1 Evaluation of sugar floatation technique for diagnosis of gastro- intestinal parasites and its evaluation as epidemiological tool for the study of Hymenolepis nana By Durria Mansour Elhussein (M.Sc. Zoology, Faculty of Science, U of K) Supervisor: Prof. Suad Mohamed Sulaiman Thesis Submitted for the Fulfillment of the degree of Doctorate of Philosophy in Zoology Faculty of Science, Department of Zoology, University of Khartoum Feb. 2005
Transcript
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Evaluation of sugar floatation technique for diagnosis of gastro-
intestinal parasites and its evaluation as epidemiological tool for
the study of Hymenolepis nana
By
Durria Mansour Elhussein
(M.Sc. Zoology, Faculty of Science, U of K)
Supervisor: Prof. Suad Mohamed Sulaiman
Thesis Submitted for the Fulfillment of the degree of Doctorate of Philosophy in
Zoology
Faculty of Science, Department of Zoology, University of Khartoum
Feb. 2005
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Dedication
To my mother and
the soul of my father To my spouse Kamal Widaa
To Dalia , Dalal & Widaa
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Acknowledgement
This research was carried out at Ahfad Biomedical Research Laboratory (ABRL), recently
established by Professor Abdella Elharith, prof. of Microbiology, Royal Tropical Institute during his
mission at Ahfad University during the period from 1999-2003.
I would like to extend my grateful gratitude and appreciation to prof. Abdella Elharith as head of
ABRL who had contributed to this research since its start in 2001.Through his close supervision,
encouragement and advice this research came to light.
I would like to thank my supervisor prof. Suad Mohamed Suleiman for her kind help, supervision,
advice and continuous support during the course of this research.
Thanks are also extended to Dr. Elzein Bashir, Veterinary Research Center for the invaluable help he
extended to me during the data analysis.
Thanks are also extended to my prof. Ahmed Abdelmageed for his continuous support and
encouragement during the course of this study.
Great appreciation is also extended to the team who helped in the data collection, Wisam Mahgoub,
Saria Elsadig, Tayseer Ahmed and Eman Omer for the great effort they had done during the field
work. Special thanks for Wisam Mahgoub, Administrative assistant at ABRL for her continuous
help during the course of this study.
I would also like to thank my colleagues at ABRL Dr. Somaya Elsayed, Dr. Eman Shamat, Mr.
Elfadil Abass and Mr .Mohamed Elmutasim for their kind support and help.
Great thanks to Ahfad University staff and any one who had been involved in this research by any
mean.
Special thanks are due to Mr. Fareed Omer Madani, General Secreatary for Kennana Sugar
Company For his kind support and interest in this research.
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Abstract
In order to gain more insight into the prevalence of gastro-intestinal (GIT) parasite infections among
Sudanese populations residing in the outskirts of Khartoum State, a sugar floatation technique (S FT)
as described by Soulsby (1963) was reproduced and optimized. Evaluation of the SFT as a
diagnostic tool for GIT parasites was carried out at a laboratory level in Omdurman Central Hospital
and in the field including residential areas in the outskirts of Khartoum, Omdurman and Khartoum
North town. Fresh and formaldehyde-preserved stool specimens from children and adults citizen
were collected according to the conventional procedures. Using specially designed questionnaire, the
relevant demographic, clinical and epidemiological data were recorded. As a first step in this
research study, reliability of the SFT, its optimization and standardization was carried out at the
Laboratory of Biomedical Research of Ahfad University for Women (Omdurman). Reliability of
SFT was evaluated by comparison versus the routinely applied direct wet mount (DWM), sodium
dodecyle sulphate sedimentation (SDS) and zinc sulphate floatation (ZFT) tests. Eleven out of 31
formaldehyde-preserved specimens were found to be positive for GIT parasite infection with the
SFT and 8/31 with SDS while all samples were negative with the DWM technique. Using 60 fresh
stool samples, SFT detected parasite infection in 47% and SDS in 12%. In comparison with ZFT
which detected only 2/11, the SFT did so in 10/11. The SFT detected also large number of cases with
wider spectrum of parasite species. In addition to higher reliability, SFT provided clear preparation
background that enabled easy recognition and identification of the parasite species involved. Also,
the test performed equally good both in fresh and formaldehyde- preserved stool specimens. The
SFT detected 9/11 of the formaldehyde-preserved samples in comparison with 7/11 of freshly
collected equivalents. These results signified the importance of employing the SFT in large-scale
surveillance procedures. The present study indicated that the minimum number of helminth eggs that
allowed recovery by SFT was 432 per gram of stool specimen. This number was used as threshold
for comparison between SFT, SDS and DWM using experimentally infected stool specimens in
which SFT showed 100% recovery and SDS 60%. None of those samples was found positive with
DWM. The minimal test incubation period for parasite recovery with SFT was 15 minutes with
maximum recovery after one hour. Attempts were made to reduce the test costs by using gum Arabic
and gelatin as alternative ingredients to replace sugar as small amounts of these reagents provide test
media with specific gravity comparable to that of sugar. The results indicated poor performance
implying that not only specific gravity but also other undetermined factors might affect floatability
of parasite eggs.
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For evaluation at the hospital level, 443 samples were collected from patients with suspicion for GIT
parasite infection. Comparison was carried out with DWM which is currently used as the only
routine test applied in Sudanese hospitals. There was significant difference between the two tests
revealing superiority in favor of SFT which detected 26% of the samples as positives by comparison
with 6% for the DWM. Furthermore, SFT detected various parasite species which included Giardia
lamblia, Entamoeba histolytica, Entrobius vermicularis, Schistosoma mansoni, Taenia saginata and
Trichuris trichiura. Only Giardia lamblia, Hymenolepis nana and Entamoeba histolytica were
detected with DWM. Statistical analysis was applied to determine the difference between SFT and
DWM in their respective reliability for detection of individual parasite species.
Gender and age profiles as well as clinical symptoms in relation to parasite species detected were
also established. Evaluation at field level was carried out in apparently healthy school children (5-17
years of age) residing in three displaced population areas (Mayo, ElHajYousif and Abuzeid). The
total number of stool samples used was 830 collected from both male and female school children.
SFT evidenced higher reliability in detecting dormant parasites and therefore can be recommended
for surveillance procedures. High GIT parasite prevalence was obtained in Mayo (31.7%) and
Abuzied (30.3%) areas in comparison with Haj Yousif (23.5%). Seven parasite species were
detected with H.nana as the most prevalent parasite species in all three study areas. Distribution of
GIT parasites was established for each area and the difference in prevalence with age was
determined. No gender difference in parasite prevalence was found between the three areas. Parasite
prevalence however, was significantly higher among children at the age group of >5≤ 8 years. The
results obtained point out towards the risk associated with high prevalence of GIT infection among
school children in displaced populations and its importance as public health problem.
Application of SFT at a large scale was implemented in an epidemiological study of H.nana
infection. In total, 1790 stool specimens were collected from school children at the age group of 6-17
years together with the demographic, clinical, socioeconomic and behavioral data. H.nana infection
prevalence rate of 30.7% was found. Parasite species other than H.nana were also detected with a
prevalence rate of 65%. Some of those species (26%).were found in association with H.nana. The
highest prevalence rate (53.8%) was found among children at the age group of >5≤ 8 years. The
infection intensity was also the highest (23%) among that age group. There was no correlation
between age, prevalence rate and intensity of H.nana parasite infections. Within the two sex groups,
H.nana parasite prevalence was comparable being 48.8% in the male and 51.2% in the female
population. Among the clinical manifestations, abdominal pain and diarrhea were the most frequent.
Significant correlation was established between the clinical manifestations and intensity of H.nana
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infections. Other clinical manifestations not directly associated with H.nana infections were
vomiting, fever, anal irritation, headache, dizziness and nausea.
The important factors affecting H.nana prevalence in children were identified. Those included age,
parent’s level of education, occupation, type of foodstuffs consumed, source of drinking water, site
of defecation and the behavior of not washing hands after defecation. Multivariate analysis applying
logistic regression was used to determine the risk factors. Child age, the behavior of not washing
hands and defecation site were identified as major factors. Children were classified into three groups
(heavy, moderate and mild) according to the intensity of H.nana infection. The Intra-family
transmission was studied in families of children with different H.nana infection intensities (heavy,
moderate and mild) and a group consisting of families belonging to children found to be free of
H.nana infection (control). Stool specimens collected from 450 family members were examined with
H.nana prevalence of 13%. The importance of H.nana intra-family transmission could not be
established in this study as there was no correlation between the prevalence rates among family
members and intensity of the infection in children. However, H.nana parasite egg count was higher
among family members of children with heavy infection. No gender difference in H.nana prevalence
was found among the family members. Gender difference however, was established among children
at the age group of >10> 15 years where males showed higher prevalence rate than females.
The zoonotic source for H.nana infection was addressed by screening cockroaches and rats for the
presence of H.nana developmental stages. The eggs were demonstrated in 5% of the cockroaches
and in 8.3% of the rodent population suggesting the potential role of these animals in H.nana
transmission.
The efficacy level of Prazaquantel and Niclosamide for treatment of H.nana was assessed. By
comparison with Niclosamide, Prazaquantel showed superiority as evidenced by the significant
higher reduction rate in egg count on day two post- treatment and the complete clearance of stool
specimens from H.nana ova on the seventh day after treatment. Prazaquantel can therefore be
applied as a drug of choice for H.nana treatment.
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Table of Content
Items Page Dedication I
Acknowledgement II
Abstract III
Table of Contents XI
List of Tables XVI List of figures XX
Chapter One: Introduction and Objective 1 1.1 Introduction 1
1.2 Objectives 5 1.2.1 General-objective 5 1.2.2 Specific objectives 5 Chapter two: Literature Review 6
2.1 Important gastro- intestinal parasites 6 2.1.1 Protozoan parasites 6
2.1.1.1.3 Prevalence 7 2.1.1.1.4 Current detection methods 8 2.1.1.1.5 Detection methods under development 8 2.1.1.2 Cryptosporidium species 8 2.1.1.2.1 Route of transmission 9 2.1.1.2.2 Clinical aspects 9 2.1.1.2.3 Prevalence 9 2.1.1.2.4 Current detection methods 10 2.1.1.2.5 Detection methods under development 10
2.1.1.3 Other coccidian parasites 11 2.1.1.4 Entamoeba histolytica 11 2.1.1.4.1 Route of transmission 12 2.1.1.4.2 Clinical aspects 12
2.1.1.4.3 Prevalence 12 2.1.1.4.4 Recent accomplishment in the field of amoebiasis 12 2.1.1.4.5 Current detection methods 12 2.1.1.4.6 Detection methods under development 13 2.1.1.5 Association of protozoan parasites with water 13 2.1.2 Helminth parasites 14 2.1.2.1 Schistosoma mansoni 14
4.4.12 H.nana prevalence among families of children with different intensities of infection
106
11
4.5 Assessment of two drugs for treatment of H.nana 113
Chapter Five: Discussion 122
Chapter Six: Conclusions and Recommendations 141
References 144
Annex
12
List of Figures Fig No. Title
Page
1 Parasite species recovered from stool specimens of patients
received at Khartoum Pediatric Hospital using SDS and SFT
49
2 Percentage of eggs recovered from deliberately infected samples.
52
3 Eggs recovered by SFT in different specimens at different time
intervals.
56
4 Percentage of gastro-intestinal parasites in 442 samples tested with SFTand DWM.
60
5 SFT and DWM results for detection of H.nana and G.lamblia at different health settings.
61
6 Symptoms appeared in patients with different parasite species.
67
7 Gastro-intestinal parasite infection among children at different age groups.
71
8 The distribution of parasites positivity according to the age groups in the three study areas.
72
9 The intensity of H.nana infection in school children of Abuzeid area.
75
10 H.nana prevalence in different age groups of school children in Abuzeid area
76
11 Intensity of infection in different age groups of school children with H.nana.
77
12 Correlation between intensity of H.nana infection and age of child population in Abuzeid area.
78
13 H.nana prevalence in children fed on different food stuffs
86
13
14 Association of abdominal pain and the intensity of H.nana in children.
94
15 Prevalence of diarrhea among children with different intensities of H.nana infection.
96
16 Intensity of H.nana infection among children with vomiting.
98
17 Other symptoms observed in children with H.nana infection.
103
18 Mean egg count in H.nana positive family members of children with different intensities of infection.
109
19 Prevalence of H.nana among family members. 111
20 H.nana prevalence in females and males family members at different age groups.
113
21 The average egg count in children treated with Praziquantel and Niclosamide.
116
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List of Tables
able No Title Page
1 Results of stool examination using SFT and SDS 48
2 Parasite recovery in 60 fresh stool specimens using SFT and SDS. 50
3 Parasite species recovered with SFT in 28 stool specimens. 50
4 SFT performance in fresh & preserved samples 51
5 Comparison of SFT, SDS and DWM performance in experimentally 53
contaminated stool specimen
6 Cross tabulation of SFT and ZSF stool examination results 54
7 Parasite species detected with Sugar and zinc sulphate floatation 54
techniques.
8 Egg recovery with SFT at different time intervals 55
9 Use of different gelatin concentrations in saline solution for 57
recovery of H.nana
10 Use of different gum concentrations in saline for recovery of 58
H.nana eggs
11 Recovery of H.nana eggs using optimal gelatin, gum arabic and sugar 59
concentrations
12 Results of stool examination of patients at health settings: 59
15
Comparison between SFT and DWM
13 Parasite species detected with DWM in 442 stool specimens 62
from patients at different age groups
14 The percentage of parasites detected using sugar floatation test 62 in faecal samples of different age groups.
15 Parasite species detected using DWM in 442 faecal samples 63 from male and female patients
16 Parasite species detected using SFT in 442 faecal samples from 63
male and female patients
17 Detection of E.histolytica using SFT and DWM. 64 18 Detection of Giardia lamblia direct wet mount using SFT & DWM 65 19 Comparative performance of SFT and DWM for detection of H.nana 66
20 Frequency of symptoms in 442 patients tested with SFT 67 21 Stool appearance in 442 samples collected at the health settings 68 22 Parasites species detected in school children at three 69 displaced population localities using SFT. 23 Prevalence of gastro-intestinal parasite infection among children 70
population in the three study areas. 24 Distribution of GIT parasite positivity among male and 72 female child population 25 Prevalence of H.nana and other parasites species among 73
School children in Abuzeid area. 26 H. nana co- infection with other parasite species in school children in 74 Abuzeid. 27 Positively for H.nana in male and female population in Abuzeid area. 79
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28 H.nana prevalence among school children based on 81 father’s level of education. 29 H.nana prevalence in school children based on mother’s. 83 level of education
30 Prevalence of H.nana in relation to father’s occupation 84 31 H.nana prevalence among children fed on different foodstuffs. 85
32 H.nana prevalence rates in relation to house construction. 88
33 H.nana prevalence according to source of drinking water. 89
34 Site of defecation and H.nana prevalence rate 90 35 Effect of hand washing after defecation on H.nana prevalence. 91
36 H.nana prevalence among school children in relation to playing in soil. 92 37 Prevalence of H.nana in children with complaints of abdominal pain. 93 38 Intensity of H.nana infection in relation to abdominal pain 94 39 Prevalence of H.nana infection among children with diarrhoea 95 40 Prevalence of H.nana in children with vomiting. 97 41 H.nana prevalence in children with fever. 99 42 Intensity of H.nana infection in relation to occurrence of fever. 100 43 The association of anal irritation and H.nana infection. 101 44 Intensity of H.nana infection in relation to occurrence of anal irritation. 102 45 H.nana prevalence in children who visited hospitals during 104 the last six months. 46 Risk factors for H.nana infection applying logistic regression. 105
47 Prevalence estimate and confidence interval using 106 the calculated design effect. 48 H.nana and other parasite species detected in family members 107 of infected children.
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49 Egg count in H.nana positive family members of 108
children with different intensity of infection intensity. 50 H.nana prevalence among family members of children 110 with different levels of infection. 51 H.nana prevalence among male and female family members. 112 52 Percentage of children under weight compared to the standard 114 53 Average egg count before and after drug administration. 115
54 Average egg count in children with different levels of infection treated 117
with Niclosamide or Praziquantel.
55 Reduction in H.nana egg count in the second day after treatment 118 56 Reduction in H.nana egg count in the 3rd day post treatment 119 57 Reduction in H.nana egg count in the forth day post treatment 120 58 Reduction in H.nana egg count one week after treatment 121
59 H.nana recovery from rats and cockroaches 121
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Chapter One
Introduction
1.1 Introduction
Gastro-intestinal parasite infections are transmitted through drinking water or
food intake. Microorganisms that are related to the two major parasitic groups,
protozoa and helminthes, cause the most important and persistent health
problems with a prevalence of particular significance among African children
(WHO , 1996).
species of helminthes have been found in association with humans 342 Over
dered as primary inhabitants of would be consi197 of which , )1999, Crompton(
of , species were considered to cause disease20 About . their gastrointestinal tract
Nector , sAscaris lumbricoide, Entrobius vermicularis(which six species
ides Strongylo and Trichuris trichiura, Anchlystoma deudenale, americanus
In addition ). 2003, Horton(affect over half of the world population ) sterocoralis
species and Taenia(to these soils transmitted nematodes there are cestodes
Clonorchis and .Fasciola sp. ,Schistosoma sp(and trematodes ) Hymenolepis nana
.e important human parasiteswhich ar, ).sp
While most of these infections amount to no more than diarrhoea or abdominal pain,
they can be fatal. Intestinal helminth infections are generally chronic, rarely causing
dramatic symptoms or signs. Therefore, health workers, whose priority lies within
acute and fatal infections, may overlook them. However, chronic infections have
profound effect on nutrition (Stephenson et al. 1993), growth (Hadju et al. 1996),
cognitive function (Simeon et al. 1995) and anemia (Dossa et al. 2001) and Torlesse
& Hodges (2001) in infants, school age children and adults. Beside their negative
effect on mental and physical health they can also be pathogenic. The high positivity
for Ascaris causes abdominal pain and discomfort, biliary disease (Sandouk et
al.1997), colic, cholangitis, calculus cholecystis or haemorrhagic pancreatitis (Bahu
et al. 2001; Saowaros et al. 1999; Kutsumi et al. 1989; Khuroo et al 1992).
Furthermore, Ascaris infection may lead to repeated upper respiratory tract infections
(Vizer, et al. 2001). The latter contribute to high mortality in children in developing
countries. These diseases are important both to individuals and to the community.
Thus community-based treatment of gastro-intestinal parasites is considered essential
according to the WHO, Assembly statement in May 2001(WHO, 2001). For
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individual treatment to be effective, diagnosis is necessary to confirm infection and
guide therapeutic choice.
Infections due to intestinal parasites are common throughout the tropics posing
serious public health problems in developing countries (Ramsay et al. 1991, Erko
&Tedla. 1993, WHO , 1986 and Roma & Worku 1997). In these parts of the world
high prevalence rate of intestinal parasites is attributed largely to the socio-economic
status, poor sanitation, inadequate medical services and absence of safe drinking
water (Erko & Tedla WHO, 1986). The low literacy of parents particularly mothers
are also considered as major factors.
According to the WHO estimates, there are 800 million cases with A.lumbricoides,
500 million with E. histolytica and 700-900 million with Anclystoma infections
(WHO, 1995).
Although mortality due to gastro-intestinal parasites is low, the absolute number of
deaths is fairly high because so many people are infected. It is estimated that in 1993,
ninety thousands people died due to Anclystoma species infection, 70,000 to
E.histolytica and 60,000 to A.lumbricoides (WHO, 1995).
The infections are rapidly spreading in slums, shantytowns and squatter settlement, a
trend that is likely to continue with the increasing and unplanned urbanization (WHO,
1995).
Laboratory investigations for parasitic diseases is of vital importance since physical
and clinical examination only is not enough to make decision on whether the patient
is really infected and what parasite species is involved. Therefore, laboratories play
an important role in establishing diagnosis of the parasitic diseases.
Reliable diagnostic techniques are also important for execution of epidemiological
studies to assess and control parasitic infections.
In Sudan, where poverty is widely spreading, population growing fast coupled with
deteriorating sanitation and environmental hygiene, one would expect high
prevalence of gastro intestinal parasitic infections. In addition, migration of large
number of people from rural areas to Khartoum state as a result of civil war and
famine has resulted in introduction of various infections including those caused by
gastro intestinal parasites. This is further worsened by migration of refugees from
neighboring countries to Sudan and their settlement in camps around Khartoum and
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Omdurman. Transmission of these parasites is further enhanced by the absence of
basic sanitary and health services in the growing slums and shantytowns. The
significant influx of displaced population have further contributed to the wide spread
of various parasitic infections. Also, the increase in the number of cases with
malnutrition, HIV/ AIDS may have contributed to the flare up of opportunistic
parasitic infection among citizens of Khartoum state.
As for other endemic diseases in Sudan, control of gastro-intestinal parasitic infection
is rendered difficult due to the absence of sensitive and practical tools for detection
and identification of these parasites. Statistical information on these parasite
infections is usually generated from data provided by hospitals and health care
centers. These institutions, however, are not in a position to monitor the specific
conditions that promote the appearance and spread of these parasites as these
phenomena are determined by specific geographical and ecological factors. In order
to gain better insight into the gravity of these infections, we have decided the
introduction of improved tools to assess the situation more accurately. This approach
is considered essential since the current methods used are insensitive, highly
expensive or advanced to be executed at moderately equipped laboratories. Of these
techniques, the direct wet mount (DWM) can allow for demonstration of parasite ova,
cyst or trophozoite,.However, microscopic examination is rendered difficult due to
unclear background. Concentration methods are considered less efficient due to
deformation or even disintegration of trophozoites.
Ethyl acetate sedimentation though recovers all types of helminth ova, larvae as well
as protozoan cysts and most suitable for formalin preserved samples, it has the
disadvantage of being flammable and expensive. Also, using this technique, more
debris appears in microscopic examination than with floatation.
Among the floatation techniques used, the most common is the zinc sulphate method.
This method is recommended both for protozoan cysts and helminths eggs. However,
this technique is expensive, unsuitable for fatty stool samples and does not allow for
the exposure of some trematode ova and tapeworms.
Simple and reliable diagnostic techniques that does not depend on special equipment
or expensive reagent are therefore of paramount importance in developing countries
such as Sudan.
The focus of this study is to introduce more sensitive techniques for detection of
gastrointestinal parasites. The floatation technique as developed by Soulsby (1968)
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and used for detection of gastrointestinal parasites in animals will be evaluated here
for detection of human parasites.
The idea behind this study has emerged from a pilot trial conducted at Khartoum
Paediatric Hospital. Thirty-one samples that have been declared negative at that
hospital were re-examined using the Sugar Floatation Technique. Another
sedimentation technique was also used in which Sodium Dodecyle Sulfate (SDS)
detergent was used. Eleven samples were found positive with different parasite
species by the sugar floatation, as the SDS recovered eight. The results obtained had
encouraged us to scale up the study for wider evaluation.
The study is also intended to explain the phenomenon of the increased H.nana
prevalence as a general observation in hospitals, health centers and private clinics.
H.nana is a cestode known as dwarf tapeworm. The parasite is one inch in length,
does not require an intermediate host and the eggs are capable of causing direct
infection in humans. The consumed eggs hatch and develop into tiny cysticercoids in
the intestinal villi. The adults emerge into the intestinal mucosa and begin laying eggs
which may continuously auto infect patients through the fecal-oral route (Robert &
Janovy 2000). The parasite produces either no symptoms or very mild disease.
However in fairly heavy infections the disease is symptomatic. Therefore few
numbers of clinical cases are reported and many other sub clinical infections may
pass undiagnosed. The clinical importance of this parasite had been reported recently
(Despommier,& Dickson 1992, Scherrchard etal. 1996 and Sirivichanyakul, et al.
2000) initiating public concern about this parasite. Appearance of important reports
on the development of immature cysticercoids in the stool of patients under
chemotherapy and the spread in body tissues confirmed importance of this parasite
(Rudrapatna, et al. 2002). Although infection caused by this parasite appears to be
common in Sudan, data on its prevalence, transmission and clinical importance is
completely lacking.
The importance of studying H.nana infection stems also from the fact that this
parasite causes serious lesions in immuno-suppressed animals. Experimental infection
in mice had profoundly been influenced by immune suppression. This immune
suppression was caused by T-cells deprivation as a result of steroid treatment
resulting in massive abdominal cysticercoids multiplication. Similar pathology was
also observed in patients with Hodgkin’s disease who received immune-suppressive
chemotherapy (WHO, 1995). Observations on the course of H.nana infection in
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immuno-suppressed mice were also reported by Lucas et al. (1980).Treatment of
these animals with hydrocortisone acetate caused super infection in the bowels with
adult worms and developing cysticercoids in the mesenteric lymph node and liver.
Infection of mice deprived of T-cells by pre adult thymectomy and antithymocyte
serum administration resulted in super infection and widespread metastasis of
aberrant cysticercoids causing death in about five minutes (Locas,etal 1980). These
findings may be of significance in immunosuppressed human subjects with H.nana
co-infection. Therefore H.nana infection is one of the conditions that should be
eliminated before applying chemotherapy in immunosuppressed patients.
The study deals also with an aspect that remained unsolved in H.nana epidemiology,
which is the role of the animal reservoir in the spread of human infection. Although
cross infection between human and animal has not been confirmed the role of animal
as disseminator for human infection has not been excluded. This study aims towards
providing base line data on the epidemiology of H.nana in Sudan including
evaluation of the role of Arthropodes (cockroaches) and rodents in the transmission
of the parasite. The purpose of this part of the study was to assess the health risk
associated with human, cockroaches and rodents interaction as they pertain to
H.nana. Given that children have less knowledge of hygiene they are under great risk
for the disease.
1.2 Objectives:
1.2.1 General
The general objective of this study is to contribute in a more accurate approach to
assessing gastrointestinal parasite infection in the Sudan.
1.2.2 Specific objectives:
1. To introduce sugar floatation technique for detection of gastrointestinal parasites.
2. To evaluate the technique at laboratory, hospital and field levels. 3. To study the epidemiology of H.nana in school children in selected areas in
Khartoum state i.e. in Abuzeid area.
4. To study the relative efficacy of two different antihelminthic drugs in the treatment
of
H.nana.
23
Chapter two Literature Review
2.1 Important gastro- intestinal parasites
2.1.1 Protozoan parasites
Several species of parasitic protozoa are transmitted through food and water. Among these, Giardia,
Entamoeba and Cryptosporidia are of most importance worldwide. Morbidity and mortality rates
due to E. histolytica are high, especially in developing countries.
2.1.1.1 Giardia lamblia
Giardia is a flagellated protozoan. The taxonomy and host specificity remained the subject of
considerable debate. The organism has been found in more than 40 animal species (Meyer, 1994).
Nowadays, five species of Giardia are established in the scientific literature, including the three
species G.muris in rodents, reptiles and birds, G.intestinalis (lamblia or duodenalis) in mammals,
rodents and reptiles and G.agilis in amphibians, G.ardae in the great blue heron (Erlandsen et al.
1990) and G.psittaci
in the budgerigar (Erlandsen & Bemrick, 1987). Giardia life cycle is simple requiring no vector or
reservoir (Feely, et al. 1990 & Meyer, 1994). The parasite is shed in faeces as cyst which then
transmitted to a second host where the trophozoite emerge and attach to the epithelial cells and feed
on them. The trophozoites detach from the epithelial cells due to the turnover of these cells and
undergo mitotic division in the intestinal lumen. During periods of diarrhoea the trophozoite may be
excreted but cannot survive long outside the host.
2.1.1.1.1Clinical Aspects
Asymptomatic carriage appears to be the most common form of infection with Giardia (Farthing,
1994). Of the infected individuals, 16-86% are asymptomatic. The mechanism by which Giardia
causes diarrhoea and malabsorption is still unclear. The organism could act as a barrier but the areas
covered by the trophozoite in the intestines are probably too small to affect absorption of nutrients.
Giardia infection appears to affect the activity of gut enzymes damaging the mucosal surface and
give rise to overgrowth of bacteria (Tomkins et al. 1978) and yeast (Naik et al. 1978) in the small
intestine.
The most prominent symptoms are diarrhoea, weakness, weight loss, abdominal pain and to a lesser
extent nausea, vomiting and fever.
Infection with Giardia is common in children of the developing countries (Farthing, 1994, Rabbani
& Islam, 1994).
2.1.1.1.2 Route of Transmission
24
Faecal-oral transfer of Giardia cysts is the major route for transmission of the disease as indicated by
the high prevalence in developing countries where poor standards of hygiene and sanitation, in day
care centers and nurseries are lacking. There was also secondary spread among households of those
who had attended the day care centers (Vande et al. 1991).
Food borne outbreaks were the result of food contamination by infected workers or household
members (Islam, 1990). The role of animal in Giardia transmission is still unclear. However,
Giardia isolates from animals and humans may be morphologically indistinguishable (Flannagan,
1992) and this had led to many reports of animal sources of human Giardiasis (Moore, 1969).
2.1.1.1.3 Prevalence
In the developed countries, prevalence peaks at the age of 1-4 years (Flannagan, 1992) and again at
20-40 years age groups, partly because of caring for young children and partly as a result of
traveling. In the developing countries, Giardia prevalence in patients with diarrhoea is about 20%
(Islam, 1990). In Brazil, Robert et al. (2001) reported infection rate of 27.4% among children.
Prevalence was 9.7% in children with diarrhoea and 7.4% in the non-diarrhoeic. He also reported
significantly lower weight and height for symptomatic children by comparison with asymptomatics.
A prevalence rate of 24% was reported by Lee (2000) in school children in Taoyuan. A recent study
in Venezuela has shown 21% Giardia prevalence among children attending day care centers (Miller
et al. 2003). In a peri-urban community from the province of Buenose Aires (Argentina) a study
revealed 26.3% prevalence of Giardia (Pezzani etal. 1996). Saygi etal. (1995) reported a prevalence
rate of 8.2% among students of adult educational centers in Sivas, Turkey. De Rezende et al. (1997)
reported 2% prevalence rate among food handlers in a public school in Brazil. In a study carried out
in Serbia, Nikolie, etal. (1988) found 6.8% of the study sample to be infected with G.lamblia and
Magambo et al. (1998) reported 9.8% prevalence rate among children in Southern Sudan.
2.1.1.1.4 Current detection methods
Examination of fresh stool for cysts and trophozoite and examination of permanent stained smears
are still the most widely used detection methods. Because the parasite mechanically adheres to the
intestinal mucosa, a series of five to six stools may be examined without success in recovering the
organism (Garcia & Buckner 1993). Procedures using Enzyme Immuno-Assay (EIA) had been
developed to detect Giardia antigen in faeces. An evaluation of a commercially available EIA kit
using kit was found to be sensitive and 100% specific. Because a single technician can read many
specimens in a short period, the EIA is less expensive than microscopic examination (Addiss et al.
1991). Recently, the Triage parasite panel introduced a new qualitative Enzyme Linked Immuno
25
Assay panel for detection of G. lamblia, E. histolytica, E.dispar & C.parvum using fresh stool
specimen.
A direct Fluorescent Antibody (DFA) method using monoclonal antibodies detected very large
numbers of organisms in a shorter period of time than that needed for examination of permanent
stained smears. No false results were reported (Garcia et al.1992).
2.1.1.1.5 Detection methods under development
Nucleic acid-based detection method for G.lamblia cysts and trophozoites in faecal specimens have
proven to be a challenge due in part to the difficulty of lysing cysts and more importantly the large
amount of other DNA and inhibitory substances present in the clinical specimens (Marilyn et al.
1997).
So far, only one reported study applied PCR for evaluation of human stool samples. The sensitivity
of the rRNA small subunit in PCR assay resulted in some false negative and false positive results by
comparison with microscopic detection (Weiss, 1995).
2.1.1.2 Cryptosporidium Species
Cryptosporidium is a coccidian parasite pathogen of the intestinal tract causing diarrhoea both in
immunocompromised and immunocompetent human subjects (Current and Garcia, 1991). C. parvum
is now considered as one of the most known pathogen in AIDS patients. Its occurrence is dependent
on age, season and demographic characteristics of population. Among children with diarrhoea, C.
parvum may be the most frequently found pathogen (Palmer, 1990).
Members of the genus Cryptosporidium are small coccidian protozoan parasites that infect the
microvillus region of epithelial cells of the digestive and respiratory tracts of vertebrates. C. parvum
is the causative species of human infections.
2.1.1.2.1 Route of transmission
Oocysts, the environmentally resistant transmission stage of the parasite are shed in the faeces of
infected hosts (Fayer & Ungar, 1986, Fayer, et al. 1997) and are readily infectious.
A major mode of infection with cryptosporidium is person-to-person transmission, as evidenced
by outbreaks in day care centers (Fayer & Ugar, 1986, Casemore, 1990 and Cordell & Addiss,
1994). Several sexual practices involving oral-anal contact also involve high risk of exposure to
the organism. Cryptosporidium can also be transmitted from mammals especially new born
animals to humans and many infections had been derived from contact with infected calves
and lambs (Casemore, 1990). Person to person or zoonotic transmission may occur through
contaminated water (Casemore etal. 1997).
2.1.1.2.2 Clinical aspects:
26
The incubation period varies greatly but is usually 7 days (Ungar, 1990). Watery diarrhoea is
the most prominent symptom of intestinal C. parvum infection (Fayer & Ungar 1986, Ungar,
1990). The copious bowl movements can cause dehydration and weight loss (Arrowood, 1997).
Other symptoms include nausea, abdominal cramps, vomiting and mild fever. In immuno
competent individuals, the infection is limited by the immune response that eventually clears
the parasite. Infections in patients with defective cellular mediated response (congenital or due
to AIDS) are persistent and heavy (Ungar, 1990). Several studies in animals suggest that the
immune response protect against reinfection (Zu et al.1992). Protective immunity in human is
indicated by the large number of asymptomatic carriers in countries with high prevalence
rates.
2.1.1.2.3 Prevalence
Prevalence of cryptosporidiosis in AIDS patients in developed countries is around 10-20% (Current
& Garcia, 1991). In the absence of an effective immune response, the infection may spread
throughout the entire intestinal tract and in other parts of the body (gall bladder, pancreas,
respiratory tract). No effective therapeutic agent has yet been found (Blagburn & Sova, 1977).
Immunotherapy with monoclonal antibodies has been reported to resolve diarrhoea at least
temporarily in AIDS patients (Riggs, 1997).
In one study in the UK, 19% of AIDS patients were thought to have died due to cryptosporidiosis
(Conolly et al. 1988). In another study compiling case reports of cryptosporidium, 46% mortality
rate was reported among AIDS patients and 29% in patients with other immunodeficiencies (Fayer
& Ungar, 1986).
In patients with gastrointestinal infections, prevalence of cryptosporidium was 1-4% in Europe
and North America and 3-20% in Africa, Asia, Australia and Southern and Central America
(Current & Garcia, 1991). High rates of asymptomatic carriage (10-13%) are common in
developing countries (Current & Garcia, 1991). The number of asymptomatic carriers as
determined by stool survey in industrialized countries was generally very low (< 1%) (Current
and Garcia, 1991). However, routine endoscopies suggests higher prevalence of asymptomatic
carriage of up to 13% in the non-diarrhoeal patients (Roberts et al. 1989). Seroprevalence
rates are generally higher than fecal carriage rates being 25-35% in industrialized countries
and up to 95% in South America (Casemore, etal. 1997). Infection increased with age (Zu et
al. 1992, Kuhls et al.1994) and is relatively high among dairy farmers and daycare centers
attendants (Kuhls et al. 1994).
2.1.1.2.4 Current detection methods
27
Routine diagnostic methods include fecal concentration with Sheather’s sugar floatation or
formalin- ethyl acetate or acid fast stained smears (Garcia & Buckner, 1993). This approach is
labor-intensive and is limited by skill of the microscopist. The need for rapid sensitive
diagnostic techniques has initiated development of commercial kits. These included EIA and
DFA techniques with various advantages and disadvantages (Marilyn et al.1997).
2.1.1.2.5 Detection methods under development
A passive haemoagglutination assay was developed by Farrington et al. (1994). The assay
involved an anti-oocyst monoclonal antibody coupled with stabilized sheep erythrocytes. It has
been reported that this procedure could be used as a screening test; but had not been evaluated
versus EIA or IFA (Arya, 1995). A variety of polymerase chain reaction (PCR) techniques
have offered an alternative to conventional diagnosis of Cryptosporidium both for clinical and
environmental specimens (George et al.1990). Although PCR is rapid, highly sensitive and
accurate, it has several limitations. False positive outcome can result from detection of naked
nucleic acids of non-viable microorganisms or environmental contamination.
2.1.1.3 Other Coccidian parasites:
There are almost one thousand Microsporidia species widely distributed in nature (Stewart &
Osborn, 1996). Microsporidia have been recognized as pathogens in fish, birds and some mammals,
but several species have recently been identified as cause of disease in several immuno-
compromised patients. These species are associated primarily with infections of the intestinal tract
although dissemination to the biliary, urinary and respiratory tracts may occur. The mode of
transmission is still unclear, but faecal-oral route is most likely. The persistence of these species in
water, their resistance to disinfectants and their small size suggest water-borne transmission.
Toxoplasma gonidii is an intracellular coccidian parasite that has been recognized as human
pathogen. Feline is the definitive host and can be infected primarily by the consumption of infected
mammals and birds, which act as secondary hosts. In secondary hosts, the parasite becomes encysted
in muscles and brain tissue. Only felines carry the parasite in the intestinal tract and shed oocysts
that sporulate in the environment. Consumption of uncooked meat or raw milk and contact with
feline faeces are the primary source of infection in human (Stewart & Obsborn, 1996).
2.1.1.4 Entamoeba histolytica
E. histolytica is one of the most important parasites of the gastrointestinal tract in man. It
belongs to the class Rhizopoda (Dey, 1964). The genus Entamoeba includes among others two
species, E. dispar, a commensal parasite of the gut and E. histolytica, which invades the
mucosa. It is difficult to differentiate between the two species morphologically. The two species
however can be differentiated by DNA probes and polymerase chain reaction (PCR).
28
The most important host of E. histolytica is man and patients with different clinical forms of
amoebiasis are the most important source of the infective agent (Ximenez, et al. 1993).
The infection is generally associated with poor socioeconomic condition, and unhygienic habits
in the developing countries (Arturo et al. 1994). The parasite is among ten most common
infections in the world (Warren & Mohamed, 1984). According to WHO reports,
approximately 500 million people suffer from amoebiasis worldwide and the annual mortality
is 40,000-110,000 (Mckerrow, 1994, Luaces & Berrett (1993). Beside travellers, groups at high
risk for amoebiasis are immigrants, immunocompromised individuals and sexually active male
homosexuals.
2.1.1.4.1 Route of transmission
Cysts are transmitted mainly by ingestion of contaminated food, water or through direct
contact. Cysts, which are excreted in the feces, are infective to other susceptible human hosts
or can remain viable under favorable environmental conditions for two months (Bruckner,
1992).
2.1.1.4.2 Clinical aspects
The incubation period varies from few days to months, depending on the area of endemicity.
Normally, it ranges from 1 to 4 months. E.histolytica is unique among other intestinal amoebae
because it is able to invade tissues. The major intestinal syndromes are acute colitis, abdominal
pain, frequent bloody stools and fever. (Reed, 1992).
2.1.1.4.3
Prevalence
Infection with E.histolytica occurs worldwide and it has been suggested that 12% of the
world’s population is infected with this parasite (Reed,.1992). Of those infected every year,
10% manifest clinical symptoms; 80 to 98% of these patients show symptoms related to
intestinal mucosa damage and in the remaining 2-20% the infection is cleared. Recurrence of
invasive colitis or amoebic abscess is unusual. (Bruckner, 1992) With the exception of malaria
and Schistosomiasis, amoebiasis causes more deaths than any other parasite (Reed, 1992).
2.1.1.4.4 Recent accomplishment in the field of amoebiasis
Biochemical, immunological and genetic data have recently indicated that there are two
different subspecies namely, E. histolytica and E. dispar. Of the two subspecies, E. histolytica is
capable for causing invasive disease (Clark & Diamonnd 1994). Proteins associated with
virulence had been identified including lectin that mediates adherence to epithelial cells, lyses
of host cells and secretion of proteases that degrade host tissues. This virulent protein and the
29
unique antigen presented on parasite surface were potential targets for antiamoebic vaccines
(WHO, 1997).
2.1.1.4.5 Current detection methods:
The routine laboratory diagnosis is by examining a minimum amount of stool specimen using
concentration or permanent stain techniques. The importance of the stained preparation
cannot be over emphasized, particularly when there are so few organisms in the stool specimen
(Bruckner, 1992).
2.1.1.4.6 Detection methods under development
With the recognition of the pathogenic E.histolytica and non-pathogenic E.dispar, nucleic
probe assay have been developed. Several genes that contain DNA sequences differentiating
between the two subspecies are the basis for DNA assays to detect and identify E.histolytica.
Two assays had been tested with patient’s samples where PCR was employed to study the
epidemiology of pathogenic and non-pathogenic strains in a rural community in Mexico. Stool
samples were used for extraction of DNA.
The PCR amplification was performed with two sets of primers that discriminated between
pathogenic and non-pathogenic strains. This assay had a sensitivity of 96% and specificity of
98% (Acuna et al. 1993). The second method was performed at room temperature and the
results were read in the same day. One trophozoite per mg of stool sample was enough for
detection and differentiation (Katzwinkel et al. 1994).
An Enzyme Immuno Assay (EIA) had been reported to directly distinguish between
pathogenic and non-pathogenic strains in stool samples (Hague, 1994). Recent studies
suggested that assays based on antigen detection were more sensitive and specific for the
diagnosis of E. histolytica than microscopy (Pillai et al.1999).
2.1.1.5 Association of protozoan parasites with water
Transmission through water is common in developing countries where much of the water
supply for drinking is untreated and faecally contaminated. The use of human faeces for
fertilization was also an important source for infection (Bruckner, 1992). In the United States,
between 1946-1980 six outbreaks were attributed to E.histolytica (Lippy and Waaltrip, 1984).
Giardia lamblia cysts had been detected in 81% of the raw water samples (LeChevallier, et al.
1992) and 17% in the filtered (LeChevallier, et al. 1991). In the United States, Giardia
continues to be the most frequent cause of water borne disease outbreaks. Outbreaks of
giardiasis were associated with the intake of unfiltered or inadequately chlorinated surface
30
water (Herwaldt, et al. 1991). Cryptosporidium oocysts were found in 87% of raw water
samples (LeChevallier, et al. 1992), 27% in drinking water (LeChevallier, et al., 1991).
Cryptosporidium was responsible for eight outbreaks associated with water intended for
dinking in the United States. (Berkelman, 1994, Joce, et al. 1991,McAnulty et al.1994).
2.1.2 Helminth parasites
Helminth parasites are related to three main distinguished classes, namely trematoda, cestoda and
nematoda. The most important gastro-intestinal helminthes are:
2.1.2.1 Schistosoma mansoni
It belongs to the group trematoda. Humans become infected usually by immersing themselves
partially or entirely in fresh water that contains cercaria which is the infective stage. Infection occurs
when the parasite penetrates through the host skin.
In endemic areas, any standing or moving untreated fresh water whether natural or man-made may
contain cercariae (Klotemetzel, 1962 and Certon, et al. 1996). The parasite inhabits the mesenteries
of the intestine and adult worms deposit their eggs in these blood vessels. The eggs migrate to the
lumen of the intestine. Not all eggs pass via the intestine but many of them swept back to the liver
where they are entrapped.
As a result of host immune reactions, granulomas are formed (Cheever, 1986).
The disease is common in tropical and subtropical areas in Southern and Central America, Africa,
Asia and South-east Asia.
The clinical signs of the disease take two different phases; an acute stage known as katayma fever
normally occurs in young children with no previous exposure and the chronic stage in which patients
experience diarrhoea and fever. In children, the disease depresses growth rate and lead to
hepatosplenomegaly, portal hypertension and granuloma formation.
2.1.2.2 Taenia saginata and Taenia solium
These two cestode parasites are among the oldest parasites causing infection in human worldwide
with marked economic impact. Taenia saginata infection is common in many developing countries
(Tanowizt, et al. 2001& Matuchansky & Lenormard, 1999). Human infection is acquired from
eating raw beef containing the viable cysticercus larvae. Cattle acquire infection by grazing on grass
polluted with sewage of human faeces containing eggs of the parasite (Hammerberg et al. 1978).
31
Man is infected by Taenia solium when eating uncooked or insufficiently cooked pork (Lucas &
Gills, 1990). The scolex lodges in the upper jejunum. Usually only one worm is present. Multiple
infections may occur and infection by both species had been described. Adult worms cause minimal
pathological changes. However, intestinal mucosal biopsy had shown inflammatory reactions.
Among the most resulting signs were abdominal pain, nausea, weakness, loss of appetite, dizziness,
diarrhoea headache and constipation.
2.1.2.3 Hymenolepis nana
This is another important cestode encountered frequently in children and is cosmopolitan in
distribution (Ichpujani & Bhatia, 1994). Hymenolepis nana infection is a common cause for
morbidity especially among immigrants in USA (Tanowitz et al. 2001). It is transmitted by the
ingestion of eggs in food or water, by hands contaminated with faeces or autoinfection, which
is rather common.
2.1.2.4 Ascaris lumbricoides
This is one of the most important helminth belonging to the class nematoda (Clinch & Stephen,
2000). It is a soil transmitted gastrointestinal worm causing high morbidity among school children in
developing countries (Savioli et al. 1992). It flourishes in warm, moist climate and in moist
temperate regions where personal hygiene and environmental conditions combining together favour
development and persistence of the infective stage in polluted soil (Paul et al. 1984). Also where
sanitation is inadequate and human faeces are used as fertilizer (Monica, 1998). Infection is acquired
by ingestion of eggs which hatch in the jejunum, penetrate the intestinal wall, migrate to the trachea
up to the pharynx and then swallowed again to reach the intestine. Most of the infected persons are
asymptomatic. Complications usually resulted from migration of adult worms causing bile duct
obstruction, peritonitis, liver abscess and appendicitis (Bar-Maor et al. 1984).
2.1.2.5 Trichuris trichiura
Known also as whip worm and inhabits the large intestine causing trichiuriasis.
T. trichiura infects approximately 800 million people. It has a worldwide distribution but most
common in tropical and subtropical regions where sanitation is low (WHO, 1987). Infection is
acquired by ingestion of infective eggs or contaminated food. 2.1.2.6 Entrobius vermicularis
Also known as pin worm and causes entrobiasis (Markel & Voges, 1999). It has a worldwide
distribution and more common in temperate than in warm climate. Children are more commonly
infected than adults (Manson & Bell 1987). Transmission is by ingestion of infective eggs and
autoinfection by swallowing infective eggs through contaminated fingers was common in children
(Franklin & Harold, 1994).
32
Prevalence in institutionalized persons is 50-100%. Pinworm infestation is rarely fatal. Death and
morbidity are from secondary infection (WHO, 1987).
2.1.2.7 Ancylostoma deudenale and Nector americanus
Generally called hookworms and they occur commonly in warm climates especially in communities
with poor sanitation and hygiene. Anemia secondary to blood loss is the most important feature of
hookworm infection. Occurrence of the disease depends on the load of infection, state of iron store
and host nutritional status (Lucas & Gills, 1990). Adult worms live in the intestine and attach to the
walls through which they suck blood. Infection is by contact with soil and larvae penetrate the skin.
It is endemic in tropical and subtropical regions including Africa, Southern East USA, Asia and the
Caribbean. In parts of West Africa, Central and South America, infection with both species may
occur. (Lucas & Gills, 1990).
2.1.2.8 Strongyloides stercoralis
This is a nematode worm that parasitizes man and other primates including dogs and, cats. It is a
parasite of tropical origin, although found in temperate regions. Having a complete life cycle, with a
free-living larval stages, the female adult worm inhabits the small intestine of its host. The eggs
usually hatch in the intestinal lumen and the hatched larvae are passed in the faeces (Roberts &
Janovry, 2000). Clinical importance of this parasite was reported by Pires & Dreyer (1993).
2.1.3 Prevalence of helminth parasites
Prevalence of helminth parasites was studied in different areas and in different target groups.
Molina et al. (1988) found 32% of Southeast Asian refugees infected with one parasite and 8%
with multiple parasites. Hookworms, Strongyloides spp and H.nana were the most found.
In Serbia, similar studies were conducted by Nikolie et al. (1988)and high prevalence of
intestinal parasites was reported including E.histolytica, G.lamblia, H.nana, E.vermicularis, A.
lumbricoides and T.trichiura and a prevalence of up to 66.7% was reported.
A cross sectional survey of intestinal parasite infection in rural community was conducted in
Kenya (Chunge et al. 1991). Prevalence rates of 81.4% were recorded for single and 72.7% for
multiple infections.
A comparative study on intestinal parasites prevalence was conducted among children living
in rural and urban settings in chennai by Fernandez et al. (2002). He found an overall
prevalence of 91% in rural location and 33% in urban settings.
The prevalence of helminth infections in Namibia was studied by Evans &Robert (1989). He
reported 6.8% with Hookworm, 2% Stronyloides sp., 1% H. nana and 0.9% of Taenia species.
Hookworms were found more common among females than males.
33
The status of intestinal parasite infection was investigated in two villages in Nepal (Young et
al.2000). A prevalence rate of 44% was found with higher prevalence among females.
Entamoeba coli was the most commonly found protozoa (21%) followed by Giardia lamblia
(13.7%). Hookworm was the most common helminth (13%) followed by T.trichiura (3%).
Intestinal helminth infections were also studied in Ethiopia in nineteen communities located in
the southern part of the country (Birrie, 1994). A prevalence rate of 10% was reported for S.
species (3.4%), T.trichiura (7.5%) and E.histolytica (8.2%). Those parasites occurred often as
multiple infections.
Intestinal parasite infection in HIV positive and negative individuals was studied in San pedro Sula,
Honduras (Lindo, et al. 1998). The results revealed that Cryptosporidium parvum and S. sterocoralis
were found exclusively in patients with HIV. By contrast, extra cellular parasites such as G.lamblia,
A.lumbricoides and T. trichiura were significantly higher in the HIV negative. The authors
concluded that HIV might selectively determine establishment of certain parasite infections. This
may be due to the fact that HIV-induced enteropathy does not favor the establishment of extra
cellular parasites. Intracellular parasites and mucosal dwelling parasites however, may benefit from
pathogenic changes and reduced local immune responses induced by the virus.
Infection with the human immunodeficiency virus (HIV) predisposes patients to several parasitic
diseases such as pneumocystis carinii, and Toxoplasma encephatilis. Among intestinal parasites, the
intracellular coccidians (Isospora belli and Cryptosporidium parvum) are most common (Fleming,
1990). The microsporidian Enterocytozoan bienusi are also significant enteric pathogens (Canning et
al.1990).
Investigations for intestinal parasitic infections in AIDS patients in Africa had focused on those with
diarrhoea. Isospora and Cryptosporidium had consistently been found in those patients at prevalence
varying rates 32% and 12% to 19% respectively ( Henry et al.1986, Colon et al. 1990 and Hunter et
al. 1992).
Some reports indicated that there was no convincing evidence on the etiological agents isolated from
African AIDS patients with diarrhoea and that their prevalence or incidence was increasing due to
HIV infection (Khumalo et al.1994).
In a study conducted by Fisseha et al. (1999), Cryptosporidium was reported to be more prevalent in
AIDS patients and that its occurrence was strongly associated with chronic diarrhoea. Association
between AIDS, parasitic infections and diarrhoea was not evident in that study. This supports the
view that common parasites such as A.lumbricoides, G.lamblia, T.trichiura, Taenia species and
E.histolytica are not opportunistic in nature.
Prevalence of intestinal parasite pathogens in HIV seropositive subjects was studied in Indian
patients (Mohandas et al. 2002). Thirty per cent of the patients were found to harbour
intestinal parasites. The most common were cryptosporidium (10.8%), G.lamblia (8.3%),
Cyclospora (3.3%) and Isospora (2.5%). E. histolytica was observed in two and hookworms in
one patient. Of the tested patients, 7% had diarrhoea. The paper emphasizes the necessity of
increasing awareness among clinicians regarding occurrence of these parasites.
2.1.9 Gastrointestinal Parasite infections in Sudan
39
Researches on gastrointestinal parasite infections in Sudan are very scarce. However since 1904
many studies had been conducted on Schistosomiasis especially in the Gezira area where the first
mortality due to Schistosomiasis was reported by Balfor (1904). The author found 17% prevalence
among school children. His finding was confirmed later by many researchers (Humphery, 1932;
Amin & Satti1973; Omer et al. 1976; Amin et al. 1982, Babiker et al. 1985; Eltom et al. 1993). Very
few studies had discussed in general the status of intestinal helminths in Sudan (Steketee and
Mulholand 1982, Reckart et al. 1985). Annual report by the Ministry of Health (MOH report 2000)
had shown an incidence rate of 27%.
In a study survey for intestinal protozan and helminthes infections carried out in a University
community in Khartoum, Ibrahim (1983) reported an overall prevalence rate of 7.7% including
G.lamblia (43.8%) E.histolytica (25.9%), S.sterocoralis (5.4%), A. lumbricoides (3.7%), Hookworms
(1.6%), H.nana (13.3%), T.saginata (1.2%), S.mansoni (4%) and S.bovis (1.2%).
An analytical study on intestinal parasite infections at University of Juba Clinic (Hakim and Mogga,
1983) was prompted by a sudden rise in the number of cases of diarrhoea associated with T.hominis,
G.lamblia and E.histolytica during the period Dec 1979 and 1980 March-June. That period coincided
with the dry season in Juba and the shortage of water for domestic use. Results of stool examination
based on direct microscopy gave an overall picture of parasite spectrum in the patients presenting
with diarrhoea. Flagellates were the commonest (46-82%), followed by E.histolytica (38.21%) and
Entamoeba coli (70%). The study indicated that intestinal parasitization was closely linked with the
poor sanitation in the town, where majority of the people has no toilets. The occurrence of flagellates
in association with diarrhoea during that period of water scarcity suggests that the flagellate may be
transmitted directly, through shaking of hands when greeting and communal eating habits as
common practices in Sudan.
A parasitological survey among refugees resided in Juba area revealed that 66% of the population
harboured intestinal parasites (Marnell et al. 1992). The most commonly found parasites were
hookworms (36%), S.mansoni (26%), Strongyloides sterocoralis (20%), H. nana (11%),
A.lumbricoides (1.2%), T.trichiura (0.8%), and Taenia species (0.4%). Many of the specimens
(42%) harboured single infection, 21% had double, 2% had triple and 1% quadriple infection. The
parasites prevalence and intensity were analyzed in relation to sex, age, religion, and occupation.
Gender prevalence profile showed differences; females showed higher prevalence of H.nana and S.
mansoni infection than males. In the same study, prevalence of parasites was also found to be higher
among Christians than Muslims. None of the parasites showed any significant difference with regard
to occupation although H nana was more prevalent among white collars and medical workers.
40
Another study was conducted in Southern Sudan to determine the prevalence of intestinal parasites
among school children (Magambo et al. 1998). In 275 children examined, 15 different species were
identified. Children at the age group 6-10 years were most affected followed by the 11-15 years
group. The infection was slightly higher in males than females. The commonest protozoan parasites
identified were G.lamblia (9.8%), E.histolytica (28.4%), E.coli (37.8%). The helminth parasites
reported were hookworm (13.1%), S.sterocoralis (3.3%), and T.trichiura (1.8%).
In a study of helminth parasite prevalence in Elgamair residential area (Omdurman town in
Khartoum State) conducted by Idris (1999), 1268 stool samples were collected from school children
and patients reported to health centers. 37.2% of the population was found to harbour intestinal
helminth. The most common parasite species were A.lumbricoides (15.7%), H.nana (11.6%),
E.vermicularis (8.1%), hookworms (4.8%), Taenia saginata (3.4%), S.mansoni (3.2%) and T.
trichiura (1.7%).
Among the school children examined, the overall prevalence was 43% with A.lumbricoides being
most prevalent and boys showed higher prevalence than girls. The result of health centers showed an
overall prevalence of 26.3% with A.lumbricoides occupying the top of the list. The study indicated
also a clear relationship between the prevalence rate and socio-economic status, personal hygiene
and lack of knowledge.
Homeida (1991) conducted a survey on the prevalence of intestinal helminth in Sudan. In his report,
240 patients in Juba area were examined revealing a prevalence of 66%. An extensive prevalence
survey was later carried out in 6 different locations in the Sudan covering different regions. Those
included Abujin village (central Sudan), Rank (upper Nile, Southern Sudan) Juba in addition to six
camps of displaced people around Khartoum. A total population of 2489 children (aged 7-14 years)
was studied. S.mansoni prevalence was found to be 29.2% ranging from 0%-64%. Fifty-three of 724
of the patients were found to harbor geohelminthes infection. Ninety four percent of the
geohelminthes infected cases were found in Juba, with 4 cases having mixed infection. Further
examination was carried out in Juba in 2789 children aged 7-14 years. Twenty-nine locations were
examined including displaced camps schools and religious institutions. S.mansoni accounted for
6.9%, geohelminthes 20.6% and co- infection of 3.5%. In his study, Homeida et al. (1994) were
unable to observe any religious related differences in the infection rate. Transmission as they had
reported depended on tribal habits and environmental conditions as both muslims and christians
lived in the same community. They reported low prevalence of geohelimthes in Sudan and
recommended that before implementing any large-scale treatment program, proper prevalence
survey should be carried out.
41
A recent study on gastrointestinal parasite infection conducted by Priscilla (2003) in school children
of Juba revealed that nearly half of the children were infected. The parasites involved were H.nana
(14%), G.lamblia (3.2%), S.sterocoralis (2%) and S.mansoni (0.4%). Some of the children had
infection with more than one parasite species. The study attributed the high worm manifestation to
the lack of clean drinking water since most of those children 62.8% drank surface water; 17.2%
drank water from the Nile and 45.6% from untreated tap water. Also most of the children walked
bare footed and that may had justified the higher prevalence of hookworm infection. The low
educational level, lack of knowledge on personal hygiene as well as poor sanitation were the most
important factors affecting parasite prevalence in that area. Regarding nutritional status as related to
parasitic infection, the study revealed that 52% of the children were mildly malnourished, 4.4%
moderately and 43.6% reasonably fed. No severe malnourished or obese children were observed and
that although high number of children was infected the parasite load was not heavy to affect the
nutritional status.
2.1.10 Conventional diagnosis of gastrointestinal parasites:
2.1.10.1 Direct Wet Mount (DWM):
Direct faecal specimen can be examined freshly or in formalin fixed using saline solution and
lugol’s iodine. Wet mount examination is valuable with specimens containing moderate or high
number of cysts, ova or larvae.
Direct wet mount is especially helpful in finding protozoan parasites, such as Giardia spp and
Entamoeba spp. The main disadvantage of this technique is its insensitivity because small specimens
may not contain enough eggs or larvae.
It is reported that direct saline smear preparation using iodine may be sufficient, particularly when
the parasite is present in large numbers (Islam, 1990 and Thomas & Smith, 1995).
In one study it was reported that direct wet microscopy is equally sensitive as concentration methods
for diagnosis of G.lamblia and Entamoeba spp (Alan, 1989). This is due to the unique morphology
of G. lamblia and Entamoeba cysts and trophozoites.
2.1.10.2 Concentration method:
It has been shown that concentration techniques are more sensitive than direct microscopy for
detection of gastrointestinal parasites (Danciger and Lopez.1975).
The floatation and sedimentation concentration methods are particularly helpful in recovering
parasite ova, cysts and larvae. The commonly used sedimentation method is the formal ether
although it is considered by other researchers as unsuitable due to deformation or even
disintegration of trophozoites and its flammable nature.
2.1.10.3 Floatation techniques:
42
This method is more accurate than the direct wet mount because it concentrates parasite eggs and
larvae. The commonly used floatation solutes are magnesium sulphate, sugar, sodium nitrate and
zinc sulphate. A specific gravity of 1.2 to 1.3 was reported optimal for floatation of most eggs
(Garcia & Buckner, 1997). Each solution has advantages and disadvantages.
-Magnesium sulphate solution
Is inexpensive but if the slide has to stay for a while before reading, the fluid crystallizes and eggs
may be distorted.
-Sodium nitrate solution
Sodium nitrate can be purchased as ready for use reagent and therefore shorten the time for
microscopy but it is very expensive.
-Sugar solution (Sheather’s method, 1923)
The prepared slides can be kept long before reading. It has been recommended for identification of
Cryptosporidium since oocysts floats easily in this medium & the rate of detection increase (Garcia
et al. 1983). Although some of the materials in the floatation are inexpensive and can be prepared in
the laboratory yet the medium should always be checked with a hydrometer to control and adjust
specific gravity. Though this procedure shows clear background during microscopic examination yet
the oocyst do not appear as pink refractile. Some authors advise to examine the remaining sediment
in the test tube after examining the supernatant. Other investigators prefer to take the supernatant
fluid and after washing in normal saline examine the sediment for oocysts. However, such washing
step results in a considerable loss of oocysts and thus a decrease in test sensitivity (Weber et al. 1991
and Garcia et al. 1983). This procedure does not suit the workflow in the laboratories and specimens
cannot easily be divided in batches to allow reading within 15 minutes after which the oocyst tends
to collapse and disappear. Moreover, presence of sugar in such high concentration inhibits staining
procedures (Weber et al. 1991).
The sucrose floatation technique was compared with the Kato Katz and Army Medical School
methods for diagnosis of intestinal parasites among 285 school children in West Java, Indonesia. The
Kato Katz method detected only two helminth species with prevalence rate of 10% while using the
sucrose nine species (31%) were detected positive and the Army Medical School Method detected
10 ( 22%) (Uga et al. 2002)
Zinc sulphate floatation:
The use of zinc sulfate solution instead of Sheather for G.lamblia diagnosis was preferred by some
authors (Mihatov, 2000). This is because in the Sheather’s floatation method, specific gravity and
the high viscosity of the solution result in distortion of Giardia cyst and trophozoites (Thomas &
Smith, 1995). Zinc sulphate is better for detection of Giardia because cysts do not distort quickly
43
(Garcia& Buckner, 1998). The zinc sulphate method however does not allow floatation of
trematodes and cestode eggs. The Zinc sulphate method was also used for detection of
Microsporidia oocysts. Compared with direct wet mount DWM, the Zinc floatation was found to be
better for detection of Giardia in Australia (Bruno et al. 1993). All faecal samples found negative by
direct wet mount DWM were found positive by the zinc floatation.
A modified zinc floatation technique using formalinized faecal specimens was compared with the
formalin ether (FE) concentration method for diagnosis of intestinal parasites. This study was carried
out (Marilyn et al. 1978) because many laboratories had difficulties in storing, using and disposing
of ether. Data as obtained on recovery of parasites from faeces preserved in formalin for less and
longer than 1 month. Whereas, the formalin-ether method was generally found to be more efficient,
the formalin ZnSo4 was less so for recovery of schistosoma eggs.
Detailed results of this study had shown that 379 out of the 505 were detected by both methods. The
formalin-ether technique detected 91% of the total positives while the formalin-ZnSo4 detected 84%.
A greatest difference between the two techniques was found in the recovery of the helminth eggs,
with the 84% being recovered by the formalin ether and 77% by the ZnS. When data pertaining to
protozoa only were considered, this difference was less (FE 94%, FZnSo4, 89%). In the same study
(Mariylin et al. 1978), the preservation time was found to have effect on detection of some
parasites. Whereas 100% recovery was attained in specimens preserved in formalin less than one
month, only 50% recovery was reported on samples stored longer than that using both techniques.
Relative efficiency of the formalin ether method increased from 50% to 100% for E.vermicularis
egg when the period of storage was prolonged. Influence of formalin preservation on floatability was
also studied by Moitinho et al. (1999). Comparison was made between fresh faecal samples
containing G.lamblia kept under refrigeration and others preserved in formalin using sucrose at
different specific gravities. The results showed that faecal samples kept under refrigeration or
preserved in formalin gave similar results.
2.1.10.4 Staining methods:
Mounts of the concentrated faecal specimens are examined in the same way as direct wet
mount using different staining techniques. Stained preparations are occasionally required for
better identification of the parasite specimens or sending it to a reference laboratory for
confirmation (WHO, 1991).
Differential staining methods including Safranin-Methylene blue stain, kinyoun or Ziehl-Neelsen,
and Dimethyle Sulfoxide and Carbol-fochsin, stain oocyst red and counter stain the background blue.
44
The modified Ziehl Neelsen acid fast staining technique is optimal for identification of oocysts in
stool samples. The oocyst appears bright red against blue green background of faecal debris and
yeast cells. (Fayer et al. 2000 Pear & Rosemery.1983 and Garcia et al. 1983).
The cold Kinyoun acid-fast stain is also used for detection and identification of Cryptosporidium and
it yields higher rate of oocyst than floatation methods. The reagent used in this technique was
inexpensive. Some investigators found the method more acceptable than Enzyme Immuno-Assay
(EIA) because of the high cost of reagent and the time required for executing the EIA (Karen et al.
1995).
2.1.10.5 Permanent stained smears:
Permanent stained preparation is routinely made whenever re-examination has to be
performed on stool specimen (Committee For Education American Society, CEASP, 1977) and
(Commission Laboratory Acrediation (CLA) 2002).It allows for detection of protozoan cysts
and trophozoites which may be destroyed or lost during concentration procedures such as in
the formalin ethyle acetate.
Certain chemicals used as fixatives are now recognized as being dangerous for human and
environment. Samples are generally preserved in polyvinyl alcohol (PVA). This fixative contains
mercuric chloride, which is potentially hazardous to laboratory personnel and presents disposal
problems. Modified PVA fixatives containing copper or zinc instead of mercury are now available,
but the quality of parasite morphology is usually not good. New alternative non-toxic fixatives were
evaluated (Jensen et al. 2000). Permanent stained stool smears preserved in three environmentally
safe fixatives (Parasafe, Ecovix and Protofix) were compared with PVA for quality of background,
clarity of parasite morphology and appearance of the parasite nucleus. The results had shown that the
background with all fixatives was clear except for the Parasafe, which gave dirty background,
making it difficult to resolve the internal structure of the parasite. Identification of parasites with the
PF and EC fixatives was comparable to PVA. The sensitivity obtained with PS was significantly
lower than that obtained with the other two fixatives.
A new approach for fixation, concentration and staining of intestinal parasites was reported (Omer &
Amin, 2000). Proto-fix TM was used in combination with a new concentration/sedimentation
reagent, (CONSED TM) to replace the formalin-ethyle acetate (FEA) using Lugol’s iodine. The
quality of the fixative and stain were found to be superior to the FEA lugol’s method and the number
of parasite was considerably higher.
2.1.3 Hymenolepis nana
Hymenolepis nana is also known as the dwarf tapeworm. The parasite causes human infection
especially in children. It is common throughout the world and has no restriction in geographical
45
distribution. Grassi (1887) between 1886-1892 had worked on the life cycle and demonstrated that
no intermediate host was involved.
2.1.3.1 Morphology
The adult worm is usually 25-50 mm long, but specimens as small as10 mm and as long as 24 mm
were found. The worm is very narrow, almost filiform and is provided with minute scolex, about 0.3
mm wide, which has 4 suckers and a proboscis, armed with a number of hooks.
There is a long slender cervical region beginning with short narrow immature proglottids (Hoeden,
1964). Those more distal in position become increasingly broader (Beaver et al. 1984). The eggs are
set free by regular disintegration of the distal most proglottids. They are spherical or sub-spherical,
hyaline, measures 30-47µm diameter and contain an onchosphere that is enclosed in an inner
envelope with two polar thickenings, from each of which arise 4 to 8 polar filaments. Within the
onchosphere are three pairs of lancet shaped hooklets.
2.1.3.2 Life Cycle
H.nana is unique in having a dual life cycle pattern that sets it apart from all other tapeworms.
Typical cestode development involves larval development in one or more obligatory intermediate
hosts before maturation in vertebrate host. An evolutionary stage has been reached in H.nana
whereby the intermediate host is not utilized, larval and adult development occurring within a single
animal. Eggs ingested by the vertebrate host hatch in the small intestine and release hexacanth larva
(oncosphere) that invades the mucosal lining and develop in the intestinal villi. Larval growth to the
cysticercoid stage occurs in the villi in about five days and the young tapeworm attaches itself by
suckers and rostellum near the iliocaecal junction. Growth of the adult strobila follows in about 8
days and eggs appear in the faeces of the host 13-24 days after initial ingestion of eggs.
The indirect life cycle of H.nana was first demonstrated by Bacigalupo (1931) who found
cysticercoid in fleas; grainbeatles and grain moth fed on H.nana eggs.
Although golden hamsters are known to harbour H.nana (stunkard, 1945), there appear to be no
reports giving the percentage development of the parasite or other host parasite relationship.
Hamsters were compared with white mice by Johns & Larsh, (1946) who studied the percentage of
development to adult stage, and the extend of the prepatent period. Their results revealed that
hamsters showed greater percentage of development to adult stage and their length was much greater
than those in mice.
2.1.3.4 Epidemiology of H.nana
An epidemiological study on H.nana was carried out in Avila Province, Cuba (Suarez et al.1998).
During the period 1981-1995, 3,108 samples were examined for parasites. Seasonal influence of this
parasitism was not detected. There was more prevalence in children than adults with infection in
46
males prevailing over females. The symptoms and signs reported were abdominal pain, diarrhoea
and anorexia.
The epidemiology of H.nana was also studied in school children in Zimbabwe (Mason and
Patterson, 1994). Stool samples were obtained from children in rural villages and high density
suburban rural towns H.nana was found in 21% of the children. Infection was higher in younger
children in the urban area and among older children in rural areas. Prevalence in the urban areas was
higher (24%) than in the rural areas (18%) and infection correlates with hygiene score and presence
of siblings. The prevalence of infection in rural communities did not correlate with availability of
water, number of households, low hygiene or presence of infected sibling. New or reinfection
occurred more frequently in households who had infected siblings in urban settings. The study
demonstrated the distinct differences in H.nana transmission in rural and urban communities. The
data suggested intrafamily transmission in urban communities particularly in households with poor
hygiene, leading to primary infection. In rural areas, prevalence of infection and incidence of
reinfection were higher among children at the school age and there was little evidence of intrafamily
transmission.
The epidemiology of H.nana was studied in 2,083 Thai orphan children in Bangkok (Chukiat et al.
2000). Where 13% of the children was found to be infected; males had statistically significant higher
prevalence than females.
A survey on prevalence of A.lumbricoides, T.trichiura and H.nana was conducted in two adjacent,
but socio-economically distant urban communities and a squatter settlement with access to modern
sewage facilities (Kosoff et al. 1989). The prevalence of these parasites was significantly higher in
the former. The squatter children were however more heavily infected with A.lumbricoides and
H.nana than adults. In Dolj County, prevalence of H.nana was studied by Marx et al. (1989) who
investigated 45,725 cases in polyclinic and hospital settings between 1985-1987 in a population of
different age and sex. They found low incidence (2.3%) of H.nana with a seasonal variation showing
maximum prevalence in autumn and minimum in winter.
In Mexico, Romero et al. (1991) studied H.nana prevalence among school children and reported this
parasite among the top five. In the majority of the cases it was found in association with other
species particularly G.lamblia.
Prevalence of H.nana among Ethiopian children under five was reported by Samson et al. (2000). In
a cross sectional study he found that H.nana to be second to E.histolytica with a prevalence rate of
17-30%.
47
H.nana was also found among intestinal parasite infections that prevailed among Asian
housekeepers working in Abha (Saudi Arabia) where high prevalence of 46.5% was reported. (Omer
et al.1991).
Hymenolepis nana was also studied in Egyptian children and its incidence rate was found to be 16%
(Khalil et al. 1991).
South African refugees residing in United States were screened for presence of intestinal parasites
(Molina et al. 1988). Of those, 32% were positive for one parasite infection and 8% for multiple
including H.nana. In those who had been found infected, H.nana was persistent for more than two
years especially among those who had acquired auto-infection or person to person transmission.
Prevalence of H.nana in Buenos Aires was found to be 7.9% (Pezzani et al.1996).
2.1.3.5 Clinical Symptoms:
H.nana presence even in large numbers is commonly well tolerated in human subjects. The
mechanism initiating symptoms was usually through allergic reaction. Donckaster & Habibe (1958)
studied development of symptoms in 43 patients and found that 86% had complaints of headache,
dizziness, insomnia, irritation in nose and anus, periodic diarrhoea and abdominal discomfort. Of
those, 65% were restless and irritable, 5% had epileptic convulsions and one third had eosinophilia.
They explained neurological symptoms such as dizziness to a poorly understood toxin produced by
the parasite. Absorption of parasite metabolites, especially in children, may result in dizziness and
insomnia. The presentation of dizziness, insomnia and convulsions was primarily anecdotal and
viewed with reservation by some researchers. (Frisby, 1995). Recent study by Magoud et al.(1991)
revealed some pathological changes in the brain of experimentally infected mice which may give
explanation of the epileptic convulsions reported by Donckaster & Habibe, (1958).
Overwhelming intestinal autoinfection had been observed in Immunosuppressed mice, with hyper
infection with cysticercoids of the intestines by adult worms, the mesenteric lymph nodes and liver.
Apparently intestinal autoinfection occurs in human and may be a complication in patients under
immunosuppressive treatment (Lucas et al.1980).
In a study carried out in Nepal (Scherchard, 1996) in children and adults with or without
abdominal discomfort, H.nana prevalence was found to be higher in the latter group.
Despommier and Dickson (1992) also reported that abdominal pain, which was not localized to
any particular area, was most common among H. nana infected individuals.
A Study on H.nana prevalence among orphan children had shown that most infected children were
asymptomatic. In symptomatic children the symptoms were mild and non specific such as pruritis,
abdominal pain, diarrhoea and dizziness. In study conducted in India (Bijay & Jyotish 2002), H.nana
was reported as a common cause of pediatric diarrhoea in urban dwellers. Of the cases studied, 0.8%
48
was malnourished and 55% grossly anemic. Symptoms such as diarrhoea and abdominal pain were
common; other symptoms included weakness and vomiting.
Clinical and laboratory studies of H.nana in children were carried out in Mexico by Romero et al.
(1991). The study involved 325 infect children. The results had shown that H.nana was among the
top five intestinal parasites. In the majority of cases H.nana was associated with protozoan,
especially with Giardia .The most important and constant clinical manifestations were abdominal
pain and irritability. In those infected with H.nana, weight loss and flatulence were common.
Giardia infection was the most frequent in this group. The paper concluded that H.nana is an update
disease in Mexico due to its high frequency among school and preschool children.
Pathological and immunological changes in albino mice experimentally infected with H.nana were
reported by Mangoud et al. (1991). Mice infected with H.nana and scarified one, three and five
months later manifested diarrhoea and loss of appetite. The liver and spleen showed some
pathological changes.Cysticercoid were seen particularly in the liver and kidneys. The kidneys
showed cloudy swelling (3/90). The brain showed oedema in (15/90). Intestinal atrophy and
ulceration were very marked 30/90 with negative reaction to Alcian blue stain. Cysticercoids were
seen in intestinal villi. The immunoglobulin in section of small intestine changed from moderate to
negative IgA or to mild IgM or from mild to marked IgG. The paper concluded that dissemination of
H.nana cysticercoids beyond its normal habitat to other vital organs, as the liver is dangerous.
Clinical symptoms of H.nana were also reported by Suarez et al. (1998) who studied epidemiology
of H.nana in school children in Cuba.
The common symptoms and signs reported were abdominal pain, diarrhoea and anorexia.
2.1.3.6 Immunity and Immune response to H.nana
Although H.nana infection is the commonest among cestode infection in man, there is scarce data
available on immunity of the infection. However, it was initially reported that the infection is
strongly immunogenic against challenge infection with the same parasite (Heyneman, 1959). Natural
49
immunity due to age was reported to be relatively effective as it adds to the host resistance against
infection. The variation in normal host infectivity emphasizes the importance of parasite strain and
the variations in environmental factors that may influence the parasite virulence that determined host
ability for resisting the infection (Heyneman, 1959).
Hymenolepiasis in adults is usually self-limiting. Whether serum antibodies against H.nana are
produced and whether they are of protective nature remains unknown. Since the parasite is attached
to the intestinal wall by its scolex, antigens (surface, somatic or metabolic) derived from this region
of the parasite could possibly be used for antibody detection. Any humoral immune response against
the parasites might also be relevant to the reliability of serodiagnosis. Recent studies had shown that
antibodies play a role in recovery from H. nana infection (Frisby, 1995).
It has been found that H.nana infection in man initiates low but detectable humoral immune
response using ELISA (Gomez et al. 1991). Although not useful for diagnostic purposes this may be
related to the serodiagnosis in other tissue cestode infection of man since cross reactivity versus
other cestode was reported.
2.1.3.7 H.nana in immunosuppressed patients:
The presence of immature H.nana worms was reported in patients with acute lymphoblastic
leukemia (LL) on anticancer treatment (Rudrapatna, et al. 2002). For management of acute LL, the
patient was put under chemotherapy for 4-week treatment period, followed by 10 day cranial
irradiation. Following treatment, the patient developed loose stool and vomiting. Microscopic
examination of the stool showed blood mucus and fragments of immature H.nana worm. The
authors attributed that to the denudation of the mucosa and thus appearance of immature worms in
the stool .To prevent worms from other tissues, patients were given abendazole (15mg/kg /day) as
recommended for cysticercosis. The complaints disappeared in few days and no worms or eggs were
detected in stool. These findings highlighted the importance of this parasite before and during
chemotherapy in cancer patients.
2.1.3.8 Abnormal development of H.nana in Immunosuppressed mice
H.nana infection in mice is profoundly influenced by immunosuppression. This is caused by T-cell
deprivation or by induced steroid treatment and thymectomy, which results in multiplication of
cysticercoids in the viscera. This suggested that H.nana could be one of the parasitic conditions,
which should be eliminated before initiation of any immunosuppressive therapy (Lucas et al. 1980).
Observations of the course of H.nana infection in Immunosuppressed mice were reported by Lucas
et al. (1980). Treatment of the host with hydrocortisone acetate caused super infection in the bowel
with worms and cysticercoids in the mesenteric lymph nodes and liver. Natural infection of mice
50
deprived of T. cells or by thymectomy and administration of antithymocyte serum resulted in super
infection and wide spread metastasis of cysticercoids causing death after about five minutes.
2.1.3.9 Role of animals in transmission of H.nana
Hymenolepis species are found in all rodents and the common species in rats are H. diminuta and
H.nana, both species infect man and other primates. These two parasites are transmitted to and
between rodents through contaminated bedding and by insects carrying eggs from one host to
another (Harkness and wagner 1983, Hsu, 1979).
Data on acquiring human infection with H.nana by pet rodents are lacking. However, a survey of pet
stores in southern Connecticut, USA, conducted on faecal specimens revealed 1. 9% prevalence in
110 samples collected weekly from cages holding the animals. Of 11 animal species, only mice and
dogs were found positive (Duclos & Richardson, 2000).
H.nana was found among the helminth parasites of rodents in Dakahahlia Governorate in Egypt
(Elshazaly et al. 1994). The paper concluded that rodents are the most important source of zoonotic
infection.
Presence of H. nana eggs in dog faeces was also reported by Bruno, et al. (1993).
2.1.3.10 Diagnosis of H.nana
Enzyme linked immunoassay was evaluated for detection of antibodies against H.nana (Gomez et
al.1991). The results revealed that all children harbouring H.nana were serologically positive.
Evaluation of the test gave sensitivity of 100%. Very high cross reactivity was obtained with
Taeniasis and cysticercosis 75% 45.8% respectively. The paper concluded that the efficiency and
simplicity of faecal examination considered as more practical than detection of serum antibodies for
H.nana diagnosis in human patients.
2.1.3.11 Treatment of H.nana
Successful treatment of H.nana and other cestode species with Praziquantel was reported by Bouree
(1991 & 1988), who treated 15 patients with H.nana using one single oral dose of Praziquantel. No
side effects were reported except for 6 patients who complained of abdominal pain and diarrhoea.
Efficacy of the drug was optimal. The author concluded that Praziquantel is the best for treatment of
adult cestode.
By comparison with Mebendazole, Praziquantel gave significant higher cure rates, being 91.1% and
97.7% after 2 & 4 weeks respectively compared with Mebendazole which gave cure rate of 50% and
59% after the same period. Both drugs were well tolerated by patients and had no side effects.
However, Praziquantel was given as a single dose and thus had the advantage of being more
effective and easily administrable (Khalil et al.1991).
51
In a study carried out by Arther et al. (1981), praziquantel was used for treatment of naturally
infected mice. H.nana reduction in mice receiving medicated ground food for seven days was higher
than in those receiving non medicated food suggesting that Praziquantel (efficacy was 87-100%) is
efficient for Hymenolepis eradication in mice.
In a study carried out by Gonenc and Sarimehmetoglu 2001), nitroscanate effect on H.nana in mice
and rats was investigated at a dose of 50 mg/kg per day for 4 days. The regimen treatment was found
to be 86% and 100% effective for treatment of H.nana in mice and rats respectively. Nitazoxanide
was also studied for treatment of common intestinal helminths and found to be effective as broad
spectrum antiprotozoal and antihelminthic drug (Herbert & Paul 2002 and Rossignol &
Maisonneuve 1984).. The drug was well tolerated with no serious adverse effects. In rats a
significant reduction (84%) in egg count was achieved.
praziquantel was also used for treatment of H.nana infection in Thai children. A single oral dose of
25 mg/kg body weight was well tolerated (Sirvichayakul, et al. 2000) and all children had
parasitological cure. Only four children reported diarrhoea and abdominal pain and two had
headache after treatment with Praziquantel.
However, all symptoms were mild and required no treatment.
52
Chapter Three
Material and Method
3.1 Study design
The major components of the study are: -
1/Laboratory development of the Sugar Floatation Techniques (SFT) as developed by Soulsby
(1968) and its evaluation for detection of gastrointestinal parasite in Sudanese patients.
The method was originally developed and used for detection of gastrointestinal parasites in
animals but had never been evaluated for the diagnosis in human infections. Evaluation was
carried out by comparison with other techniques, namely Sodium Dodecyle Sulphate (SDS)
sedimentation, Zinc Floatation (Faust, et al. 1938) and Direct Wet Mount Technique (DWM).
The last one is used widely in hospitals and health centers in Sudan.
Standardization and optimization of the technique was also carried out. These included studying
performance of the technique in experimentally contaminated stool specimens. The least number
of eggs that can be detected by SFT as well as the optimal time at which maximum recovery of
parasites occurred were to be determined. Effect of stool preservation on floatation of parasites
was also investigated. Comparing performance of the test in fresh and preserved stool specimens
did this. Use of other reagents as alternative to sugar (Gum Arabic+ gelatin) in this technique was
also evaluated.
2/ Evaluation of the sugar floatation technique at central hospital level.
Comparing SFT performance versus that of the Direct Wet Mount technique was carried out.
Stool specimens used in this study were collected from patients visiting central hospitals, health
centers and private clinics. Patients with various complains including abdominal pain, diarrhoea,
vomiting, were chosen as suspects for gastrointestinal parasite infections
3/ Field evaluation of the reliability of the sugar floatation for detection of dormant parasite
infections.
53
This was carried out in three displaced child population residing in the outskirts of Khartoum,
namely Elhag Yousif, Mayo and Abuzeid. Three hundred apparently healthy school children from
each area were enrolled in this study.
4/ Use of sugar floatation in epidemiological study of H.nana.
A cross sectional study was conducted to confirm own observation on the high H.nana prevalence
and assess in depth the epidemiology of the parasite that has completely been neglected as a
pathogen in the Sudan. The study was conducted in Abuzied, an area resided by displaced
population located in Western Omdurman. School children were chosen for this study since in
parasitic infections, children are known to represent the situation in the community as a whole.
School children are also considered as most suitable for such study since they are easily
accessible and the disease occurrence is usually better expressed among this age group.
Total coverage included screening of all male and female school children for presence of H.nana
as well as other parasite infections. Demographic and epidemiological data and information
regarding the transmission of H.nana including the role of animals were collected.
After completion of the mass screening, all children harboring H.nana were classified in to three
groups based on the level of infection. Those having egg count, <50/gm of faeces were classified
as having mild infection. Children with egg count ≤100/gm as having moderate infection and
those with more than 100egg/gm as having heavy infection. This classification was adopted to
investigate whether there is any association between the level of infection in those children and
prevalence of the parasite within their families. This will help to address the question whether
these children acquired infection form home or their school.
For executing this part of the study, ten children from each of the three classes were selected; an
additional group of 10 children was added to serve as negative control. All family members of the
selected children were included in the study. The required approval was obtained from the family
members involved, the health authority and the local government of the area.
The concerned popular committee had helped in approaching the families under study and
explaining the objectives of the study. However, two problems were encountered. First it was
difficult to collect samples from infants and secondly some of the family members especially the
men refused to give samples.
In order to explore the role of animals in the transmission of H.nana, 100 cockroaches and 15
mice were captured, dissected and their gut contents were removed and examined for presence of
H.nana.
5 /Efficacy of Niclosamide and Praziquantel for treatment of H.nana infection was evaluated. In
this part of the study 55 infected children were chosen from the three classes: 24 from the heavily
54
infected, 20 from the mild and 11 from the moderate. The effect of each drug was judged by the
reduction in egg count or total H.nana clearance of the stool specimen.
Treatment was administered at Ahfad Health Center under supervision of qualified physician
after clinical assessment and reconfirmation of H.nana infection.
3.2 Study population
Five study populations were included in this research:-
-Patients at the age of ≤3 - ≥50 years who reported to central hospitals, health centers or private
clinics in Omdurman with complaints of abdominal pain, diarrhoea, vomiting or other symptoms
suggesting gastro-intestinal parasite infections.
-School children at the age of 5-17 years belonging to three displaced population residing in
Abuzeid, El Hag Yousif and Mayo.
-School children at the age of 5-17 years from Abuzied area.
-Family members of children who had positive H.nana infection.
-Cockroaches and mice collected in or at the vicinity of the children homes.
3.3 Sample size and sampling technique
For the hospital based study, 450 outpatients with suspicion for gastrointestinal infections were
included:
-Omdurman Central Hospital (150).
-Mohammed Elamin Pediatric Hospital (100).
-Ahfad Health Center (50).
-Tropical Medicine Hospital (50).
-Private clinic (100).
For the field evaluation of the test 831 school children including both male and female population
were included.
In each of the three-study area, there were two schools, one for girls and one for boys. One
hundred and fifty samples were collected from each school through stratified random sampling.
Nineteen from each class were selected. To obtain gender balanced sample, the same procedures
were followed for both male and female schools.
For H.nana prevalence survey study, total coverage of the two existing schools in this area was
carried out with a total of 1780 children.
55
Regarding the study of H.nana transmission among families of children, sampling was based on
classification according to level of infection, which included 392 family members. Hundred
cockroaches and 15 rats were included in this study.
In the treatment part of the study, a total of 55 children were enrolled. Twenty four school
children with heavy infection, 20 with mild and 11with moderate level of infection.
3.4 Types of data
3.4.1 Clinical data
These included abdominal pain, diarrhoea, vomiting, fever, anal irritation and dehydration.
Other data included history of previous hospital visits six months before enrollment in the study
together with the type of complaints they had reported.
Abnormalities in stool specimens and presence of mucus, blood or worms were recorded.
3.4.2 Demographic data
These included administrative information such as name, sex, age, and area of residence.
3.4.3 Epidemiological data
Prevalence rate, intensity, types of parasite species other information on father’s Job, educational
level of the parents, house construction, type of food, presence or absence of latrine, water sources
as well as behavioral and self-hygiene data were recorded.
3.5 Method of data collection
During the hospital based study, a questionnaire form was completed. The part concerning clinical
examination was completed by the hospital physicians and researcher reported results of stool
investigations.(Annex1).
For the field study clinical, demographic, epidemiological and socio-economic data were collected
through specially designed questionnaire (Annex II). Questionnaires were completed with the help
of research assistants and selected school teachers. Research assistants assigned for collection of
stool specimens and proper completion of the questionnaire in the field together with school
teachers had acquired training on these two tasks.
3.6 Specimen collection:
Patients who had visited the hospitals or health centers were provided with plastic stool containers
with applicator stick and a tight fitting lid. All patients were instructed to pass a reasonable stool
specimen directly into the container without mixing the stool with urine or any other undesirable
material. The containers were clearly labeled with the patient name and date of sample collection.
56
Specimens collected in hospitals were usually examined within one hour to prevent deterioration or
change in the morphology of the parasite. Samples intended for laboratory evaluation were either
taken fresh and immediately examined after arrival to the laboratory or preserved in 4%formalin
for later examination.
For the school survey, the stool specimens were collected and preserved in formaldehyde and
transported to the laboratory.
The names and age of the children at the beginning of the school year were obtained from the
school records. The purpose of the study was explained to the children, who were then asked to go
to the school toilets and pass the required volume of feaces into plastic containers labeled with full
name of the child and date of specimen collection.
3.7 Specimen preservation
After submission of the stool specimen, formaldehyde saline solution (4%v/v) was added and
the specimen was well mixed using an applicator to break up lumps. More of the preservative
was added to cover the whole sample. The containers were tidy screwed and packed carefully
in a box and transported to the laboratory where they were stored at room temperature.
3.8 Collection of insects and rodents
Families of the school children included in this study and selected as described above were
visited by the researcher with the purpose of explaining the role of animals and insects in the
transmission of the infection. Family members were requested to help in collection of
cockroaches and mice.
The insects and mice were transported in containers with apertures on the covers to allow for
ventilation. Dissection and examination of the animals were carried out at the laboratory.
3.8.1 Screening of cockroaches
A small pieces of cotton soaked in formaldehyde was used to anesthetize the insect. Dissection was
carried out by applying a vertical cut and removing the gut. The contents after homogenization in
saline were transferred to a plastic centrifuge tube. The tube was then filled with more
formaldehyde solution and left overnight. After passing the contents through a strainer the tube was
centrifuged for 3 min at 2000 rpm. The sediment was then examined as for stool specimen in the
sugar floatation technique.
3.8.2 Screening of rodent
At first faecal sample of rodents found in the glass jars were collected and examined using the
sugar floatation technique. The mouse was anesthetized with formalin and dissected .The entire gut
was removed and chopped well in a Petri dish containing saline solution. The gut contents were
57
then strained through mesh in a plastic container. The filtrate was transferred to a test tube and
centrifuged at 3000 rpm for 3 min. After decanting the supernatant, 5ml sugar solution (58.6%w/v)
was added and after proper mixing, the tube was filled to the rim and a cover was placed on the top
of the tube. The tube was left to stand in an upright position for 45 min after which the cover slip
was removed, placed on a clean slide and examined under the microscope.
3.9 Sugar floatation
The method was applied as described by Soulsby (1968). A fixed stool specimen was properly
emulsified and centrifuged to obtain pellet. After decanting the supernatant, pellet was thoroughly
mixed in 10 ml saturated (58.6%w/v) sugar solution. The test tube is then filled to the rim with the
same media. Fresh specimens were processed directly without centrifugation. Avoiding formation
of air bubbles, a cover slip was placed on top of the test tube and the later was left to stand in
upright position for 45-60 minutes. The principle of this technique is that at such high relative
sugar solution density, parasite eggs and cysts due to relative lower specific gravity float to the
surface and thus easily recovered on the cover slip. The cover slip was carefully removed and
placed on glass slide and examined under the microscope. A drop of iodine was added when
staining was needed.
3.10 SDS sedimentation
As for the sugar floatation method, the pellet from the formaldehyde fixed stool specimen was
emulsified in 8ml medium containing Sodium Dodecyle Sulphate (0.05w/v) and sodium chloride
(0.9 % w/v). Fresh stool specimen was properly homogenized in the detergent medium, and then
passed through nylon gauze of 400 µm mesh size to remove coarse particles. The filtrate is returned
to the test tube and left to settle. After one-hour incubation, a large drop of the sediment was
transferred to a clean glass slide, covered with a cover slip and examined under the microscope.
3.11 Zinc sulphate floatation method
3.11.1 Preparation of zinc sulphate solution
The zinc sulfate solution was prepared by adding 386 grams of zinc sulphate to one liter of water.
Using hydrometer, the solution was checked and adjusted to1.18 specific gravity. The zinc sulphate
solution was stored in tightly capped container to prevent evaporation.
3.11.2 Faecal examination using zinc sulfate flotation fluid
Two to three grams of faeces were thoroughly mixed in ZnSo4 (specific gravity=1.18) and passed
through strainer into 15 ml test tube. The test tube was centrifuged for 2 minute at 1500-2000 rpm.
58
Using Pasteur pipette, the surface of the sample was siphoned on a microscopic slide, a drop of
iodine was added to stain cysts, a cover slip was placed and the specimen was examined under the
microscope.
3.1.12 Direct wet mount technique
The wet mount technique is the most simplest and easier for examination of stool specimens. It
can directly be applied to faecal material before or after concentration producers (WHO,1991).
A drop of saline solution was placed on the center of the glass slide. A drop of iodine was added to
the specimen to allow for better visualization of parasite cysts.
The specimen then covered with a cover slip. To reduce the chance of including air bubbles, the
cover slip was held at an angle touching the edge of the slide and then lowered gently onto the
slide.
The slide was examined under the microscope using x10 objective lens or with high magnification
if needed. The whole area of the cover slip was systemically examined.
3.13 Egg count
The modified Wisconsin (Corwin & Julie 1997) procedure for egg counts using floatation methods
was employed to quantify the amount of parasite eggs per gram of faeces of individual host. The
following steps were carried out:
-Two grams of faecal sample were weighed in a plastic container.
-Ten ml normal saline was added and the specimen was thoroughly mixed.
-The mixture was passed through strainer into another plastic container. The material was then
passed through strainer (400µm).
-More saline was added to rinse the container from Faecal material clinging in to the sides and
bottom. The rinsed material was passed through the strainer.
-The filtrate was then poured into a test tube (15ml) filled with more saline and centrifuged at
1500-2000 rpm for 3min.
-The supernatant was decanted and the tube was filled to half of its size with the sugar solution and
mixed with an applicator stick to insure proper homogenization of all the material.
-The test tube was filled again to the rim and covered with a cover slip, placed on the top and left to
stand in an upright position for 45 min. to 1 hour. The glass cover was removed by lifting it straight
upward and placed into a glass slide. All eggs and cysts throughout the entire cover slip were
59
counted using a low power 10x objective lens or high power if needed. To obtain the number of
eggs per gram of faeces, the total number of eggs or cysts, obtained was divided by two.
3.14 Treatment of school children
The drugs used in this study were praziquantel and niclosamide. A total of 55 school children were
enrolled in this part of the study. Of those, twenty-four were heavily infected, 11 with moderate
and 20 with mild level of infection. All children were re examined and the egg counts per gram of
stool were recorded to allow comparison for effect of treatment. The children were transported to
Ahfad Health Center in groups of 5 per day where they were seen by the responsible physician.
Before clinical examination, they were interviewed by the nutritionist in the center and assessed for
weight. This information as well as the clinical data was recorded. The children were then divided
according to gender and half of each gender group was treated with praziquantel while the other
with niclosamide. To insure that the children properly took the drugs, our research assistants
strictly supervised treatment.
Stool samples were collected starting from the second day after treatment for three successive days
and then after a week. All samples were preserved in formaldehyde and examined with the sugar
floatation technique. The number of eggs per gram of stool was recorded for each follow-up stool
collection. During the follow up period children observed for any side effect that might occur.
The data were analyzed and comparison was made for efficacies of the two drugs using reduction
in H.nana egg /gram of stool, seven days after treatment and percentage of total stool clearance
from H.nana infection
3.15 Data analysis:
Data was analyzed using two soft ware programs namely, the SPSS and STATA.
Chi-square was used for comparison of data. McNemer test (a version of chi-square) was used
when independence of the two comparison groups was not assumed .The T-test was applied in the
case of two independent variables.
Data was always checked for normality and in case of abnormal distribution log transformation
was performed before applying the appropriate statistical test.
Logistic regression was used for multivariate analysis of risk factors associated with H.nana
prevalence among school children in Abuzied.
60
61
Chapter Four
Results 4.1 Laboratory evaluation of the sugar floatation technique
4.1.1 Comparative performance of the sugar floatation versus the direct wet mount
and sodium dodecyle sulphate technique:
The Sugar Floatation Technique (SFT) (Soulsby, 1968) was evaluated versus the
sedimentation Sodium Dedocyle Sulphate (SDS) technique and direct wet mount technique
(DWM) which is currently used as routine test in hospitals and health centers in Sudan.
Thirty one stool samples declared negative at Khartoum Pediatric Hospital were used for
This evaluation. After being examined by DWM at the hospital, the remainders of those
samples were preserved in formalin (4%) and transported to our laboratory. All samples
were examined with SFT and SDS techniques.
Result of stool examination revealed that 11 out of 31 samples were positive in SFT and the
SDS detected 8. None of the cases was detected by DWM. The statistical analysis using Chi
square (Mc Nemar) showed high level of significance P< 0.001 and moderate level of
agreement between the two tests, Kappa = 0.26233 (Table1).
Table (1): Results of stool examination using SFT& SDS
Samples tested by SDS
Samples tested by SFT Positive Negative
Total
Positive 5
3 8
Negative 6
17 23
Total 11 20 31
Parasites recovered included H.nana (54%), E.vermicularis (18%), T. trichiura (18%),
E.histolytica (9%) and A. lumbricoides (45 %). Double infection with A.lumbricoides and
T.trichiura was found in 9% and a triple infection with H.nana, E.vermicularis and
A.lumbricoides was also detected in 9% of the specimens. In the SDS, H.nana was detected
in 36%, E.vermicularis in 18%, T.saginata in 9% and E.histolytica in 18% of the samples.
Double infection with H.nana and E.vermicularis was found in 9% of the samples (figure 1).
62
Figure 1: Parasite species recovered from stool specimens of patients received at
Khartoum Pediatric Hospital using SDS and SFT
0
10
20
30
40
50
60
E. vermicularis H. nana A. lumbricoides A.lumbricoides+T.
trichiura
H. nana+ E.vermicularis+ A.. lumbricoides
T. saginata E.vermicularis+A.lumbricodes
E. histolytica
Parasite spieces
(%) SFT
SDS
63
4.1.2 Comparative performance of SFT and SDS in fresh stool specimens:
To compare the sensitivity of SFT and SDS for detection of parasites in fresh samples, 60 stool
samples collected from out-patients suspected for gastro-intestinal parasite infection, in
Omdurman Pediatric Hospital, were examined with both techniques. The results are presented in
table 2.
Table (2): Parasite recovery in 60 fresh stool specimens using SFT and SDS.
Parasite recovered with SDS Parasites recovered with SFT Negative Positive
Total
Negative
32
0
32 (53%)
Positive 21
7
28 (47%)
Total
53 (88%)
7 (12%)
60 (100%)
SDS was positive in 7 (12%) out of the 60 specimens tested and all were H.nana. The SFT detected
parasite infection in 28 (47%) including species other than H.nana. Parasite species recovered with the
SFT are presented in table 3.
Table (3): Parasite species recovered with SFT in 28 stool specimens.
Parasites recovered Number of stool specimen
%
H. nana
21 75.00
T. saginata. and H. nana
1 3.57
A. deudenalis
1 3.57
E. vermicularis
1 3.57
H. nana and A. lumbricoides
1 3.57
A. lumbricoides
2 7.14
Total
28 100.00
64
The results presented in table 4 revealed high sensitivity for SFT in detecting diversity of gastro-
intestinal parasites by comparison with SDS technique. Four parasite species other than H.nana were
detected by the former technique including T. saginata, A.deudenale, E.vermicularis and A.
lumbricoides. H.nana was found to be associated with T.saginata in one of the patients and with A.
lumbricoides in another one.
The difference in parasite recovery in favour of SFT was found to be significant using Mcnemar test (P
value is <0.001). Kappa statistic showed high level of agreement in the results obtained by the two
tests regarding the positive cases KZ=0.2623.
4.1.3 Sugar loatation technique performance in fresh and preserved stool specimens:
The effect of formaldehyde in SFT performance was studied in 46 stool specimens collected from outpatients presenting abdominal pain and diarrhea, received at Omdurman Pediatric Hospital. Each sample was divided into two equal portions. One portion was examined immediately and the other was fixed in 4% formaldehyde saline for later examination at our laboratory. The results obtained in fresh and preserved samples are presented in table 4.
In the preserved samples examined, 9 were found to be positive while in the fresh 7 were positive. The
difference was not significant (p>0.05) and the level of agreement was high KZ=0.2750. This implies
the merit of preservation in large scale surveillance considering the number of fresh samples to be
examined at a shorter period of time.
4.1.4 Optimization and standardization of SFT:
4.1.4.1 Determination of the least minimum amount of ova present in stool specimen:
To obtain minimum number of eggs recoverable with SFT, ten negative stool samples were
experimentally infected with T. saginata eggs. T. saginata eggs were chosen because they can easily
be obtained by teasing of preserved worm progllottides. Eggs of T .saginata were counted using
NegativePositive
Parasite recovered inFresh samples
Total
Neg
Pin
Table (4): SFT performance in fres
65
counting chamber and the average egg number per 1 ml was obtained. The negative stool specimen
(1gm) was infected with different number of eggs as follows: 216, followed by 432, 846, 1080 and
2160. The negative specimens used were collected from healthy students at Ahfad University Medical
School who were reexamined to ensure absence of T.saginata. Each of the experimentally infected
specimens was tested with SFT. The minimum number detectable was found in specimens containing
432 ova /gm and the number of ova increased proportionally with the number of ova administered
(figure2).
Figure. (2): Percentage of egg recovered from deliberately infected samples.
Percentage of egg recovered from deliberately infected samples
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
216 432 864 1080 2160
Eggs in the sample
Pere
cent
age
of e
gg re
cove
red
4.1.4.2 Performance of SFT, SDS and DWM in experimentally infected stool specimens: To compare for sensitivity in SFT, SDS and DWM, ten negative samples (used in the above
experiment) were infected with 432 T.saginata ova /gm and employed.
Table (5): Comparison of SFT, SDS and DWM performance in experimentally infected stool
At first occasion all 10 samples recorded positive with SFT were found to be negative with DWM. In the second occasion 3 samples were found positive by DWM. SDS detected parasite ova in 6 out of ten positive samples. Four of the negatives with SDS were also negative with DWM in both occasions. These results confirmed earlier findings, which revealed that SFT is more sensitive for detection of parasite ova than SDS and DWM even at this level of experimental infection. Kappa statistic showed high level of agreement (kappa statistics= 724).
4.1.4.3 Comparative performance of SFT and Zinc Sulphate Floatation Techniques (ZSF): In This part of the study, stool specimens obtained from Omdurman Pediatric Hospital were used. Omdurman Pediatric Hospital was chosen for collection of the specimens because it is not far from our laboratory allowing therefore for quicker examination of fresh specimens. All samples were collected and examined within one hour. The results obtained in this study were summarized in table 6.
Table (6): Cross tabulation of SFT and ZSF stool examination results.
Zinc Sulphate Total Sugar floatation
Negative Positive
Negative 1 0 1 Positive 8 2 10 Total 9 2 11
The SFT was found to be superior to ZSF. Ten out of 11 samples were detected positive by SFT
including the two positive samples (E. histolytica or H. nana), which were detected by zinc sulphate.
Speciation of the parasites recovered by the two techniques is shown in table 7.
Table (7): Parasite species detected with Sugar and Zinc Sulphate Floatation techniques.
From the results presented in table 8 it can be observed that the number of eggs that emerged at the
surface of the sugar suspension increases with the length of incubation period. The highest egg
recovery was obtained after one hour. These results were consistent for all samples examined (figure3).
These finding are also of importance for quantitative performance of SFT.
(Figure.3): Eggs recovered by SFT in different specimens at different time intervals.
0
50
100
150
200
250
300
350
Egg
coun
t/ tim
e in
terv
al
1 2 3 4 5 6 7 8 9 10
Sample
Fifteen minutes
Half an hour
Forty five minutes
One hour
69
Figure 3 shows clearly that the number of eggs recovered by sugar floatation increased with time and
that the pattern was the same for all ten samples examined irrespective of egg number present in each
of them.
4.1.4.5 Use of Gum Arabic and Gelatin as alternative to sugar in the floatation technique: Performance of Gelatin and Gum Arabic was evaluated in the floatation method as an alternative to
sugar. These two substances were chosen because of the very small amounts needed to reach the
required specific gravity (1.18) for test medium and hence reduction in the technique cost. Different
concentrations of gum and gelatin were tried and the specific gravity was calculated according to the
standard procedures using the specific gravity flask. The specific gravity was calculated according to
the standard formula:
SG=mass solution /mass of equal volume of water. H.nana positive stool specimens were used in this
evaluation. Results obtained by different concentrations of gelatin and gum are shown in Table 9.
Table (9): Use of different gelatin concentrations in saline solution for recovery of H. nana.
No eggs were detected at 0.1%, 0 .2% or 0.3% concentration of gelatin in saline solution after 60
minutes. Eggs of H. nana however were detected in 3 out of 10 infected samples at 0.5% gelatin
concentration (specific gravity=1.192).
70
Table (10): Use of different gum concentrations in saline for recovery of H.nana eggs.
Samples no. Gum Arabic Concentration (w/v)
0.05% 0.1% 0.15% 0.2% 106
- ve
- ve
- ve
- ve
109 - ve - ve - ve - ve 93 - ve - ve - ve - ve 125 - ve - ve - ve - ve 31 - ve - ve - ve + ve 126 - ve - ve - ve - ve 128 - ve - ve - ve + ve 145 - ve - ve - ve - ve 14 - ve - ve - ve - ve 110 - ve - ve - ve -ve
Table 10 showed that no H.nana eggs were detected at gum concentration of 0.05%, 0.1%, and 0.15%.
However eggs of H.nana were detected in 2 out of 10 infected samples at 0.2% concentration (specific
gravity=1.18).
4.1.4.6 Use of 0.5 % gelatin and 0.2% gum concentrations in floatation:
Twenty samples were tested in the floatation techniques using gelatin, Arabic
gum or sugar at optimal concentrations of 0.5, 0.2 and 58.6% respectively. All 20 samples were
positive with SFT (100%), two (10%) were positive with gelatin and three (15%) with gum. Although
both gelatin and gum have comparable specific gravity as sugar, the results evidenced superiority of
sugar over the two substances in floatation techniques (table 11).
71
Table (11): Recovery of H.nana eggs using optimal gelatin, gum Arabic and sugar concentrations. Results of stool examination
Gelatin 0.5%
Gum Arabic 0. 2%
Sugar solution 58.6%
Positive 2 3 20
Negative 18 17 0
Total 20 20 20
4.2 Evaluation of the sugar floatation technique at health settings: Of 442 stool specimens collected from patients with abdominal complaints, 116 (26%) tested positive
in SFT and only 27 (6.1%) were positive with DWM (table 12). Of those 116, only 21 were detected
with DWM. All of the remaining 95 positive SFT samples scored negative results with DWM. Of the
326 SFT negative patients, DWM identified 6 positives. Four of those 6 patients were with G.lamblia,
one with H.nana and one with E.histolytica infections (figure 5). The difference in results of the two
tests was highly significant (P<0.001) with high level of agreement (kz=0.216) indicating that SFT was
more sensitive than DWM. The spectrum of parasites species detected was presented in figure 4.
72
Table (12): Results of stool examination of patients at health settings: Comparison between SFT
and DWM.
Figure (4): Percentage of gastro-intestinal parasites in 442 samples tested with Sugar floatation test and direct wet mount technique.
SFT for Giardia lamblia cystDW for Giardia lamblia cystSFT for H.nanaDW for H.nana
By comparison with DWM, SFT detected larger number of positive cases. However both techniques
appear to be sensitive for detection of G.lamblia, E.histolytica and H.nana, and hence this criteria was
used to compare between performance of the two techniques. Performance of DWM versus that of SFT was consistent at all 5 health setting and superiority of SFT was clearly demonstrated irrespective of the situation in health settings.
4.2.2 Application of SFT and DWM on stool specimens collected from patients at various age
groups:
74
No significant difference in the level of G.lamblia and E.histolytica infection was found by DWM
between patients at various age groups. For H.nana however, prevalence of the parasite was
significantly higher (p<0.05) among the age group >3 ≤ 10 years (table 13).
Table (13) Parasite species detected with DWM in 442 stool specimens from patients at different
age groups
Parasite ≤3
years >3≤10 years
>10≤20 years
>20≤30 years
>30≤50 years
>50 years
P-value
E.histolytica cyst
0 1.69 0 1.96 0.02 0 0.667
G.lamblia cyst 1.18 6.78 3.13 .96 1.18 4 0.196
H.nana 1.18 10.17 1.56 .96 1.18 0 0.017* *Significant difference The effect of age on parasite was demonstrated by applying SFT. H.nana and E.vermicularis showed
high prevalence among children below the age of 10 years. Age effect on H.nana confirmed the above
results obtained with DWM. G.lamblia prevalence was higher among the age group > 20 ≤30 but
declined with the increase in age. Prevalence of E. histolytica also increased with age and showed high
increase at the age group >50 (table 14). Application of the Chi –square test revealed significant
differences with age versus E.histolytica, H.nana and G.lamblia infections (p value < 0.05).
Table (14): The percentage of parasites detected using sugar floatation test in faecal samples of different age groups. Parasite ≤3
yrs ≤10 yrs
≤20 yrs
≤30 yrs
≤50 yrs
>50 yrs
P-value (P<.05)
E.histolytica
0
0
1.6
4.8
0
8
0.01*
Giardia cyst
4.7
1.7
9.4
19.2
14.1
12
003*
E.vermicularis
2.4
10.2
3.1
1.9
1.2
0
0.085
75
S.mansoni
0
1.7
0
0
0
0
-
T.saginata
0
0
1.6
0
1.2
4
-
H.nana
3.5
22
17.2
10.6
3.5
0
0.000*
*Significant difference
4.2.3 Effect of gender on parasite recovery with DWM &SFT:
No significant effect of gender on parasite recovery by DWM p > 0.05 (table15). Table (15) Parasite species detected using DWM in 442 faecal samples from male and female patients Parasite Male Female
P-value (P<.05)
No % No % E. histolytica cyst
4 1.69
1 0.49
0.379
G. lamblia cyst 6 2.53 4 1.95 0.758
H. nana 5 2.11 6 2.93% 0.761
Although wide variety of parasite species were recovered by SFT, no significant difference between
males and females was established P > 0.05 (table 16).
Table (16): Parasite species detected using SFT in 442 faecal samples from male and female
patients: Parasite Male Female
P-value (P<0.05)
No % No % E.histolytica 4 1.7 5 2.4 0.739
G.lamblia cyst 25 10.5 25 12.2 0.652
E.vremicularis 4 1.7 9 4.4 0.156
S.mansoni 1 0.4 0.0 0.0 1.00
76
T. saginata 2 0.8 1 0.5 1.00
H.nana
21 8.9 21 10.2 0.63
A comparison on the performance of DWM & SFT for detection of E.histolytica was carried out. Nine
cases were detected with SFT, 5 cases with DWM of which only one was detected by the SFT.
Considering the few number of positive cases detected, the results obtained did not allow for Kappa
statistics (table 17).
Table (17): Detection of E.histolytica using SFT and DWM.
1 8 9
11.1% 88.9% 100.0%
20.0% 1.8% 2.0% 4 429 433
.9% 99.1% 100.0%
80.0% 98.2% 98.0% 5 437 442
1.1% 98.9% 100.0%
100.0% 100.0% 100.0%
Count % within SugarFloatation Technique
% within Direct Wet
Count % within SugarFloatation Technique
% within Direct Wet
Count % within SugarFloatation Technique
% within Direct Wet
Yes
No
Sugar Floatation
Total
yes no
Direct Wet Mount Total
77
50 cases of G.lamblia infection were detected with SFT, only 10 were positive in DWM (table 18), of
them three were detected by SFT. Though there was statistically significant difference between the two
tests (P< 0.001), kappa statistic showed low level of agreement (kz.059) results are shown in table 18.
Table (18): Detection of Giardia lamblia using SFT & DWM
3 47 50
6.0% 94.0% 100.0%
30.0% 10.9% 11.3% 7 385 392
1.8% 98.2% 100.0%
70.0% 89.1% 88.7% 10 432 442
2.3% 97.7% 100.0%
100.0% 100.0% 100.0%
Count
% within Sugar Floatation
% within Direct Wet MountCount
% within Sugar Floatation
% within Direct Wet Mount
Count
% within Sugar Floatation
% within Direct Wet Mount
Yes
No
Sugar Floatation
Total
Yes No
Direct Wet Mount Total
78
Out of 442 suspected patients examined, 42 (9.5%) were detected with SFT as H.nana and 11 (2.5%)
with DWM. Ten (23.8%) of the 42 SFT positives tested also positive with DWM. There was
significant difference in the performance of the two tests P < 0.001 and high level of agreement KZ
=.2352 (table 19).
Table (19): Comparative performance of SFT and DWM for detection of H.nana
10 32 42 23.8% 76.2% 100.0%
90.9% 7.4% 9.5% 1 399 400
.3% 99.8% 100.0%
9.1% 92.6% 90.5% 11 431 442
2.5% 97.5% 100.0%
100.0% 100.0% 100.0%
Count
% within Sugar Floatation
% within Direct Wet Mount
Count
% within Sugar Floatation
% within Direct Wet Mount
Count % within Sugar Floatation
% within Direct Wet Mount
Yes
No
Sugar Floatation
Total
Yes No
Direct Wet MountTotal
79
4.2.4 Symptoms prevailing in patients having different parasite infection
Among the 442 patients received at the five health settings 275, (62%) were with abdominal pain,
followed by164 (37%) with diarrhoea, 93 (21%) with vomiting and 63 (14%) with dehydration (table
20). Table (20): Frequency of symptoms in 442 patients tested with SFT.
The majority of patients found positive with E.histolytica (77.8%), G.lamblia (72%),
E. vermicularis (92%) and H.nana (76.2%) were presented with abdominal pain. Considerable
percentage of those positive with E.histolytica (33%), G.lamblia (42%) E.vermicularis and H.nana
have diarrhoeic stool. Dehydration and vomiting were also among the symptoms which appeared in all
group with different percentages (figure 6).
Figure (6): Symptoms appeared in patients with different parasite species.
Symptoms Frequency Total %
Dehydration Vomiting Abdominal Pain Diarrhoea
63 93 275 164
14.3 21.0 62.2 37.1
80
0
10
20
30
40
50
60
70
80
90
100Pe
rcen
tage
E.cyst G.lambila Enterobius H.nana
Parasite
DehydrationVomitingAbdominal painDiarrhoea
4.2.5 Stool appearance of samples collected from patients in health settings
Fifty percent of the 442 specimens collected from patients were soft. In those specimens most of the
parasite species were identified. 13.6% of the specimens were in liquid form and in those most of the
parasites were detected. E.histolytica, G.lamblia and E.vermicularis were absent in the solid dry stool
while H.nana was present in all forms of stool specimens (table 21).
Table (21) Stool appearance in 442 samples collected at the health settings.
Stool appearance
Frequency Total %
E.histolytica %
G.lamblia %
E.vermicularis %
H.nana %
Solid dry
11
2.5
0
0
0
18.2
Firm 37 8.4 0 2.7 5.4 18.9
Liquid 60 13.6 1.7 15 1.7 8.3
Semi-liquid
55 12.4 0 9.1 3.6 5.5
Soft 221 50 3.2 12.7 3.6 9.05
Mucoid 94 21.3 1.1 7.4 0 9.6
Bloody 30 6.8 0 3.3 0 3.3 4.3 Field evaluation of the sugar floatation Technique in three displaced areas
81
The sensitivity of SFT for detection of dormant parasites was evaluated in a population of apparently
healthy school children residing in areas around Khartoum State. The results obtained revealed high
prevalence of gastro-intestinal parasite infections. In Mayo, Abuzeid and Elhaj Yousif, 31.7%, 30.3%
and 23.5% prevalence rates were recorded respectively. Seven parasite species were detected in Mayo
with varying percentages. Those were E. histolytica, G.lamblia, and T. saginata. A. lumbricoides
A.duodenale E.vermicularis and H.nana. All of those parasite species were also found in Abuzeid
with the exception of E. histolytica. In Elhaj Yousif, E .histolytica, T. saginata Ancylostoma sp.,
A.lumbricoides were not detected (table 22). H.nana infection was the most prevalent parasite in all of
the 3 areas. SFT can be considered as one of the best techniques for such survey studies since it detects
a wide range of parasites among children in endemic area (table 22).
Table (22): Parasites species detected in school children at three displaced population localities using SFT.
Parasite species ElhajYousif
% Abuzeid % Mayo %
Total
Entamoeba histolytica trophozoite
0
0
3 (1%)
3 (0.4%)
Entamoeba histolytica cyst
3 (1.2%) 0 3 (1%)
6 (0.7%)
Giardia lamblia trophozoite
1 (.4%) 0 2 (.7%)
3 (0.4%)
Giardia lamblia cyst 5 (2%) 1 (.3%) 14 (4.8%)
20(2.4%)
Entrobius vermicularis 4 (1.6%) 4 (1.4%) 4 (1.4%)
12(1.4%)
Taenia saginata 0 1 (.3%) 2 (.7%)
3 (0.4%)
Anchlystoma species 0 1 (.3%) 1 (.3%) 2 (0.24)
Ascaris lumbricoides 0 9 (3.1%) 2 (.7)
11(1.3%)
Hymenolepis nana 51 (20.3%)
77 (26.8%)
71 (24.2%)
199 (24%)
Total positive 59 (23.5%)
88 (30.3%)
93 (31.7%)
240 (29%)
Total samples tested 251 287 293 831
82
Irrespective of the study area, an overall positivity of 28.9% was found (table 23). The highest
prevalence (50%) was found among school children at the agegroup of >5 ≤ 8, followed by >8≤ 10
(31.4%). The prevalence decrease with the increase in age being 13.3% in children > 12 years (figure
7).
Table (23): Prevalence of gastro-intestinal parasite infection among child population in the three
study areas.
Age group Positive Negative Total Infected %
>5≤8 Year
44
44
88
50.0%
>8≤10 Years 111 243 354 31.4%
>10≤12 Years 71 212 283 25.1%
>12 Years 14 91 105 13.3%
Total 240 590 830 28.9%
83
Fig.(7): Gastro-intestinal parasite infection among children at different age groups.
50%
31.40%
25.10%
13.30%
0%
5%10%
15%20%
25%30%
35%40%
45%
50%
Prev
alen
ce
>5<=8 Year >8<=10 Years >10<=12 Years >12 YearsAge groups
Prevalence of gastro-intenstinal parasite in the different age groups
Gastro-intestinal parasites prevalence among the different age groups of children in the three study
areas is presented (figure 7). There was negative correlation versus age in the three areas. The
difference is statistically significant in Abuzeid P<0.05 and highly significant in Mayo P< 0.01 and
infection decreased with the increase in age. In Mayo the highest infection rate (65.8%) was in the age
group>5≤8 years and the same was true in Abuzied (50%).
In ElHaj Yousif, the prevalence among the age group >5≤8 year was lower (25%) than >8≤10 years
(28.1%), although this difference was not significant and prevalence decreased also with age (figure 8).
84
Figure (8): The distribution of parasites positivity according to the age groups in the three study areas.
0%
10%
20%
30%
40%
50%
60%
70%
Posi
tivity
ElHag Yousif Mayo Abozoid Total
Location
The distribution of parasite positivity in the different age groups in the three study areas
>5<=8 Year>8<=10 Years>10<=12 Years>12 Year
There was no effect of gender regarding gastro- intestinal parasites prevalence in the child population
in the three areas ( P>0.05) (table 24).
Table (24): Distribution of GIT parasite positivity among male and female child population.
Location Female Male P-value
Elhaj Yousif 32 (21.6%) 27 (26.2%) 0.399
Mayo 49 (33.8%) 44 (29.7%) 0.53
Abuzeid 41 (28.3%) 47 (33.1%) 0.443
Total 122 (31.04%) 118 (26.9%) 0.49
85
4.4 The use of SFT for H.nana epidemiological survey in Abuzeid area
A study on H.nana epidemiology carried out in school children of Abuzeid revealed that this parasite
occupied the top of the list for GIT parasites with a prevalence rate of 30.7%. G.lamblia, E.histolytica
and E.vermicularis were detected in 11.8%, 8.2% and 7.8% of the children respectively. The first two
species were also the most common in the previous study conducted at the health settings that included
442 patients. Other parasite species detected were: T.trichiura (0.7%), T. saginata (1.8%), Hookworm
(0.5%), A.lumbricoides (2.8%), and S.mansoni (0.3%) giving total parasite prevalence rate of 65% in
this area (table 25).
Table (25): Prevalence of H.nana and other parasites species among School children in Abuzeid
area.
Parasite species Positive Negative Prevalence %
Entamoeba histolytica trophozoite
6
1784
0.3%
Entamoeba histolytica cyst 146 1644 8.2%
G. lamblia trophozoite 5 1785 0.3%
G. lamblia cyst 211 1578 11.8%
Entrobius vermicularis 139 1651 7.8%
Trichuris trichiura 12 1778 0.7%
Schistosoma mansoni 5 1785 0.3%
Taenia saginata 32 1758 1.8%
Ancylostoma species 9 1781 0.5%
Ascaris lumbricoides 50 1740 2.8%
Hymenolepis nana 549 1241 30.7
86
Total 1164 626 65%
Table (26): H. nana co- infection with other parasite species in school children in Abuzeid area.
________________________________________________________________ Parasite species H.nana Total
Twenty six percent of the children who had tested positive for H.nana had co-infection with one or two
more parasite species. The most common co-infection was with G.lamblia (9.8%), E.vermicularis
(6.8%) and E.histolytica (8%) (table 26). At lower rates was the co-infection with G.lamblia and
E.histolytica trophozoites. Ancylostoma species, and S.mansoni were not found in association with
H.nana.
87
Intensity of H.nana infection was classified according to egg count in to three groups. Those who had
egg count of less than 50 eggs/gm of faeces were classified as mild infection, those of 50-100 as
moderate and more than 100 as heavy infection. Based on the results obtained, 73% of children had
mild infection, 10.93% moderate and 16.03% heavy infection (figure 9).
Figure (9): The intensity of H.nana infection in school children of Abuzeid area.
A high H.nana prevalence rate of 53.8% was found among school children within the age group >5≤ 8. The prevalence decreased with the increase in age (Fig10). The difference in prevalence between the
four age groups was highly significant (P<.001). A negative correlation was found between age and
Figure (10): H.nana prevalence in different age groups of school children in Abuzeid area.
16.03%
10.93%
73.04%
Less than 50 eggsLess than 100 eggsMore than 100 eggs
88
53.90%
33.40%
21.20% 20.40%
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
Prev
alen
ce
>5<=8 >8<=10 >10<=12 >12
Based on egg count, one can conclude that those who were having less than 50eggs /gm of faeces
represented the majority in each of the groups. By comparing intensity of infection in the different age
89
groups, (figure11)the highest percentage (87%) of children with mild infection was among the age
group >10≤12 years, followed by those >12 years (78%) and > 8≤10 Years (71.7%) respectively.
Among those with moderate infection, the highest percentage was within the age group >5≤ 8 (12.9%)
and >8 ≤10 (12.8%) less in those >10≤12 (4.3%) and 10.2% in those >12 years old.
Most of the children with heavy infection were at the age group >5≤8 (23%) followed by those >8≤10
(15.6%), >10≤ 12 (8.7%) and >12 (11.2%). These results revealed also that younger children (>5 ≤ 8)
were the most affected with H.nana infection in term of intensity. The difference in intensity among
the different age groups was highly significant (p<.001) (figure 11).
Figure (11): Intensity of infection in different age groups of school children with H.nana.
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
70.00%
80.00%
90.00%
Perc
enta
ge
>5<=8 >8<=10 >10<=12 >12
Age groups/Years
Egg count group >50Egg count group >50<=100Egg count group >100
The analysis of variance (ANOVA) was used to compare the intensity of infection in children
categorized by age group. When a crude age was used for analysis (ANOVA) negative correlation
between egg count and age was found with correlation coefficient of -.97 (figure 12).
Figure (12): Correlation between intensity of H.nana infection and age of child population in Abuzeid area.
90
0
100
200
300
400
500
600
700
5 6 7 7.5 8 8.5 9 10 11 12 13 14 15 16 17
Childern age
eggc
ount
/gra
m
4.4.1 H.nana prevalence in male and female child population of Abuzeid area.
Of the total child population under study, 48.8% were males and 51.2% were females. In the male
population 31.2% were detected positive with H.nana representing 49.75% of the total positives of the
female children 30.1% were found positive and contributing to 50.3% of the total positives.
No significant difference (P>0.05) was found in H.nana prevalence on basis of gender. These results
are also concordant with earlier findings in this study (table 27).
91
Table (27) : Positively for H.nana in male and female population in Abuzeid area.
4.4.2 Correlation of parent’s education and H.nana prevalence among school children in Abuzeid
The highest prevalence (45.7%) of H.nana was detected in children whose fathers are university
graduates, followed by those with khalwa level of education (41.9%), secondary (33%), illiterate
(35%) and primary level of education (29.9%). The majority of the children (42.3%) who were found
positive represented those whose fathers were of primary level of education, followed by illiterate
(35%) khalwa (9.8%), secondary&.3%), university 2.9% and intermediate level (0.4%).Variability in
the sample size in this part of the study however, did not allow for fair assessment of the education
impact. Representation of university and intermediate level were lower as compared to other groups
(Table 28).
273 601 87431.2% 68.8% 100.0%
49.7% 48.4% 48.8%
276 640 91630.1% 69.9% 100.0%
50.3% 51.6% 51.2%
549 1241 179030.7% 69.3% 100.0%
100.0% 100.0% 100.0%
Count % within sex % within children
Count % within sex % within children
Count % within sex % within children
Male
Female
Sex
Total
Yes No
H.nana Total
92
Table (28) H.nana prevalence among school children based on father’s level of education.
93
The majority of the children (57.3%) who had positive diagnosis were from illiterate mothers (Table
29), followed by those whose mothers have primary level of education (33.3%).Other groups had
192 473 66528.9% 71.1% 100.0%
35.0% 38.1% 37.2%
54 75 12941.9% 58.1% 100.0%
9.8% 6.0% 7.2%
232 544 77629.9% 70.1% 100.0%
42.3% 43.8% 43.4%
2 13 1513.3% 86.7% 100.0%
0.4% 1.0% .8%
40 79 11933.6% 66.4% 100.0%
7.3% 6.4% 6.6%
16 19 3545.7% 54.3% 100.0%
2.9% 1.5% 2.0%
13 38 5125.5% 74.5% 100.0%
2.4% 3.1% 2.8%
549 1241 179030.7% 69.3% 100.0%
100.0% 100.0% 100.0%
Count % within Father education% within microscopicexamination of preservedspecimen H.nanaCount % within Father education% within microscopicexamination of preservedspecimen H.nanaCount % within Father education% within microscopicexamination of preservedspecimen H.nanaCount % within Father education% within microscopicexamination of preservedspecimen H.nanaCount % within Father education% within microscopicexamination of preservedspecimen H.nanaCount % within Father education% within microscopicexamination of preservedspecimen H.nanaCount % within Father education% within microscopicexamination of preservedspecimen H.nanaCount % within Father education% within microscopicexamination of preservedspecimen H.nana
Illiterate
Khalwa
Primary
Intermediate
Secondary
University
Unknown
Father education
Total
yes no
microscopicexamination of
preserved specimen H.nana
Total
94
lower representation in the positive group. The same variability in the sample size of the children
father's level of education was also encountered in the mothers making it difficult to draw any
conclusion on the effect of mother’s level of education on H.nana prevalence.
Table (29): H.nana prevalence in school children based on mother’s level of education.
95
4.4.3 Correlation of parent’s occupation and H.nana prevalence among school children in Abuzeid
315 798 111328.3% 71.7% 100.0%
57.4% 64.3% 37.2%
7 14 21 33.3% 66.7% 100.0%
1.3% 1.1% 1.2%
183 349 53234.4% 65.6% 100.0%
33.3% 28.1% 29.7%
7 10 1739.5% 58% 100.0%
1.3% 8% 9%
7 26 43 53.8% 60.5% 100.0%
3.1.% 2.1% 2.4%
7 6 1353.8% 56.2% 100.0%
1.3% 5% 7%
13 38 5125.5% 74.5% 100.0%
2.4% 3.1% 2.8%
549 1241 179030.7% 69.3% 100.0%
100.0% 100.0% 100.0%
Count % within Father education% within microscopicexamination of preservedspecimen H.nanaCount % within Father education% within microscopicexamination of preservedspecimen H.nanaCount % within Father education% within microscopicexamination of preservedspecimen H.nanaCount % within Father education% within microscopicexamination of preservedspecimen H.nanaCount % within Father education% within microscopicexamination of preservedspecimen H.nanaCount % within Father education% within microscopicexamination of preservedspecimen H.nanaCount % within Father education% within microscopicexamination of preservedspecimen H.nanaCount % within Father education% within microscopicexamination of preservedspecimen H.nana
Illiterate
Khalwa
Primary
Intermediate
Secondary
University
Unknown
Mother r Education
Total
Yes No
Microscopicexamination of
preserved specimen H.nana
Total
96
Since it was difficult to assess the economic status of the family on ground of statements given by most
children we had considered father occupation as an indicator of family income, though women have
important contribution in the income and economic status of the family. The highest H.nana
prevalence was among children whose fathers had irregular occupation (35%) or lower paid jobs such
as soldiers (34.7%), drivers (34.8%) or unemployed (30.9%). The lowest prevalence was among
children whose fathers were teachers (18.8%), animal traders (22.9%) or medical assistants (25%)
(Table 30).
Table (30) Prevalence of H.nana in relation to father occupation.
Occupation
Positive Negative Total Prevalence
Teacher 2 9 11 18.18
Animals trader 22 74 96 22.92
Medical assistant 3 9 12 25.00
Butcher 12 32 44 27.27
Farmer 13 35 48 27.08 Worker 79 205 284 27.82
Unemployed 48 107 155 30.97
Soldier 16 30 46 34.78
Driver 31 58 89 34.83
Other occupation 76 136 212 35.85
4.4.4 Effect of food quality on H.nana prevalence among school children of Abuzeid
The quality of food intake was assessed versus H.nana prevalence as shown in Table 3.
97
The highest H.nana prevalence (32%) was among children who consumed cereals and vegetables only.
The effect of high nutritive food on prevalence of infection was clearly demonstrated in children who
had fed on the five food stuffs (14%).
The different rates of H.nana infection in children fed on the different food stuffs was highly
significant (P<.05) (Fig 13).
Pearson chi2 (3) = 8.2367 Pr = 0.04
Fisher's exact = 0.03
Table (31) H.nana prevalence among children fed on different foodstuffs.
Food H.nana infection
Positive Negative Total
Cereal, meat, dairy and vegetables
155 (28.4%)
390 (71.6%)
545
Cereal and vegetables
370 (32.6%)
766(67.4%)
1136
Cereals, dairy and fruits
19 (26.4%)
53 (73.6%)
72
Cereals, meat, dairy, vegetable and fruits
5 (14.3%)
30 (85.7%)
35
Fig.(13) H.nana prevalence in children fed on different food stuffs
98
0
5
10
15
20
25
30
35Pr
eval
ence
1 2 3 4Food stuffs
*Cereal and vegetables=1, Cereal, meat, dairy and vegetables=2, Cereals, dairy and fruits=3, Cereals, meat, dairy, vegetable and fruits=4
Food groups Cereal and Vegetables =1, cereal, meat, diary and vegetables =2 Cereals, diary and fruits =3 Cereals, meat, dairy, vegetable and fruits =4
4.4. 5 Effect of house construction on H.nana prevalence among school children:
99
The highest H.nana prevalence was among children who lived in houses built of straw (33.8%)
followed by those lived in houses built of sacs (31.8%) or mud and straw (31.3%) and those lived in
mud built houses (30.6%). Less infected were those who lived in houses made of bricks (27%) or mud
and bricks (23%). Despite these differences, analysis applying chi square indicated no significant
difference (P>.05) in H.nana prevalence as related to house construction (Table 32).
Table (32): H.nana prevalence rates in relation to house construction.
100
4.4.6 Effect of drinking water source on H.nana prevalence
384 871 1255
30.6% 69.4% 100.0%
22 43 65
33.8% 66.2% 100.0%
25 66 91
27.5% 72.5% 100.0%
21 45 66
31.8% 68.2% 100.0%
94 206 300
31.3% 68.7% 100.0%
3 10 13
23.1% 76.9% 100.0%
549 1241 1790
30.7% 69.3% 100.0%
Count%
Count%
Count%
Count%
Count%
Count%
Count%
Mud
Straw
Brick
Sacs
Mud+ straw
Mud + brick
House Construction
Total
Yes No
H.nana Total
101
Of the total H.nana infected children, 32.5% drank water from tanks, followed by those from tank and
river (34.4 %%). Those who drank water from river and wells represented 23.5% and
24.7%respectively (Table 33). Statistical analysis revealed significant differences between the four
groups suggesting that water source had noticeable impact on H.nana prevalence. The percentage of
positives among those who drank water from river only was lower by comparison to those drinks from
river and tanks or from tanks only. These results indicated that tanks could have had effect on H.nana
prevalence.
Table (33): H.nana prevalence according to source of drinking water.
4.4.7 Site of defecation in relation to H.nana prevalence among school children of Abuzeid area
295 614 909 32.5% 67.5% 100.0%
39 119 158 24.7% 75.3% 100.0%
66 215 281 23.5% 76.5% 100.0%
136 259 395 34.4% 65.6% 100.0%
536 1207 1743 30.8% 69.2% 100.0%
100.0% 100.0% 100.0%
Count %
Count %
Count % Count % Count %
Water tank
Well
River
Water tank and River
Water Source
Total
Yes No
H.nana
Total
102
The results obtained in this part of the study showed clearly the effect of the site of defecation on
H.nana transmission. The effect of the site of defecation on prevalence was even clearer should the
number of positives in each category be considered (Table34). Among the children who defecated in
open areas or used neighbor's latrines, 50% were H.nana positive. Of those who defecated in latrines
and sometimes in the open areas or those who had used to defecate in the open areas only, infection
rates were 42.2% and 38.8% respectively. Lower infection rates were among those who either used
their own latrines (29.1%) or who did not have latrines but used their neighbour’s latrines (28.7%).
The difference between the different groups was statistically significant (p < 0.001).
(Table (34): Site of defecation and H.nana prevalence rate.
4.4.8 Effect of hand washing after defecation on H.nana prevalence Among the children who had assured us that they used to wash their hands after defecation, 29% were
H.nana positive. Of those who did not wash their hands (42%) and among those who wash them
401 978 1379 29.1% 70.9% 100.0%
26 41 67 38.8% 61.2% 100.0%
52 129 181 28.7% 71.3% 100.0%
17 17 34 50.0% 50.0% 100.0%
49 67 116 42.2% 57.8% 100.0%
545 1232 1777 30.7% 69.3% 100.0%
Latrine
Open area
Neighbour latrine
Neighbor Latrine + Open area
Latrine + open area
Place of Defecation
Total
+ve -ve
H.nana Total
103
sometimes the rate of infection was 30.6%. The difference between the first group and the other two
was statistically significant (P< 0.05), (table 35).
Table (35): Effect of hand washing after defecation on H.nana prevalence
4.4.9 Effect of playing in soil on H.nana prevalence.
The majority of the children mentioned that they did not play in the soil. Those who admitted playing
in soil, 32.1% were positive and among those who had not played in soil, 30.1% were positive. No
infection was found among children who did play sometimes in soil. Also no significant difference
465 1108 1573 29.6% 70.4% 100.0%
62 83 145 42.8% 57.2% 100.0%
22 50 72 30.6% 69.4% 100.0%
549 1241 1790 30.7% 69.3% 100.0%
Yes
No
Sometime
Washing hands after defecation
Total
+ve -ve
H.nana
Total
104
(P>0.05) was found between the two groups who frequently did or those who never played in soil
(table 36).
Table (36): H.nana prevalence among school children in relation to playing in soil.
4.4.10 Clinical symptoms encountered within H.nana infection
Among the children studied, 51.8% had complaints of abdominal pain. 37% of them had positive
H.nana infection by comparison with 23.3% positives in the child group without complaints. In those
187 395 582 32.1% 67.9% 100.0%
358 833 1191 30.1% 69.9% 100.0%
6 6 100.0% 100.0%
545 1234 1779 30.6% 69.4% 100.0%
Frequently
Not done
Occasionally
Playing In soil
Total
+ve -ve
H.nanaTotal
105
with occasional complaints, H.nana infection was (33.3%). There was significant difference (P<0.05)
in H.nana prevalence among the three groups indicating a clear association between abdominal pain
and H.nana infection (table37).
The results obtained in this study showed clear evidence that abdominal pain in the child population of
Abuzeid area was associated with H.nana infection The highest number of children with abdominal
pain (81.8%) had also the highest intensity of infection (egg count>100/gm) (table 38).
Table (38): Intensity of H.nana infection in relation to abdominal pain
Intensity of infection (egg count)
Abdominal pain
No abdominal pain
Occasional Total
Less than 50 eggs/gm
228 (56.9%) 172 (42.9%) 1 (0.2%) 401
343 585 928 37.0% 63.0% 100.0%
62.5% 47.1% 51.8%
205 654 859 23.9% 76.1% 100.0%
37.3% 52.7% 48.0%
1 2 3 33.3% 66.7% 100.0%
.2% .2% .2%
549 1241 1790 30.7% 69.3% 100.0%
100.0% 100.0% 100.0%
Count % within abdominal pain
% within H.nana
Count % within abdominal pain
% within H.nana
Count % within abdominal pain
% within H.nana
Count % within abdominal pain
% within H.nana
Yes
No
Sometime
Abdominal Pain
Total
+ve -ve
H.nanaTotal
Table (37): Prevalence of H.nana in children with complaints of abdominal pain.
106
50-100 eggs/gm 43 (71.7%) 17 (28.3) 0 60
More than 100 eggs/gm
72 (81.8) 16 (18.2) 0 88
Fig (14): Association of abdominal pain and the intensity of H.nana in children
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
70.00%
80.00%
90.00%
Com
plai
nts
of a
bdom
inal
pai
ns
<50 50-100 > 100Intensity of Infection (egg count/gram)
Among the children with diarrhea, 38.9% were with H.nana infection, and those without diarrhoea
(26.6%). This difference was found to be highly significant (P<0.01) indicating that diarrhoea could be
one of the symptoms associated with H.nana (table 39).
Table (39) Prevalence of H.nana infection among children with diarrhoea
107
Occurrence of diarrhoea (65.9%) was high among children with heavy H.nana infection (figure 15).
Occurrence decreased with the decreased in intensity of infection being 53.3% in moderate and 34% in
the mild levels, indicating significant association between diarrhoea and intensity of H.nana infection
(P<0.01).
Figure (15): Prevalence of diarrhoea among children with different intensity of H.nana infection.
230 361 59138.9% 61.1% 100.0%
41.9% 29.1% 33.0%
319 879 119826.6% 73.4% 100.0%
58.1% 70.9% 67.0%
549 1240 178930.7% 69.3% 100.0%
100.0% 100.0% 100.0%
Count% within diarrhea
% within H.nana
Count% within diarrhea
% within H.nana
Count% within diarrhea
% within H. nana
Yes
No
Diarrhea
Total
+ve -ve
H.nana
Total
108
35%
65%
53%
47%
66%
34%
0%
10%
20%
30%
40%
50%
60%
70%
Com
plai
nts
of D
iarr
hoea
<50 50-100 >100Egg count /gm
DiarrheaNo diarrhea
H.nana was more prevalent (36.9%) among children with vomiting than in those without (28.4%)
(table 40). Although this difference was highly significant (P<0.001), the highest percentage of
positives was found among those with no complaints of vomiting (69.2%).This can be attributed to the
larger number of children under study who did not have vomiting (table 40).
109
Although occurrence of vomiting increased with intensity of H.nana infection (figure 16), the
difference is not significant (P>0.05) indicating no association between intensity of H.nana infection
and vomiting.
164 280 444 36.9% 63.1% 100.0%
29.9% 22.6% 24.8%
380 959 1339 28.4% 71.6% 100.0%
69.2% 77.3% 74.8%
5 2 7 71.4% 28.6% 100.0%
.9% .2% .4%
549 1241 1790 30.7% 69.3% 100.0%
100.0% 100.0% 100.0%
Count % within vomiting
% within H.nana
Count % within vomiting
% within H.nana
Count % within vomiting
% within H.nana
Count % within vomiting
% within H.nana
Frequent
Absent
Occasional
Vomiting
Total
-ve
H.nana Total
+ve
Table (40): Prevalence of H. nana in children with vomiting.
110
Figure (16): Intensity of H.nana infection among children with vomiting.
28%30%
40%
0%
5%
10%
15%
20%
25%
30%
35%
40%
Com
plai
nts
of v
omiti
ng
<50 50-100 >100Intensity of infection (egg count/gm faeces)
Among the children who were presented with fever, 34.2% were H.nana positive (table 41). Only
27.3% of those who had no fever had H.nana infection. The difference between the two groups was
statistically significant (p<0.05). Also the majority (53.0%) of infected children had no fever. This can
also be attributed to the same reason of having a larger number of H.nana positives without fever..
111
250 480 730 34.2% 65.8% 100.0%
45.5% 38.7% 40.8%
291 755 1046 27.8% 72.2% 100.0%
53.0% 60.8% 58.4%
8 6 14 57.1% 42.9% 100.0%
1.5% .5% .8%
549 1241 1790 30.7% 69.3% 100.0%
100.0% 100.0% 100.0%
Count % within fever % within H.nana
Count % within fever % within H.nana
Count % within fever % within H.nana
Count % within fever % within H.nana
Frequent
Absent
Occasional
Fever
Total
+ve -ve
H.nana Total
Table (41): H.nana prevalence in children with fever.
112
The results presented in table 42 shows that there was no association between the fever and intensity of
H.nana infection. Prevalence of fever was almost the same for all of the three groups, being 42.4% in
the mildly infected, 53.3% in the moderate and 54.5% in the heavily infected group. No significant
difference was found (P>0.05).
Table (42): Intensity of H.nana infection in relation to occurrence of fever.
Positivity for H.nana was higher among children with anal irritation (38%) compared to those without
irritation (29.5%). However This difference was not significant (p>0.05) (table 43).
Table (43): The association of anal irritation and H.nana infection.
Count % within Egg count group% within fever Count % within Egg count group% within fever Count % within Egg count group% within fever Count % within Egg count group% within fever
<50
50-100
>100
Egg count Group
Total
Frequent Absent Occasional Fever Total
113
No significant difference (P> 0.05) between the three groups with different infection intensity as
regard to anal irritation being 16%, 25% and 20.5% in children with mild, moderate and high intensity
levels (table 44).
97 157 254 38.2% 61.8% 100.0%
17.7% 12.7% 14.2%
452 1078 1530 29.5% 70.5% 100.0%
82.3% 87.2% 85.7%
1 1 100.0% 100.0%
.1% .1%
549 1236 1785 30.8% 69.2% 100.0%
100.0% 100.0% 100.0%
Count % within anal irritation
% within H.nana
Count % within anal irritation% within H.nana
Count % within anal irritation% within H.nana
Count % within anal irritation% within H.nana
Present
Absent
Occasional
Anal Irritation
Total
+ve -ve
H.nana
Total
Table (44): Intensity of H.nana infection in relation to occurrence of anal irritation.
114
64 337 401 16.0% 84.0% 100.0% 66.0% 74.6% 73.0%
15 45 60 25.0% 75.0% 100.0% 15.5% 10.0% 10.9%
18 70 88 20.5% 79.5% 100.0% 18.6% 15.5% 16.0%
97 452 549 17.7% 82.3% 100.0%
100.0% 100.0% 100.0%
Count % within Egg count group% within anal irritationCount % within Egg count group% within anal irritationCount % within Egg count group% within anal irritationCount % within Egg count group% within anal irritation
<50
50-100
>100
Egg count
Total
+ve -veAnal irritation
Total
115
Other symptoms also encountered among children were shown in (figure 17).
Figure (17): Other symptoms observed in children with H.nana infection:
Headache, nausea and dizziness,
10.60%No, 46.60%
Headache and dizziness, 2.70%
Nausea and dizziness, 3.50%
Dizziness, 2.60%
Headache and nausea, 10.90%
Nausea, 3.10% Headache, 20%
In the majority (63.4%) of those who had nausea and dizziness, H.nana was diagnosed, followed by
those who had triple presentation of headache, nausea and dizziness (43.3%). There was also high
H.nana prevalence among those who had dizziness only (42.45%) or those who had nausea and
headache (42.3%) (Annex III).
Of the children included in the study, 47.7% visited hospitals during the last six months and 52.3%
didn't. H.nana was detected in 32.4% of the former group that represented 50.3% of the total positives.
Among those who didn't visit hospitals, 29.1% were found positive for H.nana representing 49.7% of
the total positives. The results revealed no significant difference (P>0.05) between the two groups.
Table (45): H.nana prevalence in children who visited hospitals during the last six months.
116
4.4. 11 Risk factor for H.nana infection applying logistic regression
All variables that might have affected H.nana prevalence were screened for statistical significance
using chi-square based on the recommendation of Hosmer and Lemshew (Table 46). All variables that
may have effect on prevalence were then included in a logistic regression model and the fit of the
model was assessed using Wald test and Hosmer and Lemshew tests. Variables that had no significant
effect on H.nana prevalence or in the final fit of the model were removed. The final model was fitted
using age, house construction, defecation site, self cleaning, washing after defecation and drinking
water source. Dummy variables were created for each subgroup within each variable.
According to the logistic regression, age less than 8 years, use of neighbor’s latrine or open area as
defecation sites and the attitude of not washing hands after defecation were found to be risk factors (P-
value<0.05) for H.nana infection. Both P-value and confidence interval were used to define a risk
factor. The risk for children less than 8 years was found to be twice the risk for children between 8 and
10 (odds ratio= .44) and four times the risk for children between 10 and 12 (odds ratio= .25) and for
children more than 12 years (odds ratio=.24). The risk for children using neighbors latrine and open
276 577 853 32.4% 67.6% 100.0%
50.3% 46.5% 47.7%
273 664 937 29.1% 70.9% 100.0%
49.7% 53.5% 52.3%
549 1241 1790 30.7% 69.3% 100.0%
100.0% 100.0% 100.0%
Count % within visit hospitals
% within H.nana
Count % within visit hospitals
% within H.nana
Count % within visit hospitals
% within H.nana
Yes
No
Hospitals visit during last 6 months
Total
+ve -ve
H.nana
Total
117
area for defecation was found almost twice that for children using own latrine (odds ratio=1.76) and
the risk for children who did not wash their hands after defecation was found to be 1.5 times the risk of
children who washed their hands after defecation (odds ratio=1.48).
Table (46): Risk factors for H.nana infection applying logistic regression.
Mud & Brick 0.9958115 0.7017116 0.995 0.2502458 3.962666 Open area defecation
1.046 0.297264 0.874 0.5992771 1.825726
Neighbor's latrine 1.04192 0.2053833 0.835 0.7080202 1.533287 Latrine & open area
2.101699 0.8356696 0.062 0.9640931 4.581651
Neighbor's latrine and open area
1.724998 3705845 0.011 1.132201 2.628172
Do not wash hand after defecation
1.475581 0.2859727 0.045 1.009246 2.157391
Sometime wash hand after defecation
1.007994 1.007994 0.978 0.5765583 1.762272
Well 1.228158 0.2427854 0.299 8336556 1.809348 River 1.013641 0.1632978 0.933 0.7391884 1.389994 Water tank & river 9789443 0.1402997 0.882 0.7392069 1.296433 4.4.12 H.nana prevalence among families of children with different intensities of infection
Thais part of the study was conducted to explore correlation between intensity of H.nana infection
among children and the inter-family transmission rates. Children were grouped according to level of
infection as based on egg counts varying from mild (<50 eggs /gm), moderate (50-100 egg/gm) or
heavy (>100 egg/gm). Fifteen families from heavily and mildly infected groups were included. Ten
118
families of moderately infected children and 15 families from children free of H.nana infection were
also included.
It is well established that using a cluster sampling technique for estimation of infection prevalence has
an effect on the confidence interval. Though intracluster correlation (correlation within families) does
not affect prevalence per se, it underestimates the standard deviation and produce narrow confidence
interval. Therefore, the confidence interval for H.nana prevalence was inflated based on calculated
design effect so that the effect of cluster sampling technique (design effect) could be accounted for
because families rather than individual were sampled.
The design effect was calculated using STATA software and was found to be 1.158. The prevalence
estimate and confidence interval is shown in table 47.
Table (47): Prevalence estimate and confidence interval using the calculated design effect. Prevalence Standard
The results presented in (table 48) show prevalence of H.nana and other GIT parasites among family
members of infected children.
The overall prevalence of H.nana in the different families was found to be 13.1% with .018 standard
error and a confidence interval of (.0937-0.1166). Other GIT parasites detected were almost all of
those found earlier in the school children namely, G.lamblia,
119
Entamoeba histolytica, T.trichiura, T.saginata and Ancylostoma species. Although H.nana occupied
the top of the list (13.1%), its prevalence among families however, was lower by comparison with that
of the children (30.8%).
Table (48): H.nana and other parasite species detected in family members of infected children.
Parasite species Stool Examination Positive Negative Prevalence
Entamoeba histolytica cyst 6 386 1.5%
Giardia Lamblia cyst 12 380 3.1%
Enterobious vermicularis 5 387 1.3%
Trichuris trichiura 3 389 0.8%
Taenia saginata 7 385 1.8%
Ancylostoma species 4 388 1.0%
Ascaris lumbricoides 5 387 1.3%
Hymenolepis nana 51 341 13.1%
The number of family members found infected with H.nana was different for the four groups,
nevertheless, higher among family members of the heavily infected group (table 49).
The average egg count was higher among families of heavily infected children (34.26), followed by the
moderate (28.7) and mild (16.88) and very low in the negative control group (5). The standard
deviation in all groups was high and there was no statistical significance between the four groups
regarding egg count using both the parametric test ANOVA and the non parametric test Kruskal Wallis
test (P>0.05).
120
Table (49) Egg count in H.nana positive family members of children with different infection
intensity.
Intensity of H.nana infection*
No. of individuals tested
Mean egg count/gm
Median egg count/gm
Standard deviation
Negative 96 5 2 5.15
Heavy 110 34.26 4 69.4
Mild 120 16.88 4 26.16
Moderate 66 28.7 8 62.55
* Families were classified according to their children intensity of infection
Figure (18): Mean egg count in H.nana positive family members of children with different intensites of infection:
121
05
101520253035404550556065707580859095
100105110
Control Mild Moderate Heavy
Egg
coun
t
The number of tested family members of moderately infected children was 66, of them 10 (15.2%),
were positive for H.nana. Family members of the mildly infected children were 120 and 17 (14.2%) of
122
them were positive. The number of family members of the heavily infected children was 110 and 19
(17.3%) of them were positive. The control group included 96 family members, only 5.2% were
positive. Statistical analysis revealed no significant differences in H.nana prevalence among the
different groups examined p>0.05 suggesting no correlation between intensity of infection among
children and prevalence of H.nana among their family members (table 50).
The effect of family on H.nana transmission was explored and the data was checked for presence of
intraclass (family) relationship. No correlation between intensity of the infection in children and H.
nana prevalence within the family was established. The intraclass correlation was found to be almost
0.000.
The Kruskal Wallis test was applied to compare for the prevalence of H.nana infection within families
of children with different intensities of infections. No significant difference was found. (Chi square
=2.4).
Regarding age and prevalence of H.nana among family members, the results revealed the higher
prevalence within the age groups of >5<=10 (18%) and >15 <=10 (19%) respectively. Similar
prevalence rates were also found in the age groups <5= (14%) and >10<=15. Prevalence was however
lower among the age group of >20 (9%). These differences were not significant (P> 0.05).
56 10 66 84.8% 15.2% 100.0% 16.4% 19.6% 16.8%
103 17 120 85.8% 14.2% 100.0% 30.2% 33.3% 30.6%
91 19 110 82.7% 17.3% 100.0% 26.7% 37.3% 28.1%
91 5 96 94.8% 5.2% 100.0% 26.7% 9.8% 24.5%
341 51 392 87.0% 13.0% 100.0%
100.0% 100.0% 100.0%
Count% within Infection% within H.nanaCount% within Infection% within H.nanaCount% within Infection% within H.nanaCount% within Infection% within H.nanaCount% within Infection% within H.nana
Moderate
Mild
Heavy
Control negative
Infection level
Total
_-ve +veH.nana
Total
Table (50): H.nana prevalence among family members of children with different levels of infection.
123
Fig. (19): Prevalence of H.nana among family members
14%
18%19%
14%
9%
0%2%4%6%8%
10%12%14%16%18%20%
Prev
alen
ce
<=5 >5<=10 >10<=15 >15<=20 >20
Age group/Years
The results shown in Table 51 revealed no significant differences (p>0.05) on H.nana prevalence
between the male and female population. Twenty-six (51.0%) of the positives were males and 25 (49.0
%) were females. These results were concordant with those obtained earlier in the same study.
Table (51): H.nana prevalence among male and female family members.
124
The results presented in figure (20) showed clearly the effect of gender on H.nana prevalence at the
age class of up to 10 years. H.nana prevalence was higher among females by comparison to males. At
the age class >10<=15 years the male population scored a higher prevalence rate than females.
Figure (20): H.nana prevalence in females and males family members at different age groups:
158 26 18485.9% 14.1% 100.0%46.3% 51.0% 46.9%
183 25 20888.0% 12.0% 100.0%53.7% 49.0% 53.1%
341 51 39287.0% 13.0% 100.0%
100.0% 100.0% 100.0%
Count% within sex % within H.nana Count% within sex % within H.nana Count% within sex % within H.nana
Male
Female
Sex
Total
-ve +veH.nana
Total
125
0%
5%
10%
15%
20%
25%
30%Pr
eval
ence
<=5 years >5<=10 years >10<=15 >15<=20years
>20 years
Age/years
Males Females
4.5 Assessment of two drugs for treatment of H.nana
Assessment of the efficiency of praziquantel and niclosamide for treatment of H.nana infection was
carried out by treating the infected children. Children were divided in to three groups according to the
infection level, being heavy, moderate or mild. Each group consisted of 10 males and 10 females. One
group was put on praziquantel (single dose of 25mg/kg) and the other with niclosamide (chewable
tablets, 2g for 4-6 days). The two drugs were administered under supervision of the physician in charge
of Ahfad Health Center. The children were examined by the physician who prescribed the dose
according to weight. All children were tested and the average egg count was reported. In order to
randomize the sample, egg count post treatment was done blindly without previous knowledge of the
group to which child belonged before treatment. Analysis of results was made first to acknowledge the
reduction in egg count post treatment in the three groups and secondly to compare between the
reduction in egg count as a result of administration of the two drugs. The Third objective was to
compare efficiency of the two drugs for complete elimination of H.nana eggs in stool on the second,
Third, forth and seventh day post treatment.
Effect of H.nana on child weight:
The weight of children who had heavy infection was at the range of 20-25 kg, in those with moderate
infection 30-35 kg and in those who had mild infection, 25-30 kg
126
In order to determine whether the child weight is affected, the following formula was used : standard
weight= actual weight×2 +8.
The majority of children who have had heavy H.nana infection were under weight (83.33%). In the
moderately and mildly infected groups, those who were under weight represented 72% and 75%
respectively. Though the highest deviation from the standard weight was obtained in the heavily
infected children, statistical analysis revealed no significant difference between the three groups
(P>0.05) indicating that under weight might not only be a result of H.nana infection (table 52).
Table (52) Percentage of children under weight compared to the standard.
Level of infection
Under weight
Normal weight
Total Percentage of under weight
Percentage of normal weight
Heavy 20 4 24 83.33% 16.67%
Mild 15 5 20 75% 25%
Moderate 8 3 11 72.73% 27.27%
Total 43 12 55 78.18 21.82%
Our results showed that both drugs had positive effect in reducing H.nana egg count with time.
Although reduction in count was observed in day 2 after treatment with both drugs, the highest
reduction was found with praziquantel starting from day 3 after treatment (table 53).
Table (53): Average egg count before and after drug administration.
Niclosamide Praziquantel Period
Mean SD Min. Max Mean SD Min. Max Before treatment
374.7
894.65
5
4670
284.2
476.11
9
2221
127
Day 2 post treatment
247
939.56
0
5100
295.2
1168
0
6000
Day 3 post treatment
206.3
828.45
0
4490
22.9
53.5
0
191
Day 4 post treatment
52.5
133.54
0
665
5.2
13
0
50
Day 7 post treatment
17.3
42.615
0
185
1.9
7.15
0
35
Figure (21): The average egg count in children treated with praziquantel and Niclosamide:
128
284.23 295.15
22.92 5.23 1.96
374.69
247 206.3452.48276
17.310340
50
100
150
200
250
300
350
400
Before treatment First day aftertreatment
Second day aftertreatment
Third day aftertreatment
Week after treatment
Niclosamide
Prazequantel
Time
Egg
cou
nt
Effect of treatment on egg count. Multi-factorial ANOVA was used to assess the effect of niclosamide and praziquantel and the time
after treatment on H.nana egg count. The data was screened for normality and a clear deviation from
normality was observed. Therefore, the data was log transformed. Significant difference in the overall
model was observed. A highly significant level (P<0.01) in egg count reduction for both praziquantel
and niclosamide was found (figure.21).
Effect of treatment on H.nana different levels of infection: Although reduction in egg count after treatment was evident in the three groups, some discrepancies in
egg count were observed. There was increase in egg count in day 4 in the mild and moderate groups as
treated with niclosamide and in the heavily infected group with praziquantel on day 3 (table 54).
129
Despite the discrepancies in egg count, the effect of both drugs in reduction of egg count in all three
groups was clear. Higher reduction rate was observable with praziquantel implying superiority over
niclosamide.
Table (54): Average egg count in children with different levels of infection treated with
Niclosamide or Praziquantel.
Niclosamide Prazaquantel Period
Heavy mild Moderate Heavy mild Moderate Day 2 after treatment
902.14286
17.6
46
215.71429
8.0
40.5
Day 3 after treatment
762
5.0
37
35.51429
0.0
3.0
Day 4 after treatment
160.42857
7.4
0.0
3.4285714
0.0
11.5
Days 7 after treatment
57.857143
0.0
2.5
0.57142857
0.0
0.0
Irrespective of pretreatment level of infection, our results revealed that 15 (27%) of the children
completely cleared of H.nana eggs. Of those 8 (53.3%) were treated with niclosamide and 7 (46%)
were treated with praziquantel. No clear difference on the effect of the two drugs on egg count on the
second day after treatment was observed
(table 55).
130
Thirty five (63.6%) stool samples were cleared of H.nana eggs on the third day after treatment.
Twenty of those (57.1%) received praziquantel and 15 (42.9%) received niclosamide.
(76.9%) of those completely cleared were treatmed with praziquantel whereas 51.7% with
niclosamide. These results confirmed superiority of praziquantel over Niclosamide (table 56).
Table (56): Reduction in H.nana egg count in the 3rd day post treatment.
Table (55): Reduction in egg count in the second day after treatment.
8 7 15
53.3% 46.7% 100.0%
27.6% 26.9% 27.3%21 19 40
52.5% 47.5% 100.0%
72.4% 73.1% 72.7%29 26 55
52.7% 47.3% 100.0%
100.0% 100.0% 100.0%
Count% within Reduction inegg count in the firstday after treatment% within DRUGCount% within Reduction inegg count in the firstday after treatment% within DRUGCount% within Reduction inegg count in the firstday after treatment% within DRUG
Clearance of infection(egg count=0)
Presence of H.nana egg
Reduction in egg count
Total
Niclosamide PraziquantelDRUG Total
131
Thirty eight (69%) samples of the total samples were totally cleared of H.nana eggs at day four after
treatment. Of those 21(55%) received praziquantel and 17 (44.7%) niclosamide.
Eighty percent of those completely cleared were treated with Praziquantel while 58.6% of them were
niclosamide treated. (table 57).
Table (57): Reduction in H.nana egg count in the forth day post treatment.
15 20 35
42.9% 57.1% 100.0%
51.7% 76.9% 63.6%14 6 20
70.0% 30.0% 100.0%
48.3% 23.1% 36.4%29 26 55
52.7% 47.3% 100.0%
100.0% 100.0% 100.0%
Count
% within Reduction
% within DRUGCount
% within Reduction
% within DRUGCount
% within Reduction
% within DRUG
Complete clearance
Partial clearance
Reduction in egg count
Total
Niclosamide
Praziquantel
DRUGTotal
132
On the 7th day post treatment, 78% of the samples were freed from H.nana eggs. Of those 84 %
received praziquantel and 72.4% received niclosamide (table 58).
17 21 38
44.7% 55.3% 100.0%
58.6% 80.8% 69.1%12 5 17
70.6% 29.4% 100.0%
41.4% 19.2% 30.9%29 26 55
52.7% 47.3% 100.0%
100.0% 100.0% 100.0%
Count
% within Reduction
% within DRUGCount
% within Reduction
% within drugCount
% within Reduction in
% within drug
Complete clearance
Partial clearance
Reduction in egg count
Total
Niclosamide
Praziquantel
DRUG Total
Table (58): Reduction in egg count one week after treatment.
133
Table (59) H.nana recovery from rats and cockroaches
Typing Animals Number
Of animals dissected
No. of Positive No. of Negative
Rats
15 1 (8.3%) 14 (91.7%)
Cockroach
100 5 (5 %) 95 (95%)
2122
43
48.8% 51.2% 100.0%
72.4% 84.6% 78.2%8 4 12
66.7% 33.3% 100.0%
27.6% 15.4% 21.8%29 26 55
52.7% 47.3% 100.0%
100.0% 100.0% 100.0%
Count% within Reductionin egg count oneweek after treatment% within DRUGCount% within Reductionin egg count oneweek after treatment% within DRUGCount% within Reductionin egg count oneweek after treatment% within DRUG
Clearance of infection(egg count=0)
Presence of H.nana egg
Reduction in egg
Count
Total
Niclosamide
Praziquantel
DRUG Total
134
Chapter Five
Discussion Intestinal parasite infection is one of the major causes of morbidity in developing countries. Most of
these infections are chronic, contributing to malnutrition among children and reducing host resistance to
other diseases. Children being the victims serve also as source of infection thus contributing to
transmission. Proper diagnosis based on accurate results of stool examination is therefore of vital
importance for the control of these infections.
So far, microscopic examination is considered the gold standard for diagnosis of gastro-intestinal
parasites. In Sudan, there is an urgent need for simple and accurate methods of stool examination, since
the current methods are insensitive and as a result many parasites can be missed.
This study was undertaken to evaluate the Sugar Floatation Technique (SFT) for detection of
gastrointestinal parasites infection as diagnostic test as well as for epidemiological study of H.nana
among displaced children in Abuzied area. Results of stool examination for H.nana and other parasite
species were presented together with egg count to determine intensity of infection. Clinical
manifestations were recorded using questionnaires that included presence of symptoms and their
severity. Intra-family transmission was also investigated by examining family members of infected
children. Attempts were also made to identify the sources for H.nana infection. The role of rodents and
insects in H.nana transmission was also investigated. In one part of the study, treatment was given with
the purpose of comparing efficacy of praziquantel and niclosamide.
Evaluation of SFT was made by comparison with other concentration methods and with direct stool
examination techniques. Results obtained revealed significant differences in favour of SFT.
The SFT detected 11(35%) positive samples out of 31 declared negative with DWM at Khartoum
Pediatric Hospital. Eight (24%) were found positive by the SDS (table1). High level of agreement
between the two tests was found. Parasite species recovered by SFT included H.nana (54%),
Table (60) The prevalence of H.nana in children with other symptoms
256 732 98825.9% 74.1% 100.0%
46.6% 59.0% 55.2%
110 247 35730.8% 69.2% 100.0%
20.0% 19.9% 20.0%
17 36 5332.1% 67.9% 100.0%
3.1% 2.9% 3.0%
60 82 14242.3% 57.7% 100.0%
10.9% 6.6% 7.9%
14 19 3342.4% 57.6% 100.0%
2.6% 1.5% 1.8%
19 11 3063.3% 36.7% 100.0%
3.5% .9% 1.7%
58 76 13443.3% 56.7% 100.0%
10.6% 6.1% 7.5%
15 37 5228.8% 71.2% 100.0%
2.7% 3.0% 2.9%
549 1240 178930.7% 69.3% 100.0%
100.0% 100.0% 100.0%
Count% within others% within microscopicexamination of preservedspecimen H.nanaCount% within others% within microscopicexamination of preservedspecimen H.nanaCount% within others% within microscopicexamination of preservedspecimen H.nanaCount% within others% within microscopicexamination of preservedspecimen H.nanaCount% within others% within microscopicexamination of preservedspecimen H.nanaCount% within others% within microscopicexamination of preservedspecimen H.nanaCount% within others% within microscopicexamination of preservedspecimen H.nanaCount% within others% within microscopicexamination of preservedspecimen H.nanaCount% within others% within microscopicexamination of preservedspecimen H.nana
no
headache
nausea
Headache &nausea
Dizziness
Nausea and dizziness
Headache +nauseadizziness
headache dizziness
others
Total
yes no
microscopic examination of
preserved specimen H.nana
Total
182
Annex I Stool Examination Report Form
PATAINTS: -Name -Age
-Male/ Female -Mother educational level
-Address/Area -Father educational level
-Number -Job
- Income /year
LIVING CONSTRATION
-House construction
-Main food stuff
-Sources of drinking water
-Place of defecation
-Hand washing after defecation
-Self cleaning after defecation
-Means of hands washing after defecation
-Playing on the soil
COMPLAINS OF THE FOLLOWING DISOREERS DURING THE LAST SIX MONTHS: -Abdominal pain
-Diarrhea
-Vomiting
-Fever
-Anal irritation
-Others
DID YOU VIST HOSPTALS DURING THIS PERIOD? Yes No IF YES WHAT ARE THE DISORDERS?
183
DID YOU OBSERVED CHANGES IN THE STOOL? Yes No IF YES, WHAT WERE THESE CHANGES? APPEARANCE OF STOOL COLLECTED -Hard dry Yes……………. No…………… -Liquid Yes……………. No……………. -Soft Yes……………. No……………. -Firm Well Yes……………. No……………. - Semi liquid Yes…………….. No…………….. MICROSCOPIC EXAMINATION -Entamoeba histolytic Trophozoite Yes………….. No…………… - Entamoeba histolytic Cyst Yes…………… No……………. -Giardia Lamblia Trophozoite Yes………….. No……………. -Giardia Lamblia Cyst Yes………….. No……………. -Entrobius Vermicularis egg Yes…………. No…………….. -T.Trichiura egg Yes………… No……………… -S.mansoni egg Yes………… No……………… -Taenia Saginata (segm/eggs) Yes…………. No……………… -Hookworms eggs Yes………….. No……………… -Ascaries Lumbricoides (worm/ eggs) Yes…………. No……………… -Strongloides sterocoralis Yes…………. No……………… -H.nana Yes…………. No………………
