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Comparison of Multilocus Sequence Typing and the Xpert C. difficile/ Epi Assay for Identification of Clostridium difficile 027/NAP1/BI Tracy McMillen,a Mini Kamboj,b,c,d
N. Esther Babadya
Clinical Microbiology Service, Department of Laboratory Medicine,a Infection Control Service, Department of Medicine,b and Infectious Disease Service, Department of Medicine,c Memorial Sloan Kettering Cancer Center, New York, New York, USA; Department of Medicine, Weill Cornell Medical College, New York, New York, USAd
Clostridium difficile 027/NAP1/BI is the most common C. difficile strain in the United States. The Xpert C. difficile/Epi assay allows rapid, presumptive identification of C. difficile NAP1. We compared Xpert C. difficile/Epi to multilocus sequence typing for identification of C. difficile NAP1 and found “very good” agreement at 97.9% ( ⴝ 0.86; 95% confidence interval, 0.80 to 0.91).
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lostridium difficile is the most common bacterial cause of hospital-associated diarrhea, with an estimated 500,000 cases in 2011 (1). In the United States, C. difficile PCR ribotype 027/North American pulse-field gel electrophoresis (PFGE) type 1 (NAP1)/ restriction endonuclease analysis (REA) group BI (C. difficile 027/ NAP1/BI), is the most frequently isolated strain in community and healthcare-associated cases of C. difficile infection (CDI) (1, 2). C. difficile 027/NAP1/BI emerged as a hyper-virulent strain from its association with multiple outbreaks of severe CDI that were reported across North America and Europe (3–5). Identification of C. difficile 027/NAP1/BI has emerging implications for disease management and provides useful information for epidemiologic purposes. Molecular typing methods are crucial to understanding and monitoring circulating C. difficile strain types. Several methods are available for typing C. difficile, and each has its own set of advantages and limitations (6, 7). Multilocus sequence typing (MLST) functions by sequencing a 500- to 700-bp fragment of each of seven C. difficile housekeeping genes and detecting the presence of polymorphisms (8, 9). MLST sequence data are uploaded to the PubMLST database (http://pubmlst.org /cdifficile), and the resulting allelic combination is used to determine individual strain type (ST). PCR ribotyping of C. difficile is based on the amplification of DNA fragments in the heterogenous region between the 16S and 23S rRNA of the chromosome (10). The band pattern produced by these fragments allows comparison of unique strains (10). These two methods have moderate discriminatory power, with MLST having the advantage of data portability and the simplicity of data interpretation compared to that of the band pattern produced by PCR ribotyping where the interpretation may be subjective (6, 7). While these methods assist in the identification of phylogenic relationships between isolates, neither method is capable of producing a rapid result, as the average turnaround time is around 5 to 7 days, which includes the time for anaerobic C. difficile culture. One assay for rapid, presumptive identification of C. difficile 027/NAP1/BI directly from a stool sample is the Xpert C. difficile/Epi assay (Cepheid Inc., Sunnyvale, CA). The performance of the Xpert assay for routine diagnosis of CDI has been reported in several studies with the assay showing high sensitivity and specificity for the detection of C. difficile (11– 13). Performance of the Xpert assay for accurate identification of C. difficile NAP1 has been previously compared to that of PCR
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ribotyping, tcdC gene sequencing, pulse-field gel electrophoresis (PFGE), and restriction endonuclease analysis (REA) with high agreement (11, 14, 15). Recent reports have emerged on the misclassification of C. difficile isolates as presumptive C. difficile 027/ NAP1/BI by the Xpert assay (14, 16, 17). Laboratory detection of CDI at our hospital (Memorial Sloan Kettering Cancer Center) is performed using the Xpert C. difficile/Epi assay. All samples that are positive for C. difficile are subjected to routine typing by MLST to determine evolutionary trends and to identify hospital-based transmission. In this study, we compared the performance of the Xpert assay to that of MLST for identification of C. difficile NAP1. Discrepant strain types with these two methods were further characterized by PCR ribotyping and tcdC gene sequencing. (The abstract was presented at the Association for Molecular Pathology 2015 Annual Meeting, Austin, TX, 5 to 7 November 2015.) Specimens included in this study were collected between 1 September 2013 and 31 August 2015. These specimens were positive for C. difficile by the Xpert C. difficile/Epi assay and were received in the laboratory for routine diagnosis of CDI. Xpert PCR was performed as previously described (11), and results were reported to the physician. All Xpert-positive samples were saved, and an aliquot was stored at ⫺80°C until further analysis for routine epidemiology surveillance. Samples were typed weekly by MLST as described elsewhere (9). The allelic profile and strain type identification for each sample were completed using the PubMLST database (http://www.pubmlst.org/cdifficile). As part of this study, PCR ribotyping and sequencing of the entire tcdC gene were performed on all discrepant isolates (Cdiff 1 to 25) and on an additional 10, randomly selected concordant isolates (Cdiff 26 to 35) as previously described (11). The strain ATCC BAA-1805 (con-
Received 19 November 2015 Returned for modification 10 December 2015 Accepted 14 December 2015 Accepted manuscript posted online 23 December 2015 Citation McMillen T, Kamboj M, Babady NE. 2016. Comparison of multilocus sequence typing and the Xpert C. difficile/Epi assay for identification of Clostridium difficile 027/NAP1/BI. J Clin Microbiol 54:775–778. doi:10.1128/JCM.03075-15. Editor: A. B. Onderdonk Address correspondence to N. Esther Babady, [email protected]. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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FIG 1 C. difficile ST distribution. (A) Distribution of C. difficile ST identified by MLST. Other STs include more than 80 different STs with 1 to 20 isolates. (B) Xpert C. difficile NAP1 characterized by MLST. Not ST 1 isolates include 10 STs with 1 to 5 isolates. No data, no isolates recovered or lost samples.
firmed NAP1/027/BI) was used as a positive control (11). Additionally, C. difficile isolates were confirmed by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) (Vitek MS; bioMérieux, Durham, NC). A total of 1,573 stool samples (12% of the total samples) tested positive for C. difficile by Xpert PCR. Presumptive C. difficile NAP1 was identified in 132 samples (8.4% of all positive samples). A total of 1,222 C. difficile isolates (77.7% of all positive samples) were recovered by culture and were successfully characterized by MLST. From the 132 presumptive C. difficile NAP1 stool samples, 110 C. difficile isolates (83.4% of NAP1 stool samples) were recovered in culture and were successfully characterized by MLST. ST 1 (C. difficile 027/NAP1/BI) was identified in 85 Xpert NAP1 isolates (Fig. 1). The total percentage of agreement for identification of C. difficile NAP1 between MLST and Xpert PCR was very good at 97.9% ( ⫽ 0.86; 95% confidence interval (CI), 0.80 to 0.91). Twenty-five samples produced discordant results between MLST and the Xpert assay (Fig. 2). The number of mismatched alleles between ST profiles of the 25 isolates and the ST 1 profiles ranged from 3 alleles (ST 41 and ST 154) to all 7 alleles (ST 39). Three alleles, tpi, dxr, and glyA, were consistently different among all of the misidentified strains. PCR ribotyping revealed the presence of multiple ribotypes among the presumptive Xpert NAP1-positive samples. Fourteen of the 25 (56%) discordant samples were closely related to ribotype 027 by PCR ribotyping, and sequencing of the tcdC gene confirmed the presence of the 18-bp tcdC deletion in 11/14 (79%) samples. Eight of the 25 (32%) samples were highly dissimilar; the 18-bp tcdC deletion was identified in 4/8 (50%) of these isolates. Three of the 25 (12%) samples did not yield any data by PCR ribotyping, but 2/3 of these isolates had the18-bp tcdC deletion. Overall, sequencing of the tcdC gene confirmed the presence of an 18-bp tcdC deletion in 17/25 (68%) discordant samples. All 10 concordant isolates showed high similarity to ribotype 027, were highly similar by MALDI-TOF MS (⬎85%) (data not shown), and they all included the 18-bp tcdC deletion. Using MLST as the gold standard, the Xpert PCR assay showed an overall sensitivity of 100% (95% CI, 96% to 100%), a specificity of 98% (95% CI, 96% to 100%), a positive predictive value of 77% (95% CI, 68% to 85%), and a negative predictive value of 100% (95% CI, 100%) (P ⬍ 0.0001). When the isolates
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confirmed as NAP1 by PCR ribotyping and tcdC sequencing are included as true positives, the Xpert assay characteristics are as follows: a sensitivity of 100% (95% CI, 96% to 100%), a specificity of 99% (95% CI, 98% to 99%), a positive predictive value of 87% (95% CI, 80% to 93%), and a negative predictive value of 100% (95% CI, 100%) (P ⬍ 0.0001). C. difficile remains a significant cause of morbidity and mortality. Knowledge of circulating C. difficile strains is essential to establish an effective surveillance program. Methods available for typing C. difficile isolates vary in their level of technical complexity, cost, and discriminatory power (6, 7). PFGE and REA have high discriminatory power but are technically demanding, and data generated are not transferable across centers (6). Conversely, MLST is a relatively simple method with adequate discriminatory power and data that can be compared between different laboratories (7). The Xpert C. difficile/Epi assay provides a rapid, simple method to identify presumptive C. difficile NAP1, the most common C. difficile strain in the United States (1). However, occurrences of misclassifications have been reported. In one study, the Xpert C. difficile/Epi misclassified 5 isolates as ribotype 027, which were then identified as ribotypes 016, 019,080,176, and 046 (14). These strains differed in their PCR ribotypes from 027 by only a few bands, 1 band for ribotypes 016 and 176 and 3 to 5 bands for ribotypes 019, 046, and 080 (14). Ribotype 176 was also misclassified as 027 in another study from the Czech Republic, which confirmed that the two ribotypes differed only by one peak on the electropherogram when analyzed by PCR ribotyping and capillary electrophoresis (17). The authors of the study further showed that ribotype 176 has all of the markers used by the Xpert C. difficile/ Epi assay for the presumptive identification of 027. To our knowledge, our study is the first study to compare the performance of the Xpert C. difficile/Epi assay to that of MLST for identification of C. difficile NAP1/027. Although the concordance between the two methods was very good, several isolates with multiple STs were not confirmed as NAP1 by either PCR ribotyping or by tcdC gene sequencing. Of note, not all C. difficile ribotype 027 isolates are identified as NAP1 or BI strains (15), and similarly, not all ribotype 027 isolates may be classified as ST 1. This adds the complexity of interpreting typing data and underscores the need for a simple, standardized typing methodology. More recently,
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NAP1 Typing by Xpert C. difficile/Epi PCR and MLST
FIG 2 Summary of PCR ribotyping patterns, MLSTs, and tcdC sequencing of 25 discordant isolates (Cdiff 1 to 25) and 10 concordant isolates (Cdiff 26 to 35). NAP1A to NAP1D, replicates of control NAP1 strain; MSK1, unidentified ST.
whole-genome sequencing (and more specifically, core genome MLST [cgMLST]) has emerged as a highly discriminatory method for determining clonal relatedness. As the cgMLST scheme for C. difficile becomes established, this method may eventually become the gold standard for epidemiology studies (18). In conclusion, we showed that in addition to providing rapid and accurate diagnostic C. difficile results, the Xpert C. difficile/Epi presumptive NAP1 results have high concordance with those obtained by MLST. The method may be used as a first-line screen or a surrogate method to monitor C. difficile strain types with a change in C. difficile 027/NAP1/BI rates suggesting a change in the overall epidemiology of circulating C. difficile strain types. FUNDING INFORMATION HHS | NIH | National Cancer Institute (NCI) provided funding to under grant number P30 CA0088748. This research was funded in part through NIH/NCI Cancer Center Support Grant P30 CA008748.
REFERENCES 1. Lessa FC, Winston LG, McDonald LC, Emerging Infections Program C. difficile Surveillance Team. 2015. Burden of Clostridium difficile infection in the United States. N Engl J Med 372:2369 –2370.
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2. Aitken SL, Alam MJ, Khaleduzzuman M, Walk ST, Musick WL, Pham VP, Christensen JL, Atmar RL, Xie Y, Garey KW. 2015. In the endemic setting, Clostridium difficile ribotype 027 is virulent but not hypervirulent. Infect Control Hosp Epidemiol 36:1316 –1323. 3. Kuijper EJ, Barbut F, Brazier JS, Kleinkauf N, Eckmanns T, Lambert ML, Drudy D, Fitzpatrick F, Wiuff C, Brown DJ, Coia JE, Pituch H, Reichert P, Even J, Mossong J, Widmer AF, Olsen KE, Allerberger F, Notermans DW, Delmee M, Coignard B, Wilcox M, Patel B, Frei R, Nagy E, Bouza E, Marin M, Akerlund T, Virolainen-Julkunen A, Lyytikainen O, Kotila S, Ingebretsen A, Smyth B, Rooney P, Poxton IR, Monnet DL. 2008. Update of Clostridium difficile infection due to PCR ribotype 027 in Europe, 2008. Euro Surveill 13(31):pii⫽18942. http: //www.eurosurveillance.org/ViewArticle.aspx?ArticleId⫽18942. 4. Loo VG, Poirier L, Miller MA, Oughton M, Libman MD, Michaud S, Bourgault AM, Nguyen T, Frenette C, Kelly M, Vibien A, Brassard P, Fenn S, Dewar K, Hudson TJ, Horn R, Rene P, Monczak Y, Dascal A. 2005. A predominantly clonal multi-institutional outbreak of Clostridium difficile-associated diarrhea with high morbidity and mortality. N Engl J Med 353:2442–2449. http://dx.doi.org/10.1056/NEJMoa051639. 5. McDonald LC, Killgore GE, Thompson A, Owens RC, Jr, Kazakova SV, Sambol SP, Johnson S, Gerding DN. 2005. An epidemic, toxin genevariant strain of Clostridium difficile. N Engl J Med 353:2433–2441. http: //dx.doi.org/10.1056/NEJMoa051590. 6. Huber CA, Foster NF, Riley TV, Paterson DL. 2013. Challenges for standardization of Clostridium difficile typing methods. J Clin Microbiol 51:2810 –2814. http://dx.doi.org/10.1128/JCM.00143-13.
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7. Knetsch CW, Lawley TD, Hensgens MP, Corver J, Wilcox MW, Kuijper EJ. 2013. Current application and future perspectives of molecular typing methods to study Clostridium difficile infections. Euro Surveill 18(4): pii⫽20381. http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId ⫽20381. 8. Lemee L, Pons JL. 2010. Multilocus sequence typing for Clostridium difficile. Methods Mol Biol 646:77–90. http://dx.doi.org/10.1007/978-1 -60327-365-7_6. 9. Griffiths D, Fawley W, Kachrimanidou M, Bowden R, Crook DW, Fung R, Golubchik T, Harding RM, Jeffery KJ, Jolley KA, Kirton R, Peto TE, Rees G, Stoesser N, Vaughan A, Walker AS, Young BC, Wilcox M, Dingle KE. 2010. Multilocus sequence typing of Clostridium difficile. J Clin Microbiol 48:770 –778. http://dx.doi.org/10.1128/JCM.01796-09. 10. Bidet P, Barbut F, Lalande V, Burghoffer B, Petit JC. 1999. Development of a new PCR-ribotyping method for Clostridium difficile based on ribosomal RNA gene sequencing. FEMS Microbiol Lett 175:261–266. http://dx.doi.org/10.1111/j.1574-6968.1999.tb13629.x. 11. Babady NE, Stiles J, Ruggiero P, Khosa P, Huang D, Shuptar S, Kamboj M, Kiehn TE. 2010. 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 48:4519 – 4524. http: //dx.doi.org/10.1128/JCM.01648-10. 12. Gyorke CE, Wang S, Leslie JL, Cohen SH, Solnick JV, Polage CR. 2013. Evaluation of Clostridium difficile fecal load and limit of detection during a prospective comparison of two molecular tests, the illumigene C. difficile
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14. 15.
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and Xpert C. difficile/Epi tests. J Clin Microbiol 51:278 –280. http://dx.doi .org/10.1128/JCM.02120-12. Pancholi P, Kelly C, Raczkowski M, Balada-Llasat JM. 2012. Detection of toxigenic Clostridium difficile: comparison of the cell culture neutralization, Xpert C difficile, Xpert C. difficile/Epi, and Illumigene C. difficile assays. J Clin Microbiol 50:1331–1335. http://dx.doi.org/10.1128/JCM .06597-11. Mentula S, Laakso S, Lyytikainen O, Kirveskari J. 2015. Differentiating virulent 027 and non-027 Clostridium difficile strains by molecular methods. Expert Rev Mol Diagn 15:1225–1229. Tenover FC, Akerlund T, Gerding DN, Goering RV, Bostrom T, Jonsson AM, Wong E, Wortman AT, Persing DH. 2011. Comparison of strain typing results for Clostridium difficile isolates from North America. J Clin Microbiol 49:1831–1837. http://dx.doi.org/10.1128/JCM.02446-10. Kok J, Wang Q, Thomas LC, Gilbert GL. 2011. Presumptive identification of Clostridium difficile strain 027/NAP1/BI on Cepheid Xpert: interpret with caution. J Clin Microbiol 49:3719 –3721. http://dx.doi.org/10 .1128/JCM.00752-11. Krutova M, Matejkova J, Nyc O. 2014. C. difficile ribotype 027 or 176? Folia Microbiol (Praha) 59:523–526. http://dx.doi.org/10.1007/s12223 -014-0323-5. Hasman H, Saputra D, Sicheritz-Ponten T, Lund O, Svendsen CA, Frimodt-Moller N, Aarestrup FM. 2014. Rapid whole-genome sequencing for detection and characterization of microorganisms directly from clinical samples. J Clin Microbiol 52:139 –146. http://dx.doi.org/10.1128 /JCM.02452-13.
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Comparison of Multilocus Sequence Typing and the Xpert C. difficile/ Epi Assay for Identification of Clostridium difficile 027/NAP1/BI Tracy McMillen,a Mini Kamboj,b,c,d
N. Esther Babadya
Clinical Microbiology Service, Department of Laboratory Medicine,a Infection Control Service, Department of Medicine,b and Infectious Disease Service, Department of Medicine,c Memorial Sloan Kettering Cancer Center, New York, New York, USA; Department of Medicine, Weill Cornell Medical College, New York, New York, USAd
Clostridium difficile 027/NAP1/BI is the most common C. difficile strain in the United States. The Xpert C. difficile/Epi assay allows rapid, presumptive identification of C. difficile NAP1. We compared Xpert C. difficile/Epi to multilocus sequence typing for identification of C. difficile NAP1 and found “very good” agreement at 97.9% ( ⴝ 0.86; 95% confidence interval, 0.80 to 0.91).
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lostridium difficile is the most common bacterial cause of hospital-associated diarrhea, with an estimated 500,000 cases in 2011 (1). In the United States, C. difficile PCR ribotype 027/North American pulse-field gel electrophoresis (PFGE) type 1 (NAP1)/ restriction endonuclease analysis (REA) group BI (C. difficile 027/ NAP1/BI), is the most frequently isolated strain in community and healthcare-associated cases of C. difficile infection (CDI) (1, 2). C. difficile 027/NAP1/BI emerged as a hyper-virulent strain from its association with multiple outbreaks of severe CDI that were reported across North America and Europe (3–5). Identification of C. difficile 027/NAP1/BI has emerging implications for disease management and provides useful information for epidemiologic purposes. Molecular typing methods are crucial to understanding and monitoring circulating C. difficile strain types. Several methods are available for typing C. difficile, and each has its own set of advantages and limitations (6, 7). Multilocus sequence typing (MLST) functions by sequencing a 500- to 700-bp fragment of each of seven C. difficile housekeeping genes and detecting the presence of polymorphisms (8, 9). MLST sequence data are uploaded to the PubMLST database (http://pubmlst.org /cdifficile), and the resulting allelic combination is used to determine individual strain type (ST). PCR ribotyping of C. difficile is based on the amplification of DNA fragments in the heterogenous region between the 16S and 23S rRNA of the chromosome (10). The band pattern produced by these fragments allows comparison of unique strains (10). These two methods have moderate discriminatory power, with MLST having the advantage of data portability and the simplicity of data interpretation compared to that of the band pattern produced by PCR ribotyping where the interpretation may be subjective (6, 7). While these methods assist in the identification of phylogenic relationships between isolates, neither method is capable of producing a rapid result, as the average turnaround time is around 5 to 7 days, which includes the time for anaerobic C. difficile culture. One assay for rapid, presumptive identification of C. difficile 027/NAP1/BI directly from a stool sample is the Xpert C. difficile/Epi assay (Cepheid Inc., Sunnyvale, CA). The performance of the Xpert assay for routine diagnosis of CDI has been reported in several studies with the assay showing high sensitivity and specificity for the detection of C. difficile (11– 13). Performance of the Xpert assay for accurate identification of C. difficile NAP1 has been previously compared to that of PCR
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ribotyping, tcdC gene sequencing, pulse-field gel electrophoresis (PFGE), and restriction endonuclease analysis (REA) with high agreement (11, 14, 15). Recent reports have emerged on the misclassification of C. difficile isolates as presumptive C. difficile 027/ NAP1/BI by the Xpert assay (14, 16, 17). Laboratory detection of CDI at our hospital (Memorial Sloan Kettering Cancer Center) is performed using the Xpert C. difficile/Epi assay. All samples that are positive for C. difficile are subjected to routine typing by MLST to determine evolutionary trends and to identify hospital-based transmission. In this study, we compared the performance of the Xpert assay to that of MLST for identification of C. difficile NAP1. Discrepant strain types with these two methods were further characterized by PCR ribotyping and tcdC gene sequencing. (The abstract was presented at the Association for Molecular Pathology 2015 Annual Meeting, Austin, TX, 5 to 7 November 2015.) Specimens included in this study were collected between 1 September 2013 and 31 August 2015. These specimens were positive for C. difficile by the Xpert C. difficile/Epi assay and were received in the laboratory for routine diagnosis of CDI. Xpert PCR was performed as previously described (11), and results were reported to the physician. All Xpert-positive samples were saved, and an aliquot was stored at ⫺80°C until further analysis for routine epidemiology surveillance. Samples were typed weekly by MLST as described elsewhere (9). The allelic profile and strain type identification for each sample were completed using the PubMLST database (http://www.pubmlst.org/cdifficile). As part of this study, PCR ribotyping and sequencing of the entire tcdC gene were performed on all discrepant isolates (Cdiff 1 to 25) and on an additional 10, randomly selected concordant isolates (Cdiff 26 to 35) as previously described (11). The strain ATCC BAA-1805 (con-
Received 19 November 2015 Returned for modification 10 December 2015 Accepted 14 December 2015 Accepted manuscript posted online 23 December 2015 Citation McMillen T, Kamboj M, Babady NE. 2016. Comparison of multilocus sequence typing and the Xpert C. difficile/Epi assay for identification of Clostridium difficile 027/NAP1/BI. J Clin Microbiol 54:775–778. doi:10.1128/JCM.03075-15. Editor: A. B. Onderdonk Address correspondence to N. Esther Babady, [email protected]. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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FIG 1 C. difficile ST distribution. (A) Distribution of C. difficile ST identified by MLST. Other STs include more than 80 different STs with 1 to 20 isolates. (B) Xpert C. difficile NAP1 characterized by MLST. Not ST 1 isolates include 10 STs with 1 to 5 isolates. No data, no isolates recovered or lost samples.
firmed NAP1/027/BI) was used as a positive control (11). Additionally, C. difficile isolates were confirmed by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) (Vitek MS; bioMérieux, Durham, NC). A total of 1,573 stool samples (12% of the total samples) tested positive for C. difficile by Xpert PCR. Presumptive C. difficile NAP1 was identified in 132 samples (8.4% of all positive samples). A total of 1,222 C. difficile isolates (77.7% of all positive samples) were recovered by culture and were successfully characterized by MLST. From the 132 presumptive C. difficile NAP1 stool samples, 110 C. difficile isolates (83.4% of NAP1 stool samples) were recovered in culture and were successfully characterized by MLST. ST 1 (C. difficile 027/NAP1/BI) was identified in 85 Xpert NAP1 isolates (Fig. 1). The total percentage of agreement for identification of C. difficile NAP1 between MLST and Xpert PCR was very good at 97.9% ( ⫽ 0.86; 95% confidence interval (CI), 0.80 to 0.91). Twenty-five samples produced discordant results between MLST and the Xpert assay (Fig. 2). The number of mismatched alleles between ST profiles of the 25 isolates and the ST 1 profiles ranged from 3 alleles (ST 41 and ST 154) to all 7 alleles (ST 39). Three alleles, tpi, dxr, and glyA, were consistently different among all of the misidentified strains. PCR ribotyping revealed the presence of multiple ribotypes among the presumptive Xpert NAP1-positive samples. Fourteen of the 25 (56%) discordant samples were closely related to ribotype 027 by PCR ribotyping, and sequencing of the tcdC gene confirmed the presence of the 18-bp tcdC deletion in 11/14 (79%) samples. Eight of the 25 (32%) samples were highly dissimilar; the 18-bp tcdC deletion was identified in 4/8 (50%) of these isolates. Three of the 25 (12%) samples did not yield any data by PCR ribotyping, but 2/3 of these isolates had the18-bp tcdC deletion. Overall, sequencing of the tcdC gene confirmed the presence of an 18-bp tcdC deletion in 17/25 (68%) discordant samples. All 10 concordant isolates showed high similarity to ribotype 027, were highly similar by MALDI-TOF MS (⬎85%) (data not shown), and they all included the 18-bp tcdC deletion. Using MLST as the gold standard, the Xpert PCR assay showed an overall sensitivity of 100% (95% CI, 96% to 100%), a specificity of 98% (95% CI, 96% to 100%), a positive predictive value of 77% (95% CI, 68% to 85%), and a negative predictive value of 100% (95% CI, 100%) (P ⬍ 0.0001). When the isolates
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confirmed as NAP1 by PCR ribotyping and tcdC sequencing are included as true positives, the Xpert assay characteristics are as follows: a sensitivity of 100% (95% CI, 96% to 100%), a specificity of 99% (95% CI, 98% to 99%), a positive predictive value of 87% (95% CI, 80% to 93%), and a negative predictive value of 100% (95% CI, 100%) (P ⬍ 0.0001). C. difficile remains a significant cause of morbidity and mortality. Knowledge of circulating C. difficile strains is essential to establish an effective surveillance program. Methods available for typing C. difficile isolates vary in their level of technical complexity, cost, and discriminatory power (6, 7). PFGE and REA have high discriminatory power but are technically demanding, and data generated are not transferable across centers (6). Conversely, MLST is a relatively simple method with adequate discriminatory power and data that can be compared between different laboratories (7). The Xpert C. difficile/Epi assay provides a rapid, simple method to identify presumptive C. difficile NAP1, the most common C. difficile strain in the United States (1). However, occurrences of misclassifications have been reported. In one study, the Xpert C. difficile/Epi misclassified 5 isolates as ribotype 027, which were then identified as ribotypes 016, 019,080,176, and 046 (14). These strains differed in their PCR ribotypes from 027 by only a few bands, 1 band for ribotypes 016 and 176 and 3 to 5 bands for ribotypes 019, 046, and 080 (14). Ribotype 176 was also misclassified as 027 in another study from the Czech Republic, which confirmed that the two ribotypes differed only by one peak on the electropherogram when analyzed by PCR ribotyping and capillary electrophoresis (17). The authors of the study further showed that ribotype 176 has all of the markers used by the Xpert C. difficile/ Epi assay for the presumptive identification of 027. To our knowledge, our study is the first study to compare the performance of the Xpert C. difficile/Epi assay to that of MLST for identification of C. difficile NAP1/027. Although the concordance between the two methods was very good, several isolates with multiple STs were not confirmed as NAP1 by either PCR ribotyping or by tcdC gene sequencing. Of note, not all C. difficile ribotype 027 isolates are identified as NAP1 or BI strains (15), and similarly, not all ribotype 027 isolates may be classified as ST 1. This adds the complexity of interpreting typing data and underscores the need for a simple, standardized typing methodology. More recently,
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NAP1 Typing by Xpert C. difficile/Epi PCR and MLST
FIG 2 Summary of PCR ribotyping patterns, MLSTs, and tcdC sequencing of 25 discordant isolates (Cdiff 1 to 25) and 10 concordant isolates (Cdiff 26 to 35). NAP1A to NAP1D, replicates of control NAP1 strain; MSK1, unidentified ST.
whole-genome sequencing (and more specifically, core genome MLST [cgMLST]) has emerged as a highly discriminatory method for determining clonal relatedness. As the cgMLST scheme for C. difficile becomes established, this method may eventually become the gold standard for epidemiology studies (18). In conclusion, we showed that in addition to providing rapid and accurate diagnostic C. difficile results, the Xpert C. difficile/Epi presumptive NAP1 results have high concordance with those obtained by MLST. The method may be used as a first-line screen or a surrogate method to monitor C. difficile strain types with a change in C. difficile 027/NAP1/BI rates suggesting a change in the overall epidemiology of circulating C. difficile strain types. FUNDING INFORMATION HHS | NIH | National Cancer Institute (NCI) provided funding to under grant number P30 CA0088748. This research was funded in part through NIH/NCI Cancer Center Support Grant P30 CA008748.
REFERENCES 1. Lessa FC, Winston LG, McDonald LC, Emerging Infections Program C. difficile Surveillance Team. 2015. Burden of Clostridium difficile infection in the United States. N Engl J Med 372:2369 –2370.
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