JOURNAL OF CLINICAL MICROBIOLOGY, July 1992, p. 1783-1786
Vol. 30, No. 7
0095-1137/92/071783-04$02.00/0 Copyright ©D 1992, American Society for Microbiology
Evaluation of the Bead Enzyme-Linked Immunosorbent Assay for Detection of Cholera Toxin Directly from Stool Specimens T. RAMAMURTHY,1 S. K. BHATTACHARYA,1 YOSHIHIKO UESAKA,2 KAZUKI HORIGOME,2 MOUSUMI PAUL,1 D. SEN,' S. C. PAL,1 TAE TAKEDA,3 YOSHIFUMI TAKEDA,4 AND G. BALAKRISH NAIR"* National Institute of Cholera and Enteric Diseases, P-33, CIT Scheme XM, Beliaghata, Calcutta 700 010, India,' and Institute for Diagnostic Reagents, Nissui Pharmaceutical Co., Yuki, Ibaragi 307, 2 Department of Infectious Diseases Research, National Children's Medical Research Center,
Tokyo 154,3 and Department of Microbiology, Faculty of Medicine, Kyoto University, Kyoto 606, 4Japan Received 26 November 1991/Accepted 24 April 1992
A highly sensitive bead enzyme-linked immunosorbent assay (bead ELISA) for detection of cholera toxin (CT) was evaluated for direct detection of CT from stool specimens of patients with acute secretory diarrhea. Of the 75 stool samples examined, 59 yielded biochemically and serologically confirmed strains of Vibrio cholerae 01. The bead ELISA was positive for CT in stool supernatants in 50 (84.7%) of the 59 samples from which V. cholerae 01 was isolated. In addition, the bead ELISA was positive for three stool specimens which were negative by culture. The free CT present in 48 of the 50 stool samples positive by culture for V. cholerae 01 and for CT by bead ELISA was completely absorbed by anti-CT immunoglobulin G. All of the 59 strains of V. chokrae 01 biotype eltor isolated in this study produced in vitro CT. The concentration of CT present in the bead ELISA-positive stool samples ranged between 26 pg/mI and >100 ng/ml. This evaluation study demonstrates that the bead ELISA is a sensitive and simple method for direct detection of CT in nonsterile stool samples, and we recommend routine use of this assay for detection of CT in stool samples and culture supernatants in clinical and reference laboratories.
Over the past few years, the enzyme-linked immunosorbent assay (ELISA) has become the method of choice for detection of bacterial enterotoxins in research and reference laboratories of both developed and developing countries. This is because the ELISA is a rapid, economical, and sensitive assay that is easy to perform and interpret and is convenient to use for both large and small numbers of specimens. A sandwich ELISA (17) or a receptor-based Gm1 ganglioside ELISA (13) or minor modifications of the above two ELISAs are currently in vogue for detection of cholera toxin (CT). However, the ELISA in its current format of using microtiter plates precludes the use of this technique in peripheral less-sophisticated laboratories of less-developed countries, where quality microtiter plates are an expensive and not readily available commodity. To further augment simplicity, Oku et al. (8) have developed an alternative highly sensitive ELISA which used 6-mm-diameter polystyrene beads as the solid phase (bead ELISA) and can be performed in 3.5 h in standard laboratory test tubes for detection of CT (the bead ELISA can detect more than 60 pg of CT per ml) and other bacterial protein toxins. Detection of CT directly from stool specimens of patients with acute diarrhea has remained an attractive but hitherto unaccomplished proposition. The ability to detect CT directly in stool samples would not only circumvent the time needed for primary isolation and secondary growth of broth cultures but would also enable, perhaps, the most rapid diagnosis of the infection. Preliminary attempts, with various degrees of success, have previously been made by using the Y1 adrenal cell culture assay (2, 6, 7), ELISA (6, 7), and counterimmunoelectrophoresis (7) for detection of CT and *
Escherichia coli heat-labile enterotoxin (LT) activity in human diarrheic stools. In view of the picogram sensitivity of the bead ELISA, we decided to conduct a systematic study to evaluate the performance of this assay to detect CT directly from stool specimens of patients with acute secretory diarrhea.
MATERIALS AND METHODS Stool specimens for evaluation. From July to November 1989, 75 stool samples from patients with acute watery diarrhea admitted to the Infectious Diseases Hospital, Calcutta, India, were included in this study. Stool samples of patients who had not received antibiotics were collected upon admission to the hospital by using sterile catheters in clean, sterile screw-cap bottles and transported to the laboratory within an hour. On receipt at the laboratory, a portion of each sample was immediately processed for bacteriological examination, the remainder was transferred into fresh sterile tubes and centrifuged at 8,000 x g for 15 min at 4°C, and the clear supernatant was stored at -20°C until use. Control specimens. To assess the specificity of the CT bead ELISA, an additional set of 40 stool samples from patients with watery diarrhea from whom V. cholerae 01 could not be isolated were examined subsequent to performance of the evaluation study. Bacteriological studies. Each stool sample was processed for V. cholerae, shigellae, salmonellae, campylobacters, enterotoxigenic Escherichia coli (ETEC), and other pathogenic vibrios by using previously published methods (3, 11, 15). Direct detection of CT in stool by bead ELISA. Stool specimens obtained from all of the patients were assayed directly for free fecal CT by using the bead ELISA reported
Corresponding author. 1783
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previously (8, 16). To determine the minimum detection ability of the bead ELISA used in this study, we performed the assay (described below) by using various dilutions of purified CT (lot CVX-034; List Biological Laboratories, Campbell, Calif.), and on the basis of the results a standard curve was plotted. The above-described dilutions of CT were also run concurrently as positive controls whenever a batch of stool samples or cell-free culture supernatants were assayed by the bead ELISA. To assess the extent of crossreactivity of the CT bead ELISA with LT of E. coli, various dilutions of purified LT prepared as described previously (14) were also assayed. Each stool sample was assayed in duplicate on two different occasions by one group of investigators. To check for reproducibility of results, a second group of investigators repeated the assay 6 months later on all 75 stool samples in a blind fashion. In addition to the neat stool sample, the second group of investigators also assayed various dilutions (1:20, 1:50, 1:200, 1:500, 1:2,000, and 1:4,000) of each stool sample by the bead ELISA. The findings of the two groups were compared and checked for discordant results. To perform the assay, 0.25 ml of each neat or diluted stool sample was mixed with an equal volume of 10 mM bicine [N,N-bis(2-hydroxyethyl)glycine; Sigma Chemical Co., St. Louis, Mo.] buffer (pH 8.3) containing 0.1 M NaCl, 0.1% bovine serum albumin, and 0.1% sodium azide in glass test tubes (13 by 10 mm) and an anti-CT immunoglobulin G (IgG; raised by immunizing rabbits as described in detail by Honda et al. [4])-coated (50 ,ug/ml) bead was introduced into the mixture, which was incubated at 37°C for 1 h in a water bath and subsequently washed twice with double-distilled water. The bead was then incubated at 37°C for 1 h with 0.5 ml of Fab'-horseradish peroxidase conjugate (400 ng/ml) in 10 mM phosphate buffer containing 100 mM NaCl (pH 7.0) and 2% bovine serum albumin. After incubation, the bead was washed three times in double-distilled water and transferred to a fresh test tube. Peroxidase activity was determined by incubating the bead at 30°C for 1 h with 0.6 ml of 28 mM 3,3',5,5'-tetramethylbenzidine in 0.1 M sodium acetate buffer (pH 5.5) containing 2 mM EDTA, followed by addition of 0.2 ml of 0.02% H202. The reaction was stopped by adding 0.2 ml of 4 N H2SO4, and the resulting yellow color intensity was measured at 450 nm by using a spectrophotometer (DU-64; Beckman Instruments, Inc., Fullerton, Calif.). Absorption assay using anti-CT IgG. An absorption assay using the bead ELISA was performed to determine whether the free CT present in stool samples could be absorbed with anti-CT IgG. For this assay, a neat CT-positive stool sample or a dilution of a CT-positive stool specimen exhibiting an optical density (OD) below 1.2 was selected. In the absorption assay, a neat CT-positive stool sample or a selected dilution of each CT-positive stool sample was preincubated at 37°C for 1 h with 10 ,ug of rabbit anti-CT IgG per ml. From earlier experiments it was determined that 10 ,ug of anti-CT IgG per ml can completely absorb 500 ng of pure CT per ml. Subsequently, the coated beads were introduced into 0.5 ml of the above-described incubations and the bead ELISA was performed as detailed above. Cell-free supernatants. V. cholerae 01 and non-O1 strains recovered in this study were grown in yeast extract peptone water employing stationary (for 5 h) and shake (for 19 h) culture phases at 37°C as recommended by Iwanaga and Kuyyakanond (5) for optimal in vitro CT production. Cellfree culture supernatants were obtained by centrifugation at 8,000 x g for 15 min at 4°C and assayed for CT by the bead ELISA as described above.
E
2-
0
z 0 (U)
03E
1
10
100
1000
10000
CONCENTRATION OF TOXIN (pg/ml)
FIG. 1. Standard curve of CT and LT determined by bead ELISA. Purified CT and LT at the concentrations indicated were assayed by bead ELISA as described in the text. Each value is a mean of 10 determinations performed on 10 different days. Symbols: 0, CT; 0, LT.
RESULTS The standard curve for CT by the bead ELISA is shown in Fig. 1. None of the negative controls (buffers and media) yielded a net OD of more than 0.039 in 10 different runs performed on different days (mean, 0.026; standard deviation [SD], ±+0.009]. The mean OD for 10 pg of pure CT per ml by this assay was 0.06 (SD, +0.017), while the mean OD for 39 pg/ml was 0.17 (SD, +0.055). On this basis, an OD of 0.10 or greater was arbitrarily selected as the cutoff for evidence of a positive CT test. The least amount of CT that was consistently detected by this assay was 25 pg of pure CT per ml, which corresponded to the cutoff OD value of 0.10 (Fig. 1). The extent of cross-reactivity of the CT bead ELISA with purified LT of E. coli is shown in Fig. 1. About 10 times more LT was needed to give the OD values obtained with CT. Of the 75 stool samples examined, 59 yielded biochemically and serologically confirmed strains of V. cholerae C1. The bead ELISA was positive for CT in stool supernatants from 50 (84.7%) of the 59 samples from which V. cholerae 01 was isolated (Table 1). Of the 16 stool samples which were negative for V. cholerae 01 by culture, 13 (81.3%) showed a negative test by the bead ELISA. The bead ELISA was positive for three stool specimens which were negative for V. cholerae C1 by bacteriological testing. Significantly, the bead ELISA was negative for the two cases in which ETEC (one case was LT', and the other was LT' and
DETECTION OF CT FROM STOOL BY BEAD ELISA
VOL. 30, 1992 TABLE 1. Bead ELISA results compared with culture results of stool samples from patients with acute secretory diarrhea No. of V. cholerae 01
Total
culture results
Bead ELISA result
Positive
Negative
Positive Negative
50 9
13
53 22
Total
59
16
75
3
heat-stable enterotoxin positive [ST']) was isolated as the sole enteropathogen. Thirty-four (85%) of the 40 control stool specimens from which V. cholerae 01 was not isolated were negative for CT by the bead ELISA. Among the six control stool specimens which were positive, the etiology could not be determined. Comparison of the results of the two groups of investigators who performed the bead ELISA on stool specimens independently and at different time intervals revealed complete concordance. All of the stool samples which were positive for CT by the bead ELISA by the first group of investigators were also positive for CT when the assay was repeated 6 months later by the second group, indicating that there was no appreciable drop in the amount of CT in the stool samples during the interim period, when they were preserved at -20°C. We further analyzed the etiological agents associated with the 75 cases of acute diarrhea in relation to the results of the bead ELISA (Table 2). The etiological agents in the three stool specimens which were negative for V. cholerae 01 by culture but positive for CT by bead ELISA were STproducing ETEC and V. cholerae non-C1, and in one sample no recognized bacterial enteropathogen was detected. The free CT present in 48 of the 50 stool samples positive by culture for V. cholerae 01 and for CT by bead ELISA was completely absorbed by anti-CT IgG. In two stool specimens, the absorption assay could not be performed owing to insufficient sample amounts. Similarly, fecal CT was completely absorbed in two of the three samples which were positive for CT by bead ELISA but did not yield V. cholerae 01 by culture. However, the fecal CT present in TABLE 2. Etiological agents associated with 75 cases of acute diarrhea in relation to bead ELISA results V. cholerae
01
+ + + + + + -
Total
Etiological agent(s) other than V. cholerae 01
samples
No. of stool samples positive for CT by ELISA
None V. cholerae non-01 Campylobacter spp. ETEC (LT+) ETEC (ST+) ETEC (LT+ ST+) ETEC (LT+) ETEC (ST+) ETEC (LT+ ST+) V. cholerae non-01 Campylobacter spp. Salmonella typhimurium None
43 4 9 1 1 1 1 4 1 2 1 1 6
36 3 9 1 0 1 0 1 0 1 0 0
75
53
No. of
bead
1
1785
one of the three samples which yielded V. cholerae non-C1 was only partially absorbed by anti-CT IgG. All of the 59 strains of V. cholerae 01 biotype eltor (25 belonged to the Ogawa subtype, and 34 belonged to the Inaba subtype) isolated in this study produced in vitro CT. In contrast, none of the six V. cholerae non-C1 strains recovered either as the sole pathogen or in conjunction with other enteropathogens elaborated in vitro CT when examined by using the medium and culture conditions used for V. cholerae 01. The concentration of CT present in the bead ELISApositive stool samples was estimated from the standard curve and found to range between 26 pg/ml and >100 ng/ml. The concentrations of CT in 42.4% of the CT-positive stool samples and 57.6% of the V. cholerae 01-positive stool samples were lower than 1 ng/ml.
DISCUSSION This evaluation study demonstrates that the bead ELISA is a sensitive method for direct detection of CT in nonsterile stool samples. The results of the bead ELISA and bacteriological tests were concordant for 85% of the stool specimens. Additionally, this assay picked up CT from nine specimens (three samples in the evaluation study and six of the control stool specimens) from which V. cholerae 01 could not be isolated. What concerned us most, however, was the inability to detect CT in stool samples from nine cases in which positive isolation of V. cholerae 01 was achieved. The isolates from these cases, and also those from all of the cases in which positive V. cholerae 01 isolation was achieved, produced in vitro CT (data not shown). Free CT, if present in the stool samples from these cases, was so low that it went beyond the minimum detection ability of the bead ELISA and of any other available assay method. Inactivation of CT during processing or storage or by some component of the fecal and intestinal fluids could be responsible for our inability to detect CT in the nine samples. While there is not complete correlation between isolation of V. cholerae 01 and detection of CT, the value of this method is in the rapidity of detection of CT in stool specimens. The bead ELISA is not intended to replace bacteriological culture of stool specimens but only to assist in rapid diagnosis of cholera. Quantification of the amount of CT present in stools positive for CT by the bead ELISA provided interesting insights. The most obvious inference was that any method attempting to assay for CT in stools should be ultrasensitive because in 57.6% of the stool samples positive for V. cholerae 01, the amount of CT was below 1 ng/ml. The enterotoxin-binding effect of free gangliosides present in mucin found in stools is probably an important reason for the low or undetectable levels of enterotoxins in stools (9, 12). Direct detection of enterotoxins in stool specimens of patients with acute diarrhea represents a greatly simplified approach for the rapid diagnosis of toxin-mediated diarrheas. There is a need for rapid diagnosis of cholera, especially in epidemiological surveys and drug evaluation studies and to monitor and evaluate new vaccination programs for cholera. During the period of this study, we successfully established an outbreak of cholera despite being unable to isolate V. cholerae 01 from stool samples of the outbreak by demonstrating the presence of anti-CT IgGabsorbable fecal CT in the stools by the bead ELISA (10). The application of an assay like the bead ELISA, which can directly detect fecal enterotoxin, is therefore manifold.
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It has been documented that tissue culture assays are less sensitive than ELISA for detection of fecal CT and LT (6, 7), and the need for sterile stool filtrates further limits the universal applicability of this method. Besides, the process of passing stools through membrane filters (0.22-[im pore size) is likely to destroy or inactivate the toxin (7). A recently developed commercially available reversed passive latex agglutination assay for detection of CT (1) is a simpler alternative to ELISA, but the lower minimum detection ability (1 to 2 ng/ml) precludes its use for assay of free faecal CT in stool specimens. For instance, the latex agglutination assay would not be able to detect CT in 42.4% of the bead ELISA-positive stool samples examined in this study in which the concentration of CT was lower than 1 ng/ml. Among the existing ELISAs for detection of CT, the bead ELISA appears to be the most pragmatic method which could be routinely adapted even in peripheral clinical microbiology laboratories of less developed countries. This is because unlike the microtiter plate ELISA, the bead ELISA could be performed on single specimens without economic constraints. For a coated microtiter plate, samples have to be stored until sufficient numbers of samples are available to perform the assay. The other distinct advantage of the bead ELISA is that the assay can be performed in 3.5 h and results can be made available on the same day. Even for the latex agglutination test, which is undoubtedly simpler to perform than the ELISA, a period of 20 to 24 h is required before the results can be read (1). The need for a spectrophotometer to interpret the results of the bead ELISA can be dispensed with, because the color intensity of samples showing an OD of greater than 0.1 can be visually differentiated. The bead ELISA, therefore, represents a breakthrough in technical simplicity and enhanced sensitivity, and on the basis of this evaluation we recommend routine use of this assay for stool samples and culture supernatants in clinical and reference laboratories. The easy commercial availability of anti-CT IgG-coated beads and the conjugate (Nissui Pharmaceutical Co., Ibaragi, Japan) makes the bead ELISA the method of choice for rapid diagnosis of cholera, especially in lessdeveloped countries. ACKNOWLEDGMENTS This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan and a grant from the Japan Health Science Foundation. REFERENCES 1. Almeida, R. J., F. W. Hickman-Brenner, E. G. Sowers, N. D. Puhr, J. J. Farmer III, and I. K. Wachsmuth. 1990. Comparison of a latex agglutination assay and an enzyme-linked immunosorbent assay for detecting cholera toxin. J. Clin. Microbiol. 28:128-130.
J. CLIN. MICROBIOL. 2. Echeverria, P., L. Verheart, C. V. Ulyanco, and L. T. Santiago. 1978. Detection of heat-labile enterotoxin-like activity in stools of patients with cholera and Escherichia coli diarrhea. Infect. Immun. 19:343-344. 3. Honda, T., S. Taga, Y. Takeda, and T. Miwatani. 1981. Modified Elek test for detection of heat-labile enterotoxin of enterotoxigenic Escherichia coli. J. Clin. Microbiol. 13:1-5. 4. Honda, T., Y. Takeda, and T. Miwatani. 1981. Isolation of special antibodies which react only with homologous enterotoxins from Vibrio cholerae and enterotoxigenic Escherichia coli. Infect. Immun. 34:333-336. 5. Iwanaga, M., and T. Kuyyakanond. 1987. Large production of cholera toxin by Vibrio cholerae O1 in yeast extract peptone water. J. Clin. Microbiol. 25:2314-2316. 6. Merson, M. H., R. H. Yolken, R. B. Sack, J. L. Froehlich, H. B. Greenberg, I. Huq, and R. W. Black. 1980. Detection of Escherichia coli enterotoxins in stools. Infect. Immun. 29:108-113. 7. Morgan, D. R., H. L. Dupont, L. V. Wood, and C. D. Ericsson. 1983. Comparison of methods to detect Escherichia coli heatlabile enterotoxin in stool and cell-free culture supernatants. J. Clin. Microbiol. 18:798-802. 8. Oku, Y., Y. Uesaka, T. Hirayama, and Y. Takeda. 1988. Development of a highly sensitive bead-ELISA to detect bacterial protein toxins. Microbiol. Immunol. 32:807-816. 9. Pierce, N. F. 1972. Differential inhibitory effects of cholera toxoids and ganglioside for the enterotoxins of Vibrio cholerae and Escherichia coli. J. Exp. Med. 137:1009-1023. 10. Ramamurthy, T., A. Pal, G. B. Nair, S. C. Pal, T. Takeda, and Y. Takeda. 1990. Experience with toxin bead ELISA in cholera outbreak. Lancet ii:375-376. 11. Sen, D., M. R. Saha, G. B. Nair, P. Das, S. K. Niyogi, S. P. De, S. K. Bhattacharya, P. Datta, D. Dutta, and S. C. Pal. 1985. Etiological spectrum of acute diarrhoea in hospitalized patients in Calcutta. Indian J. Med. Res. 82:286-291. 12. Strombeck, D. R., and D. Harrold. 1974. Binding of cholera toxin to mucins and inhibition by gastric mucin. Infect. Immun. 10:1266-1272. 13. Svennerholm, A.-M., and J. Holmgren. 1978. Identification of Escherichia coli heat-labile enterotoxin by means of a ganglioside immunosorbent assay (Gmi-ELISA) procedure. Curr. Microbiol. 1:19-23. 14. Takeda, Y., T. Honda, S. Taga, and T. Miwatani. 1981. In vitro formation of hybrid toxins between subunits of Escherichia coli heat-labile enterotoxin and those of cholera enterotoxins. Infect. Immun. 34:341-346. 15. Takeda, Y., T. Takeda, T. Yano, K. Yamamoto, and T. Miwatani. 1979. Purification and partial characterization of heatstable enterotoxin of enterotoxigenic Escherichia coli. Infect. Immun. 25:978-985. 16. Uesaka, Y., Y. Otsuka, M. Kashida, Y. Oku, K. Horigome, G. B. Nair, S. C. Pal, S. Yamasaki, and Y. Takeda. 1992. Detection of cholera toxin by a highly sensitive bead-enzyme linked immunosorbent assay. Microbiol. Immunol. 36:43-53. 17. Yolken, R. H., H. B. Greenberg, M. H. Merson, R. B. Sack, and A. Z. Kapikian. 1977. Enzyme-linked immunosorbent assay for detection of Escherichia coli heat-labile enterotoxin. J. Clin. Microbiol. 6:439-444.