laboratory facilities. A coagglutination test was used to detect V. cholerae antigen in bile-peptone broth incubated with feces. In the technique, Staphylococcus ...
Vol. 25, No. 11
JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1987, p. 2204-2206 0095-1137/87/112204-03$02.00/0 Copyright C 1987, American Society for Microbiology
Rapid Diagnosis of Cholera by Coagglutination Test Using 4-h Fecal Enrichment Cultures MAHBUBUR RAHMAN,'* DAVID A. SACK,"12 SALEH MAHMOOD,1 AND ANOWAR HOSSAIN1 Laboratory Sciences and Epidemiology Division, International Centre for Diarrhoeal Disease Research, Bangladesh, Dacca 2, Bangladesh,' and Division of Geographic Medicine, Johns Hopkins University, Baltimore, Maryland 212052 Received 1 April 1987/Accepted 7 August 1987
A simple, rapid, and reliable method to detect Vibrio cholerae in fecal specimens would assist in the cases of severe diarrhea, especially since most such cases occur in areas with minimal laboratory facilities. A coagglutination test was used to detect V. cholerae antigen in bile-peptone broth incubated with feces. In the technique, Staphylococcus aureus Cowan 1 coated with anti-V. cholerae O1 antiserum was tested with cultures incubated for 4 h. When 165 specimens were tested, the sensitivity, specificity, and accuracy of the test, compared with standard culture methods, were 97, 99, and 98%, respectively. These promising results were better than those of dark-field microscopy using the same specimens, and the test was logistically easy to perform. The coagglutination test using enrichment broth culture of feces is a simple and rapid method which may be used to confirm a diagnosis of cholera. management of
Cholera is an illness of major public health importance in many developing countries, including Bangladesh (1). Conventional methods to detect Vibrio cholerae in feces, thus confirming a clinical diagnosis of cholera, require bacteriological media, supplies, and equipment, with results available in 24 to 48 h (5). These facilities are often not available in parts of the world where cholera is common. Rapid methods to detect V. cholerae in feces have been described, the most common being microscopic examination of stool with a dark-field microscope (1, 4, 6, 9, 12). This test requires a dark-field microscope and a trained technician, resources and skills which are often lacking. Coagglutination tests are widely used to detect antigens in clinical specimens (2, 3, 5, 7, 9, 11, 13). This report describes a coagglutination test which appears to satisfy the needs for a simple, rapid, cheap, and reliable diagnostic test for V. cholerae which could be used in the field as well as in laboratories.
tellurite positive) were tested for agglutination with V. cholerae O1 polyvalent antiserum (10). A dark-field microscopic examination of the bile-peptone broth was performed after 4 h of incubation. If bacteria with typical darting movements were seen, the diagnosis of V. cholerae was confirmed by inhibition of the movement with V. cholerae O1 polyvalent antiserum. Preparation of V. cholerae O1 antisera. Hyperimmune V. cholerae O1 antisera were prepared in rabbits with live classical V. cholerae O1 Ogawa 16563 and Inaba Z-26676 as antigens (14, 15). Four rabbits were immunized with each strain. A rabbit was selected only if its preimmunization serum did not contain V. cholerae antibody by tube agglutination. To prepare the antigen, a single colony of each strain was incubated overnight in TlNi (Trypticase [BBL Microbiology Systems, Gaithersburg, Md.], 1%; NaCI, 1%) broth. The undiluted culture was injected into rabbits with the following schedule. Culture fluid was injected intramuscularly and intravenously (equal volume by each route), with increasing volumes being given on subsequent days. The volumes were 0.3, 0.4, and 0.5 ml by each route on days 1, 2, and 3, respectively. On days 14, 15, and 16, the volumes injected by each route were 0.5, 0.8, and 0.8 ml. Blood was collected in week 4 by exsanguination after the titer was checked. After clotting, the serum was separated and incubated at 55°C for 30 min. Finally, the antisera were pooled to prepare V. cholerae polyvalent antiserum. The titer of the rabbit sera by tube agglutination was 1:3,200 with each homologous strain. To validate its specificity, the serum was tested by slide agglutination with seven strains of V. cholerae non-O1 and by coagglutination with TlNi broth. No nonspecific reactions were seen. Preparation of coagglutination reagents. The method of Kornvall (8) was used to prepare the coagglutination reagents. S. aureus Cowan 1 was grown overnight in Trypticase soy broth on a shaker at 37°C. The bacterial cells were washed three times with phosphate-buffered saline (PBS; 0.03 M phosphate, 0.12 M NaCI [pH 7.3]) and suspended in 0.5% formaldehyde in PBS. After incubation at room temperature for 3 h, the treated bacterial cells were washed
MATERIALS AND METHODS Clinical specimens. A total of 111 rectal swabs and 54 stool specimens were collected from patients with diarrhea who were admitted to the Diarrhoeal Treatment Centre of the International Centre for Diarrhoeal Disease Research, Bangladesh, in Dacca, Bangladesh, during 1986. These specimens were tested for V. cholerae by standard culture methods, dark-field microscopy, and coagglutination. Coagglutination tests were performed without knowledge of the results of the alternative methods. Method to diagnose V. cholerae. Fecal specimens were inoculated onto tellurite-taurocholate-gelatin agar plates and also into a tube containing 0.75 ml of bile-peptone broth (NaCJ, 1%; peptone, 1%; sodium taurocholate, 0.5% [pH 8.8]). After 4 h of incubation, this enrichment broth was used to inoculate a second tellurite-taurocholate-gelatin agar plate. Colonies resembling V. cholerae (gelatinase and *
Corresponding author. 2204
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COAGGLUTINATION TEST FOR CHOLERA
TABLE 1. Results of fecal culture, coagglutination test, and dark-field microscopy after 4 h of enrichment of feces in bile-peptone broth No. of samples
Method
Culture Coagglutination test Dark-field microscopy a
b
Positive
Negative
86 84
79
78b
81 87
Of 165 samples, 54 were stool samples and 111 were rectal swabs. All 78 were also positive for V. cholerae by culture and the coagglutina-
tion test.
again three times with PBS and diluted to a final cell concentration of 10% (vol/vol) in PBS. The cell suspension was heated at 80°C in a water bath for 1 h and then cooled quickly. Finally, the cells were washed with PBS, reconstituted to 10% (vol/vol) in PBS, and stored at 4°C. To coat the cells with antiserum, 1 ml of the 10% (vol/vol) stabilized, heat-treated S. aureus cell suspension was added to 0.2 ml of high-titer V. cholerae Q1 polyvalent antiserum. This was incubated at room temperature for 3 h with gentle hand shaking at 0.5-h intervals. Following centrifugation and washing with PBS, the cells were finally diluted to a 2% (vol/vol) suspension in PBS with 0.1% sodium azide. These cells, designated the V.C. COAG reagent, were stored at 4°C until used. A 2% (vol/vol) formaldehyde-stabilized, heat-treated S. aureus cell suspension in PBS with 0.1% sodium azide, which was not coated with antiserum, was used as a negative control and designated CONT. As a positive antigen control, V. cholerae Q1 lipopolysaccharide (List Biological Laboratories), was used. When dissolved in PBS at a concentration of 0.5 mg/ml, easily visible agglutination with V.C. COAG reagent was observed. Coagglutination test procedure. The same bile-peptone enrichment broth used for culture and dark-field microscopy was used in the coagglutination test. The V.C. COAG and CONT reagents were brought to room temperature before use. Using glass slides on which two rectangles had been drawn with a wax pencil, one drop of the incubated broth was placed in each of the rectangles. One drop of V.C. COAG reagent was then added to one rectangle, and one drop of CONT reagent was added to the other rectangle. After thorough mixing with a loop, the slides were tipped back and forth. After 2 to 3 min, the slides were observed against a dark background for agglutination. The coagglutination test was considered positive if agglutination was seen with the V.C. COAG reagent but not the CONT reagent. When coagglutination was observed with both reagents, the broth culture was heated to 70°C for 3 min, cooled, and retested. In all cases, agglutination with the CONT reagent was eliminated following this heating procedure. To determine the sensitivity of the test, V. cholerae Q1 was diluted in bile-peptone broth and used to detect the minimum concentration of bacteria necessary to produce agglutination. In addition, samples of five noncholera stools were mixed with V. cholerae Q1 in concentrations of 101, 102, 103, i04, 105, and 106 CFU/ml of stool. A 10-pul sample of each spiked stool was added to 1.5 ml of bile-peptone broth and incubated for 4 h. Each of these broth cultures was subjected to culture, dark-field microscopy, and the coagglutination test.
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RESULTS
There were no differences in results with rectal swabs or stool samples; therefore, the results were combined. The microbiological culture detected V. cholerae Q1 in 86 (52%) of 165 samples (Table 1). The coagglutination test detected V. cholerae Q1 in 84 samples, while dark-field microscopy detected V. cholerae Q1 in 78 samples. All of the specimens found positive by dark-field microscopy were also positive by the coagglutination test. Table 2 shows the correlation between the results of culture and the coagglutination test. Relative to the culture results, the coagglutination test yielded one false-positive and three false-negative results. Heating of the broth to 70°C was sometimes found necessary because 18 specimens gave nonspecific agglutination reactions with the control reagent when tested unheated. Heating as described removed this nonspecific agglutination. When the V. cholerae cells were diluted in culture broth, the test was positive when the concentration was >1.5 x 107 CFU/ml. When normal stools artificially inoculated with different concentrations of V. cholerae were incubated in bile-peptone broth for 4 h, the coagglutination test detected V. cholerae Q1 if the concentration of organisms in the original stool was at least 104 CFU/ml of stool. The corresponding concentrations for culture and dark-field microscopy were 102 and 105 CFU/ml of stool, respectively. Tests were carried out to estimate the stability of the coagglutination reagents. When the V.C. COAG reagent was stored in an incubator at 37°C for 2 weeks, it continued to detect the same positive specimens as the reagent stored at
40C. DISCUSSION We describe here a coagglutination test to detect V. cholerae in rectal swabs or stool specimens which requires little equipment or facilities and which can provide reliable results within 5 h after samples are collected. The only equipment required are tubes of enrichment broth, the V.C. COAG and CONT reagents, microscope slides, a dropper, and a means of heating the broth tubes to 70°C. The sensitivity and specificity of the test were 97 and 99%, respectively, suggesting that the new test is nearly as good a diagnostic test as the standard culture method. Other rapid methods have been described for detection of V. cholerae in feces (1, 4, 6, 9, 12). The hanging-drop method is neither sensitive nor specific and requires a microscope (1). Dark-field microscopy has been more extensively used (1); however, the microscope and an experienced microscopist are required. Suitable antisera not containing preservatives (which nonspecifically inhibit motility) are also required and are not generally available. The fluorescentantibody microscopic technique, though reliable, is not TABLE 2. Comparison of results of the coagglutination test with microbiological culture to detect V. cholerae O1 No. of cultures
Coagglutination test result
Positive
Negative
Positive
83 3
78
Negative
1
a The sensitivity was 83 (96.5%) of 86 for a positive V. cholerae culture, the specificity was 78 (98.7%) of 79 for a negative V. cholerae culture, and the accuracy was 161 (98%) of 165. The test had a positive predictive value for 83 (99%) of 84 and a negative predictive value for 78 (96%) of 81.
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RAHMAN ET AL.
generally used because of the requirements for special reagents and a UV microscope (4). Coagglutination tests for V. cholerae have recently been published. Using fecal samples, Jesudason et al. reported that one-third of the samples gave nonspecific agglutination reactions, which required mixing fecal specimens with unsensitized S. aureus cells to absorb nonspecific antibody and centrifugation (6). These additional steps make this method less practical. Selina and Lam suggested enriching V. cholerae in the fecal specimens in an agar broth biphasic medium, followed by agglutination (12). This procedure is also more laborious than that described here, and the sensitivity of the method is not known because of the low number of positive specimens. Finally, Lesmana et al. applied a coagglutination method to detect V. cholerae in alkaline peptone water cultures inoculated with feces (9). This test required 18 h to complete. In contrast to these published methods, our technique requires only a simple enrichment broth in small volume (0.75 ml in a 4-ml vial) with 4 h of incubation. Neither absorption nor centrifugation of the sample is needed. However, a water bath is needed to eliminate nonspecific agglutination with some sample. Although these experiments were carried out by incubation of the broth cultures at 37°C, preliminary studies indicate that incubation at room temperature (23 to 25°C) yields similar results, suggesting that the technique could be used in the field. Furthermore, V. cholerae lipopolysaccharide is available commercially to use as a positive control antigen, which helps to maintain the quality of the test reagent. The test has several possible applications. (i) As a rapid test, it can provide confirmation of the diagnosis of cholera, thus assisting clinicians in managing the disease and public health officials in instituting control measures. (ii) As an inexpensive test, it could replace more expensive culture methods used in many diarrhea treatment centers and hospitals. (iii) As an aid for surveillance, it can assist epidemiologists in detecting V. cholerae in areas where bacteriological laboratories are not available. Finally, as a simple test, it can provide confirmation of cholera even without fully trained microbiology technicians. ACKNOWLEDGMENTS We thank J. P. Butzler and Bart Gordts, St. Peter Hospital, Brussels, Belgium, for encouraging this research, A. Latif Mia and M. A. Salek of the International Centre for Diarrhoeal Disease Research, Bangladesh, Diagnostic Laboratory staff for assistance during the work, and M. Sirajul Islam Molla for secretarial assistance.
Funding for this project
was
obtained through
a
grant from the
United Nations Development Program and the World Health Organization. LITERATURE CITED
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