Enhanced Detection of Vaccine Type Colonization and Dual Serotype Carriage with Molecular Strategies for Identification of Streptococcus pneumoniae Colonization

Abstract

Background. Detecting Streptococcus pneumoniae (SP) carriage in children via conventional microbiological methods lacks sensitivity as high density is required for routine culture and identification of dual serotype colonization is a technical challenge. To increase understanding of post vaccine nasopharyngeal (NP) carriage, specifcally persistence of vaccine serotypes, strategies for molecular identification of SP have been developed. These methods most ofen rely on the identification of the SP autolysin gene lytA through non-quantitative PCR or semi-quantitative real-time PCR (RTPCR). Methods. We collected NP swabs from 600 healthy or sick children <5 years old at Boston Medical Center from Nov 2015-Mar 2016 and used enhanced microbio-logic culture and molecular identification strategies to characterize SP colonization. NP specimens were broth enriched for 4 hours and cultured on selective blood agar; routine microbiologic methods were used to isolate and identify SP. A second aliquot of enriched broth was used for DNA isolation. RTPCR assays were performed targeting the lytA, piaB (SP membrane permease), cpsA (SP capsule operon) genes, and 21 SP serotypes: all serotypes included in 13-valent pneumococcal conjugate vaccine and 8 additional non-vaccine serotypes. Results. In our sample, 16% of specimens were SP positive via culture and 33% were RTPCR-positive (Cq≤35) for the lytA gene. Multiplex RTPCR assays with both the lytA and piaB genes yielded a positive result for 24% of samples. Further RTPCR confirmed that 99% of the lytA/piaB positive samples were positive for cpsA (a second marker for assay validation) but only 70% of the lytA positive, paiB negative samples were cpsA positive. Serogroup 19 was the most frequently isolated vaccine serogroup using both culture and RTPCR; molecular analysis identified 6% of specimens with concurrent carriage of more than one serotype. Conclusion. Compared with enhanced culture, we found a 50% increase in SP detection using combined lytA and piaB multiplex RTPCR. Similarly, the proportion of children colonized with vaccine serotypes increased from 2% to 7%. This work is funded by an investigator initiated grant to BUMC from Pfzer.