Viability-PCR Shows That NAAT Detects a High Proportion of DNA from Non-Viable Chlamydia trachomatis
| Autor: | Christian J. P. A. Hoebe, Kevin J. H. Janssen, Mayk Lucchesi, Nicole H. T. M. Dukers-Muijrers, Petra F. G. Wolffs, Lisanne Eppings |
|---|---|
| Přispěvatelé: | MUMC+: DA MMI Toegelatenen (9), RS: CAPHRI - R4 - Health Inequities and Societal Participation, Med Microbiol, Infect Dis & Infect Prev, Promovendi PHPC, MUMC+: DA MMI Moleculaire dia (9), MUMC+: DA MMI Management (9), RS: NUTRIM - R3 - Chronic inflammatory disease and wasting |
| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Pathology lcsh:Medicine Artificial Gene Amplification and Extension Chlamydia trachomatis medicine.disease_cause Pathology and Laboratory Medicine Polymerase Chain Reaction Biochemistry law.invention Chlamydia Infection chemistry.chemical_compound 0302 clinical medicine law Propidium monoazide Nucleic Acids Medicine and Health Sciences 030212 general & internal medicine Chlamydia lcsh:Science DNA extraction Polymerase chain reaction Multidisciplinary Cell Death Bacterial Pathogens Real-time polymerase chain reaction Infectious Diseases Medical Microbiology Cell Processes Vaginal swabs Female Biological Cultures Pathogens Research Article DNA Bacterial medicine.medical_specialty 030106 microbiology Sexually Transmitted Diseases Biology Research and Analysis Methods Microbiology 03 medical and health sciences Young Adult Extraction techniques medicine Nucleic Acid Amplification Tests Humans Molecular Biology Techniques Molecular Biology Microbial Pathogens Microbial Viability Bacteria lcsh:R Organisms Biology and Life Sciences Cell Biology Cell Cultures Molecular biology chemistry lcsh:Q DNA HeLa Cells |
| Zdroj: | PLoS ONE, Vol 11, Iss 11, p e0165920 (2016) PLoS ONE PLOS ONE, 11(11):e0165920. Public Library of Science |
| ISSN: | 1932-6203 |
| Popis: | Objectives According to the current guidelines for laboratory diagnosis of sexually transmitted infections (STIs), nucleic acid amplification tests (NAATs) are the preferred diagnostic method for Chlamydia trachomatis (CT) infections. However, NAATs amplify the available target DNA without discriminating between DNA originating from viable or non-viable CT. Assessing CT viability will provide more insights in the clinical and public health relevance of a CT positive test result. The aim of this study was to technically validate and implement viability-PCR (V-PCR) to asses CT viability. Methods Technical validation of V-PCR was performed by the assessment of predefined viability ratios of CT. Samples were subjected to V-PCR which consisted of propidium monoazide (PMA) treatment prior to DNA extraction followed by quantitative PCR (qPCR) targeting the ompA gene for the detection of CT DNA. Finally, V-PCR was applied to vaginal swabs of 50 CT positive patients, as indicated by routine NAAT, collected at our outpatient STD clinics before antimicrobial treatment. Results Technical validation of V-PCR showed that PMA treatment of heat-inactivated CT culture resulted in an almost complete loss of qPCR signal. PMA treated samples of the fresh viable CT culture showed no marked reduction of PCR signal, indicating that all DNA from viable CT could be detected. Applying V-PCR to clinical samples showed that in 36% of samples (18/50) less than 1% of CT DNA originated from viable bacteria. Conclusions V-PCR showed to be a fast and easy method to assess CT viability in clinical samples, without the need of traditional challenging cell culture methods. Furthermore, V-PCR results of clinical samples have indicated that a substantial amount of the amplified CT DNA originated from non-viable cells. Although results might be influenced by cell death during transport, this study suggests that there is a potential overestimation of quantitative CT positivity by currently used NAATs. |
| Databáze: | OpenAIRE |
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