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A prospective study of two isothermal amplification assays compared with real-time PCR, CCNA and toxigenic culture for the diagnosis of Clostridium difficile infection.

ABSTRACT: New molecular methods of detecting Clostridium difficile infection (CDI) provide the routine lab with a sensitive random access method to produce results that are available in a shorter time than traditional methods.In this prospective study a total of 989 stool specimens were tested over a period of 16 months in parallel using two isothermal amplification assays, AmpliVue® (Quidel) and Illumigene® (Meridian) and the results compared to those from toxigenic culture. In addition all specimens were tested using a cytotoxic cell neutralisation assay (CCNA) and three different Real-time PCR targeting a C. difficile-specific 16S rDNA sequence or the toxin genes tcdA, tcdB/tcdB027 or cdtB.AmpliVue® was positive in 242 (24.5%) and Illumigene® in 228 (23.1%) specimens. 167 (16.9%) specimens were positive in toxigenic culture. Real-time-tcdA and -tcdB PCR was positive in 211 (21.3%) specimens, Real-time-cdtB PCR was positive in 101 (10.2%) specimens and C. difficile-PCR (16S rDNA) in 267 (27.0%) specimens.The respective sensitivity, specificity, positive predictive value and negative predictive value compared to toxigenic culture were 91, 89, 62 and 98% for AmpliVue® and 91, 91, 67 and 98% for Illumigene®.

SUBMITTER: Neuendorf M

PROVIDER: S-EPMC4751656 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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A prospective study of two isothermal amplification assays compared with real-time PCR, CCNA and toxigenic culture for the diagnosis of Clostridium difficile infection.

Neuendorf Martina M Guadarrama-Gonzalez Raquel R Lamik Birgit B MacKenzie Colin R CR

BMC microbiology 20160212

<h4>Background</h4>New molecular methods of detecting Clostridium difficile infection (CDI) provide the routine lab with a sensitive random access method to produce results that are available in a shorter time than traditional methods.<h4>Methods</h4>In this prospective study a total of 989 stool specimens were tested over a period of 16 months in parallel using two isothermal amplification assays, AmpliVue® (Quidel) and Illumigene® (Meridian) and the results compared to those from toxigenic cul ...[more]

PMID: 26868647

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Project description:Probiotics might offer an attractive alternative to prevent and control Clostridium difficile (C. difficile) infection (CDI). Limited information is available on the ability of commercially used bifidobacterial strains to inhibit C. difficile. This study examined the anti-clostridial effects of Bifidobacterium longum JDM301, a widely used commercial probiotic strain in China, in vitro and in vivo. In vitro evaluation revealed a significant reduction in C. difficile counts when JDM301 was co-cultured with C. difficile, which was correlated with the significant decrease in clostridial toxin titres (TcdA and TcdB). Furthermore, the cell-free culture supernatants (CFS) of JDM301 inhibited C. difficile growth and degraded TcdA and TcdB. Notably, the results showed that acid pH promoted the degradation of TcdA by CFS from JDM301. Furthermore, comparative studies among 10 B. longum strains were performed, which showed that the inhibitory effect of CFS from JDM301 was similar with the other 8 B. longum strains and higher than strain BLY1. However, when it was neutralized, the significant different was lost. When present together, it was suggested that the acid pH induced by probiotics not only played important roles in the growth inhibition against C. difficile resulting in the reduction of toxins titres, but also directly promoted the degradation of clostridial toxin. In vivo studies proved that JDM301 partially relieved damage to tissues caused by C. difficile and also decreased the number of C. difficile and toxin levels. In summary, our results demonstrated that the commercial strain, JDM301 could be considered a probiotic able to exert anti-toxin capability and most of the CFS from Bifidobacterium were able to inhibit the growth of C. difficile, depending on acid pH. These results highlighted a potential that JDM301 could be helpful in preventing CDI and that most of the bifidobacterial strains could (at least partially) exert protective effects by reducing toxin titres through growth inhibition against toxigenic C. difficile.

Project description:BackgroundClostridium difficile is the main cause of nosocomial diarrhea, but is also found in asymptomatic subjects that are potentially involved in transmission of C. difficile infection. A sensitive and accurate detection method of C. difficile, especially toxigenic strains is indispensable for the epidemiological investigation.MethodsTaqMan-based quantitative-PCR (qPCR) method for targeting 16S rRNA, tcdB, and tcdA genes of C. difficile was developed. The detection limit and accuracy of qPCR were evaluated by analyzing stool samples spiked with known amounts of C. difficile. A total of 235 stool specimens collected from 82 elderly nursing home residents were examined by qPCR, and the validity was evaluated by comparing the detection result with that by C. difficile selective culture (CDSC).ResultsThe analysis of C. difficile-spiked stools confirmed that qPCR quantified whole C. difficile (TcdA+TcdB+, TcdA-TcdB+, and TcdA-TcdB- types), TcdB-producing strains (TcdA+TcdB+ and TcdA-TcdB+ types), and TcdA-producing strains (TcdA+TcdB+ type), respectively, with a lower detection limit of 103 cells/g of stool. Of the 235 specimens examined, 12 specimens (5.1%) were C. difficile-positive by qPCR: TcdA+TcdB+ strain in six specimens and TcdA-TcdB- strain in the other six. CDSC detected C. difficile in 9 of the 12 specimens, and toxigenic types of the isolates from the 9 specimens were consistent with those identified by qPCR, supporting the validity of our qPCR method. Moreover, the qPCR examination revealed that the carriage rate of whole C. difficile and that of toxigenic strains in the 82 subjects over a 6-month period ranged from 2.4 to 6.8% and 1.2 to 3.8%, respectively. An average qPCR count of C. difficile detected was 104.5 cells/g of stool, suggesting that C. difficile constituted a very small fraction of intestinal microbiota.ConclusionOur qPCR method should be an effective tool for both clinical diagnosis and epidemiological investigation of C. difficile.

Dataset Information

A prospective study of two isothermal amplification assays compared with real-time PCR, CCNA and toxigenic culture for the diagnosis of Clostridium difficile infection.

Publications

A prospective study of two isothermal amplification assays compared with real-time PCR, CCNA and toxigenic culture for the diagnosis of Clostridium difficile infection.

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