1
Genetic diversity of Schistosoma haematobium parasite IS NOT
2
associated with severity of disease in an endemic area in Sudan
3
Nagla Gasmelseed1, Nhashal E. Kuramino1, Mohamed O. Abdelwahid1,
4
Anas O. Hamdoun2 Ahmed E. Elmadani2,
5
1Department
6
of Gezira, Wadmedani, Sudan
7
2Department
8
of Gezira, Wadmedani, Sudan
9
Abstract
of Molecular Biology National Cancer Institute University
of Medical Imaging, National Cancer Institute, University
10
Worldwide, more than 200 million people are infected of which the
11
higher disease rates occur in children. Eighty three students from Quran
12
School for boys in Radwan village, Gezira state, Sudan (mean: 12.45 ±
13
3.2 years; 6 - 20) were investigated for the relationship between the
14
genetic diversity of Schistosoma haematobium strains and the severity
15
of the disease. Schistosoma haematobium infection was detected by
16
urine filtration amd random amplified polymorphic DNA (RAPD)
17
methods. Disease severity was assessed by ultrasonography, . Sixty out
18
of 83 (72 .3% ) subjects had Polymerase chain Reaction product
19
indicating an infection. Varied degrees of infection were noted. Indeed
20
mild disease was predominant followed by severe disease as detected
1
21
by ultreasound.
We report here three genotypes of Schistosoma
22
haematobium in the study subjects i.e. A01-1, A01-2 and A01-3. The
23
study revealed no association between the different genotypes of S.
24
haemtobium and the severity of the disease. This study recommended
25
that examination of more samples from different areas to identify any
26
possible differences between the parasites genes and disease severity.
27
Key words: S. haematobium, genetic diversity, ultrasound, severity,
28
Gezira State, Sudan
29
Introduction
30
Schistosomiasis affects approximately 200 million people, mainly in
31
rural areas of developing countries, with an estimated 79 million people
32
at risk of the disease [1]. Both S. haematobium and S. mansoni are
33
present in Sudan. Schistosomiasis is found in many different areas in
34
Sudan especially in Gezira, Rahad, Kenana and other irrigation schemes
35
[2]; [3].The dynamics of the transmission are necessarily complicated
36
and subject to considerable variation due to many factors influencing
37
the common environment, the behavioural patterns of the definitive
38
host and the bionomic of the intermediate host.
39
The genome of Schistosoma is approximately 270 Mbp [4], which is
40
considerably about one tenth the size of the human genome. It is
41
estimated that the S. mansoni genome has a GC content of 34% [5] ,
2
42
with 4-8% highly repetitive sequence, 32-36% middle repetitive
43
sequence and 60% single copy sequence[4] . Numerous highly or
44
moderately repetitive elements have been identified and their
45
occurrence within existing sequence datasets also indicates that the
46
genome contains at least 30% repetitive sequence [6]. With genetic
47
crossing occurring between adult worms, heterogeneity of an infection
48
may strongly influence development of new variants of the parasite [7].
49
Recent studies using randomly amplified polymorphic DNA (RAPDs)
50
markers have found multiple genotype infections by S. haematobium in
51
the Planorbidae intermediate host [8] [9]. RAPDs mainly segregate as
52
dominant markers with heterozygous and homozygous dominant
53
individuals having the same banding pattern at a locus [9], while
54
homozygous recessive individuals have no band at that locus. Statistical
55
measures taking this limitation into account have been developed in
56
order to derive population parameters describing heterozygosity, gene
57
flow, linkage disequilibrium, and other factors [10]. Randomly amplified
58
polymorphic DNA (RAPD) studies of Schistosoma mansoni infection in
59
naturally infected rodent species revealed even more genetic diversity
60
per infection with as many as 28 specific genotypes per rat [11].
61
Although few specific primers for polymorphic regions exist, RAPD-
62
polymerase chain reaction (PCR) technology has made it possible to
63
conduct population-based studies of schistosomes with little prior
3
64
sequence information [12].RAPD primers have been used in several
65
studies to examine the genetic diversity among populations of
66
schistosomes in snails [13];[14] and have proved valuable despite the
67
assumptions necessary for their interpretation [15]. They have
68
demonstrated that, with S. haematobium, it is feasible to sample a
69
population of parasites conveniently and arrive at estimates of the
70
frequency of various alleles using RAPD–PCR. The method of
71
schistosome sampling, through single genetic drift, and through limited
72
numbers of infected snails, snail mortality, does play a role, increasing
73
the numbers of snails for S. haematobium [16]. Knowing the extent of
74
parasite gene flow and the nature of any barriers to such flow will be
75
important in predicting the likely washing spread of drug resistance
76
genes, if they appear [16]. RAPD primers survey a large number of loci
77
throughout the entire genome and have proven useful in characterizing
78
both inter- and intraspecific relationships [17];[18]. As very little DNA is
79
required for such analyses, cercarial or miracidial stages of
80
schistosomes can be used as a source of genetic material, thus
81
minimizing selection caused by passaging through unnatural hosts
82
[19]; [16].
83
Distribution of S.haematobium in Sudan in several irrigation schemes
84
have been constructed e.g. Gezira, Rahad Agriculture Scheme, Ginaid,
4
85
AL Gerba, Kenana and other sugar cane schemes [20], also found in
86
small part of Blue Nile, southern of Sudan [21]. Little is known about
87
the extent of genetic diversity of S. haematobium within its definitive
88
host, humans. Understanding the genetic structuring of populations at
89
each stage of the life cycle is essential to account for the creation of
90
diversity and its maintenance in natural populations of parasites [22].
91
Genetic variability among parasite populations is an important factor in
92
their potential for producing harmful effects on the human populations.
93
Since damage from schistosome infections is so closely linked to the
94
immune reaction to parasite eggs deposited in tissue, diversity of this
95
infection may play an important role in development of pathology with
96
heterogeneous versus homogeneous infections resulting in different
97
clinical outcomes. Genetic differences may also lead to some strains
98
being innately more immunogenic or fecund than others [23]. The aim
99
of this study was to identify if there is any relationship between the
100
genetic diversity of S.haematobium and pathology of the disease in
101
school children in Gezira State, Sudan.
102
Material and Method
103
Study area
104
The study was conducted in Roudwan village, located in the south west
105
of Wadmedani city. Wadmedani is the capital of Gezira State in SUDAN.
5
106
This village is a part of Gezira irrigation scheme and surrounded by
107
canals from the south and east. There is only one small health center.
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No school as most of the children study in Khalwa (Quranic schools). No
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safe water source is available, and the main source of water is from
110
canals. Students from Quranic School (Khalwa) of boys were recruited
111
for this study. The students came from different regions of Gezira state
112
and other states of Sudan such as (Blue Nile state, White Nile state,
113
Sennar, Gadarif). They stay up to 15 years to study the Quran. All the
114
students were at risk of schistosomiasis infection due to the daily
115
activities in water canals (swimming & drinking).
116
Urine Filtration Technique
117
All the students in the Quranic School were recruited to this study.
118
Urine sample (10 ml) were collected from each student and examined
119
by
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S.haematobium eggs were counted of (egg/10 m)l. The intensity of
121
infection was classified as egg count <100 eggs recorded as low level
122
infection and between 101-199 eggs as moderate and >200 eggs as
123
high intensity of infection [25].The remaining urine was centrifuged and
124
the deposit kept at -80 0C for DNA extraction. The students were
125
weighted and those found positives for S. haematobium infections
126
were treated with Praziquantel at 40 mg/kg dosage.
filtration
method
for
S.
haematobium
6
detection
[24].
127
Ultrasound examination
128
Ultrasonographic examination was done by an expert radiologist on
129
each student. Ultrasonographic examination of the urinary tract was
130
performed by Aloka (5100) with a 3.5 MHz convex probe and Shimadzu
131
with convex probe 2-5 and 5-10 MHz for all recruited study subjects
132
after adequate filling of the bladder. Urinary tract and kidneys were
133
examined according to WHO standards [26]. The bladder was evaluated
134
using a 3.5 MHz sector scan in three directions. Bladder wall thickness
135
was measured, and mucosal irregularities, masses or pseudo polyps
136
were classified as proposed by the WHO workshop [26]. Irregularities of
137
the bladder wall or thickening were considered pathological
138
abnormality in the case of more than 0.5 cm thickening. The severity of
139
S.haematobium was categorized according to the size of bladder wall
140
thickening irregularity, calcification, nodularity, dilatation and ureter
141
thickness, nodularity, dilatation, calcification and kidney dilatation, and
142
echogencity. Classification of urinary tract pathology was done as
143
normal, mild, moderate and severe disease.
144
Genotyping of S. haematobium DNA using Random Amplified PCR
145
Reaction (RAPD)
146
DNA was extracted for all collected samples using QIAamp DNA Mini kit
147
(Germany). Freezing concentrated precipitate urine samples were kept
7
148
in room temperature (RT) for 15 min. According to manufacture
149
structure DNA was extracted using QIAamp Mini spin column using
150
proteinase K for digestion in different steps. DNA was incubated at
151
room temperature (15 – 25)0C for 1 min, and centrifugation was done
152
at full speed for 1 min. DNA concentration was measured by
153
spectrophotometer.
154
genotyping of S. haematobium strain (A01, 5'-CAGGCCCTTC-3';A02, 5'-
155
TGCCGAGCTG-3';A12 , 5'-TCGGCGATAG-3';A13, 5'-CAGCACCCAC-3';Y20
156
,5'-AGCCGTGGAA-3';), primers were selected according to Shiff et al
157
[16]. Amplification reaction were carried out in a total volume of 25 µl
158
containing 5 ng of DNA; 10M dNTP ; 5 mM MgCl2 ; 10 pmol of primer;
159
0.5 unit of Taq ; 2.5 µl 10x PCR buffer and 11.2 µl deionized water.
160
Amplifications were carried out in a Perkin–Elmer Thermo Cycler and
161
the program as followed, an initial denaturation at 95 °C for 5 minm 2
162
cycles annealing 30°C for 2 min, extension 72°C for 1 min and 95°C
163
denaturation for 30 s at followed by 33cycles then end 4° C.
In this study five primers were used for
8
164
Gel Electrophoresis
165
Polyacrylamide was prepared with 4% acrylamide solution, 16.8 g urea,
166
2 mL 10X TBE buffer (Tris base, Boric acid and EDTA), and 7.5 mL 40%
167
acrylamide/bis solution (19:1). The solution was dissolved.
168
hundred and fifty μL of 10% ammonium persulfate (APS) was added to
169
the solution, then 25 μL of TEMED and 0.5 µg/ml ethidium bromide was
170
added quickly but gently was mixed. PCR product was run in the gel
171
electrophoresis apparatus. An electric field is applied across the gel,
172
causing the negatively charged nucleic acids migrate across the gel
173
towards the positive (+) electrode (anode). The power was adjusted to
174
approximately 20 watts 800 volts foe 15 min. Depending on their size,
175
each biomolecule moves differently through the gel matrix: small
176
molecules more easily fit through the pores in the gel, while larger ones
177
have more difficulty. The gel visualized by gel documentation system.
178
Hundred base pairs DNA marker was used for molecular weight
179
identification.
9
Two
180
Ethical Approval
181
Ethical clearance was obtained from National Cancer Institute Research
182
Ethical committee NCI-REC University of Gezira. Prior to conducting the
183
study, aims and all information about the study was explained to
184
community during meetings. Written consent was obtained from the
185
local leaders who are guardian and responsible of the children. All
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infected subjects were treated immediately after diagnosis.
187
Statistical Analysis
188
Data was analyzed using SPSS version 16 statistical package. Description
189
data was used for mean ± SD, age range and intensity of infection.
190
Pearson chi-square for trend was used, where appropriate, to measure
191
associations among the genotyping and severity of disease.
192
Results
193
All of the study subjects were males; the mean age was 12.45 ± 3.2 with
194
age range between 6 - 20 years. The majority of age groups were
195
between 9-11years, 36.1% (30) followed by 12-14years 26.5% (22) as
196
shown in Figure (1). About 54.2 %( 45/83) of the students were positive
197
to S.haematobium by microscopic examination (filtration method). The
198
stratification of the age range showed that 70.6 % were positive for age
10
199
17-15 yrs followed by age range 18-20 yrs and less than 8yrs while the
200
age from 9 -14 50% were positive as in Figure (2).
201
. All the age groups of the study subjects had a low intensity of
202
infection, the age group less than 8 and 18-20 had a high percentage
203
80% and 100% respectively. While the age less that 8 had a high
204
moderate intensity of infection 20% compared with the other group.
205
Age group 9-11 reported 13% of high intensity of infection compared
206
with the other groups as shown in (Figure 3).
207
Detection of urinary tract pathology by Ultrasound:
208
Eighty three of the Khalwas’ students were diagnosed by ultrasound.
209
The diagnosis included upper urinary tract (Kidneys and ureters) and
210
lower urinary tract (urinary bladder). About 85% (51) of study subjects
211
had pathological changes. The changes included 85.9% (49/51)
212
abnormal wall thickness, 3.9% (2/51) one polyp and 56.8 % (29/51)
213
multiple nodularities as shown in figure (4A) and 1.9% (1/51) wall
214
calcifications of urinary bladder. Regarding kidneys and ureters, the
215
pathological changes were 11.73% (22/51) abnormal ureteric thickness
216
and 3.9% (2/51) echogenic kidneys (Figure 4B). The age range 9-11 year
217
had a high frequency of abnormality in bladder thickness and bladder
218
masses compared with the other age groups, while 12-14 year age
11
219
group had higher frequency regarding abnormality of kidneys as shown
220
in table 1.
221
Molecular Genotyping of S. haematobium Strain:
222
RAPD primers have been used in several studies to examine the genetic
223
diversity among populations of schistosomes. DNA was extracted from
224
filtrated urine. Genotyping of S. haematobium was done using RAPD
225
test .Only A01 primer was found to have PCR product, while no product
226
was detected for the other primers (A02, A12, A13, Y20). A01 amplified
227
72.3% (60) of the total sample. Three different alleles were genotyped
228
(1, 2, and 3) in polyacrylamide gel using with different molecular weight
229
as known by 100 bp DNA marker polymorphic band in marker 700, 800,
230
900, 1100 bp (Figure 5). Genotype A01- 1 was 80% (48), A01-2 was 15%
231
(9) and A03-3was 5% (3).
232
S.haematobium infection severity and the Genotypes
233
The association between severity and genotype of S.haematobium was
234
reported using pearson Chi-Square. In genotype A01 -1 mild severity
235
was 94.4% (17) of study subjects, while moderate severity was 71.4%
236
(10) and 76.9. % (10) had severe feature of the disease. Genotype A01-
237
2 showed 28.6% (4) was moderate and 15.4.3% (2) was severe. There
238
no association was found was P-value = (0.4) between Severity of
239
S.haematobium and the genotypes (Table 2).
12
240
Forty five study subjects were positive by PCR and microscopic
241
examination; genotype A01- 1was found to be 87% (39), genotype
242
A02-2 was 9% (4) and genotype A03-3 was 5% (2). No association was
243
found between severity of infection and genotype of S.haematobium.
244
Genotype A01-1 had a high percentage than other two genotype 2 and
245
3 in the severity of the disease; the normal subjects were 83.3% (10)
246
and mild were 93.7% (15), moderate were 87.5% (7) and 77.8% (7) had
247
severe clinical feature.
248
according to the different severity, pearson Chi-Square = 4, P-value =
249
0.6, (Table 3).
250
Fifteen study subjects were positive by PCR and negative by
251
microscopic examination; 60% (9/15) was genotype A01- 1, while 33.3%
252
(5) was genotype A02-2 and 6.7% (1) weas genotype A03-3. There was
253
no
254
S.haematobium and the genotypes using pearson Chi-Square = 6, P-
255
value = (0.4) as in(Table 4).
assocaition
Genotype A01-2 and 3 low percentage
between
different
severity
of
infection
of
256
257
Discussion
258
In this study 85% of study subjects were found to have
259
ultrasonographic abnormal pathological conditions including; abnormal
260
wall thickness 85.9% (49) multiple nodularity 56.8 % (29),one polyps
13
261
3.9% (2) and wall bladder calcifications1.9% (1). In Mali a similar study
262
was conducted they found that [27] irregular bladder wall were the
263
most frequently diagnosed abnormality in 3.4% of children. The high
264
percentage of the abnormality in our study may be due to the
265
continuous exposure of the study subjects and lack of regular control.
266
Study in Nigeria
267
abnormal wall thickness (55.8%)[28] while in our study 56.8%, irregular
268
bladder wall (27.9%), masses (23.3%) and pseudo polyps 2 (4.7%) ].
269
these studies reported similar findings that most abnormal urinary
270
bladder wall recorded in children below 15 years old.
271
conducted in southern Nigeria identified about 6.7% and 1.7% of the
272
patients had the right pelvis and left pelvis of their kidney moderately
273
dilated, respectively [29]; in contrast to this study where no kidney
274
abnormalities were found probably due to duration of infection in the
275
study participants. This may be due to the low intensity of egg count in
276
our study.
277
Schistosomiasis was detected by PCR the result showed 72.3% (60)
278
were positive by PCR ,while microscopic method revealed 54.2% (45),
279
this may be due to the low sensitivity of microscopy when diagnosing
280
low infection.
reported 71% abnormal pathological conditions;
14
A study
281
In this study PCR was done by Using RAPD test for S. haematobium
282
parasite genotyping 72.3% (60) study subjects had a PCR product for
283
A01 primer, A01 primer showed different three genotypes (1, 2, and 3)
284
80% (48/60) were genotype A01-1 while 15% (9/60 ) genotype A01 - 2
285
and 5% (3) genotype A01-3, using polyacrylamide gel . Study done by
286
[16] RAPD fragments of an inbred Egyptian strain of S. haematobium
287
generated by primers G17 and A02,using miracidial isolate from a
288
particular host. Differences in alleles were recorded among the 37
289
variable bands. In this study, only 6 allels in three variable bands were
290
recorded, and polymorphic region was between 700 and 1100 bp as in
291
figure 5. The small number of variation alleles in this study refer to the
292
samples collection was from one study area, while Shiff et l studied
293
samples were collected from infected students coming from different
294
regions infected with Schistosoma[16].
295
RAPD primers have been used in several studies to examine the genetic
296
diversity among populations of schistosomes in snails [8]. Studies done
297
by [8] at different sites in Bamako, on cercariae isolated from different
298
snails and 47 different genotypes were recorded from 414 schistosome
299
individuals. Ten primers (A01, A02, A10, A13, A19, G09, G11, G17, G19,
300
G18) were used but only three primers were detected (A01, A10,
301
G19),In this study 5 primers(A01, A02, A12, Y20 and A13) were used but
302
only one primer was detected A01,the results were consistent with the
15
303
study conducted by [8]
with
respect
304
was detected in the two studies, while genotype A02 and A13were
305
used but did not give any results. Primer A01 revealed two polymorphic
306
bands in marker 1100bp, while in our study other variability in marker
307
between 700 and 900bp was noted. The findings showed more
308
different genotype when more than five primers are used or by
309
increasing the study population that increases the chances of finding
310
other genetic differences. Our results showed that all genotype was
311
A01, s may reflect that this genotype was not aggressive enough to
312
cause severe disease as most of the patients have mild disease.
313
Morbidity manifestations have great role in diagnosis S.haematobium
314
and genetic differences may lead to some strains being innately more
315
immunogenic or fecund than others, these differences may play
316
important role in disease severity. There are few studies that found the
317
relation between severity and genetic diversity[30]. This study revealed
318
no association between the severity and genotypes compared with a
319
study from Zimbabwe that used ultrasonography to characterize the
320
extent of urinary tract pathology of infected children, and random
321
genetic markers to examine the relationship between genetic diversity
322
of S. haematobium and clinical outcome. They found that parasite
323
heterogeneity did not differ; allelic frequencies at eight loci differed
324
significantly between the mild and severe groups. Parasite isolates
16
to genotype
A01, where it
325
were analyzed further using a modified cluster analysis that segregated
326
the population into 13 clusters of associated genotypes. Three clusters
327
were significantly over-represented in children with severe lesions.
328
Although preliminary parasite genetic associations may be important in
329
clinical outcome [30]. In this study the genotype A01 type one was
330
recorded as highest in all the positive subjects by PCR and microscopic
331
examination together and also in those negative by microscopic as in
332
tables 1,2, and 3. Mild severity represent the vast majority in the three
333
groups (94.4 %,100% and 93.7 % respectively), followed by severe form.
334
There was no association between morbidity and different genotype as
335
reported in a study from Zimbabwe [30]. This may be due to S.
336
haematobium infections differ under controlled circumstances, where
337
factors such as exposure to parasites, infection intensity, type of
338
parasite strains.
339
Conclusion:
340
Molecular genotype of S.haematobium in infected Sudanese children in
341
Gezira State proved that primer A01 with three different genotypes
342
(polymorphic band between 700 and 1100 bp), was the most prevalent
343
strain in study area.
344
Difference in genotypes of S.haematobium may play a major role in
345
disease severity. However, molecular characterization of strain is
17
346
needed to identify the type of strain in the area which may facilitate
347
prevention and treatment.
348
More samples from different parts of the state will be collected for
349
future molecular study of the strains.
350
Declaration on conflict of interest
351
No conflict of interest
352
Author contribution and information
353
Nagla Gasmelseed, PhD Molecular Biology (writing the manuscript and
supervising all the work) Nhashal E. Kuramino (collects the samples and
doing the lab work )
354
355
356
Mohamed O. Abdelwahid (supervising the lab work)
357
Anas O. Hamdoun; MD Radiology (doing ultrasonography)
358
and Ahmed E. Elmadani ; MD Radiology (doing ultrasonography)
359
Acknowledgement
360
Thanks for the Khalwas’ school members for their great help and
support. I would like to express my thanks to the Ministry of Health
Gezira State for providing treatment freely for infected children.
361
362
363
364
365
366
367
368
369
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Figure 1: Distribution of the study subjects by age range, N=83
457
458
Figure 2: Shows the results of study subjects age groups by filtration
459
technique, N=45
460
Figure 3: Shows result of S.haematobiumegg count by filtration
461
technique, of 45 study subjects
462
Figure 4: Shows Ultrasound Image
463
464
A: shows thickness on wall of bladder, polyps and calcification B: shows
465
thickness of ureter 6 mm andUltrasonographic examination wasdone
466
by experiencedradiologist.
467
468
Table 1 Ultrasound finding of the Study Subjects by age range
469
Figure 5: Shows S.haematobium genotypes for A01 primer in
470
polyacrylamide gel
471
Table (2) Shows Association between Severities
21
472
And Genotypes of S.haematobium, N=60
473
Table (3) Shows Association between Severity and Positive Study
474
Subject by Filtration Method and Positive by PCR, N=45
475
476
Table (4) Shows Association between Severity and Negative Study
477
Subject by Filtration Method and Positive by PCR, N=15
22