Vol. 11, 1992
References 1. van der Willigen AH, Polak-Vogelzang AA, Habbema L, Wagenvoort JHT: Clinical efficacy of ciprofloxacin
versus doxycyclinein the treatment of non-gonococcal urethritis in males. European Journal Clinical Microbiology and Infectious Diseases 1985, 5: 658--661. 2. Segreti J, Kessler HA, Kapell KS, Trenholme GM:
In vitro activity of A-56268 (TE-031) and four other antimicrobial agents against Chlamydia trachomatis. Antimicrobiai Agents and Chemotherapy 1987, 31: I00-101. 3. Slamm WE, Tam M, Koester CM, Cles L: Detection of Chlamydiatrachomatisinclusions in McCoycell cultures with fluorescein-conjugated monodonal antibodies. Journal of Clinical Microbiology1983,17: 666668. 4. Barnes RC, Wang SP, Kua CC, Slamm WE: Rapid
immunotyping of Chlarnydia trachomatis with monoclonal antibodies in a solid-phase enzyme-immunoassay. Journal of Clinical Microbiology 1985, 22: 609--613. 5. M~rflh PA, Paavonen J, Puolakkainen M: Chlamydia. Plenum Publishing, New York, 1989, p. 103-114.
6. Jones RB, van der Pol B, Martin DH, Shepard MK:
Partial characterization of Chlamydia trachomatis isolates resistant to multiple antibiotics. Journal of Infectious Diseases 1990, 162: 1309-1315. 7. Arya OP, Hobson D, Hart CA, Bartzokas C, Pratt BC: Evaluation of ciprofloxacin 500 mg twice daily
for one week in treating uncomplicated gonococcal, chlamydial and non-specific urethritis in men. Genitourinary Medicine 1986, 62: 170--174. 8. Jeskanen L, Karppinen L, Ingervo L, Reitamo $, Happonen HP, Lassus A: Ciprofloxacin versus doxycycline
in the treatment of uncomplicated urogenital Chlwnydia trachornatis infections. A double-blind comparative study. Scandinavian Journal of Infectious Diseases 1989, 60: 62--65.
E v a l u a t i o n o f an E n z y m e l m m u n o a s s a y for D e t e c t i o n o f Clostridium difficile T o x i n A
The laboratory diagnosis of enteric disease due to Clostridiurn difficile is usually established by fecal culture and/or tissue culture assay (1). We report the results of an evaluation of a new enzyme immunoassay (EIA) for the rapid detection of Clostridium difficile toxin A. Fecal specimens were spread on cycloserinecefoxitin-fructose egg yolk agar (Oxoid, UK) plates which were incubated anaerobically at 37 °C for 48 h. Isolates with the characteristic colonial morphology and odor were identified as Clostridiurn difficile with the R a p i D A N A II system (Innovative Diagnostic Systems, USA) and
563
the Serobact C. difficile latex agglutination test kit (Wellmark Diagnostics, Canada). Fecal filtrates and broth cultures of Clostridium difficile isolates were assayed f o r cytotoxin using the Toxi-titer microtiter plate system (2) with human foreskin fibroblast cells (Bartels Immunodiagnostic Supplies, USA) according to the manufacturer's instructions. The plates were examined daily for 48 hours; in positive specimens showing the characteristic cytopathic effect results were confirmed by neutralization with antitoxin.
Clostridium diffictTe toxin A was detected with the Premier C. difficile toxin A E I A (Meridian Diagnostics, USA), according to the manufacturer's instructions except that seven washes were used to remove unbound conjugate. Using only five washes, as recommended by the manufacturer, there was an unacceptably high rate ( 1 8 % ) of indeterminate results. Results were analyzed using a spectrophotometer (Whittaker M.A. Bioproducts, USA) at A45o (reading < 0.100, negative; > 0.100 to 0.149, indeterminate; > 0.150, positive). Clostridium difficile was isolated from 170 specimens from 94 patients and cytotoxin was detected in 92 specimens (65 patients). Toxigenic Clostridium difficile was recovered from 100 specimens (73 patients). Only 18 patients had symptoms associated with antibiotic-associated diarrhea. Seven (1.4 % ) fecal specimens negative in the culture and cytotoxin assay gave indeterminate results in the EIA to detect toxin A and were excluded from further analysis. The E I A results compared to those obtained in the cytotoxin assay are shown in Table 1. The toxin A E I A was very sensitive (90 %) and specific (98 %). The EIA was also positive in five of eight specimens negative in the cytotoxin assay but from which toxigenic Clostridium difficile was recovered. Although both toxin A and toxin B are probably involved in the pathogenesis of Clostridium difficile-associated diseases, toxin A is presumed to be responsible for most of the symptoms of pseudomembranous colitis as only toxin A induces gastrointestinal tissue damage and a fluid response in animal models (3-6). An E I A for detection of toxin A was first described by Lyerly et al. (7) and was subsequently found to have 100 % specificity but only 6 1 % sensitivity in patients thought to have Clostridium difficile-related enteric disease according to clinical criteria (8). Further refinements in Clostridium difficile toxin A antibody preparations have led to the development of several commercial EIAs for
564
Eur. J. Clin. Microbiol. Infect. Dis.
Table 1: Comparison of results of the toxin A EIA with results of the cytotoxin assay and isolation of toxigenic Clostridium difficil~ No. of strains
Toxin A E I A Positive
Negative
Cytotoxin assay Positive 92 Negative 391
83 (90 %)a 8
9 383 (98 %)b
Cytotoxin assay and/or isolation Positive 100 Negative 383
88 (88 %)a 3
12 380 (99 %)b
aFigure in brackets indicates the sensitivity. bFigure in brackets indicates the specificity.
rapid detection of toxin A in fecal specimens, including the test kit evaluated in this study. In the current study, correlation of test results with the clinical status was not possible because very few patients had enteric symptoms. However, our results are similar to those of Dipersio et al. (9) and indicate that the test is both sensitive and specific compared to the cytotoxin assay and/or isolation of toxigenic Clostridium difficile. There were few indeterminate test results using seven washes, and the assay was simple to perform, not requiring filtration or centrifugation of fecal specimens. Test results are available within three hours and may be obtained either by visual inspection or by spectrophotometric analysis. Therefore, this method provides a rapid and accurate alternative to tissue culture cytotoxin assay in the detection of Clostridium difficile toxins in stools.
K. Tsimidis A.E. Simor*
3. Lyerly DM, Lockwood DE, Richardson SH, Wiikins TD: Biological activities of toxins A and B of Clostridium difficile. Infection and Immunity 1982, 35: 1147-1150. 4. Lyerly DM, Krivan HC, Wilkins TD: Clostridium difficile: its disease and toxins. Clinical Microbiology Reviews 1988, 1: 1-18. 5. Taylor NS, Thorne GM, Bartlett JG: Comparison of two toxins produced by Clostridium difficile. Infection and Immunity 1981, 34: 1036--1043. 6. Borriello SP, Ketley J, Mitchell J, Barclay FE, Welch AR, Price AB, Stephen J: Clostridium difficile: a spectrum of virulence and analysis of putative virulence determinants in the hamster model of antibiotic-associated colitis. Journal of Medical Microbiology 1987, 24: 53-64. 7. Lyerly DM, Sullivan NM, Wiikins TD: Enzyme-linked immunosorbent assay for Clostridium difficile toxin A. Journal of Clinical Microbiology 1983, 17: 72-78. 8. Walker RC, Ruane PJ, Rosenblatt JE, Lyerly DM, Gieaves CA, Smith TF, Pierce PF, Wilkins TD: Comparison of culture, cytotoxicity assays, and enzymelinked immunosorbent assay for toxin A and toxin B in the diagnosis of Clostridium diffieile-related enteric disease. Diagnostic Microbiology and Infectious Diseases 1986, 5: 61-69. 9. Dipersio JR, Varga FJ, Conwell DL, Kraft JA, Kozak KJ, Willis DH: Development of a rapid enzyme immunoassay for Clostridium difficile toxin A and its use in the diagnosis of C. difficite-associated disease. Journal of Clinical Microbiology 1991, 29: 2724-2730.
Department of Microbiology, Room 602, Mount Sinai Hospital, 600 University Avenue, University of Toronto, Toronto, Ontario, M5G 1X5, Canada.
References 1. Bowman RA, Riley TV: Laboratory diagnosis of Clostridiura difficile-associated diarrhea. European Journal of Clinical Microbiology and Infectious Diseases 1988, 7." 476--484. 2. Wu TC, Gersch SM: Evaluation of a commercial kit for the routine detection of Clostridium difficile cytotoxin by tissue culture. Journal of Clinical Microbiology 1986, 23: 792-793.
Evidence of Human Herpesvirus 6 in Sjiigren Syndrome and Sarcoidosis Human herpesvirus-6 (HHV-6) has been suspected to be a causative agent of Sj6gren syndrome and sarcoidosis. Sj0gren syndrome is a disease of unknown etiology characterized by
References 1. van der Willigen AH, Polak-Vogelzang AA, Habbema L, Wagenvoort JHT: Clinical efficacy of ciprofloxacin
versus doxycyclinein the treatment of non-gonococcal urethritis in males. European Journal Clinical Microbiology and Infectious Diseases 1985, 5: 658--661. 2. Segreti J, Kessler HA, Kapell KS, Trenholme GM:
In vitro activity of A-56268 (TE-031) and four other antimicrobial agents against Chlamydia trachomatis. Antimicrobiai Agents and Chemotherapy 1987, 31: I00-101. 3. Slamm WE, Tam M, Koester CM, Cles L: Detection of Chlamydiatrachomatisinclusions in McCoycell cultures with fluorescein-conjugated monodonal antibodies. Journal of Clinical Microbiology1983,17: 666668. 4. Barnes RC, Wang SP, Kua CC, Slamm WE: Rapid
immunotyping of Chlarnydia trachomatis with monoclonal antibodies in a solid-phase enzyme-immunoassay. Journal of Clinical Microbiology 1985, 22: 609--613. 5. M~rflh PA, Paavonen J, Puolakkainen M: Chlamydia. Plenum Publishing, New York, 1989, p. 103-114.
6. Jones RB, van der Pol B, Martin DH, Shepard MK:
Partial characterization of Chlamydia trachomatis isolates resistant to multiple antibiotics. Journal of Infectious Diseases 1990, 162: 1309-1315. 7. Arya OP, Hobson D, Hart CA, Bartzokas C, Pratt BC: Evaluation of ciprofloxacin 500 mg twice daily
for one week in treating uncomplicated gonococcal, chlamydial and non-specific urethritis in men. Genitourinary Medicine 1986, 62: 170--174. 8. Jeskanen L, Karppinen L, Ingervo L, Reitamo $, Happonen HP, Lassus A: Ciprofloxacin versus doxycycline
in the treatment of uncomplicated urogenital Chlwnydia trachornatis infections. A double-blind comparative study. Scandinavian Journal of Infectious Diseases 1989, 60: 62--65.
E v a l u a t i o n o f an E n z y m e l m m u n o a s s a y for D e t e c t i o n o f Clostridium difficile T o x i n A
The laboratory diagnosis of enteric disease due to Clostridiurn difficile is usually established by fecal culture and/or tissue culture assay (1). We report the results of an evaluation of a new enzyme immunoassay (EIA) for the rapid detection of Clostridium difficile toxin A. Fecal specimens were spread on cycloserinecefoxitin-fructose egg yolk agar (Oxoid, UK) plates which were incubated anaerobically at 37 °C for 48 h. Isolates with the characteristic colonial morphology and odor were identified as Clostridiurn difficile with the R a p i D A N A II system (Innovative Diagnostic Systems, USA) and
563
the Serobact C. difficile latex agglutination test kit (Wellmark Diagnostics, Canada). Fecal filtrates and broth cultures of Clostridium difficile isolates were assayed f o r cytotoxin using the Toxi-titer microtiter plate system (2) with human foreskin fibroblast cells (Bartels Immunodiagnostic Supplies, USA) according to the manufacturer's instructions. The plates were examined daily for 48 hours; in positive specimens showing the characteristic cytopathic effect results were confirmed by neutralization with antitoxin.
Clostridium diffictTe toxin A was detected with the Premier C. difficile toxin A E I A (Meridian Diagnostics, USA), according to the manufacturer's instructions except that seven washes were used to remove unbound conjugate. Using only five washes, as recommended by the manufacturer, there was an unacceptably high rate ( 1 8 % ) of indeterminate results. Results were analyzed using a spectrophotometer (Whittaker M.A. Bioproducts, USA) at A45o (reading < 0.100, negative; > 0.100 to 0.149, indeterminate; > 0.150, positive). Clostridium difficile was isolated from 170 specimens from 94 patients and cytotoxin was detected in 92 specimens (65 patients). Toxigenic Clostridium difficile was recovered from 100 specimens (73 patients). Only 18 patients had symptoms associated with antibiotic-associated diarrhea. Seven (1.4 % ) fecal specimens negative in the culture and cytotoxin assay gave indeterminate results in the EIA to detect toxin A and were excluded from further analysis. The E I A results compared to those obtained in the cytotoxin assay are shown in Table 1. The toxin A E I A was very sensitive (90 %) and specific (98 %). The EIA was also positive in five of eight specimens negative in the cytotoxin assay but from which toxigenic Clostridium difficile was recovered. Although both toxin A and toxin B are probably involved in the pathogenesis of Clostridium difficile-associated diseases, toxin A is presumed to be responsible for most of the symptoms of pseudomembranous colitis as only toxin A induces gastrointestinal tissue damage and a fluid response in animal models (3-6). An E I A for detection of toxin A was first described by Lyerly et al. (7) and was subsequently found to have 100 % specificity but only 6 1 % sensitivity in patients thought to have Clostridium difficile-related enteric disease according to clinical criteria (8). Further refinements in Clostridium difficile toxin A antibody preparations have led to the development of several commercial EIAs for
564
Eur. J. Clin. Microbiol. Infect. Dis.
Table 1: Comparison of results of the toxin A EIA with results of the cytotoxin assay and isolation of toxigenic Clostridium difficil~ No. of strains
Toxin A E I A Positive
Negative
Cytotoxin assay Positive 92 Negative 391
83 (90 %)a 8
9 383 (98 %)b
Cytotoxin assay and/or isolation Positive 100 Negative 383
88 (88 %)a 3
12 380 (99 %)b
aFigure in brackets indicates the sensitivity. bFigure in brackets indicates the specificity.
rapid detection of toxin A in fecal specimens, including the test kit evaluated in this study. In the current study, correlation of test results with the clinical status was not possible because very few patients had enteric symptoms. However, our results are similar to those of Dipersio et al. (9) and indicate that the test is both sensitive and specific compared to the cytotoxin assay and/or isolation of toxigenic Clostridium difficile. There were few indeterminate test results using seven washes, and the assay was simple to perform, not requiring filtration or centrifugation of fecal specimens. Test results are available within three hours and may be obtained either by visual inspection or by spectrophotometric analysis. Therefore, this method provides a rapid and accurate alternative to tissue culture cytotoxin assay in the detection of Clostridium difficile toxins in stools.
K. Tsimidis A.E. Simor*
3. Lyerly DM, Lockwood DE, Richardson SH, Wiikins TD: Biological activities of toxins A and B of Clostridium difficile. Infection and Immunity 1982, 35: 1147-1150. 4. Lyerly DM, Krivan HC, Wilkins TD: Clostridium difficile: its disease and toxins. Clinical Microbiology Reviews 1988, 1: 1-18. 5. Taylor NS, Thorne GM, Bartlett JG: Comparison of two toxins produced by Clostridium difficile. Infection and Immunity 1981, 34: 1036--1043. 6. Borriello SP, Ketley J, Mitchell J, Barclay FE, Welch AR, Price AB, Stephen J: Clostridium difficile: a spectrum of virulence and analysis of putative virulence determinants in the hamster model of antibiotic-associated colitis. Journal of Medical Microbiology 1987, 24: 53-64. 7. Lyerly DM, Sullivan NM, Wiikins TD: Enzyme-linked immunosorbent assay for Clostridium difficile toxin A. Journal of Clinical Microbiology 1983, 17: 72-78. 8. Walker RC, Ruane PJ, Rosenblatt JE, Lyerly DM, Gieaves CA, Smith TF, Pierce PF, Wilkins TD: Comparison of culture, cytotoxicity assays, and enzymelinked immunosorbent assay for toxin A and toxin B in the diagnosis of Clostridium diffieile-related enteric disease. Diagnostic Microbiology and Infectious Diseases 1986, 5: 61-69. 9. Dipersio JR, Varga FJ, Conwell DL, Kraft JA, Kozak KJ, Willis DH: Development of a rapid enzyme immunoassay for Clostridium difficile toxin A and its use in the diagnosis of C. difficite-associated disease. Journal of Clinical Microbiology 1991, 29: 2724-2730.
Department of Microbiology, Room 602, Mount Sinai Hospital, 600 University Avenue, University of Toronto, Toronto, Ontario, M5G 1X5, Canada.
References 1. Bowman RA, Riley TV: Laboratory diagnosis of Clostridiura difficile-associated diarrhea. European Journal of Clinical Microbiology and Infectious Diseases 1988, 7." 476--484. 2. Wu TC, Gersch SM: Evaluation of a commercial kit for the routine detection of Clostridium difficile cytotoxin by tissue culture. Journal of Clinical Microbiology 1986, 23: 792-793.
Evidence of Human Herpesvirus 6 in Sjiigren Syndrome and Sarcoidosis Human herpesvirus-6 (HHV-6) has been suspected to be a causative agent of Sj6gren syndrome and sarcoidosis. Sj0gren syndrome is a disease of unknown etiology characterized by
