with penicillin. Antimicrobial Agents and Chemotherapy 1977, 12: 401--405. 3. Sahm DF, Tortes C: Effects of medium and inoculum variations on screening for high-level aminoglycoside resistance in Enterococcus faecalis. Journal of Clinical Microbiology 1988, 26: 250-256. 4, Yagupsky P, Pelry S, Menegus MA: Comparison of four methods for testing high-level aminoglycoside resistance in enterocoeci. European Journal of Clinical Microbiology and Infectious Diseases 1990, 9: 133-135.
5. Zervos MJ, PattersonJE, Edberg S, Pierson C, Kauffman CA, MikesellTS, SchabergDR: Single concentration broth microdilution test for detection of high-level aminoglycoside resistance in enterococci. Journal of Clinical Microbiology 1987, 25: 2443-2444. 6. Spiegel CA: Laboratory detection of high-level aminoglycoside-aminocyclitol resistance in Enterococcus species. Journal of Clinical Microbiology 1988, 26: 2270-2274. 7. Rosenlhai SL, Freundlich LF: An aminoglyeoside disk sensitivity test for use with enteroeoeci. Journal of Antimicrobial Chemotherapy 1982, 10: 459--462. 8. Sabra DF, Tortes G: High-content aminoglycoside disks for determining aminoglycoside resistance in Enterococcusfaecalis. Journal of Clinical Microbiology 1988, 26: 257-260. 9. Facklam RR, Carey RB: Streptococci and aerococci. In: Lennette EH, Balows A, Hausler WJ, Shadomy HJ (ed): Manual of clinical microbiology. American Society for Microbiology, Washington, DC, 1985, p. 154-175. 10. National Committee for Clinical Laboratory Standards: Development of in vitro susceptibility testing criteria and quality control parameters. Proposed Guideline. Document M23-E NCCLS, Villanova, PA, 1987. 11. Courvalin P, Shaw WV, Jacob AE: Plasmid-mediated mechanisms of resistance to aminoglyeoside-aminocyelitol antibiotics and to ehloramphenieol in group D streptococci. Antimierobial Agents and Chemotherapy 1978, 13: 716-725. 12. Krogstad DJ, Korfhagen TR, Moellering RC, Wennersten C, Swartz MN, Perzynsk S, Davies J: Aminoglycoside-inactivating enzymes in clinical isolates of Streptococcus faecalis: an explanation for antibiotic synergism. Journal of Clinical Investigation 1978, 62: 480-486. 13. Courvalin P, Carlier C, Collatz E: Plasmid-mediated resistance to aminocyclitol antibiotics in group D streptococci. Journal of Bacteriology 1980, 143: 541-551. 14. Ounissi H, Derlot E, Cadier C, Courvalin C: Gene homogeneity for aminoglycoside-modifying enzymes in gram-positive cocci. Antimicrobial Agents and Chemotherapy 1990, 34: 2164-2168. 15. CalderwoodSA, WennerstenC, MoelleringRC: Resistance to antibiotic synergism in Streptococcusfaecalis: further studies with amikacin and with a new amikacin derivative, 4'-deoxy, 6'-N-methy|amikaein. Antimicrobial Agents and Chemotherapy 1981, 19: 549-555. 16. Carlier C, Courvalin P: Emergence of 4', 4"-aminoglycoside nucleotidyltransferase in enterococci. Antimicrobial Agents and Chemotherapy 1990, 34: 15651569.
Eur. J. Clin. Microbiol. Infect. Dis.
Evaluation of a Commercial Enzyme Immunoassay Kit for the Detection of Clostridium difficile Toxin A S.P. Borriello 1*, T. Vale 1, J.S. B r a z i e r 2, S. H y d e 1, E. C h i p p e c k 1
A new enzyme immunoassay (EIA) kit developed for the rapid detection of Clostridium difficile toxin A in faecal specimens, Premier (Meridian Diagnostics), was evaluated using 101 faecal specimens. Sixty-nine specimens were positive for Clostridium difficile by isolation of the organism and by cytotoxicity in tissue culture. The EIA for toxin A was positive in 49 of these 69 cases. No specimen that was negative for cytotoxicity was positive by EIA. Eight of the 32 specimens negative by both EIA and cytotoxicity assay yielded Clostridium difficile by culture. In five of these cases the cytotoxigenic status of the isolate was determined, and four were positive. There was no direct relationship between cytotoxin titre and EIA reading.
Clostridium difficile causes p s e u d o m e m b r a n o u s colitis (1) and is associated with m a n y cases of antibiotic-associated diarrhoea (2). L a b o r a t o r y diagnosis is based on detection of the organism (3) and/or either of its two m a j o r toxins, toxins A and B (4). In the vast majority of cases isolation of Clostridium difficile on a selective m e d i u m (5) and detection of free toxin in the faeces by tissue culture (4) are the m e t h o d s employed. D u e to the time involved (48 h of anaerobic culture) and the diagnostic limitations of culture, i.e. it does not indicate w h e t h e r the isolate is toxigenic and, if toxigenic, w h e t h e r toxins were produced in vivo, attempts were m a d e to develop immunoassays for the rapid detection o f toxin A and toxin B (6-10). O n e of the m e t h o d s for toxin A detection was a latex agglutination kit which, although rapid, was shown not to detect toxin A (11, 12). In the present study we evaluated a new 1Microbial Pathogenicity Research Group, Clinical Research Centre, Watford Road, Harrow Middlesex HA1 3UJ, UK. 2Public Health Laboratory, Luton and Dunstable Hospital, Luton, Bedfordshire LU4 0DZ, UK.
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•kit developed for the rapid detection of toxin A in faecal specimens.
Materials and Methods. A total of 101 faecal specimens from patients with diarrhoea were analysed. Specimens consisted of those from patients of the Luton and Dunstable Hospital (LDH) and those sent directly to the Public Health Laboratory, LDH or the Clinical Research Centre for analyses over a four-month period. The majority of specimens sent to the Clinical Research Centre were specimens SUspected to be positive and sent for confirmation. The following bacteria were examined for their reactivity in the enzyme immunoassay (EIA) kit: Clostridium difficile VPI 10463, four laboratory isolates of Clostridium difficile (producing toxins A and B), Clostridium difficile 8864 (producing only toxin B) and Clostridium difficile M-1 (nontoxigenic); and Clostridium sordellii 3703 (producing toxins LT and HT; provided by Prof. Nakamura, Kanazawa University, Japan) and three non-toxigenic laboratory isolates of Clostri-
dium sordellii. Toxin A was prepared from Clostridium difficile strain VPI 10463 as described in detail previously (13). Briefly, crude culture filtrate was passed through a bovine thyroglobulin affinity (BTA) column at 4 °C and the material retained eluted at 37 °C and passed through two anion-exchange chromatographic steps by fast protein liquid chromatography (Pharmacia, Sweden) to yield pure toxin A. Toxin B was prepared by passing the material that eluted from the BTA column at 4 °C (i.e. free of toxin A) through the same two fast protein liquid chromatography anion-exchange chromatographic steps. This material still had some trace contaminants but was shown to be free of toxin A by its lack of reaction with a monoclonal antibody, PCG-4, which specifically recognises toxin A (kindly provided by Dr Lyerly, Virginia Polytechnic Institute, Blacksburg, USA). For analysis of faecal specimens, the Premier Clostridium difficile Toxin A kit (Meridian Diagnostics, USA) was used as directed by the manufacturers with the exception that for all stools an accurate five-fold dilution was made in the sample diluent provided. The test consisted of adding one drop (50/al) of enzyme conjugate followed by one drop (50 ~1) of faecal suspension, and incubation at 37 °C for 2 h. The test wells were then washed and one drop (50 !al) each of substrate A and B added and mixed. Wells were incubated for 10 min at room temperature before
the reaction was stopped by the addition of one drop of stop solution. The reaction was determined spectrophotometrically by reading the absorbance at 450 nm on an EIA plate reader (Titertek Multiskan MC, Flow Laboratories), which was blanked with 150/al of distilled water. Under these conditions any reading greater than 0.1 was considered positive. Crude culture filtrates of bacteria grown in Robertsons cooked meats (Southern Group Laboratories, UK) were analysed as if they were a faecal filtrate. Pure toxin A and purified toxin B were serially diluted ten-fold in sample diluent and each dilution analysed directly as if it were a faecal specimen. To test for stability, a semi-solid stool specimen proven positive was stored at 4 °C and tested for cytotoxicity and for reactivity in the EIA kit at six- to eight-day intervals. For the cytotoxin assay, faecal specimens were diluted ten-fold in phosphate-buffered saline and filtered through a 0.45/am pore filter (Acrodisc, Gelman, USA) before being applied to African green monkey kidney (Vero) cells as described in detail previously (4). Endpoints were recorded as the reciprocal of the last dilution giving a cytopathic effect in half of the cell sheet. Stools were cultured for Clostridiurn difficile by the alcohol-shock method as described by Borriello et al. (14) with the modification that the material was cultured on a cefoxitin-cycloserine egg yolk agar after incorporating 0.1% cholic acid, and on a non-selective fastidious anaerobe agar (Lab M, UK) incorporating 7 % defibrinated horse blood (Difco, USA). The latter medium is optimal for the fluorescence characteristic of Clostridium difficile (15). For enrichment a Robertsons cooked meat broth was also inoculated and incubated at 37 °C for 48 h. The agar plates were incubated at 37 °C in an anaerobic chamber (Don Whitley Scientific, UK) and examined after overnight incubation. Negative cultures were re-incubated and re-examined after 48 h. If Clostridium difficile was not isolated by direct culture, the cooked meat enrichment broth was subcultured on the cefoxitincycloserine egg yolk agar and the non-selective fastidious anaerobe blood agar and similarly processed. Lecithinase-negative colonies typical of Clostridium difficile were identified by their characteristic odour and chartreuse fluorescence under long-wave ultraviolet light, and by agglutination of the Clostridium difficile latex par-
Eur. J. Clin. Microbioi. Infect. Dis.
ticle slide agglutinating reagent (Microscreen; Mercia Diagnostics, UK).
Results and Discussion. The Premier EIA kit gave positive results for all five fully toxigenic strains of Clostridium difficile, the toxigenic strain of Clostridium sordellii, and a preparation of pure toxin A with a cytotoxin titre of 103; in the last case, toxin A was detected by the EIA kit following a 1:5 x 103 dilution of the preparation (OD450 = 0.108). Negative results were obtained for the non-toxigenic strain of Clostridium difficile and for the strain producing only toxin B; purified toxin B in a preparation witha cytotoxin titre of 105 was detected only at a dilution of 1:50 (OD450 = 0.360) but not at 1:500 or greater. This toxin B preparation did not react with the toxin A-specific monoclonal antibody PCG-4, showing that reactivity was not due to contaminating toxin A. The concordance between the results of the EIA for toxin A and the cytotoxin assay is shown in Table 1. Of 101 faecal specimens, 32 were negative for toxin A by both EIA and the cytotoxin assay. Clostridium difficile was present in eight of these specimens, and four of the five isolates available for analysis were cytotoxigenic. The one specimen that yielded Clostridium difficile only after enrichment belonged to this group of eight. Of the remaining 69 specimens, all were culture positive for Clostridium difficile, 49 (71%) were positive by both EIA and the cytotoxin assay, and 20 (29 %) were positive only by the cytotoxin assay. There were no cytotoxin-negative E/Apositive specimens. The mean cytotoxin titre of 45 specimens so analysed was 102.7 (range 101105; median 103) and the mean OD450 for the 49 EIA-positive specimens was 1.088 (range 0.1383.0; median 0.885). Table 1: Concordance of eytotoxin assay and Premier EIA
results based on analysis of 69 faecal specimens eulturepositive for Clostridium difficile. Cytotoxin assay
Premier E I A Positive
Figure 1: Correlation between faecal eytotoxin titre determined in Veto cells and OD450 values obtained following analysis for toxin A by EIA.
After 44 days of storage at 4 °C, both the EIA and cytotoxin tests were still positive, although the initial OD450 EIA reading of 0.786 had fallen to 0.202. After 52 days the EIA test became negative (OD450 of 0.008), but the cytotoxin test remained positive until completion of the study at 58 days. Although many EIAs have been developed and evaluated for the detection of toxins A and B of Clostridium difficile (6-10), a commercial system has not been readily available. The first system marketed to meet this requirement, a latex agglutination test, was shown to detect not toxin A but another unrelated protein (11, 12). The kit evaluated in this study does detect toxin A and is as sensitive as Vero cell tissue culture for detection of pure toxin. This is in keeping with the relative sensitivities of tissue culture and EIA reported by Laughon et al. (7). They also reported that their immunoassay, which had a sensitivity of 91.1%, correctly identified all of the faecal specimens that had a cytotoxin titre of > 103, but only 23 % of those with lower titre (7). Lyerly et al. (6) also reported that in their immunoassay there was a 100 % correlation with tissue culture when the titre was >_.103, but that only 20 % of those with a lower titre were detected. These findings imply that there is a direct correlation be-
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tween the amount of toxin B (predominantly responsible for the cytopathic activity) in a faecal specimen and the amount of toxin A. No such correlation was found in this study. The negative results for the 20 specimens that were positive in the tissue culture assay but negative by EIA were not due to low cytotoxin titres. Of the 12 specimens for which cytotoxin titres were obtained, most had cytotoxin titres of 103 (mean 102'7; r a n g e 101-104; m e d i a n 103). I n addition, the
lack of concordance between the EIA and tissue culture methods could not be correlated with the cytotoxin titre. In fact, there was no correlation between cytotoxin titre and EIA OD450 for the 45 specimens for which both cytotoxin titres and EIA readings were available (Figure 1). Firm conclusions about this observation are difficult, as both toxins contribute to cytotoxicity, the effect of toxin A becoming increasingly important as the amounts of toxin B, which is about 1000-fold more cytotoxic in this cell line, decrease. However, the implications are that faecal specimens can contain relatively high levels of toxin A and little toxin B and vice versa. The sensitivity, specificity and overall correlation of the Premier immunoassay kit compared to cytotoxicity in tissue culture indicates that it is a suitable alternative for routine diagnostic laboratories that lack access to tissue culture facilities, especially as there were no false-positive results. In those laboratories where cell lines less sensitive than Vero cells are used, the sensitivity and overall correlation of the immunoassay kit may be better than reported here. For those cases in which there is a strong suspicion of Clostridium difficile involvement but a negative result with the kit, a follow-up specimen should be requested, and if still negative, a specimen sent to a reference centre for testing by cytotoxin assay.
References 1. Larson HE, Price AB, Honour P, Bordello SP: Clostridium difficile and the aetiology of pseudomembranous colitis. Lancet 1978, ii: 1312-1314. 2. Burdon DW: Spectrum of disease. In: Borriello SP (ed): Antibiotic associated diarrhoea and colitis. Martinus Nijhoff Publishers, Boston, 1984, p. 9-24.
3. Bordello SP, Honour P: Detection isolation and identification of CIostridium difficile. In: Borriello SP (ed): Antibiotic associated diarrhoea and colitis. Martinus Nijhoff Publishers, Boston, 1984, p. 37-47. 4. Bordello SP, Welch AR: Detection of Clostridium difficile toxins. In: Bordello SP (ed): Antibiotic associated diarrhoea and colitis. Martinus Nijhoff Publishers, Boston, 1984, p, 49-56. 5. George WL, Suller VL, Citron D, Finegold SM: Selective and differential medium for isolation of Clostridium difficile. Journal of Clinical Microbiology 1979, 9: 214-219. 6. Lyedy DM, Sullivan MM, Wilkins TD: Enzyme-linked immunosorbent assay for Clostridimn difficile toxin A. Journal of Clinical Microbiology 1983, 17: 72-78. 7. Laughon BE, Viscidi RP, Gdovin SL~ Yolken RH, Bartlett JG: Enzyme immunoassays for detection of Clostridimn difficile toxins A and B in faecal specimens. Journal of Infectious Diseases 1984, 149: 781788. 8. Aronsson B, Granstriim M, Miiilby R, Nord CE: Enzyme immunoassay for detection of Clostridium difficile toxins A and B in patients with antibiotic-associated diarrhoea and colitis. European Journal of Clinical Microbiology 1985, 4: I02--107, 9. Krishnan C: Detection of Clostridium difficile toxins by enzyme immunoassay, Journal of Hygiene 1986, 96: 5-12. 10. Nguyen VK, Rihn B, Heekel C, Bisseret F, Girardot R, Monteil H: Enzyme immunoassay (ELISA) for detection of Clostridium difficile toxin B in specimens of faeces. Journal of Medical Microbiology 1990, 31: 251-257. 11. Lyerly DM, Wilkins TD: Commercial latex test for Clostridium difficile toxin A does not detect toxin A. Journal of Clinical Microbiology 1986, 23: 622-623. 12, Borriello SP, Barclay FE, Reed PJ, Welch AR, Brown JD, Burdon DW: Analysis of latex agglutination test for Clostridium difficile toxin A (D-I) and differentiation between CIostridium diJficile toxins A and B and latex reactive protein. Journal of Clinical Pathology 1987, 40: 573-580. 13. Kamiya S, Reed P J, Bordello SP: Purification and characterisation of Clostridium difficile toxin A by bovine thyroglobulin affinity chromatography and dissociation in denaturing conditions with or without reduction. Jourr~al of Medical Microbiology 1989, 30: 69-77. 14. Bordello SP, Honour P: A simplified procedure for the routine isolation of Clostridium difficile from faeces, Journal of Clinical Pathology 1981, 34: 11241127. 15. Levett PN: Effect of basal medium upon fluorescence of Clostridium difficile. Letters in Applied Microbiology 1985, 1: 75-76.