203
The ENZYMEBA test: detection of intestinal Entamoeba histolytica infection by immuno-enzymatic detection of histolysain A. L. LUACES 1 *, T. PICO1* and A. J. BARRETT 2 ! 1 2
Departamento de Parasitologia, Instituto de Medicina Tropical 'Pedro Kouri' (IPK), Ciudad de la Habana, Cuba Department of Biochemistry, Strangeways Research Laboratory, Cambridge CB1 4RN, England
(Received 21 October 1991; revised 12 March 1992; accepted 12 March 1992)
SUMMARY We describe a new, improved test for the detection of intestinal infection by Entamoeba histolytica. The test depends upon immunoadsorption of the E. histolytica cysteine proteinase, histolysain, from faecal samples, and subsequent visual detection of the enzyme by a colour reaction. With samples from 200 volunteers, results agreed closely with those obtained by the conventional microscopic technique, and there were no false positive reactions with samples containing other parasites. The test is suitable for use either in the laboratory or in the field. Key words: Entamoeba histolytica, histolysain, histolysin, diagnosis, amoebiasis, ENZYMEBA.
INTRODUCTION
The methods currently employed for the detection of intestinal amoebiasis depend upon the microscopical examination of faeces. These procedures are tedious, laborious and expensive, and the results depend heavily on the skill of the microscopist. Even experienced technicians often mistake macrophages containing ingested red blood cells for Entamoeba histolytica trophozoites (Walsh, 1986). An additional difficulty is that because of the intermittent excretion of cysts, examination of a single faecal sample detects only 40-50 % of cases, and three or more separate samples have to be examined for the detection of 80—90 % of cases (Mathur & Kaur, 1973). Immunochemical methods have been developed for the detection of E. histolytica antigens in stools (Baumann & Gottstein, 1987 ; Grundy, 1982 ; Ungar, Yolken & Quinn, 1985), but the specificity of these methods depends directly upon the specificity of the antibodies used. For this reason, a number of 'false positives' and cross-reactions with other parasites are obtained. To avoid these problems, high dilution of the samples is usually necessary, which impairs sensitivity. The limitations of the procedures based on the
detection of circulating antibodies are well known. Most cases of intra-intestinal amoebiasis occur without a detectable rise in circulating antibodies. Only when invasive amoebiasis occurs, as in severe dysentery or extra-intestinal abscess, do antibody levels increase. Unfortunately, the levels then remain relatively high for a long period, making it difficult to distinguish a recent or current infection from an earlier one. Histolysain (EC 3.4.22.35) is the major cysteine proteinase of E. histolytica. Characteristics such as substrate specificity, p H optimum, molecular weight, amino-terminal amino acid sequence and action on natural substrates have already been described (Luaces & Barrett, 1988). (Histolysain was previously known as 'histolysin', but the name has been altered to be consistent with the convention that the names of cysteine proteinases end in ' -ain '). We have developed a highly sensitive and specific method for the detection of intestinal E. histolytica infection. The method which we call ENZYMEBA, is based on immuno-enzymatic detection of histolysain in faeces.
MATERIALS AND METHODS
Reagents * Present address: CODEME S.A., Calle Cero No. 114, Miramar, C. Habana, Cuba. f Reprint requests to Dr A. J. Barrett, Strangeways Research Laboratory, Worts Causeway, Cambridge CB1 4RN, England.
Parasitology (1992), 105, 203-205
Printed in Great Britain
Benzyloxycarbonyl - L - arginyl - L - arginine 2 - (4 methoxy)-naphthylamide (Z-Arg-Arg-Mna) was obtained from Bachem, Switzerland. All other chemicals were analytical grade from Sigma or Merck.
A. L. Luaces, T. Pico and A. J. Barrett
Anti-histolysain antibodies Histolysain was purified as described previously (Luaces & Barrett, 1988). Each of two New Zealand white rabbits was immunized as follows. The enzyme (50 pig) in 0-5 ml of phosphate-buffered saline (PBS) was emulsified with an equal volume of Freund's complete adjuvant, and injected intramuscularly. Seven subsequent injections were given at approximately 3-week intervals, with a similar amount of enzyme, but mixed with Freund's incomplete adjuvant, and injected subcutaneously. Blood was taken 1 and 2 weeks after the last injection. To obtain a crude immunoglobulin preparation serum was diluted 1 :2 in PBS, mixed with an equal volume of saturated ammonium sulphate and kept for 45 min at 4°C. The suspension was centrifuged (7000 £, 30 min) and the precipitate was made up to the initial serum volume with PBS. The precipitation procedure was repeated twice and the final solution of antibodies was dialysed against PBS, and stored at - 2 0 °C.
The ENZYMEBA procedure The wells of 96-well polyvinylchloride ELISA plates (Falcon) were filled with 100/il of a 1 :100 (v:v) dilution of anti-histolysain antibodies in coating buffer (100 mM sodium carbonate-bicarbonate buffer, pH 9-6) and the plates incubated at 4 °C for 16 h. The plates were washed with 005 % Tween 20 in PBS (PBS-T), and 150 fi\ of blocking buffer (5 % bovine serum albumin in PBS) was added and the plates incubated for 2 h at 37 °C. The plates were emptied and shaken dry over filter paper. Faecal samples were diluted 1:5 with 5 % calf serum in PBS-T and 100 fi\ of the dilution were placed in contact with the sensitized surface for 3 h at 4 °C so that histolysain present in the infected stools was captured by the specific antibody. The wells were then washed free of non-histolysain material, and the enzyme was revealed by reaction with 100/(I of 100 //M Z-Arg-Arg-Mna in 50 mM glycine/HCl buffer, pH 9-5, containing 2 mM L-cysteine for 16 h at 37 °C. The reaction was stopped by addition of 100/tl of 5 mM£>-chloromercuribenzoate, 22-5 //g/ml Fast garnet, 25 mM EDTA, 1 ° 0 Tween 20, pH 6-0 (Barrett, 1972). The visual observation of red colour was considered a positive result; negatives remained vellow.
Samples The procedure was tested on 200 volunteers. In the faeces of 50 of them, parasites other than E. histolytica were found microscopically and these samples were therefore used for the study of crossreactivitv.
204
Table 1. Comparison of the results of tests by microscopical examination and the ENZYMEBA procedure ENZYMEBA
Microscopical observation
Positive Negative
Positive
Negative
28 0
4 118
Table 2. Absence of cross-reactions with other parasites Results bv ENZYMEBA Observed parasite
Negative
Positive
Trichuris trichiura Giardia lamblia Endolimax nana Entamoeba coli Blastocystis hominis Strongyloides stercoralis Ascaris lumbricoides Enterobius vermicularis
33 10 34
0 0 0 0 0 0 0 0
12 30 3 5 12
Samples from 6 separate defaecations of the subjects were examined by the conventional microscopical method (examination of one unstained and one eosin-stained smear) and by the ENZYMEBA procedure. The microscopical observations were performed by an experienced microscopist. E. histolytica cysts were identified as refractile bodies measuring approximately 10 /im in size, containing an eccentric nucleus with central karyosome. RESULTS
Table 1 summarizes the results of the analyses of samples by microscopical observation and by the ENZYMEBA test. By comparison with microscopy, the ENZYMEBA test showed 87-5 ° 0 sensitivity (4/32) and 100% specificity (0/100). Of the ENZYMEBA-positive cases that were also found to be positive by microscopy, only 50 % were detected by the microscopist at the examination of the first sample; 25 % were detected after the examination of not less than 3 serial samples, and 25 % only after the sixth sample. This illustrates the greater sensitivity of the ENZYMEBA test. Faeces from patients not harbouring E. histolytica (after microscopical analysis of 6 serial samples), but with other intestinal parasites were then analysed by the ENZYMEBA test. The results are shown in Table 2. In no case was the result of the ENZYMEBA test positive. The positive cases received anti-amoebic treatment (diloxanide 500 mg/8 h during 10 days).
Detection of Entamoeba infection
205
Faeces from 8 of them were collected 10 days after the end of treatment and analysed by ENZYMEBA. All samples were then negative.
ditions, and is expected to be suitable not only for clinic diagnosis but also for massive screening. Since the present study was made with asymptomatic volunteers, however, the full evaluation of the test as a diagnostic tool has still to be done.
DISCUSSION
We thank Dr Pam Dando for immunization of the rabbits, and our colleagues for helpful discussions.
The results of the ENZYMEBA procedure correlate well with microscopical observations. There are indications that the 4 cases found negative by ENZYMEBA and positive by microscopy resulted from errors of the microscopist, because these cases were reported positive only in the first of the series of 6 tests. Also, faeces of these individuals were cultivated in Robinson's medium in order to try to isolate E. histolytica, but from none of them was growth of parasites observed. The ENZYMEBA test has the advantage of not being dependent on the skill of a microscopist. ENZYMEBA has high specificity, benefiting from the specificity of the antibody-enzyme interaction as well as that of the enzymic reaction. It is therefore possible to adjust the conditions of the test to achieve high sensitivity without interference by non-specific interactions from, say, overconcentrated samples. This makes it possible to perform a reliable identification of intestinal amoebiasis from a single stool sample (as compared with 6 or more necessary for the microscopical diagnosis). As ENZYMEBA is based on the detection of enzymatic activity, the presence of the intact parasite (cyst or trophozoite) in the sample is not required. Depending on the substrate used, ENZYMEBA can result in a colour reaction visible by naked eye or spectrophotometry, or a fluorescent product which can be read either with a fluorimeter or by observation of the fluorescence under ultraviolet light. ENZYMEBA is suitable for use under field con-
REFERENCES BARRETT, A. j . (1972). A new assay for cathepsin Bl and other thiol proteinases. Analytical Biochemistry 47, 280-93. BAUMANN, D. & GOTTSTEIN, B. (1987). A double-antibody
sandwich ELISA for the detection of Entamoeba histolytica antigen in stool samples of humans. Tropical Medicine and Parasitology 38, 81-5. GRUNDY, M. s. (1982). Preliminary observations using a multilayer ELISA method for the detection of Entamoeba histolytica trophozoite antigens in stool samples. Transactions of the Royal Society of Tropical Medicine and Hygiene 76, 396^-00. LUACES, A. L. & BARRETT, A. j . (1988). Affinity purification
and biochemical characterization of histolysin: the cysteine proteinase of Entamoeba histolytica. The Biochemical Journal 250, 903-9. MATHUR, T. N. & KAUR, j . (1973). The frequency of
excretion of cysts of Entamoeba histolytica in known cases of non-dysenteric amoebic colitis based on 21 stool examinations. Indian Journal of Medical Research 61, 330-3. UNGAR, B. L. P., YOLKEN, R. H. & QUINN, T. C. (1985). U s e
of monoclonal antibody in an enzyme immunoassay for the detection of Entamoeba histolytica in fecal specimens. American Journal of Tropical Medicine and Hygiene 34, 465-72. WALSH, j . A. (1986). Problems in recognition and diagnosis of amebiasis: estimation of the global magnitude of morbidity and mortality. Revue of Infectious Diseases 8, 228-38.
The ENZYMEBA test: detection of intestinal Entamoeba histolytica infection by immuno-enzymatic detection of histolysain A. L. LUACES 1 *, T. PICO1* and A. J. BARRETT 2 ! 1 2
Departamento de Parasitologia, Instituto de Medicina Tropical 'Pedro Kouri' (IPK), Ciudad de la Habana, Cuba Department of Biochemistry, Strangeways Research Laboratory, Cambridge CB1 4RN, England
(Received 21 October 1991; revised 12 March 1992; accepted 12 March 1992)
SUMMARY We describe a new, improved test for the detection of intestinal infection by Entamoeba histolytica. The test depends upon immunoadsorption of the E. histolytica cysteine proteinase, histolysain, from faecal samples, and subsequent visual detection of the enzyme by a colour reaction. With samples from 200 volunteers, results agreed closely with those obtained by the conventional microscopic technique, and there were no false positive reactions with samples containing other parasites. The test is suitable for use either in the laboratory or in the field. Key words: Entamoeba histolytica, histolysain, histolysin, diagnosis, amoebiasis, ENZYMEBA.
INTRODUCTION
The methods currently employed for the detection of intestinal amoebiasis depend upon the microscopical examination of faeces. These procedures are tedious, laborious and expensive, and the results depend heavily on the skill of the microscopist. Even experienced technicians often mistake macrophages containing ingested red blood cells for Entamoeba histolytica trophozoites (Walsh, 1986). An additional difficulty is that because of the intermittent excretion of cysts, examination of a single faecal sample detects only 40-50 % of cases, and three or more separate samples have to be examined for the detection of 80—90 % of cases (Mathur & Kaur, 1973). Immunochemical methods have been developed for the detection of E. histolytica antigens in stools (Baumann & Gottstein, 1987 ; Grundy, 1982 ; Ungar, Yolken & Quinn, 1985), but the specificity of these methods depends directly upon the specificity of the antibodies used. For this reason, a number of 'false positives' and cross-reactions with other parasites are obtained. To avoid these problems, high dilution of the samples is usually necessary, which impairs sensitivity. The limitations of the procedures based on the
detection of circulating antibodies are well known. Most cases of intra-intestinal amoebiasis occur without a detectable rise in circulating antibodies. Only when invasive amoebiasis occurs, as in severe dysentery or extra-intestinal abscess, do antibody levels increase. Unfortunately, the levels then remain relatively high for a long period, making it difficult to distinguish a recent or current infection from an earlier one. Histolysain (EC 3.4.22.35) is the major cysteine proteinase of E. histolytica. Characteristics such as substrate specificity, p H optimum, molecular weight, amino-terminal amino acid sequence and action on natural substrates have already been described (Luaces & Barrett, 1988). (Histolysain was previously known as 'histolysin', but the name has been altered to be consistent with the convention that the names of cysteine proteinases end in ' -ain '). We have developed a highly sensitive and specific method for the detection of intestinal E. histolytica infection. The method which we call ENZYMEBA, is based on immuno-enzymatic detection of histolysain in faeces.
MATERIALS AND METHODS
Reagents * Present address: CODEME S.A., Calle Cero No. 114, Miramar, C. Habana, Cuba. f Reprint requests to Dr A. J. Barrett, Strangeways Research Laboratory, Worts Causeway, Cambridge CB1 4RN, England.
Parasitology (1992), 105, 203-205
Printed in Great Britain
Benzyloxycarbonyl - L - arginyl - L - arginine 2 - (4 methoxy)-naphthylamide (Z-Arg-Arg-Mna) was obtained from Bachem, Switzerland. All other chemicals were analytical grade from Sigma or Merck.
A. L. Luaces, T. Pico and A. J. Barrett
Anti-histolysain antibodies Histolysain was purified as described previously (Luaces & Barrett, 1988). Each of two New Zealand white rabbits was immunized as follows. The enzyme (50 pig) in 0-5 ml of phosphate-buffered saline (PBS) was emulsified with an equal volume of Freund's complete adjuvant, and injected intramuscularly. Seven subsequent injections were given at approximately 3-week intervals, with a similar amount of enzyme, but mixed with Freund's incomplete adjuvant, and injected subcutaneously. Blood was taken 1 and 2 weeks after the last injection. To obtain a crude immunoglobulin preparation serum was diluted 1 :2 in PBS, mixed with an equal volume of saturated ammonium sulphate and kept for 45 min at 4°C. The suspension was centrifuged (7000 £, 30 min) and the precipitate was made up to the initial serum volume with PBS. The precipitation procedure was repeated twice and the final solution of antibodies was dialysed against PBS, and stored at - 2 0 °C.
The ENZYMEBA procedure The wells of 96-well polyvinylchloride ELISA plates (Falcon) were filled with 100/il of a 1 :100 (v:v) dilution of anti-histolysain antibodies in coating buffer (100 mM sodium carbonate-bicarbonate buffer, pH 9-6) and the plates incubated at 4 °C for 16 h. The plates were washed with 005 % Tween 20 in PBS (PBS-T), and 150 fi\ of blocking buffer (5 % bovine serum albumin in PBS) was added and the plates incubated for 2 h at 37 °C. The plates were emptied and shaken dry over filter paper. Faecal samples were diluted 1:5 with 5 % calf serum in PBS-T and 100 fi\ of the dilution were placed in contact with the sensitized surface for 3 h at 4 °C so that histolysain present in the infected stools was captured by the specific antibody. The wells were then washed free of non-histolysain material, and the enzyme was revealed by reaction with 100/(I of 100 //M Z-Arg-Arg-Mna in 50 mM glycine/HCl buffer, pH 9-5, containing 2 mM L-cysteine for 16 h at 37 °C. The reaction was stopped by addition of 100/tl of 5 mM£>-chloromercuribenzoate, 22-5 //g/ml Fast garnet, 25 mM EDTA, 1 ° 0 Tween 20, pH 6-0 (Barrett, 1972). The visual observation of red colour was considered a positive result; negatives remained vellow.
Samples The procedure was tested on 200 volunteers. In the faeces of 50 of them, parasites other than E. histolytica were found microscopically and these samples were therefore used for the study of crossreactivitv.
204
Table 1. Comparison of the results of tests by microscopical examination and the ENZYMEBA procedure ENZYMEBA
Microscopical observation
Positive Negative
Positive
Negative
28 0
4 118
Table 2. Absence of cross-reactions with other parasites Results bv ENZYMEBA Observed parasite
Negative
Positive
Trichuris trichiura Giardia lamblia Endolimax nana Entamoeba coli Blastocystis hominis Strongyloides stercoralis Ascaris lumbricoides Enterobius vermicularis
33 10 34
0 0 0 0 0 0 0 0
12 30 3 5 12
Samples from 6 separate defaecations of the subjects were examined by the conventional microscopical method (examination of one unstained and one eosin-stained smear) and by the ENZYMEBA procedure. The microscopical observations were performed by an experienced microscopist. E. histolytica cysts were identified as refractile bodies measuring approximately 10 /im in size, containing an eccentric nucleus with central karyosome. RESULTS
Table 1 summarizes the results of the analyses of samples by microscopical observation and by the ENZYMEBA test. By comparison with microscopy, the ENZYMEBA test showed 87-5 ° 0 sensitivity (4/32) and 100% specificity (0/100). Of the ENZYMEBA-positive cases that were also found to be positive by microscopy, only 50 % were detected by the microscopist at the examination of the first sample; 25 % were detected after the examination of not less than 3 serial samples, and 25 % only after the sixth sample. This illustrates the greater sensitivity of the ENZYMEBA test. Faeces from patients not harbouring E. histolytica (after microscopical analysis of 6 serial samples), but with other intestinal parasites were then analysed by the ENZYMEBA test. The results are shown in Table 2. In no case was the result of the ENZYMEBA test positive. The positive cases received anti-amoebic treatment (diloxanide 500 mg/8 h during 10 days).
Detection of Entamoeba infection
205
Faeces from 8 of them were collected 10 days after the end of treatment and analysed by ENZYMEBA. All samples were then negative.
ditions, and is expected to be suitable not only for clinic diagnosis but also for massive screening. Since the present study was made with asymptomatic volunteers, however, the full evaluation of the test as a diagnostic tool has still to be done.
DISCUSSION
We thank Dr Pam Dando for immunization of the rabbits, and our colleagues for helpful discussions.
The results of the ENZYMEBA procedure correlate well with microscopical observations. There are indications that the 4 cases found negative by ENZYMEBA and positive by microscopy resulted from errors of the microscopist, because these cases were reported positive only in the first of the series of 6 tests. Also, faeces of these individuals were cultivated in Robinson's medium in order to try to isolate E. histolytica, but from none of them was growth of parasites observed. The ENZYMEBA test has the advantage of not being dependent on the skill of a microscopist. ENZYMEBA has high specificity, benefiting from the specificity of the antibody-enzyme interaction as well as that of the enzymic reaction. It is therefore possible to adjust the conditions of the test to achieve high sensitivity without interference by non-specific interactions from, say, overconcentrated samples. This makes it possible to perform a reliable identification of intestinal amoebiasis from a single stool sample (as compared with 6 or more necessary for the microscopical diagnosis). As ENZYMEBA is based on the detection of enzymatic activity, the presence of the intact parasite (cyst or trophozoite) in the sample is not required. Depending on the substrate used, ENZYMEBA can result in a colour reaction visible by naked eye or spectrophotometry, or a fluorescent product which can be read either with a fluorimeter or by observation of the fluorescence under ultraviolet light. ENZYMEBA is suitable for use under field con-
REFERENCES BARRETT, A. j . (1972). A new assay for cathepsin Bl and other thiol proteinases. Analytical Biochemistry 47, 280-93. BAUMANN, D. & GOTTSTEIN, B. (1987). A double-antibody
sandwich ELISA for the detection of Entamoeba histolytica antigen in stool samples of humans. Tropical Medicine and Parasitology 38, 81-5. GRUNDY, M. s. (1982). Preliminary observations using a multilayer ELISA method for the detection of Entamoeba histolytica trophozoite antigens in stool samples. Transactions of the Royal Society of Tropical Medicine and Hygiene 76, 396^-00. LUACES, A. L. & BARRETT, A. j . (1988). Affinity purification
and biochemical characterization of histolysin: the cysteine proteinase of Entamoeba histolytica. The Biochemical Journal 250, 903-9. MATHUR, T. N. & KAUR, j . (1973). The frequency of
excretion of cysts of Entamoeba histolytica in known cases of non-dysenteric amoebic colitis based on 21 stool examinations. Indian Journal of Medical Research 61, 330-3. UNGAR, B. L. P., YOLKEN, R. H. & QUINN, T. C. (1985). U s e
of monoclonal antibody in an enzyme immunoassay for the detection of Entamoeba histolytica in fecal specimens. American Journal of Tropical Medicine and Hygiene 34, 465-72. WALSH, j . A. (1986). Problems in recognition and diagnosis of amebiasis: estimation of the global magnitude of morbidity and mortality. Revue of Infectious Diseases 8, 228-38.
