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)

By:

Samira Mohamed Mahboub Abd El Hameed

M.B.Ch.B, Faculty of Medicine, University of Alexandria,

1998M.P.H. (Tropical Health), High Institute of Public Health,

University of Alexandria, 2004

1

High Institute of Public Health

University of Alexandria

2008

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Effectiveness of the Fasciolicidal Drug Myrrh in

Treatment of Human and Animal Fascioliasis

By:

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 doctorIn 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

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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

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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.

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CONTENTS

Chapter Title Page

i List of figures

I Introduction A- Prevalence of human fascioliasis

B- Prevalence of animal fascioliasis

C- Life cycle of Fasciola sp.

D- Characteristics of life cycle of Fasciola sp. 1- Domestic animal reservoir hosts

2- Wild animal reservoir hosts 3- Intermediate snail hosts 4- Proper Environmental Conditions 5- Mode of transmission 6- Association of fascioliasis with other

parasitic infections E- Clinical picture of human fascioliasis F-Clinical picture of animal fascioliasis

G- Diagnostic tools for fascioliasis 1- Stool examination 2- Serological methods 3- Detection of fecal antigens of Fasciola

hepatica 4- Radiological findings

H- Diagnosis of acute fascioliasis

I- Diagnosis of chronic fascioliasis

J- Differential diagnosis of primary and secondary infections

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8899

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K- Treatment of fascioliasis 1- Old lines of treatment of facsioliasis i- Bithionol (Bitin) ii- Praziquantel iii- Metronidazole 2- Currently used drug

i- Triclabendazole (TCBZ):

3- Trials using new fasciolicidal drugs

i- "3-Cyanopyridine Derivatives"

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

iii- Nitazoxanide

iv- Myrrh

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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

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List of figures

Figure No. Title Page

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

Figure (2):

Figure (3):

Figure (4):

Figure (5):

Figure (6):

Figure (7):

Figure (8):

Figure (9):

Figure (10):

Figure (11):

A diagrammatic illustration of the pre-intervention phase of the study

Prevalence of Fasciola sp. infection among different age groups

Intensity of infection with Fasciola sp.in different age groups

The parasitological cure rates one, two and three months after treatment of human cases with Mirazid

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

A diagrammatic illustration of the pre-intervention phase of the animal study

Prevalence of animal fascioliasis among different age groups

GMEC among different age groups of infected animals

Animal cure rates one, two and three months after treatment with Mirazid

GMEC in uncured animal cases of fascioliasis before and after treatment

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56

61

69

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78

81

86

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List of figures

Figure No. Title Page

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

Figure (2):

Figure (3):

Figure (4):

Figure (5):

Figure (6):

Figure (7):

Figure (8):

Figure (9):

Figure (10):

Figure (11):

A diagrammatic illustration of the pre-intervention phase of the study

Prevalence of Fasciola sp. infection among different age groups

Intensity of infection with Fasciola sp.in different age groups

The parasitological cure rates one, two and three months after treatment of human cases with Mirazid

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

A diagrammatic illustration of the pre-intervention phase of the animal study

Prevalence of animal fascioliasis among different age groups

GMEC among different age groups of infected animals

Animal cure rates one, two and three months after treatment with Mirazid

GMEC in uncured animal cases of fascioliasis before and after treatment

25

31

32

41

46

53

57

61

63

67

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The Memory of my Father

My Mother

My Beloved Husband

and My Lovely Childe

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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)

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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)

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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)

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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

15

amphibious snail, and also for the swimming cercariae. The ideal temperature range for 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

16

is diagnosed. The duration of the survival of the pathogen in human may be up to 13 years.(23)

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)

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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

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from 96 – 99.1%, However, the examination of three Kato smears from a single stool specimen is more feasible in field studies.(84)

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

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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: 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)

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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 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

21

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)

22

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

23

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

24

(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)

25

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

26

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

27

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)

28

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.

29

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

30

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

31

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

32

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.

33

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 studyB) Therapeutic trial

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

34

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).

35

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

Target populationn=1167

<5 years n=138 (excluded)

75 did not give stool samples

Study population

n=1092(93.6%)

36

Fascioliasisn=68 (6.2%)

True fasciliasis

n=64 (5.8%)

Liver free diet for 1 week, repeat stool

analysis

Total population n=1305

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

Variable NO.(n=1305) %

Age (years)<55-10-20-30-40-≥50

138141345200161170150

10.610.826.415.312.313.111.5

SexMaleFemale

642663

49.250.8

Family size≤4 members5-8 members9 or more members

280417608

21.431.946.7

37

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

Helminthic infection No.(n=1092)*

%

Free 973 89.1Helminthic infection:Schistosoma mansoniFasciola sp.Ascaris lumbricoidesHymenolepis nanaEnterobius vermicularis¶Trichuris trichiura

11976647766

10.97.05.80.60.60.50.5

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

38

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%).

39

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

Variable

Fasciola sp. Infection(n=1092) OR (95%CI)Free Infected

No. (%) No.(%)Age group5-10-20-30-®

40-≥50 years

94 (82.5%)325 (95.9%)166 (95.4%)155 (96.3%)154 (93.9%)134 (95.7%)

20 (17.5%)14 (4.1%)8 (4.6%)6 (3.7%)10 (6.1%)6 (4.3%)

5.5(1.99-15.92)*1.11(0.39-3.31)1.24(0.38-4.15)11.68(0.54-5.34)1.16(0.32-4.16)

Sex Male®

Female532 (95.7%)496 (92.5%)

24 (4.3%)40 (7.5%)

11.79(1.03-3.11)*

Family size3-4 members®

5-8 members9 or more members

261 (95.6%)333 (91.7%)434 (95.2%)

12 (4.4%)30 (8.3%)22 (4.8%)

11.96(0.94-4.14)1.1(0.51-2.41)

Household animal contactNoYes

756 (95.6%)272 (90.3%)

35 (4.4%)29 (9.7%)

12.3(1.34-3.95)*

*P<0.05

OR = Odds ratioCI = Confidence interval

40

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

Age group(in years)

Fasciola sp. infection

Fisher's Exact testMale FemaleFree

No.(%)InfectedNo.(%)

FreeNo.(%)

InfectedNo.(%)

5- 43 (78.2%)

12 (21.8%)

51 (86.4%)

8 (13.8%) P=0.32

10- 177(97.8%)

4 (2.2%)

148(93.7%)

10(6.3%) P=0.05

20- 79(100%)

0(0%)

87(91.6%)

8(8.4%) P=0.008*

30- 77(100%)

0(0%)

78(92.9%)

6(7.1%) P=0.02*

40- 77(95%)

4(5%)

77(92.8%)

6(7.2%) P=0.74

50 or more 79(95.2%)

4(4.8%)

55(96.5%)

2(3.5%) P=1.000

* p<0.05

41

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.

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

42

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).

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

43

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.

44

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 memberOnly one family memberTwo or three family members

433013

24.617.27.4

Total 175 100

45

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

Fasciola sp. Infection No.(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

46

Table (VII): Clinical picture of chronic fascioliasis

SymptomCases of fascioliasis

(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

47

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

48

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).

49

Table (IX): Some parameters associated with intensity of infection of human fascioliasisVariable Intensity of infection Monte Carlo test

Light Moderate Heavy GMECAge:5-

10-

20-

30-

40-

50 or more

10 (50.0%)

12(85.7%)

8(100.0%)

6(100.0%)

8(80.0%)

4(66.7%)

4(20.0%)

2(14.3%)

0(0%)

0(0%)

0(0%)

2(33.3%)

6(30.0%)

0(0%)

0(0%)

0(0%)

2(20.0%)0(0%)

114.46

49.77

23.21

56.61

54.83

92.90

Monte Carlo testP=0.016*

Sex:Male

Female

18(75.0%)

30(75.0%)

4(16.7%)

4(10.0%)

2(8.3%)

6(15.0%)

64.59

63.56

Monte Carlo testP=0.672

Presence of symptoms:

Asymptomatic

Symptomatic

32(80.0%)

16(66.7%)

4(10.0%)

4(16.7%)

4(10.0%)

4(16.7%)

50.66

94.28

Monte Carlo testP=0.469

Family size:≤ 4 members

5 -8 members

9 or more members

12 (100.0%)

18 (60%)

18 (81.8%)

0 (0%)

6 (20%)

2 (9.1%)

0 (0%)

6 (20%)

2 (9.1%)

42.65

92.97

47.87

Monte Carlo testP=0.027*

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

50

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.

51

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

Duration after treatment

Cases of fascioliasisCured 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

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

52

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

Variable

Cure rate after one month(n=52)

Cure rate after two months(n=54)

Cure rate after three months(n=34)

Cured cases

No. (%)

Uncured cases

No. (%)

Cured cases

No. (%)

Uncured cases

No. (%)

Cured cases

No. (%)

Uncured cases

No. (%)Age group5-10-20-30-40-50 or more

10 (55.6%)2 (16.7%)2 (100%)4 (66.7%)8 (80%)2 (50%)

8 (44.4%)10 (83.3%)

0 (0%)2 (33.3%)2 (20%)2 (50%)

18 (90%)8 (80%)6 (100%)4 (66.7%)6 (75%)2 (50%)

2 (10%)2 (20%)0 (0%)

2 (33.3%)2 (25%)2 (50%)

10 (100%)4 (100%)2 (50%)

4 (66.7%)8 (100%)2 (100%)

0 (0%)0 (0%)2 (50%)

2 (33.3%)0 (0%)0 (0%)

Test of significane Monte Carlo test P= 0.012*

Monte Carlo test P= 0.256

Monte Carlo test P= 0.031*

SexMaleFemale

8 (40%)20 (62.5%)

12 (60%)12 (37.5%)

14 (70%)30 (88.2%)

6 (30%)4 (11.8%)

8 (100%)22 (84.6%)

0 (0%)4 (15.4%)

Test of significane Fisher's Exact testP=0.156

Fisher's Exact testP=0.147

Fisher's Exact testP=0.55

Family size3-4 members5 or more members

12 (100%)16 (40%)

0 (0%)24 (60%)

12 (100%)32 (76.2%)

0 (0%)10 (23.8%)

12 (100%)18 (81.8%)

0 (0%)4 (18.2%)

Test of significane Fisher's Exact testP=0.000*

Fisher's Exact testP=0.094

Fisher's Exact testP=0.27

*p<0.05

53

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.

54

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

Variable

Cure rate after one month(n=52)

Cure rate after two months(n=54)

Cure rate after three months(n=34)

Cured cases

No. (%)

Uncured cases

No. (%)

Cured cases

No. (%)

Uncured cases

No. (%)

Cured cases

No. (%)

Uncured cases

No. (%)Presence of symptomsAsymptomaticSymptomatic

20 (62.5%)8 (40%)

12 (37.5%)12 (60%)

32 (84.2%)12 (75%)

6 (15.8%)4 (25%)

20 (100%)10

(71.4%)

0 (0%)4

(28.6%)

Test of significane Fisher's Exact testP=0.156

Fisher's Exact testP=0.459

Fisher's Exact testP=0.02*

Household animal contactNoYes

16 (53.3%)

12 (54.5%)

14 (46.7%)10 (45.5%)

28 (93.3%)

16 (66.7%)

2 (6.7%)8 (33.3%)

18 (94.7%)12 (80%)

1 (5.3%)3 (20%)

Test of significane Fisher's Exact testP=1.000

Fisher's Exact testP=0.016*

Fisher's Exact testP=0.29

Intensity of infection before treatmentLightModerateHeavy

24 (60%)0 (0%)4 (50%)

16 (40%)4 (100%)4 (50%)

36 (81.8%)2 (50%)6 (100%)

8 (18.2%)2 (50%)0 (0%)

26 (86.7%)0 (0%)

4 (100%)

4 (13.3%)0 (0%)0 (0%)

Test of significane Monte Carlo test P= 0.012*

Monte Carlo test P= 0.055

Monte Carlo test P= 1.000

GMEC before treatment 47.788 96.129 49.692 60.285 50.06 70.99

ANOVA test for GMEC F= 2.269 F=0.638 F= 0.233

*p<0.05

55

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%.

56

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

Intensity of infection Before treatment

After treatment

1 month 2 month 3 month

GMEC 96.12 34.28 30.61 44.89

Wilcoxon signed ranks testZ=-3.794* Z=-2.558* Z=-1.414

*P<0.05

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

57

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

Intensity of infection Before treatment One month after treatment

Light 1666.7%

2291.7%

Moderate 416.7%

00%

Heavy 416.7%

28.3%

Total 24100%

24100%

Percent reduction of egg count 20%

58

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

Light 880%

10100%

Moderate 220%

00%

Heavy 00%

00%

Total 10100%

10100%

Percent reduction of egg count 42%

59

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

Light 4100%

4100%

Moderate 00%

00%

Heavy 00%

00%

Total 4100%

4100%

Percent reduction of egg count 30%

60

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).

61

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

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

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

AST level Pre-intervention

Post-intervention Paired samples t-test

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

62

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).

63

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

Sample sizen= 119

Response rate =57.2%

Total animal population n=208

Did not give stool samplesn=89

64

Cases of fascioliasis

n=63(52.9%)

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

Variable NO.(n= 208) % Mean age ± S.D

(in years)

Animal species:BuffaloCowGoatSheepDonkeyTotal

5287272022208

25.041.813.09.610.6100

3.7 ±1.93.6 ±1.93.1 ±1.52.4 ±1.23.7±1.53.5 ± 1.8

Sex:MaleFemaleTotal

50158

208

24.076.0

100

2.8±1.53.7±1.83.5±1.8

65

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

Animal species Fasciola sp. infection Test of significanceFree Infected

Buffalo 11 (34.4%) 21 (65.6%)

Monte Carlo test

P= 0.04*

Cow 24 (44.4%) 30 (55.6%)

Goat 7 (87.5%) 1 (12.5%)

Sheep 5 (50%) 5 (50%)

Donkey 9 (60%) 6 (40%)

*P<0.05

66

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).

67

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

Animal species Mean age of studied animals t TestFree 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

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

68

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

Sex

Fasciola sp. infection

Chi square testFree Infected

No. % No. %

Males 13 52.0 12 48.0

X2=0.3Females 43 45.7 51 54.3

Total 56 47 63 53

p>0.05

69

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*).

70

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

VariableIntensity of infection Test of

significanceLightNo. (%)

ModerateNo. (%)

HeavyNo. (%) GMEC

Animal type

Buffalo

Cow

Goat

Sheep

Donkey

7 (33.3%)

8 (26.7%)

0 (0%)

1 (20%)

6 (100%)

2 (9.5%)

8 (26.7%)

1 (100%)

0 (0%)

0 (0%)

12(57.2%)

14(46.6%)

0 (0%)

4 (80%)

0 (0%)

304.24

243.006

312

531.27

41.71

Monte Carlo testP=0.01*

Sex

Male

Female

7 (58.3%)

15(29.4%(

0 (0%)

11(21.6%)

5 (41.7%)

25( 49.0%)

123.65

275.57

Monte Carlo testP=0.1

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

*P<0.05

71

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

72

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).

73

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

Duration after treatment

Cases of fascioliasisCured Not cured

No. % No. %1 month(n=42) 27 64.3 15 35.7

2 months(n=51) 35 68.6 16 31.4

3 months(n=40) 31 77.5 9 22.5

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

74

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

Variable

Cure rate after one month(n=42)

Cure rate after two months(n=51)

Cure rate after three months(n=40)

Cured cases No. (%)

Uncured cases

No. (%)

Cured cases

No. (%)

Uncured cases

No. (%)

Cured cases No. (%)

Uncured cases

No. (%)Animal type:BuffaloCowGoatsSheepDonkeys

10 (66.7%)13 (76.5%)1 (100%)2 (40%)1(25%)

5 (33.3%)4 (23.5%)

0 (0%)3 (60%)3(75%)

14 (73.7%)16 (76.2%)1 (100%)2 (40%)2(50%)

5 (26.3%)5 (23.8%)

0 (0%)3 (60%)2 (50%)

10 (76.9%)15 (88.2%)1 (100%)3 (60%)2(50%)

3 (23.1%)2 (11.8%)

0 (0%)2 (40%)2 (50%)

Test of significane

Monte Carlo test P= 0.206

Monte Carlo test P= 0.429

Monte Carlo test P= 0.429

Age group

Less than 1 year1-3-5 or more

2 (40%)9 (69.2%)7 (63.6%)9 (69.2%)

3 (60%)4 (30.8%)4 (36.4%)4 (30.8%)

3 (60%)11 (68.8%)12 (92.3%)9 (56.3%)

2 (40%)5 (31.3%)1 (7.7%)7 (43.8%)

2 (66.7%)10 (76.9%)11 (91.7%)8 (66.7%)

1 (33.3%)3 (23.1%)1 (8.3%)4 (33.3%)

Test of significane

Monte Carlo test P= 0.550

Monte Carlo test P= 0.094

Monte Carlo test P= 0.291

Sex

MaleFemale

4 (50%)23 (67.6%)

4 (50%)11(32.4%)

4 (40%)31 (77.5%)

6 (60%)9(22.5%)

7 (70%)24 (80%)

3 (30%)6 (20%)

Test of significane

Fisher's Exact testP=0.425

Fisher's Exact testP=0.048*

Fisher's Exact testP=0.665

*p<0.05

75

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.

76

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

Variable

Cure rate after one month(n=42)

Cure rate after two months(n=51)

Cure rate after three months(n=40)

Cured cases

No. (%)

Uncured cases

No. (%)

Cured cases

No. (%)

Uncured cases

No. (%)

Cured cases

No. (%)

Uncured cases

No. (%)Intensity of infection before treatmentLightModerateHeavy

9 (50%)4 (80%)

14 (73.7%)

9 (50%)1 (20%)

5 (26.3%)

13 (65%)5 (71.4%)17 (73.9%)

7 (35%)2 (28.6%)6 (26.1%)

14 (82.4%)6 (85.7%)11 (68.8%)

3 (17.6%)1 (14.3%)5 (31.3%)

Test of significane Monte Carlo test P= 0.231

Monte Carlo test P= 0.937

Monte Carlo test P= 0.651

GMEC before treatment 231.656 153.355 221.290 175.303 149.24 324.12

ANOVA test for GMEC F= 0.490 F=0.004 F= 5.12*

*P<0.05

77

Table (XXVII): GMEC in uncured animal cases of fascioliasis before and after treatmentIntensity of infection Before

treatmentAfter treatment

1 month 2 month 3 month

GMEC 153.35 47.34 31.38 35.64

Wilcoxon signed ranks testZ=-3.303* Z=-3.016* Z=-2.524*

*P<0.05

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

78

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%.

79

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

Intensity of infection Before treatment One month after treatment

Light 960%

1066.7%

Moderate 16.7%

320%

Heavy 533.3%

213.3%

Total 15100%

15100%

Percent reduction of egg count 56.3%

80

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

Intensity of infection Before treatment Two months after treatment

Light 743.7%

1381.3%

Moderate 212.6%

212.5%

Heavy 743.7%

16.3%

Total 16100%

16100%

Percent reduction of egg count 64.6%

81

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

Intensity of infection Before treatment Three months after treatment

Light 333.3%

777.8%

Moderate 111.1%

111.1%

Heavy 555.6%

111.1%

Total 9100%

9100%

Percent reduction of egg count 71.9%

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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)

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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,

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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)

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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

86

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)

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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)

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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

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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%.

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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%.

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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.

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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.

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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

94

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.

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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.

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السلوك في حدثت التي التغيرات كذلك و األخيرة البيئية التغيرات تسببت لقد

أعداد زيادة في و الكبدية الدودة لمرض جديدة جغرافية حدود إيجاد في اإلنساني

. المرض بهذا المصابين عدد ازداد لقد و المرض بهذا اإلصابة لخطر المعرضين

عام التي 1980منذ المناطق على قاصرة اإلنسان في به اإلصابة ازدياد تعد لم و

الحيوانات . بين كبير بشكل وجوده من تعاني ( في ( تتسبب خطيرة مشكلة تعد الفاشيوال الكبدية الدودة فإن للحيوان بالنسبة و

. هائلة اقتصادية خسارة . و المصاب للكائن البراز عينة في البويضة بإيجاد يتم المرض هذا تشخيص و

إلى تصل المرض هذا تشخيص في الكاتو طريقة فحص% 99حساسية تم ما إذا

مدى على تجميعها تم عينات ثالث من أو البراز عينة نفس من معدة شرائح ثالث

. أيام ثالثةكفاءة له يكون أن الديدان عالج في المستخدم الجيد الدواء خواص أهم من إن و

أخيرا و العائل أنسجة في دائمة كيميائية بقايا وجود في يتسبب ال و آمن و عالية

. إقتصادي بسعر التداول سهل يكون أن

الكبدية الدودة مراحل كل على القضاء في فعاال و آمنا التريكالبندازول كان لقد و

( السنوات( في تم لكن و الحيوان و اإلنسان من كل في ناضجة الغير و الناضجة

من جديدة ساللة تكون نتيجة العقار لهذا تستجيب ال حاالت اكتشاف اإلخيرة

. . فعال جديد عقار عن البحث يستدعي الذي األمر تقاومه الديدان

في الفاشيوال و البلهارسيا لعالج جديد كعقار ترخيصه تم فقد الميرازيد أما و

عام من مارس شهر في من 2002مصر نقي مستخلص يتم المروهو الذي و

. مولمول الكوميفورا شجرة من عليه الحصول

112

اإلنسان في الفاشيوال من الشفاء معدل تقييم بهدف الدراسة هذه إجراء تم قد و

و بالفاشيوال اإلصابة شدة على المر تأثير لتحديد و بالمر العالج بعد الحيوان و

العالج تلقي بعد و قبل الدم في الكبد إنزيمات مستوى في التغييرات لدراسة

المزمنة . بالفاشيوال المصاب اإلنسان في بالمر

يونيو من الفترة في الدراسة هذه إجراء تم فبراير 2005ولقد في 2006حتى

أبيس قرية من العاشرة لكل 8العزبة تعداد عمل و للمكان خريطة عمل تم حيث

. للسكان الكلي التعداد أن وجد ولقد حيوانات من يمتلكون ما و بالعزبة القاطنين

يبلغ العزبة منهم 1305في من 138شخصا أقل أعمارهم هؤالء 5طفال و سنوات

. الدراسة من استبعادهم تمو القرية في الموجودة العشبية الحيوانات كل دراسة تم للحيوان بالنسبة و

عددهم بلغ قد و الحمير و الماعز و الغنم و الجاموس و البقر .208شملت حيوانا حيوانات من يمتلكون ما و بالعزبة للمقيمين شامل مسح عمل تم قد و

. العينات عدد وبلغ الكاتو طريقة باستخدام بالفاشيوال منهم المصابين لتشخيص

فحصها تم و 1092التي السكان من فحص 119عينة تم حيث الحيوانات من 3عينة

بيض عد تم لكن و فيها الديدان من تشخيصه تم ما تسجيل و عينة كل من شرائح

بعد . بالفاشيوال المصابين الناس من أخرى عينة فحص تم قد و فقط الفاشيوال

مصابة الغير الحاالت استبعاد يتم كي الكبد من الخالية األطعمة تناول من أسبوع

. العينة من حقااستبيان الستمارة السكان في المشخصة الفاشيوال حاالت خضعت لقد و

من بالفاشيوال اإلصابة تمت ما إذا تشمل التي و بالمرض الخاصة المعلومات لجمع

. أما و قبل من للمرض عالج أخذ تم ما إذا و و بالمرض الخاصة األعراض و قبل

الحيوانات هذه مالكي من معلومات جمع تم فقد الحيوان في المشخصة الحاالت

. فضالته من التخلص كيفية و جنسه و عمره و المصاب الحيوان بنوع خاصة

113

و العالج أخذ قبل السكان من بالمرض المصابين من دم عينة أخذ تم كذلك و

. ( الدم ( في الترانزأماينيز الكبد إنزيمات نسبة لقياس أخذه من شهر بعد أخرى

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

كبسوالت ( صورة في الميرازيد عقار بإستخدام اإلنسانية الحاالت عالج تم و300 ( / بجرعة ذلك و الكبسولة / 10مجم لمدة اليوم في الجسم وزن من كج 6مجم

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الواحد اليوم في القصوى تم. 600الجرعة فقد الحيوان في للحاالت بالنسبة و مجم

الميرازيد معلق باستخدام / 600بجرعة% 10عالجها متتاليين يومين لمدة اليوم مجم

. خاوية معدة على الصباح في تؤخذشهر بعد ذلك و مرات ثالث بالميرازيد العالج بعد الشفاء معدل تقييم تم قد و

. كل من براز عينات ثالث جمع تم مرة كل في و العالج من أشهر ثالثة ثم شهرين ثم

( منها ( كل بين تفصل الحيوان و اإلنسان في بيض 24حالة وعد لتشخيص ساعة

الفاشيوال. : التالية النتائج استخالص تم و إحصائيا تحليلها و البيانات جمع تم

: اإلنسانية الدراسةاإلنسان- 1 في بالفاشيوال اإلصابة معدل هذا% 5.8بلغ أن على يدل مما

أكبر ثاني يمثل حيث أبيس منطقة في رئيسية صحية مشكلة يمثل زال ما المرض

. البلهارسيا بعد المنطقة تلك في بالطفيليات إصابة

من- 2 أقل بالفاشيوال 10األطفال لإلصابة عرضة أكثر كانوا مرات 5سنوات

ثقيلة ( إصابة نسبة أعلى لديهم كان كما سنا األكبر الفئات من أعلى%) 30أكثر و

) البراز في البيض لعدد .114.46متوسط سنا) باألكبر بالمقارنة

114

الرجال- (3 من بالفاشيوال لإلصابة عرضة أكثر كن النساء% 7.5النساء من

ب بالمقارنة بالمرض مصابين ) 4.3كن نسبة% أعلى لديهن كان كما الرجال من

بالرجال ( بالمقارنة بالمرض ثقيلة ثقيلة% 15إصابة إصابة لديهن كان المصابات من

ب ).8.3بالمقارنة المصابين% الرجال من

اإلصابة- 4 خطر من يزيد مهما عامال بالمنزل تعيش حيوانات وجود كان

التعرض أن رغم غيرهم من أكثر مرتين يعادل بما المنزل بهذا للمقيمين بالفاشيوال

. لإلنسان العدوى نقل طرق من ليس المصاب للحيوان المباشر

مرضية- (5 أعراض أي من تعاني ال كانت الفاشيوال حاالت %)64.5معظم

كانت مرضية أعراض من تعاني كانت التي الحاالت في شيوعا األعراض أكثر بينما

البطن ( في الغثيان%) (24.1ألم كان%) 4.8ثم قد يعاني% 3.2و الحاالت من فقط

. البراز في دم وجود أو الصفراء من

المصابة- 6 الحاالت في مرضية أعراض أي وجود عدم بين ارتباط هناك كان

معظم كانت حيث أخرى ناحية من خفيفة اإلصابة كون بين و ناحية من بالفاشيوال

مرضية ( أعراض من تعاني ال التي كما%) 80الحاالت بالفاشيوال خفيفة إصابة لديها

و المصابة الحاالت لدى المتوسط من أقل لديها البراز في البيض عدد متوسط كان

مرضية ( أعراض لديها ).94.28مقابل 50.66التي بالترتيب

بلغ- 7 قد و اإلنسان في الفاشيوال لعالج فعاال جديدا واعدا عالجا المر يعتبر

استخدامه بعد الشفاء .88.2معدل العالج% من أشهر ثالثة مرور بعد ذلك والمعالجة- : 8 الحالة كون كانت بالمر العالج بنجاح المرتبطة العوامل أن وجد

تكون أن و مرضية ألعراض مصاحبة غير المصابة الحالة تكون أن و الذكور من

جود و عدم أخيرا و الحجم صغيرة لعائلة منتمية الحالة تكون أن و خفيفة اإلصابة

. المنزل نفس في تعيش حيوانات

115

نقصا- 9 أظهرت قد بالمر العالج بعد شفاؤها يتم لم التي الحاالت أن وجد

العالج قبل المتوسط بلغ حيث البراز في البيض عدد متوسط في و 96.12ملحوظاإلى قل إلى 34.28قد ثم شهر كان 30.61بعد أخيرا و شهرين ثالثة 44.89بعد بعد

. المجتمع في العدوى انتشار تقليل في العامة للصحة كبيرة أهمية يعكس مما أشهر

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

: الحيوان على الدراسةقد- 1 و أبيس قرية في الحيوانات بين كبيرا انتشارا منتشرة الفاشيوال أن وجد

بها اإلصابة معدل الجاموس% (52.9بلغ بين انتشارا أكثر كانت قد ثم%) 65.6و

الماعز%) (55.6البقر ( بين إصابة نسبة أقل وجدت قد %).12.5و

إصابة- 2 لديها كان بالفاشيوال المصابة الحيوانات نصف من يقرب ما أن وجد

عاليا ( الحيوانات في البراز في البيض لعدد الكلي المتوسط كان قد و شديدةمن) ( 236.56 أقل السن الصغيرة الحيونات في نسبة أعلى المتوسط هذا بلغ قد و

بلغ) حيث .905.78عام الحيوان عمر ازدياد مع تدريجيا قل قد و

نسبة- 3 بلغت قد و المرض نقل على عالية قدرة لديها الحمير أن وجد

فيها %.40اإلصابة

مرور- 4 بعد منخفضا بالميرازيد العالج بعد الحيوانات في الشفاء معدل كان

العالج ( من أشهر الجرعة%) 77.5ثالثة نفس استخدام نتيجة هذا يكون قد و

في المصاب الحيوان وزن أخذ عدم مع المصابة الحيوانات أنواع لكافة العالجية

اإلعتبار.

116

5 : حيث- المعالج الحيوان نوع بالمرهي العالج بنجاح المرتبطة العوامل كانت

في التقدم و الخفيفة اإلصابة إلى باإلضافة البقر و الماعز بين أفضل النتائج كانت

العمر.

داللة- 6 ذو و كبير بشكل أقل كان البراز في البيض عدد متوسط أن وجد

شفاؤها ( يتم لم بالتي مقارنة شفاؤها تم التي الحاالت في مقارنة 149.24إحصائية

).321.12ب الترتيب على

117

الكبدية الدودة عالج في المر عقار فاعلية

الحيوان و اإلنسان في: من مقدمة

الحميد/ عبد محبوب محمد سميرة طعامة جراحة و طب بكالوريوس

- اإلسكندرية 1998جامعة

( العامة ( للصحة العالي المعهد حارة مناطق صحة عامة صحة ماجستير - اإلسكندرية 2004جامعة

درجة على )الدكتوراه للحصول ) حارة مناطق صحة العامة الصحة فيعلى الحكم و المناقشة لجنة

الرسالةموافقون

/ حسن. محمد عزت د أ

/ د. المصري أ أحمد ثناء

/ د. هيب أ و أحمد علي

شتات/ أ. زكريا حنان د

/ / التاريخ

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اإلشراف لجنة / حسن. محمد عزت د أ

/ د. قطقاط أ محمود أميرة

/ شتات. زكريا حنان د أ

الصحن د/ أ. الفتاح عبد أمل

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الكبدية الدودة عالج في المر عقار فاعلية

الحيوان و اإلنسان في

رسالةلشروط جزئيا إيفاء اإلسكندرية جامعة العامة للصحة العالي المعهد إلى مقدمة

( ) حارة مناطق صحة العامة الصحة في الدكتوراه درجة على الحصول

من:الحميد/ عبد محبوب محمد سميرة ط

عامة جراحة و طب بكالوريوس - اإلسكندرية 1998جامعة

( العامة ( للصحة العالي المعهد حارة مناطق صحة عامة صحة ماجستير - اإلسكندرية 2004جامعة

العامة للصحة العالي المعهد

اإلسكندرية جامعة

120

2008

121