APMIS 98: 652-654, 1990
Rapid diagnosis of Clostridium dificile =associated diarrhoea using a latex agglutination test K. JUNG and B. ARONSSON Department of Bacteriology, Stockholm County Council Central Microbiological Laboratory, Sweden
Jung, K. & Aronsson, B. Rapid diagnosis of Clostridium dificile-associated diarrhoea using a latex agglutination test. APMIS 98: 652-654, 1990. A rapid latex agglutination test, Culturette Brand CDT from Marion Laboratories, was evaluated and compared to a tissue culture assay (TCA) and isolation of Clostridium diflcile in 380 faecal specimens from 226 patients with clinically suspected Clostridium dificile-associated diarrhoea. The sensitivity and specificity of the latex test compared with the TCA were 83% and 80% respectively, and the positive and negative predictive values were 55% and 94% respectively. In patients with repeated sampling the sensitivity increased to 95%. The latex test may be useful as a screening test for negative specimens in laboratories where TCA is not available, but positive specimens have to be confirmed by TCA. Key words: Clostridium diflcile; latex agglutination test. Karin Jung, Department of Bacteriology, Stockholm County Council Central Microbiological Laboratory, P. 0. Box 70470, S-107 26 Stockholm, Sweden.
Intestinal overgrowth of C. diflcile is related to antibiotic-associated diarrhoea (AAD) and pseudomembranous colitis (PMC) (2, 3). The most common method to verify C. diflcile-associated diarrhoea (CDAD) in the laboratory is to demonstrate a cytotoxin in a tissue culture assay (TCA) (1). Recently a rapid latex agglutination test (Culturette Brand CDT, Marion Laboratories, Kansas City, Mo, USA) became available. We have evaluated this test in patients with AAD and other diarrhoea1 disorders, by comparing it with TCA and isolation of the bacterium.
MATERIALS AND METHODS Patients Three hundred and eighty consecutive faecal specimens from 226 patients sent to the laboratory for routine detection of C. dificile-cytotoxin were investigated. One
Received November 30, 1989. Accepted January 25, 1990.
652
hundred and sixty-six patients (72 males, mean age 58 years; 94 females, mean age 6 1 years) suffered from AAD according to charts, i.e. antimicrobial agents given within two months of the start of diarrhoea. Sixty patients (29 males, mean age 40 years; 31 females, mean age 55 years) had diarrhoea unrelated to antibiotic treatment. The specimens were obtained during a six-week period from the middle of April to the beginning of June 1988 and originated from outpatients (44) and inpatients ( 182) in the Stockholm area. Transportation of specimen 2-3 ml of fresh stool without additives was sent to the laboratory and immediately processed, except at weekends when the specimens were frozen at -70 "C. All tests were camed out simultaneously. Culture,for C. diffcile The faecal samples were cultured in two ways. First, faeces was inoculated directly onto selective agar (CCFA) (4) containing cefoxitin (8 microgram/ml) and cykloserine (250 microgram/ml) with a cotton swab and incubated anaerobically in Gas Pak@jars (BBL, Cockeysville, Md, USA) with AnaeroculP A (MERCK, Darmstadt, West Germany) for 48 h at 37 'C. Second, for enrichment, faeces was homogenized in 2 ml phosphate buffered saline (PBS, pH 7.4) and two
LATEX AGGLUTINATION TEST FOR C. DIFFICILE
drops were inoculated into 10 ml Peptone Yeast Broth with glucose (PYG) (National Bacteriological Laboratory, Stockholm, Sweden) with cefoxitin and cykloserine in the same concentrations as above. After anaerobic incubation for 48 h in 37 "C the broth was subcultivated onto CCFA as described above. C. ditficile were presumtively identified by typical colonial morphology and verified by the characteristic pattern of volatile fatty acids produced on gas liquid chromatography (5).
Tissue culture assay Stool specimens were diluted 1: 10 in PBS (phosphate buffered saline, pH 7.4), homogenized and centrifuged at 8000 x g for 15 minutes. Fifty microliters of the supernatant and 1:10, 1:I00 and 1:1OOO dilutions in PBS were used in a cytotoxin assay with human embryonal lung fibroblasts in microtiter plates. One hundred microliter culture medium MEM (Minimal Essential Medium, Gibco, Paisley, Scotland) with foetal calf serum 5% (Flow Laboratories, Irvine, Scotland)was added, and the cells were observed in a light microscope for typical cytotoxic effects after 18-20 h incubation in 3.5% C 0 2at 37 "C ( 1 2). For specificity reason a neutralisation test, using C. diflcile antiserum (National Bacteriological Laboratory, Stockholm, Sweden) mixed 1: 1 with diluted stool and incubated overnight, was performed (9). The strains of C. ditficile isolated from the stool specimens were grown in vitro in Peptone Yeast Broth with starch (National Bacteriological Laboratory, Stockholm, Sweden) under anaerobic conditions for 48 h at 37 "C, centrifuged at 5000 x g for 15 minutes and the supernatant was diluted and tested for cytotoxin as described above. Latex agglutination test Stool specimens were diluted 1:2 with the buffer included in the CDT latex kit and treated as described in the kit manual. The test was regarded as positive when a clear agglutination was visible within three minutes' incubation. Positive and negative controls were included. The supernatants obtained after in vitro growth of C. dijicile were also investigated for agglutination in the latex assay as described above.
TABLE 1. Results of latex agglutination and TCA in stool specimen from patients with clinically suspected CDAD Number of specimens
Number of patients
TCA+/Latex+ TCA+/Latex-
71 15
43 4
TCA-/Latex+ TCA-/ Latex-
59 235
41 138
positive in latex agglutination and TCA negative. In specimens from patients with diarrhoea not associated with antibiotics, 2 1% ( 17/82) of the specimens were positive whereas neither the bacterium nor the cytotoxin could be demonstrated. Using the TCA test as the reference test for CDAD the sensitivity and specificity of the latex test were 83% (7 1/86) and 80% (235/294) respectively (Table 1). From Table 1 the positive predictive value (PPV) 55Yo (71/130) and the negative predictive value (NPV) 94% (235/250) can be calculated. If instead of specimens, patients are considered, the sensitivity and specificity were 9 1 Yo (43/47) and 77% ( 1 38/ 179) respectively. When sampling was repeated in patients with two consecutive, known TCA positive specimens, the sensitivity of the latex test increased from 79% (1 5/ 19) in the first sample to 95% ( 18/ 19) after two samples. Ninety-two per cent (35/38) of C. dificile isolates obtained from the stool specimens were toxigenic in vitro whereas all were positive in the agglutination test.
DISCUSSION RESULTS
If all specimens are considered, the latex test appeared to be the most sensitive with 34%(1 301 380) positive tests compared to TCA with 23% (86/380) and isolation of the bacterium with 10% (38/380) positive tests. In specimens from AAD patients, the latex test was positive in 38% ( 1 131 298), the TCA test in 2990 (86/298) and isolation of C. dificile in 13% (38/298). Twenty per cent (42/2 12) of specimens from AAD patients were
Rapid laboratory diagnosis of CDAD may sometimes be imperative. Culture for C. dificile is time-consuming and as shown here less sensitive than TCA, which on the other hand is not available in all laboratories. In 1986 Marion Laboratories introduced a latex agglutination test for C. dificile antigen, which has been evaluated in different laboratories(6,10,11,13)(Table 2). Our data seem to indicate that latex agglutination is more sensitive than TCA for CDAD. However, an equal
653
JUNG & ARONSSON
TABLE 2. Comparative eficiency of latex test and TCA in specimens from patients with diarrhoea reported by direrent investigators Author(ref)
Sens" Spec" PPV" NPV" Prep No
Sherman(11) Kelly (6) Wexler(13) Presentstudy
83 67 89 83
93 94 90 80
72 58 70 55
96 96 97 94
17 11 NGb 23
201 626 320 380
asens: sensitivity, Spec: specificity, PPV: positive predictive value, NPV: negative predictive value, Prev: prevalence. bNG-not given.
portion of samples from patients with diarrhoea1 disorders unrelated to antimicrobial use had a positive latex agglutination test, indicating that these might be false positive results. The reasons for this may be that the latex test, initially claimed to detect toxin A but lately shown to detect an antigen distinct from toxin A, also demonstrates other anaerobic bacteria as well as nontoxigenic C. dificile, as shown by other investigators (7, 8). More importantly, 17% of TCA positive specimens and 9% of TCA positive patients were negative in the latex agglutination test. This indicates that repeated sampling from suspected CDAD patients increases the sensitivity of the latex test. Indeed, repeated sampling from TCA positive patients increased the sensitivity of the latex test to 959/0.The PPV, however, would remain at about 50%due to the addition of false positive specimens. Our results with the latex agglutination test are similar to those reported by other investigators, as shown in Table 2. We agree with the conclusion drawn by Sherman & co-workers (1 1) that the latex test can be useful as a screening test to identify negative specimens in laboratories where TCA is not available. All positive specimens have to be confirmed by the biological assay. The relatively low sensitivity is, however, a major drawback which has to be considered by attending physicians. Repeated kmpling may be the way to circumvent these problems. The rapidness of the latex agglutination test compared to the TCA makes this a rational approach. This study was supported by Marion Laboratories Inc., Kansas City, Mo.
654
REFERENCES 1. Aronsson, B., Mollby, R . & Nord, C.-E.: Occurrence of toxin-producing Clostridium dificile in antibiotic-associated diarrhea in Sweden. Med. Microbiol. Immunol. 170: 27-35, I98 1. 2. Bartlett, J. G.: Antibiotic-associated pseudomembranous colitis. Rev. Infect. Dis. 1: 530-539, 1979. 3. George, R. H., Symonds, J. M., Dimock, F., Brown, J. D., Arabi, Y., Shinagawa, N., Keighley, M. R. B., Alexander- Williams, J. & Burdon, D. W.: Identification of Clostridium dificile as a cause of pseudomembranous colitis. Brit. Med. J. I: 695, 1978. 4. George, W. L., Sutter, V. L., Citron, D. & Finegold, S. M.: Selective and differential medium for isolation of Clostridium diflcile. J. Clin. Microbiol. 9: 214-219, 1979. 5. Holdeman, L. V., Cato, E. P. & Moore, W. E. C. (Eds.): Anaerobe Laboratory Manual, 4th edn. Virginia Polytechnic Institute and State University, Blacksburg, Virginia 1977, p. 98. 6. Kelly, M. T., Champagne, S. G., Sherlock, C. H., Noble, M. A., Freeman, H. J. & Smith, J. A , : Commercial latex agglutination test for detection of Clostridium dificile-associated diarrhea. J. Clin. Microbiol. 25: 1244-1247, 1987. I . Lyerly, D. M., Ball, D. W., Toth, J. & Wilkins, T. D.: Characterization of cross-reactive proteins detected by Culturette Brand Rapid Latex Test for Clostridium diflcile. J. Clin. Microbiol. 26: 397-400, 1988. 8. Miles, B. L., Siders, J. A. & Allen, S. D.: Evaluation of a commercial latex test for Clostridium dificile for reactivity with Clostridium dificile and cross-reactions with other bacteria. Clin. Microbiol. 26: 2452-2455, 1988. 9. Mollby, R., Nord, C. E. & Aronsson, B.: Diagnosis of Clostridium dificile-associated enterocolitis in Sweden. Laboratory and epidemiological aspects. Scand. J. Infect. Dis. Suppl. 22: 30-36, 1980. 10. Peterson, L. R., Holter, J. J., Shanholtzer, C. J., Garrett, C. R. & Gerding, D. N.: Detection of Clostridium dificile toxins A (enterotoxin) and B (cytotoxin) in clinical specimens. Am. J. Clin. Path. 86: 208-2 1 1, 1986. 11. Sherman, M . E., DeGirolami, P. C., Thorne, G. M., Kimber, J. & Eichelberger, K.: Evaluation of a latex agglutination test for diagnosis of Clostridium dificile-associated colitis. Am. J. Clin. Path. 89: 228233, 1988. 12. Thelestam, M. & Bronnegard, M.: Interaction of cytopathogenictoxin from Clostridium dificile with cellsin tissueculture. Scand. J. Infect. Dis. Suppl. 22: 16-29, 1980. 13. Wexler, H.: Diagnosis of antibiotic-associated disease caused by Clostridium dificile. Clin. Microbi01. Newsletter I 1 (4): 25-29, 1989.
Rapid diagnosis of Clostridium dificile =associated diarrhoea using a latex agglutination test K. JUNG and B. ARONSSON Department of Bacteriology, Stockholm County Council Central Microbiological Laboratory, Sweden
Jung, K. & Aronsson, B. Rapid diagnosis of Clostridium dificile-associated diarrhoea using a latex agglutination test. APMIS 98: 652-654, 1990. A rapid latex agglutination test, Culturette Brand CDT from Marion Laboratories, was evaluated and compared to a tissue culture assay (TCA) and isolation of Clostridium diflcile in 380 faecal specimens from 226 patients with clinically suspected Clostridium dificile-associated diarrhoea. The sensitivity and specificity of the latex test compared with the TCA were 83% and 80% respectively, and the positive and negative predictive values were 55% and 94% respectively. In patients with repeated sampling the sensitivity increased to 95%. The latex test may be useful as a screening test for negative specimens in laboratories where TCA is not available, but positive specimens have to be confirmed by TCA. Key words: Clostridium diflcile; latex agglutination test. Karin Jung, Department of Bacteriology, Stockholm County Council Central Microbiological Laboratory, P. 0. Box 70470, S-107 26 Stockholm, Sweden.
Intestinal overgrowth of C. diflcile is related to antibiotic-associated diarrhoea (AAD) and pseudomembranous colitis (PMC) (2, 3). The most common method to verify C. diflcile-associated diarrhoea (CDAD) in the laboratory is to demonstrate a cytotoxin in a tissue culture assay (TCA) (1). Recently a rapid latex agglutination test (Culturette Brand CDT, Marion Laboratories, Kansas City, Mo, USA) became available. We have evaluated this test in patients with AAD and other diarrhoea1 disorders, by comparing it with TCA and isolation of the bacterium.
MATERIALS AND METHODS Patients Three hundred and eighty consecutive faecal specimens from 226 patients sent to the laboratory for routine detection of C. dificile-cytotoxin were investigated. One
Received November 30, 1989. Accepted January 25, 1990.
652
hundred and sixty-six patients (72 males, mean age 58 years; 94 females, mean age 6 1 years) suffered from AAD according to charts, i.e. antimicrobial agents given within two months of the start of diarrhoea. Sixty patients (29 males, mean age 40 years; 31 females, mean age 55 years) had diarrhoea unrelated to antibiotic treatment. The specimens were obtained during a six-week period from the middle of April to the beginning of June 1988 and originated from outpatients (44) and inpatients ( 182) in the Stockholm area. Transportation of specimen 2-3 ml of fresh stool without additives was sent to the laboratory and immediately processed, except at weekends when the specimens were frozen at -70 "C. All tests were camed out simultaneously. Culture,for C. diffcile The faecal samples were cultured in two ways. First, faeces was inoculated directly onto selective agar (CCFA) (4) containing cefoxitin (8 microgram/ml) and cykloserine (250 microgram/ml) with a cotton swab and incubated anaerobically in Gas Pak@jars (BBL, Cockeysville, Md, USA) with AnaeroculP A (MERCK, Darmstadt, West Germany) for 48 h at 37 'C. Second, for enrichment, faeces was homogenized in 2 ml phosphate buffered saline (PBS, pH 7.4) and two
LATEX AGGLUTINATION TEST FOR C. DIFFICILE
drops were inoculated into 10 ml Peptone Yeast Broth with glucose (PYG) (National Bacteriological Laboratory, Stockholm, Sweden) with cefoxitin and cykloserine in the same concentrations as above. After anaerobic incubation for 48 h in 37 "C the broth was subcultivated onto CCFA as described above. C. ditficile were presumtively identified by typical colonial morphology and verified by the characteristic pattern of volatile fatty acids produced on gas liquid chromatography (5).
Tissue culture assay Stool specimens were diluted 1: 10 in PBS (phosphate buffered saline, pH 7.4), homogenized and centrifuged at 8000 x g for 15 minutes. Fifty microliters of the supernatant and 1:10, 1:I00 and 1:1OOO dilutions in PBS were used in a cytotoxin assay with human embryonal lung fibroblasts in microtiter plates. One hundred microliter culture medium MEM (Minimal Essential Medium, Gibco, Paisley, Scotland) with foetal calf serum 5% (Flow Laboratories, Irvine, Scotland)was added, and the cells were observed in a light microscope for typical cytotoxic effects after 18-20 h incubation in 3.5% C 0 2at 37 "C ( 1 2). For specificity reason a neutralisation test, using C. diflcile antiserum (National Bacteriological Laboratory, Stockholm, Sweden) mixed 1: 1 with diluted stool and incubated overnight, was performed (9). The strains of C. ditficile isolated from the stool specimens were grown in vitro in Peptone Yeast Broth with starch (National Bacteriological Laboratory, Stockholm, Sweden) under anaerobic conditions for 48 h at 37 "C, centrifuged at 5000 x g for 15 minutes and the supernatant was diluted and tested for cytotoxin as described above. Latex agglutination test Stool specimens were diluted 1:2 with the buffer included in the CDT latex kit and treated as described in the kit manual. The test was regarded as positive when a clear agglutination was visible within three minutes' incubation. Positive and negative controls were included. The supernatants obtained after in vitro growth of C. dijicile were also investigated for agglutination in the latex assay as described above.
TABLE 1. Results of latex agglutination and TCA in stool specimen from patients with clinically suspected CDAD Number of specimens
Number of patients
TCA+/Latex+ TCA+/Latex-
71 15
43 4
TCA-/Latex+ TCA-/ Latex-
59 235
41 138
positive in latex agglutination and TCA negative. In specimens from patients with diarrhoea not associated with antibiotics, 2 1% ( 17/82) of the specimens were positive whereas neither the bacterium nor the cytotoxin could be demonstrated. Using the TCA test as the reference test for CDAD the sensitivity and specificity of the latex test were 83% (7 1/86) and 80% (235/294) respectively (Table 1). From Table 1 the positive predictive value (PPV) 55Yo (71/130) and the negative predictive value (NPV) 94% (235/250) can be calculated. If instead of specimens, patients are considered, the sensitivity and specificity were 9 1 Yo (43/47) and 77% ( 1 38/ 179) respectively. When sampling was repeated in patients with two consecutive, known TCA positive specimens, the sensitivity of the latex test increased from 79% (1 5/ 19) in the first sample to 95% ( 18/ 19) after two samples. Ninety-two per cent (35/38) of C. dificile isolates obtained from the stool specimens were toxigenic in vitro whereas all were positive in the agglutination test.
DISCUSSION RESULTS
If all specimens are considered, the latex test appeared to be the most sensitive with 34%(1 301 380) positive tests compared to TCA with 23% (86/380) and isolation of the bacterium with 10% (38/380) positive tests. In specimens from AAD patients, the latex test was positive in 38% ( 1 131 298), the TCA test in 2990 (86/298) and isolation of C. dificile in 13% (38/298). Twenty per cent (42/2 12) of specimens from AAD patients were
Rapid laboratory diagnosis of CDAD may sometimes be imperative. Culture for C. dificile is time-consuming and as shown here less sensitive than TCA, which on the other hand is not available in all laboratories. In 1986 Marion Laboratories introduced a latex agglutination test for C. dificile antigen, which has been evaluated in different laboratories(6,10,11,13)(Table 2). Our data seem to indicate that latex agglutination is more sensitive than TCA for CDAD. However, an equal
653
JUNG & ARONSSON
TABLE 2. Comparative eficiency of latex test and TCA in specimens from patients with diarrhoea reported by direrent investigators Author(ref)
Sens" Spec" PPV" NPV" Prep No
Sherman(11) Kelly (6) Wexler(13) Presentstudy
83 67 89 83
93 94 90 80
72 58 70 55
96 96 97 94
17 11 NGb 23
201 626 320 380
asens: sensitivity, Spec: specificity, PPV: positive predictive value, NPV: negative predictive value, Prev: prevalence. bNG-not given.
portion of samples from patients with diarrhoea1 disorders unrelated to antimicrobial use had a positive latex agglutination test, indicating that these might be false positive results. The reasons for this may be that the latex test, initially claimed to detect toxin A but lately shown to detect an antigen distinct from toxin A, also demonstrates other anaerobic bacteria as well as nontoxigenic C. dificile, as shown by other investigators (7, 8). More importantly, 17% of TCA positive specimens and 9% of TCA positive patients were negative in the latex agglutination test. This indicates that repeated sampling from suspected CDAD patients increases the sensitivity of the latex test. Indeed, repeated sampling from TCA positive patients increased the sensitivity of the latex test to 959/0.The PPV, however, would remain at about 50%due to the addition of false positive specimens. Our results with the latex agglutination test are similar to those reported by other investigators, as shown in Table 2. We agree with the conclusion drawn by Sherman & co-workers (1 1) that the latex test can be useful as a screening test to identify negative specimens in laboratories where TCA is not available. All positive specimens have to be confirmed by the biological assay. The relatively low sensitivity is, however, a major drawback which has to be considered by attending physicians. Repeated kmpling may be the way to circumvent these problems. The rapidness of the latex agglutination test compared to the TCA makes this a rational approach. This study was supported by Marion Laboratories Inc., Kansas City, Mo.
654
REFERENCES 1. Aronsson, B., Mollby, R . & Nord, C.-E.: Occurrence of toxin-producing Clostridium dificile in antibiotic-associated diarrhea in Sweden. Med. Microbiol. Immunol. 170: 27-35, I98 1. 2. Bartlett, J. G.: Antibiotic-associated pseudomembranous colitis. Rev. Infect. Dis. 1: 530-539, 1979. 3. George, R. H., Symonds, J. M., Dimock, F., Brown, J. D., Arabi, Y., Shinagawa, N., Keighley, M. R. B., Alexander- Williams, J. & Burdon, D. W.: Identification of Clostridium dificile as a cause of pseudomembranous colitis. Brit. Med. J. I: 695, 1978. 4. George, W. L., Sutter, V. L., Citron, D. & Finegold, S. M.: Selective and differential medium for isolation of Clostridium diflcile. J. Clin. Microbiol. 9: 214-219, 1979. 5. Holdeman, L. V., Cato, E. P. & Moore, W. E. C. (Eds.): Anaerobe Laboratory Manual, 4th edn. Virginia Polytechnic Institute and State University, Blacksburg, Virginia 1977, p. 98. 6. Kelly, M. T., Champagne, S. G., Sherlock, C. H., Noble, M. A., Freeman, H. J. & Smith, J. A , : Commercial latex agglutination test for detection of Clostridium dificile-associated diarrhea. J. Clin. Microbiol. 25: 1244-1247, 1987. I . Lyerly, D. M., Ball, D. W., Toth, J. & Wilkins, T. D.: Characterization of cross-reactive proteins detected by Culturette Brand Rapid Latex Test for Clostridium diflcile. J. Clin. Microbiol. 26: 397-400, 1988. 8. Miles, B. L., Siders, J. A. & Allen, S. D.: Evaluation of a commercial latex test for Clostridium dificile for reactivity with Clostridium dificile and cross-reactions with other bacteria. Clin. Microbiol. 26: 2452-2455, 1988. 9. Mollby, R., Nord, C. E. & Aronsson, B.: Diagnosis of Clostridium dificile-associated enterocolitis in Sweden. Laboratory and epidemiological aspects. Scand. J. Infect. Dis. Suppl. 22: 30-36, 1980. 10. Peterson, L. R., Holter, J. J., Shanholtzer, C. J., Garrett, C. R. & Gerding, D. N.: Detection of Clostridium dificile toxins A (enterotoxin) and B (cytotoxin) in clinical specimens. Am. J. Clin. Path. 86: 208-2 1 1, 1986. 11. Sherman, M . E., DeGirolami, P. C., Thorne, G. M., Kimber, J. & Eichelberger, K.: Evaluation of a latex agglutination test for diagnosis of Clostridium dificile-associated colitis. Am. J. Clin. Path. 89: 228233, 1988. 12. Thelestam, M. & Bronnegard, M.: Interaction of cytopathogenictoxin from Clostridium dificile with cellsin tissueculture. Scand. J. Infect. Dis. Suppl. 22: 16-29, 1980. 13. Wexler, H.: Diagnosis of antibiotic-associated disease caused by Clostridium dificile. Clin. Microbi01. Newsletter I 1 (4): 25-29, 1989.
