.9 ± 0.4 for women; men, on the other hand, go from 6.3 ±
km/h, and after exercise, it is 5.2 ± 0.6 km/h. 0.8 km/h to 6.6 ± 0.8 km/h.
When we separate women and men, we obtain a

128
Table 07. Effect of exercise on performance at the anaerobic threshold
Speed Speed Difference P
reached reached between
before after before and
exercise exercise after
(km/h) (km/h) exercise
Obese Women (n=12) 5.5 ± 0.4
6.2 ± 0.3 + 12.9% 0.06
Obese Men (n=8) 6.5 ± 0.5
7.4 ± 0.5 + 8.3 % 0.21
Women en Men Obese
(n=20) 6.1 ± 0.3
7.2 ± 0.3 + 11.8 % 0.02
Obese Women (n=07) 6.9 ± 0.2 7.2 ± 0.2 +2.1% 0.69

Obese Men (n=05) 8.5 ± 0.4 9.2 ± 1.0 + 9.0 % 0.06

Women en Men Obese 7.6 ± 0.4 8.2 ± 0.5 + 5.2 % 0.21

(n=12)

km/h before exercise and 7.4 ± 0.5 km/h after

The data were available for 20 obese subjects (12
women and 8 men) and 12 control subjects (7
men and 5 women). Before exercise, obese
subjects reached the anaerobic threshold at a
speed of 6.1 ± 0.3 km/h. After exercise, this speed
significantly increased to 7.2 ± 0.3 km/h. When
we divide the data by gender (not shown), there
is a trend towards significance in women: a speed
of 5.5 ± 0.4 km/h before exercise and 6.2 ± 0.3
km/h after exercise. However, for men, the
variation is not significant: a speed of 6.5 ± 0.5
exercise. Let's now turn to the control subjects.
When considering all 12 subjects as a whole,
their speed at the anaerobic threshold changes
from 7.6 ± 0.4 km/h to 8.2 ± 0.5 km/h, which is
not a significant modification. For women
observed individually, the speed changes from
6.9 ± 0.2 km/h to 7.2 ± 0.2 km/h, and for men, it
changes from 8.5 ± 0.4 km/h to 9.2 ± 1.0 km/h.
Thus, there is a variation that tends towards
significance in the latter. A significant decrease in
heart rate is observed in obese men.

129
 Effect of physical exercise on physiological characteristics at a speed of 6 km/h
Table 08. Effect of physical exercise on heart rate at the 6 km/h threshold
HR before HR after Difference
exercise exercise between P
(heartbeats (heartbeats before and
per per after
minute) minute)
Obese Women (n=12) 139.1 ± 3.9
138.8 ± 3.8 - 0.3 % 0.91
Obese Men (n=8) 128.2 ± 7.4
120.8 ± 7.2 - 5.6 % 0.05
Women en Men Obese 133.8 ± 3.6
(n=20) 132.1 ± 3.8 - 1.5 %
0.43
Obese Women (n=07) 128.2 ± 5.5
124.4 ± 3.5 - 2.6 % 0.54
Obese Men (n=05) 109.3 ± 5.7
111.1 ± 13.1 + 0.6 % 0.96
Women en Men Obese 120.8 ± 4.4
(n=12) 123.1 ± 5.2
- 1.8 % 0.56

men. Regarding the control subjects, when

The data were collected from 20 obese subjects considering both sexes, their heart rate
(12 females and 8 males) and 12 control subjects decreased from 123.1 ± 5.2 bpm to 120.8 ± 4.4
(7 females and 5 males). Prior to exercise, obese bpm, also indicating a non-significant variation.
subjects had a heart rate of 133.8 ± 3.6 bpm, When examining the sexes separately, a decrease
which decreased to 132.1 ± 3.8 bpm after in heart rate was observed in female controls
exercise, representing a non-significant decrease from 128.2 ± 5.5 bpm to 124.4 ± 3.5 bpm, while
of -1.5% (p=0.43). When examining the sexes male controls experienced a slight increase from
separately, it was observed that obese women 109.3 ± 5.7 bpm to 111.1 ± 13.1 bpm. Therefore,
experienced a slight decrease from 139.1 ± 3.9 a significant decrease in heart rate was only
bpm to 138.8 ± 3.8 bpm, while obese men observed in obese men.
experienced a significant decrease from 128.2 ±
7.4 bpm to 120.8 ± 7.2 bpm. Thus, a significant
decrease in heart rate was only observed in obese
Table 09. Effect of exercise on oxygen consumption at a 6 km/h threshold.
VO2 before VO2 after Difference
exercise (in exercise (in between P
ml/kg/min) ml/kg/min) before and
after
Obese Women (n=12) 19.8 ± 0.6 20.8 ± 0.8 +6.1% 0.20

Obese Men (n=8) 20.8 ± 1.8 22.4 ± 1.2 +5.2% 0.52

Women en Men Obese 20.1 ± 0.5 21.7 ± 0.6 +5.3 % 0.05

(n=20)
Obese Women (n=07) 19.8 ± 0.4 21.8 ± 0.5 +9.2% 0.33

Obese Men (n=05) 19.8 ± 1.9 17.9 ± 2.5 -11.1% 0.49

Women en Men Obese 19.8 ± 0.4 21.6 ± 0.9 +4.1% 0.44

(n=12)

significant variation. When examining the sexes

Obese subjects had an oxygen consumption of separately, obese women had an oxygen
20.1 ± 0.5 ml/kg/min before exercise, which consumption of 19.8 ± 0.6 ml/kg/min before
increased to 21.7 ± 0.6 ml/kg/min after exercise. exercise, which increased to 20.8 ± 0.8
This indicates a clear tendency towards a ml/kg/min after exercise, while obese men had

130
an oxygen consumption of 20.8 ± 1.8 ml/kg/min
before exercise, which increased to 22.4 ± 1.2
ml/kg/min after exercise. Regarding the control
subjects, regardless of sex, their oxygen
consumption increased from 19.8 ± 0.4
ml/kg/min to 21.6 ± 0.9 ml/kg/min. When
separating women from men, the following
results were obtained: control women had an
oxygen consumption of 19.8 ± 0.4 ml/kg/min
before exercise, which increased to 21.8 ± 0.5
ml/kg/min after exercise, while men had an
oxygen consumption of 19.8 ± 1.9 ml/kg/min
before exercise, which decreased to 17.9 ± 2.5
ml/kg/min after exercise.
Thus, we observe an increase close to the
threshold of significance only in obese subjects
regarding oxygen consumption.
DISCUSSION
This study reveals that our participants
significantly improved after the program in terms
of heart rate, aerobic fitness, functional capacity,
exercise tolerance, total body fat, strength, and
muscle mass.
When comparing the pre-exercise fitness
characteristics, such as speed, heart rate, and
oxygen consumption at the fat and anaerobic
thresholds, between our obese subjects and
control subjects, we observed non-significant
differences at the fat threshold, except for
ventilation in women. However, at the anaerobic
threshold, we observed significant differences in
both speed (in both sexes) and oxygen
consumption (only in women). Additionally, in
men, there is a trend towards a significant
difference in heart rates.
In other words, our results indicate that obese
subjects reach the anaerobic threshold with less
effort than control subjects, which means they
oxidize fats for a shorter duration. These
observations are consistent with the hypothesis
put forward by Wade et al. (Wade, Marbut and
Round, 1990) that body composition influences
the preference for oxidized substrates. According
to this hypothesis, obese subjects, who have a
higher percentage of body fat and a lower
proportion of slow-twitch muscle fibers (type 1),
would be less efficient in fat oxidation. However,
it is worth mentioning that other studies,
including the one by Geerling et al. (Krassas et al.,
2001), have questioned this notion. Furthermore,
it is important to note that the initial training
level may also play a role in the observed
differences, as it influences the combustion of
energy substrates, and it is possible that the
control subjects had a higher initial training level.
Effect of Physical Exercise on Body
Composition
One of the most notable and remarkable
conclusions of this study is that the examined
physical exercise program leads to a significant
reduction in weight, body fat, and abdominal
circumference.
Our results demonstrate an average weight loss
of -0.14 ± 0.03 kg per week for our obese
subjects, regardless of gender. This weight loss is
nearly twice as high as that reported by Wilmore
in an analysis of 46 studies (Wilmore, 1995)
(Wilmore, 1996), where the average duration of
exercise was 16.3 weeks. Furthermore, the
weight loss in our subjects is 11 times higher
than that in the HERITAGE study (Wilmore et al.,
1999), which is one of the most significant and
recent studies in the field of weight management
through physical activity. In this 20-week study,
subjects underwent progressive endurance
training. When considering both genders
separately, we observe a weight loss of -0.08 ±
0.03 kg per week in the women of our study,
whereas the HERITAGE study showed a weight
loss of only -0.005 kg per week in women. For
men, the weight loss in our cohort is -0.19 ± 0.06
kg per week, nearly three times higher than that
in the HERITAGE study. Furthermore, our study is
also consistent with a meta-analysis of 28 studies
conducted by Garrow and Summerbell (Garrow
and Summerbell, 1995), which found slightly
lower weight loss in our obese female subjects
compared to their study (0.09 kg/week versus
0.12 kg/week after excluding two studies), while
our male participants lost nearly three times
more weight (0.26 kg/week versus 0.09
kg/week).
Additionally, it is important to highlight the
significant reduction in abdominal circumference
that we observed in obese women and men. This
observation is particularly interesting as studies
have demonstrated that high values of abdominal
circumference, reflecting significant
accumulation of abdominal fat, are associated
with an increased risk of metabolic complications
such as insulin resistance, diabetes, lipid
disorders, and atherosclerosis (Adults (US),
1998).
Although our sample size is modest compared to
large-scale studies like HERITAGE, it is important
to note that our physical activity program led to
significant improvements in body composition
that have not yet been observed in previous
studies.
Effects of Physical Activity on Performance
and Physiological Characteristics
In addition to its effects on body composition, our
physical activity program also has beneficial
effects on the performance of obese subjects at
the fat and anaerobic thresholds. After
completing the program, obese subjects require
significantly greater effort to reach these
131
thresholds. In other words, they spend more time
at the fat threshold. This results in increased fat
oxidation, thus promoting fat loss. Conversely,
these changes were not observed in the control
subjects, or at least to a lesser extent.
We also examined physiological characteristics
(heart rate, oxygen consumption, and ventilation)
measured before and after exercise at the fat and
anaerobic thresholds. Except for oxygen
consumption at the anaerobic threshold, none of
the other parameters showed significant changes,
despite a significant improvement in
performance achieved after exercise, as
mentioned earlier. Although we did not observe a
significant variation in the measured parameters
at a given speed (we chose 6 km/h), we can
conclude that obese subjects become, at least in
terms of their physiological characteristics, more
exercise-tolerant: heart rate increases less
rapidly, and ventilatory effort is reduced.
Correlation between Weight Loss and
Improvement in Physical Performance
Surprisingly, we did not observe a significant
correlation between the magnitude of weight loss
and the improvement in performance or changes
in physiological characteristics. Thus, these
parameters appear to be relatively independent
of weight variation. This finding leads us to
hypothesize that the changes induced by physical
exercise (particularly the shift in the fat threshold
in obese individuals) cannot be solely explained
by weight loss but may result from mechanisms
that are still poorly understood. It is possible that
the development of slow-twitch muscle fibers,
mentioned earlier, may play a role in promoting
better fat oxidation.
Conclusion
In this study, despite its limitations, we were able
to demonstrate significant changes in both body
composition and physiological characteristics in
obese subjects exposed to a physical exercise
program tailored to their needs, alternating
efforts at the fat threshold with efforts at the
anaerobic threshold. It is very likely that this
combination of moderate and intense efforts,
which has been scarcely investigated in previous
studies, confers superiority to our program
compared to those that only focus on a single
aspect of physical exertion.
With these observations, we can now consider
that physical exercise, even when practiced
without a strict hypocaloric diet, can be highly
beneficial for obese individuals. However, it is
crucial that this physical exercise precisely meets
the demands of obese subjects, which are likely
different from those of normal-weight
individuals. The combination of varied intensity
efforts indeed promotes a significantly higher
compliance among subjects who may struggle
with sustained intense efforts in the long term.
Many questions regarding physical activity in
obese patients in general, and in our subjects in
particular, still remain unanswered. What are the
repercussions of a physical activity program that
leads to significant weight loss on the quality of
life of an obese individual? To what degree of
compliance can we expect if such a program is
applied in the long term? Do the effects on weight
and physiological characteristics from our
program persist after the training period ends,
and if so, for how long and to what extent?
Although these questions remain open, we can no
longer ignore the importance of physical activity
in obese individuals, whether it is to achieve
weight loss or simply improve physical fitness.
Adults (US), N.O.E.I.E.P. on the I., Evaluation, and
Treatment of Obesity in (1998) Clinical Guidelines
on the Identification, Evaluation, and Treatment of
Overweight and Obesity in Adults. National Heart,
Lung, and Blood Institute.
Andersen, R.M. (1995) ‘Revisiting the behavioral
model and access to medical care: does it
matter?’, Journal of Health and Social Behavior,
36(1), pp. 1–10.
Carneiro LSF, Fonseca AM, Vieira-Coelho MA,
Mota MP, Vasconcelos-Raposo J (2015) ‘Effects of
structured exercise and pharmacotherapy vs.
pharmacotherapy for adults with depressive
symptoms: A randomized clinical trial’, J Psychiatr
Res, December, p. 71:48–55.
Garrow, J.S. and Summerbell, C.D. (1995) ‘Meta-
analysis: effect of exercise, with or without
dieting, on the body composition of overweight
subjects’, European Journal of Clinical Nutrition,
49(1), pp. 1–10.
Gupta N, Goel K, Shah P, Misra A. (no date)
‘Childhood obesity in developing countries:
epidemiology, determinants, and prevention.
Endocr Rev. 2012;33(1): 48-70.’
Krassas, G.E. et al. (2001) ‘Prevalence and trends
in overweight and obesity among children and
adolescents in Thessaloniki, Greece’, Journal of
pediatric endocrinology & metabolism: JPEM, 14
Suppl 5, pp. 1319–1326; discussion 1365.
Paffenbarger RS, Hyde RT, Wing AL, Hsieh CC. (no
date) ‘Physical activity, all-cause mortality, and
longevity of college alumni. N Engl J Med .
1986;314(10): 605-613’.
Rahim, S. and Baali, A. (2011) ‘Étude de l`obésité
et quelques facteurs associes chez un groupe de
femmes marocaines résidentes de la ville de
Smara (sud du Maroc)’, Antropo, (24), pp. 43–53.
Schuch, F. B., Vasconcelos-Moreno, M. P.,
Borowsky, C., Zimmermann, A. B., Rocha, N. S., &
Fleck, M. P (2015) ‘Exercise and severe major
depression: effect on symptom severity and
quality of life at discharge in an inpatient cohort’,
132
Journal of psychiatric research, pp. 61, 25–32.
Shephard RJ, Balady GJ. (no date) ‘Exercise as
cardiovascular therapy. Circulation. 1999;99(7):
963-972’.
Wade, A.J., Marbut, M.M. and Round, J.M. (1990)
‘Muscle fibre type and aetiology of obesity’,
Lancet (London, England), 335(8693), pp.
805–808. Available at:
https://doi.org/10.1016/0140-6736(90)90933-
v.
Wilmore, J.H. (1995) ‘Variations in physical
activity habits and body composition’,
International Journal of Obesity and Related
Metabolic Disorders: Journal of the International
Association for the Study of Obesity, 19 Suppl 4,
pp. S107-112.
Wilmore, J.H. (1996) ‘Increasing physical activity:
alterations in body mass and composition’, The
American Journal of Clinical Nutrition, 63(3
Suppl), pp. 456S-460S. Available at:
https://doi.org/10.1093/ajcn/63.3.456.
Wilmore, J.H. et al. (1999) ‘Alterations in body
weight and composition consequent to 20 wk of
endurance training: the HERITAGE Family Study’,
The American Journal of Clinical Nutrition, 70(3),
pp. 346–352. Available at:
https://doi.org/10.1093/ajcn/70.3.346.
World Health Organization (2002) Rapport sur la
santé dans le monde : 2002 : réduire les risques et
promouvoir une vie saine. Organisation mondiale
de la Santé. Available at:
https://apps.who.int/iris/handle/10665/42522
(Accessed: 17 February 2023).

133
CONFERENCE PAPER Hamid Taouil et al. 2022, 134-139

Study of Didactic Obstacles Related to the Learning of Basic Concepts in

Organic Chemistry: Case of Undergraduate Teaching University Cycle
Hamid TAOUIL1,2*, Zineb BOUMAAIZE3, Khalid AROUYA1, Abdelaziz AMINE4,5,Abderrazak NIMOUR1,
Nabil OUKOUR1, Mohamed. AHD2
1
Laboratory of Organic Chemistry, Catalysis and Environment. Faculty of Sciences, University Ibn-
Tofail, Kenitra, BP: 133, 14,000 Kenitra, Morocco.
2
Laboratory of Electronic Systems, Information Processing, Mechanics and Energy. Faculty of
Sciences, University Ibn- Tofail, Kenitra, BP: 133, 14,000 Kenitra, Morocco.
3
Faculty of science, Ibn Tofail University, Kénitra, Morocco.
4
Laboratory of condensed Matter and Renewable Energy, FST Mohammedia, University of Hassan II,
Morocco.
5
Laboratory Instrumentation Measurement and Control, Chouab Doukkali University, El Jadida,
Morocco.

A pedagogical project that takes into account the obstacles to knowledge

mastery would be an approach to diagnosis, obstacle management and
anticipation of pedagogical actions to be undertaken. Thus, whatever the
evolution of the science education system, the idea of obstacles to learning
will remain a constant in the development of quality pedagogy in science
education in particular and science teacher training in general. For this
reason, our work aims more particularly to have explanatory elements of
failure rates for first-year university students in Morocco; this study aims to
identify the state of their knowledge in organic chemistry. The results
obtained show, on the one hand, that there is a lack of basic knowledge by
creating a conceptual obstacle. On the other hand, the effect of problems
related to the change in the language of instruction may be the only source
of language barriers, which creates difficulties in learning disciplinary and
scientific concepts.

*
Corresponding Author
Name: Hamid TAOUIL INTRODUCTION
Email: taouil.hamd@gmail.com Science education in general requires several
Phone: +212 672-224847 preliminary actions and reflections that can
guarantee a form of knowledge, a presentation
ISBN : 9788367405256 and approaches likely to ensure a teaching that
DOI: 10.2478/9788367405256-018 could generate a possible learning of the concepts
and notions targeted by the educational actions
Published by Sciendo. © 2022. Hamid Taouil et al. developed. Thus, organic chemistry is often
This is an open access article licensed under the Attribution described as "a science, an art and an industry"
4.0 International
Compain (2003), and organic synthesis is one of
(https://creativecommons.org/licenses/by/4.0/)
its essential components for industry and
scientific developments.
134
The teaching must make it possible to show
students all aspects of the main purpose of organic
chemistry professions: the synthesis of organic
chemical species.
According to (Henderleiter et al., 2001; Hassan et
al. and Kind, 2004) students present
misconceptions and difficulties related to certain
concepts, such as the relationships between
electronic structure and properties of organic
chemical species. Thus Rushton et al (2008)
showed that these conceptions are persistent even
after several years of teaching. In addition,
Bhattacharyya (2008), Ferguson and Bodner
(2008) reported that students have only a
superficial knowledge of organic chemistry
concepts and models, which prevents their
transfer to new problems to be solved. Nash et al
(2000) noted that although a positive evolution in
the hierarchy of students' knowledge was
observed during their first semester of organic
chemistry. In addition, according to Cracolice et al
(2008), students do not have sufficient problem
representation and reasoning skills to solve
conceptual problems. And according to Cracolice
et al., 2008; Taagepera and Noori, 2000), students
can only solve algorithmic problems that only
require the application of memorized procedures
without the need for conceptual understanding on
the one hand and on the other hand, according to
David Lafarge (2010), students have great
difficulty considering several approaches
simultaneously in solving an organic chemistry
problem. As a result, they do not have a common
thread and the evolution of the reasoning owes
much to chance. Therefore, for students who learn
organic chemistry by heart, they seem to be more
producers than reasoners. To this end, work on
the didactics of chemistry has been carried out
(Wood, 2006, Zoller and Pushkin, 2007) on the
cognitive abilities implemented by students
according to the problems they face. However,
other work is interested in evaluating the learning
of first-year university students (general
chemistry option), based on the introduction of
multiple choice questionnaires (MCQs) in
continuous monitoring Akrim et al. (2006). Thus,
the use of MCQs as a means of measuring learning
assessment has been successful in some academic,
national and international institutions Akrim et al.
(2006). In addition, studies are being conducted to
identify learning difficulties among university
graduates in atomic chemistry (Chafiqi, F. et al.
2003). In addition, El jamali et al. (2007) raised
possible difficulties encountered by students in
the context of the continuous monitoring
introduced by the new reform. In this work, our
objective is based on the identification of some
obstacles that make it difficult for baccalaureate
students to acquire certain basic concepts in
organic chemistry such as nomenclature, isomery
and on the proposal of causes and some solutions
to minimize this paradoxical phenomenon.
METHODOLOGY
DEVELOPMENT OF THE STUDY INSTRUMENTS
Given the exploratory nature of our study, we
opted for the questionnaires as data collection
instruments, which include four themes:
(Molecular nomenclature, Configuration (R, S, E
and Z), Configuration isomer and surgical
structures).
• For the theme "Nomenclature of molecules"
has 13 items
• For the theme "Configuration (R, S, E and Z)"
has 10 items
• For the theme "Configuration isomer" has 15
items
• For the theme "Surgical Structures" has 10
items
STUDY POPULATION
The population concerned by this study is
composed of 120 first-year students (Option
S.V.T.U.) from the Ben M'sik Faculty of Science in
Casablanca, Morocco.
RESULTS AND DISCUSSION
In order to determine the conceptual difficulties
encountered by bachelor students in learning
organic chemistry, an analysis of conceptual
difficulties was carried out on four themes
(Nomenclature of molecules, Configuration (R, S, E
and Z), Configuration isomer, and Chiral
structures). Thus, in the following Figures, correct
answers are noted (RJ), wrong answers (RF) and
unanswered answers (NR).
OVERALL ANALYSIS
In order to determine the theme that poses the
most difficulties for the bachelor students
interviewed, we have grouped the responses
related to each of the four themes (Molecular
Nomenclature, Configuration (R, S, E and Z),
Configuration Isomer, Chiral Structures) in Figure
1.
135
 Figure 2: Results relating to the
"Nomenclature" theme
The examination of this Figure shows that for the
majority of the items studied, the percentages of RJ
are generally high (values higher than 50%). These
results suggest that respondents are familiar with
the nomenclature of organic compounds, but most
are unable to reinvest this knowledge in the
concrete cases of compounds:

Figure 1: Results for the four themes • CH3-CH2-CH2-CH2-CH2-CHO (item Q1f )

• CH3-CH2-OH (item Q1h)

Examination of this figure shows that the
percentage of correct answers is particularly high
for the "Nomenclature" theme (above 60%), but
this percentage remains below 42% for the other
themes. In addition, we note relatively a
quasi-equality between the percentages of correct
and false answers for the themes "Absolute
Configuration; Configuration E, Z" and "Isomeric"
and also a quasi-equality between the percentages
of correct and unanswered answers for the theme
"Chiral Structures".
These results show overall that students'
pre-acquired skills are insufficient for these three
themes. These may be due to the effect of language
problems. Indeed, previous work has shown that
the Moroccan context presents a particular
specification relating to the secondary to higher
transition Chafiqi and Chlyeh (1998) .Thus, this
transition is then accompanied by linguistic
difficulties; some related to the change in the
status of the language Alagui, et al. (1996)which
poses major problems for new students. Thus,
after this preliminary descriptive reading of the
results obtained, we sought to understand the
possible origins of the errors made and the gaps in
the knowledge of the students interviewed. For
this purpose, a detailed analysis by theme is
necessary.
• (item Q1m)
Indeed, the percentages of false responses to these
items are high (values greater than 48% for Q1f
items; Q1h and greater than 75% for Q1m item).
Moreover, item Q1f corresponds to the hexanal
compound, it is an aldehyde whose name is
terminated by the suffix "al" and not "one". For
item Q1h, it corresponds to a primary alcohol,
because the alcohol -OH function carried by a
carbon linked to a carbonaceous group + 2 H. For
item Q1m, it is a tertiary alcohol because the
functional carbon is linked to three carbons (the
structural formula of this alcohol is OH- C (CH3)3.
We conclude for these three previous items (Q1f,
Q1h, Q1m) that students have problems
identifying the functions of organic compounds,
particularly in terms of functional carbon. It is
about applying knowledge to sort and classify
these functions, which requires a high level of
scientific expertise.
THEME 2: ABSOLUTE CONFIGURATION R AND S;
CONFIGURATION E AND Z

ANALYSIS BY THEME The symbolism of organic chemistry has no clear

THEME 1: NOMENCLATURE
For this "Nomenclature" theme, we have chosen
13 items for baccalaureate students. The results
obtained are presented in Figure 2.
meaning for most undergraduate students based
on the several results obtained by American
researchers Anderson and Bodner (2008). For
this purpose, we have chosen 10 items for the
configuration theme for undergraduate students.

The results obtained are shown in Figure 3.

136
Figure 3: Results for the theme "Configuration Indeed, item Q3c corresponds to the C torque, it is
R, S, E and Z" about two conformation isomers, because a
simple rotation of the C-C bond displayed
Examination of this figure shows that among the
according to Newman.
first five items (absolute configurations R and S),
items Q2a and Q2d have relatively high
percentages of false response (RF) (greater than
50%). We conclude that these two items present
difficulties of knowledge, as the answer requires
an ability to sort and classify. Indeed, for item Q2a
corresponds to compound A: Assignment of
priority orders by classifying O, S, C and H by
atomic number: S > O > C > C > H. So priority 1 for
SH, 2 for OCH3, 3 for CH3 and 4 for H. Therefore,
the H is at the back, so you have to look at the
molecule from the front, the direction 1, 2, 3 is
turned counter-clockwise: the asymmetric carbon
has the S configuration.
For item Q2d, it is compound D which has two
C*(asymmetric carbon). We can determine the
configuration of these two C*, one is S and the Figure 4: Results related to the theme "Isomer
other is R, or we can identify the particular of conformation and configuration"
symmetry of a meso compound that will be
superimposed on its image in a mirror. However,
we know that a meso compound has a C* of Thus, for items from Q3f to Q3j (configuration
configuration R and a C* of configuration S. isomer), both items Q3f and Q3h have percentages
of false answers greater than 50%. For the two
items Q3k and Q3m, we note an equality of the
percentages of false answers (values of the order
of 50%).
These results allow us to conclude that there is a
lack of acquisition of certain concepts, especially
in the concrete cases of the previous items. So it's
These results lead to the conclusion that the about applying knowledge using a high level of
classification of chemical elements in order of scientific know-how.
priority is apparently not well understood. This is
especially true in the case of the previous items, Indeed, for item Q3f, which corresponds to two
where the student is also asked to formulate a configuration isomers (couple A), there is a
response that requires a good mastery of the conflict among students since we have even
concept and formulation, which then requires a developed a formula and a C* of different
high level of scientific know-how. We also note configuration.
that the percentages of unanswered questions are
significant (35%), and the correct answers are low
(values between 19% and 42%), for items from
Q2f to Q2j (configuration E and Z).

THEME 3: ISOMER OF CONFORMATION AND

CONFIGURATION A
For item Q3h, it is a question of two structures of
For this conformation and configuration isomeric the same compound with a meso form (couple C).
theme, we chose 15 items for undergraduate
students. The results obtained are illustrated in
Figure 4, and we note that for items from Q3a to
Q3e (conformation isomer) the only item Q3c that
has a high percentage of false response (RF) (>
50%). However, this item presents an additional
difficulty because the answer requires an ability to
master the notion of conformation.
137
 structures and optical activity"
Indeed, for item Q4g (RJ: 13%) the amine formula
does not have optical activity because the nitrogen
atom is bound to two identical alkyl rumps (-CH3).
Thus, for item Q4i (RJ: 27%) which corresponds to
the alkene molecule. This molecule has no C*, so it
is not optically active, but item Q4j (RJ: 22%)
corresponds to a chiral molecule.

Thus, for item Q3k, it is two structures 1 and 2

that are not enantiomers. For them to be
enantiomers, they must be images of each other in
a mirror and not superimposed. On the other
hand, item Q3m, compound 5 has no optical
activity, because it is not chiral (does not admit
C*). We conclude that there is a deficiency in the
acquisition of some notion of the isomeric
conformation and configuration. CONCLUSION

In this study, we identified some obstacles that

THEME 4: CHIRAL STRUCTURES AND OPTICAL
ACTIVITY
For this theme, we have chosen 10 items for
undergraduate students. The results obtained are
illustrated in Figure 5, where we see that for items
from Q4a to Q4e (Chiral Structure), the only item
Q4b has a percentage of 32% of RJs that is lower
than the percentage of RFs. Thus, the other items
from Q4f to Q4j (Optical activity) have high
percentages of RLs that are greater than 50%.
These results indicate that most respondents who
are unable to control the notion of optical activity
with respect to polarized light are unable to do so.
Indeed, that the notion of "optically active" and
"chiral" in this item is not sufficiently detailed in
teaching and therefore quickly forgotten, which
shows that the notion of optical activity is far from
being acquired.
Figure 5: Results related to the theme "Chiral
make it difficult for first-year students (Option
S.V.T.U.) at the Ben M' sik Faculty of Science in
Casablanca, Morocco, to acquire certain basic
concepts in organic chemistry. During the analysis
of the tasks present in the QCM statements, we
found that almost all of these tasks provide
information on the structure of reactive chemical
species (nomenclature, functional group,
structural formula, isomeric, chirality). This study
allowed us to find that organic chemistry presents
some difficulty for a large number of learners
when moving from one theme to another. Indeed,
the students interviewed could not answer the
questions related to the notion of chirality and
optical activity. This study also revealed a lack of
acquisition of some chemical symbols, namely the
E, Z and absolute R and S configurations.
This suggests that there may be insufficient
acquisition of certain scientific concepts and
difficulties in learning disciplinary concepts.
In addition, a reading of some of the work relating
to this notion suggests that the possible types of
difficulty are diverse, thus opening up a new
avenue of research on the origins of the difficulties
that this study has revealed.
The previous analyses led us to make proposals to
improve this teaching-learning process: restore
the importance of translation subjects among
baccalaureate science students ( Introduce the
translation subject to the national baccalaureate
examination and increase its number of hours),
restructure the content overall by focusing on the
questions of the organic chemist and proposing a
progressive approach to basic design ( Electronic
structure, number of chemical bonds for atoms,
LEWIS model...), promote the modelling strategy
138
by proposing adapted tasks (goals / conditions of
realization) and by making more operational the
models of special representations such as
NEWMAN's and reduce the number of
presentations such as Fisher's for first year
students.
ACKNOWLEDGEMENTS
Sample
REFERENCES
Compain, P. 2003. "Le pari de la simplexité. Le
simple et le complexe en synthèse organique."
L'Actualité Chimique 263-264: 129-134.
Bhattacharyya, G. 2008. "Who am I? What am I
doing here? Professional identity and the
epistemic development of organic chemists."
Chemistry Education: Research and Practice 9(2):
84-92.
Ferguson, R. et Bodner, G. M. 2008. "Making sense
of the arrow-pushing formalism among good
student who didn't 'get' organic chemistry."
Chemistry Education Research and Practice 9(2):
93-101.
Nash, J. G., Liotta, L. J. et Bravaco, R. J. 2000.
"Measuring conceptual change in organic
chemistry." Journal of Chemical Education 77(3):
333-337.
SALGADO, M. D. T. PASSOS, G. C.; RIBEIRO, A. C. D.
Método de Resolução de problemas no ensino
médio: uma proposta interdisciplinar abordando
o tema agrotóxicos. Revista Prática Docente, v. 3,
n. 2, p. 646-647, 2018.
Cracolice, M. S., Deming, J. C. et Ehlert, B. 2008.
"Concept learning versus problem solving: a
cognitive difference." Journal of Chemical
Education 85(6): 873-878.
Taagepera, M. & Noori, S. 2000. "Mapping
students' thinking patterns in learning organic
chemistry by the use of knowledge space theory."
Journal of Chemical Education 77(9): 1224-1229.
D. Lafarge.2010. ‘Analyse didactique de
l'enseignement-apprentissage de la chimie
organique jusqu'à bac + 2 pour envisager sa
restructuration’. Université Blaise Pascal.
Wood, C. 2006. "The development of creative
problem solving in chemistry." Chemistry
Education Research and Practice 7(2): 96-113.
Zoller, U. et Pushkin, D. 2007. "Matching
Higher-Order Cognitive Skills (HOCS) promotion
goals with problem-based laboratory practice in
a freshman organic chemistry course." Chemistry
Education: Research and Practice 8(2): 153-171.
H. Akrim, S. El Jamali, A. El-Bousaadani, M. Radid,
M. TALBI. 2006. ‘L’intégration des QCM dans le
contrôle continu des étudiants de 1ère année
universitaire SMP et SMC1’. Cas de la Chimie
Générale.
Chafiqi, F., Alagui, A., Bouab, O., Chlyeh, M.A., Cros,
D., Sivade, A. 2003. Identification des difficultés
en chimie chez les bacheliers scientifiques
Marocains. Res academia, volume 21, numéro 1,
p. 63-94.
S. El jamali et al. 2005. L’océan des sciences
physiques, Guide du professeur. Casablanca :
SOMADIL-SOGELIV, numéro d’accréditation du
ministère de l’Éducation nationale 08.232.105.
V. Turcotte .2020. ‘Le laboratoire par enquête
guidée pour provoquer un changement
conceptuel chez la population étudiante en
Chimie des solutions’.
Henderleiter, J., Smart, R., Anderson, J., Elian, O.
2001. "How do organic chemistry students
understand and apply hydrogen bonding?"
Journal of Chemical Education 78(8): 1126-1130.
Chafiqi, F. et Chlyeh, M.A. 1998. ‘Francais langue
véhiculaire du savoir scientifique, diagnostic des
difficultés rencontrées par les étudiants des
premières années de la faculté des sciences de
Marrakech’. In actes du colloque maghrébin
langue et enseignement scientifique au Maghreb,
bilan et perspectives, Faculté des letteres et
Sciences Humaines, Université Mohamed V,
Rabat-Maroc.
Alagui, A., Bouab, O., Bouab,W., Chafiqi, F., Chlyeh,
M.A., Cros, D. 1996. Etude des préacquis des
nouveaux bacheliers scientifiques marocains; cas
de l’atomistique. Actes des Deuxièmes journées
internationales des didactiques des sciences,
28-30 octobre, publication de l’université Cadi
Ayyad Marrakech, Maroc, 49-55.
Anderson, T. L. and Bodner, G. M. 2008. "What can
we do about 'Parker'? A case study of a good
student who didn't ‘get’ organic chemistry.
139
140
 CONFERENCE PAPER Younes IDERDAR et al. 2022, 140-143

The Contribution of Morocco to the Global Traumatic Brain Injury Research

Younes IDERDAR1*, Elmadani SAAD1, Noureddine ELKHOUDRI1, Amina IBNLFASSI2, and Mohamed
CHAHBOUNE1
1
: Hassan First University of Settat, Higher Institute of Health Sciences, Laboratory of Health Sciences
and Technologies, Settat, Morocco
2
: Hassan First University of Settat, Faculty of Sciences and Techniques, Laboratory of Engineering,
Industrial Management and Innovation, Settat, Morocco

To evaluate the contribution of Morocco to the global traumatic brain injury

research in the last two decades. A retrospective study was conducted in
2020 between (2000–2019) involving all the records in the following
databases: Scopus, Web of Science, and PubMed.
A review of Moroccan records was performed, and the results were obtained
using keywords: “Traumatic brain injury”, “Traumatic brain damage”,
“Traumatic head injury”, “Traumatic head damage”, and “Morocco”. A total
number of articles were identified between 2000 and 2019 through all the
datasets. The indices show a low contribution of Morocco to the global
Traumatic Brain Injury research, and the proportion of the total
international traumatic brain injury records that relate to Morocco is less
than 0.01%. As a conclusion, Morocco has been witnessing prolonged
growth in TBI research, and the country’s contribution to the global
literature remains small comparing to its population size and its actual
needs. Knowledge gaps are waiting to be filled.
*
Corresponding Author accidents; around 20% to 30% are caused by falls,
and 10% are caused by violence (Gururaj, 2002).
Name: Younes IDERDAR After a brain injury, many years of productive life are
Email: iderdarunes@gmail.com lost, and many people must endure years of
Phone: +212 6 53 49 43 69 disability.
ISBN: 9788367405256 Furthermore, it imposes high financial
consequences on people, families, and society.
DOI: 10.2478/9788367405256-019
Through greater prevention, many lives can be
Published by Sciendo. © 2022. Younes IDERDAR et al. spared and years of incapacity can be prevented
This is an open access article licensed under the Attribution (World Health Organization, 2006).
4.0 International
(https://creativecommons.org/licenses/by/4.0/) The mortality rate after a mild TBI is less than
1%, while 20–50% of those who suffer a severe TBI
die. The intermediate category of "moderate head
INTRODUCTION injury" represents a 2–5% mortality rate (World
Health Organization, 2006). But even after a mild
Traumatic brain injury (TBI) is a major public
event, disability is a common problem after
health concern around the world, affecting
hospitalization for TBI (Thornhill et al., 2000).
approximately 10 million individuals each year and
resulting in either death or hospitalization Since road traffic accidents cause about 85% of
(Puvanachandra & Hyder, 2009), and it will be the the deaths in low- and middle-income countries and
leading cause of mortality and disability by 2020, constitute the primary cause of TBIs, and as North
according to the World Health Organization (Hyder Africa face rapid economic transition with increasing
et al., 2007). In all parts of the world, over 60% of motorization and motorization, TBIs are becoming a
traumatic brain injuries (TBIs) are caused by traffic leading cause of mortality and morbidity (Peden,
140
2005). Road traffic crashes will have risen from were included in the search. A study was eligible for
ninth to third place in the global burden of disease inclusion if it meets the search strategy.
ranking by 2020 (World Health Organization, 2006).
1.4
This study aims to report and quantify the
progress in TBI research in Morocco during the last
two decades and compare it with the world’s
progress.
METHODS
1.1 Exclusion & inclusion criteria
On the 14th and 15th of May, 2020, the search
was conducted utilizing three academic databases:
Scopus, Web of Sciences, and PubMed. The search
strategy began with the identification of various
terms in the databases that could refer to TBI, such
as "Traumatic brain injury," "Traumatic brain
damage," "Traumatic head injury," and "Traumatic
head damage."
Both the singular and plural forms of TBI key
terms were searched for in the article title, abstract,
and keywords. Conference presentations, case
reports, interventions and facts were excluded from
the analysis. The search was limited to articles on
human subjects; articles with titles that contain
words like “Ancient”, “Rats”, “Dark ages” were
excluded.
1.2 Data sources
We defined three indicators to assess Morocco's
contribution to TBI research (Table 1 shows the
search strategy for the academic databases). These
indices correspond to the number of papers
published or data that were found by searching
three separate data sources.
The following are the indices:
Scopus and Web of Science: The number of entries
found in each database after searching for studies on
TBI in Morocco using a technique that included both
free text and field codes, as well as no language
constraints.
PubMed: The number of entries found in the
PubMed database after searching for publications on
TBI in Morocco using a technique that included free
text, field codes, and MeSH headings, as well as no
language or year constraints. The MeSH heading
'Brain Injuries, Traumatic' [MeSH Terms] was
researched to encompass all the subheadings.
December 2019 was our research's cut-off date.
To search for Moroccan studies, we included country
affiliation restrictions in all three databases.
1.3 Types of studies
Only articles published between 2000 and 2019, Between 2000 and 2019, the distribution of TBI
conducted in Morocco, and involving human subjects records published by Morocco through the three
Types of intervention
In the first step, we obtained a total number of
articles that concerns TBI all over the world in all
three academic databases. In the second step, we
limited the search to Morocco by adding the
country's name to the search key terms to obtain its
results.
After applying the search criteria, we obtained
the results shown in "table 1", which shows the
distribution of articles in Morocco and globally
according to all three academic databases. Data were
collected by searching the following keywords:
“Traumatic brain injury”, “Traumatic brain damage”,
“Traumatic head injury”, “Traumatic head damage”,
and the key terms were searched for in the article
title, abstract and keywords.
RESULTS
The results in “Table 1” show differences in terms
of the number of articles published at each level. To
quantify the contribution of Morocco to the global
records, using the three indices, we compared the
country's contribution to overall global TBI scientific
publications. We found 43543 records through
Scopus Index, 28878 records through Web of Science
Index, and 37260 records through PubMed Index
since 2000 (Table 1).
In comparison to global TBI recordings, the
percentage of Moroccan records in all databases is
negligible, with a total contribution of less than
0.01% (Table 1). The records started to appear in
1891, but in Morocco, they did not begin to appear
until 2011. 22 articles were found following the
search strategy. Only 10 matched the research
requirements and were included for a full-text
evaluation, after deleting duplicates and doing an
initial screening. One was excluded since it was
only abstract. The review comprised a total of 9
full-text papers.
Table 1. Complete search strategy for the
academic databases (2000-2019)
Set Strategy Results
"Traumatic brain injury" OR Scopus: 43543
"Traumatic brain damage" OR
#1
"Traumatic head injury" OR
WoS: 28878
"Traumatic head damage" PubMed: 37260
#2 Morocco
Scopus: 10
#1 AND #2 WoS: 8
PubMed: 4
141
databases shows a low contribution in the number in the field of TBI (Zitnay et al., 2008).
of published articles. Morocco has only ten articles in To promote research in TBI and improve
Scopus database, eight in Web of Sciences and four outcomes, we need to conduct future clinical
articles in PubMed (Fig. 1). Of the nine studies pathway studies for surveys of risk behaviours in the
included in this study, one study was on children and general community, as well as a strategy for the TBI
eight studies on adults. Sample sizes across all registries and data systems. Studies are crucial not
studies were ranging from “1” to “986” participants just for the direct advantages they provide, but also
in total. for the indirect benefits they provide, by allowing
and fueling mathematical modeling and
cost-effectiveness evaluations. TBI research, clinical
trials, and training programs account for less than
1% of overall yearly TBI expenses (Mohamed et al.,
2018; Zitnay et al., 2008).
The findings of this study showed that the
contribution of Morocco to the global TBI research
has been relatively low, and it requires effective
measures and interventions to promote and
encourage research in this field.
As a limitation of our study, we relied on reports
Figure 1: Distribution of traumatic brain injury and papers published in online databases to assess
articles in Morocco (2000 - 2019) the development of TBI research in Morocco; reports
that were not published were automatically excluded
DISCUSSION from the study.
Our findings show that Morocco's contribution to the CONCLUSION
worldwide TBI literature is insufficient, accounting
for less than 0.01%. Regardless of the index used to Despite considerable difficulties, Morocco has
measure the contribution, the results all came to the seen a very slow progress in TBI research.
same conclusion. This small contribution shows Considering this, the kingdom's contribution to
remarkable slow progress in TBI research in the world literature is insignificant in comparison to its
country. Because of the enormous and growing real demands. Encouraging academics from many
worldwide health burden of TBI, there is an urgent professions and sectors to conduct more TBI
need to accelerate the research in this field in the research will aid in preventing a high number of
Moroccan Kingdom. deaths, raising awareness, and lowering the costs of
While some nations have made progress, others TBI losses. In addition, existing research activities
remain far behind in terms of willingness and should be supported in a long-term manner to
capacity to conduct TBI research. Because of this guarantee that progress is maintained.
significant gap in TBI records, Moroccan
policymakers should push themselves to look for the ACKNOWLEDGEMENTS
reasons that prevented TBI researches from being a
field of interest. We would like to thank our colleagues from the
In certain areas, scientific efforts are underway, but Higher Institute of Health Sciences, who contributed
valuable insight and expertise to the study.
the number of TBI records in Morocco, relative to
global TBI records in the three databases, show a REFERENCES
critical deficiency in the volume of research. TBIs
occurs mainly in countries with low and middle Gururaj, G. (2002). Epidemiology of traumatic brain
income (Perel et al., 2006), indicating a structural injuries: Indian scenario. Neurological Research,
24(1), 24–28.
deficiency that stems from a number of
https://doi.org/10.1179/016164102101199503
circumstances, including low interest in TBI
academic papers, a lack of public backing, and/or Hyder, A. A., Wunderlich, C. A., Puvanachandra, P.,
insufficiency of research resources. However, Gururaj, G., & Kobusingye, O. C. (2007). The
considerable progress has been achieved in the area impact of traumatic brain injuries: A global
of prevention: airbags, seatbelts, modern vehicle perspective. NeuroRehabilitation, 22(5), 341–353.
design, and the usage of a helmet (Zitnay et al., https://doi.org/10.3233/nre-2007-22502
2008). Mohamed, W. R. A., Leach, M. J., Reda, N. A.,
Countries that are behind the curve in terms of Abd-Ellatif, M. M., Mohammed, M. A., &
research production, Morocco as an example, should Abd-Elaziz, M. A. (2018). The effectiveness of
step up their research efforts to solve the country's clinical pathway-directed care on
TBI problem. To speed up development, the hospitalisation-related outcomes in patients with
Kingdom should fund more training grants, research severe traumatic brain injury: A quasi-experimental
partnerships, as well as other training programmes
142
 study. JOURNAL OF CLINICAL NURSING,
27(5–6), E820–E832.
https://doi.org/10.1111/jocn.14194

Peden, M. (2005). Global collaboration on road traffic

injury prevention. International Journal of Injury
Control and Safety Promotion, 12(2), 85–91.
https://doi.org/10.1080/15660970500086130
Perel, P., Edwards, P., Wentz, R., & Roberts, I. (2006).
Systematic review of prognostic models in traumatic
brain injury. BMC Medical Informatics and Decision
Making, 6, 1–10.
https://doi.org/10.1186/1472-6947-6-38
Puvanachandra, P., & Hyder, A. A. (2009). The Burden of
Traumatic Brain Injury in Asia : a Call for Research.
Pak J Neurol Sci, 4(1), 27–32.
Thornhill, S., Teasdale, G. M., Murray, G. D., McEwen, J.,
Roy, C. W., & Penny, K. I. (2000). Disability in
young people and adults one year after head injury:
Prospective cohort study. British Medical Journal,
320(7250), 1631–1635.
https://doi.org/10.1136/bmj.320.7250.1631

World Health Organization. (2006). NEUROLOGICAL

DISORDERS: public health challenges.
https://apps.who.int/iris/handle/10665/43605
Zitnay, G. A., Zitnay, K. M., Povlishock, J. T., Hall, E. D.,
Marion, D. W., Trudel, T., Zafonte, R. D., Zasler, N.,
Nidiffer, F. D., DaVanzo, J., & Barth, J. T. (2008).
Traumatic brain injury research priorities: The
conemaugh international brain injury symposium.
Journal of Neurotrauma, 25(10), 1135–1152.
https://doi.org/10.1089/neu.2008.0599

143
CONFERENCE PAPER Msegued Ayam Iman et al. 2022, 144-148

Study of Moroccan cumin seeds (Cuminum cyminum L.) essential oils

Msegued Ayam Iman* , El ghadraouiLlahsen, Errachidi Faouzi

Functional Ecology and Environment Engineering Laboratory, Faculty of Science and Technology, Sidi
Mohamed Ben Abdellah University, Imouzzer street, B.P.2202, Fes, Morocco.
Errachidi faouzi : Orcid ID 0000-0001-6596-4108

This research and development work, concentrated on Cumin (Cuminum

cyminum L) grown in Morocco, was centered on essential oils
characterization and evaluating of their biological activities.
The main components of all studied essential oils are Cumin aldehyde
(32.5%), γterpinene-7-al (23.4%), α-terpinene-7-al (13%), γ-terpinene (9.5
%), β-cymene (4.2%), βpinene (4.36%) and p-mentha-1,4-dien-7-ol
(0.82%). Cumin seeds essential oil has a very high antioxidant activity, based
on scavenging capacity of DPPH (IC50 = 4.2 mg/mL) and ferric reducing
power FRAP (IC50 = 15.4 mg/mL). Results indicate that essential oils Cumin
seeds, grown in Morocco, are an excellent source of natural antioxidants,
which are used in food and pharmaceutical industries.

* has many pharmacological properties

Corresponding Author
Name: Msegued Ayam Iman
Email: iman.mseguedayam@usmba.ac.ma
Phone: +212631746524
ISBN : 9788367405256
DOI:10.2478/9788367405256-020
Published by Sciendo. © 2022. Msegued Ayam Iman et al.,
This is an open access article licensed under the Attribution
4.0 International
(https://creativecommons.org/licenses/by/4.0/)
INTRODUCTION
Cumin (Cuminum cyminum L.) is herbaceous
plant from the Umbelliferae family (Johri, 2011),
which is generally used as a spice to improve food
taste and flavor (Srinivasan, 2018). Cumin seeds
are a rich source of essential oil (2.5 to 4.5%)
(Sayed Ahmad, 2018), phytochemicals, minerals,
proteins, fatty acids and vitamins (Chen et al.,
2014 ; Merah et al., 2020). Cumin seeds are often
used in traditional medicine as stimulants,
carminatives, astringents, stomachic, diuretics,
emmenagogues, antispasmodics and
antidiarrheals (Mnif and Aifa, 2015). Also, Cumin
(Allahghadri et al., 2010).
In Morocco, Cumin occupies an important place in
Agriculture Ministry programs. In the Green
Morocco Plan program (2008-2018), efforts were
focused on improving technical management, but
in the new program (Generation Green: GG
2019-2030) objectives are set on the development
of this plant heritage of high added value. Cumin
protected geographical indication (PGI) as a
quality criterion has imposed strict regulations to
enhance the value of local products. This situation
highlights the role of physicochemical
characterization of Cumin seeds for label granting.
This work aims to determine morphometric and
physicochemical properties of Cumin seeds
belonging to two populations, one local (Rehamna)
and one imported from the Orient. The already
stated can also contribute to the recognizing of
Moroccan cumin plant genetic resources, which are
overwhelmed by varieties imported from Iran and
India.
144
 MATERIALS AND METHODS
1. SEEDS STUDIES
1.1. Morphometric study of cumin seeds
Studied batch seeds consists of two chemotypes
of Cumin composed of four local populations
From the Rehamna rural commune and four
populations of an imported variety used as a
control (planted in Sidi Ali rural commune).
To determine the Cumin seeds morphometry, 100
seeds of each chemotype were randomly selected
from a bulk batch. For each seed, three main axial
dimensions were assessed : Length (L), Width
(W) and Thickness (T) we measured these
characteristics using a caliper with a reading
accuracy of ±0.1mm. Knowledge of these axial
dimensions made it possible to determine other
seeds characteristic parameters by using the
following relationships (Abdellaoui et al., 2016):
• Arithmetic diameter (Da) and geometric
diameter (Dg) were determined using the
following procedures:
● Sphericity index (SI) was calculated according to
the following procedure.
Where L = length, l = width and e = thickness
● We calculated the area of the seed using the
following procedure.
1.2. Biochemical, Phytochemical study and
antioxidants activities
Total Phenol Content TPC was determined by
using the Folin-Ciocalteu method at 765 nm
according to (Slinkard & Singleton, 1977). We
quantified the flavonoid content (TFC) by
Aluminum chloride assay through colourimetry
(Samatha et al., 2012).
Essential oils antiradical power was tested by
DPPH (1,1-diphenyl-2-picrylhydrazyl) method
according to the protocol described by (Wang et
al., 2008). Ferric Ion Reducing Power (FRAP)
method was tested by the method described by
(Benzie and Strain, 1996). Biochemical analysis
were performed according to Official methods
(AOAC 2009).
2. Extraction and analysis of essential oils
2.1. Extraction
Essential oils extraction was carried out by
hydrodistillation, according to the protocol
recommended by the French pharmacopoeia. We
mixed 100 g of the Cumin powder with 900 ml of
distilled water. Thus, the mixture is heated for 3
hours. Essential oil yields were assaissed in g
relative to 100 g of dry plant material.
2.2. Chromatographic analyzes of essential oils
Essential oils GC analysis is carried out using a
chromatograph equipped with a flame ionization
detector (FID) and two capillary columns of
different polarities of OV type. 101 (25 m x 0.22
mm x 0.25 μm) and Carbowax 20 M (25 m x 0.22
mm x 0.25 μm). The Heluim gas carrier is used
with a flow rate at 0.8 ml/min and the oven
temperature programming under temperature
gradient from 50 to 200°C by 5°C/min. The CG/MS
coupling was carried out on a DB1 type fused
silica capillary column (25m x 0.23mm x 0.25μm)
with helium as carrier gas and a temperature
programming identical to that of the CG.
1.1. Scanning Electron Microscopy (SEM)
Cumin seeds morphology was studied by SEM
using a scanning electron microscope (JEOL
JSMIT500HR) with a magnification range of
×600.000, an accelerating voltage of 0.5 kV-30.0
kV, and a maximum sample size of 3 mm in
diameter and 1.5 mm in height.
RESULTS AND DISCUSSION
1. Morphometric study of local and imported
cumins
To inquire about the genetic differences between
local and imported varieties, we conducted a
study of seeds morphological parameters, namely:
length (L), width (l) and thickness (E), arithmetic
diameter (Da), geometric diameter (Dg),
sphericity index (SI) and surface seed (S). The
following table (Table 1) summarizes the results
obtained.
Table 1: Morpho-metric study of local and
imported varieties
Varieties L W E Da Dg SI S
4,78 1,26 0,71 2,25 1,61 0,34 7,96
Local
Imported 4,84 1,27 0,78 2,29 1,67 0,35 8,40
Length (L), width (l) and thickness (E), arithmetic
diameter (Da), geometric diameter (Dg),
sphericity index (SI) and surface seed (S)
145
Results show a significant difference in thickness Essential oils 3,2 2,24
only between local and imported varieties. This (%)
difference is noticed at the level of synthetic Fixed oils (%) 15,5 22
indices: arithmetic diameter (Da), sphericity index
(SI) and seeds surface (S). These results

Biochemical and phytochemical analysis of Cumin

agree with those of Abdellaoui and his
collaborators who studied several genotypes in
Morocco (2016).
To complete morph-metric analysis, we made a
scanning electron microscopic observation (SEM)
to detect morphological difference details of seeds
surfaces. The following figure (Figure 1)
illustrates the apparent difference in external seed
structures.
seeds prompted us to test the antioxidant activity
of the two essential oils extracted from the two
types of Cumin studied. Table 3 shows that the
evaluated C50s by the two methods are lower in
local park varieties compared to imported ones.
Table 3: Study of the antioxidant activity of
local and imported Cumin essential oils

Varieties/ CI50/DPPH IC50/FRAP

controls (mg/ml)
(mg/ml)

Local Cumin 4.2±0.89 15.4 ±0.05

Imported Cumin 5.73 ± 0.79 17.7 ±0.12

BHT(control 1) 1.29 ±0.12 0.043 ±0.31

Vitamin E (control 2) 3.15 ±1.04 0.226 .56

Figure 1: microscopic observation (SEM) of
local seeds a) and imported b) Cumin seeds.
Microscopic analysis shows that spines size 3. Essential oils comparative study of local and
distribution are not the same and agrees imported Cumin
with results illustrated in table
Gaz chromatography (GC) spectra analysis of
2. Biochemical, Phytochemical study and
cumins essential oils shows that profiles are not
antioxidants activities
identical. Imported varieties show in figure 2
Biochemical analysis carried out on local and
peaks appear after 19 min (5 ridges). Mass
imported Cumin seeds are summarized in the
spectrometry analysis allowed us to identify
following table (Table 2). Statistical analysis
essential oils chemical compounds of local and
made by student tests shows that the difference
imported Cumin. The following table (Table 4)
is significant between averages relating to fixed
summarizes obtained results from this qualitative
oils and essential oils. On the other hand,
and quantitative analysis.
differences in the means connecting to the
contents of proteins, soluble sugars, phenolic
compounds are not significant between local
varieties and imported varieties. We noticed that
the essential oil content of local types (3.2%) is
higher when compared to imported types
(2.24%).

Table 2: Biochemical and phytochemical

analysis of local and imported cumin seeds
Analysis Local Cumin Imported Cumin
Protein (%) 20,5 20,3
Soluble sugars 1,9 2,4
(%)
Phenolic 72,2 68.6
compounds
(mg/g)
Flavonoids 29,23 35
(mg/g)
146
 presence of β-Pinene (2.29%), α-Terpenene
(13%), p-Cymene (4%), Limonene (2.92%),
β-Phelandrene (1.76%), γTerpinene (9.5%),
Cumin aldehyde (32.5%), pMenthatriene
(5.33%), γ -terpinen-7-al (23.4%) and
α-terpinen-7-al (22.16%).

Compounds 1, 4-p-Menthadien-7-ol (4.08%),

Cumin alcohol (1.94%),
2-Ethylidene-6-methyl-3,5heptadienal (9.18%),
α-Terpinene (1.37%) and
αProyl-benzenemethanol (4.24%) are specific for
imported varieties.
The analyses obtained on local varieties agree
with those of (Abdellaoui et al., 2019;
Mehdizadeh et al., 2017), while the composition
of imported essential oils partially agree with
those of (Ali & Jumma, 2019).

CONCLUSION

Figure 2: Essential oils gas chromatography
of local cumin (a) and imported cumin (b)
In from this study, we notice that local types of
Cumin belonging to the Errahmna area, have very
distinct morphometric and biochemical
characteristics. Difference in essential oils
composition between local and imported Cumins
is a qualitative and quantitative

Table 4: Chemical composition of essential

oils of local and imported cumin
indicator to differentiate between Cumins
Compouds R.Time Local Impoted planted in Morocco.
Cumin Cumin Local Cumin has a copy of β-Pinene
β-pinene 3,383 2,29 0
Myrcene 9,154 0 1,59
(2.29%),αTerpenene (13%), p-Cymene (4%),
α-Phellandrene 9,281 0 0 Limonene (2.92%), β-Phelandrene (1.76%),
δ-3-Carene 10,442 0 3,16 γ-Terpinene (9.5 %), Cumin aldehyde (32.5%),
α-Terpinene 10,525 13 0 p-Menthatriene (5.33%), γ -terpinen-7-al
p-Cymene 10,653 4 4,25 (23.4%) and α-terpinen-7al (22.16%).
Limonene 11,667 2,92 0
1-8-Cineole 14,369 0 0
β-Phellandrene
REFERENCES
15,734 1,76 0
γ-Terpinene 16,707 9.5 12,73
Trans carveol 16,933 0 2,28 Abdellaoui, M., Alaoui, A., & El Rhaffari, L. (2016).
Cumin aldehyde 17,133 32,5 26,22 Propriétés physiques des graines du cumin
trans-Shisool 17,822 0 0 (Cuminum cyminum) dans la réserve de
p-Menth-2-en-7-ol trans 17,933 0 0 biosphère des oasis du Maroc/Physical
p-Menthatriene 18,052 5,33 6,71
properties of cumin seeds (Cuminum
γ -terpinen-7-al
cyminum) in biosphere reserve oasis of
18,331 23.4 5,01
Morocco. International Journal of Innovation
α-terpinen-7-al 18,454 22,16 17,24
and Applied Studies, 15(2), 412.
1,4-p-Menthadien-7-ol 19,379 0 4,08
Abdellaoui, M., Bouhlali, E. D. T., & El Rhaffari, L.
Cumin alcohol 19,985 0 1,94 (2019). Chemical Composition and
2-Ethylidene-6-methyl- 20,414 0 9,18
3,5-heptadienal
Antioxidant Activities of the Essential Oils of
α-Terpinene 20,981 0 1,37 Cumin (Cuminum cyminum) Conducted Under
α-Proylbenzenemethanol 21,391 0 4,24 Organic Production Conditions. Journal of
essential oil-bearing plants JEOP, 22,
1500‑1508.
Local cumin seeds are caracterised by the Ali, A. M., & Jumma, H. J. A. (2019). Yield, Quality

147
 and Composition of Cumin Essential Oil as 1354018
Affected by Storage Period. International
Journal of Analytical Mass Spectrometry and
Chromatography, 7(1), Merah, O., Sayed-Ahmad, B., Talou, T., Saad, Z.,
9‑17.https://doi.org/10.4236/ijamsc.2019.7 Cerny, M., Grivot, S., Evon, P., & Hijazi, A.
1002 (2020). Biochemical composition of cumin
Allahghadri, T., Rasooli, I., Owlia, P., Nadooshan, seeds, and biorefining study. Biomolecules,
M. J., Ghazanfari, T., Taghizadeh, M., & Astaneh, 10(7), 1054.
S. D. A. (2010). Antimicrobial property, Samatha, T., Shyamsundarachary, R., Srinivas, P., &
antioxidant capacity, and cytotoxicity of Swamy, N. R. (2012). Quantification of total
essential oil from cumin produced in Iran. phenolic and total flavonoid contents in
Journal of food science, 75(2), H54‑H61. extracts of Oroxylum indicum L. Kurz. Asian
Benzie, I. F., & Strain, J. J. (1996). The ferric Journal of
reducing ability of plasma (FRAP) as a Pharmaceutical and Clinical Research, 5(4),
measure of “antioxidant power”: The FRAP 177‑179.
assay. Analytical biochemistry, Slinkard, K., & Singleton, V. L. (1977). Total phenol
239(1), 70‑76. analysis : Automation and comparison with
Chen, Q., Gan, Z., Zhao, J., Wang, Y., Zhang, S., Li, manual methods. American journal of enology
J., & Ni, Y. (2014). In vitro comparison of and viticulture, 28(1), 49‑55.
antioxidant capacity of cumin (Cuminum
cyminum L.) oils and their main components. Wang, H., Gao, X. D., Zhou, G. C., Cai, L., & Yao, W. B.
LWT-Food Science and technology, 55(2), (2008). In vitro and in vivo antioxidant
632‑637. activity of aqueous extract from
Johri, R. K. (2011). Cuminum cyminum and Choerospondias axillaris fruit. Food
Carum carvi : An update. Pharmacognosy Chemistry, 106(3), 888‑895.
reviews, 5(9), 63. https://doi.org/10.1016/j.foodchem.2007.0
5.068

Mehdizadeh, L., Ghasemi Pirbalouti, A., &

Moghaddam, M. (2017). Storage stability of
essential oil of cumin ( Cuminum Cyminum L.)
as a function of temperature. International
Journal of Food Properties,
1‑9.https://doi.org/10.1080/10942912.2017.

148
CONFERENCE PAPER Karima El-Mouhdi et al. 2022, 149-156

Results of an Entomological Survey on Phlebotomine Sand flies (Diptera:

Psychodidae) Fauna of affected area of leishmaniasis in central Morocco
Karima El-Mouhdi *1,2, 3, Samia Boussaa4, Abdelkader Chahlaoui3, Chafika Faraj5 , Jawad Bouzid6 and
Mohammed Fekhaoui1
1
Scientific Institute, Mohammed V University, Rabat, Morocco
2
ISPITS- Higher Institute of Nursing Professions and Health Techniques, Ministry of Health and Social
Protection, Meknes, Morocco
3
Laboratory of Health and Environment, Faculty of Sciences, Moulay Ismail University, Meknes, Morocco
4
ISPITS- Higher Institute of Nursing Professions and Health Techniques, Ministry of Health and Social
Protection, Rabat, Morocco
5
National Institute of Hygiene; Medical Entmology, Ministry of Health and Social Protection, Rabat, Morocco
6
Higher Institute of Health Sciences, Hassan I University, Settat, Morocco

Sand flies (Diptera: Psychodidae) are small midges from temperate regions
of the world, which transmit numerous diseases, including leishmaniasis,
which is endemic in Morocco and represents a real public health problem. In
this study, we present the results of entomological surveys on sand flies in
the region of ELHajeb which is affected by leishmaniasis. The objective of
this work is to study the biodiversity and Spatio-temporal distribution of
sand flies in their ecological and epidemiological context. We studied six
stations. Bi-monthly adhesive trapping captures were performed during
2019. 14590 sand flies have been collected. In the combined collections,
seven species of Phlebotomus: Phlebotomus longicuspis (79.4%),
Phlebotomus sergenti (7.2%), Phlebotomus perniciosus (2.7%), Phlebotomus
papatasi (2.05%), Phlebotomus bergeroti (1. 1%), Phlebotomus alexandri
(0.7%), Phlebotomus ariasi (0.2%) and five species of Sergentomyia:
Sergentomyia minuta (4.42%), Sergentomyia schwetzi (1%), Sergentomyia
fallax (0.7%), Sergentomyia dreyfusi (0.5%), and Sergentomyia antennata
(0.1%) have been identified. We present for the first time entomological data
in leishmaniasis outbreaks and discuss the distribution of captured species
about biodiversity, biotopes, seasonality, and periods of high transmission
risk.
*
Corresponding Author
Name: El-Mouhdi Karima
Email: k.elmouhdi@ispitsmeknes.ac.ma Phone:+212618320461

ISBN : 9788367405256
DOI: 10.2478/9788367405256-021

Published by Sciendo. © 2022. Karima El-Mouhdi et al.

This is an open access article licensed under the Attribution 4.0 International
(https://creativecommons.org/licenses/by/4.0/)

INTRODUCTION are highly responsive in temperate climate

Sand flies (Diptera, Psychodidae, Phlebotominae) countries. They represent a human and animal
health problem in many countries of the world
149
(WHO, 2015; Depaquit and Léger, 2018; El-Mouhdi
et al., 2022). Indeed, these diseases are endemic in
most temperate countries such as Morocco, They
are transmitted to humans by small female sand
flies (WHO, 2011; El-Mouhdi, 2021).
The disease is endemic in several regions of the
country and has two main clinical forms:
cutaneous leishmaniasis (CL) and visceral
leishmaniasis (VL) (Moroccan Ministry of Health,
2010; Boussaa et al., 2010; El-Mouhdi et al., 2020).
In Morocco, sand flies are called "chniwla"
(El-Mouhdi et al., 2020a; El-Mouhdi et al., 2021;
El-Mouhdi et al., 2022).
Currently, sand fly control is a challenge for the
Moroccan government to eliminate all forms of
leishmaniasis by 2030 (Moroccan Ministry of
Health, 2010; Boussaa et al., 2016; El-Mouhdi et al.,
2020b). However, the species of sand fly vectors of
leishmaniasis in central Morocco in the El Hajeb
region have never been studied. The objective of
this work is to determine the phlebotomine fauna
circulating in the province of El Hajeb and to
provide health officials with basic data to optimize
entomological surveillance and vector control
activities in the region.
METHODS
Study area
The survey was conducted in six municipalities in
the province of El Hajeb: the municipality of Ain
Taoujdate (05°13'07.78" W; 33°57'09.33" N), the
municipality of Ait Naaman (05°19'13.79" W;
33°43'15.38" N), the municipality of Sidi Mbarek (
05°15'01. 50" W ; 33°57'59.60" N), the commune
of Aït Rbaa (05°10'10.79" W ; 33°51'04.67" N); the
commune of Aït Brahim (05°05'28.75" W ;
33°49'48. 46" N), and the commune of Aït Oufella
(05°09'31.93" W ; 33°56'20.51" N) [4] (Fig. 1.).
The province of El Hajeb has a semi-humid
Mediterranean climate, characterized by wet
winters and dry, hot summers. The average annual
rainfall is 572 mm, the average annual temperature
is 17 °C, the maximum temperature in July is 35 °C
and the minimum temperature is 4 °C in January
(Masen, 2018; El-Mouhdi, 2021).
The choice of trapping stations was based on
epidemiological justifications. In fact, we based on
the latest data of the epidemiological situation of
leishmaniasis in this region (El-Mouhdi et al.,
2019). Therefore, six stations were surveyed.
These were the Sidi Mbarek station and the Aït
Oufella station; the Aït Rbaa station; the Aït Brahim
station; the Aïn Taoujdate station, and the Aït
Naaman station.
Collection of specimens
The sampling of sand flies took place in six stations
in the province of El Hajeb in central Morocco. For
the collection of sand flies, we used adhesive traps
with A4 white paper, soaked in castor oil on both
sides during the whole night (Rioux and Golvan,
1969). These adhesive traps were placed in the
same place throughout the trapping period. Sand
flies were captured every 2 weeks at each station
during the study period. Entomological surveys
were conducted between April 2019 and January
2020, a period of high sand fly activity in Morocco
(Miri et al., 2013).
Laboratory work
The collected sand flies were transported to the
laboratory where they were sorted, counted, sexed
and preserved in 95% ethanol solution. The
morphological determination of the specimens
was carried out using the identification key of the
Moroccan Ministry of Health and Social Protection.
(Moroccan Ministry of Health, 2010).
Data processing and analysis
The faunal characteristics of the sand fly
population were identified by calculating the
following parameters:
(a) Abundance (A) corresponds to the total
number of individuals collected for each species.
(b) The relative abundance of each species (RA) is
estimated by dividing the number collected of a
given species by the total count of all species
collected multiplied by 100
AR= n/N×100 (1)
(c) Species richness (S) represents the total
number of species in the stand.
(d) The index of biodiversity of sand flies is
expressed by the Shannon index "H'" and the
Evenns index "E", where:
(2)
H’=-⅀PiLnPi
Where Pi: is the proportion of the total number of
species in all specimens.
E= H’/Hmax (3)
Where Hmax : LnS
(e) The ratio of males to females is expressed as
the sex ratio (SR):
SR= M / F (4)
(f) The density is expressed as the number of
specimens per unit area of paper (m2) per night:
D= ph/S/night (5)
150
Where:
D: density
Ph: number of sand flies
S: surface
RESULTS
Biodiversity, species richness and sex ratio of
captured sand flies
In this study, 14950 sand flies were captured and
80.3% of them were males. Table 1 (Appendix1)
shows the abundance, diversity, species richness,
and sex ratio of sand flies at six stations. The
researchers identified twelve sand fly species, and
observed differences between stations in terms of
species composition and sex ratio. The
male-to-female ratio was similar at Aït Taoujdate,
Sidi Mbarek, Aït Oufella, Aït Rbaa, and Aït Naaman,
but at Aït Brahim there were more female sand
flies than males. The commune of Aït Oufella had
the highest frequency of sand flies (46.8%),
followed by Aïn Taoujdate and Aït Rbaa. The
researchers used the Shannon diversity index and
Evenns equitability to calculate the diversity of the
sand fly population. Aït Rbaa and Sidi Mbarek had
the highest biodiversity, while Aït Brahim had the
lowest. Aït Naaman was the most diversified
station, due to its high diversity value and its
proximity to its maximum theoretical value.
Inventory of identified sand fly species
From the total of 14590 sand flies collected, 14070
underwent morphological identification, while the
remaining 520 pregnant and/or gorged females
were held for possible future molecular
identification. Table 2 (in Appendix 2) presents
the identification results for the 14070 sand flies,
with 93.3% (13128) belonging to the Phlebotomus
genus, and 6.7% (942) belonging to the
Sergentomyia genus. Morphological identification
confirmed the presence of 12 sand fly species,
including Phlebotomus (Phlebotomus) papatasi
Scopoli, P. (Phl.) bergeroti, P. (Phl.) Parrotomyia, P.
(Paraphlebotmus) serpedes, P. (Phlebotomus)
papatasi Scopoli, and P. (Paraphlebotmus) sergenti
Parrot, along with Sergentomyia (Sergentomyia)
minuta Rodani, S. (Ser.) schwetzi Adler, S. (Ser.)
fallax Parrot, S. (Ser.) antennata, and S.
(Grassomyia) dreyfusi Parrot.
Among the identified specimens, five species
represented 95.7% of the phlebotomine
population in the El Hajeb region, namely P.
longicuspis, P. sergenti, S. minuta, P. perniciosus, and
P. papatasi. The most abundant species among all
sand flies was P. longicuspis, with a relative
abundance of 79.7%. It was found in all stations,
with particularly high abundance in the Aït Oufella
(89.8%), Ain Taoujdate (89.1%), Aït Rbaa (78.5%),
and Sidi Mbarek (71.8%) localities. P. sergenti was
captured at all surveyed sites, with a relative
abundance of 7.17% among all specimens, and the
highest proportion (16.6%) in Aït Rbaa. P. papatasi
was found in a relative abundance of 2.05%, and
was not detected in Aït Naaman but was highly
abundant in Aït Brahim (54%). S. minuta was the
most abundant species among Sergentomyia sand
flies, representing 65.9% of them all. It was
present at all sites, with a particularly high
proportion (42.3%) in Aït Naaman.
Density and monthly fluctuation of fauna in El
Hajeb province
The density and monthly variation of wildlife in
the El Hajeb area is depicted in Figure 2. The data
reveals the overall density of the phlebotomine
population over time, showcasing that the highest
total density was seen during the summer and
autumn months. More specifically, July, August,
September, and November had the maximum
density levels with 31 ph/m2/night, 53
ph/m2/night, 101 ph/m2/night, and 21
2
ph/m /night, respectively.
The monthly activity of the five most common
species in El Hajeb Province, namely P. longicuspis,
P. sergenti, S. minuta, P. perniciosus, and P. Papatasi,
demonstrated varying levels of activity throughout
the year (Fig. 3). The months with the highest
activity levels were July (12.8%), August (21.7%),
September (42%), and November (8.5%), whereas
lower levels were observed in May (2.7%) and
June (3.9%). The overall sand fly catches over the
year exhibited a tri-phasic pattern, with the first
peak in early June-July, the most significant peak in
September, and the third peak in November.
In terms of their monthly activity, sand flies that
transmit visceral leishmaniasis evolved differently
based on species. P. longicuspis has three peaks
extending from April to November, with the first
one appearing in June. The number of sand flies
increases steadily until September, which is the
most significant peak. The number then decreases
in October before slightly increasing again in
November for a less significant peak. On the other
hand, P. perniciosus has two peaks in July-August
and November, but it was absent in Aït Brahim
despite being collected in other stations. However,
males captured had the typical form of this species.
Meanwhile, the vector species of cutaneous
leishmaniasis, specifically P. sergenti and P.
Papatasi, followed a biphasic pattern with peaks in
June-July and September.
151
Figure 1: Sand fly sampling sites in El Hajeb (El-Mouhdi, 2021).
DISCUSSION
In Morocco has described 24 sand fly species, five
of which are known to transmit leishmaniasis
(El-Mouhdi et al., 2020a, b; El-Mouhdi et al.,
2022a; Faraj and Himmi, 2020). Our study
identified 12 sand fly species, accounting for 50%
of Moroccan sand fly species, and found that the
five vector species were present, making up
91.6% of identified flies in 83% of survey sites.
Additionally, our results showed that three
species cohabited, a finding consistent with
previous studies in different regions of the
country (Ouanaimi et al., 2015; Guessous-Idrissi
et al., 1997; Boussaa et al., 2008, Guernaoui et al.,
2005). Despite high sand fly abundance,
leishmaniasis incidence remained low in the
studied province, a phenomenon also seen in
other regions of Morocco (El-Mouhdi et al., 2019).
This suggests that the parasite ecology influences
leishmaniasis more than vector distribution, as
previously stated by Ouanaimi et al (Ouanaimi et
al., 2015).
The identified species can be categorized into two
genera, namely Phlebotomus and Sergentomyia
[4]. Out of the identified Phlebotomus species, P.
longicuspis accounted for 79.7% of the sand flies.
This species was abundant throughout the time
period studied and in all six localities, with a
pattern of three peaks in July, September, and
November (El-Mouhdi, 2021). However, the long
active period and high abundance of this
confirmed vector of visceral leishmaniasis (VL)
poses a threat and indicates the potential for
transmission of the visceral form in the province.
These findings corroborate with studies by
Al-Koleeby et al. (2021) and Guernaoui et al.
(2005), which show similar patterns of peaks in
different regions. The explanation for such
variations is attributed to the adaptability of P.
longicuspis to the environmental conditions of
each region (El-Mouhdi, 2021). Around 53% of
this species was captured in the locality of Aït
Oufella, with an altitude of 581 m. Boussaa et al.
(2009) found that this species is abundant
between altitudes of 600 m and 799 m.
The adult P. sergenti was found in all the localities
studied and its period of activity covers the
months of April to November with a bimodal
rhythm, the first in June-July and the other in
September (El-Mouhdi, 2021). Our data
corroborate those obtained in the province of
Sefrou (Talbi et al., 2020).which showed that the
activity of this species is bimodal. This can be
explained by the climatic and geographical
conditions of the province of El Hajeb which have
similar to its neighbor Fez (Talbi et al., 2015). This
result was also obtained through surveys
conducted in the prefectures bordering our study
site of El Hajeb, where the survey also took place
from April to November (Talbi et al., 2020).
Overall, our study highlights the presence of three
important sand fly species in El Hajeb, all of which
exhibit bimodal activity patterns and are potential
vectors for leishmaniasis. These findings
emphasize the importance of integrated vector
control programs for the prevention of sand
fly-borne diseases in the region.
152
Figure 2: Monthly evolution of the total density of the phlebotomine fauna in El Hajeb

Figure 3: Monthly fluctuation of fauna in El Hajeb province by species

CONCLUSIONS

In conclusion, the present study has established

for the first time the phlebotomine fauna of the El
Hajeb region in central Morocco. It provides basic
data on the abundance, biodiversity, seasonality of
sand flies and risk periods. It was revealed that
the different species of sandflies responsible for
the transmission of leishmaniasis disease in El
Hajeb province (P. sergenti, P. papatasi, and P.
longicuspis) predominate the population of adult
sand flies collected. Entomological surveillance
actions must be carried out and accompanied by
actions to sensitize the inhabitants to prevent the
spread of leishmaniases.

153
ACKNOWLEDGEMENTS
The authors are deeply thankful to Mr El Kohli. and
Mr Lakraa of the Laboratory of Medical
Entomology of the National Institute of Hygiene of
the Ministry of Health for their support during this
study. Likewise, they are grateful to Mr El Maakol.
The authors would also like to thank the
authorities and inhabitants of the surveyed
localities for their cooperation and collaboration.
REFERENCES
Al-Koleeby Z, El Aboudi A, Aboulfadl S, Faraj C.
2021. Diversity and Bionomics of Sandflies
(Diptera: Psychodidae) of an Endemic Focus of
Cutaneous Leishmaniasis in Zagora Province,
Southeast of Morocco. J Parasitol Res. e8812691.
https://www.hindawi.com/journals/jpr/2021/881
2691/
Boussaa S, Boumezzough A, Sibold B, Alves-Pires C,
Morillas Marquez F, Glasser N, et al. 2009.
Phlebotomine sandflies (Diptera: Psychodidae) of
the genus Sergentomyia in Marrakech region,
Morocco. Parasitol Res. 104(5):1027‑33.
Boussaa S, Guernaoui S, Pesson B, Boumezzough A.
Seasonal fluctuations of phlebotomine sand fly
populations (Diptera: Psychodidae) in the urban
area of Marrakech, Morocco. Acta Trop.
2005;95(2):86‑91.
http://www.sciencedirect.com/science/article/pii
/S0001706X05001245
Boussaa, S., Boumezzough, A., Remy, P. E., Glasser,
N., & Pesson, B. 2008. Morphological and
isoenzymatic differentiation of Phlebotomus
perniciosus and Phlebotomus longicuspis (Diptera :
Psychodidae) in Southern Morocco. Acta Tropica,
106(3), 184‑189.
https://doi.org/10.1016/j.actatropica.2008.03.011
Boussaa, S., Kahime, K., Samy, A. M., Salem, A. B., &
Boumezzough, A. 2016. Species composition of
sand flies and bionomics of Phlebotomus papatasi
and P. sergenti (Diptera : Psychodidae) in
cutaneous leishmaniasis endemic foci, Morocco.
Parasites & Vectors, 9(1), 60.
https://doi.org/10.1186/s13071-016-1343-6
Boussaa, S., Neffa, M., Pesson, B., & Boumezzough,
A. 2010. Phlebotomine sandflies (Diptera :
Psychodidae) of southern Morocco: results of
entomological surveys along the
Marrakech–Ouarzazat and Marrakech–Azilal roads.
Annals of Tropical Medicine & Parasitology, 104(2),
163‑170.
https://doi.org/10.1179/136485910X126070123
74235
Depaquit J, Léger N. Chapitre 12. Les phlébotomes
(Diptera : Psychodidae : Phlebotominae). In:
Duvallet G, Fontenille D, Robert V, éditeurs. 2018.
Medical and veterinary entomology. Marseille: IRD
Éditions; p. 295‑320:
http://books.openedition.org/irdeditions/22046
El-Mouhdi K, Boussaa S, Chahlaoui A, Fekhaoui M.
2022. Prevalence and risk factors of canine
leishmaniasis in Morocco: a systematic review and
meta-analysis. J Parasit Dis. 46(4):967‑87.
https://doi.org/10.1007/s12639-022-01521-2
El-Mouhdi K, Chahlaoui A, Boussaa S, Fekhaoui M.
2020. Sand Flies Control: A Review of the Knowledge
of Health Professionals and the Local Community,
Province of El Hajeb, Morocco. Int J Environ Res
Public Health. 17(22):8448.
https://www.mdpi.com/1660-4601/17/22/8448
El-Mouhdi K, Chahlaoui A, Fekhaoui M. 2020. The
Cutaneous Leishmaniasis and the Sand Fly:
Knowledge and Beliefs of the Population in Central
Morocco (El Hajeb). Dermatol Res Prac .:e1896210.
https://www.hindawi.com/journals/drp/2020/18
96210/
El-Mouhdi K, Chahlaoui A, Lalami AEO, Bouzid J,
Omari HE, Fekhaoui M. Epidemiological Situation of
Leishmaniasis in the City of El Hajeb (Central
Morocco) During the Period 2013-2017. 2019. Eur
Sci J ESJ ;15(3):155‑168.
https://eujournal.org/index.php/esj/article/view/
11706
El-Mouhdi, K., Chahlaoui, A., & Fekhaoui, M. 2022.
Constraints and Challenges of Local
Operationalization of the National Leishmaniasis
Control Program by Health Professionals : A
Pioneer Study in Morocco. In Emerging Trends in
Disease and Health Research. Vol. 6 (p. 9‑23). Doi:
10.9734/bpi/etdhr/v6/3567E
El-Mouhdi K, Fekhaoui M, Rzeq M, Ennassih Y,
Chahlaoui A. 2021. Knowledge and behaviour of
individuals towards sandflies’ vectors of
leishmaniasis in Morocco. East Mediterr Health J.
27(9):911‑7.
https://applications.emro.who.int/EMHJ/V27/09/
1020-3397-2021-2709-911-917-ara.pdf
El-Mouhdi, K. 2021. Conceptual Scheme for the
Control of the Sand Fly Vector of Human and Canine
Leishmaniasis in Morocco (ELHajeb)”, Ph.D. thesis,
Rabat. University of Mohammed V, Rabat, Morocco,
El-Mouhdi, K., Fekhaoui, M., Elhamdaoui, F.,
Guessioui, H., & Chahlaoui, A. 2020. Knowledge and
Experiences of Health Professionals in the
Peripheral Management of Leishmaniasis in
Morocco (ELHajeb). Journal of Parasitology
Research, e8819704.
https://doi.org/10.1155/2020/8819704
Faraj C, Himmi O. 2020. Updated list of
Phlebotominae (Diptera: Psychodidae) from
154
Morocco. Bull Soc Pathol Exot.;6. =1
Guernaoui S, Boumezzough A, Pesson B, Pichon G. World Health Organization. Leishmaniasis Control
2005. Entomological Investigations in Chichaoua: Report of the WHO Leishmaniasis Control Expert
An Emerging Epidemic Focus of Cutaneous Committee Meeting. 2011.
Leishmaniasis in Morocco. J Med Entomol. 1
42(4):697‑701.
https://academic.oup.com/jme/article/42/4/697/
912354
Guessous-Idrissi N, Chiheb S, et al. 1997. Cutaneous
leishmaniasis: an emerging epidemic focus of
Leishmania tropica in north Morocco. Trans R Soc
Trop Med Hyg. 91(6):660‑3.
https://doi.org/10.1016/S0035-9203(97)90511-3
Masen MA for SE. Etude d’impact environnemental
et social de la centrale solaire photovoltaïque Noor
el Hajeb. 2018.
http://www.masen.ma/sites/default/files/docume
nts_rapport/El%20Hajeb_FESIA_27_07_2018.pdf
Miri HE, Rhajaoui M, Himmi O, Ouahabi S,
Benhoussa A, Faraj C. 2013. Etude entomologique
de cinq foyers de leishmaniose cutanée dans la
province de Sidi Kacem au nord du Maroc. Ann
Société Entomol Fr NS. 49(2):154‑9.
https://doi.org/10.1080/00379271.2013.808504
Moroccan Ministry of Health. Guide of
Leishmaniasis, 2010. DELM;
Ouanaimi F, Boussaa S, Boumezzough A. 2015.
Phlebotomine sand flies of Morocco: results of an
entomological survey along three transects from
northern to southern country. Asian Pac J Trop Dis.
5(4):299‑306.
http://www.sciencedirect.com/science/article/pii
/S2222180814607878
Rioux JA, Golvan YJ. 1969. Epidemiology of
leishmaniasis in southern France. University of
Montpellier Laboratory of Medical Ecology and
Parasitic Pathology. National Institute of Health and
Medical Research.Paris;
Talbi FZ, Faraj C, EL-Akhal F, El Khayyat F,
Chenfour D, Janati Idrissi A, et al. 2015. Diversity
and Dynamics of Sand Flies (Diptera:
Psychodidae) of Two Cutaneous Leishmaniasis
Foci in the Fes-Boulemane Region of Northern
Morocco. International Journal of Zoology. 24 déc
2015:e497169.
Talbi F, Sbai A, el Omari H, Najy M, El Ouali Lalami
A. 2020. Spatial Relation Among Incidence of
Leishmaniasis and Altitude Factor of Different
Communes of Sefrou Province: Contribution of
Geographic Information Systems. p. 1202‑10.
World Health Organization. 2015. Cutaneous
leishmaniasis control in selected countries of the
eastern Mediterranean and African regions report
of an interregional network meeting Casablanca.
https://apps.who.int/iris/bitstream/handle/1066
5/173804/9789241508773_eng.pdf;jsessionid=24
A8D7CF4ABEE9BA604D0E7CC0B25F2B?sequence
155
 Appendix1:

Table 1 . Biodiversity of sand fly fauna captured at the six stations

Station Ain Taoujdate Sidi Mbarek Aït Oufella Aït Rbaa Aït Naaman Aït Brahim
Abundance 3023 1044 6833 2083 1407 200
Males 2227 837 6093 1542 931 85
Females 796 207 740 541 476 115
Sex-Ratio (M/F) 2,8 4,04 8,23 2,85 1,95 0,73
Species richness 9 12 10 11 10 7
H' 0,596 1,173 0,611 0,894 1,751 1,539
E 0,271 0,472 0,265 0,373 0,76 0,791
E: Evenns; H’: Shannon-Wiener diversity index
Appendix2:

Table 2. Inventory of phlebotomine species identified at the six study sites.

Genus Phlebotomus Sergentomyia Total
Subgenus Phlebotomus Paraphlebotmus Larroussius Sergentomyia Grassomyi
a
Species Ph. Ph. Ph. Ph. Ph. Ph. Ph. Se. Se. Se. Se. Se.
papata bergero sergen alexand longicuspi perniciosu ariasi minut schwet fallax antennat dreyfusi
si ti ti ri s s a zi a
Station Aït M 62 63 222 22 5582 107 8 5 5 17 0 0 6093
Oufella F 46 2 82 5 373 18 1 4 2 6 0 0 539
Station Sidi M 35 16 85 5 651 9 2 18 1 4 5 6 837
Mbarek F 21 5 49 3 73 10 0 9 0 0 0 1 171
Station Aït M 0 0 97 7 267 31 15 335 82 44 5 48 931
Naaman F 0 0 19 1 81 3 2 225 40 10 1 10 392
Station Aït M 3 8 284 29 1187 11 0 3 3 7 5 2 1542
Rbaa F 6 0 44 6 365 6 0 7 0 0 1 1 436
Station Ain M 21 14 68 12 2000 105 0 3 3 1 0 0 2227
Taoujdate F 1 3 35 5 631 44 0 6 1 1 0 0 727
Station Aït M 33 26 13 0 0 0 0 4 4 5 0 0 85
Brahim F 61 12 11 0 4 0 0 2 0 0 0 0 90
Total 289 149 1009 95 11214 344 28 621 141 95 17 68 1407
0

156
 CONFERENCE PAPER Samira Jayche et al. 2022, 157-164

Monitoring system for Acute Flaccid Paralysis in Children: the case of

Kénitra Province, Morocco; between 1994 and 2018
Samira Jayche1,2,3*, Ahmed Idrissi2, Sanae Sendaoui4, Amr Loutfi1,3, Mohamed EL Hioui3,
Mohamed Lahmam1, 3, Ahmed Omar Thouhami Ahami3

1 Delegation of the Ministry of health in Kénitra, Morocco, 2 National School of Public Health
(Rabat; Morocco), 3 Departement of Biology, Faculty of Sciences, Biology And Health
Laboratory, Unit of Neuroscience And Nutrition, Ibn Tofail University, Morocco, 4 Delegation of
the Ministry of health in Agadir, Morocco

Article History:
ABstRACT

Morocco has been polio-free since 1989. The objectives of our study are to
review the existing Acute Flaccid Paralysis (AFP) surveillance system in
Kénitra province, identify the strengths and weaknesses of the existing
system, and suggest a list of actions for efficient performance. Qualitative
and quantitative evaluations are conducted in the provincial epidemiology
unit (PEU) of Kénitra. The database of AFP cases is reviewed, and relevant
stakeholder interviews are conducted, consulting guidelines formulated
by the Center for Disease Control and Prevention (CDC) in 2001 for
evaluating public health surveillance systems. The system was flexible.
The "sensitivity" of the AFP Surveillance system has reached 50%. More
than 40% of patients are notified within seven days of the onset of
paralysis. The investigation time within 48 hours of notification exceeds
70%. In Kénitra, the AFP Surveillance system is performant, flexible and
highly vigilant with a capacity for rapid response. Besides, it is vital to
sustaining the AFP Surveillance till the goal of global polio eradication
achieved.

ISBN :
DOI: 10.2478/9788367405256-022

Published by Sciendo. © 2022. Samira Jayche et al.

This is an open access article licensed under the Attribution 4.0 International
(https://creativecommons.org/licenses/by/4.0/)

INTRODUCTION of the effort to reduce polio infections in

The WHO launched Global Polio Eradication
Initiative in 125 countries when polio
paralysed over 350,000 children annually
(GPEI). Humans are the virus' only hosts,
hence vaccines are effective and safe. Efficacy
endemic nations has been shown. The WHO
eliminated wild poliomyelitis 2 in 1999. Until
this year, only wild type 1 or 3 polio cases
have been reported. However, these
serotypes persist in parts of Africa (Nigeria)
and Asia (Pakistan and Afghanistan). On

157
January 30, 2019, GPEI reported one polio
case in Afghanistan(WHO, 2019).
The WHO provides many immunisation
strategies to combat this very contagious
disease. Recently, attenuated monovalent
(mOPV1) and bivariate type 3 vaccinations
from Sabin have helped to speed up the
eradication program. All polio cases must
alsobe recorded. This was later extended to
all AFP in 1996. This Surveillance should be
carried out in each country. The WHO's
worldwide reference lab network employs
virological testing to detect AFP viruses.
Characterization of polioviruses as wild or
vaccine versions allows rapid responses and
the selection of suitable strategies to halt
viral transmission, such as vaccination
campaigns. Polio surveillance relies on AFP
surveillance. Enterovirus surveillance in
clinical specimens and environmental
poliovirus surveillance are recognised by the
WHO. Despite being eradicated in Morocco,
polio persists in some African countries.
Poliovirus surveillance entails reporting and
examining any AFP in a child under 15 years
old(MPH, 2012). Enterovirus monitoring
ensures that poliovirus is not circulating in
enterovirus samples from non-AFP
patients(M. Rhajaoui, 2015).
To increase the quality, efficiency, and
effectiveness of the AFP Surveillance System
in Morocco, it must be evaluated. The Atlanta
CDC has created a handbook to assist assess
public health monitoring systems
(qualitative, quantitative, utility and costs
involved). This plan identifies system flaws
and makes solutions for enhancing disease
monitoring(R. R. German et al 2001).
SYSTEM DESCRIPTION
AFP Surveillance is vital to the country's polio
eradication. A high-risk group of patients is
monitored closely syndromically. AFP
Surveillance is now a global active/passive
system. Surveillance is defined as the
systematic reporting of suspicious incidents.
The active Surveillance also gives a weekly
report of all negative events.
The Principal Objective
The objective of this work consists of a rapid
evaluation of the Surveillance system based
on the CDC guide and the WHO tools to
identify structural weaknesses and priority
actions to be implemented.
The Specific Objectives Include
✔ Identification, declaration and rapid
investigation of any case of AFP in children
under Fifteen years old, regardless of the
diagnostic hypothesis. As well as the
suspicious diagnosis of polio in individuals
of all ages;
✔ Timely detection and analysis of AFP
/ poliomyelitis outbreaks to rapidly
implement control measures,
✔ Monitoring and evaluation of AFP
trends;
✔ Early identification of imported or
indigenous cases of polio.
✔ Surveillance and maintenance of
polio eradication, the definitions of
suspected, confirmed, compatible,
eliminated, and associated with vaccination
have been established.
MATERIALS AND METHODS
Study Area:
Our study covers the province of Kénitra,
located in the North-West of Morocco is part
of the Region Rabat SaléKénitra (RSK). It is
one of the largest Moroccan cities with a rich
agricultural area.
Case Definitions
A suspected case is any sudden onset flaccid
motor disability in a child under the age of
15, regardless of the diagnostic hypothesis,
and to all suspected polio in people of all
ages.
158
A confirmed case is any case in which wild
poliovirus is isolated from the case or a close
relative's excretion.
A compatible case is one in which no stool
sample was taken, the sequelae occurred
within 60 days, and the patient died or was
ignored.
A rejected case has a negative result, which
was collected on time.
A case of polio associated with the vaccine
is defined as any case of acute flaccid
paralysis in which the vaccine virus was
isolated from stool samples and had sequelae
consistent with polio, occurring up to 60 days
after the onset of motor impairment.
Figure 1: Diagram of conduct to follow in a case of AFP
All AFP cases that fulfil the WHO definition will
be managed consistently, which includes
treating them as polio cases until final
classification, even if it is clear from the
beginning that this is Guillain-Barré syndrome
or another non-polio AFP (Figure 1).(MPH,
2012).
METHODS
It is a retrospective and descriptive study of
active and passive AFP surveillance in the
province of Kénitra during a 25 years period,
AFP epidemiological Surveillance system:
The AFP epidemiological Surveillance system
uses four instruments for information
collection: the AFP epidemiological
investigation form, the specimen collection
form, the AFP cases entourage collection
form, and the investigation form of a case
contact. All forms include: general data,
individual notification, residency data,
epidemiological history, clinical data, care,
laboratory results, case progress and
conclusion. The Regional Delegation and the
Department of Epidemiology and Disease
Control (DEDC) must be notified right away
from 1994 to 2018. To evaluate the AFP
Surveillance system in Kénitra province, we
used the updated and revised version2001 of
public health Surveillance systems evaluation
(CDC) guidelines (Table 1) and WHO
performance indicators of AFP Surveillance
(Table 2)(MPH, 2012, R. R. German et al 2001).
We evaluated system performance using
qualitative analysis criteria like simplicity and
flexibility, as well as quantitative analysis
features like sensitivity and representativeness
159
Table 1: Summary of AFP monitoring system attributes goals and performance indicators
according to CDC standards
Example of Performance
Attribute Goal
Indicator
As simple as possible in terms of Amount and type of data needed to
Simplicity structure and ease of confirm that the health-related
implementation event has occurred
Can adapt quickly and easily to Retrospective assessment of how
Flexibility changing information needs or the system has adapted to the new
implementation conditions demand
Completeness and validity of Percentage of unknown or missing
Data Quality
data responses for key data variables
The willingness of individuals
Acceptability and organizations to participate Completeness of reporting forms
in Surveillance
A high proportion of cases of a The percentage of cases in a defined
Sensitivity
disease or epidemic detected population under Surveillance
A high proportion of reported
Positive Predictive Evaluation of "false positives"
cases with the monitored health
Value proportion detected by Surveillance
event
Accurately describes
Comparison of characteristics (e.g.
health-related events over time
age, sex, and geographic location) of
Representativeness and their distribution in the
reported events with all actual
population by location and
events
person.

Table 2: Performance Indicators of AFP Surveillance according to WHO

Indicators Objective
Completeness of weekly sheets ≥90%

AFP-NP Ratio ≥1
Adequate specimens ≥80%

Stool condition ≥80%

Samplesshippedin 72 hours ≥80%
Non-polio enterovirus isolation ≥10%
Collection within 14 days ≥80%
Follow-up exams performed in 60 days 100%
Notification in health system ≤7 Days ≥80%
Health system investigation in 48-hour ≥ 80%

160

RESULTS
Qualitative attributes:
Simplicity:
The Surveillance system has five case
definitions. Confirmation or definitive
elimination of poliomyelitis requires
laboratory Surveillance, including of timely
stool collection and specificresearch on wild
poliovirus. Health care, surveillance, and the
environment all play a role in the monitoring
process. It requires professionals with
specialized knowledge, two neurological
evaluations (acute phase and re-visit) are
necessary. In addition, we use of complex
complementary examination and others
fewer complexes, to be done in a hospital
environment, such as a lumbar puncture
Flexibility
In Morocco, polio Surveillance and control
have evolved over time. AFP Surveillance is
integrated with the notifiable disease
epidemiological Surveillance system. This
monitoring is governed by: ⎫ Royal Decree
No. 554-65 of 17 Rabii i 1387 (June 26,
1967), enacting a law making it compulsory
to report illnesses; ⎫ The decree of the
Minister of Public Health n° 683-95 of 30
Choual 1415 (March 31, 1995) specifies
modalities of application of the royal decree.
To make polio Surveillance more effective in
Morocco, the Department of Epidemiology
and Disease Control (DEDC) of the Ministry of
Health launched in 1995 an active
Surveillance system for AFP: ministerial
circular N ° 2 / DE / 02 of 25/03/1994),
applied a national level. A manager (Mr PFA)
was selected for each PEU; he plays an
essential role in the active search for
instances of AFP in hospital departments. The
PEU manager ensures the rapid recovery of
clinical samples and epidemiological
information to be sent to the DEDC and the
National Institute of Hygiene (NHI). Since
1994, the NHI has been a reference
laboratory, and it was certified in 2001. Its
function entails evaluating all AFP cases
reported in Morocco in search of polioviruses
or other nonpolio enteroviruses responsible
for AFP seen in children under the age of 15,
as well as in the patients' entourage, if the
AFP case was not collected within 14 days of
the beginning of paralysis. The information
and the results obtained are transmitted to
the DEDC and then to the WHO.
Quantitative attributes
Sensitivity
The sensitivity of a system is its ability to
detect all cases of AFP; it’s taking the
proportion of expected cases compared to
detected cases. It enables a system with little
resources to identify any aberrant propensity
for sickness in a population in real time. The
annual number of AFP cases reported ranged
from zero (in 1999/2000) to 9 (in 2015). The
aim of 6 cases/year (2 cases / 100,000
residents under 15) met in 2002, 2004 and
2007; however, we could not surpass 4
instances in the other years. The sensitivity of
AFP’s Surveillance system founded to be
50%.
Representativeness
The under 5-age group is affected by AFP in
51% and 21% of children aged between 11
and 15 years old, and the youngest case was a
newborn aged three months. Male accounted
for 59% of patients, and rural areas 77% of
AFP notifications. Among the 23
municipalities in the province of Kénitra, the
municipality of Kénitra (the capital)
represents the highest frequency of notified
AFP cases (15/76), followed by ben Mansour
with 10 cases and Sidi Boubker El-Haj with
only one declared AFP case for 25 years.
161
DISCUSSION
In Morocco, the last endemic case was in
1989. Polio eradication, including the cases
and the virus from the world, is the current
goal (WHO, 2019). Surveillance of AFP cases
is the best method for controlling the
circulation of wild poliovirus (WPV)(WHO,
2019, MPH, 2012). For this purpose, we must
maintain the continuity of all the detection,
investigations and monitoring procedures(S.
Fontanaet al, 2021)and also the recording,
securing and results of the database. In our
study,we found that the system was not
simple, similar findings reported in others
studies(S. Fontanaet al, 2021, M. Muzondo et
al, 2018). However, it is seen as flexible.
Changes to the system, research file or
information flow require adaptations,
training, recruitments, meetings, and
publication of guides(B. Hashim et al, 2020).
The system is considered flexible. Training,
recruitments, meetings, and the printing of a
guide are all necessary when a new system,
research file or information flow is
implemented. (A. H. Kakar, 2014, R. N. Nnaji
et al, 2020).
We also found out that the AFP Surveillance
system was not sensitive(M. Muzondo, A et al,
2018, J. Masa-Calles et al, 2018). The
sensitivity was affected by the vastness of the
province of Kénitra and under-reporting of
cases. In Kénitra, the AFP Surveillance system
is generally representative. The cases
distributed geographically, and cases arrived
of several province communes(B. Hashim et
al, 2020). Kénitra is the commune that
occupies first place with 15 cases (19%). The
high population density of Kénitra commune
might be a factor in this difference. This
difference in reporting between different
areas has also observed at the national
level(I. Tadmori et al, 2010, L.S. Khuzwayo et
al 2013, A. Levitt et al 2014, M.M. Alleman et
al, 2014).
The obtained results indicate that the age of
the remarkable occurrence was 1 to 5 years
(51%) (F. Sevencan et al, 2013, J. G. T.
Doumtsop et al, 2014, D. O. Mariam et al,
2015)of AFP cases with male predominance
(59%)(J.K. Odoom et al, 2018), and the
patients came from rural areas (77%) (I.
Tadmori et al, 2010).
CONCLUSION
Active Surveillance of non-polio PFA was
shown to be useful in this investigation. The
predicted minimum reporting rates for
non-polio PFA were met in2002, 2004, 2007,
and 2015 (1 case per 100,000 children under
the age of 15). The system is considered
effective in achieving desired objectives
Efforts should be taken, however, not just to
preserve the current approach, but also to
ensure that all incidents of AFP are reported
and examinedas quickly as feasible.
Furthermore, assessing the AFP incidence
rate in various locations on a regular basis is
required in order to evaluate the monitoring
system and guarantee the continuing
eradication of poliovirus.
. RECOMMENDATIONS
• Prioritize and sensitize health professionals
to the notification of AFP cases.
• Report and investigate any cases of AFP
under the age of 15 for timely control action
immediately.
• Conduct an active search at reference
hospitals and clinics, as well as check the
medical files of children under the age of 15
who have been hospitalised with a diagnosis
of AFP.
• Increase trusted notification sources.
• Involving the private health sector in the
reporting of AFP cases
ACKNOWLEDGMENTS
The authors would like to thank all the
health staff of the Provincial Epidemiology
Unit of the Health Delegation in Kenitra,
whose participation made this study
possible, the team of the Epidemiology
Directorate and the fight against diseases
(DEDC), the virology laboratory of the
National Institute of Hygiene. They also
recognize the support of the National School
of Public Health (ENSP), especially the FETP
program to develop this work.
162
REFERENCES
Alleman, MM., Meyer SA., Mulumba
A.Nyembwe, M., RizikiY.Mbule, A., and al.
2014. Improved acute flaccid paralysis
Surveillance performance in the
Democratic Republic of the Congo,
2010–2012.J Infect Dis.;210(Suppl 1)
DoumtsopJ, G., KhalefI, T., Diakite, ML,
Boubker, N., 2014. Epidémiologie des PFA
et les performances du système de
Surveillance en Mauritanie de 2008 à
2012. Pan AfricanMedical Journal, 18(1).
Fontana, S., Buttinelli, G., Fiore, S., Amato, C.,
Pataracchia, M, Kota, M, Aćimović, J,
Blažević, M, Mulaomerović, M,
Nikolaeva-Glomb, L, Mentis, A,
Voulgari-Kokota, A., Gashi, L.,
Kaçaniku-Gunga, P., Barbara, C., Melillo, J.,
Protic, J., Filipović-Vignjevic, S., O'Connor,
PM., D'Alberto, A., Orioli, R., Siddu, A.,
Saxentoff, E., Stefanelli, P. 2021.
Retrospective Analysis of Six Years of
Acute Flaccid Paralysis Surveillance and
Polio Vaccine Coverage Reported by Italy,
Serbia, Bosnia and Herzegovina,
Montenegro, Bulgaria, Kosovo, Albania,
North Macedonia, Malta, and Greece.
Vaccines (Basel) ;10(1):44. doi:
10.3390/vaccines10010044. PMID:
35062705; PMCID: PMC8779529.
German, RR., Lee, LM., Horan, JM., Milstein, R.,
L,. Pertowski, CA., Waller, MN. 2001
Guidelines Working Group Centers for
Disease Control and Prevention (CDC).
Lignes directrices mises à jour pour
l’évaluation des systèmes de Surveillance
de la santé publique : recommandations
du Groupe de travail sur les lignes
directrices. MMWR. Recommandations et
rapports : Rapport hebdomadaire sur la
morbidité et la mortalité.
Recommandations et rapports, 50(RR-13),
1–CE7.
Hashim, B., Yahaya, M., Nyampa, B., Lukman,
S., Polycarp, O. 2020. Evaluation of Acute
Flaccid Paralysis (AFP) Surveillance
system, Kebbi State, Nigeria, 2013-2018.
10.21203/rs.3.rs-23557/v1.
Kakar, AH. 2014. Evaluation of acute flaccid
paralysis (AFP) Surveillance system in
BalochistanKakar A. International Journal
of Infectious Diseases, 21, 239.
Khuzwayo, LS., KuonzaL, R., Ngcobo, NJ. 2013.
Evaluating the acute flaccid paralysis
Surveillance system in South Africa,
2005–2009: an analysis of secondary data.
Pan Afr Med J.;14:86.
Levitt, A., Diop, OM., Tangermann, RH.,
Paladin, F., Kamgang, JB., Burns, CC., and
al. 2014. Surveillance systems to track
progress toward global polio
eradication—worldwide, 2012–2013.
MMWR Morb Mortal
WklyRep.;63:356–61.
Mariam, DO., Ange-Éric, KA., N’tah, BSC.,
Léonard, KK., Ismaila, D., Thérèse, SD.,
2015. Paralysies flasques aiguës (PFA) de
l’enfant en côte d’ivoire (CI): données
actuelles. Revue Neurologique, 171,
A138-A139.
Masa-Calles, J., Torner, N., López-Perea, N.,
Torres de Mier, MV., Fernández-Martínez,
B. , Cabrerizo, M., Gallardo-García, V,. Malo,
C., Margolles, M., Portell, M., Abadía, N.,
Blasco, A., García-Hernández, S., Marcos,
H., Rabella, N., Marín, C., Fuentes, A.,
Losada, I., Gutiérrez JG., Nieto, A., 2018.
Spanish Afp Surveillance Working Group
Acute flaccid paralysis (AFP) Surveillance:
challenges and opportunities from 18
years' experience, Spain, 1998 to
2015. Euro Surveillance: bulletin
Europeen sur les maladies transmissibles
= European communicable disease
bulletin, 23(47), 1700423.
Minister of Public Health. 2012. Guide to
epidemiological Surveillance of acute
flaccid paralysis, Morocco.
Odoom, JK., Ntim, NAA., Sarkodie, B., Addo, J.,
Minta-Asare, K., Obodai, E., and al. 2018.
Evaluation of AFP Surveillance indicators
in polio-free Ghana, 2009–2013. BMC
Public Health. 2014; 14(1):687
Muzondo, M., Shamu, A., Shambira, G., Gombe,
NT., Juru, TP., Tshimanga, M. 2018.
Evaluation of the acute flaccid paralysis
(AFP) Surveillance system in Mwenezi
163
 district, Masvingo, 2018: a descriptive
study. BMC research notes, 11(1), 875.
Nnaji, R., Osai , P., Nguku, P., Ihekweazu, C.,
Ezeudu, CC. 2020. Acute flaccid paralysis
Surveillance system evaluation-Enugu
state, Nigeria 2015–2018. International
Journal of Infectious Diseases, 101, 337.
Rhajaoui, M. 2015. Bulletin of the National
Institute of Hygiene.;4(Editorial). Rabat.
Maroc 2015 Bulletin_INH_N4.pdf
Sevencan, F., Ertem, M., Öner, H., Aras Kılınç,,
E., Köse, OÖ., Demircioğlu, S., Dilmen, F.,
Eldemir, R., Öncül, M. 2013. Acute flaccid
paralysis Surveillance in southeastern
Turkey, 1999-2010. The Turkish journal of
pediatrics, 55(3), 283-291.
Tadmori, I., Elalj, I., Chaouki, S., Bouharrou, A.,
Hida, M. 2010. P319-Les paralysies
flasques aiguës à propos de 29
cas. Archives de pédiatrie, 17(6), 129-130.
WHO. 2019. Global polio eradication initiative
annual report 2019 and semi-annual
status updates, January - June and July -
December. 9789240013117-eng.pdf
(who.int).
164
 CONFERENCE PAPER El aameri Mohammed et al.,2022, 165_173

Evaluation of dietary habits in type 2 diabetic subjects

El aameri Mohammed *1 and Taboz Youness ²
1
Laboratory of Natural Resources and Sustainable Development-Ibn Tofail University-Faculty of science
-B. P 133. (Kenitra)-Morocco.
² Laboratory of Natural Resources and Sustainable Development-Ibn Tofail University-Faculty of science
-B. P 133. (Kenitra)-Morocco.

444
Corresponding Author:
Name: Mohammed EL AAMERI
Email: laamri1900@gmail.com
Phone:2126424749099.

DOI: 10.2478/9788367405256-019
ISBN: 9788367405256

Published by Sciendo. © 2023. All rights reserved.

165
such as diabetic foot) and macrovascular
complications (vascular disease and stroke).
The type 2 diabetic patient must be able to
follow a particular dietary education with the
help of the attending physician or dietician
(ADA,2008).In type 2 diabetic patient,
management limited only to medication
(OADs: oral antidiabetic drugs and insulin)
cannot be effective without a pre-established
and rigorous diet. Most type 2 diabetics are
unaware of the good dietary habits to follow.
The objectives of the nutritional approach are
to normalize blood glucose levels by balancing
dietary intake, regulate glycated hemoglobin
and avoid as much as possible the
degenerative complications caused by type
two diabetes, which is often fatal
(cardiovascular diseases). Diabetes is a
disease that requires above all an essential
nutritional management, mainly
recommended by a nutritionist. There will be
no good clinical practice without an adapted
prescription and some dietary rules (ADA,
2019). A sedentary lifestyle, overweight, a diet
too rich in calories and saturated fats are
factors that aggravate the action of insulin and
promote insulin resistance which is
responsible for the onset of type 2 diabetes.
The nutritional management of the diabetes
disease, therefore, consists of knowing the
relationship that exists between food,
nutrients, weight and insulin resistance
(Gulbrand et al, 2012). Diabetes constitutes a
health problem because of its constantly
increasing prevalence in developed and
developing countries. Moreover, diabetes is
characterized by its severity, the essential
source of which are the long-term
complications that contribute to death,
disability and incapacity. Cardiovascular
complications are the most involved. Before
physical education and medication, the
prevention of type two diabetes relies
essentially on nutrition (ANAES ,1999) ;(Gin
and Regalia ,1999). Malnutrition may be
defined as a multi factorial syndrome that
implies to the complex role between
deficiency in the dietary intake, increased
requirements associated with disease-related
metabolic alterations and/or decreased
availability of nutrients due to processes such
as poor absorption and/or immoderate
nutrient losses (Sauders, 2010).
MATERIAL AND METHODS
The descriptive cross-sectional survey is
conducted over 6 months in the Moulay
Hassan public Hospital in Kenitra (Morocco).
The method used is a questionnaire specific
with items on diabetic eating behaviour. These
items provide important foundation for the
diabetic patient’s diet. Diabetic patients,
generally elderly, with a blood sugar level
above 1.25 g/l are included. The interviews
were conducted in a health clinic, specifically
in the diabetic department. The number of
people with diabetes counted was 300 type
two patients. The interviews respected ethical
standards. The most important parts of this
questionnaire focus on research of
hygienic-dietary measures in type two diabetic
subjects. The survey is anonymous and
respects the ethical norms. In short, the
chosen questionnaire aims to evaluate the
dietary frequency in diabetic people.
RESULTS
Out of 300 type two diabetics, the results of
our survey show a percentage of 44.6% of
men and 55.4% of women. The age of type 2
diabetics varies considerably from the
youngest (24 years) represented by 0.4% to
the oldest (89 years) represented by 0.8%.
The age of 62 years represents the highest
percentage of 5.8%, followed by the age of 47,
which is 4.2%. 77% of people with diabetes
consume less than five amounts of fruits and
vegetables and 21% down five portions of
fruits and five portions of vegetables.
Concerning the frequency of daily meals, the
highest percentage is observed in number 4
(48%). Most type two diabetics do not eat
outside their homes (62%), followed by 22%
for those who eat once a week outside their
homes. People with diabetes occasionally
snack (66%). The foods that diabetics snack
on are fruits (50%).54% don’t skip meals. In
166
addition, if diabetics skip a meal (46%), dinner
is the meal that is usually overlooked (33%).
People with diabetes prefer chicken (70%)
and red meat (20%). They also consume one
liter of water (36%). The preferred breakfast
of diabetics is type 1 (the moderation of sugar
and composed of olive oil) (62%). The
consumption of cereals: one time per week
(57%) is the most observed, the consumption
of vegetables: three times per week is about
43%. The consumption of dairy products is
three times a week (42%), meat: one time a
week (51%), fish: two times a week (52%),
eggs: two times a week (55%), fats: 3 times a
week (47%), sweet products two times a week
(42%) (see Table 4). Only 31% of people with
diabetes think that diet can be an essential
factor in the control of diabetes and 48% don’t
think so, while 64% are moderately informed
about the nutrition of diabetics. There is a
significant statistical relationship between
hypertension in diabetic patients and age
(p=0,001) and a significant relationship
between dyslipidemia and age (p=0,006)
(Table 1). 46% of patients are hypertensive.
(Figure 2).
DISCUSSION
This study shows that the diabetic patients
followed had poor dietary practices marked by
irregular meal times, frequent snacking (66%)
and consumption of foods not recommended
for people with diabetes (MG 47%: three times
per week) and (PS: 44% two times per week).
This behaviour would be related to poor
knowledge of nutritional measures exposing
them to cardiovascular complications and
glycemic imbalance. 48% of diabetics do not
think that diet can be an essential factor in
glycemic control and 64% are only moderately
informed about diabetic nutrition. The results
observed in our study show that type two
diabetic patients are not aware of the diet
designed for type 2 diabetics and undertake a
relatively unbalanced diet. In fact, their
consumption of fiber (fruits and vegetables:
three times a week) remains clearly
insufficient while the consumption of fats
(three times a week) is not advisable. It is also
essential to limit sugary products and some
products (butter, cheese) that increase the
level of bad cholesterol (HDL). We can
evaluate the patient's usual dietary behaviour
by an initial nutritional survey, which allows
us to specify the caloric level, the number of
meals taken per day, and the distribution
between the different nutrients. The diet
should be divided into three main food
intakes. Food selection and dietary pattern are
influenced by the patient’s knowledge of a
recommended diet (Sami et
al,2020).Therefore, dietary evaluation is of
utmost importance for health promotion,
disease prevention and individual treatment
plans in patients with diabetes. The American
Diabetes Association (ADA) recommends that
treatment of diabetes include lifestyle changes
such as a low-fat, low-carbohydrate diet and
reduced protein intake to reduce
cardiovascular risk factors and increase
insulin sensitivity (Nathan,2006)
;(Castaňeda-Gonzales et al,2011). The
nutritional strategy is a personalized
prescription of hygienic and dietary rules
adapted to the patient's personal history and
lifestyle. This strategy is an integral part of the
management of diabetes (Bantle et al ,2008).
Even with optimal drug treatment, a
significant reduction in HbA1c after
nutritional therapy is observed in type 2
diabetics (Connell et al , 2010);( ENTRED,
2010). It is worthwhile to encourage reducing
energy intake in the evening and favouring
vegetables in the morning and at lunchtime to
lower HbA1c and thus improve glycemic
control. Excess energy intake in the evening
increases insulin resistance (Moris and
Wylie-Rosett, 2010). Like any chronic
pathology, diabetes poses the problem of
nutrition and therapeutic factors. Our study
allowed us to determine the frequency of
nutritional status and behavioural factors in
diabetics at the Hospital Moulay Hassan health
center (Kenitra, Morocco). We identified
factors influencing social factors, the disease,
the patient and the type of treatment as well
as food consumption. The nutritional care of a
167
type 2 diabetic patient consists on the generally recommended to precede drug
knowledge of the relation between nutrients, treatments for type two diabetes by a period
food, weight and the insulin-resistance (Gin et of type two diabetes with a dietary period of a
al , 2003). few weeks or months (Inzucchi et al, 2015).
Dietary measures are useful, if not
CONCLUSION indispensable, at different stages of the
natural history of the disease (Sachon et al,
To limit micro and macro-vascular
2007). Type 2 diabetes evolves over many
complications, blood pressure must be
years through years, passing through several
perfectly controlled. To do so, all diabetic
periods: pre-diabetes, un recognized diabetes
patients must follow the "Dietary Approaches
and finally known diabetes with three
to Stop Hypertension" or DASH-style, in
schematic stages depending on whether it is
addition to the hygienic and dietary rules
free of complications or whether it is
induced by insulin resistance, to limit their
accompanied by non-disabling complications
daily sodium and intake and increase their
or disabling complications. All these stages are
potassium intake according to their renal
accompanied by insulin resistance, more or
function. The resulting dietary rules also imply
less marked glycemic disorders which worsen
weight loss if the patient is overweight,
with the progressive disappearance of residual
moderation of alcohol consumption, and
insulin residual insulin secretion. As the
intensification of physical exercise (ENTRED
disease progresses, blood pressure and lipid
,2010). As we have seen previously, it is crucial
abnormalities appear (diabetic dyslipidemia)
to limit lipid consumption, particularly
which increase the risk of vascular
atherogenic lipids, and to favor nutrients that
complications. Dietary measures such as
increase HDL cholesterol. Various studies have
dietary measures as well as medicinal
shown that medical nutrition therapy (MNT)
treatments aim to reduce insulin resistance, to
administered by a dietician or nutritionist is a
safeguard residual insulin secretion insulin
critical component in the administration of
secretion, to reduce glycemic, lipid and blood
diabetes and an essential complement to the
pressure disturbances and to avoid the
management by the physician (Post et al,
passage from a given stage of the disease to
2010) . Therapeutic Patient Education (TPE) is
the next of the disease to the next stage. As an
recommended to improve the knowledge and
example, it has been clearly demonstrated by
understanding of type two diabetic patients
several prevention studies that the risk of
and aid dietary and nutritional behaviour
converting glucose intolerance to diabetes is
change. The basic rules consist of a balanced
reduced on average by to diabetes is reduced
diet, obtaining or maintaining weight as close
by an average of half by dietary measures
as possible to a "healthy weight" and regular
combining a calorie-restricted diet and calorie
physical activity (Wheeler et al, 2012) . In
restriction and physical activity (Tuomilehto
2006, a group of experts acting on behalf of
et al, 2010).The evaluation of dietary intake is
the American Diabetes Association (American
essential because it allows the identification
Diabetes Association) and on behalf of the
and evaluation of interventions focused on
EASD (European Association for the Study of
improving nutrition in people with diabetes.
Diabetes) recommended that nutritional and
pharmacological treatment of type two
diabetes should be initiated concomitantly, as
soon as the disease was diagnosed (Shai et al
,2008). The diagnosis of the disease. This
attitude is somewhat surprising, as it is

168
 Table 1. Distribution of biological parameters of patients by gender and age.

.
biological parameters Gender (%) Age (%) P
* < 0,05
M W <40 [41-50] [51-60] [61-70] >70

< 1,26 g /l 6,6 8 1,3 1,3 4,6 4,3 3

Glycemia (g/l) [1,26–2] 25,6 27,6 5,3 12,6 15,3 11,3 8,6 P=0,724
[2 – 3] 11,3 14 2,3 4,6 6 6,3 6 P=0,410
> 3 g /l 2,3 4,3 0,3 0,6 1,6 3 1

<6 4 0,3 0,3 1,3 2 0 0

[6 – 7] 10 13,6 3,6 5,3 7,3 5,3 2,3 P=0,399
HbA1c [7 - 8] 13 14,3 1,3 7 1,3 6 8,6 P=0,531
(%) [8 – 10] 15,3 15,3 3,3 4,3 7 10 6
> 10 3,3 9,6 0,6 1,3 5,3 3 2,6

HTA Normale 27,6 26,6 6,6 13,6 15,3 11,6 7 P=0,062

(Hypertension) Abnormal 18,3 27,3 2,6 5,6 12,3 13,3 11,6 P=0,001*
(mmHg)

Dyslipidemia Normale 36,3 38,6 8,6 16 21 18,6 11 P=0,068

(Cholesrerol Abnormal 9,3 13,3 0,6 3,3 6,6 6,33 7,6 P=0,006*
HDL-LDL)

Figure 1. Distribution of patients with diabetes by BMI and age.

169
 Figure 2. Distribution of patients withs diabetes by genetic link and hypertension.

Table 2. Frequency of consumption of fish 30% 52% 10% 8%

essential foods in type 2 diabetics Eggs 15% 55% 23% 7%
fat 5% 39% 47% 9%
Food 1T/w 2T/w 3T/w More
Sugar 3% 44% 43% 10%
cereal 5% 35% 5% 3%
products
Fruits 2% 26% 49% 23%
1T/w : one time of the week.
Vegetable 4% 20% 43% 33%
Daury 8% 39% 42% 11% Table 3. Dietary and dietary information for
products type 2 diabetics
Meat 51% 38% 8% 2%

170
 Do you think that Yes 31% nutrition for Medium 64 %
diet can be an No 48% diabetes? Never 16 %
important factor in
Maybe 21%
the control of
diabetes?

Are you sufficiently Strongly 20%

informed about

Figure 3. Dietary advice for different population for the prevention and management of type 2
diabetes
(Mozaffarian ,2016)
Table 4. Eating habits of type 2 diabetic people

Yes 46 %
Do you skip certain No 54 %
meals?
Chiken 70 %
What do you usually Red meat 20 %
eat with meals?
Fish 10 %

500 ml 27 %
1L 36 %
How many liters of 1.5 L 25 %
water do you drink
per day ? More than 1.5 L
2%

outside your More than 3 times

home? 6%
One time 22 % Never 62 %
How many times a Two times 10 % Frequentley 18
week do you eat
%

171
Do you snack Time to time 66 % What food do you Sweetened drinks
between meals ? tend to snack on ? 30%
Never 16 %
Fruits 50 %
Sweets 20 %

REFERENCES
American Diabetes Association, Bantle JP,
Wylie-Rosett J, et al. Nutrition
recommendations for the treatment and
interventions for diabetes: a position
statement of the American Diabetes
Association. Diabetes Care 2008; 31 Suppl
1:S61.
American Diabetes Association. Diabetes Care.
2019 Jan;42 Supplement 1:S46–S60.
ANAES. Suivi du patient diabétique de type 2.
Recommandations. Diabètes Métab
1999;2(suppl2).
Connell KL, Kataoka M, Williams SM, Chisholm
AW, Vorgers SM, Mann IL, Nutritional
intervention in patients with type 2 diabetes
who are hyperglycemic despite optimised
drug treatment- Lifestyle Over and Above
Drugs in Diabetes (LOADD) study:
randomised controlled trial. BMJ 2010;
341:c3337
Gin H, Rigalleau V, Baillet L. Diet and physical
activity in type 2 diabetes prevention. Rev
Prat. 2003;53(10):1074–1077.
Gin H, Rigalleau V. Diététique et diabète. Encycl
Méd Chir 1999 (Elsevier SAS, Paris),
Endocrinologie, 10-366-R-10, 9p.
INVS/ Etude ENTRED: Prévalence et
incidence du diabète, et mortalité liée au
diabète en France - Synthèse
épidémiologique.2010
Inzucchi SE, Bergenstal RM, Buse JB, et al.
Management of hyperglycaemia in type 2
diabetes, 2015 : A patient
centered-approach. Update of a Position
Statement of the American Diabetes
Association and the European Association
for the Study of Diabetes. Diabetes Care
2015 ;38 : 140–9.
J Saunders T Smith Malnutrition: causes and
consequences.Clin Med
(lond)201010662427https://www.ncbi.nlm
.nih.gov/pmc/articles/PMC4951875/
LM Catsañeda-Gonzalez, M Bacardi Gascon,
and A Jimenez Cruz. Effects of low
carbohydrate diets on weight and glycemic
control among type 2 diabetes individuals: a
systemic review of RCT more significant
than 12 weeks. Nutr Hops vol.26 no6
Madrid Nov-Dec 2011.DOI : 10.1590/S0212.
Morris SF, Wylie-Rosett J. Medical Nutrition
therapy: A key to diabetes management and
prevention. Clin Diabetes, 2010;13:12-18.
Mozaffarian D.Dietary and policy priorities for
cardiovascular disease, diabetes, and
obesity: a comprehensive
review. Circulation2016;133:187-
225. doi:10.1161/CIRCULATIONAHA.115.01
8585 pmid:26746178.
Nathan DM, Buse JB, Davidson MB et al.
Management of hyperglycemia in type 2
diabetes: a consensus algorithm for the
initiation and adjustment of therapy: a
consensus statement from the American
Diabetes Association and the European
Association for the Study of Diabetes.
Diabetes Care 2006; 29: 1963- 72.
Post RE, Mainous AG III, King DE, Simpson KN.
Dietary fiber for the treatment of type 2
diabetes mellitus: a meta-analysis. J Am
Board Fam Med. 2012; 12:16-23. DOI :
10.3122/jabfm.2012.01.110148.
R Gulbrand, B Dizdar, B Buniaki, T lindstrom, M
Bachrach-Lindstrom, M Fredikson, CJ
Ostgren, and FH Nystrom. In type 2 diabetes,
randomisation to advise to follow a
low-carbohydrate diet transiently Improves
glycaemic control compared with advice to
follow a low-fat diet producing a similar
weight loss. Diabetologia, 2012; 55(8):
2118-2127.
Sachon C, Masseboeuf N, Grimaldi A.
Alimentation et insulinothérapie
fonctionnelle. Médecine des Maladies
Métaboliques 2007 ; 1 : 26–32

172
Sami W., Alabdulwahhab K.M., Ab Hamid M.R.,
Alasbali T.A., Al Alwadani F., Ahmad M.S.
Dietary Knowledge among Adults with Type
2 Diabetes—Kingdom of Saudi Arabia. Int. J.
Environ. Res. Public Health. 2020;17:858.
Shai I, Schwarzfuchs D, Henkin Y, et al., for the
Dietary Intervention Randomized
Controlled Trial (DIRECT) Group. Weight
loss with a low-carbohydrate,
Mediterranean or low-fat diet. N Engl J Med
2008 ; 359 : 229–41.
Tuomilehto J, Linstrom J, Eriksson JG, et al.
Prevention of type 2 diabetes mellitus by
changes in lifestyle among subjects with
impaired glucose tolerance. N Engl J Med
2001 ; 344 : 1343–50.
Wheeler ML, Dunbar SA, Jaacks LM, Karmally
W, Mayer-Davis EJ, et al. Macronutrients,
Food Groups, and Eating Patterns in the
Management of Diabetes: a systematic
review of the literature. Diabetes Care,
2012;12:434-445. 35(6): 1395

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 CONFERENCE PAPER Sarah Michaud et al., 2022, 174-184

First national training in public health emergency for medical students in

Morocco: A mixed method study
Sarah Michaud 1* , Saad Uakkas 2 , Saad Chaibi 3 , Chakib Nejjari 4 , Adil Mansouri 1
1Clinical Research Department, Mohammed VI University Hospital, Marrakesh, Morocco
2The London School of Hygiene & Tropical Medicine, London, United kingdom
3Faculty of Medicine and Pharmacy of Rabat, Mohammed V University, Rabat, Morocco
4Epidemiology, Clinical Research and Community Health laboratory, Fez, Morocco
4Epidemiology, Clinical Research and Community Health laboratory, Fez, Moroccon

Introduction: Faced with the COVID-19, the first training in public health
emergency management for the Moroccan medical students was
implemented. The study objective was to explore the impacts of the training
and the participants’ satisfaction. Participants and methods: We conducted a
mixed method study among Moroccan medical students, using
self-administered questionnaires to assess the students ‘knowledge and
satisfaction level before and after the training. A case study was carried out
with focus group sessions. A triangulation analysis was done. The results
were compared to the training’s pre-established success indicators of the
training. Results: We had 26 participants aged 24±2 years. The sex ratio
female/male was 3.3. The 6th and 7th year students were the most
represented (53.9%). An improvement was noted concerning the knowledge
and perceived skills related to public health emergencies. The content
quality, the horizontal exchanges were the most appreciated elements. On a
scale of 0 to 5, the average training content level of satisfaction was 4.7±0.5.
The results from the triangulation demonstrated 1) the creation of a medical
students’ network and, 2) professionals able to transfer their knowledge to
peers. Conclusion: A network of 26 medical students with broad knowledge
and skills in health emergencies was formed.

*Corresponding Author INTRODUCTION

Name: Sarah Michaud
A public health emergency is an event, an
Email: sarahmichaudd@gmail.com
imminent threat of a disease or a health problem
Phone: +212
which could potentially be caused by bioterrorism,
ISBN: 9788367405256 a natural disaster, an epidemic or pandemic, an
DOI: 10.2478/9788367405256-024 infectious agent or a new biological toxin that is
highly lethal. It represents an increased risk for
Published by Sciendo. © 2022. Sarah Michaud et al. This is the health of populations with or without
an open access article licensed under the Attribution 4.0
permanent effects, in the short or long term.
International
(https://creativecommons.org/licenses/by/4.0/) Health emergencies have a real or a potential
impact on morbidity and mortality. It may be
difficult to respond to them because of the number
of resources that must be deployed to face them
(Nelson et al., 2007). Action in these emergencies
is one of the three main priorities of the World

174
Health Organization (WHO)(World Health
Organization, 2020a; World Health Organization,
2020b2). A health emergency is differentiated
from a Public Health Emergency of International
Concern (PHEIC). A PHEIC is a formal declaration
by WHO as an extraordinary event posing a risk to
the health of populations in several states and
eventually the risk of an international spread of
the threat (Wilder-Smith and Osman, 2020).
Immediate and coordinated international actions
may be necessary to respond. These public health
emergencies are characterized by their
seriousness, suddenness, and the fact of being
unexpected with repercussions for the health of
people beyond the national borders of the affected
state (Wilder-Smith et Osman, 2020). Since 2007,
the world faced six PHEIC with the H1N1 (2009),
Poliovirus (2014), Ebola (2014-2019), Zika (2016)
and SARS-CoV-2 (2020) [WHO,2019]. This latest
PHEIC, COVID-19, represents the biggest
pandemic of the century (Wilder-Smith and
Osman, 2020). It is a respiratory infection caused
by a highly pathogenic coronavirus which
appeared in China in December 2019. Due to its
basic reproduction number and the high death
rate observed, the WHO declared it as a PHEIC on
January 30, 2020. Then, it has been declared as a
pandemic on March 11, 2020. Response measures
against COVID-19 have been deployed in
particular to manage the numerous cases.
However, it has started to affect all countries
directly or indirectly, and even those with robust
economies and with the most resilient health
systems. An increase in indirect mortality has
been documented worldwide and the disruptions
to health systems associated with the pandemic
have negatively impacted the usual health care
systems (Haldane et al, 2021; WHO 2019). Public
Health Emergency Preparedness (PHEP) may be
defined as “the capability of the public health and
health care systems, communities, and individuals,
to prevent, protect against, quickly respond to,
and recover from health emergencies, particularly
those whose scale, timing, or unpredictability
threatens to overwhelm routine capabilities.”
PHEP plays a key role in terms of health care
workers education as it clearly boosts their
engagement and management skills to respond to
public health emergencies (Nelson et al, 2007).
Indeed, most health practitioners don’t feel
comfortable leading a local initiative for the
management of health disasters due to their
insufficient training level in this area even if they
already dealt with disaster victims in their
practicum. Preparation for the management of this
emergency type is a necessity for caregivers to
foster their skills. Also, more largely it positively
influences the workforce as well as the quality of
the response (Rottman et al, 2005). No specific
training to meet this particular need of medical
students exists in Morocco despite the fact that
they represent an essential response workforce
against COVID-19 or any other health emergency
(Savoia et al., 2013). For these reasons,
WHO-Morocco and the International Federation of
Medical Student Associations-Morocco have
conjugated efforts to develop and organize the
first training pertaining to public health
emergency management for medical students in
Morocco. The event took place over 4 days in May
2021 in Rabat. The general intent of the training
was to "create a group of medical students with
adequate skills and knowledge in terms of the
management of pandemics and health
emergencies in Morocco by the end of May 2021".
In this regard, the training featured a set of topics,
namely health emergencies, epidemiological
surveillance, protective measures, infection
control and prevention, risks communication, case
management, and maintenance of basic health
services. Our hypothesis was that such training
should be positively received by medical students
and would improve their theoretical knowledge as
well as reinforce their practical skills in public
health emergencies. The objective of the study was
to explore the knowledge and satisfaction towards
the training as well as its impacts.
METHODS
Study design
We carried out a mixed-method study in May
2021. It included a quantitative descriptive part
using a cross-sectional study. A qualitative part
with a case study conducted was conducted too
following a hypothetico-deductive approach.
Ethical approval
The study protocol had a favorable consent from
the IFMSA-Morocco ethics committee. This study
was conducted according to the guidelines of the
Declaration of Helsinki. The objectives were
explained to participants and voluntary informed
consent was obtained from all of them. All
information collected from participants was kept
confidential.
Sampling
The target population was the medical students of
Morocco. The participants included in the two
parts of our study were all the students who
participated in the training. The participants’
selection has been non-probabilistic as a
convenience sample has been used to select them.
The inclusion criteria were students enrolled in
private and public Moroccan medical schools
during 2020-2021. Also, the students had to be
enrolled in the 4th year of medical study or higher.
175
The students were selected using an application
questionnaire made via Google Forms. It was
shared on social Medias such as Facebook. The
sampling aimed to select diverse profiles of
motivated medical students. More specifically, the
form focused on 1) their experiences in public
health emergencies, 2) their personal interest in
the subject, 3) their skills and knowledge in public
health, and 4) their perspectives in using their
skills following this training. A scoring scale
ranging from 0, “not relevant at all”, to 5, “totally
relevant”, was used to classify the candidates.
Firstly, we selected the candidates with the most
points. However, the final selection considered the
general background of candidates such as their
medical school or their region. In total, 26
students with diverse profiles regarding their
genders, cities, study levels and medical schools
have been selected to be part of the training. This
low number of participants is due to the Moroccan
government who didn’t authorize event with a
large group of people in order to assure the
COVID-19 protection measures at that time.
Data collections and data analyses
The quantitative data collection was done through
self-administered online questionnaires sent
before and after the training. The variables
studied were: 1) socio-demographic information,
2) general knowledge in public health, 3)
perceived knowledge and skills related to health
emergencies in Morocco and internationally, 4)
satisfaction with the training (for the post training
only). The data was put in Excel and analyzed
using SPSS (Statistical Package for Social Science
software), version 16. Quantitative variables were
presented as means and their standard deviation
(SD). Qualitative variables were presented as
numbers and percentages. A comparison of
percentage for paired series was carried out using
the McNemar's test. At the end of the training, we
carried out focus group sessions for the qualitative
perspective. It focused on the students' motivation
to participate in the training, their general
appreciation of what the training featured and the
proposals to improve it. For this part, a discussion
guide has been prepared and the Woo-Clap digital
tool was used in order to facilitate the students to
express themselves and communicate with one
another. A moderator animated each group and
observers noted all relevant elements. All data
from the focus groups have been approved by
participants before the analysis; then, we
manually did a thematic content analysis. Finally, a
triangulation analysis was done using a
convergence model, with a comparative grid of the
quantitative and qualitative perspectives.
Therefore, we compared our results to the training
indicators. Indeed, we used the training evaluation
grid which had been validated by the WHO-
Morocco office before the event during its
conception phase. The grid included the following
indicators: 1) having a group of at least 20 medical
students, 2) having 80% of participants attending
all sessions, 3) having a significant increase in the
participants' knowledge regarding public health
after the training, 4) stimulating participants'
awareness as to the usefulness of public health, 5)
dispensing relevant lectures (≥ 28 hours) and, 6)
creating an active social network group involving
at least 80% of participants.
RESULTS
Quantitative results
Socio-demographic characteristics of participants:
There were 26 participants in total. Their average
age was 24±2 year with a sex ratio female/male of
3.3. Mainly, students belonged to the 6th and 7th
years of study with, respectively, a percentage of
23.1% and 30.8%. The cities most represented
were Rabat and Marrakesh (30.8% vs 19.2%)
(Table 1).
Medical students’ knowledge and skills in public
health:
We noted an improvement in the participants’
knowledge and skills concerning public health
emergencies after the event, in particular about
national structures and organizations. The
knowledge of the National Center for Emergency
Operations in Public Health increased from 15.4%
to 42.3% and the knowledge of its activities went
from 11.5% to 23.1%. The correct answers
proportion regarding the composition of the
Provincial and Prefectural Rapid Intervention
Team was 19.2% before and 73.1% after the
training (Figure 1).
Questions on the survey focused on the level of
knowledge and skills perceived in relation to
public health. The information collected after the
training has confirmed that these elements have
been greatly improved due to the training (Table
2).
upport of e-learning activities, instructor’s attitude
towards and control of the technology, effective-
ness of information technology infrastructure, ease
of on-campus internet access, student interactive
collaboration, instructor’s teaching style, student
motivation and technical competency, and learning
course content and structure (Figure 2).
176
Table 1: Socio-demographic characteristics of participants

177
Figure 1: Knowledge of students in pre and post training
Perceptions and satisfaction of the training
All the participants recommended the training for
other students. On a Likert scale of 0 to 5 (0 = not
satisfied at all and 5 = very satisfied), the average
level of satisfaction was 4.7±0.5with the content of
the training and 5.0±0.3with the speakers’ choice.
Regarding the information acquired, almost all
participants intend to use them in the future
(5.0±0.2).
Qualitative results
Focus group sessions
The 26 students who participated in the training
took part in focus groups. Five groups have been
carried out with 5participants in 4 of them and 6
participants in the fifth group.
Motivational characteristics of participants
The medicals students had several motivational
characteristics to be part of the training. Firstly, the
participants had a holistic view of health. Indeed,
they considered health on various levels such as
biological, psychological, social, environmental and
cultural levels. Also, they had common interests
such as being involved in the associative and social
fields. On top of that, they shared an interest in
communication skills such as writing or public
speaking, which are regarded as assets necessary
for communicating effectively, and their awareness
of the contribution of soft skills in their
professional life. In addition to that, they have
shown a keen interest in the public health
discipline and particularly in epidemiology with a
focus on health education and vulnerable
populations. “The field of public health fascinates
me.” The training was a way to supplement the
participants’ standard medical training by
improving their theoretical and practical
knowledge and develop some of their individual
skills like communication or leadership. Finally,
they were very motivated by the creation of a
health professional’s network. “The pandemic has
highlighted the importance of having well-trained
health professionals in responding to health
emergencies.” Indeed, the participants aimed to
mobilize themselves to respond to possible new
pandemics in the future. “To avoid a disastrous
situation similar to the COVID-19 situation in the
future, we must prepare for the next pandemic and
take it seriously. Especially since the current global
trajectory tends towards globalization and predicts
that pandemics will multiply. We can no longer
afford to be taken by surprise again." Actively
participating in the changes that Morocco is
undergoing has represented an important source
of motivation for medical students too. They were
very motivated to participate in the training in
order to positively impact the lives of their future
patients and have the opportunity to reduce the
suffering of the Moroccan population.
“Contributing to change in our country would be a
real honor for me.”
Appreciation of the training by the students
The first national training in public health
emergencies in Morocco was highly appreciated by
the participants because of the speakers’ profiles,
the quality of their presentations and the event
organization. This training helped to implant a new
vision of public health and raised the participants’
awareness on this field. Some elements of the
training particularly challenged them owing to the
complexity of this discipline. Further, a set of
elements or areas to be improved were put forward
by the participants, such as having a completely
face-to-face training in the future and the need to
share bibliographic resources with the participants
ahead before the training (Table 3).
178
Table 3: Training appreciation by participants

179
Table 2: Level of knowledge and perceived skills related to public health.

180
DISCUSSION
Convergence of perspectives and final assessment
Our quantitative and qualitative results converge
and complement each other. An improvement in
knowledge of public health emergencies is
notable among the participants. Also, great
satisfaction emerges regarding the quality of the
training. Besides, the students wished the training
had lasted longer and featured more face-to-face
sessions. In the light of the event indicators and
our results, it appears that the training was more
than satisfactory. Its main impact was the creation
of a network including 26 students and
representing seven regions of Morocco. It was not
only a dynamic group being created but also
future doctors ready to be involved in health
emergencies. All the participants had their
knowledge regarding public health emergencies
improved (Figure 2).
Similar training courses implemented by WHO
The emergency program of the WHO has
developed training courses before the advent of
the COVID- 19 pandemic. These courses were
related to preparation for health emergencies, the
International Health Regulations, the emergency
operations, the management of infectious risks
and risk assessment (Sultan et al., 2020).
Likewise, training in health emergencies has been
specifically provided for the African region
(WHO,2020a). Since the COVID-19 pandemic, the
WHO Academy’s first European program for
training in "management of high-casualty events"
has also emerged in Greece (WHO,2020b) .
The success factors of the Moroccan training
The participants’ motivation
The medical students selected to participate at the
training were particularly motivated and
interested in developing their knowledge in public
health. The motivation influences not only the
desire to learn but also the practice and
commitment of caregivers in a context of health
emergencies. The willingness of health
professionals is considered as an essential factor
that may determine their degree of participation
in the preparation and response to public health
emergencies. A study in Yemen in 2019 found that
caregivers' self-efficacy was associated with
willingness to participate in disaster response for
any type of health emergency(WHO,2020c).
Another study carried out in 2020 in Saudi Arabia
which assessed the perceptions of the
preparedness of health professionals and their
willingness to work during public health
emergencies, showed that participants'
willingness to work during health emergencies
varied depending on their nature. Indeed, 62.0%
presented a willingness to work in natural
disasters, 43.6% in SARS/COVID-19, 28.2% in
biological events and 24.9% in a nuclear incident.
The study found that caregivers trained in public
health emergencies were more likely to work
during dangerous events (WHO,2020d).
The training sessions’ content
The training responded to a need for training in
public health emergencies for the medical
students. Key elements to respond to health
emergencies were discussed during the event.
Areas such as the basic definitions of the
discipline, epidemiological aspects, roles and
responsibilities of health and laboratory actors,
the importance of risk communication, supply
chains and risk management have been presented
in the training. It can be related to a study carried
out in the United States in 2007 that shows that
the above elements are central for preparing
public health emergencies (Al-Hunaishi et al.,
2019).
The communication from stakeholders
One of the strategies put in place during the
training was the emphasis put on communication
strategies between actors, be it verbal or
non-verbal. Indeed, the majority of speakers
adopted a horizontal way of communication
during their sessions to promote authentic
exchanges with students and generate strong
learning dynamics. The medical teachers’
involvement and their contributions throughout
the training inspired student participants. This is
in line with an Australian study that shows that
these elements of communication are very
favorable for learning complex concepts in
medical students (WHO,2021).
Elements to improve
The pre-training preparation
According to a study carried out in Germany in
2017, the preparation of medical students for
professional activities impacts their way of
learning and acting (WHO,2020). Our results
point to the same conclusion since our
participants needed documentary resources
before the event in order to better prepare for the
training.
181
Simulation exercises
The increase in the volume of learning in terms of
hours and, more particularly, in relation to
practical work was highlighted by our
participants. According to an American study,
exercises related to the management of health
emergencies are effective ways for good
preparation and response to disasters
(Wilder-Smith et al, 2020). Although two
simulations have been done during the training,
the development of more scenarios should have
been considered. In addition, working on cases in
small groups might also be an effective method
(Zaher et al, 2012).
Recommendations
To strengthen the caregivers’ workforce and to
improve the response to public health
emergencies a strong willingness to act on the
part of health professionals is a prerequisite. It is
also necessary to strengthen their education and
inclusion regarding the management of public
health emergencies. That being so, on the basis of
our results, we recommend to 1) disseminate the
knowledge and successful techniques of training
at local levels, 2) increase the volume and time of
the training sessions with more simulation
exercises, 3) provide a list of relevant
Figure 2: Synthesis of the training evaluation regarding the event success indicators
documentary resources before for the training, 4)
promote strong horizontal exchanges between
learners and trainers, and, 5) continue to
highlight the key role of caregivers in the
management of health crises.
Limits and advantages
The training took place during the COVID-19
health crisis. The number of participants was
limited due to the security measures imposed by
the Moroccan government which probably
induced a selection and an information bias. Also,
no other previous Moroccan experience is
available to compare our results. However, this
training strongly highlighted the big role and
impact of public health professionals in Morocco
and internationally. It highlighted the willingness
and capacities of the medical students regarding
dealing with public health problems in a national
context which hadn't been done before. The
methodology is also an advantage: the
quantitative perspective allowed for a quantified
training evaluation and the qualitative part made
it possible to understand more in-depth our
results. The transferability of the positive aspects
and practices of the training may be taken into
consideration in organizing upcoming training
events similar to this one.
182

CONCLUSIONS
The first training in public health emergencies in
Morocco was very satisfactory and widely
appreciated by the students. It made it possible to
fulfill a need for training for medical students in
the COVID-19 pandemic context. Also, it raised
awareness of the importance of public health in
the medical system as a whole.
ACKNOWLEDGEMENTS
We thank all the students who participated in the
training and the WHO-Morocco staff involved in
the project.
REFERENCES
Al-Hunaishi, W., Hoe, V. C., & Chinna, K. (2019).
Factors associated with healthcare workers
willingness to participate in disasters: A
cross-sectional study in Sana’a, Yemen. BMJ
Open, 9(10), e030547.
https://doi.org/10.1136/bmjopen-2019-030547
Bosch, J., Maaz, A., Hitzblech, T., Holzhausen, Y., &
Peters, H. (2017). Medical students’
preparedness for professional activities in early
clerkships. BMC Medical Education, 17(1), 140.
https://doi.org/10.1186/s12909-017-0971-7
Haldane, V., De Foo, C., Abdalla, S. M., Jung, A.-S.,
Tan, M., Wu, S., Chua, A., Verma, M., Shrestha, P.,
Singh, S., Perez, T., Tan, S. M., Bartos, M., Mabuchi,
S., Bonk, M., McNab, C., Werner, G. K., Panjabi, R.,
Nordström, A., & Legido-Quigley, H. (2021).
Health systems resilience in managing the
COVID-19 pandemic : Lessons from 28 countries.
Nature Medicine, 27(6), 964‑980.
https://doi.org/10.1038/s41591-021-01381-y
MacDonald-Wicks, L., & Levett-Jones, T. (2012).
Effective teaching of communication to health
professional undergraduate and postgraduate
students : A Systematic Review. JBI Library of
Systematic Reviews, 10(28 Suppl), 1‑12.
https://doi.org/10.11124/jbisrir-2012-327
Martin, A., Blom, I. M., Whyatt, G., Shaunak, R., Viva,
M. I. F., & Banerjee, L. (2020). A Rapid Systematic
Review Exploring the Involvement of Medical
Students in Pandemics and Other Global Health
Emergencies. Disaster Medicine and Public
Health Preparedness, 1‑13.
https://doi.org/10.1017/dmp.2020.315
Nelson, C., Lurie, N., Wasserman, J., & Zakowski, S.
(2007). Conceptualizing and defining public
health emergency preparedness. American
Journal of Public Health, 97 Suppl 1, S9-11.
https://doi.org/10.2105/AJPH.2007.114496
Rottman, S., Shoaf, K., & Dorian, A. (2005).
Development of a Training Curriculum for Public
Health Preparedness. Journal of public health
management and practice : JPHMP, Suppl,
S128-31.
https://doi.org/10.1097/00124784-200511001
-00022
Savoia, E., Preston, J., & Biddinger, P. D. (2013). A
consensus process on the use of exercises and
after action reports to assess and improve public
health emergency preparedness and response.
Prehospital and Disaster Medicine, 28(3),
305‑308.
https://doi.org/10.1017/S1049023X13000289
Sultan, M. A. S., Løwe Sørensen, J., Carlström, E.,
Mortelmans, L., & Khorram-Manesh, A. (2020).
Emergency Healthcare Providers’ Perceptions of
Preparedness and Willingness to Work during
Disasters and Public Health Emergencies.
Healthcare (Basel, Switzerland), 8(4), E442.
https://doi.org/10.3390/healthcare8040442
World Health Organization (2020). Coronavirus
disease (COVID-19) training: Online training,
https://www.who.int/emergencies/diseases/no
vel-coronavirus-2019/training/online-training
World Health Organization (2020). Questions
fréquemment posées au sujet du Comité
d’urgence du RSI, Organisation Mondiale de la
Santé.
https://www.who.int/ihr/procedures/fr_ihr_ec_f
aq.pdf?ua=1
World Health Organization (2020). Système de
gestion des incidents(Niveau1).
https://openwho.org/courses/systeme-de-gesti
on-des-incidents
World Health Organization (2020). What we do
https://www.who.int/fr/about/what-we-do
World Health Organization Europe (2021).
Strengthening health system preparedness for
mass casualty incidents: first WHO Academy
learning programme in Europe
https://www.euro.who.int/fr/health-topics/heal
th-emergencies/pages/news/news/2021/06/str
engthening-health-system-preparedness-for-mas
s-casualty-incidents-first-who-academy-learning
-programme-in-europe
WHO Director-General’s opening remarks at the
media briefing on COVID-19
(2020).https://www.who.int/director-general/s
peeches/detail/who-director-general-s-opening-
remarks-at-the-media-briefing-on-covid-19-11-
march-2020
Wilder-Smith, A., & Osman, S. (2020). Public health
emergencies of international concern : A historic
183
View publication stats

overview. Journal of Travel Medicine, 27(8),

taaa227. https://doi.org/10.1093/jtm/taaa227
Zaher, E., & Ratnapalan, S. (2012). Programmes
d’apprentissage en petit groupe basé sur la
pratique : Synthèse critique. Canadian Family
Physician, 58(6), e310‑e316.

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