Simultaneous detection and ribotyping of Clostridioides difficile, and toxin gene detection directly on fecal samples

Tessel M Van Rossen,Andries E Budding,Joffrey Van Prehn,Robin Van Houdt,Rosa Van Mansfeld,Marcel Jonges,Christina M J E Vandenbroucke-Grauls,Alex Koek,Ed J Kuijper

doi:10.1186/s13756-020-00881-9

Abstract

BackgroundClostridioides difficile is the most common cause of nosocomial diarrhea. Ribotyping of cultured strains by a PCR-based test is used to study potential transmission between patients. We aimed to develop a rapid test that can be applied directly on fecal samples for simultaneous detection and ribotyping of C. difficile, as well as detection of toxin genes.MethodsWe developed a highly specific and sensitive primer set for simultaneous detection and ribotyping of C. difficile directly on total fecal DNA. Toxin genes were detected with primers adapted from Persson et al. (Clin Microbiol Infect 14(11):1057–1064). Our study set comprised 130 fecal samples: 65 samples with positive qPCR for C. difficile toxin A/B genes and 65 C. difficile qPCR negative samples. PCR products were analyzed by capillary gel electrophoresis.ResultsRibosomal DNA fragment peak profiles and toxin genes were detected in all 65 C. difficile positive fecal samples and in none of the 65 C. difficile negative samples. The 65 samples were assigned to 27 ribotypes by the Dutch reference laboratory. Our peak profiles corresponded to these ribotypes, except for two samples. During a C. difficile outbreak, patients were correctly allocated to the outbreak-cluster based on the results of direct fecal ribotyping, before C. difficile isolates were cultured and conventionally typed.ConclusionC. difficile ribotyping directly on fecal DNA is feasible, with sensitivity and specificity comparable to that of diagnostic toxin gene qPCR and with ribotype assignment similar to that obtained by conventional typing on DNA from cultured isolates. This supports simultaneous diagnosis and typing to recognize an outbreak.

Highlights

Clostridioides difficile is the most common cause of nosocomial diarrhea
Application of direct ribotyping during an outbreak with C. difficile During the optimization phase of our direct ribotyping technique, a C. difficile outbreak was suspected in the Intensive Care Unit (ICU)
Samples of five other patients became positive for C. difficile by quantitative PCR (qPCR) or toxin enzyme immune assay (EIA)

Summary

Introduction

Clostridioides difficile is the most common cause of nosocomial diarrhea. Ribotyping of cultured strains by a PCR-based test is used to study potential transmission between patients. A commonly used typing technique for C. difficile, which is recommended for surveillance purposes, is PCR ribotyping [8] This method is based on strain-specific differences in number and length of the ribosomal 16-23S interspace regions (IS-regions). A drawback of the currently used PCR ribotyping methods is that they can only be performed on cultured C. difficile isolates [9,10,11,12,13,14]. This delays the time to results and eventual implementation of infection control measures. Janezic et al designed new primers and tested these directly on total fecal DNA; they obtained a specificity of 100% and a sensitivity of 84.8% [15]

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