- Email: [email protected]
Are Rapid Immunoassays for in vivo Detection of Toxin A Sufficient for Diagnostic Purposes of Clostridium difficile -Associated Diseases?
Anaerobe (2000) 6, 15±19 doi:10.1006/anae.1999.0312
CLINICAL MICROBIOLOGY
Are Rapid Immunoassays for in vivo Detection of Toxin A Su¤cient for Diagnostic Purposes of Clostridium di¤cile-Associated Diseases? Gayane Martirosian1*, Alex vanBelkum2, Hanna Pituch1, Piotr Obuch-Woszczatynèski1 and Felicja Meisel-Mikolajczyk1 1
Department of Medical Microbiology Institute of Biostructure, Warsaw University Medical School, 5 Chalubinski str. 02-004 Warsaw, Poland; 2 Department of Medical Microbiology & Infectious Diseases, Erasmus University Medical Center Rotterdam EMCR Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands (Received 3 March 1999; revised 7 September, 1999; accepted in revised form 21 October 1999)
One hundred and fifty-five stool specimens of patients suspected for Clostridium difficile-associated diarrhoea, colitis or pseudomembranous colitis (PMC) were investigated. All patients were pre-treated with antibiotics, suffered from watery diarrhoea and abdominal pain and were hospitalized in different hospital units. Units varied from departments of surgery, internal medicine, intensive care, paediatry, dermatology, orthopaedy to gastroenterology. Fifty C. difficile strains were isolated from the faecal samples. Clostridium difficile toxin detection was done directly in the stool samples, and also in cultured C. difficile strains (in vivo and in vitro, respectively). We observed clear differences between in vivo and in vitro toxin A detection by using commercial rapid immuno-enzymatic tests: from 25 in vivo toxin A-negative samples, 17 were positive in vitro. This observation suggests that culturing of C. difficile on selective medium is mandatory for adequate toxin detection and necessary for confirming the presence of toxin-producing C. difficile. This is especially important among patients with clinical symptoms and history of pretreatment with antibiotics and when in vivo toxin A detection is negative. It was established that toxin gene detection by PCR is optimal and PCR results were concordant with results of other in vitro assays. Genotyping by using AP-PCR and PCR ribotyping showed heterogeneity among the toxigenic C. difficile strains cultured from in vivo toxin A-negative stool samples. # 2000 Academic Press
Introduction Clostridium difficile is a Gram-positive anaerobic bacterium. Its pathogenicity is associated with the
*Corresponding author. Tel/Fax: (48-22)628-27-39. E-mail: [email protected]
1075±9964/00/010015 + 05 $35.00/0
production of two protein toxins: enterotoxin A and cytotoxin B [1,2]. Toxin A is inducing epithelial damage, fluid secretion and hemorrhage when injected into rabbit ileal loops. Toxin B is an approximately 1000-fold more potent cytotoxin than toxin A [3,4]. Toxins A and B of C. difficile act in concert, causing as a result changes in gut cyto-skeleton and epithelial fluid secretion. Toxigenic strains of # 2000 Academic Press
16
G. Martirosian et al.
C. difficile are known as an etiological agent of pseudomembranous colitis (PMC), antibiotic-associated diarrhoea (AAD) or colitis (AAC) [4,5]. On the other hand asymptomatic colonization is found in approximatly 3% of healthy adults (the number is much higher in hospitalized adults, who have been on antimicrobial therapy) and in the order of approximately 65% of healthy neonates. Recently, toxigenic C. difficile strains were described as predominant pathogen of hospital-acquired intestinal infections. Nosocomial outbreaks of C. difficile-associated diarrhoea were described in different hospital units. Also cross-infections were described and the role of hospital environment in transmission of C. difficile strains was documented. Cultivation of C. difficile strains is not difficult since selective medium was described [6]. Different typing schemes are available for study of epidemiology of C. difficile-associated infection. Molecular typing by using different PCR assays appears to be a fast and useful method for this purpose [7±10]. Measuring the cytopoathic effect on McCoy cells is a well known ``gold standard'' technique for toxigenicity study of C. difficile strains [11], but this method requires special equipment for cell culture and takes time. Many rapid immunoassays for detection of C. difficile toxins are available on the market. Especially toxin A assays are produced by different pharmaceutical companies, since this toxin is the more stable one. Most of these commercial assays are characterised by sufficient sensitivity and specificity levels. Rapid detection of toxins of C. difficile is very important for clinical reasons. Until the end of the eighties, detection of one C. difficile toxin in vivo was sufficient for diagnosis of C. difficile-associated diseases. However, the description of a strain lacking of the toxin A gene but harbouring a full toxin B gene and still highly virulent in animal model, suggests of necessity to detect both toxins of C. difficile in stool samples [12]. The aim of the present study was to assess the efficacy of in vivo versus in vitro testing for the presence of toxins of C. difficile by using different methods (rapid immuno-assays, cell culture assay and PCR). Finally, 20 strains were compared by AP-PCR and PCR ribotyping to define the existence of similarity among C. difficile strains, cultured from in vivo toxin A-negative and in vitro positive stool samples.
Materials and Methods Clinical samples Stool samples (n=155) of 155 patients, suspected for C. difficile-associated diarrhoea or colitis were
investigated. Patients were hospitalized in departments of surgery, internal medicine, paediatry, dermatology, orthopaedy or gastroenterology. A few patients were treated in intensive care units or the outpatients' department. Any other enteric pathogens were documented in these stool samples at the moment of testing for C. difficile and its toxins. Bacterial strains Fifty C. difficile strains were isolated from faecal samples of the patients mentioned above. In addition, two reference C. difficile strains were induced, a toxigenic one possessing both the toxin A and B genes (VPI 10463) and a non-toxigenic strain (NIH BRIGGS 8050). Isolation of bacterial strains. Faecal samples were cultured on the selective medium Columbia-cycloserinecefoxitin-amphotericin B agar (CCCA; bioMerieux, France), as described priviously [7]. Isolates were identified as C. difficile by characteristic morphology of colonies, green-yellow fluorescence under UV light and biochemical activity (API-20A, bioMerieux, Marcy-l' Etoile, France). Clostridium difficile toxins detection in vivo and in vitro Clostridium difficile toxins were detected directly in faecal samples (``in vivo'') and in isolated strains (``in vitro''). Toxin A detection in vivo. For the direct detection of toxin A in faecal samples the following assays were used: (1) ``Culturette Brand Toxin CD'' (BectonDickinson, Meylan, France) enzyme immunoassay; (2) ``C. difficile toxin A test'' (Oxoid, Unipath, Wesel, Germany) rapid immunoassay. Both immunoassays were performed according to the instructions of the manufacturer. Stool samples were tested as soon as possible upon receipt in the laboratory; rest of samples was stored at 7708C for further investigation. Toxin A detection in vitro: For detection of toxin A in isolated strains, the following methods were used: (1) Two rapid immunoassays: (a) ``Culturette Brand Toxin CD'' (Becton-Dickinson, Meylan, France); (b) ``C. difficile toxin A test'' (Oxoid, Unipath, Wesel, Germany); (2) PCR by using YT28 and YT29 primers combination [13,14]. Toxin B detection in vitro: (1) Cytotoxicity assay on McCoy cells. The cytopathic effect of bacterial culture supernatants (diluted 1071±1078) on McCoy cells was used for detection of toxin B [9,15]. (2) PCR by using
Rapid Immunoassays for in Vivo Detection of Toxin A YT18 and YT19 primers combination [13,14]. A protocol for PCR detection of both the C. difficile toxin A and B genes using primer combinations YT28&YT29 and YT18&YT19 were described previously [13,14]. Genotyping of Isolates by Arbitrary Primed PCR and PCR-ribotyping Arbitrary primed PCR (AP-PCR) and PCR ribotyping of the 20 C. difficile isolates and two reference strains were performed with use of the arbitrary oligonucleotides AP-2, AP-3, ERIC1/ERIC2 and SP1, SP2 as described previously [7,15,16].
Results Stool samples of 155 patients hospitalized in different hospital units were investigated for the presence of C. difficile and either of its toxins in vivo and in vitro. Directly in faecal material, toxin A was detected in 22 cases by using rapid immunoassays, 133 samples were toxin A negative. Stool samples were tested as soon as possible upon receipt in the laboratory and the rest of samples was stored at 7708C for further investigation. Both immunoassays were performed according to the instructions of manufacturer. Fifty C.
17
difficile strains were isolated from the faecal samples. These strains were derived from the 22 in vivo toxin Apositive faecal samples, and 28 in vivo toxin Anegative samples. All 50 C. difficile strains were studied for in vitro toxin A and B production (using the same rapid immunoassays, cytotoxicity assay and PCR) and the results were compared with results, obtained from in vivo testing. In 19 cases disagreement was observed: 17 samples toxin A-negative in vivo gave positive results in vitro; and in two cases toxin A was detected in vivo (in faecal samples) and not detected in vitro. In vivo toxin A detection was not concordant with in vitro data, when rapid immunoassays were performed. The PCR results of in vitro toxin A and toxin B gene detection were fully concordant with in vitro toxin detection results, obtained by using other assays: all toxin positive strains in the in vitro assays were positive in PCR for both toxin A and B genes. Twenty C. difficile strains, isolated from different stool samples (nine with disagreements of in vivo and in vitro toxin A detection and 11 with same positive or negative in vivo and in vitro results) were chosen for comparison by AP-PCR and PCR ribotyping. The results obtained by AP-PCR and PCR ribotyping revealed genetic heterogeneity among the strains isolated from in vivo negative and in vitro positive toxin A stool samples. Similarity was not observed
Table 1. Comparison of C. difficile strains by AP-PCR and PCR-ribotyping No
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. Number of types per assay
No of patient 772 701 640 2477 2649 2523 2442 1236 2785 582 1461 428 812 1259 2242 2203 1605 574 143 1720
Sex/Age
F/6 F/53 M/30 F/43 F/6 F/38 F/4 F/52 M/2 M/71 F/53 M/46 F/35 F/32 M/4 M/3 M/48 F/42 F/64 F/42
Hospital ward b
o.p.dept o.p.dept surgical surgical paediatr ICU paediatr o.p.dept paediatr internal haematol transpl. o.p.dept haematol paediatr paediatr o.p.dept internal gastroen internal
Tox. Ac in vivo
Toxins in vitro
Ribosomal spacer
nd 7 7 7 7 7 7 7 7 7 7 7
7 7 7 7 7 7
AP-PCR
Overall typea
AP2
AP3
ERIC1/2
A B C D E F G B H I I L B I M A N I I C
A B A nd nd C D nd D D nd E F A G A H A I A
A B C D E F G H G I I L B M G N O P Q R
A B C B D E C B F G H I A H J A L H M N
I II III IV V VI VII IIa VIII IX IXa X IIb XI XII Ia XIII IXb XIV XV
13
10
16
13
15 (excl. 4 subtypes)
a
Subtypes are defined in case strains share two or three identical types when the different PCR assays are concerned. (nd ± not done) o.p.dept. ± out patient department c Toxins detection: in vivo-toxin A: (TCD, Becton Dickinson&CDA, Oxoid); in vitro-toxin A: TCD, Becton Dickinson; CDA, Oxoid; PCR; in vitro-toxin B: Mc Coy cell culture assay and PCR. b
18
G. Martirosian et al.
also among the strains isolated from stool samples with concordant results (in vivo and in vitro). Results are presented in Table 1 and Figure 1.
Discussion Clostridium difficile is the most common cause of pseudomembranous colitis (PMC), antibiotic associated diarrhoea (AAD) and colitis (AAC). This bacterium currently is the most frequently reported cause of nosocomial gastro-intestinal infectious disease, but it can be treated successfully with antibiotics not applied previously in the same patient [10]. Disease caused by C. difficile, may be associated with a spectrum of severity, ranging from mild diarrhoea to life-threatening and sometimes fatal PMC. Patients with AAD may have profuse diarrhoeae and abdominal pain and distension accompanied by nausea, fever and dehydration. Patients with PMC usually have more pronounced systemic syndromes. Sigmoidoscopy reveals characteristic raised yellow plaques-patognomic of C. difficile infection [17]. In our previous studies [5,7,15] we described different methods for diagnostic purposes and for comparison of C. difficile strains isolated from different sources in a single hospital unit. We observed similarity among isolated strains using AP-PCR and PCR ribotyping methods, indicating nosocomial spread of certain types of C. difficile. In the present study, we observed disagreement in C. difficile toxin A detection directly in faecal samples Ð in vivo and in isolated strains Ð in vitro by using rapid immunoassays. The sensitivity and specificity of these assays are dependent on a number of factors such as: liability of both toxins to degradation, thermal instability, concentration of the toxins in the specimens. Both rapid tests were performed according to the instructions of the manufacturer. Stool samples were tested as soon as possible upon receipt in the laboratory. The results of the present study suggest that a negative in vivo result does not exclude the presence of viable and toxin-producing C. difficile in patient's faeces. Toxin specific PCR testing efficiently revealed the presence of C. difficile toxin A and B genes. We decided to compare strains of C. difficile by using AP-PCR and PCR ribotyping in view of recently described new toxinotyping [18]. Authors performed PFGE typing of 25 C. difficile isolates, belonging to different toxinotypes and observed, that strains belonging to the same toxinotype usually had very similar or identical PFGE patterns (although differences among the same toxinotypes were described). For this purpose we chose 20 strains: nine with
Figure 1. AP-PCR and PCR ribotyping of C. difficile strains. DNA was amplified with primers AP3 and SP1 & SP2 respectively. Strains were derived from faeces of patients suspected for PMC, AAD or AAC. Numbering of the isolates is identical to that in Table 1. First and last lanes-molecular mass markers (100-bp ladder; Promega, Leiden, The Netherlands); the 800-nucleotide fragment is highlighted.
disagreements of in vivo and in vitro results and 11 with the same positive or negative in vivo and in vitro results. The results obtained by AP-PCR and PCR ribotyping revealed genetic heterogeneity among the strains isolated from in vivo-negative and in vitropositive toxin A samples. Similarity was not observed also among the strains isolated from in vivo and in vitro concordant, positive or negative stool samples.
Rapid Immunoassays for in Vivo Detection of Toxin A In conclusion, it can be stated that C. difficile toxin detection in vivo (in faecal samples) is important for rapid diagnosis of C. difficile-associated diseases. However, cultivation of anaerobes on selective media and in vitro toxin detection seems to be useful for confirmation of diagnosis, especially when direct faecal results are negative and the patient has a clear manifestation of C. difficile-associated diseases. Toxin gene detection by PCR is the best method for confirmation of toxigenic C. difficile and is concordant with cytotoxicity assay and other tests. Genotyping by AP-PCR and PCR ribotyping did not show similarity among the C. difficile strains isolated from in vivo negative and in vitro positive toxin A samples. We observed 15 different genotypes (excluding 4 subtypes) among 20 investigated strains. It could be interesting to further screen these C. difficile isolates for changes in the genes coding for toxin A and toxin B. Acknowledgements Appreciation is expressed to Lidia Licciardello for her assistance in AP-PCR and PCR ribotyping. This work was supported in part by KBN Grant nr. 1147/P05/97/12.
References 1. Mastrantonio P., Pantosti A., Cequetti M., Moliari A. and Donelli G. (1996). C. difficile: an update on virulence mechanisms. Anaerobe 2: 337±340 2. Bartlett J.G. (1992) Antibiotic-associated diarrhoea. Clin Inf Dis 12: 243±251 3. Fiorentini C., Malorni W., Paradisi S., Guiliano M., Mastrantonio P. and Donelli G. (1990) Interaction of C. difficile toxin A with cultured cells: cytosceletal changes and nuclear polarization. Infect Immun 58: 2329±2336
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4. Borriello S.P. (1998) Pathogenesis of Clostridium difficile infection. JAC 41: 13±19 5. Meisel-Mikolajczyk F., Martirosian G., Marianowski L., Dworczynska M. and Cwyl-Zembrzurska L. (1992). C. difficile in a maternity hospital. Int J Feto-Maternal Med 5: 173±177 6. George R.H., Sutter V.L., Citron D., Finegold S.M. (1979) Selective and differential medium for isolation of C. difficile. J Clin Microbiol 9: 214±219 7. Martirosian G., Kuipers S., Verbrugh H., van Belkum A. and Meisel-Mikolajczyk F. (1995) PCR ribotyping and arbitrarily primed PCR for typing strains of C. difficile from a polish maternity hospital. J Clin Microbiol 33: 2016±2021 8. Meisel-Mikolajczyk F., Martirosian G., Tang Y. and Silva Jr. J. (1997) Genotyping of C. difficile isolates from a hospital in Warsaw: a preliminary study. Int J Infect Dis 2: 88±90 9. Martirosian G. (1997) C. difficile: epidemiology, diagnostics. Post. Mikrobiol 4: 407±418 (in Polish) 10. Brazier J.S. (1998) The epidemiology and typing of C. difficile. JAC 41: 47±57 11. Rothman S.W. (1986) Technique for measuring 50% end points in cytotoxicity assay for C. difficile toxins. J Clin Pathol 39: 672±676 12. Borriello S.P., Wren B.W., Hyde S., Seddon S.V., Sibbons P., Krishna M.M., Tabaqchali S., Manek S. and Price A.B. (1992) Molecular, immunological and biological characterization of a toxin A-negative, toxin B-positive strain of C. difficile. Infect Immun 60: 4192±4199 13. Gumerlock P.H., Tang Y.J., Weiss J.B., Silva Jr. J. (1993) Specific detection of toxigenic strains of C. difficile in stool specimens. J Clin Microbiol 31: 507±511 14. Tang T.J., Gumerlock P.H., Weiss J.B., Silva Jr. J. (1994) Specific detection of C. difficile toxin A gene sequences in clinical isolates. Mol Cell Probes 8: 463±467 15. Pituch H., Obuch-WoszczatynÂski P., Rouyan Gh., Martirosian G. and Meisel-Mikoøajczyk F. (1998) Detection of toxin producing C. difficile using rapid diagnostic methods. Med Dosw Mikrobiol 50: 55±61 (in Polish) 16. Van Belkum A. (1994) DNA fingerprinting of medically important microorganisms by use of PCR. Clin Microbiol Rev 7: 174±178 17. Tabaqchali S. and Jumaa P. (1995) Diagnosis and management of Clostridium difficile infection. BMJ 310: 1375±1380 18. Rupnik M., Avensani V., Janc M., von Eichel-Streiber C. and Delmee M. (1998) A novel toxinotyping scheme and correlation of toxinotypes with serogroups of Clostridium difficile isolates. J Clin Microbiol 36: 2240±2247
CLINICAL MICROBIOLOGY
Are Rapid Immunoassays for in vivo Detection of Toxin A Su¤cient for Diagnostic Purposes of Clostridium di¤cile-Associated Diseases? Gayane Martirosian1*, Alex vanBelkum2, Hanna Pituch1, Piotr Obuch-Woszczatynèski1 and Felicja Meisel-Mikolajczyk1 1
Department of Medical Microbiology Institute of Biostructure, Warsaw University Medical School, 5 Chalubinski str. 02-004 Warsaw, Poland; 2 Department of Medical Microbiology & Infectious Diseases, Erasmus University Medical Center Rotterdam EMCR Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands (Received 3 March 1999; revised 7 September, 1999; accepted in revised form 21 October 1999)
One hundred and fifty-five stool specimens of patients suspected for Clostridium difficile-associated diarrhoea, colitis or pseudomembranous colitis (PMC) were investigated. All patients were pre-treated with antibiotics, suffered from watery diarrhoea and abdominal pain and were hospitalized in different hospital units. Units varied from departments of surgery, internal medicine, intensive care, paediatry, dermatology, orthopaedy to gastroenterology. Fifty C. difficile strains were isolated from the faecal samples. Clostridium difficile toxin detection was done directly in the stool samples, and also in cultured C. difficile strains (in vivo and in vitro, respectively). We observed clear differences between in vivo and in vitro toxin A detection by using commercial rapid immuno-enzymatic tests: from 25 in vivo toxin A-negative samples, 17 were positive in vitro. This observation suggests that culturing of C. difficile on selective medium is mandatory for adequate toxin detection and necessary for confirming the presence of toxin-producing C. difficile. This is especially important among patients with clinical symptoms and history of pretreatment with antibiotics and when in vivo toxin A detection is negative. It was established that toxin gene detection by PCR is optimal and PCR results were concordant with results of other in vitro assays. Genotyping by using AP-PCR and PCR ribotyping showed heterogeneity among the toxigenic C. difficile strains cultured from in vivo toxin A-negative stool samples. # 2000 Academic Press
Introduction Clostridium difficile is a Gram-positive anaerobic bacterium. Its pathogenicity is associated with the
*Corresponding author. Tel/Fax: (48-22)628-27-39. E-mail: [email protected]
1075±9964/00/010015 + 05 $35.00/0
production of two protein toxins: enterotoxin A and cytotoxin B [1,2]. Toxin A is inducing epithelial damage, fluid secretion and hemorrhage when injected into rabbit ileal loops. Toxin B is an approximately 1000-fold more potent cytotoxin than toxin A [3,4]. Toxins A and B of C. difficile act in concert, causing as a result changes in gut cyto-skeleton and epithelial fluid secretion. Toxigenic strains of # 2000 Academic Press
16
G. Martirosian et al.
C. difficile are known as an etiological agent of pseudomembranous colitis (PMC), antibiotic-associated diarrhoea (AAD) or colitis (AAC) [4,5]. On the other hand asymptomatic colonization is found in approximatly 3% of healthy adults (the number is much higher in hospitalized adults, who have been on antimicrobial therapy) and in the order of approximately 65% of healthy neonates. Recently, toxigenic C. difficile strains were described as predominant pathogen of hospital-acquired intestinal infections. Nosocomial outbreaks of C. difficile-associated diarrhoea were described in different hospital units. Also cross-infections were described and the role of hospital environment in transmission of C. difficile strains was documented. Cultivation of C. difficile strains is not difficult since selective medium was described [6]. Different typing schemes are available for study of epidemiology of C. difficile-associated infection. Molecular typing by using different PCR assays appears to be a fast and useful method for this purpose [7±10]. Measuring the cytopoathic effect on McCoy cells is a well known ``gold standard'' technique for toxigenicity study of C. difficile strains [11], but this method requires special equipment for cell culture and takes time. Many rapid immunoassays for detection of C. difficile toxins are available on the market. Especially toxin A assays are produced by different pharmaceutical companies, since this toxin is the more stable one. Most of these commercial assays are characterised by sufficient sensitivity and specificity levels. Rapid detection of toxins of C. difficile is very important for clinical reasons. Until the end of the eighties, detection of one C. difficile toxin in vivo was sufficient for diagnosis of C. difficile-associated diseases. However, the description of a strain lacking of the toxin A gene but harbouring a full toxin B gene and still highly virulent in animal model, suggests of necessity to detect both toxins of C. difficile in stool samples [12]. The aim of the present study was to assess the efficacy of in vivo versus in vitro testing for the presence of toxins of C. difficile by using different methods (rapid immuno-assays, cell culture assay and PCR). Finally, 20 strains were compared by AP-PCR and PCR ribotyping to define the existence of similarity among C. difficile strains, cultured from in vivo toxin A-negative and in vitro positive stool samples.
Materials and Methods Clinical samples Stool samples (n=155) of 155 patients, suspected for C. difficile-associated diarrhoea or colitis were
investigated. Patients were hospitalized in departments of surgery, internal medicine, paediatry, dermatology, orthopaedy or gastroenterology. A few patients were treated in intensive care units or the outpatients' department. Any other enteric pathogens were documented in these stool samples at the moment of testing for C. difficile and its toxins. Bacterial strains Fifty C. difficile strains were isolated from faecal samples of the patients mentioned above. In addition, two reference C. difficile strains were induced, a toxigenic one possessing both the toxin A and B genes (VPI 10463) and a non-toxigenic strain (NIH BRIGGS 8050). Isolation of bacterial strains. Faecal samples were cultured on the selective medium Columbia-cycloserinecefoxitin-amphotericin B agar (CCCA; bioMerieux, France), as described priviously [7]. Isolates were identified as C. difficile by characteristic morphology of colonies, green-yellow fluorescence under UV light and biochemical activity (API-20A, bioMerieux, Marcy-l' Etoile, France). Clostridium difficile toxins detection in vivo and in vitro Clostridium difficile toxins were detected directly in faecal samples (``in vivo'') and in isolated strains (``in vitro''). Toxin A detection in vivo. For the direct detection of toxin A in faecal samples the following assays were used: (1) ``Culturette Brand Toxin CD'' (BectonDickinson, Meylan, France) enzyme immunoassay; (2) ``C. difficile toxin A test'' (Oxoid, Unipath, Wesel, Germany) rapid immunoassay. Both immunoassays were performed according to the instructions of the manufacturer. Stool samples were tested as soon as possible upon receipt in the laboratory; rest of samples was stored at 7708C for further investigation. Toxin A detection in vitro: For detection of toxin A in isolated strains, the following methods were used: (1) Two rapid immunoassays: (a) ``Culturette Brand Toxin CD'' (Becton-Dickinson, Meylan, France); (b) ``C. difficile toxin A test'' (Oxoid, Unipath, Wesel, Germany); (2) PCR by using YT28 and YT29 primers combination [13,14]. Toxin B detection in vitro: (1) Cytotoxicity assay on McCoy cells. The cytopathic effect of bacterial culture supernatants (diluted 1071±1078) on McCoy cells was used for detection of toxin B [9,15]. (2) PCR by using
Rapid Immunoassays for in Vivo Detection of Toxin A YT18 and YT19 primers combination [13,14]. A protocol for PCR detection of both the C. difficile toxin A and B genes using primer combinations YT28&YT29 and YT18&YT19 were described previously [13,14]. Genotyping of Isolates by Arbitrary Primed PCR and PCR-ribotyping Arbitrary primed PCR (AP-PCR) and PCR ribotyping of the 20 C. difficile isolates and two reference strains were performed with use of the arbitrary oligonucleotides AP-2, AP-3, ERIC1/ERIC2 and SP1, SP2 as described previously [7,15,16].
Results Stool samples of 155 patients hospitalized in different hospital units were investigated for the presence of C. difficile and either of its toxins in vivo and in vitro. Directly in faecal material, toxin A was detected in 22 cases by using rapid immunoassays, 133 samples were toxin A negative. Stool samples were tested as soon as possible upon receipt in the laboratory and the rest of samples was stored at 7708C for further investigation. Both immunoassays were performed according to the instructions of manufacturer. Fifty C.
17
difficile strains were isolated from the faecal samples. These strains were derived from the 22 in vivo toxin Apositive faecal samples, and 28 in vivo toxin Anegative samples. All 50 C. difficile strains were studied for in vitro toxin A and B production (using the same rapid immunoassays, cytotoxicity assay and PCR) and the results were compared with results, obtained from in vivo testing. In 19 cases disagreement was observed: 17 samples toxin A-negative in vivo gave positive results in vitro; and in two cases toxin A was detected in vivo (in faecal samples) and not detected in vitro. In vivo toxin A detection was not concordant with in vitro data, when rapid immunoassays were performed. The PCR results of in vitro toxin A and toxin B gene detection were fully concordant with in vitro toxin detection results, obtained by using other assays: all toxin positive strains in the in vitro assays were positive in PCR for both toxin A and B genes. Twenty C. difficile strains, isolated from different stool samples (nine with disagreements of in vivo and in vitro toxin A detection and 11 with same positive or negative in vivo and in vitro results) were chosen for comparison by AP-PCR and PCR ribotyping. The results obtained by AP-PCR and PCR ribotyping revealed genetic heterogeneity among the strains isolated from in vivo negative and in vitro positive toxin A stool samples. Similarity was not observed
Table 1. Comparison of C. difficile strains by AP-PCR and PCR-ribotyping No
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. Number of types per assay
No of patient 772 701 640 2477 2649 2523 2442 1236 2785 582 1461 428 812 1259 2242 2203 1605 574 143 1720
Sex/Age
F/6 F/53 M/30 F/43 F/6 F/38 F/4 F/52 M/2 M/71 F/53 M/46 F/35 F/32 M/4 M/3 M/48 F/42 F/64 F/42
Hospital ward b
o.p.dept o.p.dept surgical surgical paediatr ICU paediatr o.p.dept paediatr internal haematol transpl. o.p.dept haematol paediatr paediatr o.p.dept internal gastroen internal
Tox. Ac in vivo
Toxins in vitro
Ribosomal spacer
nd 7 7 7 7 7 7 7 7 7 7 7
7 7 7 7 7 7
AP-PCR
Overall typea
AP2
AP3
ERIC1/2
A B C D E F G B H I I L B I M A N I I C
A B A nd nd C D nd D D nd E F A G A H A I A
A B C D E F G H G I I L B M G N O P Q R
A B C B D E C B F G H I A H J A L H M N
I II III IV V VI VII IIa VIII IX IXa X IIb XI XII Ia XIII IXb XIV XV
13
10
16
13
15 (excl. 4 subtypes)
a
Subtypes are defined in case strains share two or three identical types when the different PCR assays are concerned. (nd ± not done) o.p.dept. ± out patient department c Toxins detection: in vivo-toxin A: (TCD, Becton Dickinson&CDA, Oxoid); in vitro-toxin A: TCD, Becton Dickinson; CDA, Oxoid; PCR; in vitro-toxin B: Mc Coy cell culture assay and PCR. b
18
G. Martirosian et al.
also among the strains isolated from stool samples with concordant results (in vivo and in vitro). Results are presented in Table 1 and Figure 1.
Discussion Clostridium difficile is the most common cause of pseudomembranous colitis (PMC), antibiotic associated diarrhoea (AAD) and colitis (AAC). This bacterium currently is the most frequently reported cause of nosocomial gastro-intestinal infectious disease, but it can be treated successfully with antibiotics not applied previously in the same patient [10]. Disease caused by C. difficile, may be associated with a spectrum of severity, ranging from mild diarrhoea to life-threatening and sometimes fatal PMC. Patients with AAD may have profuse diarrhoeae and abdominal pain and distension accompanied by nausea, fever and dehydration. Patients with PMC usually have more pronounced systemic syndromes. Sigmoidoscopy reveals characteristic raised yellow plaques-patognomic of C. difficile infection [17]. In our previous studies [5,7,15] we described different methods for diagnostic purposes and for comparison of C. difficile strains isolated from different sources in a single hospital unit. We observed similarity among isolated strains using AP-PCR and PCR ribotyping methods, indicating nosocomial spread of certain types of C. difficile. In the present study, we observed disagreement in C. difficile toxin A detection directly in faecal samples Ð in vivo and in isolated strains Ð in vitro by using rapid immunoassays. The sensitivity and specificity of these assays are dependent on a number of factors such as: liability of both toxins to degradation, thermal instability, concentration of the toxins in the specimens. Both rapid tests were performed according to the instructions of the manufacturer. Stool samples were tested as soon as possible upon receipt in the laboratory. The results of the present study suggest that a negative in vivo result does not exclude the presence of viable and toxin-producing C. difficile in patient's faeces. Toxin specific PCR testing efficiently revealed the presence of C. difficile toxin A and B genes. We decided to compare strains of C. difficile by using AP-PCR and PCR ribotyping in view of recently described new toxinotyping [18]. Authors performed PFGE typing of 25 C. difficile isolates, belonging to different toxinotypes and observed, that strains belonging to the same toxinotype usually had very similar or identical PFGE patterns (although differences among the same toxinotypes were described). For this purpose we chose 20 strains: nine with
Figure 1. AP-PCR and PCR ribotyping of C. difficile strains. DNA was amplified with primers AP3 and SP1 & SP2 respectively. Strains were derived from faeces of patients suspected for PMC, AAD or AAC. Numbering of the isolates is identical to that in Table 1. First and last lanes-molecular mass markers (100-bp ladder; Promega, Leiden, The Netherlands); the 800-nucleotide fragment is highlighted.
disagreements of in vivo and in vitro results and 11 with the same positive or negative in vivo and in vitro results. The results obtained by AP-PCR and PCR ribotyping revealed genetic heterogeneity among the strains isolated from in vivo-negative and in vitropositive toxin A samples. Similarity was not observed also among the strains isolated from in vivo and in vitro concordant, positive or negative stool samples.
Rapid Immunoassays for in Vivo Detection of Toxin A In conclusion, it can be stated that C. difficile toxin detection in vivo (in faecal samples) is important for rapid diagnosis of C. difficile-associated diseases. However, cultivation of anaerobes on selective media and in vitro toxin detection seems to be useful for confirmation of diagnosis, especially when direct faecal results are negative and the patient has a clear manifestation of C. difficile-associated diseases. Toxin gene detection by PCR is the best method for confirmation of toxigenic C. difficile and is concordant with cytotoxicity assay and other tests. Genotyping by AP-PCR and PCR ribotyping did not show similarity among the C. difficile strains isolated from in vivo negative and in vitro positive toxin A samples. We observed 15 different genotypes (excluding 4 subtypes) among 20 investigated strains. It could be interesting to further screen these C. difficile isolates for changes in the genes coding for toxin A and toxin B. Acknowledgements Appreciation is expressed to Lidia Licciardello for her assistance in AP-PCR and PCR ribotyping. This work was supported in part by KBN Grant nr. 1147/P05/97/12.
References 1. Mastrantonio P., Pantosti A., Cequetti M., Moliari A. and Donelli G. (1996). C. difficile: an update on virulence mechanisms. Anaerobe 2: 337±340 2. Bartlett J.G. (1992) Antibiotic-associated diarrhoea. Clin Inf Dis 12: 243±251 3. Fiorentini C., Malorni W., Paradisi S., Guiliano M., Mastrantonio P. and Donelli G. (1990) Interaction of C. difficile toxin A with cultured cells: cytosceletal changes and nuclear polarization. Infect Immun 58: 2329±2336
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4. Borriello S.P. (1998) Pathogenesis of Clostridium difficile infection. JAC 41: 13±19 5. Meisel-Mikolajczyk F., Martirosian G., Marianowski L., Dworczynska M. and Cwyl-Zembrzurska L. (1992). C. difficile in a maternity hospital. Int J Feto-Maternal Med 5: 173±177 6. George R.H., Sutter V.L., Citron D., Finegold S.M. (1979) Selective and differential medium for isolation of C. difficile. J Clin Microbiol 9: 214±219 7. Martirosian G., Kuipers S., Verbrugh H., van Belkum A. and Meisel-Mikolajczyk F. (1995) PCR ribotyping and arbitrarily primed PCR for typing strains of C. difficile from a polish maternity hospital. J Clin Microbiol 33: 2016±2021 8. Meisel-Mikolajczyk F., Martirosian G., Tang Y. and Silva Jr. J. (1997) Genotyping of C. difficile isolates from a hospital in Warsaw: a preliminary study. Int J Infect Dis 2: 88±90 9. Martirosian G. (1997) C. difficile: epidemiology, diagnostics. Post. Mikrobiol 4: 407±418 (in Polish) 10. Brazier J.S. (1998) The epidemiology and typing of C. difficile. JAC 41: 47±57 11. Rothman S.W. (1986) Technique for measuring 50% end points in cytotoxicity assay for C. difficile toxins. J Clin Pathol 39: 672±676 12. Borriello S.P., Wren B.W., Hyde S., Seddon S.V., Sibbons P., Krishna M.M., Tabaqchali S., Manek S. and Price A.B. (1992) Molecular, immunological and biological characterization of a toxin A-negative, toxin B-positive strain of C. difficile. Infect Immun 60: 4192±4199 13. Gumerlock P.H., Tang Y.J., Weiss J.B., Silva Jr. J. (1993) Specific detection of toxigenic strains of C. difficile in stool specimens. J Clin Microbiol 31: 507±511 14. Tang T.J., Gumerlock P.H., Weiss J.B., Silva Jr. J. (1994) Specific detection of C. difficile toxin A gene sequences in clinical isolates. Mol Cell Probes 8: 463±467 15. Pituch H., Obuch-WoszczatynÂski P., Rouyan Gh., Martirosian G. and Meisel-Mikoøajczyk F. (1998) Detection of toxin producing C. difficile using rapid diagnostic methods. Med Dosw Mikrobiol 50: 55±61 (in Polish) 16. Van Belkum A. (1994) DNA fingerprinting of medically important microorganisms by use of PCR. Clin Microbiol Rev 7: 174±178 17. Tabaqchali S. and Jumaa P. (1995) Diagnosis and management of Clostridium difficile infection. BMJ 310: 1375±1380 18. Rupnik M., Avensani V., Janc M., von Eichel-Streiber C. and Delmee M. (1998) A novel toxinotyping scheme and correlation of toxinotypes with serogroups of Clostridium difficile isolates. J Clin Microbiol 36: 2240±2247