Fascioliasis is a well-known parasitic disease caused by



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

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

in partial fulfillment of the requirements for the Degree of

Doctor of Public Health (Tropical Health)

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

High Institute of Public Health

University of Alexandria

2008‏

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 doctor

In Public Health (Topical Health)

|Examiner's Committee |Approved |

|Prof. Dr. Ezzat Mohamed Hassan | |

| | |

|Prof. Dr. Thanaa Ahmad El Masry | |

|Prof. Dr. Ali Ahmad Waheeb | |

| | |

|Prof. Dr. Hanan Zakareya Shatat | |

|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 | |

| | |

| | |

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.

CONTENTS

|Chapter |Title |Page |

|i |List of figures | |

|I |Introduction | |

| |A- Prevalence of human fascioliasis |1 |

| | | |

| |B- Prevalence of animal fascioliasis |3 |

| | | |

| |C- Life cycle of Fasciola sp. |4 |

| | | |

| |D- Characteristics of life cycle of Fasciola sp. |5 |

| |1- Domestic animal reservoir hosts |5 |

| |2- Wild animal reservoir hosts |5 |

| |3- Intermediate snail hosts |5 |

| |4- Proper Environmental Conditions |5 |

| |5- Mode of transmission |6 |

| |6- Association of fascioliasis with other parasitic infections |6 |

| | | |

| |E- Clinical picture of human fascioliasis | |

| | |6 |

| |F-Clinical picture of animal fascioliasis | |

| | |8 |

| |G- Diagnostic tools for fascioliasis | |

| |1- Stool examination |8 |

| |2- Serological methods |8 |

| |3- Detection of fecal antigens of Fasciola hepatica |9 |

| |4- Radiological findings |9 |

| | | |

| |H- Diagnosis of acute fascioliasis |9 |

| | | |

| |Diagnosis of chronic fascioliasis |10 |

| | | |

| |J- Differential diagnosis of primary and secondary infections |10 |

| | | |

| |K- Treatment of fascioliasis |10 |

| |1- Old lines of treatment of facsioliasis | |

| |i- Bithionol (Bitin) | |

| |ii- Praziquantel |11 |

| |iii- Metronidazole |11 |

| | |11 |

| |2- Currently used drug |11 |

| |i- Triclabendazole (TCBZ): |12 |

| |3- Trials using new fasciolicidal drugs | |

| |i- "3-Cyanopyridine Derivatives" |12 |

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

| |iii- Nitazoxanide |12 |

| |iv- Myrrh | |

| | |12 |

| | |12 |

| | | |

| | | |

| | |13 |

| | | |

| | |13 |

| | | |

| | |13 |

|II |Aim of work |18 |

|III |Subjects and methods |19 |

|IV |Results |23 |

|V |Discussion |72 |

|VI |Summary and conclusions |81 |

|VII |Recommendations |85 |

|VIII |References |86 |

|IX |Appendices | |

| | | |

| |Arabic summary | |

List of figures

|Figure No. |Title |Page |

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

| | | |

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

| | | |

| |Prevalence of Fasciola sp. infection among different age groups | |

|Figure (3): | |43 |

| |Intensity of infection with Fasciola sp.in different age groups | |

| | | |

|Figure (4): |The parasitological cure rates one, two and three months after treatment of human cases with Mirazid |53 |

| | | |

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

|Figure (5): |treatment |56 |

| | | |

| | | |

|Figure (6): |A diagrammatic illustration of the pre-intervention phase of the animal study |61 |

| | | |

| | | |

| |Prevalence of animal fascioliasis among different age groups | |

|Figure (7): | |69 |

| |GMEC among different age groups of infected animals | |

| | | |

| | | |

|Figure (8): |Animal cure rates one, two and three months after treatment with Mirazid |74 |

| | | |

| |GMEC in uncured animal cases of fascioliasis before and after treatment | |

|Figure (9): | |78 |

| | | |

| | | |

|Figure (10): | |81 |

| | | |

| | | |

|Figure (11): | |86 |

| | | |

List of figures

|Figure No. |Title |Page |

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

| | | |

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

| | | |

| |Prevalence of Fasciola sp. infection among different age groups | |

|Figure (3): | |31 |

| |Intensity of infection with Fasciola sp.in different age groups | |

| | | |

|Figure (4): |The parasitological cure rates one, two and three months after treatment of human cases with Mirazid |32 |

| | | |

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

|Figure (5): |treatment |41 |

| | | |

| | | |

|Figure (6): |A diagrammatic illustration of the pre-intervention phase of the animal study |46 |

| | | |

| | | |

| |Prevalence of animal fascioliasis among different age groups | |

|Figure (7): | |53 |

| |GMEC among different age groups of infected animals | |

| | | |

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

|Figure (8): | |57 |

| |GMEC in uncured animal cases of fascioliasis before and after treatment | |

| | | |

|Figure (9): | |61 |

| | | |

| | | |

|Figure (10): | |63 |

| | | |

|Figure (11): | | |

| | |67 |

The Memory of my Father

My Mother

My Beloved Husband

and My Lovely Childe

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 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)

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)

Life cycle of Fasciola sp.:(36)

[pic]

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)

Characteristics of life cycle of Fasciola sp.:

1- Domestic animal reservoir hosts:

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

2- Wild animal reservoir hosts:

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

3- Intermediate snail hosts:

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

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

4- Proper Environmental Conditions:

Several environmental conditions are necessary for propagation and development of both the lymnaeid snail and the developing larval forms of the fluke. Moisture is necessary for the amphibious snail, and also for the swimming cercariae. The ideal temperature range for optimal development of both snail and fluke larvae is 15-20°C. Development can occur at temperatures between 10-15 °C but it will be slightly slower in a cooler environment. The snails also prefer a slightly acid pH.(41, 44)

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

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

5- Mode of transmission:

Water has been cited as the source of human infection, whether indirectly by contaminating vegetables or kitchen utensils or directly by drinking.(48,12) In Europe, the infections related to the consumption of wild watercress or wild dandelion occur sporadically, most frequently in France, Spain, and Portugal.(49-51) In the United Kingdom, Fasciola has been identified in imported vegetables (khat) kept moist during transport.(52)In Egypt, the prevalence of infection was directely related to the frequency of eating green leafy vegetables.(42)

Experimental results suggest that humans who consume raw dishes prepared from fresh livers infected with immature flukes could become infected with fascioliasis.(53)

6- Association of fascioliasis with other parasitic infections:

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

Clinical picture of human fascioliasis:

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

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)

Clinical picture of animal fascioliasis:

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

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

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

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

Diagnostic tools for fascioliasis:

1- Stool examination:

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

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

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

2- Serological methods:

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

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

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

3- Detection of fecal antigens of Fasciola hepatica:

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

4- Radiological findings:

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

Crescent-shaped parasites could be seen in some patients; and the parasites could be recognized spontaneously moving. Postprandial sonographic examination reveals parasites adhering to the gallbladder wall. The gallbladder contents will be mobile but will not sediment downwards after patients changed position. Non-specific abnormalities may be found such as: impaired gallbladder contractility, gallbladder tenderness, debris, calculi, wall thickening and bile duct dilatation. Biliary tract abnormalities are frequently observed by ultrasonography (US), but the detection-rate of Fasciola hepatica is disappointingly low despite the parasite's relatively large size. US findings must therefore be interpreted together with other clinical measurements. The visualization of parasites being expelled through the dilated common bile duct allows the causal interpretation of post-therapeutic abdominal pain and increase of liver enzymes. When triclabendazole is given on suspicion, visualization of worm expulsion and bile duct dilatation by US may be used to confirm diagnosis.(97)

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

Diagnosis of acute fascioliasis:

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

Diagnosis of chronic fascioliasis:

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

Differential diagnosis of primary and secondary infections:

Antibody avidity refers to the strength of interaction of an antibody with a multivalent antigen. Depending upon the strength of this binding, the complex formed may or may not be dissociated. Antibody avidity is low after primary antigenic challenge, matures with time and it usually involves IgG antibodies.(104,105)

Recently, an assay measuring the antigen-binding avidity of IgG antibodies has been developed to distinguish the low-affinity antibodies produced at an early stage of infection from those with a higher-binding affinity that reflects past immunity. This IgG avidity test has been valuable with many pathogens as both a front-line assay and as a means of distinguishing primary from secondary infections [8-10].(106-108) It is also helpful in assessing the time of the initial antigenic challenge. Avidity index (AI) is an indicator of avidity. Therefore, a low index means low avidity while a high index denotes high avidity. (101)Abou Basha et al.(2000) reported that AI of 59.90% denoted acute infection and those > 59.90% denoted chronic infection.(101)

Treatment of fascioliasis

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

I- Old lines of treatment of facsioliasis:

1- Bithionol (Bitin):

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

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

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

2- Praziquantel:

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

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 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 (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)

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 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 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)

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.

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 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

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

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.

RESULTS

This study is an intervention study in which all inhabitants of satellite number 10 of Abis VIII village aged 5 years and more and their animals were screened for fascioliasis. Then all human and animal cases of fascioliasis were treated by myrrh and followed up for three months to determine its effectiveness.

The total number of human samples examined was 1092; out of them 64 cases of fascioliasis were diagnosed. The total number of animal samples examined was 119 and 63 cases of fascioliasis were diangosed.

The results of the present study are classified as following:

I- Human study:

A) Base line study

B) Therapeutic trial

II- Animal study:

A) Base line study

B) Therapeutic trial

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).

[pic]

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

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

|Variable |NO. |% |

| |(n=1305) | |

|Age (years) | | |

| ................
................

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