Contents

Contents......................................................................................................................................................0

1.0 Introduction...........................................................................................................................................1

2.0 Purpose of Study...................................................................................................................................2

2.1 Aim....................................................................................................................................................2

2.2 Objectives..........................................................................................................................................2

Rationale.....................................................................................................................................................2

3.0 Research Methodology..........................................................................................................................3

3.1 Primary Study Approach...................................................................................................................3

3.1.1 Quantitative Tools......................................................................................................................3

3.1.2 Qualitative Tools........................................................................................................................3

3.1.4 Length of study and Study site....................................................................................................5

3.1.5 Ethical Approval.........................................................................................................................5

3.2 Secondary Study Approach...............................................................................................................5

3.2.1 Search terms........................................................................................................................5

Malaria resista.....................................................................................................................................6

3.3 Research Method Limits....................................................................................................................6

4.0 Data Analysis.........................................................................................................................................6

5.0 Expected contribution to knowledge.....................................................................................................6

6.0 Conclusion.............................................................................................................................................6

References...................................................................................................................................................6

Abbreviations..............................................................................................................................................8

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 Qualitative Assessment of the Resistance Genes of Falciparum Malaria through the
Potential Diagnosis from Mouth Vapor Using Non-invasive Approach to Determine
Prevalence in Ota, South-West, Nigeria.

1.0 Introduction

The World Health Organization reports Malaria as the topmost on the list of deadly diseases,
with no major scientific advances having been made in recent years to keep its trend at bay. This
has continuously positioned Malaria as a foremost Public health problem across the globe
(WHO, 2022). A dynamic element responsible for the failed strategies in the eradication of this
vector-borne disease is the incessant emergence of new resistant genes to all currently
recognized antiplasmodial remedies (Olasehinde, 2010, Olasehinde et al, 2015). Currently, the
falciparum parasite infects over half a billion people across the globe, resulting in mortality cases
of about one million people respectively annually (Gupta et al, 2014). This disease has defiled all
control strategies, keeping resistance on a steady rise. Fortunately, Malaria is not always a death
sentence, as preventive and curative measures still exist through the combined efforts of Joint
taskforces and stakeholders to improve on the current trend (WHO, 2015). The rise in
antiplasmodial resistance has resulted in the search for options such as vaccine advances in order
to establish prevention, thus leading to a decline in resistant cases (Olasehinde et al, 2015).

Some of the biological factors responsible for antimalarial drug resistance include the
development of efflux strategies by the malaria parasite, and alteration in the genomics of the
parasite target site resulting in the loss of attraction of the drugs to the binding site of the parasite
(Shweta, 2014). The introduction and demonstration of the efficacy of Artemisinin Combination
Therapies (ACTs) in the early 2000 birthed new hopes against the already prevailing outcomes
of malaria resistance, nevertheless, even this has been trodden down by the emergence of new
antiplasmodial resistances (Ollairo, 2003).

In detecting resistance, the use of PCR for in vitro analysis is characteristically employed in the
identification of point mutations in malaria parasitic genes, as well as to determine new strains
and their variations (Shweta et al., 2014). For instance, the Plasmodium falciparum Chloroquine
Resistance Transporter (Pfcrt) which undergoes mutation on its codon K72T and K76T DNA
segments has been discovered to be the most reliable in marking genes and this could be
amplified and detected using PCR (Ojurongbe et al., 2007).
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 Other antimalarial resistance common to Nigeria includes the Plasmodium falciparum Multidrug
Resistance (Pfmdr-1) which results from a chromosomal replacement on its N86Y codon. Its
resistance manifests through antimalarial drugs in the first-line treatment category such as
Chloroquine (Pickard et al., 2003; Dorsey et al., 2001).

Self-medication is a key player in promoting the trend in antimalarial resistance. And this could
be due to a number of reasons. Among which is the invasiveness of malaria diagnosis, which
causes discomfort, fear, and pain. Across the globe, researchers have tried to establish the use of
body fluids such as saliva, sweat, and urine for non-invasive diagnosis of infections and diseases,
in order to quickly detect malaria parasitic activities following signs of infection (Pfaffe et al,
2011), with proof of resistant genes found in saliva samples (Diji-geske et al., 2018). Body fluids
pose to be rich in proteins and DNA and are therefore certified as significant malaria diagnostic
samples that can conveniently replace blood sample collection routines (Kenji et al., 2017).

2.0 Purpose of Study

2.1 Aim

This study aims to qualitatively evaluate the presence of Plasmodium falciparum resistance
genes in humans through biomarkers in mouth vapor.

2.2 Objectives

1. To Establish the occurrence of Plasmodium falciparum in mouth vapor.

2. To establish the presence of falciparum malaria resistance gene markers in mouth vapor isolates.

3. To ascertain the diagnosis of malaria infection through a non-invasive approach from mouth
vapor.

4. To determine the effectiveness of Rapid Diagnostic Test tools in diagnosing falciparum malaria
from mouth vapor.

5. To establish mouth vapor as a non-invasive point of care diagnosis.

Rationale

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Invasive diagnostic approach through finger pricking and phlebotomy has been known to cause a
series of reactions due to reasons ranging from ineffectively blood collection procedures carried
out by inadequately trained personnel thus resulting in tissue damage and pain, to the risk of
infections from wrong needles usage (Aninagyei et al., 2021). Based on these positions, a
number of studies have recommended the use of non-invasive methods which is far less
demanding in its approach, for malaria diagnosis (Waits and Paetkau, 2005).

Hence, the continued existence of antimalarial drug resistance against all interventions informs
the reason for this study and provides the pre-requisite for the improvement in diagnosis.

3.0 Research Methodology

3.1 Primary Study Approach

Participants’ samples for this study will be collected random using convenience sampling to
deliver recruit survey forms to establish participants’ interest.

Where the expected number of samples to be collected is N = 800 (n = 200 per experimental
study group and control respectively).

The study will comprise of four groups viz; one control group and three study groups. Group A,
B & C consisted of individuals who have tested positive for falciparum malaria (A= males,
B=Females & C=Children, n =200 respectively with parasitemia level ≥ 2000) (Diji-geske et al.,
2018), while the control group will comprise of individuals who tested negative to falciparum
malaria (n=200).

This investigation will engage both qualitative and quantitative design approaches in its
collection of information and analyses of data findings.

3.1.1 Quantitative Tools

Sample collection, experimentations, surveillance, data grouping, Microscopy, PCR, and

software analysis will be engaged.

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 3.1.2 Qualitative Tools

For participants’ sample collection and monitoring experimental groups,

1. Questionnaires
2. Ethnographic research approach to determine the effect of community on the prevalence of
Falciparum malaria

  Malaria Questionnaire
1 Name  
2 Age 18- 30yrs / 31- 50 years / 51 & Above
3 Gender Male/Female
4 Kind of apartment lived in 1 bedroom/2 bedroom/ 3bedroom &above
5 No of people living in your apartment 2-5/ 6-10/ More than 10
Rate your understanding of the malaria
6 infection. Excellent /Good /Fair /Poor
What preventive measures do you take Use of insecticides/Mosquito nets/Door and window
7 to avoid an infection? nets/Clean environment (Pick all that apply)
What is your level of exposure to
8 mosquito bites? High/Medium/Low/No exposure
How long since you last visited the
9 hospital for a malaria test? 3mths - 1 year /1 - 2yrs /3yrs & above
How often do you have malaria
10 symptoms? Once a month/3 Monthly/Twice a year/Hardly
Do you wait to have symptoms before
11 treatment or self-medication Yes/No
Do you take self-medication for malaria
12 treatment? Yes/No
13 How often do you take self-medication? Frequently/Hardly/Never
Do you patronize herbal mixture
14 vendors? Frequently/Hardly/Never
15 Have you heard of drug resistance? Yes/No

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 Sample questionnaire for this research

3.1.3 Sample collection

The use of a microscopy slide to collect mouth vapor, and a non-invasive Rapid Diagnostic Test
kit (RDT) will be engaged for participants’ sample collection.

3.1.4 Length of study and Study site

This investigation will cover Ota, Ado-Odo LGA, South West Ogun State for a period of one
year.

3.1.5 Ethical Approval

The ethical consent for this research work will be acquired from the Institution’s Health &
Ethical Committee.

3.1.5.1 Informed consent

Consent will be acquired from prospective experimental individuals through non – cohesive
means.

3.1.5.2 Inclusion criteria

The eligible participants for the study will comprise of individuals

1. Who have agreed to sign the consent form.

2. Having parasitemia levels ≥ 2,000 parasites/ul of blood (Diji-geske., 2018)

3. Minors with guardians’ approval.

3.1.5.3 Exclusion criteria

Babies, and people who are not willing to give informed consent.

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 3.2 Secondary Study Approach

Web searches on Google Scholars using Google search web is the designated database collection
site.

3.2.1 Search terms

Malaria resistance genes, non-invasive approach, point of care malaria diagnosis, epidemiology
of falciparum malaria.

3.3 Research Method Limits

Anticipated limits to the research methodology include;

1. Low level of response from potential study candidates.

2. High expenses incurred from RDT kits for experimental participants.

3. Difficulty in mouth vapor collection process.

4.0 Data Analysis

The information processed from the study data will be analyzed using Microsoft Excel and SPSS
tools. It will examine the association of prevalence to the resistance between males and females
and adult versus children by Chi-square test with a 95% confidence interval.

5.0 Expected contribution to knowledge

This study is expected to

1. Establish the prevalence of malaria parasite and its resistance genes in mouth vapor.
2. Guide health care manufacturers on the need for simpler non-invasive diagnostic tools.

6.0 Conclusion

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This research poses to be timely, as the graph of antiplasmodial resistance hits the roof leaving
both policymakers and stakeholders hands-tied regarding the next point of action. Research work
must remain in top gear if the situation must be curtailed and humanity sustained.

References

Aninagyei, E., Abraham, J., Atiiga, P., Antwi, S. D., Bamfo, S. and Acheampong, D. O. (2020).
Evaluating the potential of using urine and saliva specimens for malaria diagnosis in suspected
patients in Ghana. Malar J; 19(349)

Diji-geske, R.I., Olasehinde, G.I., Fadinad, I., Arogundade, D. and Darby, P. Epidemiological
data of falciparum malaria in Ado-Odo/Ota, Southwest Ogun State, Nigeria. Data in Brief 19
(2018) 1398–1402

Dorsey, G., Kamya, M. R., Singh, A. and Rosenthal, P. J. (2001). Polymorphisms in the
Plasmodium falciparum pfcrt and pfmdr-1 genes and clinical response to chloroquine in
Kampala, Uganda. Journal of Infectious Diseases 183:1417-1420.

Gupta, P., Singh, R., Khan, H., Raza, A., Yadavendu, V., Bhatt, R. M. and Singh, V. (2014).
Genetic Profiling of the Plasmodium falciparum Population Using Antigenic Molecular Markers.
The Scientific World Journal. Volume,

Kenji, O. M., Samuel, T. Y., Livo, F. E., Obase, N. B., Jessica, Y., Jean, C. D., Calixt, D. M.,
Diane, W. T., Vivek, R. N., Rose, F. L. (2017 ). Detection of Plasmodium falciparum DNA in
saliva samples stored at room temperature: potential for a non-invasive saliva-based diagnostic
test for malaria. Malaria Journal; 16:434

Ojurongbe, O., Ogungbamigbe, T. O., Fagbenro-Beyioku, A. F., Fendel, R., Kremsner, P. G.,
Kun, J. F. (2007). Rapid detection of Pfcrt and Pfmdr1 mutations in Plasmodium falciparum
isolates by FRET and in vivo response to chloroquine among children from Osogbo, Nigeria.
Malaria Journal; 6: 41.

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Olasehinde, G. I. (2010). In- vitro and molecular studies on the resistance of P. falciparum to
antimalarial drugs in Ogun state, south- western Nigeria. Lambert Academic publishing; 978-3-
8433-7825-3.

Olasehinde, G. I., Ojurongbe, D. O., Akinjogunla, O.J., Egwari, L.O. and Adeyeba, A.O. (2015).
Prevalence of Malaria and Predisposing Factors to Antimalarial Drug Resistance in
Southwestern Nigeria. Research Journal of Parasitology; 10 (3): 92-101.

Olliaro, P. L. and Taylor, W.R. J. (2003). Antimalarial compounds: from bench to bedside.
Journal of Experimental Biology;206: 3753-3759.

Pfaffe, T., Cooper-White, J., Beyerlein, P., Kostner, K. and Punyadeera, C. (2011). Diagnostic
potential of saliva: current state and future applications. Clinical Chemistry; 57 (5): 675- 687.

Pickard, A. L., Wongsrichanalai, C., Purfield, A., Kamwendo, D., Emery, K., Zalewski, C.,
Kawamoto, F., Miller, R. S. and Meshnick, S. R. (2003). Resistance to antimalarials in Southeast
Asia and genetic polymorphisms in pfmdr1. Antimicrobial Agents and Chemotherapy; 47:2418–
2423.

Shweta, S., Bikash, M. and Rakesh, S. (2014). Challenges of drug resistant malaria. Parasite; 21:
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Waits, L. P. and Paetkau, D. Noninvasive genetic sampling tools for wildlife biologists: a review
of applications and recommendations for accurate data collection. J Wildl Manag.
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World Health Organization. (2015). Global technical strategy for malaria 2016-2030, Geneva.
http://who.int.global-technical-strategy-for-malaria-2016-2030/. Accessed June 04, 2022.

World Health Organization. (2022). Malaria.

https://www.who.int/news-room/fact-sheets/detail/malaria. Accessed June 04, 2022.

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Abbreviations

PFCRT Plasmodium falciparum chloroquine resistance transporter

PFMDR Plasmodium falciparum multi-drugs resistance

PCR Polymerase chain reaction

ACT Artemisinin-based combination therapy

RDT Rapid diagnostic test

WHO – World Health Organization