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Serodiagnosis of amoebiasis
Serodiagnosis
and Immunotherapy
in Infectious Disease (1988) 2, 79-84
Leading articles Serodiagnosis of amoebiasis
infection, involving only luminal colonisation of the gut, accounts for the vast majority of human infections (possibly 80-90%). S.ymptomatic infection may be limited to gastromtestinal tract, most commonly presenting as amoebic colitis (diarrhoea or dysentery), or have extraintestinal manzyestations, notably amoebic liver abscess. The diagnosis of amoebic infection currently relies on the demonstration of(i) E. histolyticu forms (cysts or trophozoites) in faeces or (ii) specific anti-E. histolytica antibodies in serum. Both methods have inherent problems and it is for this reason that other diagnostic approaches such as detection of E. histol#ca antigens in serums or faece+’ and the development of specific DNA probes are underway. For the present we must depend on the mom conventional diagnostic techniques. Faecal microscopy to detect E. histolytica forms is the most widely used method of diagnosis. Trophozoites may also be detected microscopically in scrapings of exudate from the rectal mucosa or in rectal mucosal biopsy specimens. These techniques are time-consuming and require considerable experience from the observer. There are sometimes difficulties in interpretation of the microscopic findings. The presence of cysts, for example, may occur in both asymptomatic carriage and invasive infection. In addition, the shedding of cysts can be intermittent and thus a negative stool examination does not exclude infection. It is recommended that at least three or more stool specimens on separate days are examined in order to detect more than 80.90% of infections. Furthermore, it can be difficult to differentiate motile trophozoites containing ingested red cells which are indicative of invasive disease from macrophages containing ingested red cells. One study in North America indicated that polymorphonuclear leukocytes were also commonly mistaken for trophozoites. Microscopic diagnosis may similarly elude the clinician in cases of extraintestinal disease, particularly amoebic liver abscess, when material aspirated from the abscess is unlikely
The enteric protozoan pathogen. Entamoeba histolvtica is found worldwide and produces an enormous burden of illness with substantial mortality. Although prevalence data remain imprecise it has been estimated that there are nearly 500 million amoebic infections worldwide, about 10% of which produce clinically evident disease, predominantly in Asia, Africa and Latin America. Accepting the conservatively estimated mortality of 75,000 annually (0.2% of illnesses), amoebiasis becomes the third leading parasitic cause of death worldwide, following only malaria and schistosomiasis’. Amoebiasis, however, is no longer an exotic disease limited to the tropics and subtropics but is endemic in many countries of the industrialised world including the U.K. and IJSA. where cases occur in individuals who have never travelled abroad. Up to 30% of homosexual men carry this parasite although it rarely causes symptomatic disease. The parasite was first observed in faecal specimens in 1869 by Lewis and in 1875. amoebic dysentry was described in a patient from St. Petersburg, Russia by Losch’. Robert Koch, 2 years later noted the invasion of tissue by amoebae and in 1903 the organism was named Entamoeba histolytica by Schaudinn because of its ability to lyse tissue. Entamoeba histolytica primarily affects humans but a few other animal species. particularly macaque monkeys, can be naturally or experimentally infected’,‘. Humans, however, continue to be the major reservoir of infection. Although the pathogenetic mechanisms involved in mucosal invasion and cell destruction are becoming clearer, the reason why there should be such a diverse host response to the parasite is still uncertain. There is now evidence that virulence varies between E. histolvtica strains, and that isoenzyme patterns (zymodemes) may act as virulence markers4. In addition, host factors are also likely to play a part. Several clinical syndromes result from the varied host-parasite interaction. Asymptomatic 79
80
Leading Articles
to yield trophozoitesx. Thus, although the gold standard for diagnosis of invasive amoebic infection is still the demonstration of erythrophagic trophozoites in body fluids, there is increasing dependence on serological responses to this parasite to confirm clinically suspected amoebic colitis and liver abscess. Specific anti-E. histolytica serum IgG, IgM, IgA and IgE responses have been reported in amoebiasis but generally only occur when the parasite leaves the intestinal lumen and invades the bowel wall*-I>. A variety of techniques have been used to detect specific antibody in the serum, including indirect haemagglutination (IHA), immunodiffusion (ID), counterimmunoelectrophoresis (CIE), indirect immunofluorescence used in the so-called fluorescent amoebic antibody test (FAT) and enzymelinked immunosorbent assay (ELISA). In general, the sensitivities of serologic testing are 95-l 00% in amoebic liver abscess and 85595% in invasive intestinal disease. The complement fixation test (CFT), which was the earliest serologic test for amoebiasis, has been superseded by others of greater sensitivity and specificity. The amoebic FAT was introduced in the 1960s using antigen sources from non-axenic cultures. It showed high rates of positivity in extraintestinal disease (> 90%) compared with disease limited to the intestine (c. 70%)i4. Asymptomatic carriers and patients without amoebiasis had titres of 1 in 32 or lower compared with titres of I in 64 or higher in active or recent infection. The antibodies detected by FAT were shown to persist for 8-10 months unlike IHA tires which remain high for up to 5 years’sm’7. Amoebic IHA titres have been found to be positive in 100% of amoebic patients with liver abscess, 98% in amoebic dysentry and 66% of asymptomatic cyst passers’“. False positives can occur in pregnancy and in the presence of heterophile antibodies. The amoebic IHA test remains positive, with titres greater than 1: 128 in about 50% of the patients for approximately 5 years following an episode of invasive disease (hepatic abscess and severe colitis)‘9 “. FAT and CIE become negative in more than half of the individuals within one year following acute amoebic infection?‘.“. Therefore one expects that the prevalence of active infection as indicated by FAT and CIE would be lower than the prevalence obtained by IHA testing which appears to indicate both present and past infections. In recent years ELISA has been widely used for serodiagnosis of infectious diseases, includ-
ing amoebiasis. Bos et al. evaluated ELISA in amoebic infection and found high titres of 1 in 10,000 or more in patients with amoebic liver abscess, although titres were low in intestinal amoebiasis?. In Northern India, Tandon et a/. evaluated ELISA in intestinal and extraintestinal amoebiasis and confirmed its diagnostic value, including its superiority over IHA. The ELISA was both more specific, with no false positives, and more sensitive. Similar findings have been observed by other workers26ZY. Modifications of solid-phase ELISA in the form of stick ELISA has been used successfully in both intestinal and extraintestinal amoebiasis’O. The ELISA inhibition test, with E. histol.vtica antigen, has been used to block positive ELISA readings and thereby increase the specificity of the tesP. Thus all or nearly all patients with invasive amoebiasis develop circulating IgG antibodies specific for E. histolytica. Individuals with asymptomatic infection, the majority of whom have only luminal colonization, less frequently develop a serum IgG antibody response. The presence of IgG antibodies for E. histolytica is regarded as being indicative of current parasite invasion or previous invasive disease”. The anti-E. histolytica IgM response is much shorter lived than the IgG response and may prove useful in distinguishing current from past infection”. Despite an IgG antibody response that may last for many years, there is no evidence that the titre of antibody to E. histotytica correlates with clinical outcome and invasive amoebiasis may occur even in the presence of high antibody titres”-‘?. Acknowledgements A.S.A. and M.J.G.F. gratefully acknowledge financial support from The Wellcome Trust. M.J.G.F. is a Wellcome Trust Senior Lecturer. A. S. ARVIND* N. SHETTY MD? M. J. G. FARTHING* *Department of GastroenteroiogJ St Bartholomew’s Hospital West Smithfield London ECIA 7BE tDepartment of Microbiology. St John’s Medical College, Bangalore, India
Leading Articles References I Guerrant RL. Amebiasis: introduction, current status, and research questions. Rev Infect Dis 1986; 8: 218.-28. hosts and natural foci of 2 Hoare CA. Reservoir human protozoa1 infection. Acta Trop 1962: 19: 281 317. 3 Dobell C. Researches on the intestinal protozoa of monkeys and man IV. An experimental study of the his~o/.vtica-like species of Entamoeha living naturally in macaques. Parasitology 1931: 23: I 72. 4 Sargeaunt PG, Williams JE. Electrophoretic isoenzyme patterns of the pathogenic and nonpathogenic intestinal amoebae of man. Trans R Sot Trop Med 1979; 13: 225--l. 5 Pillai S, Mohimen A. A solid phase radioimmunoassay for E. histol.vtica proteins and the detection of circulating antigens in amoebiasis. Gastroenterology 1982; 83: 121@6 Randall CR, Goldsmith RS, Shek J, Mehalko S, Heyneman D. Use of enzyme-linked immunosorbent assay (ELISA) for detection of Entamoebu histolytica antigen in faecal samples. Trans R Sot Trop Med 1984; 78: 593-595. I Grundy MS. Voller A, Warhurst D. An enzymelinked immunosorbent assay for the detection of Entamoeha histo!,~tica antigens in faecal material. Trans R Sot Trop Med Hyg 1987; 81: 627-32. 8 Healy GR. Immunologic tools in the diagnosis of amoebiasis: Epidemiology in the United States. Rev Infect Dis. 1986; 8: 23946. 9 Dasgupta A. Immunoglobulins in health and disease III. Immunoglobulins in the sera of patients with amoebiasis. Clin Exp Immunol 1974; 16: 163 -8. IO Harris WG, Friedman MJ, Bray RS. Serial measurement of total and parasite-specific IgE in an African population infected with Entamoeba histolvtica. Trans R Sot Trop Med Hyg 1978: 72: 427 -30. II Nilsson LS. Petchclai B, Elwing H. Application of thin layer immunoassay (TIA) for demonstration of antibodies against Entamoeba histolyticrr. Am J Trop Med Hyg 1980; 29: 524 -9. 12 Osisanya JOS, Warhurst DC. Specific anti-amoebic immunoglobulins and the cellulose acetate precipitin test in Entamoeba histolytica infection. Trans R Sot Troo Med Hvp 1980: 74: 605%8. 13 Jackson TFHG, AndersonCB. Simjee AE. Serological differentiation between past and present infection in hepatic amoebiasis. Trans R Sot Med Hyg 1984; 78: 342-5. 14 Garcia LS. Bruckney DA, Brewer TC, Shimizu R-Y. Comparison of indirect fluorescent antibody amoebic serology with counter immunoelectrophoresis and indirect hemagglutination amoebic serologies. J Clin Microbial 1982: 15. 603 -5.
81
M, Healy GR, Sharbot JM. Serologi15. Patterson cal testing for amebiasis. Gastroenterology 1980: 78: 136-41. 16 Healy GR, Kraft SC. The indirect hemagglutination test for amoebiasis in patients with inflammatory bowel disease. Am J Dig Dis 1972; 17: 97~ 104. 17 Healy GR. Visvesvara GS, Kagan IG. Observations on the persistence of antibodies to E. histolyticu. Arch Invest Med (Mex) 1974; suppl 2: 495-500. 18 Kessel JF. Lewis WP. Pasquel CM, Turner JA. Indirect hemagglutination and complement lixation tests in amebiasis. Am J Trop Med Hyg 1965; 14: 540-50. 19 Krupp IM. Powell SJ. Antibody response to invasive amebiasis in Durban, South Africa. Am J Trop Med Hyg 1971: 20: 41420. 20 Stramm WP, Ashley MJ. Bell K. The value ot amoebic serology in an area of low endemicity. Trans R Sot Troo Med Hve. 1976: 70: 49%53. 21 Juniper K Jr. Worrell CL,-Munshew MC. Roth LS. Cypert H. Lloyd RE. Serologic diagnosis of amebiasis. Am J Trop Med Hyg 1972; 21: I57 68. RC, Dutta DV, Chitkasa NL. Agarwal 22 Mahajan SC, Chhuttani PN. Sequential changes of immunofluorescence titres in hepatic amebiasis. Indian J Med Res 1974: 62: 7-10. 23 Kagan IG. Serodiagnosis of parasitic diseases. In: Rese NR. Friedman H eds. Manual of clinical immunology. Washington DC: American Society for Microbiology, 1980: 573 -604. 24 Bos HJ, Van den Eijk AA, Steerenberg PA. Application of the enzyme-linked immunosorbent assay in amoebiasis. Trans R Sot Trop Med Hyg 1975; 69: 440. immunosorbent assay 25 Tandon A. Enzyme-linked for amoebiasis. Trans R Sot Trop Med Hyg 1981; 75: 574. 26 Yang J. Kennedy MT. Evaluation of the ELISA for the serodiagnosis of amoebiasis. J Clin Microbiol 1979: 10: 778. 27 Williams JE, Robinson DM. Trans R Sot Trop Med Hyg. 1982; 76: 280. 28 Baveja UK. Warhurst DC, Voller A. Enzvmelinked immunosorbent assay in amoebiasis. Indian J Med Res 1984; 80: 529. 29 Das P. Pal S. Pal SC. Evaluation of the micro ELISA IHA and IFA techniques for serodiagnosis of amoebiasis. J Diarrh Dis Res 1984; 5: 238. 30. Feigner P. Serological diagnosis of extraintestlnal amoebiasis. a comparison of stick ELISA and other Immunological tests. Tropenmed Paras1tol 1977; 28: 491. 31. Trissl D. Immunology of Entumoeba histo/~rlco in human and animal hosts. Rev Infect Dis 1982; 4 1154 84. 32. Krupp IM. Antibody response in intestmal and extraintestinal amoebiasis. Am J Trop Med 1970; 19: 57 62.
and Immunotherapy
in Infectious Disease (1988) 2, 79-84
Leading articles Serodiagnosis of amoebiasis
infection, involving only luminal colonisation of the gut, accounts for the vast majority of human infections (possibly 80-90%). S.ymptomatic infection may be limited to gastromtestinal tract, most commonly presenting as amoebic colitis (diarrhoea or dysentery), or have extraintestinal manzyestations, notably amoebic liver abscess. The diagnosis of amoebic infection currently relies on the demonstration of(i) E. histolyticu forms (cysts or trophozoites) in faeces or (ii) specific anti-E. histolytica antibodies in serum. Both methods have inherent problems and it is for this reason that other diagnostic approaches such as detection of E. histol#ca antigens in serums or faece+’ and the development of specific DNA probes are underway. For the present we must depend on the mom conventional diagnostic techniques. Faecal microscopy to detect E. histolytica forms is the most widely used method of diagnosis. Trophozoites may also be detected microscopically in scrapings of exudate from the rectal mucosa or in rectal mucosal biopsy specimens. These techniques are time-consuming and require considerable experience from the observer. There are sometimes difficulties in interpretation of the microscopic findings. The presence of cysts, for example, may occur in both asymptomatic carriage and invasive infection. In addition, the shedding of cysts can be intermittent and thus a negative stool examination does not exclude infection. It is recommended that at least three or more stool specimens on separate days are examined in order to detect more than 80.90% of infections. Furthermore, it can be difficult to differentiate motile trophozoites containing ingested red cells which are indicative of invasive disease from macrophages containing ingested red cells. One study in North America indicated that polymorphonuclear leukocytes were also commonly mistaken for trophozoites. Microscopic diagnosis may similarly elude the clinician in cases of extraintestinal disease, particularly amoebic liver abscess, when material aspirated from the abscess is unlikely
The enteric protozoan pathogen. Entamoeba histolvtica is found worldwide and produces an enormous burden of illness with substantial mortality. Although prevalence data remain imprecise it has been estimated that there are nearly 500 million amoebic infections worldwide, about 10% of which produce clinically evident disease, predominantly in Asia, Africa and Latin America. Accepting the conservatively estimated mortality of 75,000 annually (0.2% of illnesses), amoebiasis becomes the third leading parasitic cause of death worldwide, following only malaria and schistosomiasis’. Amoebiasis, however, is no longer an exotic disease limited to the tropics and subtropics but is endemic in many countries of the industrialised world including the U.K. and IJSA. where cases occur in individuals who have never travelled abroad. Up to 30% of homosexual men carry this parasite although it rarely causes symptomatic disease. The parasite was first observed in faecal specimens in 1869 by Lewis and in 1875. amoebic dysentry was described in a patient from St. Petersburg, Russia by Losch’. Robert Koch, 2 years later noted the invasion of tissue by amoebae and in 1903 the organism was named Entamoeba histolytica by Schaudinn because of its ability to lyse tissue. Entamoeba histolytica primarily affects humans but a few other animal species. particularly macaque monkeys, can be naturally or experimentally infected’,‘. Humans, however, continue to be the major reservoir of infection. Although the pathogenetic mechanisms involved in mucosal invasion and cell destruction are becoming clearer, the reason why there should be such a diverse host response to the parasite is still uncertain. There is now evidence that virulence varies between E. histolvtica strains, and that isoenzyme patterns (zymodemes) may act as virulence markers4. In addition, host factors are also likely to play a part. Several clinical syndromes result from the varied host-parasite interaction. Asymptomatic 79
80
Leading Articles
to yield trophozoitesx. Thus, although the gold standard for diagnosis of invasive amoebic infection is still the demonstration of erythrophagic trophozoites in body fluids, there is increasing dependence on serological responses to this parasite to confirm clinically suspected amoebic colitis and liver abscess. Specific anti-E. histolytica serum IgG, IgM, IgA and IgE responses have been reported in amoebiasis but generally only occur when the parasite leaves the intestinal lumen and invades the bowel wall*-I>. A variety of techniques have been used to detect specific antibody in the serum, including indirect haemagglutination (IHA), immunodiffusion (ID), counterimmunoelectrophoresis (CIE), indirect immunofluorescence used in the so-called fluorescent amoebic antibody test (FAT) and enzymelinked immunosorbent assay (ELISA). In general, the sensitivities of serologic testing are 95-l 00% in amoebic liver abscess and 85595% in invasive intestinal disease. The complement fixation test (CFT), which was the earliest serologic test for amoebiasis, has been superseded by others of greater sensitivity and specificity. The amoebic FAT was introduced in the 1960s using antigen sources from non-axenic cultures. It showed high rates of positivity in extraintestinal disease (> 90%) compared with disease limited to the intestine (c. 70%)i4. Asymptomatic carriers and patients without amoebiasis had titres of 1 in 32 or lower compared with titres of I in 64 or higher in active or recent infection. The antibodies detected by FAT were shown to persist for 8-10 months unlike IHA tires which remain high for up to 5 years’sm’7. Amoebic IHA titres have been found to be positive in 100% of amoebic patients with liver abscess, 98% in amoebic dysentry and 66% of asymptomatic cyst passers’“. False positives can occur in pregnancy and in the presence of heterophile antibodies. The amoebic IHA test remains positive, with titres greater than 1: 128 in about 50% of the patients for approximately 5 years following an episode of invasive disease (hepatic abscess and severe colitis)‘9 “. FAT and CIE become negative in more than half of the individuals within one year following acute amoebic infection?‘.“. Therefore one expects that the prevalence of active infection as indicated by FAT and CIE would be lower than the prevalence obtained by IHA testing which appears to indicate both present and past infections. In recent years ELISA has been widely used for serodiagnosis of infectious diseases, includ-
ing amoebiasis. Bos et al. evaluated ELISA in amoebic infection and found high titres of 1 in 10,000 or more in patients with amoebic liver abscess, although titres were low in intestinal amoebiasis?. In Northern India, Tandon et a/. evaluated ELISA in intestinal and extraintestinal amoebiasis and confirmed its diagnostic value, including its superiority over IHA. The ELISA was both more specific, with no false positives, and more sensitive. Similar findings have been observed by other workers26ZY. Modifications of solid-phase ELISA in the form of stick ELISA has been used successfully in both intestinal and extraintestinal amoebiasis’O. The ELISA inhibition test, with E. histol.vtica antigen, has been used to block positive ELISA readings and thereby increase the specificity of the tesP. Thus all or nearly all patients with invasive amoebiasis develop circulating IgG antibodies specific for E. histolytica. Individuals with asymptomatic infection, the majority of whom have only luminal colonization, less frequently develop a serum IgG antibody response. The presence of IgG antibodies for E. histolytica is regarded as being indicative of current parasite invasion or previous invasive disease”. The anti-E. histolytica IgM response is much shorter lived than the IgG response and may prove useful in distinguishing current from past infection”. Despite an IgG antibody response that may last for many years, there is no evidence that the titre of antibody to E. histotytica correlates with clinical outcome and invasive amoebiasis may occur even in the presence of high antibody titres”-‘?. Acknowledgements A.S.A. and M.J.G.F. gratefully acknowledge financial support from The Wellcome Trust. M.J.G.F. is a Wellcome Trust Senior Lecturer. A. S. ARVIND* N. SHETTY MD? M. J. G. FARTHING* *Department of GastroenteroiogJ St Bartholomew’s Hospital West Smithfield London ECIA 7BE tDepartment of Microbiology. St John’s Medical College, Bangalore, India
Leading Articles References I Guerrant RL. Amebiasis: introduction, current status, and research questions. Rev Infect Dis 1986; 8: 218.-28. hosts and natural foci of 2 Hoare CA. Reservoir human protozoa1 infection. Acta Trop 1962: 19: 281 317. 3 Dobell C. Researches on the intestinal protozoa of monkeys and man IV. An experimental study of the his~o/.vtica-like species of Entamoeha living naturally in macaques. Parasitology 1931: 23: I 72. 4 Sargeaunt PG, Williams JE. Electrophoretic isoenzyme patterns of the pathogenic and nonpathogenic intestinal amoebae of man. Trans R Sot Trop Med 1979; 13: 225--l. 5 Pillai S, Mohimen A. A solid phase radioimmunoassay for E. histol.vtica proteins and the detection of circulating antigens in amoebiasis. Gastroenterology 1982; 83: 121@6 Randall CR, Goldsmith RS, Shek J, Mehalko S, Heyneman D. Use of enzyme-linked immunosorbent assay (ELISA) for detection of Entamoebu histolytica antigen in faecal samples. Trans R Sot Trop Med 1984; 78: 593-595. I Grundy MS. Voller A, Warhurst D. An enzymelinked immunosorbent assay for the detection of Entamoeha histo!,~tica antigens in faecal material. Trans R Sot Trop Med Hyg 1987; 81: 627-32. 8 Healy GR. Immunologic tools in the diagnosis of amoebiasis: Epidemiology in the United States. Rev Infect Dis. 1986; 8: 23946. 9 Dasgupta A. Immunoglobulins in health and disease III. Immunoglobulins in the sera of patients with amoebiasis. Clin Exp Immunol 1974; 16: 163 -8. IO Harris WG, Friedman MJ, Bray RS. Serial measurement of total and parasite-specific IgE in an African population infected with Entamoeba histolvtica. Trans R Sot Trop Med Hyg 1978: 72: 427 -30. II Nilsson LS. Petchclai B, Elwing H. Application of thin layer immunoassay (TIA) for demonstration of antibodies against Entamoeba histolyticrr. Am J Trop Med Hyg 1980; 29: 524 -9. 12 Osisanya JOS, Warhurst DC. Specific anti-amoebic immunoglobulins and the cellulose acetate precipitin test in Entamoeba histolytica infection. Trans R Sot Troo Med Hvp 1980: 74: 605%8. 13 Jackson TFHG, AndersonCB. Simjee AE. Serological differentiation between past and present infection in hepatic amoebiasis. Trans R Sot Med Hyg 1984; 78: 342-5. 14 Garcia LS. Bruckney DA, Brewer TC, Shimizu R-Y. Comparison of indirect fluorescent antibody amoebic serology with counter immunoelectrophoresis and indirect hemagglutination amoebic serologies. J Clin Microbial 1982: 15. 603 -5.
81
M, Healy GR, Sharbot JM. Serologi15. Patterson cal testing for amebiasis. Gastroenterology 1980: 78: 136-41. 16 Healy GR, Kraft SC. The indirect hemagglutination test for amoebiasis in patients with inflammatory bowel disease. Am J Dig Dis 1972; 17: 97~ 104. 17 Healy GR. Visvesvara GS, Kagan IG. Observations on the persistence of antibodies to E. histolyticu. Arch Invest Med (Mex) 1974; suppl 2: 495-500. 18 Kessel JF. Lewis WP. Pasquel CM, Turner JA. Indirect hemagglutination and complement lixation tests in amebiasis. Am J Trop Med Hyg 1965; 14: 540-50. 19 Krupp IM. Powell SJ. Antibody response to invasive amebiasis in Durban, South Africa. Am J Trop Med Hyg 1971: 20: 41420. 20 Stramm WP, Ashley MJ. Bell K. The value ot amoebic serology in an area of low endemicity. Trans R Sot Troo Med Hve. 1976: 70: 49%53. 21 Juniper K Jr. Worrell CL,-Munshew MC. Roth LS. Cypert H. Lloyd RE. Serologic diagnosis of amebiasis. Am J Trop Med Hyg 1972; 21: I57 68. RC, Dutta DV, Chitkasa NL. Agarwal 22 Mahajan SC, Chhuttani PN. Sequential changes of immunofluorescence titres in hepatic amebiasis. Indian J Med Res 1974: 62: 7-10. 23 Kagan IG. Serodiagnosis of parasitic diseases. In: Rese NR. Friedman H eds. Manual of clinical immunology. Washington DC: American Society for Microbiology, 1980: 573 -604. 24 Bos HJ, Van den Eijk AA, Steerenberg PA. Application of the enzyme-linked immunosorbent assay in amoebiasis. Trans R Sot Trop Med Hyg 1975; 69: 440. immunosorbent assay 25 Tandon A. Enzyme-linked for amoebiasis. Trans R Sot Trop Med Hyg 1981; 75: 574. 26 Yang J. Kennedy MT. Evaluation of the ELISA for the serodiagnosis of amoebiasis. J Clin Microbiol 1979: 10: 778. 27 Williams JE, Robinson DM. Trans R Sot Trop Med Hyg. 1982; 76: 280. 28 Baveja UK. Warhurst DC, Voller A. Enzvmelinked immunosorbent assay in amoebiasis. Indian J Med Res 1984; 80: 529. 29 Das P. Pal S. Pal SC. Evaluation of the micro ELISA IHA and IFA techniques for serodiagnosis of amoebiasis. J Diarrh Dis Res 1984; 5: 238. 30. Feigner P. Serological diagnosis of extraintestlnal amoebiasis. a comparison of stick ELISA and other Immunological tests. Tropenmed Paras1tol 1977; 28: 491. 31. Trissl D. Immunology of Entumoeba histo/~rlco in human and animal hosts. Rev Infect Dis 1982; 4 1154 84. 32. Krupp IM. Antibody response in intestmal and extraintestinal amoebiasis. Am J Trop Med 1970; 19: 57 62.