CHAPTER-I - - An Introduction INTRODUCTION

1.1. The term amoebiasis denotes the condition of harbouring B,histolytica with or without clinical manifestations (WHO, 1969). The aetiological agent of amoebiasis is Entamoeba histolytica.

1,2• Epidemiology

Amoebiasis has been reported practically from all over the world and has been estimated to affect 10% of the world population although its prevalence and severity may differ from area to area (WHO, 1969). Thus, it is a public health problem of International importance. In India, the prevalence of amoebiasis varies from 1-58% (Arora et al., 1977) . The highest prevalence rate among the asyrrptomatic people has been recorded to be 47,4% (Greval, 1968), and that in symptomatic patients 58% (Patel/ 1945). Prevalence rates of this disease in the states of Himachal Pradesh, Jammu and Kashmir, Orissa and Sikkim have been reported to be less than 10%. Prevalence rate ranging from 1-20% have been reported from Assam, Delhi, Gujarat and Madhya Pradesh. The states of Andhra Pradesh, Kerela, Rajasthan, U.P. and West Bengal showed prevalence rates upto 30% while in Maharashtra, Tamil Nadu and Chandigarh the rates were more than 30% (Arora et al., 1977). Although E.histolytica is generally more prevalent in the tropics and subtropics. the higher temperature and humidity are not thought to be directly responsible since the higher temperatures are likely to have an adverse effect on cysts in the environment. The increased prevalence in tropical countries is generally considered to reflect poor sanitation and health education rather than climatic conditions. Prevalence of E,histolytica is associated with unsanitary conditions in temperate and even in arctic regions (WHO, 1969).

The unhygeinic working and living conditions, insanitary surroundings, improper handling of food, contami­ nation of food by cockroaches and flies and refervoir or hosts such as dogs, cats etc. (which are natural hosts of E,histolytica) play a very important role in the spread of the disease. Water, food and vegetables are potential carriers of thd disease.

1,3. Classification

Kingdom - Protista Subkingdom - Protozoa Phylum - Sarcomastigophora Subphylxim - Sarcodina Superclass - Rhizopoda Class - Lobosa Subclass - Gymnamoebia Order - Amoebida 3

Suborder - Tubulina

Family - Entamoebidae

Genus and - Entamoeba histolytica species (Schaudinn, 1903).

Of the seven species of amoebae which are natural parasites of man, only E.histolytica is truely pathogenic.

Of current special interest is the recently discovered fact that free living amoebae of the genus Acanthamoeba (Synonym

Harmane11a; Page, 1976) and Naegleria are facultative parasites of man and may be highly pathogenic when invading the meninges

and brain via the nasal cavity (Singh, 1975)

1.4. Life cycle

The life cycle of E»histolytica involves the, encystment of a trophozoite, followed by release of the tropho­

zoite from the cyst when the conditions are appropriate. The trophozoite undergoes encystment only within the bowel, usually

associated with conditions in the lumen which are not ideal

for continued activity of the trophozoite.

The young cyst is uninuclear and it contains one

large glycogen vacuole and chromatoid body. It is the non motile resting stage. It is round or oval measuring 2,5 to

3,5 p in diameter. The cyst matures either within the lumen

of the bowel or outside the body in appropriately moist

surroundings. The mature cyst contains four nuclei (the

result of 2 nuclear divisions) and a number of chromatoid 4'

bodies. When this cyst is ingested, another nuclear division occurs during the cyst 's transit through the small intestine and the resulting eight trophozoites escape through a hole created in the wall of the cyst. Trophozoites migrate to the colon where they persist, undergoing binary fission every eight hours in the trophozoite stage. Encystment of these organisms occur again when the trophozoites are excreted outside the body and environmental conditions not so favour­ able to continued trophozoite multiplication, the life cycle is complete (Wilcocks et al., 1971).

The trophozoites are facultative anaerobes which require complex media for growth. The environment for optimum growth of amoeba in the colon includes a pH 6.0-6.5, a low redox potential, nutrients occuringi found in the colon particularly carbohydrates and the presence of bacteria. The trophozoite is the vegetative, motile stage measuring frcan 15-40 u. This stage is not responsible fot the spread of the disease since it is rapidly destroyed by the gastric pH and enzymes. The encysted stage readily passes this barrier. Cysts may remain viable for weeks or months in appropriate moist surroundings. The cyst, therefore, is the primary reason for the extensive prevalence of the infection throughout the world. The conditions that allow the amoebae to become invasive are not clear, however, virulence has been promoted in experimental animals by irritating the bowel with abrasives, chemicals or invasive bacteria (Lesh (Losch) 1975, Wittner et ^., l97o) . Trophozoites have been shovm to breakdown cement substance between epithelial cells of the intestine and to gain access to the submucosal region by moving between these cells (Tekeuchi et al., 1975). The events that trigger this process are not known but a number of factors in the host will contribute to Invasion such as high cholesterol, low ascorbic acid diet, high carbohydrate, low protein diet, the absence of previous exposure to amoebae, as well as climatic, emotional and genetic factors (Perez - Tamayo et al., 1971).

1,5. History

Losch in 1875 discovered motile amoeba with ingested red blood cells in the dysenteric stools of a patient. Later at the autopsy of the patients he detected colonic ulcers containing active amoebae. He is credited with documenting amoeba to be pathogenic by inducing lesions in a dog with dysenteric stool. He named the protozoan Amoeba coli; but he did not regard the organism as the actual cause of the disease.

After the discovery of Losch, discussion arose as to whether the amoeba was the aetiological agent of dysentery and this question remained unanswered for a long time. Kartulis, in Egypt in 1886, settled the role of amoeba as a fe

cause of intestinal and hepatic lesions in patients with diarrhoea. Koch and Gaffky (1887) and Osier (1890) confinned the findings. Councilman and Lafleur (1891) published a complete study of 15 cases of amoebic dysentery. They named the parasite Amoeba dysenteriae.

In 1893, Quincke and Roos demonstrated that cats could be infected either by feeding them with stool containing cysts or by injecting stool containing cysts, per anum. They distinguished amoebae other than "Amoeba coli Losch" by their lack of pathogenicity in cats. These they called "Amoeba coli mitis", found in mild dysentery; and "Amoeba intestinl vulgaris" found in the stools of healthy people.

Casagrandi and Barbagallo (1897) described Entamoeba coli from healthy people, thus establishing the generic name Entamoeba as distinct from Endamoeba Leidy, 1897.

Strong and Musgrave (1900) differentiated the pathogenic amoeba from non pathogenic one in the human intestine, They observed that in cats typical ulcers were produced by pathogenic amoeba, whereas a non-pathogenic amoeba produced no such lesions in these animals.

Hxiber (1903) demonstrated quadrinucleate cysts, but thought that it was a different species other than Entamoeba coli, so he called it Entamoeba tetraqena. Schaudinn (1903) distinguished non pathogenic E.colJ. and the pathogenic haenratophagous E.histolytica. The name 'tetragena ' caused confusion. The concept that the quadranucleate cysts were those of some species other than S.histolytica remained imquestioned for a dozen years. Huber (1909), Hartmann (1908) and others accepted that

E.tetragena was a separate species. Viereck (1907) stated that

E.tetragena was a strain of E.cqli. Elmassian (1909) gave the name E.minuta for the trophozoites associated with quadranucleated cysts.

Craig (1905) considered that the correct title should be B.dysenteriae. Thus in 1910, there were 4 names dysenteriae, tetragena, minuta and histolytica, for what is now considered to be a single entity. The accepted name for the parasite is Entamoeba histolytica (Schaudinn, 1903).

Walker and Sellards (1913) demonstrated conclusively that man could be infected with the cysts of E.histolytica.

In human volunteers fed with E.coli cysts, no such disease developed; thus it was shown experimentally for the first time, in man that E.histolytica was pathogenic to man.

1.6. Pathogenesis and pathology

Commensal trophozoites thrive adjacent to the colonic mucosa where the oxygen tension is moderately low and the pH is between 6.0 and 6.5. Host tissue appears to be damaged only by direct contact with amoebae; lyso-somal enzymes are 8

released, possibly by activating a contact-sensitive surface

organelle. The relatively anaerobic and acid conditions of

cell necrosis favour further penetration of amoebae into the

tissue. The role of intestinal bacteria^ in pathogenesis is

still uncertain. They may act mainly by creating suitable

conditions for the growth of amoebae.

1.6.1, Amoebic ulceration

It is most common in the caecxim and rectosigmoid

region, but may affect any part of the large intestine,

appendix and terminal ileum. The initial lesions are small,

discrete, superficial mucosal erosions which may then extend

more deeply through the muscularis mucosae and spread lateraly, producing ulcers that are flasTc-shaped in cross section.

Lesions may coalesce, causing extensive mucosal loss. Blood vessel involvement usually results in local thrombosis.

Penetration may extend through the muscle coats, causing peritonitis, the most dangerous complication of intestinal

amoebiasis.

Ulcers heal rapidly after treatment and persistent

scarring is uncommon. Sometimes, localised bowel lesions

show a more vigorous host response with oedema and a marked

inflammatory reaction producing an amoebic granuloma or

amoeboma. These are most common in the caecum and rectosigmoid

and may be multiple. f

Although probability of tissue invasion may be influenced by strain virulence, host factors are also important. Women in late pregnancy and the puerperium (the lying-in period) after child birth usually regarded as lasting a month) are especially susceptible as are patients on cytotoxic or corti­ costeroid therapy. Colonic cancers may be secondarily Invaded by E.histolytica. Trichuris trichura infections and intestinal schistosomiasis may also encourage invasion. The high carbohydrate and low protein diets of developing countries perhaps enhance host susceptibility.

1.6,2, Hep at ic involvement

In many patients with invasive intestinal amoebiasis, amoebae may reach the liver via the portal vein, but most are destroyed. Enlargement of liver is not xincommon in acute amoebic dysentery. Established infection of the liver usually causes progressive cytolytic lesions that extend in all directions, with trophozoites proliferating near the advancing edge. Perhaps, these lesions begin at the sites of venour embolism or thrombosis. Secondary bacterial infection is rare and the content of such a 'liver abscess' is necrotic liquified liver tissue. The abscess is sharply demarcated from surrounding liver, which shows oedema, hyperemia and a narrow zone of lymphocytic and polymorpho-nuclear infiltration a fibroblastic reaction occurs in longstanding lesions. Most liver abscesses 10

are solitary and occur long after any clinically evident intestinal ulceration; but both solitary and the rarer multiple abscesses may develop during or shortly after an attack of dysentery.

1,7, Clinical manifestations

The most common manifestation of infection with

E.histolytica is completely normal, healthy asymptomatic person. The contraversy over whether these so called 'healthy carriers' have in their intestinal tract a commensural microbe or whether they undergo small, easily controlled damages to the bowel mucosa continues. However, all agree that these persons are symptomless.

In studying amoebiasis it is convenient to classify the clinical cases into two groups :

a) Intestinal amoebiasis

b) Extraintestinal amoebiasis

1.7.1, intestinal amoebiasis

The most common illness experienced with amoebic disease is colonic irritation, occassionally without diarrhoea, usually felt as colicky lower abdominal painfe and alteration in bowel habits. The stool may be loose and mucous or blood may be present. These symptoms may be mild and may continue for months. On physical examination, lower quadrant tenderness may be elicited. 11

When colonic involvement is more extensive, patients

display a more symptomatic picture, including bloody frequent dysenteric stools, moderate abdominal pain and tenderness and

right upper quadrant signs and symptoms. The liver may be

enlarged and exhibit percussion tenderness. During severe

colonic disease, m.arked abdominal pain, tenesmus and fever are

seen. When diarrhoea is marked the secondary signs of fluid

loss and electrolyte imbalance may be observed (Adams and

MacLeod, 1977),

Occasionally, the complete picture of acute abdomen

(abdominal distension, absence of bowel sound and vomitting)

occur. This can be related to peritoneal irritation or to

bowel perforation and acute amoebic peritonitis. The presence

of amoebae plus bacteria in the peritoneum is a serious

complication of amoebiasis resulting in very high mortality.

Peritonitis occurs in 3-4 % of the patients with amoebic

dysentery (Adams and MacLeod, 1977).

A chronic irritative bowel syndrome may follow

acute amoebic colitis for many months even after the amoebae

can no longer be demonstrated. This illness usually subsides

spontaneously. A more serious but similar illness is that

referred to as ulcerative post dysenteric colitis (Adams and

MacLeod, 1977). This is not common. It has a pattern similar

to ulcerative colitis with recurrent signs and symptoms of

mucous and bloody diarrhoea that is unresponsive to antiamoebic 12

therapy but is associated with high antibody titers against amoebae.

Cutaneous amoebiasis may result from extension of severe bowel disease to the skin.

1•7•2. Extra-intestinal amoebiasis

The most common form of extra-intestinal amoebiasis is amoebic liver abscess. Hepatic amoebiasis may arise within days after the onset of dysentery, or it may develop months or years after an illness consistent with amoebic bowel infection. Over 60% of the patients with hepatic amoebic abscess however, give no history of previous or current intestinal amoebiasis (Adam and MacLeod, 1977^).

Patients most often have pain in the right upper quadrant in the right side of the chest, or in the epigastrixim; tenderness in the right upper qnadrant; and a palpable liver and fever. Rapid weight loss is common with loss of 10-15 kgs in a few weeks.

The hepatic lesions occur more often in the right lobe of the liver than in the left. Point tenderness on palpation between the ribs may be present. When the abscess is close to the diaphragm the most common signs are referred pain to the right shoulder and elevation of the right diaphragm. 13

The complications of amoebic liver abscess are associated with extension of the liver abscess into adjacent organs (Adam and KacLeod, 1977) . The most common complication is extension into the pleural spane. The most serious compli­ cations are ruptures into the pericardium and into the peri­ toneum. Occasionally patients will have only the pleural or pericardial signs without clues to the presence of hepatic disease. Brain abscess has occured as a complication of extra-intestinal amoebiasis.

1.7,3, Amoebic lesions in other organs

a) Pieuro-pulmonary amoebic infection

It may be primary due to haematogenous spread, or secondary as an extension from a liver abscess.

These are often multiple, small abscesses in one or both lungs. The infection in such a case is likely to occur through the inferior haemorrhoidal veins and inferior vena cava and finally to the lungs.

ii) Seconday lesions

These lesions occur as a complication and extension of a liver abscess through the diaphragm. There is an intense pain due to the involvement of the diaphragmatic pleura accompanied by persistent xinproductive cough. There may be 14

dyspnoea, if a considerable part of the lung is involved along with the syndrome of liver abscess. When the lesion breaks through a bronchus, a typical chocolate coloured pus may be coughed out.

b) Brain

Abscess of brain is very rare complication of extra­ intestinal amoebiasis. Several cases of amoebic brain abscess have been reported. This condition should not be confused with primary amoebic meningoencephalitis caused by free-living amoebae (Naeqleria and Hartmanella).The abscess is almost always single, small in size. The site is usually in the cerebral hemisphere with evidence of destructive brain lesion. The case terminates fatally In 7~lO days and cannot be speci­ fically diagnosed until autopsy,

c) Skin

i) Amoeboma (amoebic granuloma) may be produced in the skin round the site of the opening of a liver abscess or it may develop round a fistulous opening of an ulcer in the region of the caecum. This may stimulate the epithelioma but the tissue will show presence of trophozoite form of E,histolytic

ii) Amoebiasis cutis is usually seen in the perianal region with development of an ulcer. It is characterised by a tender swollen area with an elevated and indurated margin, dark brown in colour and centrally forming an abscess pocket with semilicfuid content of foetid odour. 15

ill) Amoebic infection is sometimes associated with

leucoderma affecting the skin or lip.

iv) There may be a condition of allergic amoebic dermatosis

including anal pruritus, utricaria or acne rosacea.

d) Urinoqenital tract

E.histolytica may involve the urinogenital tract e, through a rectovesical or rectovaginal fistula producing cystitis or vaginitis.

1.7.4. Amoeboma

It is a condition of ulcerating granuloma affecting

a short sector of colon or rectum caused by the invasion of

the gut wall by E.histolytica. This results in a mass of

granulation tissue, pseudoepitheliomatous in character, projecting into the lumen of the gut as an ulcerating fungatlng mass or as an annyial constricting band. It contains the trophozoite form of E.histolytica. Biopsy from such a case will show the presence of the organism. The comrnenest sites

of amoeboma are the caecum, flextures of the colon, rectum at

the rectosigmoid junction where they may form round a fistulous

opening in the skin draining the pus of an amoebic abscess,

1,8, Cultivation of E,histolytica

Boeck and Drobohlev (1924) are credited for successful cultivation of E.histolytica in vitro for the 16

first time. They used a diphasic medium in which the solid part was prepared from the whole egg thoroughly emulsified with Locke ' solution. The overlay consisted of horse serum diluted with Locke's solution.

Dobell and Laidlaw (1926) used inspissated horse serum as a solid constituent covered with Ringer's solution or buffered saline with inactivated horse serum in which sterile rice starch was added. Even today, it is widely used for the routine maintenance of amoebae and for clinical laboratory diagnosis.

Craig (1926) prepared a monophasic medium consisting of Locke's solution with human, horse or rabbit serum in proportion of 7:1.

Cleveland and Collier (1930) devised a liver infusion agar medium. Tsuchiya (193 2) cultivated E.histolytica in nutrient broth, along with sterile rice starch and animal charcoal, Balamuth (1946) used a monophasic medium of egg yolk, liver extract and starch. Jones (1946) prepared a simple modification of Pavlova's medium (1938) consisting of buffered saline - marmite - horse serum with rice starch. Shaffer et al.

(1948) found that 24 hours cultures of streptobacillus In fluid thioglycolate - dextrose - rice flour medium when diluted

1:2 with isotonic solution and normal horse serum had supported excellent growth of amoebae. 17

Woolfe (1957) prepared a medium called as L.M.S. medium which was a modified Pavlova's medium. The medium contained liver extract/ buffered saline, horse serum and rice starch. He found that growth of amoebae in this medium was much heavier and more consistent. Youssef (1965 a & b) succeeded in growing amoebae on the surface of nutrient agar covered with egg white* agar and rice powder. He modified this medium and used for drug assay.

All the above media when used contained a number of bacteria along with amoebae. It was formerly believed that the amoebae required the bacteria for their growth and if bacteria were eliminated the amoebae cannot survive. The presence of heterogenous bacterial flora in amotebic cultures and -their variation from strain to strain induced serious fallacies in the accurate determination of results. Therefore/ attempts were, made to grow amoebae in absence of bacteria.

Jacobs (1947) and Hansen and Anderson (1948) grew amoebae in association with clostridittm perfrinqens and organism 't" respectively.

Philips and Rees (1950) prepared the monoaxenic culture of amoeba by eliminating bacteria from amoeba cultures and replacing them with Trypanosoma cruzi. After this achieve­ ment attempts were made to axenise the amoeba completely, i,e. growing amoebae in the complete absence of any microbial associate. 18

Diamond (1961) was the first to succeed in growing

amoebae axenically i,n vitro. He devised a special medium for this purpose and used crithidia (tryj^anosoma) as microbial

associate with amoeba, to initiate axenic culture. In past,

use of amoeba-trypanosoma culture was limited because of the

infectious nature of the trypanosomatid associate. Therefore,

Diamond used amoeba-crithidia cultures as crithidia is not

infectious to vertebrates. Diamond (1968) further simplified

his medium and descrined a clear liquid medium for initiation,

maintenance and mass cultivation of amoebae.

1.8, Prevention of amoebiasis

Amoebic infection is prevented by preventing conta­ mination of food and water with human faeces. The most commonly

contaminated foods are fresh vegetables such as lettuce.

Amoebae are not killed by low doses of chlorine and iodine/

so the water should be boiled for 10 minutes to ensure the

absence of amoebae from drinking water. Vegetables should be

treated with a strong detergent soap and then soaked in acetic

acid or vinegar for 10-15 minutes to ensure eradication of

amoebae.

To reduce endemicity of amoebiasis, mass treatment

could be given with a direct acting amoebicide or possibly with metonidazole and prophylactically di-iodohydroxyquinoline, lodohydroxyquinoline or glycobiarsol could be used (WHO 1969). 19

Intermittent mass chemotherapy with entamide furoate and has been successfully attempted in Mexico and elsewhere where invasive disease is common; it may also be useful in mental institutions in temperate countries.

Personal chemoprophylaxis by visitors to the tropics is not recommended and it is better for such persons to avoid eating uncooked foods and to have their stools examined on return

(Knight, 1975).

AIMS AND OBJEI:CTIVES

Incidence of amoebiasis is very high, about 10% of the world population is being estimated to be affected with the disease (WHO 1969), It is usually refractile to the therapy of a single drug. Though there are a number of effective and cheap available drugs like fluoate, derivatives of 8-hydroxyguinolines, arsenicals like carbarsone

and acetarsone, entobex, paramomycin etc., they act in intestinal amoebiasis only and are of no use in the therapy of extra-intestinal amoebiasis. Their value is also limited because of their toxicity. Antibiotics like tetracycline

are to be used as adjuvants along with amoebicides because it

acts indirectly by killing the bacterial flora which is

essential for the growth of amoeba. If these antibiotics are

used alone, the relapse may occur after apparent cure.

acts effectively in both forms of amoebiasis - intestinal and

extra-intestinal but because of its cardiovascular toxicity ;^u

and low safety margin, its value in the treatment is seriously affected. Metronidazole is a drug of choice and it approximates the ideal amoebicide in that acting on both the forms of amoebia- sis. Nevertheless, it has certain serious drawbacks; it fails to cure hepatic amoebiasis in some cases. It has been found to produce malignant lymphomas in experimental animals. All this leads to a continuous search for a new amoebicide which will be administered easily and which will have a very low or no toxicity.

In literature, many plants have been described to possess antiainoebic activity. A few of them have been used successfully; emetine is a classical enample of this, it is an alkaloid from the plant Cephaelis ipecacynha or acuminata karsten. Some of these plants have been used with limited success, e.g. Quassin, glaucarubln, Yatantsu etc. In ayurveda also, many plants have been stated to possess anti- dysenteric properties. Dashmula, an Ayurvedic preparation is being used in India since ancient times which contains powder of 10 plant roots. One of these plants is Shyonaka (Botanical name - Proxylum indicum Vent). Mishra and Sharma (1973) have clinically shovm the antiamoebic activity of the decoction of roots of O.indicum. Although the plant has been studied widely to isolate a number of chemicilly pure substances, no attempt has so far been made to corelate the biology and the chemistry to isolate a new antiamoebic nucleus in a pure form. 21

The present studies are undertaken to isolate antiamoebic principle from the plant O.lndicum, in chemically pure form and to study its antiaraoebic activity and pharmacology in vitro and in vivo. The studies therefore, are divided clearly into 2 parts - the chemical isolation of the pure compound and the biology of the pure compound and its comparison with a nvmiber of known antiamoebic drugs.