Standardisation and validation of an in-house quantitative real-time polymerase chain reaction (qPCR) assay for the diagnosis of Clostridioides difficile infection

Maestri, Adriane Ceshin; Raboni, Sônia Mara; Cogo, Laura Lúcia; Rossi, Marisa V; Nogueira, Keite da Silva

doi:10.1016/j.mimet.2021.106399

Cited by 5 publications

(1 citation statement)

References 36 publications

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“…The difference between the qPCR results and isolation can be explained by the high sensitivity of the technique (Maestri et al 2022). We believe that the use of BHI without a previous alcohol shock would have reduced the sensitivity of the isolation method.…”

Section: Discussionmentioning

confidence: 99%

Molecular epidemiology of Clostridioides difficile obtained from fecal samples of wild animals in Brazil

Lima,

Basso,

Cerri

et al. 2024

Pesq. Vet. Bras.

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Clostridioides difficile is a strictly anaerobic, spore-forming Gram-positive bacterium associated with diarrhea, known as C. difficile infection (CDI). In domestic animals, C. difficile is considered an important pathogen mostly in pigs and horses, but there are also reports in other domestic species. In wild animals, the epidemiology of C. difficile is largely unknown, and the role of the bacterium as a cause of diarrhea is unclear. The aim of this study was to determine the prevalence of C. difficile in the feces of wild animals referred to the Center of Medicine and Research in Wild Animals (CEMPAS). Fecal samples obtained from 100 animals of 34 different species were subjected to qPCR for the detection of the C. difficile 16S rRNA gene and two major toxin genes (tcdA and tcdB) and to anaerobic bacterial isolation. A total of 63 animals (63%) were positive for C. difficile by qPCR, and 16 isolates were recovered. The opossum (Didelphis spp.) had the highest number of positive animals in both tests (from 21 samples, 19 were qPCR positive, and four isolates were recovered). Three toxigenic strains (RT 002, 004, and 014), all previously described as infecting humans and animals, were isolated in the following species: bearded dragon (Pogona vitticeps), pampas fox (Lycalopex vetulus), and marmoset (Callithrix sp.). The presence of C. difficile in the feces of wild animals highlights the importance of wildlife as potential carriers of infection for production animals or humans.

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Section: Discussionmentioning

confidence: 99%

Molecular epidemiology of Clostridioides difficile obtained from fecal samples of wild animals in Brazil

Lima,

Basso,

Cerri

et al. 2024

Pesq. Vet. Bras.

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Laboratory diagnosis of Clostridioides difficile infection in symptomatic patients: what can we do better?

et al. 2023

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Development and clinical application of a rapid, visually interpretable polymerase spiral reaction for tcdB gene of Clostridioides difficile in fecal cultures

Yin,

Song,

Wang

et al. 2023

FEMS Microbiology Letters

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In the surveillance of outbreaks of Clostridioides difficile infection, the rapid detection and diagnosis of C. difficile remain a major challenge. Polymerase spiral reaction (PSR) is a nucleic acid amplification technique that uses mixed primers and the strand displacement activity of Bst DNA polymerase to achieve a pair of primers and a single enzyme in an isothermal environment. The primer design is simple, the reaction is efficient, and a color indicator can be used to visualize the result. In this study, we developed a rapid and visually interpretable PSR to detect C. difficile by analyzing artificially contaminated feces samples and clinical isolates from patient feces samples. We designed two pairs of primers for a PSR that specifically targeted the conserved tcdB gene of C. difficile. The amplification results were visualized with the chromogenic dye hydroxynaphthol blue. The entire process was accomplished in 50 min at 64°C, with high specificity. The limit of detection of C. difficile with PSR was 150 fg/μL genomic DNA or 2 × 10 CFU/mL in artificially contaminated feces samples. With this method, we analyzed four clinical isolates and also compared the PSR with an isolation-and-culture detection method, PCR, and the Sanger sequencing. The four clinical isolates were found positive for tcdB, which confirmed the high specificity of the primers. The positive rates of tcdB in toxigenic C. difficile detected with PSR, PCR, and Sanger sequencing were 100%. The proportions of toxin types in these clinical C. difficile strains were 50% tcdA+tcdB+CDT− and 50% tcdA+tcdB+CDT+. The assay described should extend our understanding of the incidence of C. difficile. This may allow the rapid diagnosis and screening of C. difficile-related disease outbreaks in the field.

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Standardisation and validation of an in-house quantitative real-time polymerase chain reaction (qPCR) assay for the diagnosis of Clostridioides difficile infection

Cited by 5 publications

References 36 publications

Molecular epidemiology of Clostridioides difficile obtained from fecal samples of wild animals in Brazil

Molecular epidemiology of Clostridioides difficile obtained from fecal samples of wild animals in Brazil

Laboratory diagnosis of Clostridioides difficile infection in symptomatic patients: what can we do better?

Development and clinical application of a rapid, visually interpretable polymerase spiral reaction for tcdB gene of Clostridioides difficile in fecal cultures

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