Fascioliasis Is a Well-Known Parasitic Disease Caused By

Effectiveness of the Fasciolicidal Drug Myrrh in Treatment of Human and Animal Fascioliasis

Thesis submitted to the High Institute of Public Health, University of Alexandria

in partial fulfillment of the requirements for the Degree of

Doctor of Public Health (Tropical Health)

Samira Mohamed Mahboub Abd El Hameed M.B.Ch.B, Faculty of Medicine, University of Alexandria, 1998 ,M.P.H. (Tropical Health), High Institute of Public Health

University of Alexandria, 2004

1 High Institute of Public Health University of Alexandria

2 Effectiveness of the Fasciolicidal Drug Myrrh in Treatment of Human and Animal Fascioliasis

Samira Mohamed Mahboub Abd El Hameed M.B.Ch.B, Faculty of Medicine, University of Alexandria, 1998 M.P.H. (Tropical Health), High Institute of Public Health, University of Alexandria, 2004

For the degree of doctor In Public Health (Topical Health)

Examiner's Committee Approved

Prof. Dr. Ezzat Mohamed Hassan

Prof. Dr. Thanaa Ahmad El Masry

Prof. Dr. Ali Ahmad Waheeb

Prof. Dr. Hanan Zakareya Shatat

3 Advisor's Committee Approved

Prof. Dr. Ezzat Mohamed Hassan

Prof. Dr. Amira Mahmoud Kotkat

Prof. Dr. Hanan Zakareya Shatat

Prof. Dr. Amel Abd Al Fattah El Sahn

4 ACKNOWLEDGMENT

First of all, I thank GOD for helping me to accomplish this work and I ask him to accept my effort.

My deepest thanks, sincere indebtedness and appreciation to Prof. Dr. Ezzat M. Hassan, Professor of Tropical Health, High Institute of Public Health, University of Alexandria, for his whole hearted guidance, valuable instructions and above all for choosing the topic of this work.

No words can adequately express my deepest gratitude and great appreciation to Prof. Dr. Amira M. Kotkat, Professor of Tropical Health, High Institute of Public Health, University of Alexandria, for her valuable guidance, fruitful efforts and continuous encouragement through out the whole work.

No words can adequately express my deepest gratitude and great appreciation to Prof. Dr. Hanan Z. Shatat, Professor of Tropical Health, High Institute of Public Health, University of Alexandria, for her valuable instructions, great advice which helped me to overcome many obstacles.

I wish to express my deepest thanks to Prof. Dr. Amel A. El Sahn, Professor of Parasitology and Medical Entomology, High Institute of Public Health, University of Alexandria, for her laborious effort and encouragement.

I owe special thanks to Pharco Pharmaceutical Company for its generous cooperation in the intervention phase of this study.

I owe special ever lasting thanks and unlimited gratitude to my family, without her love and support this work would not be accomplished.

5 CONTENTS

Chapter Title Page

i List of figures

I Introduction A- Prevalence of human fascioliasis 1

B- Prevalence of animal fascioliasis 3

C- Life cycle of Fasciola sp. 4

D- Characteristics of life cycle of Fasciola sp. 5 1- Domestic animal reservoir hosts 5 2- Wild animal reservoir hosts 5 3- Intermediate snail hosts 5 4- Proper Environmental Conditions 5 5- Mode of transmission 6 6- Association of fascioliasis with other 6 parasitic infections

E- Clinical picture of human fascioliasis 6

F-Clinical picture of animal fascioliasis 8

G- Diagnostic tools for fascioliasis 8 1- Stool examination 8 2- Serological methods 9 3- Detection of fecal antigens of Fasciola 9 hepatica 4- Radiological findings 9

H- Diagnosis of acute fascioliasis 10

I- Diagnosis of chronic fascioliasis 10

J- Differential diagnosis of primary and secondary 10 infections

6 K- Treatment of fascioliasis 11 1- Old lines of treatment of facsioliasis 11 i- Bithionol (Bitin) 11 ii- Praziquantel 11 iii- Metronidazole 12

2- Currently used drug 12

i- Triclabendazole (TCBZ): 12

3- Trials using new fasciolicidal drugs 12 12 i- "3-Cyanopyridine Derivatives"

ii- Compound "Alpha" (5-chloro-2- 13 methylthio-6-(1-napthyloxy)1H-benzimidazole)

iii- Nitazoxanide 13

iv- Myrrh 13

II Aim of work 18

III Subjects and methods 19

IV Results 23

V Discussion 72

VI Summary and conclusions 81

VII Recommendations 85

VIII References 86

IX Appendices

Arabic summary

7 List of figures

Figure No. Title Page

Figure (1): Life cycle of Fasciola sp. 6

Figure (2): A diagrammatic illustration of the pre-intervention phase of 38 the study

Figure (3): Prevalence of Fasciola sp. infection among different age 43 groups

Figure (4): Intensity of infection with Fasciola sp.in different age 53 groups

Figure (5): The parasitological cure rates one, two and three months 56 after treatment of human cases with Mirazid

Figure (6): The geometric mean of egg count (GMEC) in uncured 61 human cases of fascioliasis before and after treatment

Figure (7): A diagrammatic illustration of the pre-intervention phase of 69 the animal study

Figure (8): Prevalence of animal fascioliasis among different age 74 groups

Figure (9): GMEC among different age groups of infected animals 78

Figure (10): Animal cure rates one, two and three months after treatment 81 with Mirazid

Figure (11): GMEC in uncured animal cases of fascioliasis before and 86 after treatment

8 List of figures

Figure No. Title Page

Figure (1): Life cycle of Fasciola sp. 4

Figure (2): A diagrammatic illustration of the pre-intervention phase of 25 the study

Figure (3): Prevalence of Fasciola sp. infection among different age 31 groups

Figure (4): Intensity of infection with Fasciola sp.in different age 32 groups

Figure (5): The parasitological cure rates one, two and three months 41 after treatment of human cases with Mirazid

Figure (6): The geometric mean of egg count (GMEC) in uncured 46 human cases of fascioliasis before and after treatment

Figure (7): A diagrammatic illustration of the pre-intervention phase of 53 the animal study

Figure (8): Prevalence of animal fascioliasis among different age 57 groups

Figure (9): GMEC among different age groups of infected animals 61

Animal cure rates one, two and three months after treatment Figure (10): with Mirazid 63

Figure (11): GMEC in uncured animal cases of fascioliasis before and after treatment 67

9 The Memory of my Father

My Mother

My Beloved Husband

and My Lovely Childe

10 INTRODUCTION

Fascioliasis (liver fluke disease) is an infection caused by one of two species: Fasciola hepatica or Fasciola gigantica. Both are major pathogens of cattle, sheep and other livestocks as well as humans.(1, 2)

Morphometric studies by computer image analysis showed that it is sometimes difficult, and in certain cases even impossible, to differentiate between F. hepatica and F. gigantica, so that several specimens were classified as intermediate forms. Traditional methods of identification of Fasciola species have relied on the morphological characteristics of adults and eggs. The adult stage of F. gigantica is much larger and slightly narrower than F. hepatica.(3)

The geographical distribution of these species differs. Although F. hepatica has a worldwide distribution, it predominates in temperate climates, whereas F. gigantica is found primarily in tropical regions of Asia and Africa.(4, 5)

Most of the areas with a high endemicity of human fascioliasis involve F. hepatica. However, in Asia the distribution of F. hepatica and F. gigantica overlaps and this makes it difficult to identify the particular species involved, which is often referred to simply as Fasciola sp. This especially occurs in China (Province of Taiwan), Japan, the Republic of Korea, and the Philippines. A similar problem occurs in Egypt, where both F. hepatica and F. gigantica as well as intermediate forms have been found (6).

Fascioliasis is recognized as a serious public health problem by the World Health Organization.(7) High prevalence of fascioliasis in humans does not necessarily occur in areas where fascioliasis is a major veterinary problem.(6)

Although the mortality due to fascioliasis is generally low, yet massive or repeated infections can lead to considerable morbidity.(8)

Prevalence of human fascioliasis:

The epidemiological picture of human fascioliasis has changed in recent years as a result of declining socioeconomic conditions, lack of improved sanitation, increasing availability of aquatic foods through wider distribution networks often without proper food inspections and the international trade of animals and their products. The people at higher risk of zoonoses are those living in the countryside, both in developed and developing countries.(9,10)

The numbers of reported clinical cases of human fascioliasis as well as of infected persons identified during epidemiological surveys have increased significantly since 1980.(6) There are about 17 million people infected worldwide.(11) Surveys in several regions indicate that there are areas with true endemic human fascioliasis, ranging from low to very high prevalence and

11 intensity.(12) Low prevalences of fascioliasis in which it is less than 1%, are present in France (13); Corsica (14); and Chile (15). Examples of intermediate levels, in which the prevalence ranges from 1% to 10%, are Portugal (16); Egypt (17); and Puerto Rico (18). High prevalences, in which the prevalence is more than 10%, are pesent in the Puno region (19) and the Mantaro valley (20), both in Peru.

The highest human prevalences have been reported in the Bolivian Altiplano: up to 66.7% detected using coprological techniques and up to 53% using immunological methods (21).

In the Eastern Mediterranean Region, only in the Libyan Arab Jamahiriya have no confirmed cases of human infection been reported, although animal fascioliasis is widespread and the potential for human infection exists. In Iraq, Lebanon, Morocco, Tunisia and Yemen fewer than 100 cases have been documented. It is possible that the problem has not yet received enough attention in these countries. (22)

In Islamic Republic of Iran, the estimated number of people having fascioliasis is 10,000.(23) Several outbreaks of this disease occurred in the Gilan province of Northern Iran.(24)

In Egypt, Fasciola eggs have been detected in a mummy, confirming that human fascioliasis has existed since Pharaonic times. In 1928, two cases were reported and then in 1958, 11 cases were reported. Until 1978 only sporadic cases were diagnosed. Since then, the problem has received increasing attention. Now, different laboratories and clinical services have diagnosed human fascioliasis in all provinces of the Delta, in some provinces of Upper Egypt, and in the city of Alexandria. Studies in some villages in the Delta have revealed prevalence rates varying between 2% and 17%. The population at risk in Egypt is considered to be 27 million. Accordingly, based on an estimated overall prevalence of 3%, the number of infected cases amounts to 830 000 individuals.(22)

Human fascioliasis is increasing in the Nile Delta particularly in Dakahlia Governorate, where it reached 7.47%.(25)

Coprologic surveys were carried out in villages of the Beheira Governorate in the Nile Delta of Egypt to characterize the epidemiologic features of human fascioliasis caused by Fasciola hepatica and F. gigantica in this lowland endemic area .The fascioliasis prevalences detected ranged between 5.2%-19.0%, with a mean of 12.8% are the highest reported in Egypt. All ages appeared to be susceptible, but prevalence and intensities were lower before and after school age.(26) In a study conducted in a village in Alexandria (Abis I), prevalence of fascioliasis was found to be 5.5% and more than half of the cases were children (59.1%).(27)

12 Prevalence of animal fascioliasis:

Fascioliasis is an economically important disease of ruminants world-wide, and especially in Europe, North America, Asia and Africa, where it causes significant morbidity and grave economic loss.(28) Results of parasitological examinations of faecal samples from ruminants between 1998 and 2002 in Germany revealed that, the prevalence of Fasciola hepatica was 1.7% in sheep and 0.6% in cattles. (29) A cross-sectional coprological survey of Fasciola hepatica in Italy was conducted on 81 bovine farms and 197 ovine farms between June 1999 and March 2000. Cattle of 9 of the 81 (11.1%) farms and sheep of 8 of the 197 (4.1%) farms were positive for F. hepatica.(30) Much higher rates were reported in Spain, where the prevalence of fascioliasis in sheep kept under field conditions reached about 83.3%.(31)

In Tunisia, a high infection rate in sheep (54.8%) was noted.(32) In Algeria, in 2002 and 2003, high prevalence of fascioliasis were found in the cattles (27.0% in slaughtered cattle and 27.3% in cattle from farms).(33)

In Egypt, the prevalence of animal fascioliasis in different governorates in Egypt was reported by Haseeb et al. (2002), where low prevalences were found among cattles in desert governorates like North Sinai (3%), Red Sea (6%), New Valley (7.8%) and Mersa Matrouh (9.5%). However high prevalences were found in Dakahlia (59.5%) and Monoufia (51.2%). (23)In Kafr El Sheikh, a percentage of 14.5%, 26.6%, 12.28% 12.3% and 5.4% from the examined Buffaloes, sheep, donkeys, cows and goats respectively were found to be infected with fascioliasis. The incidence was high in summer and autumn than the other seasons.(34,35)In Alexandria, the percentages of fascioliasis in sheep, cattle and buffaloes as indicated by stool examination were 30%, 25% and 25% respectively.(23)

13 Life cycle of Fasciola sp.:(36)

Figure (1): Life cycle of Fasciola sp.

Immature eggs (1) are discharged in the biliary ducts and in the stool. Eggs become embryonated (2) in water, eggs release miracidia (3) , which invade a suitable snail intermediate host , including many species of the genus Lymnae (4). In the snail the parasites undergo several developmental stages (sporocysts (4a), rediae (4b), and cercariae (4c)). The cercariae (5) are released from the snail and encyst as metacercariae (6) on aquatic vegetation or other surfaces. Mammals acquire the infection by eating vegetation containing metacercariae. Humans can become infected by ingesting metacercariae- containing freshwater plants, especially watercress. Human infection can also occur through drinking contaminated water. After ingestion, the metacercariae excyst (7) in the duodenum and migrate through the intestinal wall, the peritoneal cavity, and the liver parenchyma into the biliary ducts, where they develop into adults (8). In humans, maturation from metacercariae into adult flukes takes approximately 3 to 4 months. The adult flukes (Fasciola hepatica: 20-30 mm by 8-15 mm; F. gigantica: 25-75 mm by 5-12 mm) reside in the large biliary ducts of the mammalian host. Fasciola hepatica infect various animal species, mostly herbivores.(36)

14 Characteristics of life cycle of Fasciola sp.:

1- Domestic animal reservoir hosts:

In the Bolivian Altiplano, studies of fascioliasis showed that beside sheep and cattle, pigs and donkeys are efficient reservoirs of the parasite. Recent studies have, moreover, demonstrated that eggs shed by pigs and donkeys are viable, i.e. able to infect a lymnaeid snail, and that the metacercariae subsequently produced are infective for another definitive host. This was the first occasion that the need to take pigs and donkeys into account in preventive and control measures against human fascioliasis has been pointed out. (6, 37)

2- Wild animal reservoir hosts:

In Corsica, where the level of endemicity of human fascioliasis is low, infected lymnaeids snails were identified with the absence of any livestock present. Helminthological surveys showed that black rats (Rattus rattus) were repeatedly infected by liver flukes.(38,39) Brown rats (Rattus norvegicus) and mice (Mus musculus) are commonly used as definitive hosts for experimental purposes; but they seem to be only sporadically naturally infected: only one mouse infection has been described, in Corsica and a brown rat infection has been observed in Iraq .(40)

3- Intermediate snail hosts:

Lymnaeid Snail: Numerous lymnaeid snail species which are distributed worldwide can act as the intermediate host of Fasciola sp. The snails are amphibious, easily capable of surviving out of water when the relative humidity is high. They are capable of withstanding summer drought or winter freezing for several months by aestivating, which means hibernating deep in the mud. Permanent habitats include banks of ditches and streams, and the edges of ponds. Following periods of rainfall, temporary habitats can include hoof-prints and tire ruts. Lymnaeid snails are hermaphroditic. In a three-month period a single snail is capable of producing up to 100,000 descendants.(41) In Egypt, Shehata et al. reported two peaks of snail density; one in late autumn and the other in spring.(42)

The first case of transmission of a Fasciola species by a snail not belonging to the Lymnaeidae family (Biomphalaria alexandrina, Planorbidae) was reported in Egypt. The importance of this discovery for the transmission of fascioliasis remains, however, to be evaluated.(43)

4- Proper Environmental Conditions:

Several environmental conditions are necessary for propagation and development of both the lymnaeid snail and the developing larval forms of the fluke. Moisture is necessary for the amphibious snail, and also for the swimming cercariae. The ideal temperature range for

15 optimal development of both snail and fluke larvae is 15-20°C. Development can occur at temperatures between 10-15 °C but it will be slightly slower in a cooler environment. The snails also prefer a slightly acid pH.(41, 44)

The miracidia of Fasciola hepatica show positive phototactic responses bringing them to the surface, while those of Fasciola gigantica are exactly the opposite (negative phototactic response) keeping them in deeper water.(45)

One of the singular epidemiological characteristics of human fascioliasis is the link of the hyperendemic areas to very high altitude regions, at least in South America. The Northern Bolivian Altiplano, located at very high altitude (3800-4100 m), presents the highest prevalence and intensities of human fascioliasis known.(46)The Puno region (in Peru), located at a very high altitude of 3910 m in the Peruvian Altiplano proved to be hyperendemic for human fascioliasis with a prevalence of Fasciola hepatica up to 53%, and infection intensities of up to 2496 eggs per gram of faeces (epg).(47). This means not only that snail and parasite were able to colonize successfully extreme conditions of very high altitude but also that they have been able to develop different adaptation strategies which permit higher parasite transmission rates.(47)

5- Mode of transmission: Water has been cited as the source of human infection, whether indirectly by contaminating vegetables or kitchen utensils or directly by drinking.(48,12) In Europe, the infections related to the consumption of wild watercress or wild dandelion occur sporadically, most frequently in France, Spain, and Portugal.(49-51) In the United Kingdom, Fasciola has been identified in imported vegetables (khat) kept moist during transport.(52)In Egypt, the prevalence of infection was directely related to the frequency of eating green leafy vegetables.(42) Experimental results suggest that humans who consume raw dishes prepared from fresh livers infected with immature flukes could become infected with fascioliasis.(53)

6- Association of fascioliasis with other parasitic infections: Esteban et al. (2002) reported that there was significant association of F. hepatica with G. intestinalis which agrees with that found between the same organisms in the northern Bolivian Altiplano. This finding in the Asillo zone of the Puno region, located at a very high altitude of 3910 m in the Peruvian Altiplano suggests a similar transmission route and oral infection primarily through drinking water.(47, 54)

Clinical picture of human fascioliasis: The incubation period usually ranges between 3-4 months until the appearance of eggs in stool. The earliest symptoms are related to the liver migration occur as early as two weeks after exposure to infection. However, months or years may pass before the infection is diagnosed. The duration of the survival of the pathogen in human may be up to 13 years. (23)

16 Clinically, there are two stages of fascioliasis: the acute stage and the chronic stage. (55) Patients are classified according to the duration of their symptoms and the ultrasonographic findings. If the duration of symptoms is less than 4 months and there are no motile echogenic images in the gallbladder on admission, it is classified as acute. If symptoms persist for more than 4 months or there are motile echogenic images in the gallbladder, it is classified as chronic.(56)

The acute stage is associated with the migration of the larvae through the liver and is characterized by high fever, marked eosinophilia, and hepatosplenomegaly (acute eosinophilic febrile syndrome). Pain is usually present in the right hypochondrium. The patient may suffer from malaise, nausea, and vomiting. Myalgia, urticaria, and other allergic reactions are common. In severe illness, jaundice, cachexia, and bleeding episodes caused by the erosion of the liver capsule or bile ducts may occur. Laboratory findings include anemia, hypergammaglobulinemia, and elevated liver enzyme transaminases. When the worm burden is low, infection is mild or not apparent. The clinical suspicion is raised by hypereosinophilia in a patient coming from an endemic area or having special dietary habits, such as consuming watercress or self-collected or untreated vegetables.(51,57)

The chronic stage begins after 2 to 3 months and may persist in humans for more than 15 years because of the longevity of the parasite. The adult fluke remains in the gallbladder and slowly deposits eggs in the bile ducts that result in inflammation and may subsequently present as biliary obstruction.(58)The symptoms are usually related to biliary obstruction by the parasites, such as biliary colics, bacterial superinfection with acute cholangitis, obstructive pancreatitis and bleeding from ulcers in the biliary tree. (51,59) Fascioliasis should be included in the list of the differential diagnosis for colicky abdominal pain, eosinophilia and bile duct dilatation. The condition could be concurrently diagnosed and treated by endoscopic retrograde cholaogiopancreatography (ERCP).(60) Parasites or parasite fragments may act as nuclei for gallstone formation with secondary obstructive phenomena. Contrary to infections caused by the small flukes as Clonorchis and Opistorchis, there is no evidence of an association between F. hepatica infection and cholangiocarcinoma.(51,61)

Patients with chronic disease experience few nonspecific symptoms or do not recall any symptom.(55) One of the remarkable clinical characteristics of human fascioliasis in endemic areas is the relative absence of gastrointestinal symptoms.(62)

Aberrant or ectopic sites of infection are common and include the lungs (presenting with respiratory symptoms such as bronchospasm, pneumothorax, pyothorax or haemoptysis), brain, skin, heart and intestinal wall, presenting as visceral larva migrans.(51,63-65)

Acute nasopharyngeal inflammation (pharyngeal fascioliasis) may follow eating raw animal livers infected with Fasciola sp. It is thought to be an allergic reaction to larval flukes. (23)

17 Clinical picture of animal fascioliasis:

The clinical presentation of F. hepatica infections is highly variable and quantitively related to the level of infection. Animals infected with a low number of parasites frequently show no clinical evidence of disease, whereas heavily parasitized animals most commonly present with symptoms of anorexia and lethargy.(66)

Both acute and chronic forms of liver fluke infections have been reported. Acute infections result from the immature flukes tunneling through the liver parenchyma with extensive tissue damage and haemorrhage that culminate in severe clinical disease with high mortality in grazing sheep in Africa.(67-69)

The chronic disease practically results from the adult flukes, often in pairs, lodging within the bile duct, causing duct wall hyperplasia, progressive occlusion and ultimate calcification of the duct wall with characteristic chronic wasting syndromes and various hepatopathies.(70,71) Other economic production parameters observable in chronic disease include reduced conception/pregnancy rate, reduced lambing rate (72-74), delayed onset of puberty(75), reduced birth weight and reduced multiple birth rates which were reported to be sequela to the low mineral and protein supply to the bone marrow as a result of extensive liver damage characteristic of chronic fascioliasis.(76,77) However, the less dramatic, but considerable long- term deleterious effects include the associated chronic waste, which generally remains undetected, and yet, may significantly reduce productivity.(78)

The infection causes severe economic losses due to mortality, liver condemnation, reduction in milk and meat production.(40) In Egypt , according to the General Organization of Veterinary Services, Ministry of Agriculture in June 1998, the lost in meat and milk was 30% per year (= one milliard Egyptian Pounds).(23)

Diagnostic tools for fascioliasis:

1- Stool examination:

Diagnosis of Fasciola sp. infection has traditionally relied on detecting the presence of eggs in fecal samples.(79) The rapid, low cost and reproducible cellophane thick-smear technique (Kato–Katz)(80) has been used for diagnosis in most studies on fascioliasis.(81-83)

Intra and inter-specimen variability of Fasciola egg counts was investigated with the Kato–Katz technique. The sensitivity of the technique in the diagnosis of human fascioliasis was also studied and it was found that the sensitivity of the Kato–Katz test for diagnosing Fasciola infection with three Kato slides from the same specimen or on different days ranged from 96 – 99.1%, However, the examination of three Kato smears from a single stool specimen is more feasible in field studies.(84)

18 Egg concentration techniques may be used such as formol ether technique, but Kato–Katz technique is preferred for field surveys as it is more feasible.(85)

2- Serological methods:

The coprodiagnosis of fascioliasis is often unreliable because the eggs of the parasite are not found during the prepatent period. Even at later times, eggs are only intermittently released. Serologic diagnosis is preferred, particularly since antibodies to Fasciola can be detected as early as two weeks after infection and can facilitate early treatment.(86,87)

At present, the routine diagnosis of human fascioliasis is based on the detection of antifluke antibodies in serum. Methods such as immunoelectrophoresis(88) and counterimmunoelectrophoresis(89), although they are very specific, have limited sensitivity. The diagnosis was improved by the development of enzyme-linked immunosorbent assay (ELISA), using crude extracts(90), excretory-secretory products (91) and purified or recombinant molecules such as cathepsin L-1(92) and by the detection of circulating antigens and coproantigens by sandwich ELISA.(93) It is reasonable to assume that the micro-ELISA could be applied as a screening test when a large number of samples are involved, because of its low consumption of reagents (especially antigens and second-antibody conjugates).(79)

In a study in 2001, ELISA and micro-ELISA were evaluated for their ability to detect anti-Fasciola hepatica antibodies in humans by using excretory-secretory antigen. The sensitivity of each method was 100%, but the specificity was 100% for ELISA and 97% for micro-ELISA. The micro-ELISA could be used as a screening assay and ELISA could be used as a confirmatory method for the serodiagnosis of human fascioliasis.(79) On the other hand, indirect haemaglutination test (IHAT) was less sensitive and less specific.(94)

3- Detection of fecal antigens of Fasciola hepatica: Using a Western blot assay technique with hyperimmune serum obtained from excretory-secretory antigens of adult F. hepatica, it was found that there are certain antigens in the patients' feces of possible diagnostic interest. These polypeptides could be antigens common to both parasitic stages (mature and immature).(95)

4- Radiological findings: Sonography can be useful in the detection and follow-up of hepatic lesions in human fascioliasis and can facilitate the diagnosis of this condition, particularly in areas where it is endemic.(96) Sonographically, the hepatic lesions may appear as focal areas of increased echogenicity in the right lobe, multiple nodular or irregular lesions of variable echogenicity in both lobes, or a single complex mass in the right hepatic lobe.(96)

Crescent-shaped parasites could be seen in some patients; and the parasites could be recognized spontaneously moving. Postprandial sonographic examination reveals parasites adhering to the gallbladder wall. The gallbladder contents will be mobile but will not sediment downwards after patients changed position. Non-specific abnormalities may be found such as:

19 impaired gallbladder contractility, gallbladder tenderness, debris, calculi, wall thickening and bile duct dilatation. Biliary tract abnormalities are frequently observed by ultrasonography (US), but the detection-rate of Fasciola hepatica is disappointingly low despite the parasite's relatively large size. US findings must therefore be interpreted together with other clinical measurements. The visualization of parasites being expelled through the dilated common bile duct allows the causal interpretation of post-therapeutic abdominal pain and increase of liver enzymes. When triclabendazole is given on suspicion, visualization of worm expulsion and bile duct dilatation by US may be used to confirm diagnosis.(97)

In animal fascioliasis, both computerized tomography (CT) and magnetic resonence image (MRI) showed nodular lesions and MRI could also detect early tracks in the sub- capsular area. Ductal dilatation was shown by ultrasound, CT and MRI, although MRI was inferior to CT in depicting a mild ductal dilatation. Moving echogenic forms in the dilated bile ducts were observed by ultrasound from 9 weeks postinfection.(98) Treatment of fascioliasis will result in complete remission.(96)

Diagnosis of acute fascioliasis: In humans, the parasitic incubation (pre-patent) period has been estimated as 4 months. During this period, the immature parasites affect the liver and form necrotic areas that end in fibrosis.(99,100) Diagnosis of the acute phase of fascioliasis is important, as treatment is effective during this stage and can prevent the harmful sequelae of the disease.(101) Early in infection, eosinophilia, high antibody titres and high circulating antigen values are the means of diagnosis. With the appearance of ova in the stools, these values are significantly lowered. (102,103) During the acute phase, stool studies for ova are unhelpful, since the parasites cannot produce eggs before invasion of the biliary tree.(56)

Diagnosis of chronic fascioliasis: A definitive diagnosis of chronic fascioliasis can be made by finding characteristic eggs in feces. Repeated stool examinations may be necessary to find eggs. Serologic tests are essential to diagnose acute and chronic fascioliasis. These tests include the ELISA, indirect hemagglutination and complement fixation.(63)

Differential diagnosis of primary and secondary infections:

Antibody avidity refers to the strength of interaction of an antibody with a multivalent antigen. Depending upon the strength of this binding, the complex formed may or may not be dissociated. Antibody avidity is low after primary antigenic challenge, matures with time and it usually involves IgG antibodies.(104,105) Recently, an assay measuring the antigen-binding avidity of IgG antibodies has been developed to distinguish the low-affinity antibodies produced at an early stage of infection from those with a higher-binding affinity that reflects past immunity. This IgG avidity test has

20 been valuable with many pathogens as both a front-line assay and as a means of distinguishing primary from secondary infections [8-10].(106-108) It is also helpful in assessing the time of the initial antigenic challenge. Avidity index (AI) is an indicator of avidity. Therefore, a low index means low avidity while a high index denotes high avidity. (101)Abou Basha et al.(2000) reported that AI of 59.90% denoted acute infection and those > 59.90% denoted chronic infection.(101)

Treatment of fascioliasis

The most important criteria for a good anthelmentic drug are high efficacy, safety to the target individual, absence of persistent chemical residues in the host tissue and ease of application at an economic price.(109)

I- Old lines of treatment of facsioliasis:

1- Bithionol (Bitin):

Bithionol (2,2-thiobis 4,6-dichlorophenol) is the drug of choice in the treatment of paragonimiasis. It was used in treatment of fascioliasis in 1980s. (110)

For animals, the dose was 25 mg/kg body weight every other day for five doses.(1) for human cases, the recommended dose was daily dose of 30mg/kg body weight divided in three doses after meals and it was administered on alternate days for a total of five doses in 10 days. On such a regimen the Fasciola ova disappeared after the 2nd or 3rd dose giving a cure rate of 100%.(110) It is seemed to be acting through causing abnormalities in testes and egg production of the fluke.(111)

Side effects of bitionol therapy were mainly pain in the right hypochondrium, abdominal colic, nausea, vomiting, diarrhea, pruritis and urticaria.(110) Bihtionol also exerted a toxic effect on the liver cells during its metabolism. Abou Basha et al have reported that Bithionol administered to the non infected rabbits induced liver cell hypertrophy and early signs of necrosis. There was also increased serum transaminase activity more marked in SGOT, denoting early hepatic cell injury.(112)

2- Praziquantel:

Praziquantel was reported to have a broad antiparasitic effect, particularly on trematodes and cestodes. However, studies investigating its efficacy as a fasciolicidal drug revealed that it does not have good therapeutic results in fascioliasis.(113) At dose levels ranging from 40mg/kg body weight to 75mg/kg body weight it produced cure rate equals to 20%.(113)

21 3- Metronidazole:

The efficacy of metronidazole in treating fascioliaisis was studied, and the results revealed that at a dose of 1.5 g/day orally for three weeks the cure rate reached about 81% two months after end of therapy.(114) Most frequent side effects of metronidazole were metallic taste, headache and nausea.(114)

II- Currently used drug:

Triclabendazole (TCBZ):

Triclabedazole is a bezimidazole derivative that has been proved to be the most safe and effective anthelmentic for all stages of Fasciol sp. in animal and human infections.(115, 116)

It is worthy to note that the sulfoxide and the sulfone metabolites are the active species. (117, 115) It acts through inhibition of fumarate enzyme activity.(115)It can penetrate into the liver fluke through transtegumentary absorption leading to strong inhibition of motility.(115)

The cure rate was very high one month after treatment with a single oral dose of triclabendazole (10 mg/kg body weight). Taking the absence of eggs as the sole indicator of cure, the cure rate amounted to 94%. Considering both the absence of eggs and the absence of worms from the biliary passages by the ultrasonography, the cure rate was 76.5%.(118)

Triclabendazole and its two main metabolites (sulfoxide and sulfone) showed some oral toxicity, slower weight gain, lower haemoglobin level and elevated levels of plasma enzymes and liver cholesterol.(119-121)

The development of resistance against the most widely used product triclabendazole has been reported. Boray (122) was able to select on resistance of liver fluke against TCBZ in 1990. Resistance of liver fluke against TCBZ in sheep under practical conditions was initially reporte by Overend et al (1995)(123) in Australia with subsequent reportes from Irland (124,125) and Scotland(126) in 1998. This situation justifies the search for new effective drugs.(8)

III- Trials using new fasciolicidal drugs:

1- "3-Cyanopyridine Derivatives:"

Two series of 3-cyano-2-alkoxypyridine Va-h and 3-cyano-2-aminopyridine Vla-j derivatives carrying various substituents at position 4 and 6 were synthesized. These compounds were evaluated for fasciolicidal activity against Fasciola hepatica. They exhibited pronounced activity compared with reported data on triclabendazole.(115) In vetro exposure of

22 adult flukes to the tested compounds induced ultrastructural tegumental damage. The extent of damage was proportional to the concentration of the tested compound and to exposure time. (115)

2- Compound "Alpha" (5-chloro-2-methylthio-6-(1-napthyloxy)1H- benzimidazole):

It is a white powder with light characteristic odor. It is a novel compound related to the bencimidazole derivatives. Data suggest that the sulfoxide metabolite is the active principle responsible for its fasciolicidal activity in vivo.(127) It is formulated as a 10% drench suspension.(128) Ibarra et al (2004) reported that its efficacy in treating naturally and experimentally infected cattle can reach 100% in a dose of 12mg/kg/p.o.(129)

3- Nitazoxanide:

Nitazoxanide was originally discovered in the 1980s by Jean François Rossignol at the Pasteur Institute.(130) Initial studies demonstrated activity versus tapeworms. In vitro studies demonstrated much broader activity.(131). There have been several controlled trials of nitazoxanide for treatment of infection with intestinal helminths.(132) As shown in the study by Diaz, nitazoxanide is effective against Ascaris, Trichuris, and Hymenolepis.(133) However, some patients require repeated dosing. Other controlled trials have demonstrated some activity against chronic fascioliasis.(8)However, the response rates are lower than those described with triclabendazole (60% among adults and 40% among children(8). In all studies, nitazoxanide has been extremely well tolerated with adverse effects similar to placebo.(130)

4- Myrrh :

Myrrh is an olio gum resin obtained from the stem of Commiphora molmol (family: Burseraceae), a tree that grows in northeast Africa and the Arabian Peninsula. Myrrh contains 7-17% volatile oil, 25-40% resin, 57-61% gum and 3-4% impurities.(134)

Traditionally, Myrrh has been used by Sumerians and Greeks to treat "worms", by Chinese to relieve pain and swelling due to traumatic injury and by Somalians to treat stomach complaints, diarrhea and wounds.(135-137)In modern times, tincture of Myrrh is used for therapy of aphthous ulcer and for reduction of cholesterol and triglycerides.(135,138,139)Myrrh has also anticarcinogenic potentials.(140)

Myrrh is approved by the US Food and Drug Administration (FDA) for food use (21 Code of Federal Registration-CFR 172.510) and was given generally recognized as safe

23 (GRAS) status as flavor ingredient (no. 2765) by the Flavor Extract Manufacture's Association (FEMA).(141,142) The Council of Europe included Myrrh in the list of plants that are acceptable for the use in foods.(142,143).

Purified extract of Myrrh from C. molmol tree (Mirazid ®), a new herbal schistomicidal and fasciolicidal drug has been licensed in Egypt since March, 2002.(144)

In experimental studies on Swiss albino mice, Myrrh from C. molmol showed no mutagenicity, and was found to be a potent cytotoxic drug against Ehrlich solid tumor cells with no clastrogenic effect. The anti-tumor potential of C. molmol was comparable with that of the standard cytotoxic drug cyclophosphamide.(145)

Purified and characterized 8 sesquiterpene fractions from Commyphora molmol were extracted. In particular, a mixture of furanodiene-6-one and methoxyfuranoguaia-9-ene-8-one showed antibacterial and antifungal activity against standard pathogenic strains of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans, with minimum inhibitory concentrations ranging from 0.18 to 2.8 micrograms/ml. These compounds also had local anaesthetic activity, blocking the inward sodium current of excitable mammalian membranes.(146)

Antiparasitic effects of myrrh:

The efficacy of Myrrh was evaluated in treating sheep naturally infected with Moniezia expansa. Total doses of one, two or three capsules (300 mg each) were given for one, two, three, four, five, six, seven and eight successive days on an empty stomach an hour before offering their breakfast. Every day the stools of the three groups were examined microscopically and macroscopically for eggs and/or gravid segments. When the stool was negative, the treatment was stopped. The sheep group was examined macroscopically after being slaughtered for adult worms in intestine. A total dose of 3600 mg given as three capsules per days for four days gave a cure rate of 100.0% with no clinical side effects. A dose of 4800 mg given as two capsules per day for eight days gave a cure rate of 100.0% with no clinical side effects. On the other hand, a total dose of 2400 given as one capsule per day for eight days gave a cure rate 40.0%. Consequently, Myrrh extract of the medicinal plant, Commiphora molmol (Mirazid) proved to be safe and very effective in sheep monieziasis expansa.(147)

24 Assessment of Myrrh as a schistomicidal drug:

Contradictory results were reported as regard the schistomicidal effect of myrrh. While some researches reported its high efficacy, others reported low cure rate of Mirazid especially when compared to Praziquantel.(148)

Shatat et al conducted a controlled study in which three groups of recruits were treated for S. mansoni using Mirazid 600 mg for three consequative days, Mirazid 600 mg for six consequative days or preziquantel 40mg/kg body weight as a single dose. Cure was monitored 3 and 6 weeks post-treatment by parasitological stool examination using Kato-Katz and formol detergent techniques. Cure rates were 55.6% for 3 days Mirazid, 45.8% for 6 days Mirazid and 94.3% for Praziquantel when assessed 6 weeks post-treatment. (148)

Even lower cure rate was reported by Botros et al (2005), where Mirazid (in a dose of 300 mg/day for three consecutive days) showed low cure rates of 9.1% and 8.9% in S. mansoni-infected school children and household members, respectively, compared with cure rates of 62.5% and 79.7%, respectively, in those treated with praziquantel.(149)

In an experimental study of the potential antischistosomal activity of Myrrh, different derivatives of the resin, including the commercial preparation Mirazid, were tested at different doses in mice and hamsters infected with Schistosoma mansoni. In mice infected with the Egyptian (CD) strain of S. mansoni, four of six groups treated with Mirazid did not show significant worm reduction, while the remaining groups showed significant but trivial reductions. In mice infected with the Puerto Rican (Mill Hill) strain of S. mansoni, a Mirazid solution was toxic for mice at high doses and produced modest or no worm reduction at lower doses. In hamsters and mice infected with Puerto Rican (NMRI) and Brazilian (LE) strains of S. mansoni and treated with the crude extract of Myrrh in doses ranging from 180 to 10,000 mg/kg, no signs of antibilharzial activity were observed. Total tissue egg load and egg developmental stages were not affected by any of the treatment regimens.(150)

On the other hand, high cure rates were reported in other studies; seventy cases of schistosoma haematobium with different intensities were treated with Mirazid as 10 mgm/Kg. However, eight of them were unable to swallow the drug. The cure rate was 91.9% after two months follow up and reached 95.2% on the third month post-Mirazid treatment.(151)

In another study, the cure rate of 91.7% was reported after using Mirazid in a dose of 10 mg/kg of body weight/day for three days. Non responding cases were re-treated with a dose of 10 mg/kg of body weight/day for six days and gave a cure rate of 76.5%, increasing the overall cure rate to 98.09%.(152)The drug was well tolerated, and side effects were mild and

25 transient. Twenty cases provided biopsy specimens six months after treatment and none of them showed living ova.(152)

Therapeutic dose and cure rate of Myrrh in treatment of fascioliasis:

In humans:

A study was carried out and included 7 patients who were passing Fasciola eggs in their stools and treated with Myrrh. The drug (a formulation consisting of 8 parts of resin and 3.5 parts of volatile oils, all extracted from Myrrh) was given in a dose of 12 mg/kg per day for 6 consecutive days in the morning on an empty stomach. Patients were followed for 3 months. The therapy proved to be effective, with pronounced improvement of the general condition and amelioration of all symptoms and signs. A dramatic drop in the egg count was detected at the end of treatment. Eggs were no longer detectable in the feces 3 weeks after treatment and after a follow-up period of 3 months. High eosinophilic counts, elevated liver enzymes, and Fasciola antibody titers returned to nearly normal. No signs of toxicity or adverse effects were observed.(134)

In animals:

The efficacy of Mirazid was evaluated in sheep naturally infected with fascioliasis. Total doses of one or two capsules (300 mg each) were given for one, two or three successive days on an empty stomach an hour before breakfast. A total dose of 600 mg gave a cure rate of 83.3%, while a total dose of 900 to 1200 mg gave a complete cure rate (100%), with no clinical side effect. The cure rate was achieved by stool examination and/or macroscopically on slaughtering the sheep. Mirazid proved to be safe and very effective in sheep fascioliasis in Gharbia Governorate.(153)

Side effects of Myrrh :

Side effects of Myrrh 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, and abdominal pain or discomfort. Myrrh had no significant effects on liver functions, serum creatinine, or electrocardiographic findings, nor did it have any significant effects on liver and kidney functions in healthy volunteers. (152)

Effect on snails, eggs and shedding rate: Myrrh has molluscicidal effect on infected Bulinus truncatus and Biomphalaria alexandrina snails at low concentrations (10 & 20 ppm respectively) after 24 hours exposure. The number of dead-snails increased with prolongation of exposure time. One day-old egg masses were more susceptible to the ovicidal effect of Myrrh than the five-day old ones. Both types of eggs were more resistant to the effect of Myrrh than the adult snails, embryogenesis began to stop at 20 ppm and eggs were all killed at 60 & 80 ppm. Shedding of cercariae of

26 Schistosoma mansoni from infected B. alexandrina stopped at 1 ppm and was suppressed at 0.8 ppm. Snail fecundity decreased at 1 ppm.(154)

The molluscicidal properties of the oil extract of Commiphora molmol (Myrrh) were tested against Egyptian snail species: Biomphalaria alexandrina, Bulinus truncatus and Limnaea cailliaudi. The impact of the extract on the egg cluches of B. alexandrina and L. cailliaudi was also evaluated. The present laboratory studies demonstrated that Myrrh has a molluscicidal effect on the snail intermediate hosts, particularly on their eggs.(155)

Effect on mosquito larvae: Myrrh proved to have insecticidal activity against mosquito larvae. The oil extract of Myrrh possesses median lethal activity against 2nd, 3rd and 4th instar larvae of Culex pipiens. Histological examinations of Myrrh treated mosquito larvae showed great pathological effects on their fat, muscles, gut and nervous tissues.(156) The mosquitocidal plant extracts of the Myrrh, Commiphora molmol namely; oil and oleo-resin, were proved to demonstrate larvicidal activity against Culex pipiens larvae. Oleo- resin was found to induce significant higher toxic action than oil. Solvent (cremophore EL) did not perform any toxic activity. The Impact of both oleo-resin and oil extracts on the protein profile of the treated larvae was evaluated in order to explain their mode of action. Electrophoretic analysis of total proteins, lipoproteins and glycoproteins revealed inhibitory action of the used plant extracts on the protein contents. Larvicidal activity of the oleo-resin and oil was explained as to be related to the loss of certain enzymes inhibited by these extracts which affect the metabolic processes.(157)

27 AIM OF THE WORK

General objective: To study the effectiveness of the fasciolicidal drug Myrrh in treatment of human and animal fascioliasis.

Specific objectives: 1- To estimate the cure rate of human and animal fascioliasis treated with Myrrh.

2- To determine the effect of Myrrh on the intensity of Fasciola infection.

3- To investigate changes in serum levels of human liver transaminases in patients with chronic fascioliasis before and after receiving treatment with Myrrh.

28 SUBJECTS AND METHODS

(A) Study setting: Satellite number 10 of Abis VIII village was selected for the present study. The village is located 15 km south east of Alexandria city and it includes 12 satellites. Satellite number 10 was selected by simple random sampling.

(B) Study duration: The present study was carried out in the period from June 2005 to February 2006.

(C) Study design: An intervention study: Inhabitants of satellite number 10 of Abis VIII village aged 5 years and more and their animals were screened for fascioliasis. Then all human and animal cases of fascioliasis were treated by myrrh and followed up for three months to determine its effectiveness.

(D) Study procedure: Mapping was done by the researcher with the help of a cartographer. Houses, streets, mosques and landmark sites were all located on the village map. Houses were numbered serially and a census updating was carried out by house-to-house visits. The total population amounted to 1305 individuals, 138 of them were children under 5 years and were excluded from the study. All herbivorous animals present in the selected area were included in the present study (cows, baffaloes, sheep, goats and donkeys) .Total animal population was 208 animals.

Human study:

1- Data collection: a- Questionnaire: A predesigned questionnaire was filled for all study population. The questionnaire included: - Personal and socio-demographic data as age, sex and family size. - Presence of household animal contact - Data concerning Fasciola sp. infection as past history of infection, past history of any treatment of fascioliasis and presence of suggestive symptoms of fascioliasis as abdominal colic, fever, nausea, jaundice and presence of blood in stool.

b- Stool examination for screening of fascioliasis: All inhabitants of the selected satellite aged 5 years or more were screened for fascioliasis. A stool sample was collected from each individual in a pre-labeled tight proof plastic cups. The cups were distributed on one day and collected on the next day. Collected samples were transported to the parasitological lab at the High Institute of

29 Public Health (HIPH). Total number of human samples examined was 1092. Three Kato- Katz thick smears each of 41.7 mg were prepared from each sample and examined microscopically. Helminthic ova were recorded, but only Fasciola eggs were counted.(158) Number of eggs/gm stool (epg) was calculated as mean number of eggs in the three slides and multiplied by 24.(158)

To exclude false fascioliasis, positive cases were asked for a second stool sample after one week of liver free diet. Samples were examined by Kato-Katz technique (three slides from each sample).(158)

c) Blood investigations: Two ml of blood sample was collected from diagnosed cases of fascioliasis before and one month after treatment to measure serum levels of ALT and AST.(159)

2- Therapeutic intervention: a- Determination of sample size: To evaluate the effectiveness of the new drug Myrrh against fascioliasis, the required sample size was calculated to be 60 positive human cases based on 10% percision and 85% cure rate as reported by Massoud et al.(134) Inclusion criteria in the therapeutic study were patients infected with fascioliasis who had not received, within the past 4 weeks any drug with anthelminthic activity. b- Treatment of human cases of fascioliasis with Myrrh : Sixty four cases of fascioliasis diagnosed in Satellite number 10 of Abis VIII village were treated using Myrrh. Mirazid capsules (300mg/capsule) were used in a dose 10mg/kg body weight per day for 6 consecutive days in the morning on an empty stomach. The patients were instructed not to have breakfast for one hour after receiving treatment. The maximum single dose was considered as 600mg/day.(160) c- Assessment of cure rate of Myrrh and follow up of treated cases: The cure rate of Myrrh was assessed on three occasions; after one, two and three months post treatment. On each occasion, three stool samples were collected from each treated case with 24 hours interval to increase the sensitivity of the Kato-Katz technique.(84) The end-point used for evaluating the drug effectiveness was negative parasitological stool examination.

The percent change of egg count was calculated for subjects still passing Fasciola eggs applying the following formula: {No. of epg before treatment - No. of epg after treatment}/ No. of epg before treatment X 100

30 Animal study:

1- Data collection: a- Questionnaire: Data concerning species, age, and sex of each animal and method of disposal of animal excreta were collected from animal owners. b- Stool examination for screening for fascioliasis: All herbivorous animals present in the selected satellite were screened for fascioliasis. Animal stool samples were collected during field visits by a laboratory worker through rectal examination and were placed in a tight proof plastic cups labeled by house number, animal species and any obvious mark on each animal. Collected samples were transported to the Parasitology laboratory at the HIPH. Three Kato-Katz thick smears each of 41.7 mg were prepared from each sample and examined microscopically and helminthic ova were recorded, but only Fasciola eggs were counted.(158) Number of eggs/gm stool (epg) was calculated as mean No. of eggs in the three slides and multiplied by 24. Total number of animal samples examined was 119.

2- Therapeutic intervention: a- Determination of the sample size: To evaluate the effectiveness of the new drug Myrrh against fascioliasis, the required sample size was calculated to be 60 positive cases of herbivorous animals based on percision equal 10% and a cure rate of 85% as reported by Haridy F et al., in studying the effectiveness of this drug in animals.(153) b- Treatment of animal cases of fascioliasis with Myrrh : Sixty three animal cases of fasciolaisis were treated using Mirazid suspension 10% in a dose 600mg/day for 2 consecutive days in the morning on an empty stomach.(153) c- Assessment of cure rate of Myrrh : The cure rate of Myrrh was assessed on three occasions; after one, two and three months post treatment. On each occasion, three stool samples were collected from each treated animal case with 24 hours interval for detecting and counting Fasciola sp. eggs.(8) The percent change of egg count was calculated for animals still passing Fasciola eggs applying the following formula: {No. of epg before treatment - No. of epg after treatment}/ No. of epg before treatment X 100

31 E- Statistical analysis: Data collected were coded; tabulated and statistical analysis was done using the statistical package of social science (SPSS) version 9.0 and Epi-info version 6.4. The applied tests were Chi-square, Monte Carlo, Fisher's Exact, t-test, ANOVA, paired samples t-test and crude odds ratio (OR). An adjusted odds (OR) with a 95% confidence interval (CI) that did not include 1.0 was considered significant.

For non-parametric data Wilcoxon signed ranks test was used.

0.05 level was used as a cutoff point of significance.

32 RESULTS

This study is an intervention study in which all inhabitants of satellite number 10 of Abis VIII village aged 5 years and more and their animals were screened for fascioliasis. Then all human and animal cases of fascioliasis were treated by myrrh and followed up for three months to determine its effectiveness. The total number of human samples examined was 1092; out of them 64 cases of fascioliasis were diagnosed. The total number of animal samples examined was 119 and 63 cases of fascioliasis were diangosed.

The results of the present study are classified as following: I- Human study: A) Base line study B) Therapeutic trial

II- Animal study: A) Base line study B) Therapeutic trial

33 I- Human study

A- Base line study:

Figure (2) shows a diagram of the pre-intervention phase of the study. It illustrates that the total population of the village was 1305 individuals; 138 of them were excluded from the study, as they were less than 5 years of age. Accordingly, the target population was 1167 individuals; 93.6% of the target population (1092 individuals) cooperated in the present study and the remaining 75 individuals did not submit stool samples. After exclusion of false positive fascioliasis, stool analysis revealed that the prevalence of chronic fascioliasis was 5.8%.

Table (I) demonstrates some characteristics of the population of satellite 10, Abis 8 village, Alexandria Governorate. Nearly 1/5 of the village inhabitants were children aged less than 10 years. Adolescents constituted 26.4%, while old aged individuals of 50 years or more were only 11.5% of that population. Male to female ratio was nearly 1:1. As regards family size, about 1/2 of the population belonged to large families of 9 or more members.

Table (II) displays the distribution of helminthic infection among the target population, where 10.9% were found to harbour helminthic ova. The most prevalent infection was S. mansoni (7%) followed by Fasciola sp. (5.8%). Other helminthic infections (Ascaris lumbricoides, Hymenolepis nana, Enterobius vermicularis and Trichuris trichiura) had low prevalences in the target population (less than 1% each).

34 noitalupop latoT 5031=n

<5 years n=138 (excluded)

Target population n=1167

did not give stool samples 75

Study population

)n=1092(93.6%

Fascioliasis )n=68 (6.2% Liver free diet for 1 week, repeat stool analysis eurT sisailicsaf 46=n %8.5))

Figure (2): A diagrammatic illustration of the pre-intervention phase of the study

35 Table (I): Demographic characteristics of the population of satellite 10, Abis 8

NO. Variable % (n=1305) Age (years) <5 138 10.6 5- 141 10.8 10- 345 26.4 20- 200 15.3 30- 161 12.3 40- 170 13.1 ≥50 150 11.5

Sex Male 642 49.2 Female 663 50.8

Family size ≤4 members 280 21.4 5-8 members 417 31.9 9 or more members 608 46.7

36 Table (II): Distribution of helmonthic infection among the target population of satellite 10, Abis 8 village

Helminthic infection No. % (n=1092)* Free 973 89.1 Helminthic infection: 119 10.9 Schistosoma mansoni 76 7.0 Fasciola sp. 64 5.8 Ascaris lumbricoides 7 0.6 Hymenolepis nana 7 0.6 Enterobius vermicularis¶ 6 0.5 Trichuris trichiura 6 0.5

 Data are not mutually exclusive * Children <5 years were excluded from the study ¶ This prevalance is expected to be underestimated due to the use of non specific technique for Enterobius vermicularis

37 Table (III) demonstrates some characteristics of cases infected with Fasciola sp. As regard age, it was found that the highest prevalence of fascioliasis infection was among children 5-<10 years (17.5%), who were significantly at higher risk of having fascioliasis by 5.5 times than other age groups, while the least prevalence was among adults 30-<40 years old (3.7%). As regard sex, females were significantly more affected with fascioliasis than males (7.5% of females had fascioliasis versus 4.3% of males) and they had significant increased risk of having fascioliasis of about two folds than males (OR=1.79, CI (1.03-3.11), p<0.05). As regards family size, it was found that the highest prevalence of fascioliasis infection was among large families of 5-8 members (8.3%). It was also found that those having household animal contact had significant 2 folds increased risk of having fascioliasis than those who do not have household animal contact (OR= 2.3, CI (1.34-3.95), P<0.05).

Table (IV) demonstrates distribution of fascioliasis according to age and sex. It was found that in adults aged 20-<40 years all cases of fascioliasis were females. This was statistically significant. However, the highest prevelance of fascioliasis was found among male children aged 5-<10 years (21.8%).

38 Table (III): Some characteristics of cases infected with Fasciola sp.

Fasciola sp. Infection (n=1092) Variable OR (95%CI) Free Infected No. (%) No.(%) Age group 5- 94 (82.5%) 20 (17.5%) 5.5(1.99-15.92)* 10- 325 (95.9%) 14 (4.1%) 1.11(0.39-3.31) 20- 166 (95.4%) 8 (4.6%) 1.24(0.38-4.15) 30-® 155 (96.3%) 6 (3.7%) 1 40- 154 (93.9%) 10 (6.1%) 1.68(0.54-5.34) ≥50 years 134 (95.7%) 6 (4.3%) 1.16(0.32-4.16)

Sex Male® 532 (95.7%) 24 (4.3%) 1 Female 496 (92.5%) 40 (7.5%) 1.79(1.03-3.11)* Family size 3-4 members® 261 (95.6%) 12 (4.4%) 1 5-8 members 333 (91.7%) 30 (8.3%) 1.96(0.94-4.14) 9 or more members 434 (95.2%) 22 (4.8%) 1.1(0.51-2.41)

Household animal contact No 756 (95.6%) 35 (4.4%) 1 Yes 272 (90.3%) 29 (9.7%) 2.3(1.34-3.95)* *P<0.05

OR = Odds ratio CI = Confidence interval

39 Table (IV): Distribution of fascioliasis according to age and sex

Fasciola sp. infection

Age group Male Female Fisher's Exact test (in years) Free Infected Free Infected No.(%) No.(%) No.(%) No.(%)

43 12 51 8 5- P=0.32 (78.2%) (21.8%) (86.4%) (13.8%)

177 4 148 10 10- P=0.05 (97.8%) (2.2%) (93.7%) (6.3%)

79 0 87 8 20- P=0.008* (100%) (0%) (91.6%) (8.4%)

77 0 78 6 30- P=0.02* (100%) (0%) (92.9%) (7.1%)

77 4 77 6 40- P=0.74 (95%) (5%) (92.8%) (7.2%)

79 4 55 2 50 or more P=1.000 (95.2%) (4.8%) (96.5%) (3.5%)

* p<0.05

40 Figure (3) illustrates the prevalence of Fasciola sp. infection among different age groups. It shows that the peak of prevalence of fascioliasis occurred at age group 5-<10 years (17.5%) and it decreased as the age increases till becoming 4.3% among old age group aged 50 or more years.

20 17.5 % 15

10 4.6 4.1 6.1 5 3.7 4.3 0 5- 10- 20- 30- 40- 50 or more Age groups (in years)

Figure (3): Prevalence of Fasciola sp. infection among different age groups

41 Figure (4) illusterates intensity of infection with Fasciola sp. in different age groups. It was found that there were two peaks of the GMEC at the two extreme age groups; the highest GMEC was among age group 5-<10 years (114.46 epg) and the other peak was among old age group aged 50 years or more (90.2 epg).

GMEC 140

100 114.46 8092.9

56.61 54.83 40 49.77 20 23.21

0 5- 10- 20- 30- 40- 50 or more

Age groyps (in years)

Figure (4): Intensity of infection with Fasciola sp.in different age groups

42 Table (V) demonestrates distribution of families living in the satellite No.10, Abis 8 village according to the presence of Fasciola sp. infection. It was found that out of the 175 families living in the village, 1/4 of them (43 families) had at least one Fasciola sp. infected member. It was also found that about 1/3 of infected families (13 families) had two or three members infected with fascioliasis.

Table (VI) shows distribution of Fasciola sp. infected cases according to the presence of other helminthic infections. It was found that most cases of fascioliasis (59.4%) had single infections, while 37.5% of cases had co-infection with S. mansoni and only 3.1% were co- infected with E. vermicularis.

Table (VII) shows clinical picture of chronic fascioliasis. It was found that most cases were asymptomatic (64.5%). The most common symptoms in descending order of frequency were abdominal colic (24.1%), nausea (4.8%), while only 3.2% of cases had jaundice or blood in stool.

Table (VIII) demonstrates distribution of Fasciola infected cases according to intensity of infection and geometric mean of egg count (GMEC) before treatment. It was found that most cases (75%) had light infection (1-100 egg per gram (epg)), while the remaining 25% were equally distributed between moderate and heavy intensity of infection (12.5% each). The geometric mean of egg count (GMEC) of all cases was 63.94.

43 Table (V): Distribution of families living in the satellite No.10, Abis 8 village according to the presence of Fasciola sp. infection

Families living in the village No. %

Free 132 75.4

Fasciola sp. infected: One or more family member 43 24.6 Only one family member 30 17.2 Two or three family members 13 7.4

Total 175 100

44 Table (VI): Distribution of Fasciola sp. infected cases according to the presence of other helminthic infection

No. Fasciola sp. Infection % (n=64)

Fasciola sp. 38 59.4

Fasciola sp. and S. mansoni 24 37.5

Fasciola sp. and E. vermicularis¶ 2 3.1

¶ The rate may be underestimated because of using insensitive technique for E. vermicularis diagnosis

45 Table (VII): Clinical picture of chronic fascioliasis

Cases of fascioliasis Symptom (n=62)* No. %

Asymptomatic 40 64.5

Colic 15 24.1

Nausea 3 4.8

Jaundice 2 3.2

Blood in stool 2 3.2

* There was missing data for 2 cases

46 Table (VIII): Distribution of Fasciola sp. cases according to intensity of infection and geometric mean of egg count (GMEC) before treatment

Intensity of infection (epg)* No. (%) GMEC

Light (1-100) 48 (75%) 34.12

Moderate (101-400) 8 (12.5%) 177.48

Heavy (>400) 8 (12.5%) 998.34

Total 64 (100%) 63.94

* epg= egss/ gram stool

47 Table (IX) demonstrates some parameters associated with intensity of infection of human fascioliasis in satellite 10 in Abis 8 village. As regard age, it was found that the highest prevalance of heavy infection was among children aged 5-<10 years (30%) who also had the highest geometric mean of egg count (GMEC) of 114.46 compared to other age groups, while the least GMEC was found in adults aged 20-<30 years old (23.21) and all of them had light infections. These differences were statistically significant (Monte Carlo test P=0.016). As regards sex, it was found that heavy infection with fascioliasis was more prevalent among females than males (15% of infected females had heavy infection versus 8.3% of infected males). However, this difference was not statistically significant (Monte Carlo test P=0.672). As regards presence of symptoms, it was found that most of asymptomatic cases were lightly infected with fascioliasis (80%), they also had a lower GMEC compared to symptomatic cases (50.66 versus 94.28 respectively). However this difference was not statistically significant. Finally, it was found that all small families (≤4 members) had light infection and they had the least GMEC (42.65). This was statistically highly significant (Monte Carlo test P=0.000).

48 Table (IX): Some parameters associated with intensity of infection of human fascioliasis Variable Intensity of infection Monte Carlo test Light Moderate Heavy GMEC Age: 5- 10 (50.0%) 4(20.0%) 6(30.0%) 114.46

10- 12(85.7%) 2(14.3%) 0(0%) 49.77 Monte Carlo test 20- 8(100.0%) 0(0%) 0(0%) 23.21 P=0.016* 30- 6(100.0%) 0(0%) 0(0%) 56.61

40- 8(80.0%) 0(0%) 2(20.0%) 54.83 0(0%) 50 or more 4(66.7%) 2(33.3%) 92.90

Sex: Male 18(75.0%) 4(16.7%) 2(8.3%) 64.59 Monte Carlo test P=0.672 Female 30(75.0%) 4(10.0%) 6(15.0%) 63.56

Presence of symptoms: Monte Carlo test Asymptomatic 32(80.0%) 4(10.0%) 4(10.0%) 50.66 P=0.469

Symptomatic 16(66.7%) 4(16.7%) 4(16.7%) 94.28

Family size: ≤ 4 members 12 (100.0%) 0 (0%) 0 (0%) 42.65 Monte Carlo test 5 -8 members 18 (60%) 6 (20%) 6 (20%) 92.97 P=0.027*

9 or more members 18 (81.8%) 2 (9.1%) 2 (9.1%) 47.87

*P<0.05 Results of ANOVA test for GMEC were: For age F=1.73 For sex F=0.459 For presence of symptoms F= 0.171 For Family size F=2.278

49 B- Therapeutic trial:

Table (X) and figure (5) show the parasitological cure rates one, two and three months after treatment of cases with Mirazid. The cure rate increased from 53.8% in the first month to 81.5% in the second month and reached its maximum in the third month to become 88.2%.

Table (XI) shows some sociodemographic factors associated with parasitological cure in human cases treated with Mirazid after one, two and three months of treatment. As regard age, it was found that except for adults aged 20-<40 years all other age groups revealed continous increase in the cure rate throughout the three months of follow up and reached 100% after three months of treatment. As regard sex, it was found that both males and females revealed continous increase in the cure rate throughout the three months of follow up, however, males had better response to the treatment regimen than females as all males involved in the study were cured after three months versus 84.6% of females, however these differences were not statistically significant (Fisher's Exact Test P=0.55). As regard family size, it was found that small family size had a good impact on treatment outcome as all cases belonged to small families of 3-4 members only were completely cured after one month of treatment, while those belonged to large families had cure rate of only 40% after one month and it increased gradually to become 81.8% after three months.

50 Table (X): Parasitological cure rates one, two and three months after treatment with Mirazid

Duration after Cases of fascioliasis treatment Cured Not cured No. % No. % 1 month (n=52) 28 53.8 24 46.2

2 months (n=54) 44 81.5 10 18.5

3 months (n=34) 30 88.2 4 11.8

100 90 cure rate 88.2 80 70 81.5 60 53.8 50 40 30 20 10 0 1 Month 2 Months 3 Months Duration after treatment

Figure (5): The parasitological cure rates one, two and three months after treatment of human cases with Mirazid

51 Table (XI): Some sociodemographic factors associated with parasitological cure in human cases treated with Mirazid after one, two and three months of treatment

Cure rate after one Cure rate after two Cure rate after three month months months (n=52) (n=54) (n=34) Variable Cured Uncured Cured Uncured Cured Uncured cases cases cases cases cases cases No. (%) No. (%) No. (%) No. (%) No. (%) No. (%) Age group 5- 10 (55.6%) 8 (44.4%) 18 (90%) 2 (10%) 10 (100%) 0 (0%) 10- 2 (16.7%) 10 (83.3%) 8 (80%) 2 (20%) 4 (100%) 0 (0%) 20- 2 (100%) 0 (0%) 6 (100%) 0 (0%) 2 (50%) 2 (50%) 30- 4 (66.7%) 2 (33.3%) 4 (66.7%) 2 (33.3%) 4 (66.7%) 2 (33.3%) 40- 8 (80%) 2 (20%) 6 (75%) 2 (25%) 8 (100%) 0 (0%) 50 or more 2 (50%) 2 (50%) 2 (50%) 2 (50%) 2 (100%) 0 (0%) Monte Carlo test Monte Carlo test Monte Carlo test Test of significane P= 0.012* P= 0.256 P= 0.031* Sex Male 8 (40%) 12 (60%) 14 (70%) 6 (30%) 8 (100%) 0 (0%) Female 20 (62.5%) 12 (37.5%) 30 (88.2%) 4 (11.8%) 22 (84.6%) 4 (15.4%) Fisher's Exact test Fisher's Exact test Fisher's Exact test Test of significane P=0.156 P=0.147 P=0.55 Family size 3-4 members 12 (100%) 0 (0%) 12 (100%) 0 (0%) 12 (100%) 0 (0%) 5 or more members 16 (40%) 24 (60%) 32 (76.2%) 10 (23.8%) 18 (81.8%) 4 (18.2%) Fisher's Exact test Fisher's Exact test Fisher's Exact test Test of significane P=0.000* P=0.094 P=0.27 *p<0.05

52 Table (XII) demonestrates different parameters associated with parasitological cure in human cases treated with Mirazid after one, two and three months of treatment. As regard presence of symptoms, it was found that all cases revealed continous increase in the cure rate throughout the three month of follow up till reached its maximum in the third month; however, being clinically free was a good prognostic sign as all asymptomatic cases were completely cured after three months of treatment versus 71.4% of symptomatic cases. As regard household animal contact, it was found that the maximum cure rate was obtained after three months of treatment. At the third month, it was found that 94.7% of cases that had no household animal contact were completely cured versus 80% in those who had, but this difference was not statistically significant (Fisher's Exact Test P=0.29). Finally, as regard intensity of infection before treatment it was found that after three months of treatment the GMEC before treatment was lower among cured cases than those uncured throughout the three months of follow up (47.78 versus 96.12 in the first month, 49.69 versus 60.28 in the second month and 50.06 versus 70.99 in the third month respectively), but this was not statistically significant.

53 Table (XII): Different parameters associated with parasitological cure in human cases treated with Mirazid after one, two and three months of treatment

Cure rate after one Cure rate after two Cure rate after three month months months (n=52) (n=54) (n=34) Variable Cured Uncured Cured Uncured Cured Uncured cases cases cases cases cases cases No. (%) No. (%) No. (%) No. (%) No. (%) No. (%) Presence of symptoms 20 32 20 (100%) 0 (0%) 12 (37.5%) 6 (15.8%) Asymptomatic (62.5%) (84.2%) 10 4 12 (60%) 4 (25%) Symptomatic 8 (40%) 12 (75%) (71.4%) (28.6%) Fisher's Exact test Fisher's Exact test Fisher's Exact test Test of significane P=0.156 P=0.459 P=0.02* Household animal contact No 16 28 18 Yes (53.3%) 14 (46.7%) (93.3%) 2 (6.7%) 1 (5.3%) (94.7%) 12 10 (45.5%) 16 8 (33.3%) 3 (20%) 12 (80%) (54.5%) (66.7%) Fisher's Exact test Fisher's Exact test Fisher's Exact test Test of significane P=1.000 P=0.016* P=0.29 Intensity of infection before treatment Light 36 26 4 24 (60%) 16 (40%) 8 (18.2%) Moderate (81.8%) (86.7%) (13.3%) 0 (0%) 4 (100%) 2 (50%) Heavy 2 (50%) 0 (0%) 0 (0%) 4 (50%) 4 (50%) 0 (0%) 6 (100%) 4 (100%) 0 (0%) Monte Carlo test Monte Carlo test Monte Carlo test Test of significane P= 0.012* P= 0.055 P= 1.000 GMEC before 47.788 96.129 49.692 60.285 50.06 70.99 treatment ANOVA test for F= 2.269 F=0.638 F= 0.233 GMEC *p<0.05

54 Table (XIII) and figure (6) show the geometric mean egg counts (GMEC) of uncured cases of fascioliasis before and after treatment. It reveals that, for uncured cases there was a marked reduction in GMEC throughout the three months of follow up from 96.12 before treatment to 34.28 in the first month, 30.61 in the second month and 44.89 in the third month and these differences were significant in the first and second month.

Table (XIV) shows the intensity of infection of uncured human cases before and one month after treatment. It reveals that among the 24 uncured cases there were 16.7% who had moderate intensity of infection before treatment and all of them became lightly infected one month after treatment. As regard heavily infected cases before treatment, half of them became lightly infected. Percent reduction of egg count of uncured cases was 20%.

Table (XV) shows the intensity of infection of uncured human cases before and two months after treatment. It reveals that 20% of uncured cases had moderate intensity of infection before treatment and all of them became lightly infected two months after treatment. Percent reduction of egg count of uncured cases was 42%.

Table (XVI) shows the intensity of infection of uncured human cases before and three months after treatment. It reveals that all uncured cases were lightly infected before and three months after treatment with percent reduction of egg count equals 30%.

55 Table (XIII): Geometric mean egg counts (GMEC) of uncured cases of fascioliasis before and after treatment

After treatment Before Intensity of infection treatment 1 month 2 month 3 month

GMEC 96.12 34.28 30.61 44.89 Wilcoxon signed ranks test Z=-3.794* Z=-2.558* Z=-1.414 *P<0.05

100 96.12

C 75 E M

G 50 34.28 44.89

25 30.61

0 Pretreatment 1 Month 2 Months 3 Months Duration

Figure (6): The geometric mean of egg count (GMEC) in uncured human cases of fascioliasis before and after treatment

56 Table (XIV): Intensity of infection of uncured human cases before and one month after treatment

Intensity of infection Before treatment One month after treatment

16 22 Light 66.7% 91.7% 4 0 Moderate 16.7% 0% 4 2 Heavy 16.7% 8.3% 24 24 Total 100% 100%

Percent reduction of egg count 20%

57 Table (XV): Intensity of infection of uncured human cases before and two month after treatment

Intensity of infection Before treatment After two months of treatment

8 10 Light 80% 100% 2 0 Moderate 20% 0% 0 0 Heavy 0% 0% 10 10 Total 100% 100%

58 Table (XVI): Intensity of infection of uncured human cases before and three month after treatment

Intensity of infection Before treatment After three months of treatment

4 4 Light 100% 100% 0 0 Moderate 0% 0% 0 0 Heavy 0% 0% 4 4 Total 100% 100%

59 Table (XVII) demonstrates ALT level before and one month after treatment in cases of fascioliasis according to presence of symptoms. It reveals that ALT level was within normal values before treatment in both symptomatic and asymptomatic cases with a non-significant reduction in all cases 1 month after treatment.

Table (XVIII) demonstrates AST level before and one month after treatment in cases of fascioliasis according to presence of symptoms. It reveals that AST level was within normal values before treatment in both symptomatic and asymptomatic cases with a significant reduction 1 month after treatment in asymptomatic cases from 35.4±1.07 to become 32.4±11.1 (t=2.3, P<0.05).

60 Table (XVII): ALT level before and one month after treatment in cases of fascioliasis according to presence of symptoms

Pre- ALT level Post-intervention Paired samples t-test intervention

Asymptomatic cases 34.24±9.4 33.6±10.5 t=0.48

Symptomatic cases 33.45±17.8 30.7±14.3 t=1.1

Total 33.9±13.3 32.4±12.1 t=1.2

P >0.05

Table (XVIII): AST level before and one month after treatment in cases of fascioliasis according to presence of symptoms

Pre- Post- AST level Paired samples t-test intervention intervention

Asymptomatic cases 35.4±1.07 32.4±11.1 t=2.3* Symptomatic cases 32.1±13.1 31.2±14 t=0.52

Total 34.1±11.9 31.9±12.2 t=2*

*P<0.05

61 II- Animal study

A- Base line study:

Figure (7) shows a diagrammatic illustration of the pre-intervention phase of the animal study. Total animal population present in the village was 208 animal; 57.2% of them (119) were subjected to stool analysis; the remaining 42.8% were not involved because their owners refused to cooperate in the study. The prevalence of animal fascioliasis in the study sample was 52.9%.

Table (XIX) demonstrates the characteristics of animal population in Abis VIII village. It was found that most of them were cows (41.8%), 1/4 of them were buffaloes, 13% were goats, 10.6% were donkeys and only 9.6% were sheep. In that village there were no camels. As regard age, it was found that buffaloes and donkeys were older than other types (mean ages were 3.7 ±1.9 and 3.7±1.5 respectively) and sheep had the lowest mean of age (2.4 ±1.2). As regard sex, as expected, most of them were females (76%) older than males (3.7±1.8 versus 2.8±1.5).

Table (XX) demonstrates percentage of fascioliasis among animal species in satellite 10, Abis 8 villge. It was found that the highest percentage of fascioliasis was among buffaloes (65.6%) followed by cows (55.6%) and the least prevalence was among goats (12.5%). This difference was statistically significant (Monte Carlo test, P=0.04).

62 Total animal population n=208

Did not give stool samples n=89

Sample size n= 119 Response rate =57.2%

Cases of fascioliasis )n=63(52.9%

Figure (7): A diagrammatic illustration of the pre-intervention phase of the animal study

63 Table (XIX): Characteristics of animal population in Abis VIII village

Variable NO. Mean age ± S.D % (n= 208) (in years) Animal species: Buffalo Cow Goat 52 25.0 3.7 ±1.9 Sheep 87 41.8 3.6 ±1.9 Donkey 27 13.0 3.1 ±1.5 Total 20 9.6 2.4 ±1.2 22 10.6 3.7±1.5 208 100 3.5 ± 1.8

Sex: Male 50 24.0 2.8±1.5 Female 158 76.0 Total 3.7±1.8 3.5±1.8 208 100

64 Table (XX): percentage of fascioliasis among animal species in satellite 10, Abis 8 villge, Alexandria

Fasciola sp. infection Animal species Test of significance Free Infected

Buffalo 11 (34.4%) 21 (65.6%)

Cow 24 (44.4%) 30 (55.6%)

Monte Carlo test Goat 7 (87.5%) 1 (12.5%) P= 0.04*

Sheep 5 (50%) 5 (50%)

Donkey 9 (60%) 6 (40%)

*P<0.05

65 Table (XXI) demonstrates Fasciola sp. infection according to mean age of different animal sp. It reveals that infected animals were generally younger than non-infected animals (mean age was 3.2±1.8 versus 3.4±1.9 respectively), however this was not statistically significant.

Figure (8) illustrates prevalence of animal fascioliasis among different age groups. It demonstrates that the highest prevalence was found among younger age group of less than one year old (83.3%) and the prevalence decreased with increasing age, however this was not statistically significant ( Monte Carlo Test P= 0.42).

Table (XXII) demonstrates distribution of Fasciola sp. infected animals according to animal sex. It reveals that female animals were more affected with fascioliasis than males (54.3% of them were infected versus 48%% of males, X2=0.3, P>0.05).

66 Table (XXI): Fasciola sp. infection according to mean age of different animal sp.

Mean age of studied animals Animal species t Test Free Fasciola sp. infection

Buffalo 3.2±1.9 3.1±1.7 t=0.05

Cow 3.5±2.2 3.6±1.9 t=0.00

Goat 3.4±1.3 3.0 t=0.08

Sheep 2.0±0.7 2.2±1.7 t=0.05

Donkey 4.2±1.4 3±1.7 t=2.04

90 85 83.3 80 75 70 65 % 60 53.8 55 54.8 50 47.7 45 40 35 30 <1 year 1- 3- 5 years or more

Figure (8): Prevalence of animal fascioliasis among different age groups

67 Table (XXII): Distribution of Fasciola sp. infected animals according to animal sex

Fasciola sp. infection Free Infected Sex Chi square test No. % No. %

Males 13 52.0 12 48.0

Females 43 45.7 51 54.3 X2=0.3

Total 56 47 63 53 p>0.05

68 Table (XXIII) shows intensity of infection of animal fascioliasis according to animal type and sex. As regard animal type it was found that the highest prevalence of heavy infection was among sheep (80%) that also had the highest geometric mean of egg count (GMEC) compared to other species (531.27), while the least GMEC was found among donkeys (41.71) that all had light infection. All these differences were statistically significant (Monte Carlo test P=0.01). As regard sex, it was found that females were more affected than males as 70.6% of them had moderate or heavy infection versus 41.7% of males and they also had higher GMEC compared to males (275.57 versus 123.65 respectively), however, these differences were not statistically significant (Monte Carlo test P=0.1 , ANOVA test F=1.35, P>0.05).

Figure (9) shows the GMEC among different age groups of infected animals. It was found that there was a significant association between younger age groups and higher GMEC, where the highest GMEC was among animals <1 year old (905.78) and it decreased significantly as the age increased till becoming 188.93 among animals aged 5 years or more (ANOVA test for GMEC : F=3.5*).

69 Table (XXIII): Intensity of infection of animal fascioliasis according to animal type and sex

Intensity of infection Test of Variable Light Moderate Heavy GMEC significance No. (%) No. (%) No. (%)

Animal type

Buffalo 7 (33.3%) 2 (9.5%) 12(57.2%) 304.24 Cow 8 (26.7%) 8 (26.7%) 14(46.6%) 243.006 Monte Carlo test Goat P=0.01* 0 (0%) 1 (100%) 0 (0%) 312 Sheep 1 (20%) 0 (0%) 4 (80%) 531.27 Donkey 6 (100%) 0 (0%) 0 (0%) 41.71

Sex 7 (58.3%) 5 (41.7%) Monte Carlo test Male 0 (0%) 123.65 P=0.1 15(29.4% 25( 49.0% Female 11(21.6%) 275.57 ( )

Results of ANOVA test for GMEC: For animal type: F=1.88 For sex: F= 1.35

*P<0.05

1000 900 905.78 800 700 600 GMEC 500 400 273.49 300 170.46 200 188.93 100 0 <1 year 1- 3- 5 years or more

Figure (9): GMEC among different age groups of infected animals

71 B- Therapeutic trial:

Table (XXIV) and figure (10) show the animal cure rates one, two and three months after treatment with mirazid. The cure rate was continuously increasing throughout the three months follow up; it increased from 64.3% in the first month to 68.6% in the second month and reached its maximum in the third month to become 77.5%.

Table (XXV) shows different parameters associated with cure in animal cases after one, two and three months of treatment with Mirazid. As regard animal sp., it was found that the cure rate increased continuously during the three months of follow up and reached its maximum in the third month for all animals. In the third month, the highest cure rate was among goats (100%) followed by cows (88.2%) and the lowest cure rate was among donkeys (50%), however, these differences were not statistically significant (Monte Carlo test, P=0.3). As regard different age groups, it was found that after three months of treatemnt, animals aged 3- <5 years had the highest cure rate (92.3%), while the lowest cure rate was found among animals at the two extreme age groups (very young or very old groups; less than one year and 5 years and more (66.7% each). As regard sex, it was found that the highest cure rate was obtained after three months of treatment and females had better response than males through out the three months of follow up. After three months, 80% of females were cured versus 70% of males, again this was not statistically significant (Fisher's Exact Test P=0.6).

72 Table (XXIV): Animal cure rates one, two and three months after treatment with mirazid

Cases of fascioliasis Duration after Cured Not cured treatment No. % No. % 1 month 27 64.3 15 35.7 (n=42) 2 months 35 68.6 16 31.4 (n=51) 3 months 31 77.5 9 22.5 (n=40)

100 95 90 85 80 77.5 % 75 70 65 68.6 60 64.3 55 50 1 month 2 monthes 3 monthes

Figure (10): Animal cure rates one, two and three months after treatment with Mirazid

73 Table (XXV): Different parameters associated with cure in animal cases after one, two and three months of treatment with Mirazid

Cure rate after one month Cure rate after two Cure rate after three (n=42) months months (n=51) (n=40) Variable Uncured Cured Uncured Uncured Cured cases Cured cases cases cases cases cases No. (%) No. (%) No. (%) No. (%) No. (%) No. (%) Animal type: Buffalo 10 (66.7%) 5 (33.3%) 14 (73.7%) 5 (26.3%) 10 (76.9%) 3 (23.1%) Cow 13 (76.5%) 4 (23.5%) 16 (76.2%) 5 (23.8%) 15 (88.2%) 2 (11.8%) Goats 1 (100%) 0 (0%) 1 (100%) 0 (0%) 1 (100%) 0 (0%) Sheep 2 (40%) 3 (60%) 2 (40%) 3 (60%) 3 (60%) 2 (40%) Donkeys 1(25%) 3(75%) 2(50%) 2 (50%) 2(50%) 2 (50%)

Test of Monte Carlo test Monte Carlo test Monte Carlo test significane P= 0.206 P= 0.429 P= 0.429 Age group 3 (60%) 3 (60%) 2 (40%) 2 (66.7%) 1 (33.3%) Less than 1 year 2 (40%) 4 (30.8%) 11 (68.8%) 5 (31.3%) 10 (76.9%) 3 (23.1%) 1- 9 (69.2%) 4 (36.4%) 12 (92.3%) 1 (7.7%) 11 (91.7%) 1 (8.3%) 3- 7 (63.6%) 4 (30.8%) 9 (56.3%) 7 (43.8%) 8 (66.7%) 4 (33.3%) 5 or more 9 (69.2%) Test of Monte Carlo test Monte Carlo test Monte Carlo test significane P= 0.550 P= 0.094 P= 0.291 Sex 4 (50%) 4 (50%) 4 (40%) 6 (60%) 7 (70%) 3 (30%) Male 23 (67.6%) 11(32.4%) 31 (77.5%) 9(22.5%) 24 (80%) 6 (20%) Female Test of Fisher's Exact test Fisher's Exact test Fisher's Exact test significane P=0.425 P=0.048* P=0.665 *p<0.05

74 Table (XXVI) demonstrates the effect of intensity of fascioliasis infection before treatment on the cure rate during the three months of follow up. It was found that after three months of treatment the highest cure rate was among moderately infected cases (85.7%) and the lowest cure rate was among heavily infected cases (68.8%), but this was not statistically significant (Monte Carlo test P= 0.651). As regard the GMEC it was found that after three months of treatment it was significantly lower among cases that cured than those that did not (149.24 versus 324.12 respectively) and this was statistically significant (ANOVA, F=5.12, P<0.05).

Table (XXVII) and figure (11) show GMEC in uncured animal cases of fascioliasis before and after treatment. It reveals that, for uncured cases there was a marked reduction in GMEC throughout the three months of follow up from 153.35 before treatment to 47.34 in the 1st month, 31.38 in the 2nd month and 35.64 in the 3rd month and all these differences were statistically significant throughout the three months.

75 Table (XXVI): Effect of intensity of fascioliasis infection before treatment on the cure rate during the three months of follow up

Cure rate after one Cure rate after two Cure rate after three month months months (n=42) (n=51) (n=40) Variable Cured Uncured Cured Uncured Cured Uncured cases cases cases cases cases cases No. (%) No. (%) No. (%) No. (%) No. (%) No. (%) Intensity of infection before treatment Light 9 (50%) 9 (50%) 13 (65%) 7 (35%) 14 (82.4%) 3 (17.6%) Moderate 4 (80%) 1 (20%) 5 (71.4%) 2 (28.6%) 6 (85.7%) 1 (14.3%) Heavy 14 (73.7%) 5 (26.3%) 17 (73.9%) 6 (26.1%) 11 (68.8%) 5 (31.3%) Monte Carlo test Monte Carlo test Monte Carlo test Test of significane P= 0.231 P= 0.937 P= 0.651 GMEC before 231.656 153.355 221.290 175.303 149.24 324.12 treatment ANOVA test for F= 0.490 F=0.004 F= 5.12* GMEC

*P<0.05

76 Table (XXVII): GMEC in uncured animal cases of fascioliasis before and after treatment Intensity of infection Before After treatment treatment 1 month 2 month 3 month

GMEC 153.35 47.34 31.38 35.64 Wilcoxon signed ranks test Z=-3.303* Z=-3.016* Z=-2.524*

*P<0.05

160 153.35 140 120 100 GMEC 80 60 47.34 31.38 35.64 40 20 0 pre-treatment 1 month post- 2 month post- 3 month post- treatment treatment treatment

Figure (11): GMEC in uncured animal cases of fascioliasis before and after treatment

77 Table (XXVIII) demonstrates intensity of infection of uncured animal cases before and one month after treatment. It reveals that the percentage of heavily infected cases decreased from 33.3% before treatment to become 13.3% one month after treatment. The percent reduction of egg count of uncured cases was 56.3%.

Table (XXIX) demonstrates intensity of infection of uncured animal cases before and two months after treatment. It reveals that about 1/2 of uncured cases (43.7%) were heavily infected before treatment and this percent decreased after two months to become only 6.3%. The percent reduction of egg count of uncured cases was 64.6%.

Table (XXX) demonstrates intensity of infection of uncured animal cases before and three months after treatment. It reveals that more than half of uncured cases (55.6%) were heavily infected before treatment and this percent decreased after three months to become only 11.1%. The percent reduction of egg count of uncured cases was 71.9%.

78 Table (XXVIII): Intensity of infection of uncured animal cases before and one month after treatment

Intensity of infection Before treatment One month after treatment

9 10 Light 60% 66.7% 1 3 Moderate 6.7% 20% 5 2 Heavy 33.3% 13.3% 15 15 Total 100% 100%

Percent reduction of egg count 56.3%

79 Table (XXIX): Intensity of infection of uncured animal cases before and two months after treatment

Intensity of infection Before treatment Two months after treatment

7 13 Light 43.7% 81.3% 2 2 Moderate 12.6% 12.5% 7 1 Heavy 43.7% 6.3% 16 16 Total 100% 100%

80 Table (XXX): Intensity of infection of uncured animal cases before and three months after treatment

Intensity of infection Before treatment Three months after treatment

3 7 Light 33.3% 77.8% 1 1 Moderate 11.1% 11.1% 5 1 Heavy 55.6% 11.1% 9 9 Total 100% 100%

81 DISCUSSION

Human fascioliasis is a significant world-wide health problem.(8) It seems that the rate of infection of human fascioliasis is increasing in many countries worldwide.(161)There are about 17 million people infected worldwide. Although the mortality due to fascioliasis is generally low, massive or repeated infections can lead to considerable morbidity.(8) In animals, fasciolosis is a very serious parasitic disease which is responsible for heavy economic losses especially in sheep that are highly susceptible to F. hepatica infection, even when intensity of infection is very low. It also affects cattle production in many countries of the world.(162, 163)

Many drugs (tetrachloride, tetrachlorethylene, bithionol) are nowadays considered either not especially effective, too toxic or both.(8) A possible replacement (triclabendazole (TCBZ)), an effective drug against immature and adult flukes alike, is a potent fasciolicide with a better bioavailability.(162) It is proved to be highly effective in field study with a cure rate of 95.7% after only 5 weaks of treatment. Moreover, the uncured cases showed a percent reduction of egg count reached 85.1%. (42)Inspite of its high efficacy, TCBZ resistant strains were isolated.(22, 164)This situation justifies the search for new effective drugs.(8) Myrrh is an olio gum resin obtained from the stem of Commiphora molmol tree that has a bitter taste; its name is derived from Hebrew murr or maror, meaning bitter. Purified extract of myrrh (Mirazid®), a new herbal schistomicidal and fasciolicidal drug has been licensed in Egypt in March,2002.(134)

Human study: Prevalance of fascioliasis: WHO considered Egypt one of the intermediate prevalent areas as regard human fascioliasis with 7.3% infection rate in Nile Delta.(23) In the present study, a lower prevalence of 5.8% among a rural population near Alexandria in the North-Western region of Nile Delta was found. Fascioliasis was the second most common parasite in that area after S.mansoni. The geometric mean of egg count (GMEC) of all cases of fascioliasis was 63.94 epg.

An earlier study was implemented by Shehata A in 1998 in the same village, and a nearly equal prevalence of 5.3% was reported. (42) Similar findings were also reported by Abou-Basha et al. (2000) who conducted a study in another endemic village in Alexandria (Abis I). The prevalence of fascioliasis was found to be 5.5 %.(27)These continous increasing prevalences in the last 8 years in such an endemic area denote that fascioliasis is still a major public health problem in Abis villages despite the efforts done to control the infection and indicate that continuous monitoring of human fascioliasis in that area is a must. As regard the situation in other Governorates in Egypt, even higher prevalence rates were reported in Sharkia Governorate. An earlier study in 9 different villages in Sharkia was done in 1995 to investigate the real situation of endemicity of fascioliasis in that area. The total number of Fasciola positive cases by ELISA and stool analysis were 147 cases among 1350 children indicating a prevalence of 10.9% among school children in Sharkia Governorate.(165)

82 Some socio-demographic characteristics of Fasciola sp. infected cases: As regard age, the highest prevalence of fascioliasis infection in the present study was among children 5-<10 years (17.5%), who were significantly at higher risk of having fascioliasis by 5.5 times than other age groups, while the least prevalence was among adults 30<40 years old (3.7%). It was also found that the highest prevalence of heavy infection was among children aged 5-<10 years (30%) who also had the highest GMEC (114.46) compared to other age groups, while the least GMEC was found in adults aged 20-<30 years old (23.21) and all of them had light infection.The increasing prevalence and intensity of infection in children may be due to the higher probabilities of repetitive infection in this age group which may be related to both behavioral patterns, such as chewing plants or playing in transmission foci, and an immature immunologic state that is partially related to nutritional deficiencies. Similar findings to our study were reported by Abou-Basha et al. (2000) who conducted her study in Abis I, where the prevalence of fascioliasis was found to be 5.5% and more than half of the cases were children 5-14 years (59.1%). Furthermore, a greater intensity of infection was detected in children, as confirmed by the significant inverse relation between age and egg count.(27) In the study conducted in Ezbet El-Bakly (Al- Fayoum Governorate), the sociodemographic characteristics of the cases were similar to the present study, where the highest prevalence was among children <15 years (47.1%), while the least prevalence was among older age group ≥45 years old (11.8%).(166) In agreement with the present study, Trueba G et al (2000) reported that all of the ELISA-positive cases of fascioliasis detected in Cuturivı ´ Grande, an Ecuadorian villge, were children between the ages of 9 and 12, except for one sample obtained from a 41-year–old woman. The age distribution of the serologically positive cases may indicate an occupational association with the infection. Children of the ages of 9 through 12 may come in contact with Fasciola hepatica metacercaria by drinking contaminated water or eating vegetables growing near ditches because they are responsible for taking herds of sheep to grazing fields.(167) On the contrary to the present work, an earlier study in the same village in 1998 reported that fascioliasis was more prevalent among adults aged 35-<45 years compared to other age groups (8.1%). Surprisingly, in the present study this age group (35-<50 years) had a prevalence of fascioliasis similar to that was reported 8 years ago in the same village (7.7%). This may be explained by the fact that those adults were more exposed to infection through eating green vegetables frequently during their extended hours of work out doors.

As regard sex, females were significantly more affected with fascioliasis than males (7.5% of females had fascioliasis versus 4.3% of males) and they had significant increased risk of having fascioliasis of about two folds than males. They also had higher intensity of infection than males (15% of infected females had heavy infection versus 8.3% of infected males). It was also found that all adult cases of fascioliasis were females. This may be due to the fact that adult females are more liable to get fascioliasis due to their exposure to a high dose of metacercariae during washing of clothes and kitchen utensils in large canals where transmitting lymnaeids are present and during meal preparation in houses and management of freshwater plants that potentially carry attached metacercariae. Similarly, the study of Ezbet El-Bakly (Al- Fayoum Governorate), it was found that females were more affected with fascioliasis than males (52.9% of cases were females). (166) In agreement with the present study,

83 Esteban J. et al (2003) conducted a coprologic surveys in villages of the Behera Governorate in the Nile Delta region and reported that females were significantly more affected with fascioliasis than males (8.8% of males had fascioliasis versus 14.8% of females).(26) On the other hand the earlier study in the same village in 1998 reported that among cases of fascioliasis male to female ratio was nearly 1:1 with higher intensity of infection among females (72.6 versus 45.7 epg respectively).(42)

As regard household animal contact, the present study declared that those having household animal contact had twice the risk of having Fasciola sp. infection than those who do not have. Although direct contact is not the mode of transmission between infected animals and human, but this may be due to the fact that infected animals are the main source for human fascioliasis.(35) i.e. the presence of infected animals causes contamination of local farms so the owners of these animals are more frequently exposed to infection during farming, animal grazing or gathering food for their animals. These results are consistent with Curtale et al. (2003) who reported an increasing risk in the presence of cows (OR 3.2), buffaloes (OR 3.0) and goats (OR 2.6) together with the habit of taking animals to the canal for bathing and/or drinking (OR 3.2).(168) In agreement with the present study, Shehata A reported that presence of animals especially if infected increased the risk of their owners to get fascioliasis by about three times.(42)

Clinical picture of fascioliasis: The symptoms associated with human fascioliasis have been studied mostly in hospitalized subjects. Very little is known about clinical signs and symptoms associated with infections in humans in endemic zones.(168) It was found that most cases of chronic fascioliasis were asymptomatic (64.5%). The results from this study revealed that there is a link between presence of symptoms and intensity of infection. In the present study, it was found that most of asymptomatic cases were lightly infected with fascioliasis (80%), who also had a lower GMEC compared to symptomatic cases (50.66 versus 94.28 respectively), while the most common symptom among symptomatic cases was abdominal colic (24.1%) followed by nausea (4.8%), while only 3.2% of cases had jaundice or blood in stool. Similarly, Shehata A (42) reported the presence of significant association between fascioliasis and complaining of abdominal pain (39.6%), fever (31.3%), nausea and vomiting (22.9%). Another study in 2004 reported that about 23.5% of the fascioliasis patients were asymptomatic. The most frequent symptoms were abdominal distension and flatulence (76.5%), right hypochondrial pain (17.6%) and epigastric pain (17.6%).(166) Aksoy D et al (2006) reported similar findings in his study where the most frequent symptoms of fascioliasis were abdominal pain (35.3%) and fever up to39°C (29.4%).(169) These findings are similar to a previous study implemented among the rural population of three endemic foci in the Nile Delta, Egypt by Curtale F et al (2003). The most important complaints associated with the infection, were right abdominal pain (O.R. 20), epigastric burning (O.R. 16) and nausea (O.R. 8).(168)

84 Therapeutic trial using Mirazid (myrrh): Herbal products are not only safe, but can also have effective antihelminthic activity. (152) Myrrh is obtained from the stem of Commiphora molmol tree, which is a thorny tree growing in a height of about nine feet, preferring well-drained soil in the sun. Myrrh resin is collected from this tree as a thick, strongly aromatic yellow liquid from natural cracks or cuts in the tree bark. The oil which is distilled from myrrh resin is typically thick, pale yellow to orange-brown, with a warm, balsamic, sweet, spicy, and sharp aroma.(170)

In the present study the cure rate of human fascioliasis treated with Mirazid increased over the three months of follow up; it increased from 53.8% in the first month to 81.5% in the second month and reached its maximum in the third month to become 88.2%.

High cure rate was related to different factors; intensity of infection before treatment, gender, family size, presense of symptoms, household animal contact and age group.

As regard intensity of infection before treatment, it was found that the GMEC before treatment was lower among cured cases than those uncured throughout the three months of follow up (47.78 epg versus 96.12 epg in the first month, 49.69 epg versus 60.28 epg in the second month and 50.06 epg versus 70.99 epg in the third month respectively).

As regard gender, it was found that males had better response than females as all males involved in the study were cured after three months of treatment versus 84.6% of females. This may be due to the fact that infected females had higher prevalence of heavy of infection than infected males (15% of infected females had heavy infection versus 8.3% of infected males).

It was also found that small family size had a good impact on treatment outcome as all cases belonged to small families of 3-4 members only were completely cured after one month of treatment, while those belonged to large families had cure rate of only 40% after one month and it increased gradually to become 81.8% after three months. This may be attributed to higher intensity of infection among cases living in large families than cases living in small families (the lowest GMEC was among cases living in families of 3-4 members (42.65) and all of them were lightly infected) .

As regard presence of symptoms, it was found that being clinically free was a good prognostic sign as all asymptomatic cases were completely cured after three months of treatment versus 71.4% of symptomatic cases and this difference was statistically significant. Again, this may be due to the fact that most of asymptomatic cases were lightly infected with fascioliasis (80%) and they also had a lower GMEC compared to symptomatic cases (50.66 versus 94.28 respectively).

As regard household animal contact, it was found that after three months of treatment, 94.7% of cases that had no household animal contact were completely cured versus 80% in

85 those who had. This may be due to the fact that presense of household animal contact was a significant risk factor of getting fascioliasis.

It was also found that except for adults aged 20-<40 years, all other age groups revealed better response to the given regimen of treatment reaching 100% after 3 months. This may be due to the fact that all uncured cases of that age group were symptomatic females living in large families of 5 or more members and they had higher pre-treatment GMEC compared to both cured cases of the same age group and cured cases of all other age groups (70.9 versus 41.7 and 50.06 respectively).

This high cure rate of myrrh was documented by another study conducted in the Unit of Infectious Diseases and Malnutrition, Mansoura University Children's Hospital, Dakahlia from May 2002 to June 2003. A total of 21 children with fascioliasis (8 males and 13 females) with mean age of 10.4±2 years were treated with Myrrh (Mirazid) in the same dose of the present study and the dose was repeated after 4 weeks for the non-responders. Clinical evaluation and stool analysis were done at 2, 4 and 12 weeks post-treatment to evaluate cure. Parasitological cure was 90.9% at 4 weeks post-treatment. After the second dose, all non- responders were cured. (160)

Similarly, Hassan M et al. (2004) performed a study on 90 young patients with fascioliasis aged 13-27 years (mean age 21.4±4.5) attending the Outpatient Clinic Of Tropical Medicine Department, Zagazig and Mansoura University Hospitals. Patients received Mirazid as 2 tablets (600mg) for 6 days and were followed up for 3 months. One month after treatment, 91.1% of patients (82/90) were cured and remained so through the follow-up period. None of the non-responders in the 1st follow-up were cured in the 2nd or the 3rd follow- up. (171) In agreement with the present study, a field survey was done in Ezbet El-Bakly, Al- Fayoum Governorate, Egypt to assess the efficacy and safety of Mirazid in the treatment of human fascioliasis. All cases (17 cases) were treated by Mirazid as two capsules (600 mg) on an empty stomach an hour before breakfast for six consecutive days and followed up clinically and parasitologically. The parasitological cure rate, two and three months after treatment was 88.2% and 94.1% with an overt clinical cure without any side-effects.(166) These results are consistent with Motawea et al. (2001b) in Dakahlia, who studied a total of 292 fascioliasis patients. Mirazid was given in a dose of 10 mg/kg on an empty stomach an hour before breakfast for six consecutive days, as a capsule for adults and a suppository for children. Parasitologically and clinically cure rate was 98.6% at 4 weeks after treatment.(172) A preliminary study of therapeutic efficacy of myrrh was done by Massoud A. et al. in which 7 patients who were passing Fasciola eggs in their stools were treated with myrrh in a dose of 12 mg/kg per day for 6 consecutive days in the morning on an empty stomach. Eggs were no longer detectable in the feces of all patients 3 weeks after treatment and after a follow-up period of 3 months.(134) This was in agreement with the findings of another study carried out in Departments of Tropical Medicine, Al Azhar University Hospitals in the period from March 2002 to November 2003. This study reported that a total of 35 chronic fascioliasis patients were treated with myrrh in the same dose of our study, and the cure rate was 94.3% at 1 month and reached 100% after 3 months.(144)

86 Effect of myrrh on intensity of infection in uncured human cases: The results of the present study revealed that, for uncured human cases there was a significant reduction in GMEC throughout the first and second months of follow up from 96.12 epg before treatment to 34.28 epg and 30.61 epg respectively. It was noticed that there was a slight non-significant increases in GMEC in the third month to be 44.89 epg. It is worth mentioning that all uncured cases with pretreatment moderate intensity of infection became lightly infected after the first and the second month of treatment. Percent reduction of egg count of uncured cases was highest in the second month (20%, 42% and 30% respectively). Similarly, in the field survey done in Ezbet El-Bakly, Al-Fayoum Governorate the cases not completely responding to a single course of treatment showed a significant reduction of the egg count after 2 and 3 months of treatment (from 222±22.9 epg before treatment to 12±13.9 and 6±12 epg after 2 and 3 months respectively). (166) This was in agreement with the findings of the study carried out in Departments of Tropical Medicine, Al Azhar University Hospitals in the period from March 2002 to November 2003. This study reported that among the 2 cases which were still passing eggs after 1 month of therapy, the egg count decreased from 240 epg before treatment to 48 epg.(144) Massoud A. et al.(2001) reported a dramatic drop in the egg count detected at the end of treatment (from 36 epg before treatment to 6.43 epg at the end of treatment to two epg one week post treatment and 1.43 epg two weeks post treatment).(134)

Changes in ALT and AST levels one month after treatment: The effect of Mirazid on liver enzymes was investigated in this study. It was found that levels of both ALT and AST were within normal values before (33.9 and 34.1 respectively) and one month after treatment (32.4 and 31.9 respectively). The results confirm that these enzymes are not significantly affected by the drug. This was documented by Massoud A. et al (2001), who reported that ALT and AST levels were within normal ranges in patients before and 2 weeks after receiving myrrh (for ALT it decreased from 25.4± 6.1 to 24.8± 5.7 and AST decreased from 21.8± 7.2 to 21.2± 6.2)(134) Similarly, the study conducted in Department of Tropical Medicine, Al Azhar University Hospitals in the period from March 2002 to November 2003 reported that levels of liver enzymes were not disturbed in the fascioliasis patients and did not differ significantly after 3 months of treatment with myrrh.(144)

87 Animal study: Prevalance of animal fascioliasis: In the present study, the prevalence of animal fascioliasis was 52.9% which is a very high rate denoting that fascioliasis is a major public health problem in that endemic area. These findings are similar to the previous study in the same village that reported a prevalence rate of 47.8%. (42) This study was carried out 8 years ago in the same area, which means that the prevalence is increasing dispite the efforts done to control the problem. Haseeb et al (2002) reported in his review of fascioliasis in Egypt similar high prevalence in Monoufia Governorate (51.2% among cattle and 53% among sheep) and even higher rates in Dakahlia Governorate (59.5% among cattle and 78% among sheep).(23) All these high figures denote the importance of the problem throughout Egypt particularly in the Nile Delta region and not only Alexandria Governorate.

Prevalence and intensity of infection among animals according to animal sp. , Age and gender: As regard prevalence and intensity of infection among animals it was found that the highest prevalence of fascioliasis was among buffaloes (65.6%) followed by cows (55.6%) and the least prevalence was among goats (12.5%). This may be due to differences in the grazing practices. In her study, Shehata reported that donkeys had the highest percentage of infection (69.2%), followed by buffaloes (54.4%) and the lowest infection rate was among cows (31.8%)(42)

In the present study, it was also found that the highest prevalence of heavy infection was among sheep (80%) that also had the highest geometric mean of egg count (GMEC) compared to other species (531.27), while the least GMEC was found among donkeys (41.71) that all had light infection. All these differences were statistically significant. The high intensity of infection among sheep may be due to the fact that sheep are allowed to graze along the banks of water courses with subsequent exposure to higher doses of infection with metacercariae.

The presence of heavy infection among most animals in our study (47.6% of all animals had heavy infection) can be explained by rearing practices which depends mainly on grazing outdoors in such an endemic area. Besides, animals can support enormous worm burdens without developing serious disease in contrary to humans.(173) In agreement with the present work, Mas-Coma N (1998) reported in a study conducted in an endemic area in Latin America that the number of egg per gram (epg) in sheep was about 1000.(174) However, a study conducted in an endemic village in Dakahlia in 2002 reported fewer numbers of mean epg in infected animals, where it was 8.9 in goats, 13.6 in buffaloes, 22 in cows and 148.3 in sheep. (175)

As regard age, in the present study it was found that younger age group was more affected with fascioliasis, where the highest prevalence of fascioliasis was found among younger age group of less than one year old (83.3%) and the prevalence decreased with increasing age till becoming 54.8% among age group of 5 years or more. Our study also

88 revealed that there was a significant association between younger age groups and higher GMEC, where the highest GMEC was among animals aged less than one year (905.78) and it decreased significantly as the age increased till becoming 188.93 among animals aged 5 years or more. This may be due to immature immune system in younger age groups which increases the risk of infection. This was documented by Khalil who reported that cattle and buffaloes aged less than two years presented by maximal infection rates (60.2% and 52.6% respectively), then the prevalence gradually decreased with increased age to a minimum of 30.8% and 35.5% for cattle and buffaloes aged 5 years or more respectively.(176)

In the present study, it was found that female animals were more affected with fascioliasis than males (54.3% of them were infected versus 48%% of males), it was also found that female animals had higher prevalence of heavy infection than males as 70.6% of them had moderate or heavy infection versus 41.7% of males and they also had higher GMEC compared to males (275.57 versus 123.65 respectively), however, these differences were not statistically significant. This may be due to the fact that male animals are frequently raised for slaughtering so they are usually kept indoors for fattening, while females are allowed for graizing outdoors with subsequent increased risk of exposure to infection. In agreement with the present study, Shehata reported that female animals were more affected with fascioliasis than males (61.7 % versus 45.5% respectively).(42)

It is worth mentioning that, the potential reservoir role of donkeys in fascioliasis was evaluated in this study. It was found that, 40% of donkeys were infected with fascioliasis. In agreement with the present work, Shehata A (42) reported in her survey in the same village that donkeys had the highest percentage of infection of fascioliasis (69.2%). This was documented by a preliminary coprologic examination of donkeys done in eight centers of Gharbia governorate. The overall rate of infection in donkeys was 3.03%. Donkeys showed infection rates of 4.6%, 7.6% and 9.09% in the centers of Santa, Zefta and El Mahala El Kobra respectively.(177) Interestingly, Valero M. et al (2000) reported that metacercarial viabilities of donkeys are similar to viabilities of metacercariae of the main reservoir host (cattle and sheep).(178)

From all these studies we can conclude that donkeys have a high transmission potential capacity and it can be considered a secondary reservoir host of fascioliasis in endemic areas, so any preventing measure should put treating donkeys as well as other herbivorous animals into consideration to give the hoping outcome.

Myrrh in animals: Treatment of animal fascioliasis is a must. Apart from the great economic losses, they are the main hosts for both Fasciola species, thus passing the infection to man.(23) In the present study, it was found that the cure rate of animal fascioliasis treated with myrrh was continuously increasing throughout the three months follow up; it increased from 64.3% in the first month to 68.6% in the 2nd month and reached its maximum in the 3rd month to become 77.5%.

89 As regard different parameters associated with cure in animal cases after 3 months of treatment, it was found that the highest cure rate was among goats (100%) followed by cows (88.2%) and the lowest cure rate was among donkeys (50%). The 100% cure rate among goats dose not necessay mean that the dose was efficient as there was only one goat treated and cured in the present study.

As regard intensity of infection before treatment, it was found that pre-treatment GMEC was significantly lower among cured cases than those uncured (149.24 versus 324.12 respectively) and this was statistically significant.

As regard age it was found that after three months of treatemnt, animals aged 3-<5 years had the highest cure rate (92.3%), while the lowest cure rate was found among animals at the two extreme age groups; less than one year and 5 years and more (66.7% each). The higher cure rate amon adult animals may be due to their mature immune system besides; the adult animals at that age group (3-<5 years) had significant lower intensity of infection than other age groups (the highest GMEC was among animals aged less than one year (905.78) and the lowest GMEC was among animals aged 3-<5 years (170.46)). Haridy et al. (2003) studied the efficacy of Mirazid in sheep naturally infected with fascioliasis. Total doses of one or two capsules (300 mg each) were given for one, two or three successive days on an empty stomach an hour before breakfast. A total dose of 600mg gave a cure rate of 83.3%, while a total dose of 900 to 1200mg gave a complete cure rate (100%), with no clinical side effect. The cure rate was assessed by stool examination and/or macroscopically on slaughtering the sheep. Thus Mirazid proved to be safe and very effective in sheep fascioliasis in Gharbia Governorate.(153) This discrepancy between cure rates in our study and that study of Haridy et al. may be due to the fact that he studied only one species of animals (sheep) and tried different regimens in their treatment till reached the effective dose, but in our study the dose used for treating fascioliasis was the same for all animals regardless animal species or body weight of infected animals, however, the cure rate among sheep in our study was definitely less than that reported by Haridy et al. as it was just 60% although the same dose was used in both studies. This may de due to the small sample size of sheep in our study (only 5 infected sheep were diagnosed in the selected satellite), while he studied 30 infected sheep.

Effect of myrrh on intensity of infection in uncured animal cases: The results of the present study revealed that, for uncured animal cases treated with myrrh, there was a marked reduction in GMEC throughout the three months of follow up from 153.35 before treatment to 47.34 in the 1st month, 31.38 in the 2nd month and 35.64 in the 3rd month and all these differences were statistically significant throughout the three months. It was also found that percent reduction of egg count of uncured animals was continuously increasing throughout the 3 months of follow up from 56.3% in the 1st month to 64.6% in the 2nd and finally it reaches its maximum in the 3rd month to be 71.9%.

90 SUMMARY

Recent environmental changes and modifications in human behavior are defining new geographical limits of fascioliasis and increasing the populations at risk.(173) The number of reports of humans infected with Fasciola hepatica has increased significantly since 1980. High prevalence of fascioliasis in humans does not necessarily occur in areas where fascioliasis is a major veterinary problem.(23)

In animals, fascioliasis is a very serious parasitic disease which is responsible for heavy economic losses.(162,163)

Diagnosis of Fasciola sp. infection is achieved by locating the ova in fecal samples. (161) The sensitivity of Kato–Katz technique in the diagnosis of human fascioliasis with three Kato slides from the same specimen or on different days ranged from 96.0%–99.1%.(84)

The most important criteria for a good anthelmentic drugs are high efficacy, safety to the target individual, absence of persistent chemical residues in the host tissue and ease of application at an economic price.(109) Triclabedazole has been proved to be very safe and effective anthelmentic for all stages of Fasciola life cycle (mature and immature stages) in animal and human infections.(115, 116)However, the development of resistance against it has been reported.(122,123,126) This situation justifies the search for new effective drugs.(8)

Purified extract of Myrrh (Mirazid ®), a new herbal schistomicidal and fasciolicidal drug has been licensed in Egypt in March, 2002.(134) This study was carried out to estimate the cure rate of human and animal fascioliasis treated with Myrrh, to determine the effect of Myrrh on the intensity of Fasciola infection and to investigate changes in serum levels of human liver transaminases in patients with chronic fascioliasis before and after receiving treatment with Myrrh.

This work was carried out during the period from June 2005 to February 2006 in Satellite number 10 of Abis VIII village. Mapping of the selected satellite was done, then a census updating was carried out by house-to-house visits. The total population amounted to 1305 individuals, 138 of them were children under 5 years and were excluded from the study. All herbivorous animals were included in the present study (cows, buffaloes, sheep, goats and donkeys). Total animal population was 208 animals. All inhabitants of the selected satellite aged 5 years or more and their herbivorous animals were screened for fascioliasis. 3 Kato- Katz thick smears each of 41.7 mg were prepared from each sample and examined microscopically and helminthic ova were recorded, but only Fasciola eggs were counted. A second stool sample was collected from positive human cases one week later after ensuring liver free diet during this period and examined by Kato-Katz technique to exclude false positive cases. Total number of human samples examined was 1092, and that of animal was 119.

91 Human cases of fascioliasis were subjected to a predesigned questionnaire to collect data about past history of fascioliasis, its symptoms and whether or not any treatment of fascioliasis was received. As regard animal cases of fascioliasis, data concerning type, age, sex of each animal and fate of animal excreta were collected from animal owners.

A blood sample was collected from each human case before treatment and a second blood sample was collected one month after receiving treatment to measure serum levels of ALT and AST

The human sample included 64 cases and animal sample included 63 cases

For human cases, Mirazid capsules (300mg/capsule) were used for treatment in a dose 10mg/kg body weight per day for 6 consecutive days in the morning on an empty stomach. The maximum single dose was considered as 600mg/day. Animal cases of fascioliasis were treated using Mirazid suspension 10% in a dose 600mg/day for 2 consecutive days in the morning on an empty stomach.

The cure rate of Myrrh was assessed on three occasions; after one, two and three months post treatment. On each occasion, three stool samples were collected from each treated case (human and animal) with 24 hours interval for detecting and counting Fasciola sp. eggs.

Data collected were analysed using the statistical package of social science (SPSS) version 9.0 and Epi-info version 6.4 and results revealed the following:

Human study: 1- Out of 1092 individuals examined for helminthic infection, fascioliasis prevalence was 5.8% which denotes that it is still a major public health problem in Abis village as it is the 2nd most prevalent helminthic infection in that area after S.mansoni .

2- Children under 10 years old were significantly at higher risk of having fascioliasis by 5.5 times than other age groups and they also had the highest prevalence of heavy infection (30%) and highest geometric mean of egg count (114.46) compared to other age groups.

3- Females were significantly more affected with fascioliasis than males (7.5% of females had fascioliasis versus 4.3% of males) and they had significant increased risk of having fascioliasis of about two folds than males. They also had higher levels of heavy infection than males (15% of infected females had heavy infection versus 8.3% of infected males).

4- Those having household animal contact had significant 2 folds increased risk of having fascioliasis than those who do not although direct contact is not the mode of transmission between infected animals and human.

92 5- Most cases of Fasciola sp. infection were asymptomatic (64.5%), while the most common presenting symptom of symptomatic cases was abdominal colic (24.1%) followed by nausea (4.8%), while only 3.2% of cases had jaundice or blood in stool.

6- There was an association between being asymptomatic case of fascioliasis and having light infection as most of asymptomatic cases (80%) were lightly infected with fascioliasis; they also had a non significant lower GMEC compared to symptomatic cases (50.66 versus 94.28 respectively).

7- Myrrh is a promising effective new drug for treatment of human fascioliasis with a high cure rate (88.2%) after three months of treatment. 8- Factors associated with good response to treatment with Myrrh were: male gender, asymptomatic cases, light intensity of infection, living in small family and absence of house-hold animal contact.

9- For uncured cases treated with Myrrh, there was a marked reduction in GMEC from 96.12 before treatment to 34.28 in the 1st month, 30.61 in the 2nd month and 44.89 in the 3rd month and these differences were significant in the 1st and 2nd month which is of a great public health importance in decreasing infection transmission in the community.

10- Liver enzymes (ALT and AST) were not significantly affected after one month of administration of Myrrh.

Animal study: 1- Fasciola sp. infection is highly prevalent among animals in Abis village with a prevalence reaching 52.9%. The highest prevalence of fascioliasis was among buffaloes (65.6%) followed by cows (55.6%) and the least prevalence was among goats (12.5%).

2- About 1/2 of infected animals were heavily infected and the total GMEC of all infected animals was high (236.56). The highest GMEC was among animals <1 year old (905.78) and it decreased significantly as the age increased.

3- Donkeys had a high transmission potential capacity and it can be considered a secondary reservoir host of fascioliasis in endemic areas as 40% of donkeys were infected with fascioliasis.

4- Cure rate after treatment of animals with Mirazid was not high even after three months of follow up (it increased from 64.3% in the first month to 68.6% in the second month and reached its maximum in the third month to become 77.5%) this may be due to the fact that in our study the dose used for treating fascioliasis was the

93 same for all animals, regardless animal species or body weight of infected animals. In the third month, the highest cure rate was among goats (100%) followed by cows (88.2%) and the lowest cure rate was among donkeys (50%). 5- Factors associated with good response to treatment with Myrrh were:goats and cow species, light intensity of infection, older age group, 6- Cured cases had a significant lower GMEC than non cured cases (149.24 versus 324.12 respectively).

Thus to conclude, although Mirazid is a herbal safe drug with minimal side effects not affecting the liver enzymes, yet it is less effective than TCBZ in treatment of both human and animal fascioliasis due to the following causes: 1- Cure rate of Mirazid reached its maximum after 3 months of treatment (88.2%), while TCBZ induced a higher cure rate (95.7%) after only 5 weeks of treatment. 2- Mirazid needs multiple doses (6 consequative doses in human and 2 doses in animals) causing a compliance problem. 3- Mirazid should be taken on an empty stomach and the patient should avoid eating or drinking for an hour after treatment thus adding to the problem of patient compliance. However, it should be considered as an alternative fasciolicidal drug in areas with TCBZ resistant.

94 RECOMMENDATIONS

1- Continuous monitoring of human and animal fascioliasis in Abis area.

2- Treatment of animal fascioliasis is a must. A part from the great economic losses, they are the main hosts for both Fasciola speceis, thus passing the infection to man.

3- Donkeys can be considered a secondary reservoir host of fascioliasis in endemic areas, so control measures should put treating donkeys as well as other herbivorous animals into consideration to give the desired outcome.

4- Children and females should be given special interest in the control of the disease as they are considered high risk groups for fascioliasis.

5- Health education should be given to individuals living in endemic areas stressing on modes of transmission and preparation of healthy food.

6- Further studies are recommended to reach the effective dose of Mirazid and suitable therapeutic regimen for different animal species.

7- Extensive experimental studies for separation and identification of all active ingredients of myrrh.

8- Search for new drugs and altenative durable integrated control strategies should be intensified as chemotherapy alone could not eliminate transmission.

95 REFERENCES

1 -Bunnag D, Cross J, Bunnag T. Liver fluke infections. In: Strickland G. Hunter's tropical medicine and emerging diseases. 8th edition. Philadelphia, London, Toronto, Montreal, Sydney, Tokyo: WB Saunders Company; 2000. 840-7.

2- Irving J, Spithill T, Pike R, Whisstock J, Smooker P. The evolution of enzyme specificity in Fasciola spp. J Mol Evol 2003; 57:1–15.

3- Cringoli G, Rinaldi L, Veneziano V, Capelli G, Malone JB. A cross-sectional coprological survey of liver flukes in cattle and sheep from an area of the southern Italian Apennines. Vet Parasitol 2002; 108(2):137-43.

4- Moghaddam J, Massoud M, Mahmoodi H, Mahvi V, Periago P, Artigas V, et al. Human and animal fascioliasis in Mazandaran province, northern Iran. Parasitol Res 2004; 94: 61–9.

5- Tantrawatpan C, Maleewong W, Wongkham C, Wongkham S, Intapan P, Nakashima K. Serodiagnosis of human fascioliasis by a cystatin capture enzyme-linked immunosorbent assay with recombinant Fasciola gigantica cathepsin l antigen. Am J Trop Med Hyg 2005; 72(1): 82–6.

6- Mas-Coma MS, Esteban JG, Bargues MD. Epidemiology of human fascioliasis: a review and proposed new classification. Bull World Health Organ.1999;77(4):340-6.

7- World Health Organization. 1995. Control of foodborne trematode infections. WHO Tech Rep Ser 849:1-157.

8- Favennec L, Ortiz J, Gargala G, Chegne N, Ayoub A, Rossignol J.Double-blind, randomized, placebo-controlled study of nitazoxanide in the treatment of fascioliasis in adults and children from northern Peru. Aliment Pharmacol Ther 2003; 17: 265–70.

9- Antoniou M, Economou I, Wang X, Psaroulaki A, Spyridaki I, Papadopoulos B, et al. Fourteen-year seroepidemiological study of zoonoses in a Greek village. Am J Trop Med Hyg 2002; 66(1): 80–5.

10-Keiser J, Utzinger J. Chemotherapy for major food-borne trematodes: a review. Expert Opin Pharmaco ther 2004; 5(8):1711-26.

11- Adachi S, Kotani K, Shimizo T, Tanaka , Shimizo T, Okada K. Asyptomatic fascioliasis. Internal Medicine 2005; 44(9):1013:15.

12- Mas-Coma S, Bargues MD, Esteban JG. Human fasciolosis. In: Dalton JP, ed. Fasciolosis. St. Albans, Herts, England, Commonwealth Agricultural Bureaux (in press). reviews in parasitology 1997; 57: 33-7.

96 13- Cadel S. Eighteen cases of human fascioliasis in Basse-Normandie, 1994±1995. Bulletin de la SocieÂ teÂ FrancË aise de Parasitologie 1996; 14: 39-43 (in French).

14- Gil-Benito A. Human fascioliasis on the island of Corsica. PhD thesis, Faculty of Medicine and Dentistry, University of Valencia, Valencia, Spain, 1994: 1:326 (in Spanish).

15- Apt W. Prevalence of fascioliasis in humans, horses, pigs and wild rabbits in three provinces of Chile. BoletõÂn de la Oficina Sanitaria Panamericana 1993; 115: 405-14 (in Spanish).

16- Sampaio Silva ML. Summary of a report on F. hepatica infection in northern Portugal. In: Chen MG, Mott KE. Progress in assessment of morbidity due to Fasciola hepatica infection: a review of recent literature. Trop Dis Bull 1990; 87: R38.

17- Farag HF. A focus of human fascioliasis in the Nile Delta, Egypt. Trop Med Hyg 1979; 82: 188:190.

18- BendezuÂ P, Frame A, Hillyer GV. Human fascioliasis in Corozal, Puerto Rico. Journal of parasitology 1982; 68: 297-9.

19- SaÂ nchez C, Aparicio W, Hurtado C. Distomiasis of the liver in the human population of the Asilo-Azangaro-Puno river system. Libro de resuÂ mes del XI Congreso Latinoamericano de Para- sitologõÂa y I Congreso Peruano de ParasitologõÂa , Lima, PeruÂ , 1993: 50 (in Spanish).

20- Stork MG. An investigation of endemic fasciolasis in Peruvian village children. Trop Med Hyg 1973; 76: 231-5.

21- Mas-Coma S. Human fascioliasis in Bolivia: a general analysis and a critical review of existing data. Research and reviews in parasitology 1995; 55: 73-93.

22- Control of food-borne trematode infections. Report of a WHO Study Group. Geneva, World Health Organization, 1995 (Technical Report Series, No. 849).

23- Haseeb AN, El-Shazly AM, Arafa MA, Morsy AT. A review on fascioliasis in Egypt J Egypt Soc Parasitol 2002; 32(1):317-54.

24- Rokni MB, Massoud J, O'Neill SM, Parkinson M, Dalton JP. Diagnosis of human fasciolosis in the Gilan province of Northern Iran: application of cathepsin L-ELISA. Diagn Microbiol Infect Dis 2002 ;44(2):175-9.

97 25- El-Shazly AM, Soliman M, Gabr A, Haseeb AN, Morsy AT, Arafa MA, et al. Clinico- epidemiological study of human fascioliasis in an endemic focus in Dakahlia Governorate. Egypt. J Egypt Soc Parasitol 2001 ;31(3):725-36.

26- Esteban JG, Gonzalez C, Curtale F, Munoz-Antoli C, Valero MA, Bargues MD, et al. Hyperendemic fascioliasis associated with schistosomiasis in villages in the Nile Delta of Egypt. Am J Trop Med Hyg 2003; 69(4):429-37.

27- Abou Basha L, Salem A, Osman M, El-Hefni S, Zaki A. Hepatic fibrosis due to fascioliasis and or schistosomiasis in Abis I village, Egypt. Eastern Medetreanean Health Journal 2000; 6 (5): 870-8.

28- Tekelye B, Bruns E, Kasali O, Woldemariam W. Association of endoparasites and productivity of highland sheep in Ethiopia. Prev Vet Med 1992; 13: 103-11.

29- Epe C, Coati N, Schnieder T. Results of parasitological examinations of faecal samples from horses, ruminants, pigs, dogs, cats, hedgehogs and rabbits between 1998 and 2002. Dtsch Tierarztl Wochenschr 2004; 111(6):243-7.

30- Cringoli G, Rinaldi L, Veneziano V, Capelli G, Malone JB. A cross-sectional coprological survey of liver flukes in cattle and sheep from an area of the southern Italian Apennines. Vet Parasitol 2002; 108(2):137-43.

31- Paz-Silva A, Andrade R, Sua´ rez J, Pedreira J, Arias M, Lo´ pez C, et al. Prevalence of natural ovine fasciolosis shown by demonstrating the presence of serum circulating antigens. Parasitol Res 2003; 91: 328–31.

32- Jemli MH, Rhimi I, Jdidi A. La fasciolose ovine dans la re´ gion de Sejnane (Nord de la Tunisie). Rev Med Vet (Toulouse) 1991; 142:229–35.

33- Mekroud A, Benakhla A, Vignoles P, Rondelaud D, Dreyfuss G. Preliminary studies on the prevalence of natural fascioliasis in cattle, sheep, and the host snail (Galba truncatula) in north-eastern Algeria. Parasitol Res 2004; 92: 502–5.

34- EL- Baiy M. Fasciliasis among animal, snail and human hosts in Kafr El- Sheikh Governorate with special reference to species infecting humans. Vet Med J Giza 1997; (45) 2:187-209.

35- El-Shazly AM, El-Wafa SA, Haridy FM, Soliman M, Rifaat MM, Morsy TA. Fascioliasis among live and slaugthered animals in nine centers of Dakahlia Governorate. J Egypt Soc Parasitol 2002; 32(1):47-57.

98 36-http://www.dpd.cdc.gov/dpdx/images/ParasiteImages/A-F/Fascioliasis /Fasciola_LifeCycle.gif

37- Mas-Coma S. Secondary reservoir role of domestic animals other than sheep and cattle in fascioliasis transmission in the Northern Bolivian Altiplano. Research and reviews in parasitology 1997; 57: 39-46.

38- Mas-Coma S. Consequencies of phenomena ascribed to insularism in parasitic diseases. The liver fluke (Fasciola hepatica) and Muridae in Corsica. Bulletin de la SocieÂ teÂ NeuchaÃ teloise des Sciences Naturelles 1987; 110: 57-62 (in French).

39- Mas-Coma S. Small mammals as natural definitive hosts of the liver fluke, Fasciola hepatica (Trematoda: Fasciolidae): a review and two new records of epidemiologic interest on the island of Corsica. Rivista di parassitologia 1988; 5: 73-8.

40- Ménard A, Agoulon A, Hostis M, Rondelaud D, Collard S, Chauvin A. Myocastor coypus as a reservoir host of Fasciola hepatica in France. Vet Res 2001; 32 : 499–508.

41- Caruthers D. The Liver fluke (Fasciola hepatica): life cycle, epidemiology, pathology, clinical signs, effect on productivity, treatment and control. Training Bulllletin 2000; 5:14-20.

42- Shehata A. An Epidemiological study of human fascioliasis in Abis VIII village. Doctorate Thesis .University of Alexandria. High Institute of Public Health. 2001.

43- Farag HF, El Sayad MH. Biomphalaria alexandrina naturally infected with Fasciola gigantica in Egypt. Transactions of the Royal Society of Tropical Medicine and Hygiene 1995; 89: 36-9.

44- Soulsby EL. Helminths, Arthropods & Protozoa of Domesticated Animals. Baltimore 1999; Williams and Wilkins Company: 23-33.

45- Lotfy W, Hillyer G. Fasciola species in Egypt. Bullgarian Academy of Sceinces 2003; 9- 22.

46- Mas-Coma S, Funatsu IR, Bargues MD. Fasciola hepatica and lymnaeid snails occurring at very high altitude in South America. Parasitology. 2001;123Suppl:S115-27.

47- Esteban JG, Gonzalez C, Bargues MD, Angles R, Sanchez C, Naquira C, et al. High fascioliasis infection in children linked to a man-made irrigation zone in Peru. Trop Med Int Health. 2002 ;7(4):339-48.

48- Chen MG, Mott KE. Progress in assessment of morbidity due to Fasciola hepatica infection: a review of recent literature. Trop Dis Bull 1990; 87: 1-38.

99 49- Mas-Coma MS, Esteban JG, Bargues MD. Epidemiology of human fascioliasis: a review and proposed new classification. Bull World Health Organ 1999; 77:340–6.

50- Chen MG, Mott KE: Progress in assessment of morbidity due to Fasciola hepatica infection: a review of recent literature. Trop Dis Bull 1990; 87: 1–38.

51- Arjona R, Riancho JA, Aguado JM: Fascioliasis in developed countries: a review of classic and aberrant forms of the disease. Medicine 1995; 74:13–23.

52- Doherty JF, Price N, Moody AH, et al.: Fascioliasis due to the consumption of imported khat. Lancet 1995; 345:462.

53- Taira N, Yoshifuji H, Boray JC. Zoonotic potential of infection with Fasciola spp. by consumption of freshly prepared raw liver containing immature flukes. International journal for parasitology 1997; 27: 775-9.

54- Esteban J, Flores A, Aguirre C, Strauss W, Angles R & Mas-Coma S. Presence of very high prevalence and intensity of infection with Fasciola hepatica among Aymara children from the Northern Bolivian Altiplano. Acta Tropica 1997a; 66:1-14.

55- Richter J, Knipper M, Göbels K, Häussinger D. Fascioliasis. Current Treatment Options in Infectious Diseases 2002; 4:313–7.

56- Saba R, Korkmaz M, Inan D, Mamikolu L, Turhan O, Günseren F, et al. Human fascioliasis. Clinical Microbiology & Infection 2004; 10 (5): 385 - 90.

57- De Carneri I. Parassitologia Generale e Umana, edn 11. Milan, Italy: Casa Editrice Ambrosiana; 1993:193–5.

58- Khalid Al-Mekhaizeem K, Al-Mukhaizeem F, Habib M. Cases report: Fasciola hepatica infestation presenting as biliary obstruction 11 years after open cholecystectomy and CBD exploration. Kuwait Medical Journal 2004; 36 (4):293-5.

59- Acuna-Soto R, Braun-Roth G. Bleeding ulcer in the common bile duct due to Fasciola hepatica. Am J Gastroenterol 1987; 82:560–2.

60- Bafandeh Y, Daghestani D, Rad S. Biliary tract obstruction due to Fasciola hepatica managed by ERCP. Iran J Med Sci 2003; 28 (1):43-45.

61- Chapman RW. Risk factors for biliary carcinogenesis. Ann Oncol 1999; 10:308–11.

62- Marcos L, Maco V, Terashima A, Samalvides F, Gotuzzo E. Características clínicas de la infección crónica por Fasciola hepatica en niños. Rev Gastroent Perú 2002; 22: 228-33.

100 63- Noyer C, Coyle C, Werner C, Dupouy-Camet J, Tanowitz H, Wittner M. Hypereosinophilia and liver mass in an immigrant. Am J Trop Med Hyg 2002; 66(6): 774–6.

64- Montembault S, Serfaty L, Poirot JL. Hemorrhagic ascites disclosing massive Fasciola hepatica infection. Gastroenterol Clin Biol 1997; 21:785–8.

65- Perez C, Vives R, Montes M, Ostis S. Recurrent eosinophilic panniculitis associated with Fasciola hepatica infection. J Am Acad Dermatol 2000; 42:900–2.

66- Hamir A, Smith B. Severe biliary hyperplasia associated with liver fluke infection in an adult alpaca. Vet Pathol 2002; 39:592–4.

67- Njau B, Kasali O, Scholtens R, Degefa M. Review of sheep mortality in the Ethiopian highlands, 1982 to 1986. ILCA Bulletin International Livestock Center for Africa 1988; 31: 19-22.

68- Guobardia E. An abattoir and sheep markets survey of fascioliasis in sheep in Delta, Edo and Oyo States of Nigeria. Nig Vet J 1998; 19: 101-12.

69- Uzoukwu M, Ikeme M. Hepatic changes in natural F. gigantica infection in the Fulani zebu. Bull Anim Hlth Prod Africa 1978; 26: 161-7.

70- Sewell M. The pathogenesis of fascioliasis. Vet Rec 1966; 78: 98-109.

71- Ogunrinade A, Okon E, Fasanmi R. The prevalence of bovine fascioliasis in Nigeria. A 5- year analysis of abattoir records. Bull Anim Health Prod Afr 1981; 29 (4): 381-7.

72- Mage, C, Loisel J, Bonnand P. Fasciola hepatica infection and fertility problems in dairy herds. De Revue de Medecine Veterinaire 140 (10): 929-931, 1989.

73- Cawdary M. The effect of fascioliasis on ewe fertility. Brit Vet J 1976; 132: 568-75.

74- Cawdary M. The effect of regime therapy on fascioliasis II: The comparison of four drugs in ewes. Ir Vet J 1972; 26 (7): 137-43.

75- Mukasa-Mugerwa E, Kasali O, Said A. Effects of nutrition and endoparasite treatment on growth, onset of puberty and reproductive activity in merz ewe lambs. Theriogenology 1991; 36 (2): 319-28.

76- Mellor D. Nutritional and placental determinants of foetal growth rate in sheep and consequences for the new born lambs. Bri Vet J 1983; 139: 307-10.

77- Sinclair K. Studies on the anaemia of ovine fascioliasis. Bri Vet J 1964; 120: 212-22.

101 78- Okewole A, Ogundipe T, Adejinmi O, Olaniyan O. Clinical evaluation of three chemoprophylactic regimes against ovine helminthosis in a Fasciola-endemic farm in Ibadan, Nigeria. Israel Journal of Veterinary Medicine 2000; 56(1): 45-9.

79- Carnevale S, Rodríguez M, Santillán G, Labbé J, Cabrera M, Bellegarde E, et al. Iimmunodiagnosis of human fascioliasis by an enzyme-linked immunosorbent assay (ELISA) and a Micro-ELISA. Clinical and Diagnostic Laboratory Immunology 2001; 8(1): 174-7.

80- Katz N, Chaves A, Pellegrino J. A simple device for quantitative stool thick-smear technique in Schistosoma mansoni. Revista de Instituto de Medicina Tropical de São Paulo 1972; 14 :397–402.

81- Esteban JG, Mas-Coma S. Presence of very high prevalence and intensity of infection with Fasciola hepatica among Aymara children from Northern Bolivian Altiplano. Acta tropica 1997; 66:1–14.

82- Esteban JG . A population based coprological study of human fascioliasis in a hyperendemic area of the Bolivian Altiplano. Trop Med Int Health 1997; 2:695–9.

83- El-Sahn F, Farghaly A, El Masry A, Mandil A, Gad A, El Morshedy H. Human fascioliasis in an Egyptian village: prevalence and some epidemiological determinants. Journal of the Egyptian Public Health Association 1995; 70:(5):541–57.

84- El-Morshedy H, Shehab A, Zaki A, Farag H. Intra-specimen and day-to-day variations of Fasciola egg counts in human stools. Eastern Medetreanean Health Journal 2002; 8: 54-9.

85- Shehata AI. A study on the changing pattern of schistosomiasis infection in two villages, El Behiera Governorate. Thesis, MD, Alexandria University, High Institute of Public Health, 1995.

86- Tantrawatpan C, Maleewong W, Wongkham C, Wongkham S, Intapan P, Nakashima K. Serodiagnosis of human fascioliasis by a cystatin capture enzyme-linked immunosorbent assay with recombinant Fasciola gigantica cathepsin l antigen. Am J Trop Med Hyg 2005; 72(1): 82–6.

87- Chaisiri Wongkham C, Tantrawatpan C,Intapan P, Maleewong W, Wongkham S, Nakashima K. Evaluation of immunoglobulin g subclass antibodies against recombinant Fasciola gigantica cathepsin L1 in an enzyme-linked immunosorbent assay for serodiagnosis of human fasciolosis. Clinical and Diagnostic Laboratory Immunology 2005; 12 (10): 1152-6.

88- Capron A, Biguet J, Tran Vansky P, Rose G. Possibilities nouvelles dans le diagnostic immunologique de la distomatose humaine a `Fasciola hepatica'. Mise en evidence d'anticorps seriques par immunoelectrophorèse. Presse Med. 1964; 72:3103-7.

102 89- Hillyer G, Capron A. Immunodiagnosis of human fascioliasis by counterimmunoelectrophoresis. J Parasitol 1976 ; 62:1011-3.

90- Knobloch J, Delgado A, Alvarez G, Reymann U, Bialek R. Human fascioliasis in Cajamarca/Perú. I. Diagnostic methods and treatment with praziquantel. Trop Med Parasitol 1985; 36:88-90.

91- Espino A, Dumenigo B, Fernandez R, Finlay C. Immunodiagnosis of human fascioliasis by enzyme-linked immunosorbent assay using excretory-secretory products. Am J Trop Med Hyg 1990; 37:605-8.

92- O'Neill S, Parkinson M, Strauss W, Angles R, Dalton J. Immunodiagnosis of Fasciola hepatica infection (fascioliasis) in a human population in the Bolivian Altiplano using purified cathepsin L cysteine proteinase. Am J Trop Med Hyg 1998; 58:417-23.

93- Espino A, Díaz A, Pérez A, Finlay C. Dynamics of antigenemia and coproantigens during a human Fasciola hepatica outbreak. J Clin Microbiol 1998; 36:2723-6.

94- Haseeb AN, El-Shazly AM, Arafa MA, Morsy AT. Evaluation of excretory/secretory Fasciola (Fhes) antigen in diagnosis of human fascioliasis. J Egypt Soc Parasitol. 2003; 33(1):123-38.

95- Espino AM, B2rges A, Dumenigo BE. Fecal antigens of Fasciola hepatica potentially useful in the diagnosis of fascioliasis. Rev Panam Salud Publica 2000; 7(4):225-31.

96- Cosme A, Ojeda E, Poch M, Bujanda L, Castiella A, Fernandez J. Sonographic findings of hepatic lesions in human fascioliasis. J Clin Ultrasound. 2003;31(7):358-63.

97- Richter J, Freise S, Mull R, Millán J. Fascioliasis: sonographic abnormalities of the biliary tract and evolution after treatment with triclabendazole. Trop Med Int Health 1999; 4 (11): 774-6.

98- Gonzalo-Orden M, Millan L, Alvarez M, Sanchez-Campos S, Jimenez R, Gonzalez- Gallego J, et al. Diagnostic imaging in sheep hepatic fascioliasis: ultrasound, computer tomography and magnetic resonance findings. Parasitol Res 2003; 90(5):359-64.

99- Sampaio-Silva ML. Antigenic components of excretory-secretory products of adult Fasciola hepatica recognized in human infections. Am J Trop Med Hyg 1996; 54(2):146-8.

103 100- Abou Basha LM, Salem AI, Fadali GA. Ultrastructure of the liver before and after bithionol treatment. J Egypt Soc Parasitol 1990; 20:541-8. 101- Abou-Basha L, Shehab A, Osman , Farag H. Specific IgG avidity in acute and chronic human fascioliasis . Eastern Medetreanean Health Journal 2000; 6: 919-25.

102- Shehab AY. Detection of circulating E/S antigens in the sera of patients with fascioliasis by IELISA: a tool of serodiagnosis and assessment of cure. Trop Med Int Health 1999; 4(10):686-90.

103- Knobloch J. Human fascioliasis in Cajamarca/Peru. II. Humoral antibody response and antigenaemia. Tropical medicine and parasitology 1985; 36(2):91-3.

104- Rousseau S, Hedman K. Rubella infection and reinfection distinguished by avidity of IgG. Lancet 1988; 1(8594): 1108-9.

105- Enders G, Knotek F. Rubella IgG total antibody avidity and IgG subclass specific antibody avidity assay and their role in the differentiation between primary rubella and rubella reinfection. Infection 1989; 17(4):218-26.

106- Hedman K. Recent primary toxoplasma infection indicated by a low avidity of specific IgG. Journal of infectious diseases 1989; 159(4):736-40.

107- Hedman K, Vaheri A, Brummer-Korvenkontio M. Rapid diagnosis of hantavirus disease with an IgG-avidity assay. Lancet 1991; 338(8779):1353-6.

108- El-Zayyat E. Evaluation of specific immunoglobulin G avidity enzyme-linked immunosorbent assay (IgG avidity ELISA) in diagnosis of early and chronic schistosomiasis. J Egypt Soc Parasitol 1998; 28(3): 739-52.

109- Rapic D, Dzakulla N, Richard R. Comparative efficacy of triclabendazole, nitroxynil and rafoxanide against immature and mature Fasciola hepatica in natively infected cattle. Vet Rec 1988; 125: 59-62.

110- Ueno H, Alvarez JM, De Mergen AM, Cabral AF, Bodden HA. Field trials with rafoxanide, bithionol sulfoxide and niclofolan against Fasciola hepatica in cattle in the dominican republic. Natl Inst Anim Health Q 1973; 13 (2):69-74.

111- Abo El-Hoda M, Abo Shousha S. IL-1 Activity in human fascioliasis before and after treatment with bithionol and dehydroemetine. Journal of the Medical Research Institute 1997; 18(1): 74-82.

112- Abou Basha L, Farag H, El-Hefni S, Michael A, Hosni K, Salman Z. Rabbit fascioliasis under bithionol treatment. Bul. Alex. fac. Med. 1983; 19(4):983-92.

104 113- Farag H, Ragab M, Salem A, Sadek N. A short note on prazequantel in human fascioliasis. Trop Med Hyg 1986; 89: 79-80.

114- Mansour-Ghanaei F, Shafaghi A, Fallah M. The effect of metronidazole in treating human fascioliasis. Med Sci Monit 2003; 9(10): 127-30.

115- Saudi M, Elsayad M, Abou El-Hoda A. 3-Cyanopyridine derivatives: novel fasciolicidal candidates. Alex J Pharm Sci 2002; 16(2): 75-82.

116- Allam A, Elsayed M, Shehata M, Abd El-Reheem W. Effect of trichlabendazole treatment on lipid peroxides and erythrocyte glutathione reductase in acute and chronic human fascioliasis. Journal of Medical Research Institute 1998; 19(1):42-51.

117- Helmy M, Osman M, Sedki, Fayed H, Abbas O. Serum proteins and glycoproteins in acute fascioliasis before and after triclabendazole treatment. Journal of the Medical Research Institute 2002; 23(3): 70-9.

118- Osman M, Ismail Y, Aref T. Human fascioliasis: A study on the relation of infection intensity and treatment to hepatobiliary affection. J Egypt Soc Parasitol 1999; 29(2): 353-63.

119- Abd El Mawla A, El Sayed M, Hishmat M. Comparative studies on the effect of Bithionol, praziquantel and triclabendazole on rabbit's fascioliasis: Histopathological and histochemical studies of the liver. Journal of the Medical Research Institute 1998; 19(2): 105- 16.

120- Eckert J, Schneiter G, Wolfk A. Fasinex (triclabendazole) a new fasciolicide. Teit Wschr 1984; 91: 349-56.

121- Robinson C. Triclabendazole. Medica Actual./Drugs Today 1985; 21(5): 277-333.

122- Boray J, Rolfe P. Chemoprophylaxis of Fasciola hepatica in sheep. Bull Soc Franc Parasitol 1990; 8(suppl. 1): 461-9.

123- Overend J, Bowen L. Resistance of Fasciola hepatica to trichlabendazole. Aust Vet J 1995;72: 275-9.

124- Lane G. Anthelmintic resistance. Vet Rec 1998; 143: 232-6.

125- Massoud A, El-Ashmawy I, Nasr M, Salama O. Mirazid ; Anew schistomicidal agent derived from Myrrh : studies on its influence on male reproductive organs and bile flow. Alex J Pharm Sci 2002; 16(2) :82-90.

126- Mitchell B, Maris L, Bonniwell A. Trichlabendazole- resistant liver fluke in Scottish sheep. Vet Rec 1998; 143: 399-402.

105 127- Vertiz S. Evaluacion farmacocinetica de alfa-biof10 en Ganado vacuno. Tesis de Maestria en Farmacia. Faculted de Quimica, Universidad Nacional Autonoma de Mexico, 2000. 128- Ibarra V, Montenegro C, Flores C, Hernandez C, Castello B. Evaluacion de cuatro vehiculos para formular un fasciolicidal experimental. Vet Mex 2000; 31:47-51.

129- Ibarra F, Vera Y, Quiroz H, Canto J, Castillo R, Hernandez A, et al. Determination of the effective dose of an experimental fasciolicide in naturally and experimentally infected cattle. Veterinary Prasitology 2004; 120: 65-74.

130- White C. Nitazoxanide: An important advance in anti-parasitic therapy. Am J Trop Med Hyg 2003; 68(4): 382–3.

131- Rossignol JF, Maisonneuve H. 1984. Nitazoxanide in the treatment of Taenia saginata and Hymenolepis nana infections. Am J Trop Med Hyg 1984; 33: 511–2.

132- Gilles HM, Hoffman PS. Treatment of intestinal parasitic infections: a review of nitazoxanide. Trends Parasitol 2002; 18: 95–7.

133- Diaz E, Mondragon J, Ramirez E, Bernal R. Short report: epidemiology and control of intestinal parasites with nitazoxanide in children in Mexico. Am J Trop Med Hyg 2003; 68: 384–5.

134- Massoud A, El-Sisi S, Salama O, Massoud A. Preliminary study of therapeutic efficacy of a new fasciolicidal drug derived from Commiphora molmol (Myrrh ). Trop Med Hyg 2001; 65 (2): 96-9.

135- Michie C, Cooper E. Frankincense and myrrh as remedies in children. J R Soc Med 1991; 84:602–6.

136- Lee T, Lam T. Allergic contact dermatitis due to a Chinese orthopedic solution tieh ta yao gin. Contact Dermatitis 1993; 28: 89–90.

137- Cleason P, Andersson R, Samuelsson G. T. cadinol, a pharmacologically active constituent of scented myrrh: introductory pharmacological characterization and high field 1Hand 13C-NMR data. Planta Med 1991; 54: 352–6.

138- Pesko L. Aphthous ulcers. Am Drug 1990; 202: 90-5.

139- Jain S. Ethnobotany and research on medicinal plants in India. Ciba Foundation Symposium 1994; 185: 153–68.

140- Al-Harbi M, Qureshi S, Raza M, Ahmed M, Giangreco A, Shah A. Anticarcinogenic effect of Commiphora molmol on solid tumors induced by Ehrlich carcinoma cells in mice. Chemotherapy 1994; 40: 337–47.

106 141- Hall L, Oser L. Recent progress in the considration of flavoring ingredients under the food additives amendment. 3 GRAS substance. Food Technol 1965; 19: 151-97. 142- Ford A, Api M, Letizia S. Monographs on fragrance raw materials. Food Chem Toxicol 1992; 30: 1-38.

143- Council of Europe: Partial agreement in the social and public health field: flavouring substances and natural sources of flouverings. List N2. No. 150. Strusbourg: Editeur Maisonneuve SA. 1981.

144- Massoud A, El Kholy N, El Shennawy F, Farag R. Study of some immune aspects in patiants with fascioliasis before and after Chommiphora molnol (Mirazid ) treatment. J Egypt Parasitol 2004; 34(1):315-32.

145- Al Harbi M, Qureshi S, Ahmed M, Rafatulla S, Shah A. Effect of Commiphora molmol (oleogum-resin) on the cytological and biochemical changes induced by cyclophosphamide in mice. Am J Chin Med 1994; 22: 77- 82.

146- Dolara P, Corte B, Ghelardini C, Pugliese AM, Cerbai E, Menichetti S,et al. Local anaesthetic, antibacterial and antifungal properties of sesquiterpenes from Myrrh . Planta Med 2000; 66(4):356-8.

147- Haridy FM, Dawoud HA, Morsy TA. Efficacy of Commiphora molmol (Mirazid) against sheep naturally infected with Monieziasis expansa in Al-Santa Center, Gharbia Governorate, Egypt. J Egypt Soc Parasitol 2004; 34(3):775-82.

148- Shatat H, El-Sahn A. Effectiveness of myrrh in the treatment of Schistosoma mansoni infection: a controlled trial. Bulletin of High Institute of Public Health 2004; 34 (2): 425-36.

149- Botros S, Sayed H, El-Dusoki H, Sabry H, Rabie I, El-Ghannam M, et al. Efficacy of Mirazid in comparison with praziquantel in Egyptian Schistosoma Mansoni–Infected School Children and Households. Am J Trop Med Hyg 2005; 72(2): 119-23.

150- Botros S, William S, Ebeid F, Cioli D, Katz N, Day TA, et al. Lack of evidence for an antischistosomal activity of Myrrh in experimental animals. Am J Trop Med Hyg 2004; 71(2):206-10.

151- El Baz MA, Morsy TA, El Bandary MM, Motawea SM. Clinical and parasitological studies on the efficacy of Mirazid in treatment of Schistosomiasis haematobium in Tatoon, Etsa Center, El Fayoum Governorate. J Egypt Soc Parasitol 2003; 33(3):761-76.

152- Sheir Z, Nasr A, Massoud A, Salama O, Badra G, El-Shennawy H, et al. A safe, effective, herbal antischistosomal therapy derived from Myrrh . Am J Trop Med Hyg 2001; 65(6): 700–4.

107 153- Haridy FM, El Garhy MF, Morsy TA. Efficacy of Mirazid (Commiphora molmol) against fascioliasis in Egyptian sheep. J Egypt Soc Parasitol. 2003; 33(3):917-24.

154- Massoud AM, Habib FS. The effects of Myrrh (Commiphora molmol) on the infected snails of Schistosoma sp. and their egg masses: effect on shedding of cercariae and on snail fecundity. J Egypt Soc Parasitol 2003; 33(2):585-96.

155- Allam AF, El-Sayad MH, Khalil SS. Laboratory assessment of the molluscicidal activity of Commiphora molmol (Myrrh) on Biomphalaria alexandrina, Bulinus truncatus and Lymnaea cailliaudi. J Egypt Soc Parasitol 2001; 31(3):683-90.

156- Massoud AM, Labib IM. Larvicidal activity of Commiphora molmol against Culex pipiens and Aedes caspius larvae. J Egypt Soc Parasitol 2000;30(1):101-15.

157- Massoud AM, Labib IM, Rady M. Biochemical changes of Culex pipiens larvae treated with oil and oleo-resin extracts of Myrrh Commiphora molmol. J Egypt Soc Parasitol 2001;31(2):517-29.

158- Peters PA, El-Alamy M, Warren KS, Mahmoud AA. Quick Kato smear for field quantification of Schistosoma mansoni eggs. Am J Trop Med Hyg 1980; 29(2): 217-9.

159- Moss D, Henderson R. Clinical enzymology. In: Burtis C, Ashwood E. Tietz textbook of clinical chemistry. 3rd Edition. Philadelphia: WB Saunders Company; 1999. 617-721.

160- Soliman OE, El-Arman M, Abdul-Samie ER, El-Nemr HI, Massoud A. Evaluation of Myrrh (Mirazid ) therapy in fascioliasis and intestinal schistosomiasis in children: immunological and parasitological study. J Egypt Soc Parasitol 2004; 34(3):941-66.

161- Zali RM, Ghaziani T, Shahraz S, Hekmatdoost A, Radmehr A. Liver, spleen, pancreas and kidney involvement by human fascioliasis: imaging findings. BMC Gastroenterology 2004; 4:15-7.

162- Campos AH, Velarde FI, Montenegro YV, Fernández NR, Castillo R. Synthesis and Fasciolicidal Activity of 5-Chloro-2-methylthio-6- (1-naphthyloxy)-1H-benzimidazole. Chem Pharm Bull 2002; 50(5): 649—52.

163- Chauvin A, Moreau E, Boulard C. Responses of Fasciola hepatica infected sheep to various infection levels.Vet Res 2001; 32 : 87–92.

164- Gaasenbeek CP, Moll L, Cornelissen JB, Vellema P, Borgsteede FH. An experimental study on triclabendazole resistance of Fasciola hepatica in sheep. Vet Parasitol 2001; 95(1): 37-43.

108 165- Hassan MM, Moustafa NE, Mahmoud LA, Abbaza BE, Hegab MH. Prevalence of Fasciola infection among school children in Sharkia Governorate, Egypt. J Egypt Soc Parasitol 1995; 25(2):543-9.

166- Abo-Madyan AA, Morsy TA, Motawea SM, Morsy AT. Clinical trial of Mirazid in treatment of human fascioliasis, Ezbet El-Bakly (Tamyia Center) Al-Fayoum Governorate. J Egypt Soc Parasitol 2004; 34(3):807-18.

167- Trueba G, Guerrero T, Fornasini M, Casariego I, Zapata S, Ontaneda S et al. Detection of fasciola hepatica infection in a community located in the Ecuadorian Andes. Am J Trop Med Hyg 2000; 62(4): 518.

168- Curtale F, Mas-Coma S, Hassanein YA, Barduagni P, Pezzotti P, Savioli L. Clinical signs and household characteristics associated with human fascioliasis among rural population in Egypt: a case-control study. Parassitologia 2003; 45(1):5-11.

Ven S, Arslan S, Nal S, et al. Fasciola hepaticaـ Aksoy D, Kerimoglu U, Oto A, Erg 169- infection: Clinical and computerized tomographic findings of ten patients. Turk J Gastroenterol 2006; 17 (1): 40-5.

170- Baser K, Demirci B, Dekebo A, Dagne E. Essential oils of frankincense, Myrrh and opopanax. Flavour Fragr J 2003; 18: 153-6.

171- Hassan M, Abbaza B, El-Karamany I, Dyab A, El Sharkawy E, Ismail F, et al. Detection of anti-Fasciola isotypes among patients with fascioliasis before and after treatment with Mirazid . J Egypt Soc Parasitol 2004;34(3):857-64.

172- Motawea S, El Gilany A, Gaballah M, Emara F, El Shazly A. Control of Fasciola in an Egyptian endemic rural area by a new, safe, effective fasciolicidal, herbal drug. J Environm Sci 2001b; 21(5):85-104.

173- Savioli L, Chitsulo L, Montresor A. New opportunities for the control of fascioliasis. Bull World Health Organ 1999; 77 (4):300-5.

174- Mas-Coma S. Human fascioliasis in Europe and Latin America. In: Infectious Diseases and Public Health. A Research and Clinical update. Mario Angllico and Giovanni Rocchi, Editors. Balaban Publishers 1998; Philadelphia, L'Aquila.

175- El Shazly A, Abo El Wafa S, Haridy F, Soliman M, Rifaat M, Morsy T. Fascioliasis among live and slaughtered animals in the centres of Dakahlia. J Egypt Soc Parasitol 2002a; 32(1):47-57.

176- Khalil MZ. A study of fascioliasis in farm animals and man. Thesis, MPH, Alexandria, Egypt: Alexandria University, High Institute of Public Health. 1979.

109 177- Haridy FM, Morsy TA, Gawish NI, Antonios TN, Abdel Gawad AG. The potential reservoir role of donkeys and horses in zoonotic fascioliasis in Gharbia Governorate, Egypt. J Egypt Soc Parasitol 2002; 32(2):561-70.

178- Valero M, Mas-Coma S. Comparative infectivity of Fasciola hepatica metsacercariae from isolates of the main and secondary reservoir animal host species in the Bolivian Altiplano high human endemic region. Folia Parasitologica 2000; 47:17-22.

110 لقد تسببت التغيرات البيئية الخيرة و كذلك التغيرات التي حدثت في السلوك النساني في إيجاد حدود جغرافية جديدة لمرض الدودة الكبدية و في زيادة أعداد المعرضين لخطر الصابة بهذا المرض. و لقد ازداد عدد المصابين بهذا المرض منذ عام 1980 و لم تعد ازدياد الصابة به في النسان قاصرة على المناطق التي تعاني من وجوده بشكل كبير بين الحيوانات . و بالنسبة للحيوان فإن الدودة الكبدية (الفاشيول) تعد مشكلة خطيرة تتسبب في خسارة اقتصادية هائلة. و تشخيص هذا المرض يتم بإيجاد البويضة في عينة البراز للكائن المصاب. و حساسية طريقة الكاتو في تشخيص هذا المرض تصل إلى 99% إذا ما تم فحص ثلث شرائح معدة من نفس عينة البراز أو من ثلث عينات تم تجميعها على مدى ثلثة أيام. و إن من أهم خواص الدواء الجيد المستخدم في علج الديدان أن يكون له كفاءة عالية و آمن و ل يتسبب في وجود بقايا كيميائية دائمة في أنسجة العائل و أخيرا أن يكون سهل التداول بسعر إقتصادي.

و لقد كان التريكلبندازول آمنا و فعال في القضاء على كل مراحل الدودة الكبدية (الناضجة و الغير ناضجة) في كل من النسان و الحيوان و لكن تم في السنوات الخيرة اكتشاف حالت ل تستجيب لهذا العقار نتيجة تكون سللة جديدة من الديدان تقاومه. المر الذي يستدعي البحث عن عقار جديد فعال.

و أما الميرازيد فقد تم ترخيصه كعقار جديد لعلج البلهارسيا و الفاشيول في مصر في شهر مارس من عام 2002 وهو مستخلص نقي من المر و الذي يتم الحصول عليه من شجرة الكوميفورا مولمول.

و قد تم إجراء هذه الدراسة بهدف تقييم معدل الشفاء من الفاشيول في النسان و الحيوان بعد العلج بالمر و لتحديد تأثير المر على شدة الصابة بالفاشيول و لدراسة التغييرات في مستوى إنزيمات الكبد في الدم قبل و بعد تلقي العلج بالمر في النسان المصاب بالفاشيول المزمنة .

111 ولقد تم إجراء هذه الدراسة في الفترة من يونيو 2005 حتى فبراير 2006 في العزبة العاشرة من قرية أبيس 8 حيث تم عمل خريطة للمكان و عمل تعداد لكل القاطنين بالعزبة و ما يمتلكون من حيوانات. ولقد وجد أن التعداد الكلي للسكان في العزبة يبلغ 1305 شخصا منهم 138 طفل أعمارهم أقل من 5 سنوات و هؤلء تم استبعادهم من الدراسة. و بالنسبة للحيوان تم دراسة كل الحيوانات العشبية الموجودة في القرية و شملت البقر و الجاموس و الغنم و الماعز و الحمير و قد بلغ عددهم 208 حيوانا. و قد تم عمل مسح شامل للمقيمين بالعزبة و ما يمتلكون من حيوانات لتشخيص المصابين منهم بالفاشيول باستخدام طريقة الكاتو. وبلغ عدد العينات التي تم فحصها 1092 عينة من السكان و 119 عينة من الحيوانات حيث تم فحص 3 شرائح من كل عينة و تسجيل ما تم تشخيصه من الديدان فيها و لكن تم عد بيض الفاشيول فقط . و قد تم فحص عينة أخرى من الناس المصابين بالفاشيول بعد أسبوع من تناول الطعمة الخالية من الكبد كي يتم استبعاد الحالت الغير مصابة حقا من العينة. و لقد خضعت حالت الفاشيول المشخصة في السكان لستمارة استبيان لجمع المعلومات الخاصة بالمرض و التي تشمل إذا ما تمت الصابة بالفاشيول من قبل و العراض الخاصة بالمرض و و إذا ما تم أخذ علج للمرض من قبل. و أما الحالت المشخصة في الحيوان فقد تم جمع معلومات من مالكي هذه الحيوانات خاصة بنوع الحيوان المصاب و عمره و جنسه و كيفية التخلص من فضلته. و كذلك تم أخذ عينة دم من المصابين بالمرض من السكان قبل أخذ العلج و أخرى بعد شهر من أخذه لقياس نسبة إنزيمات الكبد (الترانزأماينيز) في الدم.

و قد بلغ عدد الحالت المشخصة في السكان 64 حالة و في الحيوان 63 حالة.

و تم علج الحالت النسانية بإستخدام عقار الميرازيد في صورة كبسولت (300 مجم/ الكبسولة) و ذلك بجرعة 10 مجم/كج من وزن الجسم في اليوم لمدة 6 أيام متتالية تؤخذ في الصباح قبل الفطار بساعة على معدة خاوية و قد تم إعتبار الجرعة القصوى في اليوم الواحد 600مجم. و بالنسبة للحالت في الحيوان فقد تم علجها باستخدام معلق الميرازيد 10% بجرعة 600 مجم/اليوم لمدة يومين متتاليين تؤخذ في الصباح على معدة خاوية.

112 و قد تم تقييم معدل الشفاء بعد العلج بالميرازيد ثلث مرات و ذلك بعد شهر ثم شهرين ثم ثلثة أشهر من العلج. و في كل مرة تم جمع ثلث عينات براز من كل حالة (في النسان و الحيوان) تفصل بين كل منها 24 ساعة لتشخيص وعد بيض الفاشيول. تم جمع البيانات و تحليلها إحصائيا و تم استخلص النتائج التالية: الدراسة النسانية: 1- بلغ معدل الصابة بالفاشيول في النسان 5.8% مما يدل على أن هذا المرض ما زال يمثل مشكلة صحية رئيسية في منطقة أبيس حيث يمثل ثاني أكبر إصابة بالطفيليات في تلك المنطقة بعد البلهارسيا.

2- الطفال أقل من 10 سنوات كانوا أكثر عرضة للصابة بالفاشيول 5 مرات أكثر من الفئات الكبر سنا كما كان لديهم أعلى نسبة إصابة ثقيلة (30%) و أعلى متوسط لعدد البيض في البراز( 114.46) بالمقارنة بالكبر سنا.

3- النساء كن أكثر عرضة للصابة بالفاشيول من الرجال (7.5% من النساء كن مصابين بالمرض بالمقارنة ب 4.3% من الرجال) كما كان لديهن أعلى نسبة إصابة ثقيلة بالمرض بالمقارنة بالرجال (15% من المصابات كان لديهن إصابة ثقيلة بالمقارنة ب 8.3% من الرجال المصابين).

4- كان وجود حيوانات تعيش بالمنزل عامل مهما يزيد من خطر الصابة بالفاشيول للمقيمين بهذا المنزل بما يعادل مرتين أكثر من غيرهم رغم أن التعرض المباشر للحيوان المصاب ليس من طرق نقل العدوى للنسان.

5- معظم حالت الفاشيول كانت ل تعاني من أي أعراض مرضية (64.5%) بينما أكثر العراض شيوعا في الحالت التي كانت تعاني من أعراض مرضية كانت ألم في البطن (24.1%) ثم الغثيان (4.8%) و قد كان 3.2% فقط من الحالت يعاني من الصفراء أو وجود دم في البراز.

113 6- كان هناك ارتباط بين عدم وجود أي أعراض مرضية في الحالت المصابة بالفاشيول من ناحية و بين كون الصابة خفيفة من ناحية أخرى حيث كانت معظم الحالت التي ل تعاني من أعراض مرضية (80%) لديها إصابة خفيفة بالفاشيول كما كان متوسط عدد البيض في البراز لديها أقل من المتوسط لدى الحالت المصابة و التي لديها أعراض مرضية (50.66 مقابل 94.28 بالترتيب).

7- يعتبر المر علجا واعدا جديدا فعال لعلج الفاشيول في النسان و قد بلغ معدل الشفاء بعد استخدامه 88.2% و ذلك بعد مرور ثلثة أشهر من العلج. 8- وجد أن العوامل المرتبطة بنجاح العلج بالمر كانت : كون الحالة المعالجة من الذكور و أن تكون الحالة المصابة غير مصاحبة لعراض مرضية و أن تكون الصابة خفيفة و أن تكون الحالة منتمية لعائلة صغيرة الحجم و أخيرا عدم و جود حيوانات تعيش في نفس المنزل.

9-وجد أن الحالت التي لم يتم شفاؤها بعد العلج بالمر قد أظهرت نقصا ملحوظا في متوسط عدد البيض في البراز حيث بلغ المتوسط قبل العلج 96.12 و قد قل إلى 34.28 بعد شهر ثم إلى 30.61 بعد شهرين و أخيرا كان 44.89 بعد ثلثة أشهر مما يعكس أهمية كبيرة للصحة العامة في تقليل انتشار العدوى في المجتمع.

10-وجد أن انزيمات الكبد لم تتأثر بعد مرور شهر على العلج بالمر.

الدراسة على الحيوان: 1-وجد أن الفاشيول منتشرة انتشارا كبيرا بين الحيوانات في قرية أبيس و قد بلغ معدل الصابة بها 52.9% و قد كانت أكثر انتشارا بين الجاموس (65.6%) ثم البقر (55.6%) و قد وجدت أقل نسبة إصابة بين الماعز (%12.5).

2- وجد أن ما يقرب من نصف الحيوانات المصابة بالفاشيول كان لديها إصابة شديدة و قد كان المتوسط الكلي لعدد البيض في البراز في الحيوانات عاليا (236.56) و قد بلغ هذا المتوسط أعلى نسبة في الحيونات الصغيرة السن (أقل من عام) حيث بلغ 905.78 و قد قل تدريجيا مع ازدياد عمر الحيوان.

114 3- وجد أن الحمير لديها قدرة عالية على نقل المرض و قد بلغت نسبة الصابة فيها %40.

4- كان معدل الشفاء في الحيوانات بعد العلج بالميرازيد منخفضا بعد مرور ثلثة أشهر من العلج (77.5%) و قد يكون هذا نتيجة استخدام نفس الجرعة العلجية لكافة أنواع الحيوانات المصابة مع عدم أخذ وزن الحيوان المصاب في العتبار.

5- كانت العوامل المرتبطة بنجاح العلج بالمرهي: نوع الحيوان المعالج حيث كانت النتائج أفضل بين الماعز و البقر بالضافة إلى الصابة الخفيفة و التقدم في العمر.

6- وجد أن متوسط عدد البيض في البراز كان أقل بشكل كبير و ذو دللة إحصائية في الحالت التي تم شفاؤها مقارنة بالتي لم يتم شفاؤها (149.24 مقارنة ب 321.12 على الترتيب).

115 فاعلية عقار المر في علج الدودة الكبدية في النسان و الحيوان

مقدمة من: ط/ سميرة محمد محبوب عبد الحميد بكالوريوس طب و جراحة عامة جامعة السكندرية- 1998 ماجستير صحة عامة (صحة مناطق حارة) المعهد العالي للصحة العامة جامعة السكندرية- 2004

للحصول على درجة الدكتوراه في الصحة العامة(صحة مناطق حارة) لجنة المناقشة و الحكم على الرسالة موافقون أ.د/ عزت محمد حسن أ.د/ ثناء أحمد المصري أ.د/ علي أحمد و هيب أ.د/ حنان زكريا شتات

التاريخ / /

116 لجنة الشراف أ.د/ عزت محمد حسن أ.د/ أميرة محمود قطقاط أ.د/ حنان زكريا شتات أ.د/ أمل عبد الفتاح الصحن

117 فاعلية عقار المر في علج الدودة الكبدية في النسان و الحيوان

رسالة مقدمة إلى المعهد العالي للصحة العامة جامعة السكندرية إيفاء جزئيا لشروط الحصول على درجة الدكتوراه في الصحة العامة(صحة مناطق حارة)

من: ط/ سميرة محمد محبوب عبد الحميد بكالوريوس طب و جراحة عامة جامعة السكندرية- 1998 ماجستير صحة عامة (صحة مناطق حارة) المعهد العالي للصحة العامة جامعة السكندرية- 2004

المعهد العالي للصحة العامة

جامعة السكندرية