- Email: [email protected]
Diagnostic accuracy of a multiplex real-time PCR to predict Clostridium difficile ribotype 027
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Anaerobe xxx (2013) 1–3
Contents lists available at SciVerse ScienceDirect
Anaerobe journal homepage: www.elsevier.com/locate/anaerobe
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55
Letter to the Editor
56 57 58 59 60 61 form each specimen was frozen at 80 C for further analysis (see 62 below). 63 To confirm all positive GeneXpertÒ specimens, samples were 64 enriched for toxigenic culture as previously described [15]. All as65 says were done three independent times. The PCR ribotype of 66 each specimen was determined by performing PCR on DNA 67 from BHI culture and analyzed as previously described [15]. To 68 quantify the citotoxicity of presumptive ribotype 027 specimens, 69 a Vero cell toxicity assay was done as mentioned elsewhere 70 [15]. The toxin end-point titre was determined by measuring 71 the cytopathic effect of the highest dilution with a citopathic ef72 fect >50%, and calculated by the following formula: end-point 73 titre ¼ 1/[(percentage of rounded cells of the highest dilution at 74 which >50% of the cells are round) (the highest dilution at 75 which >50% of the cells are rounded)]. All assays were done three 76 independent times. 77 A total of 75 stool samples collected between August 2011 to 78 June 2012 were positive for C. difficile by PCR GeneXpertÒ and all 79 were confirmed to be positive for C. difficile by toxigenic culture. 80 Of these, 18 (24%) were designated “presumptive positive for ribo81 type 027” by GeneXpertÒ (Table 1). All 18 isolates were positive for 82 CDT. However, when the 18 “presumptive positive ribotype 027” 83 samples were analyzed by PCR ribotyping, 10 (56%) were confirmed 84 to be ribotype 027 (Fig. 1). Of the remaining 57 C. difficile positive 85 specimens analyzed by PCR ribotyping, none was identified as ribo86 type 027 (Table 1 and data not shown). In summary, the analysis of 87 the PCR GeneXpertÒ results indicates that it has a sensitivity of 88 100% and a specificity of 88%. The positive predictive value (PPV) 89 and negative predictive value (NPV) for presumptive ribotype 027 90 were estimated in 56% and 100%, respectively. 91 We evaluated if the deletion in tcdC detected by GeneXpertÒ in 92 the 18 presumptive ribotype 027 specimens correlated with 93 elevated cytotoxicity in Vero cells. Two of all 10 truly PCR ribotype 94 027 specimens had slightly lower cytotoxicity than the epidemic 95 reference C. difficile strain R20291 (ribotype 027) but this was Q1 96 higher than that of the historical C. difficile strain 630 (ribotype 97 012), known to be a low toxin producer strain (Fig. 1A,B). Two of 98 the 8 non-ribotype 027 strains, but presumptive for a deletion in 99 tcdC, produced slightly lower cytotoxicity in Vero cells than the 100 reference strain R20291 (ribotype 027), while the remaining non101 ribotype 027 that are presumptive for a deletion in tcdC produced 102 similar or slightly higher cytotoxic levels in Vero cells than strain 103 R20291 (ribotype 027) (Fig. 1A,B). 104 In response to the rapid emergence of high toxin producing 105 strains, faster detection methods have been developed. It is 106 notable that the PCR-based GeneXpertÒ assay not only detects 107 the presence of C. difficile but also predicts if the C. difficile 108 109 110
Diagnostic accuracy of a multiplex real-time PCR to predict Clostridium difficile ribotype 027 Keywords: Clostridium difficile Ribotyping Real time PCR
Clostridium difficile infections (CDI) have become a major concern in hospital settings, were they account for nearly 20% of antibiotic associated diarrhea with mortality and relapse rates of w5 and 20%, respectively [1–3]. Recent epidemiological changes of CDI have been associated with the appearance of epidemic PCR ribotype 027 [4]. Outbreaks caused by this ribotype 027 have mortality and relapse rates that can reach up to w15 and 30%, respectively [5,6]. Recent developments of rapid and sensitive alternatives for the detection of C. difficile in stools are based on real-time PCR technology [7–10], which have the ability to detect the specific mutations including the well conserved deletion of 18-bp and the mutation at position 117 of the tcdC gene, allowing an early detection of the epidemic PCR ribotype 027 [11]. A recently available PCR commercial kit (GeneXpertÒ, Cepheid, Sunnyvale, CA) is able to identify not only the presence of toxigenic C. difficile by using specific probes against tcdB, but is also able to detect the deletion in tcdC and the binary toxin genes (cdtA and cdtB), thus, allowing rapid identification of potential epidemic ribotype 027 in clinical settings. Few studies have evaluated the performance of GeneXpertÒ in fresh stools specimens primarily due to the low prevalence of this clinically relevant ribotype [12–14]. Therefore, the aim of this study was to investigate the ability of GeneXpertÒ to identify PCR ribotype 027 from stool samples of patients with CDI symptoms. This study was approved by the Bioethics Committee of Hospital Clínico de la Pontificia Universidad Católica de Chile. The clinical setting was Hospital Clínico de la Pontificia Universidad Católica de Chile, a University tertiary-level health center, with 450 beds, of which 60 belong to critical care services. Samples were collected during an 11 month study period (August 2011–June 2012). Liquid or soft stool specimens from hospitalized patients suspected of having CDI were prospectively subjected to diagnostic test by multiplex real time PCR GeneXpertÒ C. dificile (Cepheid, CA, Sunnyvale, U.S.A.). All samples were tested within 24 h of receipt with PCR GeneXpertÒ C. difficile assay (Cepheid, Sunnyvale, U.S.A.) being part of the hospital’s routine diagnostic routine, which was performed directly on stool specimens as described by manufacturer’s instructions. In addition, 1-ml aliquot 1075-9964/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.anaerobe.2013.04.002
Please cite this article in press as: Barra-Carrasco J, et al., Diagnostic accuracy of a multiplex real-time PCR to predict Clostridium difficile ribotype 027, Anaerobe (2013), http://dx.doi.org/10.1016/j.anaerobe.2013.04.002
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Letter to the Editor / Anaerobe xxx (2013) 1–3
Table 1 Discordant analysis of “presumptive ribotype 027” detected by GeneXpertÒ. Parameter
True positive (no.) True negative (no.) False positive (no.) False negative (no.) Sensitivity (%) Specificity (%) PPV (%)b NPV (%)c a b c d
Result of GeneXpertÒ and PCR Ribotypinga PCR GeneXpertÒ
PCR Ribotyping
10 57 8 0 100 88 56 100
18 57 0 0 100 100d 100d 100d
n ¼ 75. PPV: Positive Predictive Value. NPV: Negative Predictive Value. P < 0.05 (Fisher test).
isolate(s) present in the stool sample belongs to the epidemic ribotype 027. Although our work reports good sensitivity, specificity and NPV, we observed a low PPV (56%), which was much lower
than that observed by Huang et al. (86%) [12]. However, several differences might explain the difference in PPV values observed between both studies: i) differences in sample populations which could be associated with prevalence of different ribotypes; ii) in one sample population in Huang et al. [12], the prevalence could have been overestimated due to the inclusion of several positive samples which might have affected the PPV. Our results demonstrate that PCR GeneXpertÒ did not fail to detect clinical isolates that were highly cytotoxic, because although they did classify as “presumptive ribotype 027” some clinical isolates clearly do not belong to this ribotype. This is expected since the tcdC deletion(s) is not unique to ribotype 027 but may also be present in other PCR ribotypes and therefore affects the real-time PCR amplification curve [16]. A recent study also demonstrated that three additional mutations could alter the PCR amplification curve of tcdC, which may also lead to “presumptive ribotype 027” results [14]. In conclusion, although we did not further confirm this deletion, we suggest that specimens with a “presumptive ribotype 027” by PCR GeneXpertÒ should be treated as specimens able to produce high toxin titres and ensure that adequate treatment should follow to safeguard the patient’s health.
Fig. 1. Results of in vitro cytotoxicity and PCR ribotyping of the presumptive positive “Ribotype 027” by GeneXpertÒ. A) Monolayers of Vero cells were infected with filteredsterilized culture supernatant of each “presumptive ribotype 027” detected by GeneXpertÒ and incubated for 24 h and toxin end-point titre was measured as described in methods. Values represent the mean of three independent experiments and error bars are standard errors of the mean. B), PCR ribotyping results of the C. difficile clinical isolates show that the band pattern of the ribosomal intergenic space regions of several clinical strains are identical to those of the reference strain, R20291. R, indicates strain R20291. Positive and negative symbols indicate that the band pattern is identical or different to R.
Please cite this article in press as: Barra-Carrasco J, et al., Diagnostic accuracy of a multiplex real-time PCR to predict Clostridium difficile ribotype 027, Anaerobe (2013), http://dx.doi.org/10.1016/j.anaerobe.2013.04.002
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Letter to the Editor / Anaerobe xxx (2013) 1–3
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Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments This work was supported by grants from MECESUP UAB0802, Comisión Nacional de Investigación en Ciencia y Tecnologia (FONDECYT REGULAR 1110569) and from the Research Office of Universidad Andres Bello (DI-35-11/R) (to D.P.-S); by grants from FONDECYT REGULAR 1100971 to M.A.-L.; and from the Medical Research Center, Facultad de Medicina of the Pontificia Universidad Católica de Chile, Resident Research Project (PG-20/11) to C. H.-R.
References [1] Zilberberg MD, Shorr AF, Kollef MH. Increase in adult Clostrdium difficilerelated hospitalizations and case-fatality rate, United States, 2000–2005. Emerg Infect Dis 2008;14:929–31. [2] Karas JA, Enoch DA, Aliyu SH. A review of mortality due to Clostridium difficile infection. J Infect 2010;61:1–8. [3] Deneve C, Janoir C, Poilane I, Fantinato C, Collignon A. New trends in Clostridium difficile virulence and pathogenesis. Int J Antimicrob Agents 2009;33(Suppl. 1):S24–8. [4] O’Connor JR, Johnson S, Gerding DN. Clostridium difficile infection caused by the epidemic BI/NAP1/027 strain. Gastroenterology 2009;136:1913–24. [5] Pepin J, Alary ME, Valiquette L, Raiche E, Ruel J, Fulop K, et al. Increasing risk of relapse after treatment of Clostridium difficile colitis in Quebec, Canada. Clin Infect Dis 2005;40:1591–7. [6] Kuijper EJ, Coignard B, Tull P. Emergence of Clostridium difficileassociated disease in North America and Europe. Clin Microbiol Infect 2006;12(Suppl. 6):2–18. [7] Belanger SD, Boissinot M, Clairoux N, Picard FJ, Bergeron MG. Rapid detection of Clostridium difficile in feces by real-time PCR. J Clin Microbiol 2003;41:730–4. [8] Peterson LR, Manson RU, Paule SM, Hacek DM, Robicsek A, Thomson Jr RB, et al. Detection of toxigenic Clostridium difficile in stool samples by real-time polymerase chain reaction for the diagnosis of C. difficile-associated diarrhea. Clin Infect Dis 2007;45:1152–60. [9] Stamper PD, Alcabasa R, Aird D, Babiker W, Wehrlin J, Ikpeama I, et al. Comparison of a commercial real-time PCR assay for tcdB detection to a cell culture cytotoxicity assay and toxigenic culture for direct detection of toxinproducing Clostridium difficile in clinical samples. J Clin Microbiol 2009;47: 373–8. [10] van den Berg RJ, Kuijper EJ, van Coppenraet LE, Claas EC. Rapid diagnosis of toxinogenic Clostridium difficile in faecal samples with internally controlled real-time PCR. Clin Microbiol Infect 2006;12:184–6. [11] McDonald LC, Killgore GE, Thompson A, Owens Jr RC, Kazakova SV, Sambol SP, et al. An epidemic, toxin gene-variant strain of Clostridium difficile. N Engl J Med 2005;353:2433–41. [12] Huang H, Weintraub A, Fang H, Nord CE. Comparison of a commercial multiplex real-time PCR to the cell cytotoxicity neutralization assay for diagnosis of Clostridium difficile infections. J Clin Microbiol 2009;47:3729–31. [13] Babady NE, Stiles J, Ruggiero P, Khosa P, Huang D, Shuptar S, et al. Evaluation of the Cepheid Xpert Clostridium difficile Epi assay for diagnosis of Clostridium difficile infection and typing of the NAP1 strain at a cancer hospital. J Clin Microbiol 2010;48:4519–24.
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292 293 294 295 296 297 298 299 300 301 Jonathan Barra-Carrasco Q2 302 303 Laboratorio de Mecanismos de Patogénesis Bacteriana, 304 Departamento de Ciencias Biológicas, Facultad de Ciencias Biológicas, 305 Universidad Andrés Bello, Santiago, Chile 306 307 Cristian Hernández-Rocha 308 Departamento de Gastroenterología, Facultad de Medicina, 309 Pontificia Universidad Católica de Chile, Santiago, Chile 310 Camila Miranda-Cárdenas 311 Laboratorio de Mecanismos de Patogénesis Bacteriana, 312 Departamento de Ciencias Biológicas, Facultad de Ciencias Biológicas, 313 Universidad Andrés Bello, Santiago, Chile 314 315 Manuel Álvarez-Lobos 316 Departamento de Gastroenterología, Facultad de Medicina, 317 Pontificia Universidad Católica de Chile, Santiago, Chile 318 319 Ana María Guzmán Durán** 320 Departamento de Laboratorio Clínico, Facultad de Medicina, 321 Pontificia Universidad Católica de Chile, Santiago, Chile 322 Daniel Paredes-Sabja* 323 Laboratorio de Mecanismos de Patogénesis Bacteriana, 324 Departamento de Ciencias Biológicas, Facultad de Ciencias Biológicas, 325 326 Universidad Andrés Bello, Santiago, Chile 327 Department of Biomedical Sciences, College of Veterinary Medicine, 328 Oregon State University, Corvallis, USA 329 330 * Corresponding author. Laboratorio de Mecanismos de 331 Patogénesis Bacteriana, Departamento de Ciencias Biológicas, 332 Universidad Andrés Bello, República 217, Santiago, Chile. 333 334 Tel.: þ56 02 770 3225. 335 E-mail address: [email protected] (D. Paredes-Sabja) 336 337 ** Corresponding author. Tel.: þ56 02 3543288. 338 E-mail address: [email protected] (A.M. Guzmán Durán) 339 340 11 December 2012 341 Available online xxx 342
[14] Kok J, Wang Q, Thomas LC, Gilbert GL. Presumptive identification of Clostridium difficile strain 027/NAP1/BI on Cepheid Xpert: interpret with caution. J Clin Microbiol 2011;49:3719–21. [15] Hernandez-Rocha C, Barra-Carrasco J, Alvarez-Lobos M, Paredes-Sabja D, Guzman-Duran AM. Prospective comparison of a commercial multiplex real-time polymerase chain reaction and an enzyme immunoassay with toxigenic culture in the diagnosis of Clostridium difficile-associated infections. Diagn Microbiol Infect Dis 2013;75:361–5. [16] Goorhuis A, Bakker D, Corver J, Debast SB, Harmanus C, Notermans DW, et al. Emergence of Clostridium difficile infection due to a new hypervirulent strain, polymerase chain reaction ribotype 078. Clin Infect Dis 2008;47:1162–70.
Please cite this article in press as: Barra-Carrasco J, et al., Diagnostic accuracy of a multiplex real-time PCR to predict Clostridium difficile ribotype 027, Anaerobe (2013), http://dx.doi.org/10.1016/j.anaerobe.2013.04.002
Anaerobe xxx (2013) 1–3
Contents lists available at SciVerse ScienceDirect
Anaerobe journal homepage: www.elsevier.com/locate/anaerobe
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55
Letter to the Editor
56 57 58 59 60 61 form each specimen was frozen at 80 C for further analysis (see 62 below). 63 To confirm all positive GeneXpertÒ specimens, samples were 64 enriched for toxigenic culture as previously described [15]. All as65 says were done three independent times. The PCR ribotype of 66 each specimen was determined by performing PCR on DNA 67 from BHI culture and analyzed as previously described [15]. To 68 quantify the citotoxicity of presumptive ribotype 027 specimens, 69 a Vero cell toxicity assay was done as mentioned elsewhere 70 [15]. The toxin end-point titre was determined by measuring 71 the cytopathic effect of the highest dilution with a citopathic ef72 fect >50%, and calculated by the following formula: end-point 73 titre ¼ 1/[(percentage of rounded cells of the highest dilution at 74 which >50% of the cells are round) (the highest dilution at 75 which >50% of the cells are rounded)]. All assays were done three 76 independent times. 77 A total of 75 stool samples collected between August 2011 to 78 June 2012 were positive for C. difficile by PCR GeneXpertÒ and all 79 were confirmed to be positive for C. difficile by toxigenic culture. 80 Of these, 18 (24%) were designated “presumptive positive for ribo81 type 027” by GeneXpertÒ (Table 1). All 18 isolates were positive for 82 CDT. However, when the 18 “presumptive positive ribotype 027” 83 samples were analyzed by PCR ribotyping, 10 (56%) were confirmed 84 to be ribotype 027 (Fig. 1). Of the remaining 57 C. difficile positive 85 specimens analyzed by PCR ribotyping, none was identified as ribo86 type 027 (Table 1 and data not shown). In summary, the analysis of 87 the PCR GeneXpertÒ results indicates that it has a sensitivity of 88 100% and a specificity of 88%. The positive predictive value (PPV) 89 and negative predictive value (NPV) for presumptive ribotype 027 90 were estimated in 56% and 100%, respectively. 91 We evaluated if the deletion in tcdC detected by GeneXpertÒ in 92 the 18 presumptive ribotype 027 specimens correlated with 93 elevated cytotoxicity in Vero cells. Two of all 10 truly PCR ribotype 94 027 specimens had slightly lower cytotoxicity than the epidemic 95 reference C. difficile strain R20291 (ribotype 027) but this was Q1 96 higher than that of the historical C. difficile strain 630 (ribotype 97 012), known to be a low toxin producer strain (Fig. 1A,B). Two of 98 the 8 non-ribotype 027 strains, but presumptive for a deletion in 99 tcdC, produced slightly lower cytotoxicity in Vero cells than the 100 reference strain R20291 (ribotype 027), while the remaining non101 ribotype 027 that are presumptive for a deletion in tcdC produced 102 similar or slightly higher cytotoxic levels in Vero cells than strain 103 R20291 (ribotype 027) (Fig. 1A,B). 104 In response to the rapid emergence of high toxin producing 105 strains, faster detection methods have been developed. It is 106 notable that the PCR-based GeneXpertÒ assay not only detects 107 the presence of C. difficile but also predicts if the C. difficile 108 109 110
Diagnostic accuracy of a multiplex real-time PCR to predict Clostridium difficile ribotype 027 Keywords: Clostridium difficile Ribotyping Real time PCR
Clostridium difficile infections (CDI) have become a major concern in hospital settings, were they account for nearly 20% of antibiotic associated diarrhea with mortality and relapse rates of w5 and 20%, respectively [1–3]. Recent epidemiological changes of CDI have been associated with the appearance of epidemic PCR ribotype 027 [4]. Outbreaks caused by this ribotype 027 have mortality and relapse rates that can reach up to w15 and 30%, respectively [5,6]. Recent developments of rapid and sensitive alternatives for the detection of C. difficile in stools are based on real-time PCR technology [7–10], which have the ability to detect the specific mutations including the well conserved deletion of 18-bp and the mutation at position 117 of the tcdC gene, allowing an early detection of the epidemic PCR ribotype 027 [11]. A recently available PCR commercial kit (GeneXpertÒ, Cepheid, Sunnyvale, CA) is able to identify not only the presence of toxigenic C. difficile by using specific probes against tcdB, but is also able to detect the deletion in tcdC and the binary toxin genes (cdtA and cdtB), thus, allowing rapid identification of potential epidemic ribotype 027 in clinical settings. Few studies have evaluated the performance of GeneXpertÒ in fresh stools specimens primarily due to the low prevalence of this clinically relevant ribotype [12–14]. Therefore, the aim of this study was to investigate the ability of GeneXpertÒ to identify PCR ribotype 027 from stool samples of patients with CDI symptoms. This study was approved by the Bioethics Committee of Hospital Clínico de la Pontificia Universidad Católica de Chile. The clinical setting was Hospital Clínico de la Pontificia Universidad Católica de Chile, a University tertiary-level health center, with 450 beds, of which 60 belong to critical care services. Samples were collected during an 11 month study period (August 2011–June 2012). Liquid or soft stool specimens from hospitalized patients suspected of having CDI were prospectively subjected to diagnostic test by multiplex real time PCR GeneXpertÒ C. dificile (Cepheid, CA, Sunnyvale, U.S.A.). All samples were tested within 24 h of receipt with PCR GeneXpertÒ C. difficile assay (Cepheid, Sunnyvale, U.S.A.) being part of the hospital’s routine diagnostic routine, which was performed directly on stool specimens as described by manufacturer’s instructions. In addition, 1-ml aliquot 1075-9964/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.anaerobe.2013.04.002
Please cite this article in press as: Barra-Carrasco J, et al., Diagnostic accuracy of a multiplex real-time PCR to predict Clostridium difficile ribotype 027, Anaerobe (2013), http://dx.doi.org/10.1016/j.anaerobe.2013.04.002
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Letter to the Editor / Anaerobe xxx (2013) 1–3
Table 1 Discordant analysis of “presumptive ribotype 027” detected by GeneXpertÒ. Parameter
True positive (no.) True negative (no.) False positive (no.) False negative (no.) Sensitivity (%) Specificity (%) PPV (%)b NPV (%)c a b c d
Result of GeneXpertÒ and PCR Ribotypinga PCR GeneXpertÒ
PCR Ribotyping
10 57 8 0 100 88 56 100
18 57 0 0 100 100d 100d 100d
n ¼ 75. PPV: Positive Predictive Value. NPV: Negative Predictive Value. P < 0.05 (Fisher test).
isolate(s) present in the stool sample belongs to the epidemic ribotype 027. Although our work reports good sensitivity, specificity and NPV, we observed a low PPV (56%), which was much lower
than that observed by Huang et al. (86%) [12]. However, several differences might explain the difference in PPV values observed between both studies: i) differences in sample populations which could be associated with prevalence of different ribotypes; ii) in one sample population in Huang et al. [12], the prevalence could have been overestimated due to the inclusion of several positive samples which might have affected the PPV. Our results demonstrate that PCR GeneXpertÒ did not fail to detect clinical isolates that were highly cytotoxic, because although they did classify as “presumptive ribotype 027” some clinical isolates clearly do not belong to this ribotype. This is expected since the tcdC deletion(s) is not unique to ribotype 027 but may also be present in other PCR ribotypes and therefore affects the real-time PCR amplification curve [16]. A recent study also demonstrated that three additional mutations could alter the PCR amplification curve of tcdC, which may also lead to “presumptive ribotype 027” results [14]. In conclusion, although we did not further confirm this deletion, we suggest that specimens with a “presumptive ribotype 027” by PCR GeneXpertÒ should be treated as specimens able to produce high toxin titres and ensure that adequate treatment should follow to safeguard the patient’s health.
Fig. 1. Results of in vitro cytotoxicity and PCR ribotyping of the presumptive positive “Ribotype 027” by GeneXpertÒ. A) Monolayers of Vero cells were infected with filteredsterilized culture supernatant of each “presumptive ribotype 027” detected by GeneXpertÒ and incubated for 24 h and toxin end-point titre was measured as described in methods. Values represent the mean of three independent experiments and error bars are standard errors of the mean. B), PCR ribotyping results of the C. difficile clinical isolates show that the band pattern of the ribosomal intergenic space regions of several clinical strains are identical to those of the reference strain, R20291. R, indicates strain R20291. Positive and negative symbols indicate that the band pattern is identical or different to R.
Please cite this article in press as: Barra-Carrasco J, et al., Diagnostic accuracy of a multiplex real-time PCR to predict Clostridium difficile ribotype 027, Anaerobe (2013), http://dx.doi.org/10.1016/j.anaerobe.2013.04.002
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Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments This work was supported by grants from MECESUP UAB0802, Comisión Nacional de Investigación en Ciencia y Tecnologia (FONDECYT REGULAR 1110569) and from the Research Office of Universidad Andres Bello (DI-35-11/R) (to D.P.-S); by grants from FONDECYT REGULAR 1100971 to M.A.-L.; and from the Medical Research Center, Facultad de Medicina of the Pontificia Universidad Católica de Chile, Resident Research Project (PG-20/11) to C. H.-R.
References [1] Zilberberg MD, Shorr AF, Kollef MH. Increase in adult Clostrdium difficilerelated hospitalizations and case-fatality rate, United States, 2000–2005. Emerg Infect Dis 2008;14:929–31. [2] Karas JA, Enoch DA, Aliyu SH. A review of mortality due to Clostridium difficile infection. J Infect 2010;61:1–8. [3] Deneve C, Janoir C, Poilane I, Fantinato C, Collignon A. New trends in Clostridium difficile virulence and pathogenesis. Int J Antimicrob Agents 2009;33(Suppl. 1):S24–8. [4] O’Connor JR, Johnson S, Gerding DN. Clostridium difficile infection caused by the epidemic BI/NAP1/027 strain. Gastroenterology 2009;136:1913–24. [5] Pepin J, Alary ME, Valiquette L, Raiche E, Ruel J, Fulop K, et al. Increasing risk of relapse after treatment of Clostridium difficile colitis in Quebec, Canada. Clin Infect Dis 2005;40:1591–7. [6] Kuijper EJ, Coignard B, Tull P. Emergence of Clostridium difficileassociated disease in North America and Europe. Clin Microbiol Infect 2006;12(Suppl. 6):2–18. [7] Belanger SD, Boissinot M, Clairoux N, Picard FJ, Bergeron MG. Rapid detection of Clostridium difficile in feces by real-time PCR. J Clin Microbiol 2003;41:730–4. [8] Peterson LR, Manson RU, Paule SM, Hacek DM, Robicsek A, Thomson Jr RB, et al. Detection of toxigenic Clostridium difficile in stool samples by real-time polymerase chain reaction for the diagnosis of C. difficile-associated diarrhea. Clin Infect Dis 2007;45:1152–60. [9] Stamper PD, Alcabasa R, Aird D, Babiker W, Wehrlin J, Ikpeama I, et al. Comparison of a commercial real-time PCR assay for tcdB detection to a cell culture cytotoxicity assay and toxigenic culture for direct detection of toxinproducing Clostridium difficile in clinical samples. J Clin Microbiol 2009;47: 373–8. [10] van den Berg RJ, Kuijper EJ, van Coppenraet LE, Claas EC. Rapid diagnosis of toxinogenic Clostridium difficile in faecal samples with internally controlled real-time PCR. Clin Microbiol Infect 2006;12:184–6. [11] McDonald LC, Killgore GE, Thompson A, Owens Jr RC, Kazakova SV, Sambol SP, et al. An epidemic, toxin gene-variant strain of Clostridium difficile. N Engl J Med 2005;353:2433–41. [12] Huang H, Weintraub A, Fang H, Nord CE. Comparison of a commercial multiplex real-time PCR to the cell cytotoxicity neutralization assay for diagnosis of Clostridium difficile infections. J Clin Microbiol 2009;47:3729–31. [13] Babady NE, Stiles J, Ruggiero P, Khosa P, Huang D, Shuptar S, et al. Evaluation of the Cepheid Xpert Clostridium difficile Epi assay for diagnosis of Clostridium difficile infection and typing of the NAP1 strain at a cancer hospital. J Clin Microbiol 2010;48:4519–24.
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292 293 294 295 296 297 298 299 300 301 Jonathan Barra-Carrasco Q2 302 303 Laboratorio de Mecanismos de Patogénesis Bacteriana, 304 Departamento de Ciencias Biológicas, Facultad de Ciencias Biológicas, 305 Universidad Andrés Bello, Santiago, Chile 306 307 Cristian Hernández-Rocha 308 Departamento de Gastroenterología, Facultad de Medicina, 309 Pontificia Universidad Católica de Chile, Santiago, Chile 310 Camila Miranda-Cárdenas 311 Laboratorio de Mecanismos de Patogénesis Bacteriana, 312 Departamento de Ciencias Biológicas, Facultad de Ciencias Biológicas, 313 Universidad Andrés Bello, Santiago, Chile 314 315 Manuel Álvarez-Lobos 316 Departamento de Gastroenterología, Facultad de Medicina, 317 Pontificia Universidad Católica de Chile, Santiago, Chile 318 319 Ana María Guzmán Durán** 320 Departamento de Laboratorio Clínico, Facultad de Medicina, 321 Pontificia Universidad Católica de Chile, Santiago, Chile 322 Daniel Paredes-Sabja* 323 Laboratorio de Mecanismos de Patogénesis Bacteriana, 324 Departamento de Ciencias Biológicas, Facultad de Ciencias Biológicas, 325 326 Universidad Andrés Bello, Santiago, Chile 327 Department of Biomedical Sciences, College of Veterinary Medicine, 328 Oregon State University, Corvallis, USA 329 330 * Corresponding author. Laboratorio de Mecanismos de 331 Patogénesis Bacteriana, Departamento de Ciencias Biológicas, 332 Universidad Andrés Bello, República 217, Santiago, Chile. 333 334 Tel.: þ56 02 770 3225. 335 E-mail address: [email protected] (D. Paredes-Sabja) 336 337 ** Corresponding author. Tel.: þ56 02 3543288. 338 E-mail address: [email protected] (A.M. Guzmán Durán) 339 340 11 December 2012 341 Available online xxx 342
[14] Kok J, Wang Q, Thomas LC, Gilbert GL. Presumptive identification of Clostridium difficile strain 027/NAP1/BI on Cepheid Xpert: interpret with caution. J Clin Microbiol 2011;49:3719–21. [15] Hernandez-Rocha C, Barra-Carrasco J, Alvarez-Lobos M, Paredes-Sabja D, Guzman-Duran AM. Prospective comparison of a commercial multiplex real-time polymerase chain reaction and an enzyme immunoassay with toxigenic culture in the diagnosis of Clostridium difficile-associated infections. Diagn Microbiol Infect Dis 2013;75:361–5. [16] Goorhuis A, Bakker D, Corver J, Debast SB, Harmanus C, Notermans DW, et al. Emergence of Clostridium difficile infection due to a new hypervirulent strain, polymerase chain reaction ribotype 078. Clin Infect Dis 2008;47:1162–70.
Please cite this article in press as: Barra-Carrasco J, et al., Diagnostic accuracy of a multiplex real-time PCR to predict Clostridium difficile ribotype 027, Anaerobe (2013), http://dx.doi.org/10.1016/j.anaerobe.2013.04.002