Density Distribution of Pharyngeal Carriage of Meningococcus in Healthy Young Adults

Abstract

Background: Improved understanding of Neisseria meningitidis (Nm) carriage biology and better methods for detection and quantification would facilitate studies of potential impact of new vaccines on colonization and transmission in adolescents. Methods:We performed plate cultures on 107 oropharyngeal swabs stored frozen in STGG broth and previously positive for Nm. We compared quantitative (q)PCR detection of Nm in 601 STGG-swabs with culture. Using qPCR (n=87), a log-phase broth culture standard curve and semi-quantitative plate cultures (n=68), we measured density of carriage. We compared qPCR genogrouping of DNA extracts from STGG-swabs and from plate culture lawns (n=110) with purified isolates (n=80).Results: Swab storage resulted in only 10% loss of culture sensitivity. Direct sodC qPCR Nm detection yielded more positives (87/601, 14.5%) than culture (80/601, 13.3%). Most samples (57/110) positive by culture were also positive by qPCR and vice versa, but discrepancies (single positives) were frequent among low density samples. sodC qPCR was positive in 79/80 isolates but in only 65 by ctrA qPCR. Density both by culture and qPCR varied across 4 orders of magnitude with the majority being low (<50 bacteria-gene copies/mL) and a minority being high (>1000). Genogrouping qPCRs yielded more positive results when performed on DNA extracts from lawn cultures. Conclusions:We provide the first description of the distribution of Nm carriage density. This could be important for understanding transmission dynamics and population-level effectiveness of adolescent vaccine programs. Storage of swabs frozen in STGG for batched laboratory analysis facilitates carriage studies and direct sodC qPCR for Nm combined with qPCR genogrouping of lawn culture extracts provides accurate, detailed description of colonization.

B Part of It protocol: a cluster randomised controlled trial to assess the impact of 4CMenB vaccine on pharyngeal carriage ofNeisseria meningitidisin adolescents

“…Quantitative PCR will be applied to the positive screen samples for estimation of the density of carriage of the Neisseria species. 23 A standard curve will be generated allowing comparison of crossing point values from the specimen analysis with the standard curve allowing the estimation of Neisseria density in the specimen. Samples will be stored long term in STGG broth at −80°C for future whole genome sequencing.…”

B Part of It study: a longitudinal study to assess carriage of Neisseria meningitidis in first year university students in South Australia

“…14 However, the time from swab collection to freezing may be an important factor in the isolation of Neisseria species. 14 Saliva collection has been rarely used in meningococcal carriage studies, although this method is now being used by a group in the UK. 15,16 Our study is the first to estimate carriage prevalence and risk factors of N. meningitidis in Australian university students.…”

Viable Neisseria meningitidis is commonly present in saliva in healthy young adults: Non-invasive sampling and enhanced sensitivity of detection in a follow-up carriage study in Portuguese students

“…meningitidis (Nm, meningococcus) has been the subject of increased recent study due to recognition that, although licensed on the basis of immunogenicity and inferred direct protection in recipients, the principal and most easily-exploited mechanism of action of meningococcal conjugate vaccines is their impact on transmission at the population level [1] and consequent interest in whether novel protein antigen vaccines may have analogous effects. We have reported advances in sampling and laboratory methodology which simplify the logistics and greatly reduce the costs of performing carriage studies and have used them to describe a previously unknown distribution of colonisation density, heavily skewed with a large majority of carriers having small numbers of bacteria and a minority of around 10–15% carrying 2–4 orders of magnitude more organisms in a cross-sectional population sample [2].…”

“…Introduction of a culture step prior to DNA extraction and PCR might both indicate the presence of viable organisms when signals were compared to those from PCR of uncultured samples and might increase the sensitivity of detection among the large number of low density carriers if viable bacteria were present, as reported recently for an overnight incubation step following sampling [6]. We conducted a study in healthy students at the University of Coimbra in Portugal, a population we have studied previously [2, 7]. By taking paired oropharyngeal swabs (OPS) and saliva samples from each subject, we were able to compare the sensitivity of both techniques and also evaluate to what extent it could be enhanced by analysing both samples taken at the same time.…”