Acta Tropica 178 (2018) 142–147

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Acta Tropica
journal homepage: www.elsevier.com/locate/actatropica

Identification of an area predominantly endemic for childhood and T

adolescent visceral leishmaniasis in central Sudan
Abdelhafeiz Mahamouda, Hussam Ali Osmana, Elfadil Mustafa Abassb, Atif el Agibc,
⁎
Rubens Riscala Madid, Saul J. Semiao-Santose, Abdallah el Haritha,
a
Laboratory of Biomedical Research, Ahfad University for Women, P.O. Box 167, Omdurman, Sudan
b
Department of Clinical Laboratory Science, College of Applied Medical Sciences, Imam Abdulrahman Bin Faisal University, P.O. Box 2435, Dammam, Saudi Arabia
c
Tropical Medicine Research Institute, P.O. Box 1304, Khartoum, Sudan
d
Post-graduate Program in Health and Environment, University of Tiradentes, Aracaju, Brazil
e
Department of Medicine and Nursing, University of Tiradentes, Sergipe, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Although widely spread throughout Sudan, visceral leishmaniasis (VL) is predominantly endemic in the Gedaref,
L. donovani southern Blue-Nile, and Umrimta areas located in the eastern, southern, and central regions, respectively.
Antigenic variation Regardless of form (endemic or epidemic), VL occurrence follows similar patterns as all ages and both sexes are
Central Sudan affected. From January 2005 to May 2016, we received a total of 563 patients with high suspicion for VL from
Demography
various endemic areas; 159 were children and adolescents (0.5–18 years) from Umrimta (central Sudan). A
Childhood
Adolecence
significant observation during this 11-year period of uninterrupted monitoring using a standard liquid direct
agglutination test (LQ-DAT) version was the exclusive VL occurrence (100%) in the child and adolescent po-
pulations of Umrimta when compared with other endemic areas (27.3%–48.0%). Among 12 child and adolescent
suspects who initially tested marginal in the standard LQ-DAT, 6 scored unequivocally positive readings both in
an improved LQ-DAT version (based on an autochthonous Leishmania donovani strain) and rK28 VL reference
test. None of the 4 (2.5%) VL adult suspects (≥19 years) referred had positive outcomes in the improved LQ-
DAT version or the VL reference freeze-dried direct agglutination and rK28 tests. Further incorporation of an-
tigens derived from autochthonous L. donovani strains from Umrimta (central Sudan) or Gedaref (eastern Sudan)
in LQ-DAT significantly increased the agglutination titer levels in the respective VL homologous sera
(p = 0.0263 T = 505 and p = 0.2814 T = 219), suggesting possible antigenic variation within the predominant
Sudanese L. donovani complex. Additional research is required to determine characteristics other than the ser-
ologically-based ones reported for the L. donovani strain involved.

1. Introduction
Sudan and the newly established Republic of South Sudan are
among the 6 countries that contribute to > 90% of the worldwide an-
nual incidence of visceral leishmaniasis (VL). The 3 major VL endemic
areas in Sudan are in the eastern, central, and southern regions; several
other areas of sporadic occurrence are scattered in Kordovan state and
in the central and western parts of Darfur state Following the devas-
tating outbreak of 1989–1995 and the subsequent mass migration of the
affected Neur tribe from the Bentiu area in the former state of Upper-
Nile to the capital Khartoum ( ± 900 km northwards) seeking treat-
ment, the disease has spread along this migration route to areas pre-
viously unknown as VL endemic (de Beer et al., 1991). VL infections in
Sudan seem to have no demographic preference; both sexes and all age
classes are affected. In comparison with adults, infected children were
reported to have a higher grade fever and splenomegaly (Zijlstra and el-
Hassan, 2001).
In 1988, a group of > 100 patients in different age classes from 4
villages on the west banks of the White-Nile state with high grade fever
were reported to have died before cause of death was identified as being
VL (Khalil et al., 2002 ; Ahmed et al., 1988). Eighteen years later, a
group of 150 patients consisting of mostly children in the same area
developed similar grade fevers that were unresponsive to anti-malarials
or antibiotics; 100 were diagnosed as VL cases by either bone-marrow
or lymph-node aspiration (Khalil et al., 2008). In addition to VL, this
area is also known to be endemic for cutaneous leishmaniasis due to

⁎
Corresponding author.
E-mail addresses: mabdelhafeiz@gmail.com (A. Mahamoud), hussomco@gmail.com (H.A. Osman), emabass@iau.edu.sa (E.M. Abass), atifelagib@hotmail.com (A. el Agib),
rrmadi@gmail.com (R.R. Madi), saulix@gmail.com (S.J. Semiao-Santos), abdallah.elharith@gmail.com (A. el Harith).

https://doi.org/10.1016/j.actatropica.2017.11.010
Received 13 September 2017; Received in revised form 12 November 2017; Accepted 20 November 2017
Available online 26 November 2017
0001-706X/ © 2017 Elsevier B.V. All rights reserved.
A. Mahamoud et al.
Leishmania major (el-Safi and Peters, 1991).
In January 2005, our Laboratory for Biomedical Research at Ahfad
University for Women had started receiving patients with suspicion of
VL from the various hospitals and health centers across Sudan. At the
request of the physicians in charge, patients were tested for the pre-
sence of anti-L. donovani antibodies by a locally produced liquid version
of the well-known direct agglutination test (LQ-DAT) (de Beer et al.,
1991). Until May of last year, a total of 563 patients were seen of whom
387 were from the 3 major endemic areas namely Gedarif (Eastern
state), Umrimta (White-Nile state), and the Southern part of the Blue-
Nile state; 176 other cases were from areas of relatively lower en-
demicity scattered throughout the country.
A significant observation though during our 11-year monitoring
period was the exclusive VL occurrence in Umrimta children and ado-
lescents (0.5–18 years), as compared to that in the other endemic areas
of Sudan (27.3%–48.0%). It is of relevance to mention that of the total
VL suspects referred (163) from Umrimta during this period only 4
(2.5%) were adults with ages varying between 25 and 61 years.
In this phase of our study, we intended to investigate whether this
selective VL occurrence in the children and adolescents of Umrimta
(central Sudan) was associated with prevalence of an L.donovani strain
that differs serologically from the one causing the disease in Gedaref
(Eastern Sudan). This area is the most well-known and it’s the one of
stable endemicity in Sudan (Zijlstra and el-Hassan, 2001). Since the
Umrimta area is also known to be endemic for cutaneous leishmaniasis
due to L. major, the possibility of mixed infection with L. donovani and
L. major was not excluded. Based on this information and the knowl-
edge that L. major tends to visceralize under conditions of malnutrition
or impaired immunity, sera from some of the VL affected children and
adolescents were also assessed for the presence of agglutinating anti-L.
major antibodies (Soliman, 2005; Babiker et al., 2014).
2. Material and methods
Since January 2005, the laboratory of Biomedical Research at Ahfad
University for Women was recognized as the reference laboratory for
VL (sero)-diagnosis in Sudan. Patients with suspicion for VL were re-
ferred for application of a locally produced liquid version (LQ-DAT) of
the well-known direct agglutination test. Most of those patients were
aware of the disease and the problems that it can cause if left untreated.
Therefore, they do their utmost to meet the expenses related to tra-
veling, diagnosis, and eventual treatment. Patients are advised to pre-
sent themselves first at any polyclinic section in the various public or
private hospitals in Khartoum, Khartoum-North, or Omdurman from
where they will be referred to our laboratory. Almost all VL sus-
pects < 19 years were referred from the Omdurman Emergency
Hospital for Children. Of the 563 patients received, 333 were children
and adolescents and 230 were adults of 0.5–18 and 19–61 years of age,
respectively (Table 1). Almost half (47.7%) of the referred child and
adolescent populations was from the Umrimta locality in the White-Nile
state (central Sudan).
Table 1
Positive liquid direct agglutination test (LQ-DAT) readings in 563 patients with high suspicion for visceral leishmaniasis (VL) referred from various endemic areas in Sudan to Ahfad
Laboratory for Biomedical Research during the period 2005–2015.
Endemic area/state Number of suspects referred
Umrimta locality White-Nile state 163
Gedaref locality Eastern state 133
Blue-Nile state 91
Other endemic states 176
Total 563
Acta Tropica 178 (2018) 142–147
2.1. Umrimta area
Umrimta is one of 8 localities in the White-Nile state, lies about 150
kilometers south-west of the capital Khartoum with 160,000 in-
habitants. The habitats in this locality resemble those of areas generally
known to be endemic for VL in Sudan characterized by presence of
acacia and balanites trees favoring sand-fly breeding. In addition to VL,
the area is also known to be endemic for cutaneous leishmaniasis,
malaria, brucellosis, and tuberculosis. Most of the inhabitants in this
area are well aware of VL signs and symptoms. Patients who are sus-
pected to have the disease were advised to travel either to the nearby
major state hospital in Aldweem or for the few who can afford it, to
hospitals with better facilities in Omdurman, Khartoum, or Khartoum
North.
2.2. Ethical approval
The study was approved by the ethical committees of Ahfad
University for Women, Omdurman and the Federal Ministry of Health,
Khartoum (Walieldeen et al., 2010). Since the study involved a retro-
spective analysis of routine data, informed patient's consent was not
applicable. All collected blood and serum samples remained anon-
ymous.
2.3. VL diagnosis
Almost all of the children and adolescents who presented with
suspicion for VL at Omdurman Emergency Hospital for Children were
referred to our laboratory for diagnosis of the disease. This was largely
encouraged by the availability of a non-invasive diagnostic procedure
such as LQ-DAT, the highly reputed efficiency of this procedure for VL
diagnosis and the privilege of its free of charge application. The criteria
for VL suspicion included residency or visit to this or similar other
endemic areas, presentation of signs and symptoms indicative of VL,
such as presence of fever for periods > 2 weeks, and exclusion of ma-
laria and typhoid.
Prior to LQ-DAT application, a file was created for the registration
of the relevant administrative and demographic information, history,
nature, and type of complaints. A blood sample (2–5 ml) obtained by
venous puncture were collected from adults (≥ 19 years) and older
children (≥ 10 years) or by finger prick (1–2 spots) onto filter-paper
(Whatman no 2 or 3) from younger children and severely anemic pa-
tients. The separated serum was either used immediately for LQ-DAT
application or stored frozen at −20 °C until needed. After thorough air-
drying the blood samples on filter paper were handled in a similar way.
Due to the instability of antibody content on the filter paper in com-
parison with serum under situations of frequent electric failure, filter-
paper collected blood samples were considered invalid for LQ-DAT
testing if the length of the storage period at −20 °C exceeded 3 months.
LQ-DAT positives among suspects referred LQ-DAT positives per age class (years)
0.5–18 19–61
70 (42.9%) 70 (100%) 0 (0%)
49 (36.8%) 20 (40.8%) 29 (59.2%)
33 (36.3%) 9 (27.3%) 24 (72.7%)
25 (14.2%) 12 (48.0%) 13 (52.0%)
177 (31.4%) 111(62.7%) 66 (37.3%)
143
A. Mahamoud et al.
2.4. Serological tests
2.4.1. Leishmania strains
Two strains of L. donovani isolated by bone-marrow aspiration either
from an adult or child VL patients who resided in Gedaref (Eastern
state) and Umrimda (White-Nile state, central Sudan), respectively, and
one of the L. major strains from a cutaneous ulcer of an adult patient
from Khartoum state was kindly provided by Dr. M. Mukhtar, Institute
of Endemic Diseases, University of Khartoum. The three Leishmania
strains were maintained by regular subculture every 7–8 days in liver
infusion tryptose (LIT) (Osman et al., 2016).
2.4.2. LQ-DAT
For preparation of antigens from the three Leishmania isolates, mass
cultures of the respective promastigotes were initiated in LIT medium
(without hemin), to which fetal calf serum (10%), penicillin (100 IU),
and streptomycin (100 μg/ml) were added. Following treatment with β-
mercaptoethanol and fixation in formaldehyde/Locke’s solution the
antigens were stained with Coomassie Brilliant Blue and re-suspended
in citrate saline/formaldehyde solution (el Harith et al., 1988; el Harith
et al., 1995). As in previous studies with other LQ-DAT batches, similar
quality control procedures were followed for the finished antigen sus-
pensions (Osman et al., 2016). With the exception of periods during
electric power failures, especially common in the summer season
(March–July), all 3 ready-for-use antigen batches were stored at 4 °C.
As standard procedure, the antigen employed in the initial (routine)
testing of all referred VL suspects was prepared from strains of L. do-
novani isolated in Gedaref (eastern state). Two other antigens from an L.
donovani strain isolated in Umrimda (White-Nile state) or from an L.
major in Khartoum were also included.
In the initial (routine) procedure, serum samples were tested in
twofold serial dilutions starting at 1:100. Titers are expressed as mul-
tiples of the cut-off titer (1:3200) from 1:100 (0.03125) up to 1: 102400
(32.0). For filter-paper spotted blood samples, a 5-mm disc was pun-
ched and eluted overnight in 125 μl normal saline at 4 °C; samples were
tested at 1:50 (0.015625) up to 1:51200 (16.0). For comparison of re-
activity between the antigens derived from L. donovani (Gedaref), L.
donovani (Umrimda), or L. major (Khartoum state) isolates, serum
samples were tested through a full range of dilutions starting from
1:100 (0.03125 x cut-off) up to 1: 209715200 (65536 x cut-off) or as
the number of twofold serial dilution starting from 1:100 (dilution 1) up
to 1: 655 36 00 (well no 23) in order to determine the agglutination
reaction end-point. All serum and filter-paper spotted blood samples
were tested in duplicate including positive and negative reference
serum samples. Location of the end-point titer was according to the
standard protocol previously described. For both serum and filter-paper
spotted blood samples, a titer ≥ 1:3200 was considered indicative of VL
(el Harith et al., 1988).
The outcome of LQ-DAT for each of the adult patients was reported
to the hospital from which the patient had been referred. Results from
almost all of the remaining patients who were < 19 years were sent to
Omdurman Emergency Hospital for Children.
2.4.3. FD-DAT
ITMA-DAT kits (lot 11D1B1) were purchased from the Institute of
Tropical Medicine, Antwerp (ITMA, Belgium). The freeze-dried antigen
based on L. donovani strain MHOM/SD/68/1-S was reconstituted by
adding 2.5 ml buffer to each antigen vial. The test was executed on 96
V-shaped micro-titer plates (Greiner Bio One, Frickenhausen, Germany)
according to the manufacturer‘s instructions. As in LQ-DAT, serum
samples were tested through full-out titration starting from 1:100. The
DAT titers are the highest dilutions at which agglutination was still
visible. Titers are expressed as multiples of the cut-off value (1:3200)
ranging from 1:100 (0.03125 x cut-off) to 1: 209715200 (65536x cut-
off) or as the number of twofold serial dilution starting from 1:100
(dilution 1) up to 1: 655 36 00 (well no 23) to determine the
144
Acta Tropica 178 (2018) 142–147
agglutination reaction end-point.
2.4.4. RK28 (improved rK39) strip rapid test
rK28 is a multi-epitope, recombinant chimeric protein produced by
fusing the L. infantum K9 gene with repeat units of the K39 and K26
genes. The test with the brand name OnSite Leishmania rK39-Plus was
performed according to manufacturer's instructions (CTK Biotech, Inc.
San Diego, CA, USA). Fifty microlitres of serum were transferred into
the sample well. Following addition of the diluent (50 μl), the test result
was read after 15 min of incubation at laboratory temperature
(25 °C–27 °C).
2.4.5. VL treatment
The response to VL treatment in the 66 adults who tested positive in
LQ-DAT will be published in a future report. Of the 111 children and
adolescents who scored positive LQ-DAT readings, 88 responded
promptly and favorably to the standard first-line anti-leishmanial
treatment with sodium stibogluconate. In 2 children with bacterial co-
infection, a combined pentostam/ceftriaxone (samoxin) delivered the
desired outcome. After successful completion of a 20-day full course
treatment at Omdurman Emergency Hospital for Children, all 90 chil-
dren and adolescents were discharged in good condition. Treatment
results in the remaining 21 children and adolescents are not available.
Following disclosure of the positive LQ-DAT outcome, parents or
guardians of the VL diagnosed children, due either to family situations
or financial difficulties preventing them from staying in Omdurman,
had chosen to pursue treatment in the major hospitals in their own
states.
2.4.6. Study design
All VL suspects referred to our laboratory from January 2005 to May
2016, were assessed for VL infection employing a locally produced LQ-
DAT version. As standard procedure, only antigen batches processed
from L. donovani strains isolated in Gedaref (being the most important
and adequately studied endemic area in Sudan) were used.
In order to assess possible missed or over VL diagnosis in the suspect
population referred from Umrimta (163), serum samples (filter-paper
spotted blood samples were not included due to their limited validity)
from 12 patients who initially had marginal LQ-DAT titer readings,
were retested in both the VL reference FD-DAT and rK28 tests. To in-
vestigate whether use of the corresponding autochthonous L. donovani
strain as antigen in LQ-DAT enhanced the sensitivity for VL detection in
comparison with the standard (un-autochthonous) strain from Gedaref,
all available sera from children and adolescents with (24) or without
(44) VL and adults (2) from Umrimta were retested using an improved
LQ-DAT version in which the antigen was processed from an auto-
chthonous L.donovani strain isolated in Umrimta. As a control experi-
ment, all available sera from children and adolescents (17) who initially
tested positive in LQ-DAT in addition to adults (22) from Gedaref in
whom VL was excluded, were also retested against the autochthonous
(Gedaref) and the un-autochthonous antigen from Umrimta.
Assuming that this uncommon VL form is associated with pre-
valence of an L. donovani strain that serologically differs from the
Gedaref strain and others that cause the disease elsewhere in Sudan, 18
sera from the VL affected children and adolescents of Umrimta were
compared for agglutinating reactivity against antigens derived from
L.donovani strains isolated either in the same area of Umrimta (central
Sudan), Gedaref (eastern Sudan), or the former state of Upper-Nile
(southern Sudan).
L. major is known to be endemic in the Umrimta area, and oppor-
tunistic infections with the same species in conditions of malnutrition
or impaired immunity can occur. For these reasons and in order to
evaluate possible mixed infection with L. major, all available VL sera
(18) from Umrimta were challenged for presence of agglutinating anti-
L. major antibodies (Soliman, 2005).
A. Mahamoud et al.
2.4.7. Data analysis
The titration data were transformed logarithmically and the
Wilcoxon Matched Pair Test was applied to compare LQ-DAT titers
(Umrimta x Gedarif and Gedarif x Umrimta). This is a non-parametric
free-distribution test for 2 paired samples, whose data should be mea-
sured at least at the ordinal level. For the verification of effectiveness of
LQ-DAT on FD-DAT, we used the Cochran Q test, which tests several
related samples, in which the same individuals are observed in three or
more steps. The scores are measured at nominal or ordinal level, and
the results are dichotomized: 1 (positive) or 0 (negative) (Ayres et al.,
2007). All tests were applied with a 5% confidence interval, using
statistical software Statistica 7.1 (Stat Soft Inc., Tulsa) and BioEstat 5.3
(Mamirauá Foundation, Belém).
3. Results
Despite modest laboratory infrastructure and difficulty in procuring
reagents as a result of ongoing sanctions, our laboratory at Ahfad
University for Women has nonetheless succeeded in achieving local
production of a liquid version (LQ-DAT) of the well-known DAT. Until
May 2016, the test had successfully been routinely applied to 563 VL
suspects referred from the various endemic areas in Sudan who other-
wise would have undergone invasive and rather risky bone-marrow
aspiration (Table 1). Out of those 563 referred, 177 (31.4%) tested
positive for VL using the standard LQ-DAT; 111 (62.7%) of them were
children and adolescents at ages varying between 6 months and 18
years. As many as 70 (63.1%) of those 111 were from the Umrimta
locality in central Sudan (Table 1). In contrast to other areas in which
the LQ-DAT positive rate among this age group varied between 27.3%
and 48.0%, it was 100% in the same age group from Umrimta. Different
also from the other endemic areas was the extremely low number of VL
adult suspects (4/163, 2.45%) referred from this area (Umrimta). None
of them had positive results in the initial LQ-DAT testing with the
standard antigen (Gedaref origin). All of those 4 adult suspects also
tested negative in the improved LQ-DAT version based on Umrimta
autochthonous L. donovani strain as well as in the VL reference FD-DAT
and rK28 tests (results not shown). The exclusive VL occurrence in
Umrimta children and adolescents was further supported by the out-
come of the improved LQ-DAT version and the VL reference rK28 strip
test in 12 of the suspects who initially tested marginal in the standard
LQ-DAT (Table 2).
In comparison with the standard from Gedaref, the autochthonous
antigen from Umrimta had significantly (p = 0,0263 T = 505) en-
hanced the agglutination reaction with the corresponding VL sera
(Table 3). A comparable significant increase (p = 0, 2814 T = 219) in
titer levels was also recorded for the autochthonous antigen from Ge-
daref with the homologous corresponding VL sera. Regardless however
of endemic area, LQ-DAT titers remained unequivocally negative in all
non-VL control samples included (Table 3).
Table 2
Reassessing of marginal readings initially obtained with liquid direct agglutination test (LQ-DAT) in 12 out of 163 visceral leishmaniasis suspects from Umrimta (central Sudan) by an
improved LQ-DAT version, VL reference freeze-dried direct agglutination (FD-DAT) and rK28 strip tests.
LQ-DAT (Gedaref Ag)a LQ-DAT (Umrimta Ag)a
Titre Positives Titre
1: 800 0/6 1:400 (3)
1:800 (1)
1:1600 (1)
1:3200 (1)
1: 1600 0/1 1:800 (1)
1: 3200 5/5b 1:6400 (2)
1:25600 (3)
a
Standard LQ-DAT antigen (Ag) based on L. donovani strain from Gedaref and an improved test version based on an autochthonous from Umrimta.
b
Six (children and adolescents) tested positive in LQ-DAT improved version and VL reference rK28 test, five in the standard LQ-DAT and only one in the FD-DAT.
Acta Tropica 178 (2018) 142–147
The possible existence of antigen- or sero-variants within members
of the L. donovani complex that is prevalent in Sudan is shown in
Table 4; the relative agglutination reactivity of Umrimta VL sera was
measured against antigens derived from L. donovani strains isolated
either in the same area (central Sudan), Gedaref (eastern Sudan), or the
former state of Upper-Nile (southern Sudan). In comparison with the 2
last mentioned endemic areas, the autochthonous antigen from central
Sudan demonstrated significant higher titers (p < 0.0001 Q = 32,44)
versus the corresponding VL sera. In none of the 18 VL sera from
Umrimta were titers below the cut-off seen against the autochthonous
antigen, but negative values were respectively recorded in one and 5 of
them against the 2 un-autochthonous antigens (Table 4). Also, in 5 of
those 18 VL sera, the autochthonous antigen reacted at titers exceeding
those of the maximum recorded for either un-autochthonous antigen by
1–6 fold.
Contrary to the results obtained with the 3 L. donovani antigens,
those with L. major clearly indicated absence of infection or co-infection
with the L. major species in Umrimta children (Table 4). In the 18 VL
sera tested, no titers > 1:200 were found with L. major, while in at least
12 sera, values ≥ 1:3200 were recorded for each of the 3 L. donovani
strains.
4. Discussion
Among the different age classes of VL endemic populations in
Sudan, children are generally known to be the most severely affected
(Nail and Imam, 2013). Infection rates of 30–60% were reported for this
age group, both in cross-sectional survey studies and outbreaks (de Beer
et al., 1991; Khalil et al., 2008). Although similar infection rates were
previously reported in this current study area of Umrimta, the exclusive
VL occurrence in children and adolescents has only become evident
mainly because of our 11-year period of uninterrupted monitoring and
registering of the suspects referred during this period. We also think
that the main reason for the missing data on the frequency and pattern
of VL occurrence in this area has been due to absence of a proper re-
gistration system and lack of reliable diagnostics. The poor quality of
health services together with the ignorance and financial limitations of
citizens in pursuing differential diagnosis for infections mimicking VL
(such as malaria, brucellosis, and schistosomiasis) has greatly under-
estimated the magnitude of its occurrence among children.
The success in achieving local LQ-DAT production has significantly
encouraged both physicians and patients in all VL endemic areas in
Sudan to request its application as a replacement for the rather risky
and painful bone-marrow aspiration procedure. With the exception of
21 cases that were treated in hospitals other than Omdurman
Emergency Hospital for Children, the great majority (81.1%) of the
children and adolescents identified by LQ-DAT in our laboratory as VL
cases (111), including the 70 from Umrimta, responded readily to the
VL first-line standard treatment with sodium stibogluconate.
FD-DAT rK28 strip test
Positives Titre Positives Result Positives
b
1/6 1:200 (2) 0/6 Negative (5) 1/6b
1:400 (2) Positive (1)
1:800 (1)
1:1600 (1)
0/1 1: 400 (1) 0/1 Negative (1) 0/1
5/5b 1:400 (1) 1/5b Positive (5)b 5/5b
1:1600 (3)
1:3200 (1)
145
A. Mahamoud et al. Acta Tropica 178 (2018) 142–147

Table 3
Performance of antigens derived from Leishmania donovani strains isolated in Umrimta (central Sudan) or Gedaref (eastern Sudan) in liquid direct agglutination test (LQ-DAT) on 109 sera
from visceral leishmaniasis (VL) suspects resided either in the same or alternate endemic area.

VL suspect population (Number)a L. donovani antigen origin Number of sera tested at titre range expressed as multiple of cut off (1:3200)

≤0,0625 0.125−0.5 1.0−4.0 8–32 64–256 512–2048 ≥4096

Umrimta (70) Umrimta 27 19 5 9 2 2 6

Gedaref 18 8 5 4 4 1
Gedaref (39) Gedaref 16 6 2 6 6 1 2
Umrimta 6 3 7 5 2 0

a
VL suspects from Umrimta (central Sudan) consisted of 68 children and 2 adults; 39 other VL suspects from Gedaref (eastern Sudan) included 17 children and adolescents and 22
adults.

Table 4 (p = 0,0263 T = 505) when compared with the un-autochthonous

Reactivity of antigens derived from three geographically different isolates of L. donovani (Gedaref, eastern Sudan) implies some degree of dissimilarity between
and one of L. major in liquid (LQ-DAT) or freeze-dried (FD-DAT) direct agglutination test
these two strains. This was also further verified by the comparable in-
with sera from 18 visceral leishmaniasis (VL) patients from Umrimta locality in central
Sudan.
crease in titer level measured for the latter antigen in the corresponding
VL sera from Gedaref (p = 0,2814 T = 219), again suggesting a certain
Titre Origin of L. donovani antigens in LQ-DAT and FD- LQ-DAT antigen of level of serological dissimilarity between these 2 strains. One more
rangea DAT L. major Khartoum piece of evidence also favoring this assumption is the significantly
Central Sudan
lower titers (p < 0.0001 Q = 32,44) obtained in Umrimta VL sera
Umrimta Gerdaref Upper-Nile
Central Eastern Southern when tested against a second un-autochthonous antigen from southern
Sudan Sudan Sudanb Sudan (Table 4). Lower titer levels in agreement with those that were
also reported for this last mentioned antigen were observed in a recent
<1 0 0 0 18
diagnostic study done in eastern Sudan (Osman et al., 2016).
2–5 0 1 5 0
6–10 9 7 5 0 The importance of variable antigen types in the successful diagnosis
11–14 3 3 5 0 of diseases caused by hemoflagellates other than L. donovani has been
15–18 2 6 3 0 demonstrated in experimental Trypanosoma brucie gambiense infections
19–22 3 1 0 0 (Magnus et al., 1978). Possibly by following similar mechanisms to
> 23 1 0 0 0
evade host immune responses, a switch in the pattern of promastigote
a
Titres are expressed as the number of twofold serial dilution starting from 1:100 or amastigote surface epitopes may have occurred in which the Um-
(dilution 1) up to 1: 655 36 00 (well no 23). rimta L. donovani variant has developed the required virulence to se-
b
FD-DAT based on L. donovani strain isolated in the former state of Upper-Nile lectively infect children in this area. As was the case with variable
(southern Sudan). antigen types in trypanosomiasis, the identification of bacterial ser-
otypes has also improved our understanding of the variations reported
Whether the determinant factor (s) of this VL form in Umrimta is in virulence, pathogenicity, and disease presentation. Besides academic
host or parasite-related is difficult to conclude based on our findings. interest, use of bacteria serotypes has also contributed to the estab-
We can nonetheless speculate that even though the L. donovani strain lishment of reliable diagnostics and the adoption of protocols that ef-
seems incapable of causing disease in adults, it was pathogenic in the fectively manage infections caused by Salmonella, Escherichia, Shigella,
child and adolescent populations of this area. Based on similar reasons, and Vibrio species (Gal-Mor et al., 2014).
we also considered the possibility of opportunistic L. major viscero- Whether the serological differences observed between the 3 L. do-
tropic infections as has previously been demonstrated in experimental novani strains is to be attributed to L. donovani antigen variant types or
studies (Soliman, 2005). However, the absence of LQ-DAT titers in- serotypes cannot be concluded from these results. However, since age
dicative of infection with this species when using a homologous an- proved to be a determinant of VL susceptibility in this area, we think
tigen, has excluded this as a possibility. Furthermore, the similarity in that this deviation in normal VL pattern is phenotypic in nature and
prompt and favorable responses to the first-line standard treatment thus the term L. donovani “antigen variable type” rather than “serotype”
between Umrimta-affected children/adolescents and their counterparts seems appropriate.
in other areas had also dismissed any possible infection with the L. Despite our study limitations of not being able to determine the
donovani strain that phylogenetically differs from those constituting the reason underlying occurrence of this peculiar form of VL, we hope that
predominant L. donovani complex in Sudan (le Blancq et al., 1986). we have at least succeeded in highlighting its importance as a health
Based, however, on its apparent inability to cause VL in adults but problem in the Umrimta locality. We also think that the diagnosed VL
the potential to do so in children and adolescents, the Umrimta L. cases represent only a fraction of a much larger population from which
donvani strain seems to bear similarity with L. infantum. Although ap- only families of those 70 were able to meet with the expenses related to
plied to only 12 VL suspected sera (Table 2) in this study, the multi- diagnosis and treatment in Omdurman. Our efforts although limited,
epitope rK28 antigen bearing L. infantum K9 and K26 genes, proved to are directed toward establishment of a small unit in one of the 3 ex-
be as sensitive as the improved LQ-DAT version (based on Umrimta isting hospitals to allow for local LQ-DAT application in an attempt to
autochthonous antigen) (Mukhtar et al., 2015). To gain better insight as spare parents the financial burden of VL diagnosis.
to which level the Umrimta variant equates with patterns character- An effective strategy to control this VL form in this area is expected
izing either L. infantum or L. donvani, it would also be relevant to to include a cross-sectional survey study to support the findings re-
evaluate at which level a recently developed single-epitope re- ported in this study and also incorporate appropriate molecular tools to
combinant (rKLO8) antigen (derived from an autochthonous L. dono- determine more accurately at which level this Umrimta strain matches
vani strain from eastern Sudan) would react with VL sera from this the L. donovani zymodeme pattern previously reported in Sudan (le
study area (Abass et al., 2013). Blancq et al., 1986).
The significantly higher agglutination reactivity of Umrimta auto-
chthonous (central Sudan) antigen with the VL homologue sera

146
A. Mahamoud et al.
5. Conclusion
Differently observed during 11-year of continuous monitoring than
in Gedaref and southern Blue-Nile located respectively in eastern and
southern Sudan, VL occurrence in Umrimta area (central Sudan) was
confined to the child and adolescent populations (≤ 18). Employing L.
donovani isolates from two different geographical regions in Sudan as
antigens in a direct agglutination test has evidenced significant in-
creases in the agglutination reactivity against the corresponding
(homologous) autochthonous VL sera suggesting possible existence of
variable antigen or sero-types within the broader L. donovani complex
prevalent in Sudan.
Competing interest
The authors declare that they have no competing interests.
Acknowledgments
We thank Dr. D. Mansour, Mr. M. Mutasim, Ms. W. Hassan and Mr.
Y. Mahamoud, Laboratory for Biomedical Research, Ahfad University
for Women, Omdurman for their valuable help in LQ-DAT initial testing
of the referred VL suspects and Dr. M. Mukhtar, Institute of Endemic
Diseases, University of Khartoum, Khartoum, Sudan, for providing the
endemic L. donovani and L. major strains. We also thank Dr. M. Swar, his
medical team and the directorate of Omdurman Emergency Hospital for
Children, Omdurman for their help to view the treatment results.
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