Increasing Prevalence of Toxin A-Negative, Toxin B-Positive Isolates of Clostridium difficile in Korea: Impact on Laboratory Diagnosis

ABSTRACT Guidelines recommend the use of an algorithm for the laboratory diagnosis of Clostridium difficile infection (CDI). Enzyme immunoassays (EIAs) detecting C. difficile toxins cannot be used as standalone tests due to suboptimal sensitivity, and molecular tests suffer from nonspecificity by detecting colonization. Sensitive immunoassays have recently been developed to improve and simplify CDI diagnosis. Assays detecting CD toxins have been developed using single-molecule array (SIMOA) technology. SIMOA performance was assessed relative to a laboratory case definition of CDI defined by positive glutamate dehydrogenase (GDH) screen and cell cytotoxicity neutralizing assay (CCNA). Samples were tested with SIMOA assays and a commercial toxin EIA to compare performance, with discrepancy resolution using a commercial nucleic acid-based test and a second cell cytotoxicity assay. The SIMOA toxin A and toxin B assays showed limits of detection of 0.6 and 2.9 pg/ml, respectively, and intra-assay coefficients of variation of less than 10%. The optimal clinical thresholds for the toxin A and toxin B assays were determined to be 22.1 and 18.8 pg/ml, respectively, with resultant sensitivities of 84.8 and 95.5%. In contrast, a high-performing EIA toxin test had a sensitivity of 71.2%. Thus, the SIMOA assays detected toxins in 24% more samples with laboratory-defined CDI than the high performing toxin EIA (95% [63/66] versus 71% [47/66]). This study shows that SIMOA C. difficile toxin assays have a higher sensitivity than currently available toxin EIA and have the potential to improve CDI diagnosis.

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Infection with Toxin A-Negative, Toxin B-Negative, Binary Toxin-Positive Clostridium difficile in a Young Patient with Ulcerative Colitis

Journal of Clinical Microbiology ◽

10.1128/jcm.01810-15 ◽

2015 ◽

Vol 53 (11) ◽

pp. 3702-3704 ◽

Cited By ~ 25

Author(s):

Grace O. Androga ◽

Julie Hart ◽

Niki F. Foster ◽

Adrian Charles ◽

David Forbes ◽

...

Keyword(s):

Ulcerative Colitis ◽

Clostridium Difficile ◽

Young Patient ◽

Diagnostic Methods ◽

Binary Toxin ◽

Toxin A ◽

Toxin B ◽

Content Type ◽

Severe Diarrhea ◽

Clinically Significant

Large clostridial toxin-negative, binary toxin-positive (A−B−CDT+) strains ofClostridium difficileare almost never associated with clinically significantC. difficileinfection (CDI), possibly because such strains are not detected by most diagnostic methods. We report the isolation of an A−B−CDT+ribotype 033 (RT033) strain ofC. difficilefrom a young patient with ulcerative colitis and severe diarrhea.

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A Nosocomial Outbreak of Diarrhea Caused by Toxin A-negative, Toxin B-positive Clostridium difficile in A Cancer Center Hospital

Kansenshogaku zasshi ◽

10.11150/kansenshogakuzasshi1970.78.312 ◽

2004 ◽

Vol 78 (4) ◽

pp. 312-319 ◽

Cited By ~ 15

Author(s):

Hiroko SATO ◽

Haru KATO ◽

Kenji KOIWAI ◽

Chikara SAKAI

Keyword(s):

Clostridium Difficile ◽

Cancer Center ◽

Nosocomial Outbreak ◽

Toxin A ◽

Toxin B ◽

Center Hospital

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Nucleic Acid Amplification Test Quantitation as Predictor of Toxin Presence in Clostridium difficile Infection

Journal of Clinical Microbiology ◽

10.1128/jcm.01316-17 ◽

2017 ◽

Vol 56 (3) ◽

Cited By ~ 13

Author(s):

M. J. T. Crobach ◽

N. Duszenko ◽

E. M. Terveer ◽

C. M. Verduin ◽

E. J. Kuijper

Keyword(s):

Nucleic Acid ◽

Clostridium Difficile ◽

Characteristic Curve ◽

Nucleic Acid Amplification Test ◽

Nucleic Acid Amplification ◽

Toxin A ◽

Toxin B ◽

Content Type ◽

Quantitative Results ◽

Testing Algorithm

ABSTRACT Multistep algorithmic testing in which a sensitive nucleic acid amplification test (NAAT) is followed by a specific toxin A and toxin B enzyme immunoassay (EIA) is among the most accurate methods for Clostridium difficile infection (CDI) diagnosis. The obvious shortcoming of this approach is that multiple tests must be performed to establish a CDI diagnosis, which may delay treatment. Therefore, we sought to determine whether a preliminary diagnosis could be made on the basis of the quantitative results of the first test in algorithmic testing, which provide a measure of organism burden. To do so, we retrospectively analyzed two large collections of samples ( n = 2,669 and n = 1,718) that were submitted to the laboratories of two Dutch hospitals for CDI testing. Both hospitals apply a two-step testing algorithm in which a NAAT is followed by a toxin A/B EIA. Of all samples, 208 and 113 samples, respectively, tested positive by NAAT. Among these NAAT-positive samples, significantly lower mean quantification cycle ( C q ) values were found for patients whose stool eventually tested positive for toxin, compared with patients who tested negative for toxin (mean C q values of 24.4 versus 30.4 and 26.8 versus 32.2; P < 0.001 for both cohorts). Receiver operating characteristic curve analysis was performed to investigate the ability of C q values to predict toxin status and yielded areas under the curve of 0.826 and 0.854. Using the optimal C q cutoff values, prediction of the eventual toxin A/B EIA results was accurate for 78.9% and 80.5% of samples, respectively. In conclusion, C q values can serve as predictors of toxin status but, due to the suboptimal correlation between the two tests, additional toxin testing is still needed.

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Identification of Toxin A-Negative, Toxin B-Positive Clostridium difficile by PCR

Journal of Clinical Microbiology ◽

10.1128/jcm.36.8.2178-2182.1998 ◽

1998 ◽

Vol 36 (8) ◽

pp. 2178-2182 ◽

Cited By ~ 237

Author(s):

Haru Kato ◽

Naoki Kato ◽

Kunitomo Watanabe ◽

Naoichi Iwai ◽

Haruhi Nakamura ◽

...

Keyword(s):

Cell Culture ◽

Clostridium Difficile ◽

Pcr Amplification ◽

Enzyme Linked Immunosorbent Assay ◽

Pcr Assay ◽

Toxin A ◽

Toxin B ◽

Cell Monolayers ◽

Toxigenic Strains ◽

Cell Culture Assay

Toxigenic strains of Clostridium difficile have been reported to produce both toxins A and B nearly always, and nontoxigenic strains have been reported to produce neither of these toxins. Recent studies indicate that it is not always true. We established a PCR assay to differentiate toxin A-negative, toxin B-positive (toxin A−, toxin B+) strains from both toxin-positive (toxin A+, toxin B+) strains and both toxin-negative (toxin A−, toxin B−) strains as an alternative to cell culture assay and enzyme-linked immunosorbent assay (ELISA). By using the PCR primer set NK11 and NK9 derived from the repeating sequences of the toxin A gene, a shorter segment (ca. 700 bp) was amplified from toxin A−, toxin B+ strains compared to the size of the segment amplified from toxin A+, toxin B+ strains (ca. 1,200 bp), and no product was amplified from toxin A−, toxin B− strains. We examined a total of 421 C. difficile isolates by PCR. Of these, 48 strains showed a shorter segment by the PCR, were negative by ELISAs for the detection of toxin A, and were positive by cell culture assay. Although the cytotoxin produced by the toxin A−, toxin B+ strains was neutralized by anti-toxin B serum, the appearance of the cytotoxic effects on Vero cell monolayers was distinguishable from that of toxin A+, toxin B+ strains. By immunoblotting, the 44 toxin A−, toxin B+ strains were typed to serogroup F and the remaining four strains were serogroup X. Pulsed-field gel electrophoresis separated the 48 strains into 19 types. The PCR assay for the detection of the repeating sequences combined with PCR amplification of the nonrepeating sequences of either the toxin A or the toxin B gene is indicated to be useful for differentiating toxin A−, toxin B+ strains from toxin A+, toxin B+ and toxin A−, toxin B− strains and will contribute to elucidation of the precise role of toxin A−, toxin B+ strains in intestinal diseases.

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Detection of antibiotic resistance toxigenic Clostridium difficile in processed retail lettuce

Food Quality and Safety ◽

10.1093/fqsafe/fyx032 ◽

2018 ◽

Vol 2 (1) ◽

pp. 37-41 ◽

Cited By ~ 2

Author(s):

Yi Han ◽

Joan King ◽

Marlene E Janes

Keyword(s):

Public Health ◽

Antibiotic Resistance ◽

Clostridium Difficile ◽

Antimicrobial Treatment ◽

Retail Stores ◽

Toxin A ◽

Toxin B ◽

Health Concerns ◽

Ribotype 027 ◽

Infectious Diarrhoea

Abstract Objectives: Clostridium difficile is the major cause of infectious diarrhoea in humans after antimicrobial treatment. Clostridium difficile has been isolated from food animals and meat. The main purpose of this study was to characterize C. difficile isolated from retail lettuce and determine the antibiotic resistance using five common clinical-selected antibiotics (metronidazole, vancomycin, clindamycin, erythromycin, and cefotaxime). Materials and Methods: Lettuce samples (grown in California, Arkansas, and Louisiana) were purchased from retail stores. Results: Toxigenic C. difficile was isolated from 13.8 per cent (41/297) of the lettuce samples. Among the toxigenic isolates, only 82.9 per cent (34/41) produced toxin B, 17.1 per cent (7/41) produced both toxin A and toxin B, and two of the Louisiana C. difficile isolates were identified as ribotype 027. Under the treatment of the five antibiotics, the virulence C. difficile isolates were identified as having antibiotic resistance to metronidazole, vancomycin, and erythromycin. Conclusion: The present study reports the highest prevalence of toxigenic C. difficile in US retail lettuce. The antibiotic resistance to metronidazole, vancomycin, and erythromycin of the isolated C. difficile from retail lettuces could lead to public health concerns.

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