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Use of the ProSpecT® microplate enzyme immunoassay for the detection of pathogenic and non-pathogenic Entamoeba histolytica in faecal specimens
TRANSACTIONS OFTHE ROYAL SOCIETYOFTROPICALMEDICINEANDHYGIENE(1996)90,248-249
Use of the ProSpecTB microplate enzyme immunoassay for the detection of pathogenic and non-pathogenic Entamoeba histo/ytica in faecal specimens Shiou-Jeng Ong, Mei-Ying Cheng, Kuo-Hui Liu and Chi-Byi Horng Division of Malariology and ParasitoloB, Institute of Preventive Medicine, Department of Health, Executive Yuan, Taiwan, Republic of China Abstract
A commercial microplate enzyme immunoassay (ProSpecTB EIA; Alexon Inc., Sunnyvale, CA 94089, USA) was compared with conventional microscopy for the diagnosis of Entamoeba histolytica infection. Using specimens known to be infected, the sensitivity of the ProSpecTB EIA was 78% and its specificity was 99%. No cross reaction with other intestinal parasiteswas observed.The ProSpecTB EIA and conventional microscopy (using merthiolate-iodine-formaldehyde direct wet mounts and concentration techniques) were then used to detect E. histolytica infections in 431 patients in a mental hospital in Taiwan. Using single stool specimens, microscopy detected infection in 10.9%of the patients, compared with 16.9%detected by ProSpecTB EIA. The latter method was simple and quick, but more expensive, and could be used to complement microscopy if a prompt diagnosis is desired clinically. However, ProSpecTB EIA cannot differentiate between pathogenic E. histo&ica and non-pathogenic strains (= E. d&par), which limits its usefulness. Keywords:amoebiasis, Enramoeba histolytica, diagnosis,enzyme immunoassay Introduction
The laboratory diagnosis of intestinal amoebiasis has mainlv denended on findina Entamoeba hktolvtica* cvsts or trophoioites in faecal sp&imens by microscopical”examination. However, careful collection of faeces,the use of adequateexamination methods, and experience on the part of the microscopist are required because amoebic cysts may mimic polymorphonuclear leucocytes and macrophagesmay be mistaken for amoebic trophozoites in wet nrenarations (BRUCKNER. 1992). Moreover. the sporadic excretion of organisms’(STAlciM, 1957) makes serial collection of 3 faecal specimens necessaryin order to reach 75% sensitivity (LUACES et al., 1993).Therefore, there is a need for better examination methods with high sensitivity and specificity. LUACES et al. (1992) developed the ENZYMEBA test for intestinal E. histolytica infection, an enzyme immunoassay (EIA) to detect histolysain in stools. The test had 875% sensitivity and 100% specificity (LUACES et al., 1993). Another EIA, the ProSpecTB microplate immunoassay detects E. histolytica specific antigen (EHSA) in faecal specimens.We have evaluated the feasibility of using this commercial kit for clinical diagnosis by comparing conventional microscopy of stool specimenswith the ProSpecTB EIA. Materials Subjects
and Methods
In-patients of a mental hospital in eastern Taiwan were screenedby faecal microscopy. Faecesof confirmed positive and negative caseswere then tested using the ProSpecTB EIA. Confirmation of positive cases
Following examination of direct wet mounts or concentrated specimens of faeces preserved in merthiolate-iodine-formaldehyde (MIF) (SAPERO& LAWLESS, 1953), positive results were all confirmed by Heidenhain’s iron haematoxylin staining (MELVIN & BROOKE, 1982). Confirmation of negative cases
After preliminary screening, 250 in-patients with no
E. histolytica cyst or trophozoite detected in their faeces
were further examined as follows. Three faecal specimens were collected at 2 d intervals and examined microscopically as described above. Patients with 3 consecutive negative results were subjected to the *Throughout this paper,the nameE. histolytica is used to refer to both uathoaenicand non-nathoaenicstrainsof the oarasite; someworkers-regardthe latier as> separatespecies,2. dispa; (seeDiamond.L. S. & Clark.C. G., 1993: 7ournal of Eukarvotic kimobiology, 40, 340-344) [editor].
’
-
.
ProSpecTB EIA. If the result was positive, another 3 serial faecal specimens were examined microscopically for confirmation of the infection. ProSpecTB microplate immunoassay
As recommended by the manufacturer (Alexon Inc., 1190 Borregos Avenue? Sunnyvale, CA 94089, USA) faecal specimens were either stored at 2-8°C and tested within 48 h or frozen at -20°C to -70°C and tested within 1 week after collection. Liquid stools were vortexed vigorously to ensure uniform distribution of EHSA. To 1 g of formed or semiformed specimens, 1 mL of sterile water was added and the specimens were emulsified thoroughly. One mL of the homogenized faecal suspension was diluted with one mL of specimen dilution buffer (included in the kit). The rest of the procedures were performed as recommended by the manufacturer. The-test was done in microplate wells coated with rabbit anti-EHSA antibodies. After 60 min incubation, the reaction was completed by adding peroxidase-labelled rabbit anti-EHSA and 3: 3: 5: 5’ -tetramethvl benzidine chromoaen. in that order,‘with thorough washing in between. Positive results were indicated by the development of a yellow colour at the end of the reaction; test results were read visually. Positive and negative controls were included in each test. Survey of E. histolyticaprevalence Taking advantage of a preventive survey conducted during the sorina of 1995 at a mental hosnital in southern T~iwan,~fae&l specimens of the patients were examined by both conventional microscopy (including direct wet mounts and concentration of MIF specimens) and the ProSnecTB EIA. The Y’ test was annlied to assess _~ the difference in infection rates. Results Sensitivity and specificity
Among 74 microscopically confirmed cases,58 were positive in the ProSpecTB EIA, a sensitivity of 78%; 190 ‘negative’ individuals were subjected to the ProSpecTB EIA and all except 11 of them gave a negative result. Microscopical examination of another 3 faecal specimens from each of these 11 individuals revealed cysts or trophozoites of E. histolytica in 10 of them, indicating that the ProSpecTB EIA had a specificity of at least 99%. Cross reactivity
Among 180 individuals microscopically free of E. histolytica, 61 were infected with at least one other parasite but no cross reaction was observed. The other infections included Entamoeba coli (2), Entamoeba hartmanni (l),
Endolimax nana (23), Giardia duodenalis (= G. lamblia)
PROSPECT@ IMMUNOASSAY FOR AMOEBIASIS (6), Trichuris
trichiura
(4), Enterobius
vermicularis
249
(l), and-
Clonorchis sinensis (2). Survey
The results of the comparative survey of 431 patients (Table) indicated that ProSpecT@ EIA could find more positive casesthan conventional microscopy. Table. Comparison of methods for detection of Entumoeba histdytica in single stool specimens from 431 mental hospital patients
Method MIFa EIA”
No. positive 47 73
Infection rate (%) 10.9c 16.9~
aBoth direct wet mounts and concentrated specimens preserved with merthiolate-iodine-formaldehyde. bProSpecT@microplate enzyme immunoassay. CThe difference between infection rates was significant (~2=7.19,P
Discussion
According to the information provided by the manufacturer, EHSA is produced during multiplication of both pathogenic and non-pathogenic strains of E. histolytica. The ProSpecTB EIA can detect as little as 40 ng/mL of EHSA in stools, so that the intermittent passage of cysts or trophozoites in faeces is of no consequence. Considering the sensitivity of the ProSpecT@ EIA, to be 78%, it can be calculated that the true prevalence of E. histolytica in the hospital was about 22%. This suggests that microscopical examination of single samples detected only 50% of the infections, which is consistent with the result reported by LUACES et al. (1993). Using the MIF technique, both wet mounts and concentrated specimens must be examined microscopically, becausethe former are more effective in detecting trophozoites and the latter in detecting cysts (ONG et al., 1995). The ProSpecTB EIA is therefore less laborious than conventional microscopy and misidentification of the organisms, which has been reported for microscopy (KROGSTAD et al., 1978), would not occur with ProSuecTB EIA. The techniaue is simnle and has hiah specihcity, 2 major advantages, but iis cost is a drawback. Also, non-pathogenic E. histolytica (=E. dispar) cannot be distinguished from pathogenic strains by the EIA, a further limitation of its usefulness. To eliminate misdiagnosis by morphological observation, positive caseswere all confirmed by use of permanent stain preparations in this study. For the confirmation of negative cases, three stools collected at 2 d intervals for one to 2 rounds might overcome the problem of cyclic excretion of the organisms.
One patient, apparently confirmed as being uninfected by 6 successive negative stool examinations by microsconv. save a uositive result in the ProSnecTB EIA. It ispo&ible that EHSA might be present-in the faeces of patients suffering from hepatic amoebiasis; however, serological testing (indirect haemagglutination) gave a negative result in this case.Further examinations of this individual would be necessary conclusively to exclude the possibility of infection, due to the insensitivity of microscopical examination. In conclusion, the sensitivity and specificity of the ProSpecTB EIA are much higher than conventional microscopy. To complement morphological observation of organisms, the EIA can be used for detection of EHSA in faecal specimens if an immediate laboratory diagnosis of intestinal amoebiasisis required clinically. Acknowledgements We thank S. Y. Lee. L. P. Chow. T. L. Chene and C. F. Lan for their skilful technical assistance and Dr G. R. Wang for helpful discussion. The support of Taiwan Provincial Yu-li Mental Hospital (TPYMH) and Taiwan Provincial Health Department, and the assistance of the staffs of TPYMH and the Health Bureau of Tainan County in collecting faecal specimens, are gratefully acknowledged. Special thanks are due to Professor J. D. Smyth for reading the manuscript.
References Bruckner, D. A. (1992). Amebiasis. Clinica Microbiolom Reviews, 5; 356369. ’ Krogstad, D. J., Spencer, H. C., Healy, G. R., Gleason, N. N., Sexton, D. J. & Herron, C. A. (1978). Amebiasis: epidemiologic studies in the United States, 1971-1974. Annals of Internal Medicine, g&89-97. Luaces, A. L., Pica, T. & Barrett, A. J. (1992). The ENZYMEBA test: detection of intestinal Entamoeba histoiytica infection by immuno-enzymatic detection of histolysain. Parasitology, 105, 203-205. Luaces, A. L., Osorio, L. M. & Barrett, A. J. (1993). A new test for infection by Entamoeba hiswlytica. Parasitology Today, 9, 69-71. Melvin, D. M. & Brooke, M. M. (1982). Laboratory Proceduresfm the Diagnosis of Intestmal Parasites, 3rd edition. Georgia: US Department of Health and Human Services, Public Health Service, Centers for Disease Control, pp. 84-86, 108-109, 120-122. Ong, S. J., Cheng, M. Y., Liu, K. H., Lee, S. Y., Chow, L. P., Cheng, T. L., Lan, C. F., Chang, Y. S. & Horng, C. B. (1995). Parasitic infection in a psychiatric institution in Taiwan, with special reference to Entamoeba histolytica. Epidemiology Bulletin (Department of Health, Republic of China), 11, 107-110. [English edition.] Sapero, J. J. & Lawless, D. K. (1953). The ‘MIF’ stain-preservation technique for the identification of intestinal protozoa. AmericanJournal of Tropical Medicine and Hygiene, 2,613-619. Stamm, W. P. (1957). The laboratory diagnosis of clinical amoebiasis. Transactions of the Royal Soci&y of Tropical Medicine and Hygiene, 51,306-312. 23 October 199.5; revised 19 December 1995; acceptedfor publication 20 December199.5
Received
Use of the ProSpecTB microplate enzyme immunoassay for the detection of pathogenic and non-pathogenic Entamoeba histo/ytica in faecal specimens Shiou-Jeng Ong, Mei-Ying Cheng, Kuo-Hui Liu and Chi-Byi Horng Division of Malariology and ParasitoloB, Institute of Preventive Medicine, Department of Health, Executive Yuan, Taiwan, Republic of China Abstract
A commercial microplate enzyme immunoassay (ProSpecTB EIA; Alexon Inc., Sunnyvale, CA 94089, USA) was compared with conventional microscopy for the diagnosis of Entamoeba histolytica infection. Using specimens known to be infected, the sensitivity of the ProSpecTB EIA was 78% and its specificity was 99%. No cross reaction with other intestinal parasiteswas observed.The ProSpecTB EIA and conventional microscopy (using merthiolate-iodine-formaldehyde direct wet mounts and concentration techniques) were then used to detect E. histolytica infections in 431 patients in a mental hospital in Taiwan. Using single stool specimens, microscopy detected infection in 10.9%of the patients, compared with 16.9%detected by ProSpecTB EIA. The latter method was simple and quick, but more expensive, and could be used to complement microscopy if a prompt diagnosis is desired clinically. However, ProSpecTB EIA cannot differentiate between pathogenic E. histo&ica and non-pathogenic strains (= E. d&par), which limits its usefulness. Keywords:amoebiasis, Enramoeba histolytica, diagnosis,enzyme immunoassay Introduction
The laboratory diagnosis of intestinal amoebiasis has mainlv denended on findina Entamoeba hktolvtica* cvsts or trophoioites in faecal sp&imens by microscopical”examination. However, careful collection of faeces,the use of adequateexamination methods, and experience on the part of the microscopist are required because amoebic cysts may mimic polymorphonuclear leucocytes and macrophagesmay be mistaken for amoebic trophozoites in wet nrenarations (BRUCKNER. 1992). Moreover. the sporadic excretion of organisms’(STAlciM, 1957) makes serial collection of 3 faecal specimens necessaryin order to reach 75% sensitivity (LUACES et al., 1993).Therefore, there is a need for better examination methods with high sensitivity and specificity. LUACES et al. (1992) developed the ENZYMEBA test for intestinal E. histolytica infection, an enzyme immunoassay (EIA) to detect histolysain in stools. The test had 875% sensitivity and 100% specificity (LUACES et al., 1993). Another EIA, the ProSpecTB microplate immunoassay detects E. histolytica specific antigen (EHSA) in faecal specimens.We have evaluated the feasibility of using this commercial kit for clinical diagnosis by comparing conventional microscopy of stool specimenswith the ProSpecTB EIA. Materials Subjects
and Methods
In-patients of a mental hospital in eastern Taiwan were screenedby faecal microscopy. Faecesof confirmed positive and negative caseswere then tested using the ProSpecTB EIA. Confirmation of positive cases
Following examination of direct wet mounts or concentrated specimens of faeces preserved in merthiolate-iodine-formaldehyde (MIF) (SAPERO& LAWLESS, 1953), positive results were all confirmed by Heidenhain’s iron haematoxylin staining (MELVIN & BROOKE, 1982). Confirmation of negative cases
After preliminary screening, 250 in-patients with no
E. histolytica cyst or trophozoite detected in their faeces
were further examined as follows. Three faecal specimens were collected at 2 d intervals and examined microscopically as described above. Patients with 3 consecutive negative results were subjected to the *Throughout this paper,the nameE. histolytica is used to refer to both uathoaenicand non-nathoaenicstrainsof the oarasite; someworkers-regardthe latier as> separatespecies,2. dispa; (seeDiamond.L. S. & Clark.C. G., 1993: 7ournal of Eukarvotic kimobiology, 40, 340-344) [editor].
’
-
.
ProSpecTB EIA. If the result was positive, another 3 serial faecal specimens were examined microscopically for confirmation of the infection. ProSpecTB microplate immunoassay
As recommended by the manufacturer (Alexon Inc., 1190 Borregos Avenue? Sunnyvale, CA 94089, USA) faecal specimens were either stored at 2-8°C and tested within 48 h or frozen at -20°C to -70°C and tested within 1 week after collection. Liquid stools were vortexed vigorously to ensure uniform distribution of EHSA. To 1 g of formed or semiformed specimens, 1 mL of sterile water was added and the specimens were emulsified thoroughly. One mL of the homogenized faecal suspension was diluted with one mL of specimen dilution buffer (included in the kit). The rest of the procedures were performed as recommended by the manufacturer. The-test was done in microplate wells coated with rabbit anti-EHSA antibodies. After 60 min incubation, the reaction was completed by adding peroxidase-labelled rabbit anti-EHSA and 3: 3: 5: 5’ -tetramethvl benzidine chromoaen. in that order,‘with thorough washing in between. Positive results were indicated by the development of a yellow colour at the end of the reaction; test results were read visually. Positive and negative controls were included in each test. Survey of E. histolyticaprevalence Taking advantage of a preventive survey conducted during the sorina of 1995 at a mental hosnital in southern T~iwan,~fae&l specimens of the patients were examined by both conventional microscopy (including direct wet mounts and concentration of MIF specimens) and the ProSnecTB EIA. The Y’ test was annlied to assess _~ the difference in infection rates. Results Sensitivity and specificity
Among 74 microscopically confirmed cases,58 were positive in the ProSpecTB EIA, a sensitivity of 78%; 190 ‘negative’ individuals were subjected to the ProSpecTB EIA and all except 11 of them gave a negative result. Microscopical examination of another 3 faecal specimens from each of these 11 individuals revealed cysts or trophozoites of E. histolytica in 10 of them, indicating that the ProSpecTB EIA had a specificity of at least 99%. Cross reactivity
Among 180 individuals microscopically free of E. histolytica, 61 were infected with at least one other parasite but no cross reaction was observed. The other infections included Entamoeba coli (2), Entamoeba hartmanni (l),
Endolimax nana (23), Giardia duodenalis (= G. lamblia)
PROSPECT@ IMMUNOASSAY FOR AMOEBIASIS (6), Trichuris
trichiura
(4), Enterobius
vermicularis
249
(l), and-
Clonorchis sinensis (2). Survey
The results of the comparative survey of 431 patients (Table) indicated that ProSpecT@ EIA could find more positive casesthan conventional microscopy. Table. Comparison of methods for detection of Entumoeba histdytica in single stool specimens from 431 mental hospital patients
Method MIFa EIA”
No. positive 47 73
Infection rate (%) 10.9c 16.9~
aBoth direct wet mounts and concentrated specimens preserved with merthiolate-iodine-formaldehyde. bProSpecT@microplate enzyme immunoassay. CThe difference between infection rates was significant (~2=7.19,P
Discussion
According to the information provided by the manufacturer, EHSA is produced during multiplication of both pathogenic and non-pathogenic strains of E. histolytica. The ProSpecTB EIA can detect as little as 40 ng/mL of EHSA in stools, so that the intermittent passage of cysts or trophozoites in faeces is of no consequence. Considering the sensitivity of the ProSpecT@ EIA, to be 78%, it can be calculated that the true prevalence of E. histolytica in the hospital was about 22%. This suggests that microscopical examination of single samples detected only 50% of the infections, which is consistent with the result reported by LUACES et al. (1993). Using the MIF technique, both wet mounts and concentrated specimens must be examined microscopically, becausethe former are more effective in detecting trophozoites and the latter in detecting cysts (ONG et al., 1995). The ProSpecTB EIA is therefore less laborious than conventional microscopy and misidentification of the organisms, which has been reported for microscopy (KROGSTAD et al., 1978), would not occur with ProSuecTB EIA. The techniaue is simnle and has hiah specihcity, 2 major advantages, but iis cost is a drawback. Also, non-pathogenic E. histolytica (=E. dispar) cannot be distinguished from pathogenic strains by the EIA, a further limitation of its usefulness. To eliminate misdiagnosis by morphological observation, positive caseswere all confirmed by use of permanent stain preparations in this study. For the confirmation of negative cases, three stools collected at 2 d intervals for one to 2 rounds might overcome the problem of cyclic excretion of the organisms.
One patient, apparently confirmed as being uninfected by 6 successive negative stool examinations by microsconv. save a uositive result in the ProSnecTB EIA. It ispo&ible that EHSA might be present-in the faeces of patients suffering from hepatic amoebiasis; however, serological testing (indirect haemagglutination) gave a negative result in this case.Further examinations of this individual would be necessary conclusively to exclude the possibility of infection, due to the insensitivity of microscopical examination. In conclusion, the sensitivity and specificity of the ProSpecTB EIA are much higher than conventional microscopy. To complement morphological observation of organisms, the EIA can be used for detection of EHSA in faecal specimens if an immediate laboratory diagnosis of intestinal amoebiasisis required clinically. Acknowledgements We thank S. Y. Lee. L. P. Chow. T. L. Chene and C. F. Lan for their skilful technical assistance and Dr G. R. Wang for helpful discussion. The support of Taiwan Provincial Yu-li Mental Hospital (TPYMH) and Taiwan Provincial Health Department, and the assistance of the staffs of TPYMH and the Health Bureau of Tainan County in collecting faecal specimens, are gratefully acknowledged. Special thanks are due to Professor J. D. Smyth for reading the manuscript.
References Bruckner, D. A. (1992). Amebiasis. Clinica Microbiolom Reviews, 5; 356369. ’ Krogstad, D. J., Spencer, H. C., Healy, G. R., Gleason, N. N., Sexton, D. J. & Herron, C. A. (1978). Amebiasis: epidemiologic studies in the United States, 1971-1974. Annals of Internal Medicine, g&89-97. Luaces, A. L., Pica, T. & Barrett, A. J. (1992). The ENZYMEBA test: detection of intestinal Entamoeba histoiytica infection by immuno-enzymatic detection of histolysain. Parasitology, 105, 203-205. Luaces, A. L., Osorio, L. M. & Barrett, A. J. (1993). A new test for infection by Entamoeba hiswlytica. Parasitology Today, 9, 69-71. Melvin, D. M. & Brooke, M. M. (1982). Laboratory Proceduresfm the Diagnosis of Intestmal Parasites, 3rd edition. Georgia: US Department of Health and Human Services, Public Health Service, Centers for Disease Control, pp. 84-86, 108-109, 120-122. Ong, S. J., Cheng, M. Y., Liu, K. H., Lee, S. Y., Chow, L. P., Cheng, T. L., Lan, C. F., Chang, Y. S. & Horng, C. B. (1995). Parasitic infection in a psychiatric institution in Taiwan, with special reference to Entamoeba histolytica. Epidemiology Bulletin (Department of Health, Republic of China), 11, 107-110. [English edition.] Sapero, J. J. & Lawless, D. K. (1953). The ‘MIF’ stain-preservation technique for the identification of intestinal protozoa. AmericanJournal of Tropical Medicine and Hygiene, 2,613-619. Stamm, W. P. (1957). The laboratory diagnosis of clinical amoebiasis. Transactions of the Royal Soci&y of Tropical Medicine and Hygiene, 51,306-312. 23 October 199.5; revised 19 December 1995; acceptedfor publication 20 December199.5
Received