Improving Bordetella pertussis isolation from PCR positive samples: Impact of modified Regan-Lowe agar, sample Ct value and transport time on culture positivity

Objectives: Pertussis diagnosis relies mainly on PCR tests due to their higher sensitivity and faster results than culture methods.This led to a shortage of clinical isolates for resistance testing and genomic surveillance.Data on factors affecting strain recovery and on culture medium optimization are limited.This study validated the sensitivity of a modified Regan-Lowe agar for Bordetella recovery and assessed the impact of transport medium, transport time and Ct values on culture positivity.Methods: 500 PCR-positive respiratory samples from Austria and Germany were prospectively cultured on conventional Regan-Lowe agar and an in-house prepared variant supplemented with vancomycin.All plates were examined for Bordetella-suspicious colonies and confirmed by MALDI-TOF MS.Transport time, Ct values and transport media were compared between culture-positive and -negative samples.Results: Bordetella spp.were isolated from 167/500 (33 %) samples.82 isolates (16 %) were recovered from the conventional agar, whereas 160 isolates (32 %) were obtained from the plates with vancomycin.Mean transport time for culture-positive samples was 3.9 days vs. 7.9 days for culture-negative samples; mean Ct values 23.1 and Ct 29.2, respectively.Flocked swabs in Amies transport medium (eSwab) showed the highest recovery.For short transport times, Universal Transport Medium (UTM) was a valid alternative.Multivariable analysis confirmed Ct value, transport time and transport medium as independent predictors of culture-positivity. Conclusion:The addition of vancomycin to Regan-Lowe agar doubled the probability of isolation of Bordetella from PCR-positive samples.Our findings define key parameters to optimize culture recovery for molecular epidemiology and surveillance.