Gut, 1978, 19, 940-953

Progress report Intestinal protozoa The pathogenic species of protozoa that infect the human gut are now receiving greater attention from both parasitologists and gastroenterologists. Clinical awareness of these infections is growing and there have been several diagnostic and therapeutic advances. Progress on the biomedical side has also been considerable and some interesting new techniques have been applied. The host-parasite relationship is delicately balanced in these infections and small changes in the host's immunological responsiveness may affect the clinical outcome. The purpose of this paper is to assemble some of the information in this field that has been published since 1973, in the hope that further interest will be stimulated. Three previous reviews" 2,3 have covered earlier publications in a similar way. Amoebiasis By way of introduction several more general works should be mentioned. A book edited by Jeon4 deals with the large free-living amoebae but also covers many aspects of general biological interest to the parasitologist. The pathogenic mechanisms and ecology of Entamoeba histolytica and free-living pathogenic soil amoebae (Naegleria, etc.) are compared in a monograph by Singh5. A new multi-author book edited by Padilla and Padilla6 covers various aspects of the parasitology of E. histolytica and its clinical effects, with special reference to work in Venezuela. Amoebiasis is of great importance in Mexicp from the point of view of public health and a great deal of exciting cliniical and laboratory work is being done in that country. Anyone seriously contemplating further studies in this subject must consult the supplements to Archivos de Investigacion (Mexico) where the proceedings of the annual Amoebiasis Seminars, held in Mexico City since 1969, have been published. In 1975, an international centenary conference was held in Mexico to celebrate the original discovery of E. histolytica by Losch. The conference proceedings have been published as a book edited by Sepulveda and Diamond7 and Losch's original publication has now been reprinted in English8. PATHOGENESIS

The mechanism of host tissue destruction has been further clarified by studies of the ultrastructure of the rectal tissue from dysentery patients9 and the caecal mucosa of infected guinea-pigs'0"'. Amoebae cause contact-dependent lysis of epithelial cells, and are chemotactic to neutrophils that are themselves killed on contact. Neutrophil degranulation probably contributes to the local tissue damage that includes endothelial damage leading to thrombosis of 940

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capillaries and venules in the lamina propria. Using an isotope-labelled cell monolayer as an in vitro substrate, cell damage was proportional to duration of contact and to the number of amoebae; extracts of amoebae caused no damage12. The ultrastructure of amoeba-cell contact in this system suggests that membrane damage precedes lysis13. Amoebic virulence in vitro can also be assayed using guinea-pig leucocytes'4. The fine structure of E. histolytica has been further elucidated with the elegant freeze-etching technique'5; recent electron micrographs16 have shown paranuclear bodies but no evidence of trigger lysosomes; it appears that membrane-bound cytolytic enzymes are released from dendritic plasmalemmal processes'". Diamond and Mattern'8 have reviewed the exciting new work on the viruses of E. histolytica. All the axenic strains studied carried cytoplasmic icosahedral virus, and some also had a filamentous intranuclear virus or beaded intranuclear virus-like particles. All these agents lyse susceptible amoebic hosts, but the relevance of these viruses to amoebic virulence is uncertain. Quite different structures, resembling rhabdo-viruses, have been seen in the electron micrographs of several strains including the low temperature Laredo and also E. invadens' 9. STRAIN CHARACTERISATION

Strain virulence in man has again been shown to correlate with virulence and infection rate in the weanling rat caecum20; and there appears to be a correlation between strain virulence, ease of cultivation, and growth rate2'. Amoeba cultivated without bacteria do not infect the rodent caecum22, but large inocula of crithidia-associated23 or axenic strains24 will infect and produce lesions in the hamster liver. New-born mice or hamsters can be infected with axenic E. histolytica by direct intracerebral25 or intrahepatic26 infection, and these techniques promise to be useful in chemotherapeutic and strain comparison studies. The ease with which a strain may be axenised appears to be related to its virulence in the hamster liver, and this may decline rapidly after axenisation14. Efficient methods of strain cryopreservation have been described27; at 4°C trophozoites may survive for up to 12 days and cysts for up to nine weeks28. Selective agglutination of pathogenic strains by concanavalin A has been reported29'30, and deserves further study. HOST SUSCEPTIBILITY

Several workers have studied host susceptibility in experimental systems. Protein deficiency predisposes to caecal ulceration31; and carbohydrate supplementation of the protein-deficient diet favours infection but reduces tissue invasion. Mice infected with Schistosoma mansoni32 or Trichuris muris33 are more susceptible to amoebic tissue invasion, possibly because of local tissue damage or immunosuppression. Hamster livers damaged by the intraportal injection of glass particles or ligation of a lobar branch of the portal vein, are more likely to develop amoebic abscesses34. Inbred rat strains differ in their susceptibility to caecal invasion35. Blood lymphocytes from patients with invasive amoebiasis will undergo blast transformation in the presence of amoebic antigen38 37. Patients with liver abscess may show a temporary depression of lymphocyte transformation to PHA or amoebic antigen38. There is no definite evidence of protective immunity in man, but protection has been shown in guinea pigs following injection of amoebic antigen39. Attempts have been made to detect coproantibodies in human faeces, and further work is needed in this field40'41.

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Autoantibodies to human colon have been demonstrated in the sera oL amoebic colitis patients using haemagglutination and gel diffusion techniques42. A series of 92 fatal cases of fulminant colitis documented from Senegal43 emphasises the diagnostic and therapeutic aspects of this dangerous condition. The gross and microscopic pathology of the liver that occurs in colitis patients has been classified and discussed44. Invasive amoebiasis may complicate infections with Strongyloides stercoralis with fatal consequences45, and among Malayan aborigines heavy infections with Trichuris trichiura may be complicated by amoebic dysentery46. Double infections with Shigella and E. histolytica are reported47 but they are probably under-recognised. The significance of the finding of amoebae in duodenal aspirates48 remains uncertain. CLINICAL FEATURES

Further cases of amoeboma have been reported49 and also the unusual complication of multiple stricture50. Amoebiasis of the skin and genitalia is being increasingly recognised and is reported from Uganda5' and Papua New Guinea52. Homosexual venereal transmission has been documented in one patient53 and perianal gangrene due to amoebiasis has been described in a diabetic54. Amoebiasis may secondarily infect haemorrhoidectomy wounds and produce lesions resembling rectal carcinoma55. The clinical56 and radiological features5' of abscesses of the left lobe of the liver have been described in detail, and also the dangerous complication of amoebic pericarditis58 59. An important paper by Barbour and Juniper60 compares amoebic abscesses of the liver with pyogenic ones. In a study of 30 patients61, the pH of amoebic pus ranged between 5-2 and 6'7; this finding may be of diagnostic value when an empyema is aspirated. Amoebic liver abscess has been described in a patient with situs inversus62 and also the abnormalities in fibrinogen metabolism and fibrinolytic activity in a series of patients63. Amoebic abscesses may occasionally contain Salmonella enterididis64 or S. typhi65. Scragg66 has described the diagnosis and management of hepatic amoebiasis in children, with reference to a series of 350 cases. The often difficult medical and surgical management of pleuropulmonary amoebiasis has also received attention67. DIAGNOSTIC METHODS

The diagnostic sensitivity of faecal examinations has beenl discussed together with mathematical methods for the interpretation of epidemiological data68. The variable and irregular cyst excretion patterns of different subjects have again been documented 69. Amoebae in stools or cultures may be identified as E. histolytica using immunofluorescence and specific antisera70; they may also be recognised in tissue sections by the same method7l, and trypan blue is a useful counter stain72. A stool fixative containing sodium acetate, acetic acid, and formalin (SAF) 73 promises to be of value in clinical practice as it allows the same preserved specimen to be used for both cyst concentration and premanent staining of cysts and trophozoites. The poor sensitivity of routine histology for the recognition of amoebae is emphasised in a retrospective analysis of 11 patients from Uganda74 and in a detailed clinicopathological study of eight patients in South Carolina75; fresh wet preparations must always be examined. Skin tests using an axenic strain extract (histolyticin) have been further evaluated76'77'78; some control subjects

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may give a delayed response and it is possible that the test itself induces an immunological response. The most promising new serological developments are counter-current immunoelectrophoresis79 80, and the enzyme-linked immunosorbent test81; the former is rapid, relatively simple to perform, and almost as sensitive as indirect haemagglutination. Counter-immunoelectrophoresis has also been applied to the detection of antigen in amoebic pus82; the technique appears to be much more sensitive than the detection of amoebae in liver aspirate or biopsy. The relative merits of three serological tests have been reported by Stamm et al. 83. Liver abscess patients do not give positive serological tests for a-fetoprotein84. The relative merits of isotope scans and hepatic arteriography have been discussed85 and the scintigrams of 247 liver abscess patients are reported from South Africa86. Ultrasound is another useful technique and its value has been compared with isotopic scanning8 7. Hypaque given for intravenous pyelography may also delineate a liver abscess88. TREATMENT

Metronidazole continues to give good results in both amoebic dysentery and liver abscess. However, five patients have developed liver abscess one to three months after treatment of amoebic colitis with metronidazole89. Fisher90 has described a patient with liver abscess who developed a second noncontiguous abscess while on metronidazole and refers to eight other treatment failures in the literature. Treatment failure and death at 48 days has been reported after multiple drug therapy for liver abscess9l; another patient had three episodes of hepatic amoebiasis despite multiple drug therapy92, but this was perhaps due to repeated reinvasion from the gut. Griffin93 has pointed out that the best results with metronidazole have been from centres where therapeutic aspiration is frequently practised; a comparative trial of metronidazole with and without aspiration has not been reported. In a trial of 66 patients given either chloroquine 500 mg daily for 10 weeks or metronidazole 750 mg tds for 10 days there were three 'drug failures' and resolution times were very similar94; in another trial metronidazole gave the same results as chloroquine plus emetine95. Parenteral metronidazole is now available and will be valuable in cases of amoebic colitis with perforation or ileus; its use has been documented in one patient96. Tinidazole, like metronidazole, is a nitroimidazole derivative. Its pharmacology and use in trichomoniasis, giardiasis, and amoebiasis has been extensively reviewed9 7. So far it has no documented advantage over metronidazole for amoebiasis. The results in amoebic colitis are similar98; Scragg99 however, has reported particularly good results in children. The treatment of symptomless infections remains a problem, especially in the USA where some of the halogenated quinolines have been withdrawn and furamide is not available'00. None of the nitroimidazoles gives particularly good parasitological cure rates and side-effects may be troublesome'01. A trial carried out in Washington DC has further demonstrated the effectiveness of furamidel02. Paromomycin is a useful alternative and possibly also dichloracetamidel03. Powell'04 advocated the use of furamide in all abscess patients treated with metronidazole. The finding that nitroimidazoles have mutagenic effects on some bacteria105, 106 and can also induce lymphomas and lung tumours in mice when given in large doses'07 has raised some doubts about the wisdom of using

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these drugs for symptomless or mild amoebic infections. It is reassuring that a follow-up of the offspring of 1469 women, treated in pregnancy over a 20-year period, has shown no damage108. The clinical use and toxicity of metronidazole in man and experimental animals has been reviewed by Roe109; prolonged use of this drug, as in Crohn's disease, may cause chromosomal abnormalities in circulating lymphocytes110 and occasionally peripheral neuropathy"l. Giardiasis

Giardia lamblia is the most frequently reported gut parasite in the United Kingdom"12. Transmission occurs in temperate climates and epidemics"13"'4 may occur among campers"5, or when domestic water supplies are contaminated"16. American visitors to the Soviet Union, especially Leningrad, are frequently infected and their symptomatology has been fully described"1. Wolfe 118 has given a useful account of the clinical features of this infection. PATHOGENESIS

Physiological studies of this protozoon have been held back by the difficulty of in vitro culture but Iyer and Gaitonde"19, and Meyer120 have now described their methodology. The fine structure of the encysted parasite has been described'1 . Scanning electron microscopy has shown that in mice an impression of the ventral aspect of the trophozoite remains on the microvilli at the site of contact between parasite and epithelial cell'22 23. In humans it has been shown that the microvilli and fuzzy coat are damaged at these sites123"124. A functional correlation of these observations has been the observation of reduced levels of brush border enzymes in the jejunal mucosa of patients with giardiasis'2 . Roberts-Thomson et al.'26 fed cysts of G. muris to mice and showed that reduction in villous crypt ratio was dose dependent. Changes regressed over the next 25 days, and during this period there was spontaneous loss of the infection. The weight gain of infected mice was significantly lower than controls. The same group of workers studied mixed infections with G. muris and Trichinella spiralis in mice; the intestinal phase of T. spiralis was associated with reduced counts of Giardia trophozoites and cysts12 7. Sehgal et al.'28 were able to demonstrate patent infection in albino rats fed cysts of G. lamblia. In athymic nude mice, an increased mortality was found in animals infected with G. muris and the related flagellate Hexamita129; treatment with anti-protozoal drugs reduced mortality. The inability of these animals to control gastrointestinal infections may have contributed to their wasting disease. CLINICAL FEATURES

In endemic areas children have been the main group reported to be symptomatic and to have malabsorption. Blanco Rabassa et al.'30 documented malabsorption in 31 out of 50 infected children. Tewari and Tandon'3' were able to show impaired absorption of fat and d-xylose in Indians of varying ages. Wright et al.'32 studied 40 expatriate adult patients, most of whom had travelled in India and South East Asia, and found malabsorption of two or three test substances (fat, xylose, and Vitamin B.2) in 20. The finding of impaired Vitamin B,2 absorption has been only occasionally

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documented before'33"134. Brandborg'35 has referred to the difficulties of interpreting absorption studies in parasitic disease among patients from areas where sprue or tropical enteropathy is endemic. However, in the study by Wright et al.132 there was a relative lack of response to tetracycline for four weeks while treatment with metronidazole for three days or mepacrine for 10 days produced consistent functional and morphological improvement. Tandon et al.'36 report free bile acids in jejunal aspirate from their patients with malabsorption and an abnormal jejunal microflora comprising mainly enterobacteria. Similar changes in microflora were found by Wright et al.'32, who, however, obtained normal 14C glycocholate breath tests in 13 of 14 patients with malabsorption. IMMUNOLOGICAL ASPECTS

Eastham et al.137 found an increase in intraepithelial lymphocytes in the jejunal mucosa of two patients. In a more extensive study'38 a significant increase in these cells was found in patients with impaired absorption compared with either infected patients with normal absorption or controls. It is possible that immunologically mediated mechanisms contribute to mucosal damage. Webster'39 has reviewed the relationship between variable hypogammaglobulinaemia and giardiasis; and Fisher et al.140 have reviewed the radiological changes on barium follow-through examinations in patients with normal and abnormal immunological status. Other immunodeficiencies, including selective IgA deficiency, do not appear to be associated with giardiasis'4 ; however, jejunal IgA levels were reduced when 10 symptomatic patients were compared with 10 controls'42. In tropical countries, giardiasis is common in marasmic children'43 and it is possible that reduced secretory IgA favours infection. In two patients in the USA with chronic alcoholic pancreatitis, mepacrine therapy for coexistent giardiasis rapidly reversed the malabsorption and oedema144. Among a series of 22 symptomatic infections in Sweden, five had coexistent pancreatic disease'45. A preponderance of blood group A has been found in Australian children with giardiasis'46. Human serum antibodies to Giardia have been demonstrated by Ridley and Ridley'14 . The indirect fluorescent antibody test was positive only in those patients who had impaired intestinal absorption; cysts were used as

antigen'48. DIAGNOSIS

Mahmoud and Warren'49 have suggested a systematised method for the diagnosis of giardiasis. Confirmation depends upon the demonstration of cysts or trophozoites in the stool or trophozoites in jejunal aspirates, impression smears, or biopsies'50. The Enterotest capsule is an efficient method of obtaining upper gastrointestinal juice without intubation'51. Cyst excretion is irregular and unpredictable'62, the frequency of positive stool examinations is not related to the presence or absence of malabsorption'32. TREATMENT

In the treatment of giardiasis the choice lies between mepacrine, metronidazole, the other 5-nitroimidazoles, and furazolidine. Wolfe"18 reported a 95 % cure rate with mepacrine (100 mg thrice daily for seven days) and 81 % with furazolidine. The criterion for cure was the absence of cysts from three

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consecutive stool samples one month after treatment. In another study G. lamblia was not eradicated from six out of nine patients given mepacrine at this dosage for 10 days132. Using the examination of jejunal aspirate and mucosal impression smears as well as stools, metronidazole in a single daily dose of 2-0 g for three days cured 27 out of 31 patients153, and a second course cured two of the remaining four. Using the same regimen, Green et a1.154 report that a group of 22 patients were all cured. Trials using tinidazole have been reviewed97. Muller and Lindmarkl'5 have shown that metronidazole is reduced inside anaerobic organisms allowing more drug to diffuse passively into the organism, so producing very high intracellular concentrations. The site of action of the reduced drug upon anaerobic metabolism has not been defined.

Isosporiasis The two species, Isospora belli, and L hominis, have for many years been classed together as the causes of human isosporiasis; a relatively uncommon condition normally recognised by the presence of infective oocysts or sporocysts in the stool. Experimental work has now shown that these parasites are quite distinct, while L belli has a simple direct transmission cycle in man, L hominis is now regarded as a species of Sarcocysts in which man is the definitive host and the cow or pig the obligate intermediate hosts. The taxonomic problems raised by recent work on this group of Sporozoa have been discussed by Frenkel'66. Sporocysts of . hominis appear in the human stool nine days or more after the ingestion of sarcocyst-infected meat and excretion of sporocysts may persist for 100 days or more, apparently without producing symptoms. Antibodies have been demonstrated by immunofluorescence, using S. fusiformis in cattle heart as antigen, among volunteers fed raw meat167 and German sanatorium patients fed regularly on beefsteak tartare'58; there is no cross-reaction with Toxoplasma antigens'59. The infection is often overlooked but may be surprisingly common in countries such as Holland, Germany, or Kenya where incompletely cooked meat is commonly eaten. All stages of the life cycle are not yet described, however development in the intermediate hosts is probably similar to that of S. muris in the mouse'60, and development in man is likely to be similar to Sarcocystis spp. in cats161. In Sarcocystis, unlike Isospora, there is no schizogony in the gut cycle within the definitive host, which proceeds directly to gametogony. It should be noted that man uncommonly acts as intermediate host for another species of Sarcocystis-namely, S. lindemanni-a muscle parasite that sometimes causes myositis or systemic manifestations'62. Isospora belli is spread directly by the faeco-oral route and is usually reported from the tropics, but it also occurs in mental institutions and accidental laboratory infections continue to be reported'63. Repeated schizogonic cycles occur within the epithelial cells of the small bowel causing villous shortening and functional changes; gametes are also formed and soon after oocysts appear in the stool. Many infections are symptomatic and a prolonged debilitating sprue-like illness may result from this underrecognised condition'64; other clinical features are fever, blood eosinophilia, and stool Charcot-Leyden crystals. Treatment requires further study; however, cotrimoxazole 2 g daily for three weeks was successful in a patient in Israel'66,

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as was a 49-day course of pyrimethamine and sulphonamide in an American negro who was originally infected in North Africa in 1944164. Nitrofurantoin is used to treat coccidiosis in domestic animals and deserves further study in man. Balantidiasis

Pigs appear to be the main reservoir of infection and infection rates may reach 20% in New Guinea where human contact with pigs is particularly close166. Infections in Moslem countries and mental institutions result from direct transmissiom or a rodent source167. The fuJly documented outbreak in Micronesia'68 followed a typhoon which contaminated surface -water supplies with pig faeces; the outbreak was self-limited and multifocal, suggesting that person to person infection was unusual. The rare fatal cases may show extensive ulcerative enteritis'69. Concurrent Trichuris infection may favour tissue invasion'6 . Metronidazole was used successfully to treat 20 infections in Venezuela' 70, but it failed to cure three out of four asymptomatic infections in Micronesia'68. Nitrimidazine (Naxogin (R)) cured three symptomatic cases in Mexico'67, and 12 out of 17 children in Columbia who received 500 mg daily for five days' 71. The ultrastructure of this parasite has now been described'72. Other parasites

A rectal biopsy from a 3 year old girl, who presented in Tennessee with a five-day history of severe diarrhoea and vomiting, showed parasites resembling Cryptosporidium attached to crypt epithelial cells; they measured 2-4 nm and were demonstrated by toluidine blue staining and by electron microscopy 73. The illness was severe but self-limiting; a barium enema had shown spiculated ulceration of the rectosigmoid and descending colon. This parasite should be looked for again in similar situations. The amoeboflagellate, Dientamoeba fragilis, was found in 4-2 % of the preserved faecal specimens from 43 029 subjects in Canada' 4. The disputed pathogenicity of this parasite is fully discussed in this paper; suggestive symptoms are diarrhoea or loose stools, abdominal pain, and anal pruritus. A positive association was found in this survey between D. fragilis and threadworm (Enterobius vermicularis), thus supporting the hypothesis that the egg of this worm acts as a mechanical vector for the protozoan. R. KNIGHT1 AND S. G. WRIGHT

Department of Tropical Medicine Liverpool School of Tropical Medicine Liverpool Department of Clinical Tropical Medicine Hospital for Tropical Diseases London 'Address for reprint requests: Dr. R. Knight, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 4QA. Received for publication 11 April 1978 References

"Marsden, P. D., and Hoskins, D. W. (1966). Intestinal parasites: a progress report. Gastroenterology, 51, 701-720.

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2Marsden, P. D., and Schultz, M. G. (1969). Intestinal parasites. Gastroenterology, 57, 724-750. 3Knight, R., Schultz, M. G., Hoskins, D. W., and Marsden, P. D. (1973). Intestinal parasites. Gut, 14, 145-168. 4Jeon, K. W. (ed.) (1973). The Biology of Amoeba. Academic Press: London. 'Singh, B. N. (1975). Pathogenic and Non-pathogenic Amoebae. Macmillan: London. "Padilla y Padilla, C. A., and Padilla, G. M. (ed.) (1974). Amebiasis in Man: Epidemiology, Therapeutics, Clinical Correlations and Prophylaxis. Charles C. Thomas: Springfield, Illinois. 7Sepulveda, B., and Diamond, L. S. (1976). Proceedings of the International Conference on Amoebiasis, Mexico City, 27-29 October 1975. Instituto Mexicano del Seguro Social: Mexico. 'L6sch, F. A. (1975). Massive development of amebas in the large intestine. Translation from German text, 1875. American Journal of Tropical Medicine and Hygiene, 24, 383-392. "Griffin, J. L. (1972). Human amebic dysentery. Electron microscopy of Entamoeba histolytica contacting, ingesting and digesting inflammatory cells. American Journal of Tropical Medicine and Hygiene, 21, 895-906. "Takeuchi, A., and Phillips, B. P. (1975). Electron microscope studies of experimental Entamoeba histolytica infection in the guinea pig. I. Penetration of the intestinal epithelium by trophozoites. American Journal of Tropical Medicine and Hygiene, 24, 34-48. "Takeuchi, A., and Phillips, B. P. (1976). Electron microscope studies of experimental Entamoeba histolytica infection in the guinea pig. II. Early cellular and vascular changes accompanying invasion in the lamina propria Virchows Archiv Abteilung B. Zellpathologie., 20, 1-13. "Knight, R. (1977). An in vitro model for measuring the cytopathic effect of Entamoeba histolytica. Journal of Parasitology, 63, 388-389. "Knight, R., Bird, R. G., and McCaul, T. F. (1975). Fine structural changes at Entamoeba histolytica rabbit kidney cell (RK13) interface. Annals of Tropical Medicine andParasitology, 69, 197-202. '4Bos, H. J., and Van de Griend, R. J. (1977). Virulence and toxicity of axenic Entainoeba histolytica. Nature (Lond.), 265, 341-343. "Henley, G. L., Lee, C. M., and Takeuchi, A. (1976). Freeze-etching observations of trophozoites of pathogenic Entarnoeba histolytica. Zeitschrift fur Parasitenkunde, 48, 181-190. "Proctor, E. M., and Gregory, M. A. (1972). The ultrastructure of axenically cultivated trophozoites of Entamoeba histolytica with particular reference to an observed variation in structural pattern. Annals of Tropical Medicine and Parasitology, 66, 335-338. 7Deas, J. E., and Miller, J. H. (1977). Plasmalemmal modifications of Entamoeba histolytica in vivo. Journal of Parasitology, 63, 25-31. "Diamond, L. S., and Mattern, C. F. T. (1976). Protozoal viruses. Advances in Virus Research, 20, 87-112. "Bird, R. G., and McCaul, T. F. (1976). The rhabdovirus of Entamoeba histolytica and Entamoeba invadens. Annals of Tropical Medicine and Parasitology, 70, 81-93. 2"De, P. K., Bhatia, V. N., Ray, B. G., and Agarwal, D. S. (1973). Virulence of E. histolytica strains isolated from cases of amoebiasis in New Delhi, India. Indian Journal of Medical Research, 61, 45-50. 2"Latter, V. S. (1972). Correlation between strain virulence and evaluation of growth in monoxenic culture of Entamoeba histolytica. Transactions of the Royal Society of Tropical Medicine and Hygiene, 66, 13. 22Phillips, B. P., Diamond, L. S., Bartgis, I. L., and Stuppler, S. A. (1972). Results of intraceacal inoculation of germ-free and conventional guinea pigs and germ-free rats with axenically cultivated Entamoeba histolytica. Journal of Protozoology, 19, 498-499. 23Raether, W. (1971). Intrahepatikale Infektions versuche am Goldhamster mit Entamoeba histolytica-Crithidia-Kulturen mit und ohne Beteiligung von Bakterien. Zeitschrift fidr Parasitenkunde, 36, 335-345. 24Diamond, L. S., Phillips, B. P. and Bartgis, I. L. (1973). Virulence of axenically cultivated E. histolytica. Archivos de Investigacion Medica, 4, Suppl. I, 99-104. 2"Mattern, C. F. T., and Keister, D. B. (1977). Experinmental amebiasis I. Pathogenicity of axenically cultured Entamoeba histolytica in the brain of the newborn mouse. American Journal of Tropical Medicine and Hygiene, 26, 393-401. 26Mattern, C. F. T., and Keister, D. B. (1977). Experimental amebiasis II. Hepatic amebiasis in the newborn hamster. American Journal of Tropical Medicine and Hygiene, 26, 402-411. 27Neal, R. A., Latter, V. S., and Richards, W. H. G. (1974). Survival of Entamoeba and related amoebae at low temperature. II. Viability of amoebae and cysts stored in liquid nitrogen. International Journal of Parasitology, 4, 353-360. 2"Neal, R. A. (1974). Survival of Entamnoeba and related amoebae at low temperature. I. Viability of Entamoeba cysts at 4'C. International Journal of Parasitology, 4, 227-229. 2"Martinez-Palomo, A., Gonzalez-Robles, A., and De la Torre, M. (1973). Selective agglutination of pathogenic strains of Entamoeba histolytica induced by con A. Nature New Biology, 245, 186-187. 3"Silva, P. P. da, and Martinez-Palomo, A. (1974). Induced redistribution of membrane particles, anionic sites and con A receptors in Entamoeba histolytica. Nature (Lond.), 249, 170-171 "Ross, G. W., and Knight, R. (1973). Dietary factors affecting the pathogenicity of Entamoeba histolytica in rats. Transactions of the Royal Society of Tropical Medicine and Hygiene 67

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560-567. 32Knight, R., and Warren, K. S. (1973). The interaction between Entamoeba histolytica and Schistosoma mnansoni infections in mice. Transactions of the Royal Society ofTropical Medicine and Hygiene, 67, 644-651. 33Knight, R., and Chew, L. H. (1974). The interaction between Entamoeba histolytica and Trichuris muris infections in mice. Amnerican Journal of Tropical Medicine and Hygiene, 23, 590-594. 34Gogler, H., and Knight, R. (1974). The effect of hepatic injury upon the development of amoebic liver abscess in hamsters. Annals of Tropical Medicine and Parasitology, 68, 177-185. 3"Neal, R. A., and Harris, W. G. (1975). Attempts to infect inbred strains of rats and mice with Entamoeba histolytica. Transactions of the Royal Society of Tropical Medicine and Hygiene, 69, 429-430. 36Savanat, T., Viriyanond, P., and Nimitmongkol, N. (1973). Blast transformation of lymphocytes in amebiasis. American Journal of Tropical Medicine and Hygiene, 22, 705-710. "Harris, W. G., and Bray, R. S. (1976). Cellular sensitivity in amoebiasis-preliminary results of lymphocytic transformation in response to specific antigen and to mitogen in carrier and disease states. Transactions of the Royal Society of Tropical Medicine and Hygiene, 70, 340-343. 38Ortiz-Ortiz, L., Zamacona, G., Sepulveda, B., and Capin, N. R. (1975). Cell-mediated immunity in patients with amebic abscess of the liver. Clinical Immunology and Imntnunopathology, 4, 127-134. 39Krupp, I. M. (1974). Protective immunity to amebic infection demonstrated in guinea pigs. American Journal of Tropical Medicine and Hygiene, 23, 355-360. 4"Shaalan, M., and Baker, R. P. (1970). Detection of coproantibodies in amebiasis of the colon: a preliminary report. Amnerican Journal of Clinical Pathology, 54, 615-617. 4'Mahajan, R. C., Agarwal, S. C., Chhuthani, P. N., and Chitkara, N. L. (1972). Coproantibodies in intestinal amoebiasis. Indian Journal of Medical Research, 60, 547-550. 42Salem, E., Zaki, S. A., Moneim, W. 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