Tessel M van Rossen1, Joffrey van Prehn2, Alex Koek3, Marcel Jonges3, Robin van Houdt3, Rosa van Mansfeld3, Ed J Kuijper2, Christina M J E Vandenbroucke-Grauls3, Andries E Budding4. 1. Department of Medical Microbiology and Infection Control, Amsterdam Infection and Immunity Institute, Amsterdam UMC, Vrije Universiteit Amsterdam, PK 2X132, De Boelelaan 1117, Amsterdam, The Netherlands. t.vanrossen@amsterdamumc.nl. 2. Center for Infectious Diseases, Department of Medical Microbiology, Leiden University Medical Center, Albinusdreef 2, Leiden, The Netherlands. 3. Department of Medical Microbiology and Infection Control, Amsterdam Infection and Immunity Institute, Amsterdam UMC, Vrije Universiteit Amsterdam, PK 2X132, De Boelelaan 1117, Amsterdam, The Netherlands. 4. inBiome B.V., Science park 106, Amsterdam, The Netherlands.
Abstract
BACKGROUND: 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. 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. METHODS: We 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. RESULTS: Ribosomal 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. CONCLUSION: C. 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.
BACKGROUND: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. 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. METHODS: We 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. RESULTS: Ribosomal 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. CONCLUSION:C. 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.
Authors: Christian Quast; Elmar Pruesse; Pelin Yilmaz; Jan Gerken; Timmy Schweer; Pablo Yarza; Jörg Peplies; Frank Oliver Glöckner Journal: Nucleic Acids Res Date: 2012-11-28 Impact factor: 16.971