Development of a viability real time PCR assay for Bordetella pertussis, B parapertussis and B holmesii

Abstract

Background Real time PCR provides rapid and accurate laboratory confirmation of clinically suspected pertussis and pertussis like illness (PLI). However, it does not inform the viability as it detects bacterial DNA from both viable and degraded bacterial cells. We report development of platinum based viability real time PCR assays for Bordetella pertussis, B.parapertussis and B.holmesii which cause pertussis and PLI. Material and MethodsATCC strains of the three organisms were grown on selective media, and cell counts determined. Untreated (control) and Sodium dodecyl sulphate (SDS) lysed cultures were treated with varying concentrations of platinum chloride (PtCl4) and genomic DNA extracted. Real time PCR was performed for the targets of B.pertussis (IS481), B.parapertussis (pIS1001) and B.holmesii (hIS1001) respectively, and quantitative results obtained. threshold cycle values after PtCl4 exposure and before PtCl4 exposure ({Delta} Cq) were calculated for both untreated and SDS treated cultures. ResultsFor all three species, increase in {Delta}Cq values was noted with corresponding reduction in DNA copy numbers, as PtCl4 concentration was increased, in untreated and SDS treated cultures. Untreated cultures of all three species exposed to PtCl4showed increase in Cq values, suggesting that liquid cultures contain a mixture of live and dead bacteria. ConclusionPerformance of viability q PCR assay incorporating PtCl4 at a concentration range of 6 to 10 mM would inform the viability of the infecting organism in the clinical sample. This assay would improve the diagnostic reliability, help select samples for culture as well as adoption of mitigation strategies among contacts of pertussis cases. ImportanceO_LIPertussis and pertussis like illness (PLI) are highly contagious respiratory tract infections for which Real time PCR is the frontline diagnostic tool. C_LIO_LIThis test however does not inform the viability of the infecting pathogen, as it detects nucleic acid which can be present even after degradation of the cell. C_LIO_LIPerforming a viability qPCR incorporating platinum chloride (PtCl4) at a concentration range of 6 to 10 mM would inform the viability of B. pertussis, B. parapertussis and B. holmesii in the clinical sample. C_LIO_LIIt is critical to know the viability of the infecting organism to determine which cases of pertussis and PLI which are actually infectious, to adopt mitigation strategies including prophylactic antibiotics among close contacts C_LIO_LIA viability assay can be used for quantitative measurement of bacterial number and viability and will help select the correct specimens for culture and isolation of the pathogen. C_LI