M.aziz master thesis 2014

1
Study of the efficacy of Nitazoxanide, Myrrh Total Oil and
Mirazid in comparison with Praziquantel in experimental
Schistosomiasis mansoni
Thesis
Submitted to the Medical Research Institute
University of Alexandria
In Partial Fulfillments of the
Requirements for the degree of
Master of Science
In
Applied & Molecular Parasitology
By
Mohammad Aziz Nawar Al-Kazzaz
Bachelor of Veterinary Medical Sciences
Faculty of Veterinary Medicine, University of Cairo, 1997
2014

2
Study of the efficacy of Nitazoxanide, Myrrh Total Oil and Mirazid in
comparison with Praziquantel in experimental Schistosomiasis mansoni
Prepared by
Mohammad Aziz Nawar Al-Kazzaz
Bachelor of Veterinary Medical Sciences
Faculty of Veterinary Medicine, University of Cairo, 1997
For the degree of
Master of Science in Applied & Molecular Parasitology
Examiner’s Committee Approved
Prof. Dr. Mona Hassan El-Sayad
Professor, Department of Parasitology
Medical Research Institute
Prof. Dr. Sanaa Ahmed El-Masry
Professor, Department of Tropical Health
High Institute of Public Health
Prof. Dr. Mostafa Abo El-hoda Mohamed
Assist. Prof. Dr. Hend Ali El-Taweel
Assistant Professor, Department of Parasitology
Date 15 / 10 / 2014

3
SUPERVISORS
Prof. Dr. Mona Hassan El-Sayad
Assist. Prof. Dr. Hend Ali El-Taweel
Assist. Prof. Dr. Sahar Ahmed Abu-Helw

4
‫بسى‬‫هللا‬ًٍ‫انرح‬‫انرحيى‬
‫وتعايل‬ ‫تبارك‬ ‫هللا‬ ‫قال‬:
}‫عهيى‬ ‫عهى‬ ‫ري‬ ‫كم‬ ‫وفىق‬ ‫َشبء‬ ٍ‫ي‬ ‫درجبت‬ ‫َرفع‬{
‫اية‬ ‫يوسف‬ ‫(سورة‬ ‫كرمي‬‫ان‬‫ر‬‫ق‬٦٧)

5
Dedication
TO
The spirit of my father
My mother
My brothers & sisters
My wife

6
Acknowledgement
First of all, I wish to thank God, the most gracious, the most merciful for helping me to
complete this work.
I would like to express my sincere thanks to Professor Dr. Mona Hassan El-Sayad,
professor of Parasitology, Medical Research Institute (MRI), University of Alexandria for
her precious advice, valuable guidance and great help to complete this work.
My deepest gratitude to Dr.Hend Ali El-Taweel, assistant professor of Parasitology,
Medical Research Institute, University of Alexandria for her keen supervision , constructive
guidance and unlimited cooperation to complete this work.
Many thanks to Dr.Sahar Ahmed Abou-Helw , assistant professor of Parasitology,
Medical Research Institute ,University of Alexandria for her continuous advice and
encouragement throughout this work.
I want to express my great sympathy to Dr.Mostafa Yakoot, the Medical Director of
Pharco Corporation for his support in the practical part of this work.
Also I want to express my great appreciation to all members in the Department of
Parasitology, Medical Research Institute,University of Alexandria who paid a lot of efforts in
the practical part of this work.
Last but not least, I warmly thank with sincere gratitude my family for their endless
support, care and continuous encouragement throughout this work.

7
LIST OF CONTENTS
Chapter Page
LIST OF CONTENTS……………………………………….…………..........................................I
LIST OF TABLES………………………………………………………………………………....II
LIST OF FIGURES………………...………………………………..……………………...….....III
LIST OF ABBREVIATIONS……………….……………………………………………...........VII
I. INTRODUCTION……………………………………………………......................….…....1
II. AIM OF THE WORK………………………………………...…………...............................19
III. MATERIALS AND METHODS……………………………...………....……………….....20
IV. RESULTS…………………………………………………..……………………………......32
V. DISCUSSION………………………………………………….……………………..….......71
VI. SUMMARY AND CONCLUSION………………………………………………..……......89
VII. RECOMMENDATIONS…………………………………....................................................93
VIII. REFERENCES……………………………………………….………..................................94
IX. PROTOCOL
X. ARABIC SUMMARY

8
LIST OF TABLES
Table (I) Egg counts in the stool of S. mansoni-infected mice under different
treatments compared to non-treated mice.
33
Table (II) Egg counts in stool of different groups of S. mansoni-infected mice under
different treatments compared to non-treated infected mice.
34
Table (III) Worm burden in S.mansoni-infected mice treated and non-treated groups
by time (weeks).
35
Table (IV) Percentage of change in male and female worm distribution in different
S.mansoni-treated mice groups in different periods of follow up.
37
Table (V) Body length of S.mansoni worms recovered from different treatments
compared to non-treated mice at different follow up periods.
39
Table (VI) The tissue egg count in the liver and intestine of S. mansoni-infected
mice under different treatments at different periods of follow up.
41
Table (VII) The oogram pattern (percentage egg developmental stages) in the
intestine of S. mansoni-infected mice under different treatments in
different follow up periods.
44
Table (VIII) Erythrocytes and their related red blood cell indices in S. mansoni-
infected mice under different treatments at different follow up periods.
53
Table (IX) Total and Differential Leucocytic Counts in S. mansoni-infected mice
under different treatments at different follow up periods.
59
Table (X) Platelet counts in S.mansoni-infected mice under different treatments at
63
Table (XI) Liver function tests in S. mansoni-infected mice treated with different
drugs at different times.
65
Table (XII) Kidney function tests in S. mansoni-infected mice under different
treatments at different follow up periods.
68
Table (XIII) Blood Acetylcholinesterase level in S.mansoni-infected mice under
different treatments at different periods of follow up.
70

9
LIST OF FIGURES
Figure.1 Structural formula of praziquantel 11
Figure.2 Structural formula of Nitazoxanide 16
Figure.3 Steps of mice infection with S.mansoni cercariae 22
Figure.4 Perfusion pump machine 25
Figure.5 Mice perfusion 25
Figure.6 Measurement of female S. mansoni body length under dissecting
microscope with ordinary ruller.
26
Figure.7 Egg developmental stages 28
Figure.8 Blood collection from a mouse 30
Figure.9 Egg counts in stool of different groups of S. mansoni-infected mice
under different treatments compared to non-treated infected mice.
33
Figure.10 Percentage faecal egg count reduction in different groups of S. mansoni-
infected mice under different treatments at different periods of follow
up.
34
Figure.11 Percentage reduction in the mean total worm burden in different groups
under different treatments at different periods of follow up.
36
Figure.12 Percentage reductions in female worm burden in S.mansoni-infected
mice under different treatments at 1, 2 and 4 WPT.
38
Figure.13 Percentage reductions in male worm burden in S.mansoni-infected mice
under different treatments at 1, 2 and 4 WPT.
38
Figure.14 Percentage reductions of the body length of male S. mansoni worms
recovered from different treated groups at 1, 2 and 4 WPT.
40
Figure.15 Percentage reduction of the body length of female S.mansoni worms
recovered from different treated groups at 1, 2 and 4 WPT.
40
Figure.16 Percentage reduction in the mean hepatic egg counts in S. mansoni-
infected mice under different treatments at 1, 2 and 4 WPT.
42
Figure.17 Percentage reduction in the mean intestinal egg counts in S.mansoni-
infected mice under different treatments at 1, 2 and 4 WPT.
42
Figure.18 Percentage egg developmental changes in S.mansoni-infected mice
45
Figure.19 Scanning electron micrographs of S.mansoni worms recovered from 47

10
infected non-treated mice showing normal tegument of male (A) and
female worms (B). Normal ventral sucker of male worms (C) and oral
sucker of female worms (D).The inner surface of the gynecophoric
canal of male (E) worms.
Figure.20 Effect of Praziquantel on the dorsal surface of female schistosoma
worms (F) and the male worms (G) recovered at 2 WPT.
48
Figure.21 Effect of Mirazid on dorsal aspects of the tegument of female (H), male
(I) S.mansoni worms and ventral sucker of male worms (J) recovered 2
WPT.
49
Figure.22 Scanning electron micrographs of S.mansoni worms recovered from
NTZ-treated mice showing normal tegument of female worms (K), the
tegument of male worms (L) ,the oral sucker of male worms (M), the
worm couple (N) and the gynecophoric canal of the male (O).
50
Figure.23 Scanning electron micrographs of the dorsal surface (P), oral and ventral
suckers (Q) of male S.msnsoni worms recovered from MTO-treated
mice at 2 WPT.
51
Figure.24 Mean RBCs counts in S.mansoni-infected mice under different
54
Figure.25 Mean Haemoglobin levels in the blood of S. mansoni-infected mice
54
Figure.26 Mean Packed cell volumes in the blood of S.mansoni-infected mice
55
Figure.27 Mean MCV of the RBCs in S.mansoni-infected mice under different
55
Figure.28 Mean MCH in S. mansoni-infected mice under different treatments at
56
Figure.29 Mean MCHC in S.mansoni-infected mice under different treatments at
56
Figure.30 Mean total leucocytic counts (TLC) in S.mansoni-infected mice under
different treatments at different follow up periods.
60
Figure.31 Mean Lymphocyte counts in S.mansoni-infected mice under different
60
Figure.32 Mean Neutrophils counts in S.mansoni-infected mice under different 61

11
Figure.33 . Mean Esinophils counts in S.mansoni-infected mice under different
61
Figure.34 Mean Monocytes counts in S.mansoni-infected mice under different
62
Figure.35 Mean Basophils counts in S.mansoni-infected mice under different
62
Figure.36 Mean Platelet counts in S.mansoni-infected mice under different
63
Figure.37 Liver functions tests {ALT (A), AST (B), ALP(C)} activity in
S.mansoni-infected mice under different treatments at different follow
up periods.
66
Figure.38 Kidney functions {Blood urea (A) and Serum creatinine (B)} in
S.mansoni-infected mice under different treatments at different follow
up periods.
69
Figure.39 Mean blood acetylcholinesterase levels in S.mansoni-infected mice
70

12
LIST OF ABBREVIATIONS
AChE:
ALP:
ALT:
AST:
CBC:
CNS:
DALYs:
DLC:
ECG:
EDA:
ELISA:
EMR:
EPG:
FDA:
GMEC:
HB:
HCT:
HCV:
IC:
IHA:
KOH:
LC:
LD50:
MCH:
MCHC:
MCV:
MEO:
MOHP:
MRI:
MTO:
MVO:
Acetylcholinesterase Activity
Alkaline Phosphatase
Alanine Aminotransaminase
Aspartate Aminotransaminase
Complete Blood Count
Central Nervous System
Disability Adjusted Life Years
Differential Leucocytic Count
Electrocardiography
Egyptian Drug Authority
Enzyme-Linked Immunosorbent-Assay
Eastern Mediterranean Region
Egg per gram
Food And Drug Administration
Geometric Mean Egg Count
Haemoglobin
Haematocrit Value
Hepatitis C Virus
Inhibition Concentration
Indirect Haemagglutination
Potassium Hydroxide
Lethal Concentration
Lethal Dose 50
Mean Corpuscular Haemoglobin
Mean Corpuscular Haemoglobin Concentration
Mean Corpuscular Volume
Myrrh Essential Oil
Ministry of Health and Population
Myrrh Total Oil
Myrrh Volatile Oil

13
MZD:
NTZ:
PCR:
PPM:
PT:
PZQ:
RBCs:
S/C :
SBSC:
SCE
SD:
SEAs:
SEM:
TB:
TBRI:
TGR:
US:
WHO:
WPI :
WPT:
µl:
μg :
Mirazid
Nitazoxanide
Polymerase Chain Reaction
Part Per Million
Post-Treatment
Praziquantel
Red Blood Cells
Subcutaneous
Schistosome Biologic Supply Center
Serum cholinesterase
Standard Deviation
Soluble Egg Antigens
Scanning Electron Microscopy
Tuberculosis
Theodore Bilharz Research Institute
Thioredoxin-Glutathione Reductase
United States
World Health Organization
Weeks Post-Infection
Weeks Post-Treatment
Microliter
Microgram

15
INTRODUCTION
Schistosomiasis is a parasitic disease caused by the digenetic trematodes of the genus
Schistosoma (commonly known as blood flukes) (1)
. The disease is one of ten tropical diseases
especially targeted for prevention and control by the special programs for research and training in
tropical diseases of the United Nations development program, the World Bank and the World
Health Organization (WHO). It also represents one of the major communicable diseases of public
health and socio-economic importance in the Eastern Mediterranean Region (EMR) (2)
.
Schistosomiasis ranked second only to malaria and is the most important parasitic disease in terms
of prevalence, morbidity and mortality rates especially in rural areas of developing countries (3)
.
TAXONOMY OF SCHISTOSOMES:
Kingdom: Animalia
Phylum: Platyhelminthes
Class: Trematoda
Subclass: Digenea
Order: Strigeidida
Family: Schistosomatidae
Subfamily: Schistosomatinae
Genus: Schistosoma {Schisto= cleft & soma = body} (Weinland, 1858) (4)
.
Genus Schistosoma: There are 23 identified species of Schistosoma infecting man, mammals and
birds (5)
.
Human Schistosomes :
S. mansoni, S. hematobium and S. japonicum are the most important species from the
medical point of view that can infect humans (6)
. S. mansoni is found in Africa, South America,
Caribbean and Middle-East. Fresh water snails of the Biomphalaria are an important intermediate
host for this trematode. Among final hosts, humans are most important. S. haematobium,
commonly referred to as the bladder fluke, originally found in Africa, the Near East, and the
Mediterranean basin, Freshwater snails of the Bulinus are an important intermediate host for this
parasite. S. japonicum is found widely spread in Eastern Asia and the Southwestern Pacific
region. Fresh water snails of the Oncomelania are an important intermediate host for S.
japonicum. S. mekongi and S. intercalatum are considered human blood flukes of minor
importance from the medical point of view (7)
.

16
LIFE CYCLE AND BIOLOGY OF SCHISTOSOMA MANSONI:
Schistosomes are characterized by a complex life cycle involving two phases; (1) Sexual
phase in which sexual reproduction by adult worms occur in humans (definitive host), (2)
Asexual phase in specific aquatic snails (intermediate host, Biomphalaria species). Schistosomes
develop through successive stages: egg, miracidium, sporocyst, cercaria, schistosomula and adult.
S. mansoni eggs are oval with lateral spine. Each fertilized female worm releases many eggs each
day. The eggs of S. mansoni are released singly and may remain alive up to 3 weeks after
oviposition. It contains a single miracidium. Hatching and survival of the miracidia are dependent
on fresh water contact at a temperature between 200
C-300
C. In optimal conditions; miracidia will
survive for 5-6 hours (7)
.
When S. mansoni eggs reach fresh water, usually with faeces, they hatch and release tiny
miracidia. Although miracidia of schistosome do not have eye spots, they apparently have
photoreceptors and they are positively phototropic, they also display negative geotaxis and
possess chemotactic factors. At water flow rate of about 700 cm/ minute, they are stimulated to
swim more rapidly and change direction much more frequently, thus increasing their chances of
encountering the specific snail (Biomphalaria alexandrina) and attach to its soft part. Lytic
substances secreted from miracidial glands aid penetration. After penetration of a snail, the
miracidia lose their cilia, and become non-motile sac which metamorphoses into two generations
of sporocysts. The latter migrate to the digestive gland of the snail after about two weeks. The
mother sporocyt continues producing daughter sporocysts for up to 6-7 weeks. The daughter
sporocysts migrate to and grow in the hepatic and gonadal tissue of the snail. Sporocysts mature
into hundreds of infective larval forms of the parasite (cercariae) (7)
.
Cercariae start leaving the snail 4 to 6 weeks post-infection. They migrate through the
vascular sinuses and exit from the edge of the snail’s mantle. Cercariae are unisexual, fork-tailed,
free swimming and measure 400-600 µm in length. Cercariae may survive in fresh water up to 48-
72 hours but gradually begin to lose infectivity after 12 hours. Their activity in water alternates
between active movement towards the surface and slow sinking towards the bottom. A snail
infected by one miracidium can shed thousands of cercariae every day for months. Infection
occurs when humans come into contact with fresh water containing cercariae. Cercariae attach
themselves to the skin by their ventral and oral suckers assisted by mucoid secretions from the
postacetabular glands, they penetrate the skin. Following penetration, cercariae transform into
schistosomulae and develop a double-lipid bilayer tegument that helps in protecting the worm
from immune attack. Schistosomula secrete lytic enzymes and migrate through the dermis in
search for a vein, then travel through the blood stream within several days (7, 8)
.

17
The worms migrate along the pulmonary capillaries to enter the left side of the heart and
systemic circulation. Schistosomules are carried with the arterial blood flow through the aorta to
the mesenteric arteries, splanchnic capillaries and portal veins to reach the liver. The
schistosomules transversing the skin and pulmonary capillaries are the parasite stage most
susceptible to immune attack by the host. Only about 40 % of cercariae that penetrate the skin
eventually become viable adult worms. Survival is inversely related to the host-acquired
immunity to schistosomes (7)
. The worms mature within 4-6 weeks in the portal circulation .They
differentiate into male and female worms, mate in the small vasculature of the liver and migrate to
the inferior mesenteric veins of large intestine (draining intestines) against the blood flow.
Oviposition commences 4-7 weeks post infection and female worms produce 100-300 eggs per
day (8)
.
EPIDEMIOLOGY OF SCHISTOSOMIASIS MANSONI:
The epidemiology of schistosome parasites is based on their complex life cycle. The elegant
adaptational skill that allows these organisms to parasitize snails and humans also restricts their
geographic distribution.
Geographic distribution and global burden:
Schistosomiasis transmission has been documented in 78 countries. However those
requiring treatment targeted at most at-risk population groups live in 52 countries (9)
. S.mansoni is
present in 8 Eastern Mediterrean Region (EMR) countries including Egypt, Libya, Sudan, KSA,
Oman, Yemen, Djibouti and Somalia. During the past 20 years, schistosomiasis was eliminated in
Iran, Morocco, Lebanon and Tunisia (2)
.The geographical distribution of the different schistosome
species depends mainly on the ecology of their snail intermediate hosts (7)
. It was revealed that the
global burden of schistosomiasis and its consequences had been underestimated (10)
. This
underestimation of burden is attributed to multiple factors, including the chronic and
asymptomatic nature of most infections, non-specificity of some signs and symptoms, and low
sensitivity of parasitological diagnosis (10,11)
.
Impact of Schistosomiasis on Human Health:
In 2005, the weight of evidence from a meta-analysis of 135 interventional and
observational studies indicated that human schistosomiasis is significantly associated with chronic
symptoms of pain, diarrhea, fatigue, anaemia, impaired growth and exercise intolerance. These
frequently unacknowledged disease outcomes were substantially more prevalent than the
advanced ‘classic’ schistosomiasis-related disease outcomes, such as liver fibrosis, portal
hypertension, hepatosplenomegaly, or urinary tract obstruction. Although the former, more subtle

18
outcomes are less visible in their clinical presentation, they may actually represent the greatest
part of chronic disease burden associated with schistosomiasis (10,11)
.
Schistosomiasis negatively impacts on school performance in children due to long-term
developmental and cognitive effects as well as social and economic developments in heavily
affected areas (3, 8)
.
The concept of Disability Adjusted Life Years (DALYs) was introduced by Murray and
Lopez (1996) "to assess and refine estimates of the global burden of diseases". DALY is a
population health metric that combines the years lost from premature death and the years of life
lived with disability.It can be thought of as one lost year of healthy life. This index is calculated
from disease-specific prevalence, mortality, and disability weights of a certain disease (12)
. The
report of the WHO Expert Committee (2002) (13)
on the prevention and control of schistosomiasis
estimated 1.7 million DALYs were present. The burden of disease assigned to schistosomiasis-
associated disability was estimated to be 0.5%. However, a subsequent meta-analysis re-assessing
the chronic disease with a more robust measure of morbidity determined a schistosomiasis-
associated disability of about 2-15%(11)
.
There is a consensus that schistosomiasis-specific mortality occurs only in a small
percentage of individuals who develop a chronic disease. However, on revising the global burden
of schistosomiasis ; there were about 280,000 deaths per year was estimated in sub-Saharan
Africa alone where 150,000 per year due to non-functioning kidney from S.hematobium and
130,000 per year due to hematemesis from S.mansoni were detected(10)
.
Prevalence of Schistosomiasis and Human Host Factors:
Although schistosomiasis is highly prevalent, the associated morbidity is often variable
according to :
1-Age: No age is exempted from bilharziasis but higher disease rates among age groups from 15-
70 years(22.7%) followed by children in those between 5-14 years old(19.6%) (14)
.
2-Occupation: Schistosomiasis is considered an occupational disease related to water contact of
farmers or fishermen and also an environmental hazard. Agricultural workers and their families in
endemic areas that have continuous exposure to schistosoma-infested water through farming
,washing, bathing, and water recreation have great difficulty and perhaps no practical means of
remaining free of recurrent infection(8)
.
3-Socioeconomic level: Watts (2005) (15)
reported that the majority of schistosomiasis cases are
prevalent among poor people in Sub-Saharan Africa who lack access to health services, safe
water, sanitation, and education. Furthermore, the disease helps keep them poor by lessening their
ability to work, learn, and contribute to their communities. In Egypt ,the same findings were

19
noticed for the first time by Farooq et al (1966) (16)
and also by EL-Koby et al.,(2000)(17)
as lower
socioeconomic status among those who live in rural areas, who are more likely to be employed in
agriculture and have less convenient access to medical care (and treatment for schistosomiasis).
4-Sex: Males are more infected than females with schistosomiasis .This may be due to
occupational exposure to infected water canals in agriculture or by swimming (14, 17)
.
5-Education: It has been found to impact health-seeking behaviour, which may have an effect
on prevalence of infection (18)
. It also provides the impetus behind the success of deworming
programmes, preventing the contamination of the environment, and hence transmission (19)
.
Health education implies a long-term commitment and should ideally be integrated in the general
education system (20)
.
6-Hygienic measures or sanitation: The fundamental reason for the transmission of
schistosomiasis is the low level of sanitation in endemic areas, with the result that fecal material
containing viable schistosome eggs reach natural water bodies infested with fresh water snails
susceptible to infection (21)
. So provision of clean water supplies reduces exposure to cercariae and
sanitary disposal of excreta reduces the succession of the life cycle by supplying indoor water and
toilets. Communities with improved living standards were more likely to have satisfactory results
in eradication of schistosomiasis (7)
.
Relation of schistosomiasis to the environment:
Perennial irrigation is the modern system for irrigation in Egypt which ensures a water
supply all the year round, an abundant and an unbroken succession of crops but conversion from
basin to perennial irrigation resulted in an increase in the prevelance and intensity of schistosomal
infection due to flushing of snails. Both environmental changes that result from the development
of water resources and the growth or migration of populations can facilitate the spread of
schistosomiasis .The presence of Aswan high Dam in Egypt has led to the virtual elimination of S.
haematobium from the Nile Delta but has brought about the establishment of S. mansoni in upper-
Egypt (17)
.
Reservoir Hosts of Schistosomiasis mansoni:
S.mansoni infections have been found in rodents, baboons and insectivores in Africa and
South America which may constitute a health hazard as they may act as carriers after elimination
of human infection (7)
.
Intermediate Hosts of Schistosoma mansoni:
Endemic human schistosomiasis is ecologically most dependent on the presence of the snail
intermediate host and the deposition of human and reservoir host excreta into warm fresh water
habitat. Biomphalaria snails belong to the family Planorbidae, class Gastropoda. In Africa and the

20
Middle East; are divided into four species groups: B. alexandrina, B. pfeifferi, B. choanomphala,
and B.sudanica which act as the intermediate host for S.mansoni (7)
. B. alexandrina has
historically been implicated in the transmission of S. mansoni in Egypt (21)
. These fresh water
snails are characterized by their disk or lens-shaped shells, non-operculated, hermaphrodite,
vascularized mantle and haemocael. These snails live in lightly shaded, slow-flowing (15
m/minutes), shallow (less than 2m) waters (7)
.
Current Status of Schistosomiasis in Egypt:
According to the report of Schistosomiasis Working Group (2005) (22)
on schistosomiasis in
Egypt, it has been reported that S. hematobium is prevalent in the Upper Egypt governorates while
S.mansoni is prevalent in the Nile Delta governorates; By the end of 2004, both infections had
been greatly reduced to rates below 2 .WHO report (2007)(2)
cleared that the prevalence of S.
hematobium decreased to 1.2 % and S. mansoni to 1.5 % in Egypt. Fenwick (2011) reported
much decline in the prevalence for both S.mansoni and S. hematobium allover Egypt to less than
0.5 % in the year 2010(23)
. With the concept that schistosomiasis is present in high prevalence
rates in hot spots ; Khalil (2013)(24)
found among 100 school children in a village in Kafr El-
Sheikh Governorate; that the overall prevalence was 16% by percoll or 12 % by kato-katz
technique. Taman et al., (2014) (25)
found 26.6% overall prevalence rate among fishermen in Al-
Manzala lake.
Prevalence of Schistosomiasis mansoni and its Status in Alexandria, Egypt:
In the study of Abou-Basha et al., (2000) (14)
in Abis I village, the overall prevalence rate of
S.mansoni was 19.1 %. Hussein et al.,(2000)(26)
found that the prevalence of S. mansoni infection
in Abis 7 and 8 was 24.2 %, 37.8 % respectively and concluded that drinking water supply,
sanitary sewage disposal and proper disposal of animal wastes are still deficient in some houses of
the two villages.The prevalence of S.mansoni in a surveyed community (El-prince Village, EL-
Montazaa district, Alexandria Governorate) was found to be 15.4 % in the year 2002 while it was
78.4 % in 1985 and decreased to 24 % in the following year after chemotherapy with
praziquantel(27)
. Zaki et al., (2003) carried out a study in Abis 4 villages where the prevalence of
S.mansoni was 20.5 % being lower among females and children below 5 years. S.haematobium
was absent from urine samples (28)
.
Allam et al., (2009) (29)
examined stool samples of school-children in Abis 4 and Abis 8
villages and reported that the overall prevalence of S.mansoni in the 2 villages was 5.72%.The
Health Administrative Authority of the Egyptian Ministry of Health and population (MOHP) in
Alexandria Governorate performed a survey on 3782 school-children in the period from March to
April 2009 in different districts of the governorate by examining random stool samples, the study

21
revealed that overall prevalence of S.mansoni infection in Alexandria governorate was 1.3 % in
spite of the very low or nill infection rate in Biomphalaria snails in this period of the year .Hot
spots in some rural areas of Alexandria are present ,with prevalence of schistosomiasis mansoni
ranged from 1.5-7%. Even after mass treatment with PZQ, e.g.in Abis 8 villages, the prevalence
was still about 3.5 % (30)
. Hassan (2013) surveyed 420 children in Abis 8 village and reported that
the overall prevalence of S. mansoni by the kato-katz was 2.13% with GMEC of 16 epg among
infected persons with more light infection and none of them showed heavy intensity, while by
serologic tests ; prevalence was 5.7% by IHA and 21.9% by ELISA test (31)
.
ANIMAL MODELS OF SCHISTOSOMIASIS MANSONI:
Experimental S.mansoni infection of laboratory animals has frequently been used to study
the anatomical, pathological and physiological features of the infection in humans as well as for
the study of immunity and chemotherapy (32)
. The complex nature of the schistosome parasite and
its interaction with the mammalian host necessitate the continued use of live intact animal models
in schistosomiasis research (33)
. Schistosome infections in experimental animals are less complex,
or at least more readily studied, than infections in humans (34)
.
A variety of animal models have been used in schistosomiasis research as mice, rat,
hamster, rabbit, chimpanzee and baboons. These hosts may be classified into two types according
to their susceptibility to schistosomal infection into permissive or non- permissive. Permissive
hosts are those animal hosts in which schistosome parasites can reach maturity as mice and
hamsters which are among the most susceptible host species while rats are known to be non-
permissive hosts (35)
.
Although the rat is a non-permissive host for schistosomiasis, it has been extensively
studied as it provides an immunological model for successful parasite immune-mediated rejection
studies (36)
. Schistosomes do not reach sexual maturity in the rat, being spontaneously eliminated
in the third week following infection. This schistosome attrition is immune-mediated and the
antibody dependent cell-mediated cytotoxicity mechanism plays a major role (37)
.The laboratory
rat (Rattus norvegicus) is considered a semipermissive host in that the majority of worms are
removed before reaching maturity in the portal tract in a self-cure around day 28. Although the
schistosome larvae are faster in this host than in the mouse, only 25% to 30% reach the liver, and
IgE has been directly implicated in this phenomenon (37, 38)
. On the other hand, the black rat
(Rattus rattus), which is a natural host for S. mansoni, is considered a fully permissive host (39)
.
Cioli et al. (1977) studied the survival, growth, and egg laying capacity of S.mansoni worms
surgically transplanted from mice into rats or from rats into hamsters. They found that in the rat,

22
worms were stunted, localized in the liver, and laying nonfertile eggs in small numbers. When
transferred to the hamster, they increased in size approaching normal hamster-grown worms
within 3 weeks following transplantation, were localized in the mesenteric veins, and produced
large numbers of eggs. Conversely, when adult mouse worms were injected into rats, they
regressed in size, remained in the liver, and produced small numbers of incompletely developed
eggs (40)
.
The baboon is the most frequently used non-human primate in schistosomiasis research
because of a multiplicity of qualities that make them more relevant models than rodents (41)
.
Baboons maintain natural infections in the wild (7)
and are highly susceptible to experimental
infections (42)
. They are a good model for vaccine efficacy studies but constraints limiting the use
of baboons in schistosomiasis research include the high costs involved in trapping and
maintaining monkeys in captivity. There may also be variation in data obtained from wild-caught
baboons due to their heterogeneous genetic background. In addition, some specific immunological
reagents suitable for baboon work may not be currently available (33)
.
Murine schistosomiasis has been the most studied experimental model in many aspects of
the disease as the progress of schistosomiasis in mice is approximately similar to that in humans.
Mice have tended to be the animals of choice because of their easy availability, high fertility and
susceptibility to experimental infection (43)
. Female mice are more susceptible to infection with
S.mansoni cercariae with higher mortality rate (80%) than male as fewer worms develop in male
than in female when exposed to the same number of cercariae indicating that schistosomula are
more successful in developing into adult worms in female mice (43,44)
.
PATHOLOGICAL ASPECTS OF SCHISTOSOMIASIS MANSONI :
The main immunopathlogy of the disease is the granulomatous inflammatory and
fibrosing reaction against tissue-trapped parasite eggs in the liver and intestine or other tissues (45)
.
Granuloma formation is a manifestation of cell-mediated, delayed-type hypersensitivity to soluble
egg antigens (SEAs) released by eggs that peak at the eighth week post-infection (46)
. Granuloma
formation is beneficial for the host because it blocks the hepatotoxic effects of the antigens
released from parasite eggs. However, this process may lead to fibrosis with excessive
accumulation of collagen and other extracellular matrix proteins in the periportal space (46, 47)
. Egg
granulomas activate antigen-specific CD4+T-helper cells, i.e.,Th-1 and Th-2, inducing the release
of specific immunomodulating antifibrogenic and fibrogenic cytokines (46, 48)
.
Acute pathology: Acute schistosomiasis occurs in immunologically naive, previously uninfected
people, such as immigrants. It is a toxemic disease characterized by hyper-reactivity to

23
schistosome worm and egg antigens. It is usually seen as an acute febrile illness three to four
weeks after exposure coincident with the oviposition onset. The intestinal mucosa becomes
edematous and hyperemic with small hemorrhages, early granulomas as well as shallow ulcers.
The anatomic features of the acute disease in humans include a massive dissemination of
granulomas around the eggs, especially in the liver, lung, pancreas and lymph nodes (49)
.
Chronic pathology: Infection with S. mansoni results in a relatively tolerable chronic disease
(most chronically infected individuals have few or no symptoms), however, 5-10% of patients
suffer from a severe form that leads to severe hepatic fibrosis, portal hypertension, ascites, portal
systemic shunting, gastrointestinal haemorrhage and death(45,48)
.Chronic schistosomiasis mainly
affects people born and residing in endemic areas (45)
. In heavy infections, about 50% of the eggs
are trapped in the mucosa and submucosa of the colon, resulting in colonic polyposis with the
formation of pseudotubercles, granulomas and pseudopapillomas. Although chronic liver disease
develops in only 4-8% of individuals with schistosomiasis; hepatic schistosomiasis is one of the
leading causes of liver disease internationally (12)
. The granulomatous inflammation due to the
sustained chronic infection and ongoing immune responses may cause anemias of chronic
inflammation and iron-deficiency, caloric undernutrition, growth stunting (11)
. Several studies
have suggested that chronic S.mansoni infection can increase the susceptibility to and progression
of many diseases (50-53)
. Chronic infection can increase the susceptibility to frequent falciparum
malaria attacks among children. The incidence rate of malaria has been found to be higher in those
with concomitant S. mansoni infection (51)
. On the other hand, there has been found an association
between the progression of active TB among those infected with HIV-1 and co-infected with S.
mansoni (52)
. Similarly, a significant increase has been reported in the progression rate of HCV-
mediated fibrosis in patients co-infected with schistosomiasis (52, 53)
.
Complications: Hepatosplenic complications are the most serious and life-threatening
consequences of schistosomiasis mansoni (54)
. Typically, schistosomiasis mansoni is the most
common cause of portal hypertension worldwide (55)
. Esophageal varices subsequent to portal
hypertension as a consequence of extended periportal fibrosis is the major cause of morbidity and
mortality associated with the disease (13,56)
. In schistosomiasis, fibrosis is restricted to the portal
area, with preservation of the lobular architecture of the liver where the macroscopic appearance
may show large fibrous septa, referred to as the Symmers' clay "pipestem" fibrosis (57)
. In S.
mansoni infection, fibrosis develops over five to fifteen years in comparison to S. japonicum
infection, in which it may progress more rapidly. Besides being developed in a small percentage
of infected people, the incidence of periportal fibrosis has been correlated with the age and gender

24
of the patient as well as the intensity of infection. It is more common among males than females,
and increases with age (58)
.
CHEMOTHERAPY OF SCHISTOSOMIASIS:
General overview:
Schistosomiasis control can be achieved through health education, sanitation, snail control,
immunization and chemotherapy. The chemotherapy of schistosomiasis is considered the most
effective tool for control of schistosomal morbidity in human (59)
. These chemotherapeutic drugs
had been developed and categorized into old drugs and new ones ,The old drugs could be
classified into two groups, Antimonial (Tarter emetic, Fouadin, Astiban, Anthiomaline) and Non-
antimonial compounds (Leucanthone, Hycanthone, Niridazole, Oltipraz)(60)
. The toxicity and
repeated intravenous injections of antimonials were a major limitation for considering them as a
treatment option, especially for mass therapy (61)
. The non-antimonials were abandoned because of
their toxicity to the liver, kidney, heart and carcinogenicity(62)
.
The new antischistosomal drugs include Metrifonate, Oxamniquine and Praziquantel .With
the advent of these drugs, they could be administered in a single oral dose, with good therapeutic
activity and less intense side effects than old antischistosomal drugs, it was possible to initiate
control programs in various endemic areas(61)
. Metrifonate, an organophosphorous drug which
was used firstly as an insecticide in the early 1960s, exhibits activity against S.haematobium only
by inhibition of cholinesterase of the worm (63)
. Oxamniquine is effective only against S. mansoni.
The drug was used on a large scale only in Brazil (64, 65)
. Following a half-century search for an
effective antischistosomal drug, the development of PZQ in the mid-1970s and its wide use since
the 1980s was essential feature for the great reductions in morbidity and mortality due to
schistosomiasis(63,65)
. A lot of novel therapeutic approaches under research performed to discover
new schistosomicidal agents either chemically designed (e.g., praziquantel derivatives) or
naturally (e.g., artemisinin and myrrh derivatives) (65-67)
.
The Current Antischistosomal Therapy of Schistosomiasis Mansoni:
Praziquantel is available all-over the world since 1980 in the form of 600 mg tablets (65)
and
Mirazid is present only in Egypt since 2002 as an alternative to PZQ in the form soft gelatin
capsules containing 300 mg oleoresin extract of Commiphora molmol or myrrh .

25
1-Praziquantel (PZQ):
Figure.1. Structural formula of praziquantel (C19H24N2O2) (63)
.
The antiparasitic activity of PZQ was observed in the early 1970s at the laboratories of Bayer and
E.Merck, Germany, when a large series of pyrazino-isoquinoline compounds were synthesized as
potential tranquilizers (68)
.
Antischistosomal Properties of PZQ:
In experimental animals, the therapeutic dose of PZQ depends mainly on the host species.
The dose ranges from 200 to 1000 mg/kg body weight for mice and from 100 to 500 mg/kg body
weight for hamsters (69-71)
. Experimental studies have shown that the activity of PZQ is stage-
dependent. Immature (2-4 weeks old) worms are less susceptible to PZQ than larval stage (1-2
weeks old) or adult (5 weeks old or older) worms. Hence, doses of drug that are curative against
larval or mature adult infections are sub-curative against developing worms (72)
. In man, several
regimens of PZQ treatment have been reported for the different species of schistosomes. The
standard dose of PZQ safely used for mass treatment leading to a decrease in the prevalence of
schistosomiasis mansoni is a single oral dose of 40 mg/kg body weight of PZQ (73)
. Higher doses
(60 mg/kg) (74,75)
but without significant efficacy advantage over the standard dose (75)
.
By 1989, the distribution of PZQ doses, free of charge, to all diagnosed schistosomiasis
cases was implemented through different health facilities including the network of rural health
units. In 2007, the MOHP has decided to move the control programme forward to achieve
elimination of schistosomiasis from Egypt. To accomplish the goal of elimination , the programme
plans to implement several rounds effective mass chemotherapy (1-2 rounds/ year) in '' hot spot"
areas using PZQ (2)
.
Mechanism of action of PZQ as anti-schistosomal drug: Despite the high success of PZQ in
treatment of schistosomiasis and reduction of its prevalence all-over the world, the mechanism of
action of PZQ is not known precisely and remains unresolved three decades following its
introduction.The detailed molecular mechanism of action has not been elucidated (72)
.A number of
researchers have been studied the mechanism of schistosomicidal action of PZQ (65,73,76-80)
, some

26
observations were noticed; it may induce violent muscle contraction that is linked to calcium
influx and results in shortening of the worm(76)
. But Pica-Mattoccia et al., (2008) (77)
observed that
“calcium accumulation by itself, at least as measured by whole parasites maintained in vitro.The
drug appears to damage the tegumental membrane disrupting the active immune evasion and
exposing surface antigens that were previously masked. This exposes the worm to the host
humoral immune attack which leads to worm death by host immune-mediated mechanisms. It has
also been suggested that PZQ exerts its effect by reducing schistosomal glutathione
concentrations (78)
. PZQ may bind to schistosomal Actin leading to disruption of the tegumentof
the worm (79)
or inhibiting adenosine receptors uptake (80)
. A number of metabolic alterations have
been observed in schistosomes exposed to PZQ; glucose uptake, lactate excretion and glycogen
content are all decreased (78)
.
Advantages of praziquantel in treatment of schistosomiasis:
PZQ is characterized by high efficacy, excellent tolerability, few and transient side effects,
simple administration, and competitive cost. The drug is equally suited for individual or large
scale treatment (65)
. So PZQ deserves to be included in the WHO model list of essential drugs (81)
.
Drawbacks of praziquantel in treatment of schistosomiasis:
1-Schistosomal Resistance: Even though PZQ efficacy is generally high, reported cure rates are
variable ranging from 60 to 95% (82)
. There are increasing concerns about the development of
resistance to the drug, but most published discussions of this topic conclude that convincing
evidence for the clinically relevant emergence of PZQ resistance in the field is still lacking (83-92)
.
2-PZQ is not used for prophylaxis: as PZQ is not active against immature stages of schistosomes
(schistosomula) (93)
.
3-PZQ is not ovicidal: it was reported that PZQ has no ovicidal properties (63)
.
Pharmacovigilance of praziquantel:
Abdominal discomfort, diarrhea, malaise, headache and dizziness are common side effects
of PZQ observed in a relatively large percentage of patient (30-60%), but also these are usually
mild and transient disappearing within 24 h (92,94)
. Some recipients of PZQ manifest allergic
symptoms with fever, rash, pruritis and eosinophilia in response to released worm antigens (95)
.
2-Mirazid (MZD):
Mirazid is a pharmaceutical natural preparation introduced to the Egyptian market by
Pharco pharmaceuticals (Alexandria, Egypt). The Egyptian drug authority (EDA) of MOHP has
registered this product for treatment of schistosomiasis (Reg.No.21655/2002).

27
Antischistosomal Properties of Mirazid:
MZD has been investigated, both experimentally and clinically against schistosomiasis with
controversy regarding its efficacy (96)
. Regarding the effect of MZD in vitro on S. mansoni adult
worms, Hassan et al., (2003) exposed the worms to various concentration of MZD from 100-400
μg/ml. It elicited maximal somatic muscle contraction at the highest concentration (400μg/ml) by
muscle tension method (97)
. Also Sharaf (2004) (98)
and Bakr et al., (2007) (99)
showed strong lethal
effect of MZD at both concentrations (100 and 200μg/ml) after 24 hrs of exposure of S. mansoni
adult males .Karamustafa et al., (2011) (100)
showed that MZD had antischistosomal activity
against S.mansoni larvae in vitro (IC50=7.18-32.69 µg/ml).
In experimental animals, Massoud et al., (1996) (101)
and Massoud (1999) (102)
started the
evaluation of myrrh (crude, fractions of oil or resins or combination of oil and resins) on S.
mansoni-infected hamster. They found that a combination of volatile oil and resins in special
formula was more effective as antischistosomal than the crude myrrh or separate volatile oil or
resins. Massoud et al., (2004) (103)
, Hamed and Hetta (2005) (104)
, Bakr et al. (2009)(99)
used MZD
in dose ranged from 250-600mg/kg for 3-5 days in S.mansoni infected-mice; the drug had a
valuable schistosomicidal effect against different maturation stages of S. mansoni worms (the rate
of worm reduction ranged from 81.1%-98.4%). The results of the previous experimental studies
were greatly conflicting with the following disappointing studies as Badria et al.,(2001)(105)
,
Guirguis and Mahmoud (2003) (106)
, Botros et al., (2004) (107)
, Ebeid et al .(2005) (108)
, Emam et
al., (2009) (109)
, El-Gamal et al., (2009) (110)
, Ramzy et al., (2010) (111)
, Abdul-Ghani et al., (2010)
(112)
, Lotfy et al., (2013) (113)
and EL-Malky et al., (2013) (114)
found low worm burden reduction
rate varied from (0% to 75%) either in S.mansoni or S. heamatobium or S. japonicum–treated
mice or hamster with MZD oral doses from 250-500 mg/kg for 2-5 days .
Efficacy of MZD as human anti-schistosomiasis drug was evaluated by Massoud et al.,
(1998) (115)
, Sheir et al. (2001) (116)
, Gaballah et al. (2001) (117)
, Abo-Madyan et al., (2004) (118)
,
Soliman et al.,(2004) (119)
and Massoud et al., (2010) (120)
. They enrolled 365 schistosomiasis-
infected patients (adults or children) treated with MZD in a dose of 10-11.5 mg/kg for 3-6 days.
At 2-3 months post-treatment, the drug effectiveness was assessed either parasitologically (faecal
egg count), clinically, biopsy or sigmoidoscopically. MZD achieved parasitological cure rate
varied from 80.7-100% with non-significant side effects on the liver and kidney functions .On the
other hand, many clinical studies showed that MZD has little or no beneficial activity in treatment
of schistosomiasis; as Botros et al.,(2005) (121)
, Barakat et al.,(2005) (122)
and Osman et al.,(2010)
(123)
enrolled 206 patients (adults or children) orally administered the drug in a dose of 300mg for

28
3 days, 600 mg for 3 days and 6oo mg for 6 days, respectively. MZD resulted in parasitological
cure ranged from 3.7%-15.6% at 4-8 weeks after treatments.
Mechanism of action of MZD as anti-schistosomal:
Although the exact mechanism of the schistosomicidal action of MZD has not been known.
It has been attributed to the permanent musculature loss of worms leading to unpairing of male
and female couples and their shift to the liver where subsequent destruction takes place (97)
, this
may be related to the ability of some constituents to block the inward sodium current in
membranes leading to smooth muscle relaxing action and loss of attachment between male
worms and the inner linning of the blood vessels (117)
or to the increase of intra-parasite calcium
level (98)
. MZD caused destruction, deformity and blunting of spines on the tubercles of male
worm tegument, including the lateral margin of the gynecophoric canal (99)
. It was attributed that
the change in oogram pattern produced by MZD to an early interruption of egg laying capacity in
the intestinal wall or most probably blocking the development of reproductive organs (105)
.
Pharmacovigilance of Mirazid:
MZD possesses high safety margins in human application as it has no significant effects on
liver and kidney functions in healthy volunteers. It can be given for patients with
hepatosplenomegaly as the liver enzymes nearly returned to the normal level 8 weeks after
treatment (115,116)
. MZD has no arrhythmogenic activity as it had no siginificant effect on the ECG
parameters.Side effects reported to MZD administration were transient and mild and occurred in
only 11.8% of the treated cases and in none of the healthy volunteers. The most frequently
reported side effects were giddiness, somnolence, mild fatigue, abdominal pain or discomfort (116)
.
Drug Discovery and Development for Novel Treatments of Schistosomiasis
The fear for possible emergence of drug tolerance or appearance of new resistant strains to
PZQ especially with reinfection and re-treatment makes the search for new antischistosomal drugs
an essential target either from synthetic or natural origins.
A-Synthetic compounds:
1-Praziquantel derivatives and its combinations: Commercially produced PZQ is a racemic
mixture of levo (-) and dextro (+) enantiomers, only the levo enantiomer showed schistosomicidal
activity (124)
. Adoption of an enantioselective method of synthesis should therefore theoretically
provide drug that can be administered at higher dose without any increase in toxicity or adverse
events (125)
. Intense efforts are now directed to have a single drug for such a dreadful infection via
synthesizing derivatives of PZQ (68)
. Many variants of PZQ but were less active than the parent
compound (73)
.Combination of PZQ with other substances has been attempted for the treatment of

29
schistosomiasis mansoni aiming to reduce the PZQ dose, potentiate its schistosomicidal action,
and alleviate side effects (126)
. A lot of compounds either anthelmintics as Albendazole (127)
,
Artesunate (128)
, Artemether (129)
, Oxamniquine(130)
, or non-anthelmintics as Coenzyme-Q10(131)
,
Zinc (132)
, N-Acetyl-L-Cysteine (133)
, DDB (134)
, Dexamethasone (134)
, Pentoxifylline (135)
and
Silymarin (136)
were used.
2-Oxadiazoles (Furoxan derivatives): Oxadiazoles have been found to possess inhibitory
activity against S. mansoni and S. japonicum redox protein thioredoxin-glutathione reductase
(TGR).Oxadiazole-2-oxide surpassed criteria established by the WHO for potential lead
compounds for schistosomiasis in its effectiveness in experimental studies (137,138)
.
3-Cysteine Protease Inhibitors: Abdulla et al., (2007) introduced a novel chemotherapy of
human schistosomiasis through targeting cysteine proteases by phenyl vinyl sulfone or
(K11777).The inhibition of these schistosome specific enzymes resulted in a significant reduction
in parasite burden and pathology (139)
.
4-Trioxaquines: They were initially developed against malaria and exhibit a dual mode of action:
alkylation of heme with its trioxane entity, and stacking with heme due to its aminoquinoline
moiety, thus explaining their potent anti-S.mansoni activity in vitro and in vivo (140)
.
5-Trioxolanes (secondary ozonides): Trioxolanes isomers (OZ-78, OZ-209 and OZ-288) showed
significant schistosomicidal activity in vitro and in vivo against S. mansoni and S. japonicum.
High worm burden reductions (71.7 to 86.5%) were observed after administration of single 200-
mg/kg doses of OZ-78 and OZ-288 to hamsters infected with either juvenile or adult S.mansoni
and 94.2 to 100% in S.japonicum (141)
.
6- Imidazolidines: They had broad biological anti-microbial and anti-fungal activities, were also
used for treatment of schistosomiasis because of their potent in vitro schistosomicidal effects (142)
.
7-Benzimidazole derivatives: Triclabendazole (143)
, Flubendazole (144)
, Albendazole (145)
and
Mebendazole (146)
showed some promising anti-schistosomal activity in vitro and/or in vivo.
8- Thiazoles e.g., Nitazoxanide (NTZ):
Figure. 2. Structural formula of Nitazoxanide (C12H9N3O) (147)

30
Antiparasitic activity of Nitazoxanide:
NTZ was originally discovered in the 1980s at the Pasteur Institute. NTZ is a broad-
spectrum antiparasitic drug with activity against protozoa, nematodes and trematodes.The US
Food and Drug Administration (FDA) approved oral suspension of NTZ at December 2002 for the
treatment of diarrhea caused by Cryptosporidium species and Giardia intestinalis in pediatric
patients 1-11 years of age, and in July 2004, NTZ was approved for treatment of diarrhea caused
by G. intestinalis in adults (148)
. Two reports assessed the antischistosomal activity of NTZ against
S.mansoni in experimentally infected mice with controversy results as Abdel-Rahman et al.,
(1997) (149)
proved that the drug succeeded to reduce 59.91 % of worm load but Abdulla et al.,
(2009)(150)
proved that NTZ failed to affect worm burden .
Mechanism of Action of Nitazoxanide as anti-parasitic: The mechanism of Nitazoxanide’s
activity against helminths is unknown but it interfered with the pyruvate-ferredoxin
oxidoreductase enzyme-dependent electron transfer reaction which is essential to anaerobic
energy metabolism (151)
.
Pharmacovigilance of Nitazoxanide:
NTZ is generally well tolerated, and no significant adverse events have been noted in
human trials. Adverse events have been mild and transient and principally related to the
gastrointestinal tract, such as abdominal pain, diarrhea, and nausea. Adverse events occurring in
11% of more than 2000 patients participating in clinical trials included anorexia, flatulence,
increased appetite, enlarged salivary glands, fever, infection, malaise, elevated creatinine levels,
elevated serum ALT levels, pruritus, sweat, pale yellow sclerae, rhinitis, dizziness, and discolored
urine. In addition, there have been no significant changes in results of electrocardiography, vital
signs, or hematologic, clinical chemistry, or urinalysis parameters in patients treated with NTZ, it
has been well tolerated up to the maximum dose of 4 g when taken with or without food, but the
frequency of gastrointestinal side effects increases significantly with the dose level (152)
.
9-Miscellaneous synthetic drugs: A lot of synthetic compounds were examined experimentally
for anti-schistosomal activity eg; Oxamniquine derivatives (153)
, Ro 15-5458 (154)
,Antox(155)
,
pegylated tartar emetic (156)
, Adenine derivative(157)
, Thiazolo-Derivatives(158)
,Ro-354(159)
, Nano-
compounds(160)
, Tribendimidine(161)
, Clorsulon (162
, Benzothiazoles(163)
, Ozone(164)
, Nucleoside
phosphonates(165)
,Mefloquine(166)
,Substituted Pyrimidinedione derivatives (167)
, Anti-androgens(168)
,
Arachidonic acid (169)
,Interferon(170)
, Miltefosine(171)
, Thioxo-imidazolidine compounds(172)
,
endoperoxide N-89 (173)
, Licarin (174)
, Benzodiazepines (175)
, Aryl Ozonides (176)
, Imatinib (177)
and
Ivermectin (178)
.A considerable number of these compounds were tested and proved promising

31
anti-schistosomal activities, the majority of them were consigned to the museums of history, but
few succeeded in reaching more advanced developmental phases of clinical trials.
B-Natural products or naturally derived compounds:
As considerable efforts are ongoing to develop novel schistosomicidal agents, many natural
compounds with promising antischistosomal properties have been identified.
1-Artemisinin derivatives: Artemisinin is a sesquiterpene lactone with a peroxide group derived
from the leaves of the Chinese wormwood (Artemisia annua L.) which belongs to the family
Asteraceae, Artemether and artesunate are the most common Artemisinin derivatives (179)
. These
compounds are commonly used as antimalarial agents. In the early 1980s, it was discovered that
artemisinins exhibit antischistosomal properties .A comparative evaluation between artemether
and artesunate was performed by Utzinger et al., (2002), It revealed that artemether shows
consistently higher schistosomicidal activity than artesunate due to differences in the rates of
metabolism of the drugs (180)
.These artemisinin derivatives were found to be active against all
human schistosome species (181)
.
Artemether treatment in S.mansoni infected mice 4-6 week post-infection (WPI) with
doses ranging from (100 to 800 mg/kg for 2 to 4 days) resulted in worm load reduction varies
from 40 % to 61 %. Artemether shows its highest activity against the juvenile stages of the three
major human schistosome,so broadly defined as chemoprophylactic for schistosomiasis and exerts
ovicidal activity (180-185)
. S. mansoni immature worms exposed to artemether in vitro and
experimentally in mice resulted in high worm reduction (97-100%) between days 7 and 28 post-
infection (182)
. In fact, this is the period when praziquantel and other antischistosomal drugs are
less effective (73)
. Utzinger et al., (2000) (185)
reported the prophylactic activity of oral artemether
on S.mansoni in a randomized, double-blind placebo-controlled trial in western Côte d'Ivoire. The
incidence of infection was 50% lower in children who received artemether rather than placebo,
and the intensity of infection among those uncured was also reduced.
2-New Myrrh-Derivatives:
Myrrh oil can be prepared from the crude myrrh either by steam distillation or solvent
extraction (petroleum ether). It is named myrrh essential oil (MEO) or myrrh volatile oil (MVO)
or myrrh total oil (MTO). Allam and El-Sayad (2001) (186)
found molluscicidal activity in
B.alexandrina and the lethal concentration (LC 50) of the oil was 155 ppm in 24 hrs. While El-
Ashry et al., (2003) (187)
found (LC 50) was 6-7 ppm for 24 hrs. in the same snail. Oral lethal dose
(LD50) of MEO in rats was 1650 mg/Kg (188)
. Two reports assessed the antischistosomal activity
of MTO against experimental S.mansoni infection .The oil showed promising antischistosomal

32
activity by Massoud et al. (1999) (102)
and Abo-El-Maaty (2002) (189)
in hamster or mice without
hepatic hazard. In addition, MTO was tested as anticestodal drug and proved that 75 % of H.nana-
infected rats orally treated with 834 mg/kg were cured (190)
.
3-Miscellaneous Natural products:
Likewise, research on other natural products and natural product-derived compounds
against schistosomes has been performed by many groups. Accordingly, several natural products
with antischistosomal properties have been described in the literature; Citrus reticulata(104)
,
Curcumin (Curcuma longa)(191,192)
,Ginger (Zingiber officinale)(193)
,Nigella sativa(194)
, Garlic
(Allium sativum)(194)
, piplartine (Piper) (195)
,Holothuria polii (196)
, propolis (197)
, Ailanthus altissima
(198)
, Ziziphus spina christi (198)
, Camel milk(199)
, Ferula assafoetida(200)
, Cleome droserifolia(201)
,
Chenopodium ambrosioides(202)
,Conyza dioscorides(202)
, Sesbania sesban(202)
, Balanites
aegyptiaca(203)
, Euphorbia schimperiana (204)
, Carica Papaya (205)
, Pomegranate (Punica
granatum) (206)
and Baccharis trimera (207)
.
In Egypt, large scale surveys were done on hundreds of natural products and found strong
in vitro antischistosomal activity against Schistosoma mansoni for the extracts of 30 species
which are (Agave Americana var. marginata and A. lophantha) , Furcraea selloa, Calotropis
procera, Pergularia tomentosa , Asclepias sinaica, Alkanna orientalis , Khaya grandifoliola,
Swietenia mahogany, Pimenta racemosa, Pinus canariensis, Verbascum sinaiticum ,(Solanum
elaeagnifolium, Solanum nigrum), Brachychiton rupestris, (Callistemon viminalis, C. rigidus , C.
speciosus , C. citrinus) , (Eucalyptus citriodora , E. rostrata, Eugenia edulis , E. javanica) ,
(Melaleuca leucadendron, M. stypheloides), Cryptostegia grandiflora , Zilla spinosa , Ficus
trijuja , Fagonia mollis and Nerium oleander (208-210)
. Most of the extracts or natural compounds
were only evaluated in vitro studies; it is expected that they will be evaluated using in vivo
experimental models and finally various phases of clinical trials should be followed to find the full
data of their effectiveness.

34
AIM OF THE WORK
The aim of the study is to assess efficacy of Nitazoxanide, Myrrh Total Oil and the
commercially available product of Myrrh (Mirazid) in comparison with Praziquantel in treatment
of schistosoma mansoni infected mice.

36
MATERIALS AND METHODS
MATERIALS
I.Experimental animals: The study included 120 Eight-week-old female Swiss albino mice (Mus
musculus) of the CD-1 strain weighing 18-25 gm. The animal groups were bred in separate
stainless steel wire-mesh cages under controlled conditions (Temperature 18-25°C, humidity 30-
70%, 12 hours light and 12 hours dark cycles). Animals were fed a standard pellet diet and water
ad libitum.
II.Parasite strain: Laboratory-bred B.alexandrina snails infected with miracidiae of Egyptian
(CD) strain of Schistosma mansoni were obtained from the Schistosome Biologic Supply Center
(SBSC), Theodore Bilharz Research Institute (TBRI). Cercariae shedding out of infected snails
were used to infect the experimental mice.
III.Drugs: The drugs under investigation were:
 Nitazoxanide was purchased from a pharmacy in Alexandria as Nitazode powder for oral
suspension produced by Sigma pharmaceutical company for Al-Andalus Medical
Company, Batch No: 21581.
 Mirazid capsules were obtained as free medical samples from Pharco Pharmaceuticals,
Batch No: 296.
 Myrrh total oil was obtained from Safepharma.
 Praziquantel was purchased as Biltricide tablets manufactured in Alexandria Company
for pharmaceutical and chemical industries, Batch No: 9118014.
IV. Chemicals:
 Iodine solution Cremophore EL Distilled water
 KOH (potassium hydroxide Petroleum ether Saline
V. Equipement:
 Animal house Oesophageal syringe Markers
 Sensitive balance Electricity supply Aquarium
 Black plastic Glass conical flasks Electric pump
 White fluorescent light lamp Magnetic rod Magnetic plate
 Stereobinocular microscope Dissecting microscope Glass test tubes
 Plastic pippetes (1ml) Glass slides Beakers

37
 Volumetric flasks A pair of scissors Dissecting board
 Ordinary Ruller Plastic small tubes Vortex mixer
 Incubator Tray Forceps
 Epindorf Micropipette Centrifuge
 Vacutainers Diagnostic kits Capillary tubes
 Haematology automated cell counter Haemocytometer
 Chemistry analyser Critical Point Dryer Fine coater
 Scanning electron microscope Statistical programme Computer
METHODS:
І-Mice infection with S.mansoni (figure 3):
*Cercarial shedding and counting of cercariae in the suspension (211)
:
Infected Biomphalaria alexandrina snails were washed with dechlorinated water and kept at
an aquarium in an aerated (by using electric pump), dark place (covering the glass bath with black
plastic bag). Before use, snails were rinsed gently with small volume of water to remove faeces
and other debris, then resuspended in water (1 ml /1 snail) and left uncovered in a glass test tube
under white fluorescent light for a period of 30-60 min to release cercariae .After shaking gently
to ensure homogenous distribution of cercariae, 1 ml of cercarial suspension was pipetted and
placed on glass slides, a drop of iodine was added to each slide to kill and stain the cercariae.
With the aid of a stereobinocular microscope, the number of cercariae was counted in each slide.
Generally 3 counts were made in 3 ml cercarial suspension and the average number per 1 ml was
calculated.
* Infection of mice (212)
:
Mice were allowed to urinate and defecate by its exposure to fresh water in a glass bath.
Mice were then infected using paddling technique .Each mouse was exposed separately to about
100 S.mansoni cercariae for one hour at room temperature (22-28O
C) in a glass conical flask
containing 10 ml dechlorinated water mixed with the cercarial suspension .Infected mice were
then segregated in groups of 10 in separate stainless steel wire-mesh cages.The date of infection
was recorded. Mice received a standard well balanced diet and water. Stool examination was
performed 50 days post-cercarial infection to investigate the presence of S.mansoni eggs.

38
Figure.3. Steps of mice infection with S.mansoni cercariae (1-cercarial shedding,
2- cercarial counting, 3- cercarial inoculation)
3
2
1

39
II-Preparation of Drugs Suspensions:
1- Preparation of Praziquantel Suspension (99)
:
Fresh suspension was prepared by dissolving the tablet (600mg) in 6 ml of 4% Cremophore
EL (4 ml cremophore EL+ 96 ml sterile distilled water). Each mouse (20g) requires 0.1 ml
solution. A magnetic rod was placed into the flask, and then the flask was put on a magnetic plate.
The mixture was stirred for 30 minutes to ensure complete homogeneity of the drug suspension.
The suspensions were dispensed into sterile labeled tubes with tight stoppers.
2- Preparation of Mirazid suspension (103)
:
Each capsule of the drug (300 mg) was evacuated in a flask containing 3 ml of 4 %
cremophore EL. Each mouse requires 0.1 ml solution.
3- Preparation of Nitazoxanide suspension (213)
:
Nitazoxanide After reconstitution with distilled water, each 5 ml suspension contains 100
mg NTZ. Each mouse (20g) requires 0.1 ml solution.
4- Preparation of Myrrh Total Oil Suspension (214)
:
0.3ml (100 mg by weight) of the oil was mixed with 27 ml Cremophor EL 4 %. Each mouse
(20g) requires 0.1 ml solution.
N.B. Cremophor EL is a castor oil derivative used as an emulsifying and solubilising agent for the
production of aqueous preparations containing volatile oils and other hydrophobic substance.
N.B. Drug suspensions were freshly prepared within the week of the performance of experiments,
and put in the refrigerator until use. Doses equivalent to those predetermined in the dosing
regimen were then calculated as mentioned and orally administered to each mouse using
eosophageal syringe.
III. Study Grouping:
The study was carried out on six groups of 20 mice each .The mice were housed in a room
with a controlled adequate environmental temperature .Groups of 10 mice in each cage were
allowed free access to water and food. They were acclimatized for 1 week before test and only
healthy mice were assigned to the present study. Mice of all groups were randomly allocated
through treatment and control groups, just prior to drug administration. In these groups, treatment
started 50 days post infection. The drugs were administered after overnight fasting and eating was
allowed after one hour as shown in the following:
Group1: infected and treated orally with MZD 500 mg/kg bw/day for 5 consecutive days (103,107)
.
Group 2: infected and treated orally with MTO 18 mg /kg bw/day for 3 days (102,214)
.
Group 3: infected and treated orally with NTZ 100 mg/kg bw/day for 7 consecutive days (213)
.

40
Group 4: infected and treated orally with PZQ 500 mg/kg bw/day for 2 consecutive days (99)
.
Group 5: infected and non-treated (+ control G).
Group 6: normal non-infected and non-treated (- control G).
N.B. Infected non-treated control and normal non-infected non-treated mice were given only the
vehicle (4% Cremophor EL).
IV- Drug Evaluation:
Evaluation of efficacy was based on the following parameters:
I.Parasitological Studies:
They were performed to assess the efficacy of the different treatments on fecal egg counts,
worm burdens, sexes and lengths, tissue egg loads and oogram patterns.
a-Feacal Egg Count : Eggs of S.mansoni were counted in mice stool {each pellet was weighed ,
thoroughly mixed with saline and spread on glass slide then eggs were counted every other day
starting two days post-treatment and continued till mice sacrifice (215)
.
b-Recovery of adult worms: Perfusion technique was done in experimentally infected mice (G1-
G5).After mice sacrifice 1, 2 and 4 weeks post-treatment as follow:
Perfusion technique
1. Mice were sacrificed by cervical dislocation (99)
.
2. Blood samples were collected immediately.
3. Their bodies were skinned, washed with tap water to remove any adherent hair.
4. Mice were fixed to an inclined dissecting board, laid on a stainless steel pan in which the
perfusate was collected. The abdominal muscles and peritoneum were opened to expose the
internal organs. The portal vein was quickly ligated and closed to its entrance to the liver to
prevent shift of the parasites.
5. perfusion was done according to the technique of Smithers and Terry (1965) (212)
, using
perfusion pump machine (figure 4), A twenty liter glass beaker with outlet of rubber tubing and
20 gauge needle, containing citrated solution, was put in the perfusion machine. The pressure
required for the perfusion was provided by a rotator peristaltic pump. The needle connected to
automatic machine was inserted into inferior vena cava for pumping citrated saline into the liver.
The portal vein was then cut and the perfusate flowing from it was collected .Perfusion was
continued until the fluid coming from the animal was free of blood. The needle was then removed
while the pump was still operating and inserted into the thoracic aorta downward to perfuse the
mesenteric vessels. The perfusate flew out of the portal vein. Coils of the intestine were lift and
washed down in order to dislodge any worms adhering to them. The viscera with surrounding fat
deposits,were searched thoroughly for worms.

41
Figure.4. Perfusion pump machine
Figure.5. Mice perfusion
Worm Burden :
-The worms coming out with the perfusates of the liver and mesenteries were collected.Then, the
sediment was transferred into a Petri dish using a Pasteur pipette for worms to be counted and
sexed under a stereoscopic microscope using low-power magnification (x10).
-The percentage reduction in the total worm burden were calculated by comparing the number of
worms recovered from the treated mice with those recovered from the corresponding control
according to Tendler et al.,(1986)(216)
with the following formula:

42
% Worm reduction(R) =
Mean worm count non-treated group(c)-Mean worm count treated group(t) x100
Mean worm count non-treated group(c)
Or % R= C-T/C X100
Worm length:
Random samples of collected worms from each group were classified and their length were
measured using ordinary ruller and dissecting binocular microscope (143)
(figure 6).
Figure.6. Measurement of female S. mansoni body length under dissecting microscope with
ordinary ruller.
c-Tissue egg count :
To evaluate the number of eggs present in the tissues of the liver and small intestine
according to Cheever et al., (1968) (217)
.Tissues were frozen but not formalized till examination.
KOH digestion technique:
This technique was performed in the following steps:
- Fragments of the small intestine were slit-opened and washed with saline to remove any faecal
matter present in the lumen. Also fragments of the liver were taken and washed with saline.
- The specimens of the hepatic and the intestinal tissues were weighed separately (about 1g)
before digestion.
- These weighted fragments were placed in a test-tube containing 5 ml KOH solution (5%) at
37°C overnight in an incubator till complete digestion of tissues.

43
-After overnight incubation, the test tubes containing the tissue emulsion were mixed thoroughly
in a vortex mixer.
-The digest was well shaken and three samples, (each = 0.1ml or 100 µL) were pipetted out from
each tube by micropipette and placed on slides.
-The samples were then examined microscopically with low magnification (10 X) and the number
of ova in the 3 samples was recorded.
Calculation of tissue egg loads:
* Average number of ova in 0.1ml = Total number of eggs in the three samples/3
* Total number of ova in the examined tissue (in 5 ml) = Average number of ova in 0.1ml × 5(5
ml KOH solution) / 0.1
* Number of ova/g tissue (epg) = Total number of ova in 5ml /weight of liver or intestine in grams
recorded before digestion.
Calculation of tissue egg load reduction:
Reduction in tissue egg loads was calculated by comparing the mean tissue egg loads in the
treated groups with those of the corresponding control group using the Following formula:
% Tissue epg reduction=
Mean epg in non-treated(c) - Mean epg in the treated (t) x100
Mean epg in non-treated group(c)
d. Oogram pattern (percentage of egg developmental stages) (218)
:
For studying the oogram (figure 7); eggs were classified according to their viability (dead
or viable). Generally, dead eggs appear semitransparent, granular or darkened with retracted
embryo. If death takes place at immature stages, whereas those dying after maturation appear
roughly granulated, disintegrated, calcified or as egg-shells. The viable eggs were then divided
according to their stages of maturation (immature or mature).
Oogram technique was performed in the following steps:
1-The small intestine of the perfused mouse was separated and transferred into a Petri dish
containing normal saline. The intestinal contents were removed by squeezing the intestine gently.
2- About 10 centimeters of the middle part of the small intestine were opened longitudinally with
scissors and rinsed with saline after removing excess mucus.
3- Three fragments (1-cm pieces) were cut off, slightly dried with filter paper and placed between
two slides to be examined under microscope using low-power magnification (x10).
4- One hundred S. mansoni ova were counted in each fragment and subsequently classified
according to their developmental stages into: dead or viable (mature or immature).

44
5- The mean numbers and percentages of various egg developmental stages as well as dead eggs
were calculated for each group of mice.
Figure.7. Egg developmental stages (218)
[Dead eggs of S. mansoni (immature) (A, darkened eggs, B, granular egg, C and D, semi -
transparent eggs, E, egg with retracted embryo) and Viable eggs of S. mansoni (F) immature egg,
(G) mature egg].

45
2. Scanning Electron Microscpic Studies:
Scanning electron microscopy (SEM) was utilized to examine the effect of the drug on the
tegument or external surface of the worm. This technique was applied according to Anderson
1951(219)
with some modifications according to Bricker et al. (1983) (220)
.
Technique:
1- Fixation: Two worms (one male and one female) were removed from each group then
fixed with 3% glutaraldehyde buffered with 0.1 M phosphate buffer pH 7.4 at 40
C.
2- Dehydration (to remove water): Washing of worms with phosphate buffer to remove the
fixative then worms were dehydrated in graded concentration series of Acetone
30%,40%,50% each for 15 min, then the worms were kept in 70% acetone until the time of
examination. Before examination, worms were washed for three times, the first and second
were for 30 min in 80% and 90% acetone respectively, while the last wash was for 1 hour
in 100% acetone.
3- Drying: worms were transferred to liquid CO2 at the critical point for drying in critical
point dryer.
4- Coating: The dried specimens were mounted on metal stabs then coated with gold (to make
the surface more conductive for electrons).
5- Imaging: Specimens were examined in Joel JSM-5300 scanning electron microscope in the
electron micrcoscpy unit, Faculty of science, Alexandria University, Egypt).
3. Hematological Studies:
Blood samples obtained before sacrification of mice using capillary tubes introduced into the
medial retro-orbital venous plexus (figure 8) , a part of blood (about 300 ul) was collected into
vacutainer tubes containing an anticoagulant [ethylene diamine tetra-acetic acid (EDTA)] for
determination of Complete Blood Count (CBC) (221)
by using haematology fully automated cell
counter (Mindray BC-3200) .

46
Figure.8. Blood collection from a mouse
4. Biochemical studies:
Another part of the blood (about 1ml) was collected in tubes without anticoagulant,
centrifuged at 3000 rpm for 5 minutes for collection of serum then estimation of biochemical
parameters using commercial kits. The liver function tests were assessed using alanine
aminotransaminase (ALT), aspartate aminotransaminase (AST,Diasys diagnostics) according to
Reitman and Frankel (1957) (222)
and alkaline phosphatase (ALP,Tecno diagnostics) according to
Kind and King(1954)(223)
. Blood urea and serum creatinine were used to assess kidney functions
using urea and creatinine kits (Diamond Diagnostics) according to Fawcett and Scott (1960) (224)
,
Husdon and Rapoport (1964) (225)
respectively. The previous tested parameters were counted by
photometer 5010 (fully-automated chemistry analyser, India), Cholinesterase (ChE) level was
selected to assess the neurotoxic potential in mice blood using Spinreact chemistry analyser
/Spinlab(Spain) according to the colorimetric method of Ellman et al.(1961)(226)
.
ETHICAL CONSIDERATIONS:
The study protocol was reviewed and approved by the ethics committee of the medical
research institute (MRI), University of Alexandria.

47
STATISTICAL ANALYSIS
The data were coded , collected, tabulated and analysed using one way analysis of variance
(ANOVA) followed by independent two-sample t-test or Student’s t test for comparison of means
of two corresponding groups using Minitab statistical software version 14. Descriptive statistics
were expressed as arithmetic mean ± Standard Deviation (SD) as measures of central tendency
and dispersion respectively. As regards the level of significance (P > 0.05 was considered
statistically non-significant while p<0.05 and p<0.01were considered statistically significant and
highly significant, respectively). Data were presented using Microsoft excel sheet for graphical
presentation.
The % of change between non-treated, non-infected and treated groups was calculated as follow:
% change in infected =
Mean values in non-infected (c) - Mean values in infected non-treated (n) × 100
Mean values in non-infected(c)
% change in treated =
Mean values in non-treated(c) - Mean values in treated (t) × 100
Mean values in non-treated (c)

49
RESULTS
1. PARASITOLOGICAL STUDIES
a. Egg count in stool
In S.mansoni-infected mice, it was noticed that the prepatent period lasted 49 days, as the
first patch of eggs was found in the stool of infected mice on day 50 post-infection. Non-
significant change in faecal egg count was found either in infected non-treated mice or treated
with different regimens before the 7th
day of follow up. There was gradual rise in egg count of
infected non-treated mice starting from the 8th
WPI till the 10th
week with fluctuation from day to
day in the follow up period (table I and figure 9, 10). Treatment of infected mice with PZQ caused
highly significant % reduction in the faecal egg counts, 1WPT (63%, p<0.01) as compared to the
infected non-treated group. Eggs were not detected in the faeces of infected treated mice at 2
WPT (egg reduction 100%, p<0.01) and this continued throughout the rest of the follow up period
till 4 WPT.
The results of MZD-treated S.mansoni -infected group showed non-significant % reduction
in the number of eggs 1WPT (4.5%, p>0.05), but MZD caused highly significant reduction
(39.5%, 69.6%, p< 0.01) at 2 and 4 WPT. As regards the effect of NTZ on egg count in stool of
S.mansoni -infected mice, it caused non-significant reduction (4.9%, p>0.05) at 1WPT but it
resulted in significant % reduction (22.5%, p< 0.05) at 2 WPT and highly significant reduction
(50.6 %, p< 0.01) at 4 WPT. MTO treatment resulted in insignificant change in faecal egg count
% reduction (1% and 9.8%, p>0.05) at the 1st
and 2nd
PT. The oil significantly reduced (19.4%,
p< 0.05) at the 4th
weeks of treatment (table I, II and figures 9, 10).

50
Table (I): Mean egg counts per gram stool of S. mansoni-infected mice under different
treatments compared to infected non-treated mice.
NTZ = Nitazoxanide, MTO= Myrrh total oil, MZD=Mirazid, PZQ= praziquantel , PT: post-treatment,
epg= egg count per gram , the statistical test was done by independent two-sample t-test ,Values were
expressed as mean ± SD,
a: Statistically significant at P.value < 0.05.
A : Statistically highly significant at P.value < 0.01.
Figure.9. Egg counts in stool of different groups of S. mansoni-infected mice under different
treatments compared to non-treated infected mice.
Days
(PT)
Groups
Egg counts (epg)
3 5 7 9 11 14 16 18 20 22 24 26 28
NTZ 184.00
±30.50
272.00
±67.97
538.25
±39.47
638.00
±119.6
639.00
±135a
592.50
±90.69a
632.50
±126.0a
550.00
±111.80a
426.67
±118.4A
452.50
±113.2A
395.00
±70.4A
350.00
±84.85A
355.00
±28.07A
MTO 202.50
±47.87
292.50
±93.59
560.00
±66.58
655.67
±83.27
733.33
±85.05
690.00
±57.00
696.67
±65.06
583.33
±61.10
597.33
±15.53
511.00
±205.0a
580.08
±95 a
509.00
±155 a
580.0
±77.5a
MZD 177.33
±18.62
226.67
±50.86
540.00
±42.74
558.33
±108.70
556.00
±26.17A
462.50
±39.09A
533.80
±70.05A
494.33
±127.1A
392.50
±94.54A
320.00
±113.5A
255.00
±77.2A
225.00
±63.64A
218.75
±12.97A
PZQ 178.33
±66.76
279.17
±76.97
204.0
±15.7A
156.00
±81.12A
70.00
±18.71A
0.00
±0.00 A
0.00
±0.00 A
0.00
±0.00 A
0.00
±0.00 A
0.00
±0.00 A
0.00
±0.00A
0.00
±0.00A
0.00
±0.00 A
Infected
non-
treated
178.00
±20.49
220.90
±78.81
566.00
±55.05
624.00
±79.25
745.00
±113.28
765.00
±70.83
735.00
±54.47
657.50
±80.98
657.50
±83.42
810.00
±127.67
821.00
±83.86
770.00
±81.85
720.00
±62.11

51
Table (II): Effect of different types of treatments on mean egg counts among different
groups of S. mansoni-infected mice compared to non-treated infected mice according to
WPT.
WPT
Mice groups (infected and treated)
Infected
non-treatedNTZ MTO MZD PZQ
1
538.25 ±39.47
(-4.9%)
560.00 ±66.58
(-1%)
540.0±42.74
(-4.5%)
204.0 ±15.7A
(-63.9%)
566.00 ±55.05
2
592.50 ±90.69a
(-22.5%)
690.00 ±57.00
(-9.8%)
462.50±39.09A
(-39.5%)
0.00±0.00A
(-100%)
765.00 ±70.83
4
355.00 ±28.07A
(-50.6%)
580.0 ±77.5a
(-19.4%)
218.75±12.97A
(-69.6%)
0.00±0.00A
(-100%)
720.00±62.11
NTZ = Nitazoxanide,MTO= Myrrh total oil, MZD=Mirazid, PZQ= praziquantel, WPT=weeks post-
treatment, the statistical test was done by independent two-sample t-test ,Values were expressed as
mean ± SD, Numbers in parentheses indicate the percentage of change compared to the infected non-
treated group .
Figure. 10. Percentage faecal egg count reduction in different groups of S. mansoni-infected
mice under different treatments at different periods of follow up.

52
b. Worm Burden, Sex and Length:
Treatment of S. mansoni-infectbed mice with PZQ caused pronounced and a highly
significant reduction (83%,94 %,97%, P<0.01) in the mean number of total worms at 1,2 and 4
WPT, respectively as compared with the infected control group. MZD caused significant reduction
in the total worm burden 34% (P<0.05), 50% and 71% (P<0.01) at 1, 2 and 4 WPT, respectively.
NTZ-treated group showed insignificant reduction in total number of worms (26%, P>0.05) at 1
WPT. There was significant reduction 45% and 65% (P<0.01) at 2 and 4 WPT, respectively. MTO
gained non-significant rate of total worm reduction 9% and 27% (P>0.05) at 1 and 2 WPT but the
oil possessed significant rate of worm reduction 29 % (P<0.05) at 4 WPT (table III and figure 11).
Table (III): Worm burden in S.mansoni-infected mice treated and non-treated groups by
time (weeks).
Mice groups WPT
Total worm burden
(Mean ± SD)
TWR
%
NTZ
1 14.60±1.81 26
2 15.75±0.95 A 45
4 10.00±1.82 A 65
MTO
1 18.00±4.83 8.5
2 20.67±3.21 27
4 20.33±2.08a 29
MZD
1 13.00±2.53 a 34
2 14.25±2.63 A 50
4 8.25±1.50 A 71
PZQ
1 3.33±1.75 A 83
2 1.6±0.54 A 94
4 1.00±0.70 A 97
Infected
non-treated
1 19.70±2.86 -
2 28.34±5.51 -
4 28.67±4.73
WPT: weeks post-treatment, TWR: Total worm reduction, NTZ = Nitazoxanide, MTO= Myrrh total oil,
MZD=Mirazid, PZQ= praziquantel, the statistical test was done by independent two-sample t-test.
Values were expressed as mean ± SD.

53
Figure.11. Percentage reduction in the mean total worm burden in different groups under different
treatments at different periods of follow up.
In this study, PZQ showed equal sensitivity on the numbers of both sexes as there was
significant reduction 83.3%, 93.5% and 95.7% (P<0.01) in male worms and 82.6%, 95.8% and
98% (P<0.01) of female worms respectively at 1, 2 or 4 WPT. MZD affected males more than
females at 1, 2 and 4 WPT as the drug killed 38.4%, 57.1% and 77.2% (P<0.01) of the male
worms and 25.3%, 35.3% (P<0.05) and 60% (P<0.01) of females. NTZ affected male worms
more than females as there was significant reduction 27.6% (P<0.05), 47.7% and 67.8% (P<0.01)
of male worms and 22.3% (P>0.05), 37.9 % (P<0.05) and 60% (P<0.01) of females at 1, 2 or 4
WPT. MTO resulted in significantly reduced males at 2 and 4 WPT 35.7% (P<0.05) and 35.7%
(P<0.01) more than females (10.3%, and 16.7%, P>0.05) (table IV, fig.12, 13).

54
Table (IV): Percentage of change in male and female worm distribution in different
S.mansoni-treated mice groups in different periods of follow up.
WPT: weeks post-treatment, NTZ =Nitazoxanide, MTO= Myrrh total oil, MZD=Mirazid, PZQ=
praziquantel, the statistical test was done by independent two-sample t-test. Values were expressed as
mean ± SD.
a: Statistically significant at P.value < 0.05,
A: Statistically highly significant at P.value < 0.01.
Mice groups WPT Total male Total female
NTZ
1 9.40±1.14a (27.6%) 5.20±0.83 (22.3%)
2 9.75±0.95A (47.7%) 6.00±0.81a (37.9%)
4 6.00±1.41A (67.8%) 4.00±0.81A (60%)
MTO
1 11.30±3.37 (13%) 6.70±1.82 (0%)
2 12.00±1.00a (35.7%) 8.67±2.31 (10.3%)
4 12.0±1.00A (35.7%) 8.33±1.15 (16.7%)
MZD
1 8.00±1.41A (38.4%) 5.00±1.89a (25.3%)
2 8.00±1.63A (57.1%) 6.25±1.25a (35.3%)
4 4.25±1.25A (77.2%) 4.0±1.41A (60%)
PZQ
1 2.16±1.60A (83.3%) 1.16±0.4A (82.6%)
2 1.20±0.44A (93.5%) 0.40±0.54A (95.8%)
4 0.80±0.44A (95.7%) 0.20±0.44A (98%)
Infected
Non-treated
1 13.00±2.24
-
6.70±2.59
-
2 18.67±3.21
-
9.67±3.06
-
4 18.67±2.08
-
10.00±2.65
-

55
Figure.12. Percentage reductions in female worm burden in S.mansoni-infected mice under
different treatments at 1, 2 and 4 WPT.
Figure.13. Percentage reductions in male worm burden in S.mansoni-infected mice under different
treatments at 1, 2 and 4 WPT.
The effect of the studied medications on the body length of both male and female worms
after recovery indicated that PZQ caused equal sensitivity in shortening of worm length as it
caused significant reduction in the body length of female worms 26.1%, 45.2% (P<0.05), 65.6%,
(P<0.01) and 25%, 50% and 61% (P<0.01) of male worms at 1, 2 and 4 WPT, respectively. MZD
decreased body length of male worms 11.8% (P>0.05), 40.4% and 41% (P<0.01) than in female
worms 3% (P>0.05), 20.5% (P<0.05) and 33.6% (P<0.01) respectively at 1, 2 and 4 weeks after

56
treatment. NTZ and MTO had negligible effect on worm length of both sexes as there was non-
significant decrease in the body length of worms recovered at different times 1,2 or 4 weeks post-
treatment (6.5%, 9.5% and 5.2% for males and 0%,6.8% and 5.6% for female respectively in
NTZ-treated worms, also in MTO (2.6%,4.7% and 0% for males and 0% ,5.9% and 12% for
females at 1,2 and 4 WPT) (table V and figure 14,15).
Table (V): Body length of S.mansoni worms recovered from different treatments compared
to non-treated mice at different follow up periods.
Worms
sex
WPT
The body length of S.mansoni worms recovered (mm)
Infected Treated Mice Infected
Non-treatedNTZ MTO MZD PZQ
Male
1 7.10±0.58
(-6.5%)
7.40±1.15
(-2.6%)
6.7±1.53
(-11.8%)
5.70±0.10a
(-25%)
7.60±0.55
2 7.60±1.15
(-9.5%)
8.00±1.00
(-4.7%)
5.00±1.00a
(-40.4%)
4.20±0.58a
(-50%)
8.40±1.50
4 9.00±1.41
(-5.2%)
9.50±0.71
(0%)
5.60±1.53a
(-41%)
3.70±1.15A
(-61%)
9.50±0.70
Female
1 13.00±1.73
(0%)
13.0±0.58
(0%)
12.60±0.58
(-3%)
9.60±0.58a
(-26.1%)
13.00±1.00
2 10.9±0.58
(-6.8%)
11.00±1.73
(-5.9%)
9.30±0.58a
(-20.5%)
6.40±0.58a
(-45.2%)
11.70±1.50
4 11.80±0.71
(-5.6%)
11.00±1.41
(-12%)
8.30±1.1A
(-33.6%)
4.30±0.58A
(-65.6%)
12.50±0.71
NTZ=Nitazoxanide,MTO=Myrrh total oil, MZD=Mirazid, PZQ= praziquantel, WPT=weeks post-
treatment , the statistical test was done by independent two-sample t-test ,Values were expressed as
mean ± SD, Numbers in parentheses indicate the percentage of reduction compared to the infected group.
a : Statistically significant at P value < 0.05.
A : Statistically highly significant at P value < 0.01.

57
Figure.14. Percentage reductions of the body length of male S. mansoni worms recovered from
different treated groups at 1, 2 and 4 WPT.
Figure.15. Percentage reduction of the body length of female S.mansoni worms recovered from
different treated groups at 1, 2 and 4 WPT.
c. Tissue egg count
The infected non-treated mice were loaded with higher number of eggs in the intestinal
tissues than the hepatic ones. PZQ-treated mice showed significant reduction in tissue egg load in
both liver and intestine as it was able to reduce the intestinal egg load (69.9%, 79.1% and 88%,
P<0.01) more than the reduction in the hepatic tissues (64.4%,69.2 % and 85.8%, P<0.01) at 1,2
and 4 WPT, respectively.

58
MZD produced significant reduction (28.9% ,49.3% and 66%,P<0.01) at 1,2 and 4 WPT in
the intestinal egg loads and non-significant reduction in the hepatic tissue egg load at
1WPT(22.2%,P>0.05) but it was able significantly to reduce the egg count later on in the 2nd
and
4th
week after therapy (42.8% and 65.3%,P<0.01). NTZ reduced the intestinal egg count (22.1%,
45.2% and 46.6%, p<0.01) at 1, 2 and 4 WPT. It was unable to reduce hepatic tissue egg load at 1
WPT (20%, p>0.05) but significant reduction was achieved (23.8%, p< 0.05 and 30.7%, p<0.01)
at 2 and 4 WPT, respectively. On the other hand, MTO resulted in significant reduction in the
intestinal egg count (12.2%, p<0.05), (23.2% and 31.5%, p< 0.01) at 1, 2 or 4 WPT .The oil
insignificantly reduced the hepatic tissue egg count (7.7% and 26%, p<0.05) at 1 and 2 WPT but
significant reduction was achieved (42.3%, p<0.01) at 4 WPT (table VI and figure 16, 17).
Table (VI): The tissue egg count in the liver and intestine of S. mansoni-infected mice under
different treatments at different periods of follow up.
Mice
groups
Tissue egg count(epg) x103 (% R)
Intestine (% Reduction) Liver (% Reduction)
1st
week 2nd
week 4th
week 1st
week 2nd
week 4th
week
NTZ 8.00±1.1A
(-22.1%)
8.4±1.6A
(-45.2%)
8.8±0.7A
(-46.6 %)
3.6±1.4
(-20%)
4.8±0.8a
(-23.8%)
5.4±0.5A
(-30.7%)
MTO 9.02±1.06a
(-12.2%)
11.2±0.7A
(-23.2%)
11.3±1.7A
(-31.5%)
4.15±1.18
(-7.7%)
4.66±1.4
(-26%)
4.5±1.9a
(-42.3%)
MZD 7.30±1.3A
(-28.9%)
7.4±0.5A
(-49.3%)
5.6±1.00A
(-66%)
3.5±1.6
(-22.2%)
3.6±0.8A
(-42.8%)
2.7±1.0A
(-65.3%)
PZQ 3.09±0.16A
(-69.9%)
3.05±0.9A
(-79.1%)
1.96±0.4A
(-88%)
1.6±0.7A
(-64.4%)
1.94±0.8A
(-69.2%)
1.1±0.19A
(-85.8%)
Infected
Non-
treated
10.28±0.4 14.6±0.3 16.5±0.6 4.5±0.7 6.3±0.6 7.8±1.3
WPT:weeks post-treatment, NTZ=Nitazoxanide, MTO=Myrrh total oil, MZD=Mirazid, PZQ=
praziquantel, the statistical test was done by independent two-sample t-test ,Values were expressed
as mean ± SD.
a: Statistically significant at P value < 0.05.
A : Statistically highly significant at P value < 0.01.

59
Figure.16. Percentage reduction in the mean hepatic egg counts in S. mansoni-infected mice under
different treatments at 1, 2 and 4 WPT.
Figure.17. Percentage reduction in the mean intestinal egg counts in S.mansoni-infected mice
under different treatments at 1, 2 and 4 WPT.
d. Oogram pattern (percentage of egg developmental stages):
In the present work, the oogram pattern showed that about 60% of eggs in the infected non-
treated group were immature whereas dead eggs constituted only 7-12% and the mature eggs
formed 24-32% of the total eggs at different follow up periods. PZQ induced marked changes in
the oogram pattern in comparison to the non-treated infected group, it produced a highly
significant increase in the percentage of dead eggs to 79.87 % at 1 WPT, 83.2% at 2 WPT and

60
85.75% at 4 WPT as well as highly significant reduction in immature eggs where they constituted
only 3.67% at 1 WPT, 2.6% at 2 WPT, and 1.5% at 4 WPT, respectively. As regards mature eggs,
PZQ induced significant reduction in their percentage as 16-13% of the eggs in the oogram were
mature.
MZD resulted in significant increase in the percentage of dead eggs. However, the changes
were less marked than that induced by PZQ (24.83 %, at 1 WPT), (37% and 37.33%, p<0.01 at 2
and 4 WPT). MZD also induced significant reduction in immature eggs starting from the 2nd
WPT
(23.25 %) and continued to the 4th
WPT (10.77%). MZD produced a significant increase in the
percentage of mature eggs compared to the non-treated infected mice and at 4 WPT, more than
50% of the eggs were mature. NTZ treatment resulted in significant increase in the percentage of
dead eggs to 19.50%, 22.25% and 30.5% at 1 ,2 and 4 WPT , respectively with progressive
increase in the mean percentage of total mature eggs (27.25% , p<0.01) at 1 WPT ,(34.25%,
p<0.01) at 2 WPT and (51%, p<0.01) at 4 WPT, respectively as well as progressive reduction in
immature eggs (56%, p<0.05), (43.25% and 18.50%, p<0.01) at 1,2 and 4 WPT , respectively. In
comparison to the non-treated infected mice, MTO resulted in initial and significant increase in
immature eggs to 63.25%, p<0.05 at 1WPT followed by highly significant reduction to 47% at 2
WPT and 24.5%, p<0.01) at 4 WPT. MTO also induced an initial and significant reduction in
mature eggs to 20.25% at 1 WPT followed by significant increase to (33% and 49% p<0.01) at 2
and 4 WPT, respectively. The mean percentage of the dead eggs increased significantly (16.50%,
20%, 26.5%, p<0.01) at 1, 2 and 4 WPT, respectively (table VII, figure 18).

61
Table (VII): The oogram pattern (percentage egg developmental stages) in the intestine of S.
mansoni-infected mice under different treatments in different follow up periods.
Type of egg WPT
Mice Groups
NTZ MTO MZD PZQ
Infected
Non-treated
Immature
1 56.00±0.35a 63.25±1.99a 55.50±4.42 3.67±0.03A 59.60±2.28
2 43.25±2.50A 47.00±0.16A 23.25±0.99A 2.60±0.05A 61.50±3.00
4 18.50±0.71A 24.50±0.54A 10.77±0.52A 1.50±0.09A 62.50±0.71
Mature
1 27.25±0.50a 20.25±0.99A 19.37±2.16A 16.67±0.84A 32.60±1.99
2 34.25±1.02A 33.00±1.72A 39.75±1.71A 14.20±0.76A 27.00±0.16
4 51.00±1.41A 49.00±4.24A 52.00±3.00A 13.00±1.16A 24.50±0.12
Dead
1 19.50±0.89A 16.50±1.08A 24.83±2.62A 79.87±5.20A 7.80±0.59
2 22.25±0.99A 20.00±0.16A 37.00±2.55A 83.20±5.54A 11.50±0.38
4 30.50±0.71A 26.50±0.71A 37.33±2.52A 85.75±3.30A 12.00±0.41
WPT=weeks post-treatment, NTZ = Nitazoxanide, MTO= Myrrh total oil, MZD=Mirazid, PZQ=
praziquantel. The statistical test was done by independent two-sample t-test. Values expressed as
mean percentage ± SD.

62
Figure.18. Percentage egg developmental changes in S.mansoni-infected mice under different
treatments at different periods of follow up.

63
II. Scanning electron microscopic studies:
In non-treated infected mice, there is a difference between male and female teguments as
male tegument shows hill-shaped tubercles covered with pointed spines and convoluted surface of
the tegumental membranes between tubercles but female tegument does not have tubercles
(smooth) with fines spines on the surface especially on the dorsal aspect.Oral and ventral suckers
showed intact features and organization. The gynecophoric canal of male worms showed fine
spines in its inner aspect (figure 19).
PZQ showed a pronounced tegumental damage by scanning electron microscopic
examination of S.mansoni worms recovered from treated mice 2 WPT in the form of extensive
tegumental damage with rupture of tubercles and loss of spines in wide areas in male worms.
Moreover, a marked ulceration in the tegument was detected in the outer surface of female worms.
Some teguments showed severe erosion or even sloughing of tegumental membranes exposing the
underlying muscle layers. The male tegument was more affected than females (figure 20).
MZD showed mild tegumental damage in female and male S. manoni worms without any
obvious deeper effects as the changes were topically confined to the outer surface. There was
focal erosion and ulceration with shrinkage of the outer surface in the female tegument. The
tegumental damage in male worms manifested by rupture of tubercles with marked loss of spines
and if present, lost their sharpness .There was higher sensitivity in the tegumental damages in
males than females.The oral sucker still intact (figure 21). NTZ resulted in mild tegumental
damaging effect manifested by focal lesions in the inter-tubercular ridges, disorganization of the
oral sucker of male worm and loss of spines in the gynecophoric canal (figure 22). MTO resulted
only in oedematous swelling of both oral and ventral suckers without detectable alteration in the
tegument (figure 23).

64
Figure.19.Scanning electron micrographs of
S.mansoni worms recovered from infected non-
treated mice showing normal tegument of male
(A) and female worms (B). Normal ventral
sucker of male worms (C) and oral sucker of
female worms (D).The inner surface of the
gynecophoric canal of male (E) worms.
(T = Tubercles, S= Spines, ITR= Inter-Tubercular
Ridge, VS= Ventral Sucker, OS= Oral Sucker).
T
S
ITR
VS
VS
S
S
S
X
S

65
Figure.20. Effect of Praziquantel on the dorsal surface of female schistosoma worms (F) and the
male worms (G) recovered at 2 WPT.
(T = Tubercles, S= Spines, ITR= Inter-Tubercular Ridge, X= Ulceration and erosion).

M.aziz master thesis 2014

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