Comparison of BD GeneOhm Cdiff and Seegene Seeplex ACE PCR assays using toxigenic Clostridium difficile culture for direct detection of tcdB from stool specimens.

We evaluated the performances of 2 PCR assays (BD GeneOhm and Seegene ACE) for direct detection of tcdB from stool specimens. The concordance rate between BD and Seegene was 96.3%. The sensitivities, specificities, positive predictive values (PPVs), and negative predictive values (NPVs) of BD and Seegene were 95.7%, 96.5%, 91.8%, and 98.2% and 90.0%, 97.1%, 92.6%, and 96.0%, respectively.

Comparison of VIDAS CDAB and CDA immunoassay for the detection of Clostridium difficile in a tcdA- tcdB+ C. difficile prevalent area.

Enzyme immunoassays for TcdA and/or TcdB are widely used for diagnosis of C. difficile infection. This study compared the performance of the new VIDAS C. difficile Toxin A & B assay (CDAB) with that of the existing VIDAS C. difficile Toxin A II assay (CDA) in a tcdA(-)tcdB(+) prevalent area. A total of 555 fecal samples were cultured and tested using CDAB and CDA. C. difficile was isolated in 150 samples and the concordance rate was 81.8% (454/555) between CDAB and CDA. PCR assays for tcdA and/or tcdB were used as a confirmatory test on C. difficile strains recovered from culture positive cases (n=150) and on fecal specimens in culture negative/CDAB positive or equivocal cases (n=27). The number of tcdA(+)tcdB(+), tcdA(-)tcdB(+), and tcdA(-)tcdB(-) strains on culture positive isolates (n=150) were 75 (50.0%), 41 (27.3%), and 34 (22.7%), respectively. PCR assays for tcdB gene alone in stool specimens (n=27) showed positivity in five cases. The sensitivity of VIDAS CDAB was higher than that of VIDAS CDA (65.3% vs. 29.8%), by more than 2-fold. The specificity of CDAB was almost the same as CDA (93.8% vs. 94.5%). Toxigenic culture of C. difficile isolates in culture positive/VIDAS CDAB negative cases (n=62) additionally detected 22 VIDAS CDAB positive and 9 VIDAS CDAB equivocal cases. The VIDAS CDAB assay detects more tcdA(+)tcdB(+) strains (60% vs. 45.3%) and tcdA(-)tcdB(+) strains (70.7% vs. 0%) compared with VIDAS CDA.

Algorithm combining toxin immunoassay and stool culture for diagnosis of Clostridium difficile infection.

Enzyme immunoassays (EIA) to detect glutamate dehydrogenase or toxins A (TcdA) and B (TcdB), a cytotoxicity assay, and bacteriologic culture have disadvantages when applied individually to diagnosis of Clostridium difficile infections. Stool specimens (n = 1,596) were subjected to toxin detection via an enzyme-linked fluorescent immunoassay (ELFA; Vidas CDAB assay) and bacteriologic culture for toxigenic C. difficile in a three-step algorithm with additional toxigenic culture. Isolates (n = 163) from ELFA-negative stool specimens were examined via ELFA for toxin production. We amplified tcdA and tcdB from C. difficile isolates and tcdB from stool specimens that were ELFA positive or equivocal and culture negative, and we compared the results to those obtained with the three-step algorithm. More than 26% of stool specimens (419/1,596) were culture positive, yielding 248 isolates (59.2%) with both toxin genes (tcdA- and tcdB-positive isolates), 88 isolates (21.0%) with either tcdA or tcdB, and 83 (19.8%) that had no toxin genes (tcdA- and tcdB-negative isolates). Among 49 (culture-negative/ELFA-positive or -equivocal) stool specimens, 53.1% (26/49) represented tcdB-positive isolates. Therefore, the total number of PCR-positive cases was 362, and 27.1% (98/362) of these were detected through toxigenic culture. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 63.3%, 96.7%, 90.5%, and 92.4% (ELFA alone); 92.8%, 93.3%, 80.2%, and 97.8% (culture); and 70.7%, 91.4%, 95.5%, and 100% (three-step algorithm ELFA and bacterial culture with toxigenic culture), respectively, with culture and PCR for tcdA and tcdB as the standards. Thus, sensitivity and specificity were highest using culture and ELFA, respectively, but we recommend the three-step algorithm comprising EIA to detect both toxins and toxigenic culture for C. difficile as a practical method for achieving better PPV and NPV.

Multicentre study of the prevalence of toxigenic Clostridium difficile in Korea: results of a retrospective study 2000-2005.

The prevalence of toxigenic Clostridium difficile in Korea has been reported to be approximately 60-80%. Although the prevalence of the tcdA(-)tcdB(+) C. difficile strain was less then 5% prior to the year 2000, it has become an emerging nosocomial pathogen in Korea. Therefore, we have attempted to determine the multicentre nationwide prevalence of tcdA(+)tcdB(+) and tcdA(-)tcdB(+) C. difficile for epidemiological purposes. C. difficile strains (n=724, 30 from 2000, 80 from 2001, 74 from 2002, 76 from 2003, 179 from 2004, 285 from 2005) were obtained retrospectively from January 2000 to December 2005 from in-patients at 6 hospitals, all of whom were suspected of having C. difficile-associated disease (CDAD), colitis or pseudomembranous colitis. The numbers of participating hospitals varied yearly (1 in 2000, 2 in 2001-2003, 3 in 2004, 5 in 2005). The hospitals were located in Seoul (n=4), Kyunggi Province (n=1) and Busan (n=1), Korea. PCR assays for tcdA and tcdB genes were conducted using 724 unduplicated C. difficile isolates. The mean prevalence of tcdA(+)tcdB(+) and tcdA(-)tcdB(+) C. difficile strains over the 6 years was 51.8 % (38.4-59.3%) and 25.8%(10-56.0%), respectively. The mean prevalence of tcdA(-)tcdB(+) C. difficile strains was less than 7% until 2002, but began to increase in 2003 (13.2%) and achieved a peak in 2004 (50.3%). In 2005, the mean prevalence of tcdA(+)tcdB(+) and tcdA(-)tcdB(+) C. difficile strains was 47.7% (30.9-60.3%) and 27.0% (17.6-54.8%), respectively. This nationwide epidemiological study showed that tcdA(-)tcdB(+) C. difficile strains have already spread extensively throughout Korea, and our results provide basic data regarding the controversies currently surrounding the toxigenicity of tcdA(-)tcdB(+) C. difficile. The use of enzyme immunoassays capable of detecting both TcdA and TcdB is strongly recommended for the diagnosis of CDAD in microbiology laboratories, in order to control the spread of the tcdA(-)tcdB(+) strains of C. difficile.

Emerging toxin A-B+ variant strain of Clostridium difficile responsible for pseudomembranous colitis at a tertiary care hospital in Korea.

Sixty percent to 80% of Clostridium difficile isolates in Korea have been reported to be toxigenic. However, over 1 year, we encountered a high number of tcdA-tcB+ strains associated with pseudomembranous colitis (PMC). C. difficile was isolated from 224 of 471 specimens (47.6%) from 371 patients. A subset of the culture-positive specimens (n = 106), containing no duplicate cases, was randomly selected for tcdA and tcdB polymerase chain reaction (PCR) assays. PCR results showed that tcdA+tcdB+ and tcdA-tcdB+ strains accounted for 39.6% (42/106) and 50.9% (54/106), respectively. Endoscopy, performed on 55/106 patients, revealed 29 with PMC, 5 with colitis, 14 with other colon diseases, and 7 normal cases. Among the 29 PMC cases, 21 (72.4%) were associated with tcdA-tcdB+ strains (P = 0.0016). These results revealed the possible emergence of tcdA-tcdB+ C. difficile strains in Korea, and these variant strains could evoke a higher rate of PMC than tcdA+tcdB+ strains.

Detection of 12 respiratory viruses with two-set multiplex reverse transcriptase-PCR assay using a dual priming oligonucleotide system.

BACKGROUND: We intended to evaluate the diagnostic usefulness of a multiplex reverse transcriptase- PCR (mRT-PCR) assay kit under dual priming oligonucleotide system (DPO) for the childhood acute respiratory tract infections. METHODS: Two hundred nasopharyngeal aspirates were taken from children < or =5 yr old admitted due to acute respiratory infections in 2004. Direct fluorescent antibody (FA) assays were performed with fresh specimens; then, mRT-PCRs for the detection of 12 respiratory viruses (Seeplex RV detection kit, SeeGene, Seoul, Korea) were tested with frozen specimens. RESULTS: FA assays for five common respiratory viruses showed positive results in 66 patients (33.0%), while mRT-PCR detected causative viruses in 112 patients (56.0%), including 16 co-infected cases (8.0%). A total of 129 viruses were identified: respiratory syncytial virus A/B (38.0%/7.8%), influenza virus A/B (10.1%/5.4%), parainfluenza virus 1/2/3 (7.0%/3.1%/7.8%), coronavirus 229E or NL63 (6.2%), human metapneumovirus (4.7%), adenovirus (4.7%), rhinovirus (3.9%), and coronavirus OC43 (1.6%). CONCLUSIONS: DPO-based mRT-PCR was found as a sensitive tool for the detection of the viruses that cause childhood respiratory infections. Clinical significances of the agents detected by mRTPCR need further evaluations.

[Characterization of a Toxin A-Negative, Toxin B-Positive Variant Strain of Clostridium difficile.].

BACKGROUND: Clostridium difficile is one of the most important pathogens responsible for nosocomial diarrhea. Recently, we have frequently experienced culture positive, toxin A enzyme immunoassay negative strains. Therefore, we evaluated the strains with several PCR primer sets to characterize them. METHODS: A total of 351 stool specimens were examined for toxin A using enzyme linked fluorescent immunoassay (ELFA) and also cultured for C. difficile using cycloserine cefoxitine fructose agar incubated under anaerobic conditions. Spore stain and Vitek ANA identification card (BioMerieux, France) were used for identification of C. difficile. We amplified toxin A and toxin B genes in 81 isolates using primers NK1- NK2, NK3-NK2, NK9- NK11, and NK104-NK105. RESULTS: The concordance rate between ELFA and culture was 65.2% (229/351). PCR for the toxin A gene using NK1-NK2, NK3-NK2 and for the toxin B gene using NK104-NK105 showed almost the same results. However, toxin A gene PCR using NK9-NK11 showed that 45.7% (37/81) of the evaluated strains were toxin A (-)/ toxin B(+) variant strains; thus, the corrected sensitivity and specificity of the ELFA based on the PCR results for toxin A and B genes were 65.6% and 100%, respectively. CONCLUSIONS: The low sensitivity of the ELFA results for toxin A was due to the toxin A(-)/toxin B(+) variants of C. difficile, suggesting that the prevalence of the variant strains could be higher in Korea than was expected.