Population density profiles of nasopharyngeal carriage of 5 bacterial species in pre-school children measured using quantitative PCR offer potential insights into the dynamics of transmission.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked FilesBacterial vaccines can reduce carriage rates. Colonization is usually a binary endpoint. Real time quantitative PCR (qPCR) can quantify bacterial DNA in mucosal samples over a wide range. Using culture and single-gene species-specific qPCRs for Streptococcus pneumoniae (lytA), Streptococcus pyogenes (ntpC), Moraxella catarrhalis (ompJ), Haemophilus influenzae (hdp) and Staphylococcus aureus (nuc) and standard curves against log-phase reference strain broth cultures we described frequency and peak density distributions of carriage in nasopharyngeal swabs from 161 healthy 2-4 y old children collected into STGG broth. In general, detection by qPCR and culture was consistent. Discordance mostly occurred at lower detection thresholds of both methods, although PCR assays for S. pyogenes and S. aureus were less sensitive. Density varied across 5-7 orders of magnitude for the 5 species with the abundant species skewed toward high values (modes: S. pneumoniae log3-4, M. catarrhalis & H. influenzae log4-5 CFU/ml broth). Wide ranges of bacterial DNA concentrations in healthy children carrying these bacteria could mean that different individuals at different times vary greatly in infectiousness. Understanding the host, microbial and environmental determinants of colonization density will permit more accurate prediction of vaccine effectiveness.ESPID research fellowshi

The Effects of LAIV on Nasopharyngeal Bacteria in Healthy 2-4 Year Olds:a Randomized Controlled Trial

Rationale: Viral infections of the upper respiratory tract may influence the commensal nasopharyngeal bacteria. Changes in the bacterial niche could affect transmission dynamics. Attenuated vaccine viruses can be used to investigate this empirically in humans. Objectives: To study the effects of mild viral upper respiratory infections on nasopharyngeal bacterial colonization using live attenuated influenza vaccine (LAIV) as a surrogate. Methods: We used trivalent LAIV to evaluate the effects of viral infection on bacterial carriage and density of Streptococcus pneumoniae, Moraxella catarrhalis, Haemophilus influenzae, and Staphylococcus aureus. A total of 151 healthy children were randomized 1:1 to receive the vaccine starting either at recruitment (n = 74) or 28 days later (n = 77) in a stepped wedge fashion, allowing comparisons between recipients and nonrecipients as well as whole-group comparisons pre- and postvaccination. Bacterial carriage and density were determined using quantitative polymerase chain reaction assays. Measurements and Main Results: A total of 151 children were recruited, 77 in the LAIV group and 74 in the control group. LAIV recipients (n = 63 analyzed) showed an apparent transient increase in H. influenzae carriage but no further significant differences in carriage prevalence of the four bacterial species compared with controls (n = 72 analyzed). S. pneumoniae density was substantially higher in vaccine recipients (16,687 vs. 1935 gene copies per milliliter) 28 days after the first dose (P < 0.001). Whole-group multivariable analysis (prevaccine, after one dose, and after two doses) also showed increases in density of other species and H. influenzae carriage prevalence. Conclusions: In the absence of any safety signals despite widespread use of the vaccine, these findings suggest that bacterial density, and thus transmission rates among children and to people in other age groups, may rise following attenuated influenza infections without associated clinical disease. LAIV could therefore be used as an experimental tool to elucidate the dynamics of transmission of nasopharyngeal bacteria

High Density Bacterial Nasal Carriage in Children is Transient and Associated With Respiratory Viral Infections - Implications for Transmission Dynamics

To access publisher's full text version of this article click on the hyperlink belowBACKGROUND: This longitudinal study describes the associations between respiratory viral infections, rhinitis and the prevalence and density of the common nasopharyngeal bacterial colonizers, Streptococcus pneumoniae (Sp), Moraxella catarrhalis (Mc), Haemophilus influenzae (Hi) and Staphylococcus aureus. METHODS: In an observational cohort study, 161 children attending day care centers in Bristol, United Kingdom, were recruited. Monthly nasopharyngeal swabs were taken and stored frozen in Skim-milk, tryptone, glucose and glycerin broth (STGG) broth. Quantitative polymerase chain reaction was used for detection of respiratory viruses and 4 bacterial species. t tests and logistic regression models were used for analysis. RESULTS: The frequent colonisers, Sp, Mc and Hi were more frequently found at high density in contrast to Staphylococcus aureus although temporally, high-density carriage was short lived. Respiratory viral infections and symptoms of rhinitis were both independently and consistently associated with higher bacterial density with an observed 2-fold increase in density for Sp, Mc and Hi (P = 0.004-0.017). CONCLUSIONS: For Sp and Hi, the association between young age and higher bacterial DNA density was explained by more frequent viral infection and increased nasal discharge, while the associations between some viral specie's and some bacterial species' density appear to be stronger than others. Increased colonization density and rhinitis may promote transmission of these commonly carried organisms

Viable <i>Neisseria meningitidis </i>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

INTRODUCTION AND AIMS:Improved sensitivity and efficiency of detection and quantification of carriage of Neisseria meningitidis (Nm) in young people is important for evaluation of the impact of vaccines upon transmission and associated population-wide effects. Saliva collection is quick, non-invasive and facilitates frequent sampling, but has been reported to yield low sensitivity by culture. We re-evaluated this approach in a follow-up cross sectional study using direct and culture-amplified PCR. MATERIAL/METHODS:In April 2016 we collected paired oropharyngeal swabs (OPS) and saliva samples from 1005 healthy students in Portugal into STGG broth and stored them at -80°C until DNA extraction and batched qPCR analysis. Samples were also cultured on GC agar plates for 72h and PCR done on DNA extracts from overall growth. Nm isolates were also sought from a selection of 50 samples. qPCR amplification targets were superoxide dismutase sodC and capsular locus/genogroup-specific genes (B, C, W, X and Y) and, for cultured isolates only, porA. Cycle threshold values of ≤36 were considered positive. RESULTS:556 tests (460 samples, 363 subjects, 36.1%) were positive for Nm (sodC) and 65 (45, 36, 3.6%) for MenB. More salivas were positive by direct sodC qPCR (211, 21.0%) than OPS (126, 12.5%) but fewer were positive by culture-amplified qPCR (94 vs. 125). For both sample types, many that were negative on direct qPCR came positive on culture-amplification and Nm was consistently isolated from salivas in which culture amplified the PCR signal. Using both methods on both samples yielded 36.1% Nm and 5.5% encapsulated Nm carriage rates while direct qPCR on OPS alone detected 12.5% and 2.2%. CONCLUSIONS:Detectable MenB carriage rates (2.9%) were lower than 4 years earlier (6.8%) in this population (p = 0.0003). Viable meningococci were often present in saliva. Although evidence of encapsulated Nm was less frequent in saliva than OPS, collection is more acceptable to subjects allowing more frequent sampling. Use of culture-amplification increases detection sensitivity in both sample types, especially when combined with direct PCR. Combining these samples and/or methodologies could greatly enhance the power of carriage studies to detect the impact of vaccines upon carriage and transmission

Pneumococcal Serotypes Colonise the Nasopharynx in Children at Different Densities.

Prevalence of pneumococcal serotypes in carriage and disease has been described but absolute serotype colonisation densities have not been reported. 515 paediatric nasal swab DNA extracts were subjected to lytA qPCR and molecular serotyping by microarray. Absolute serotype densities were derived from total pneumococcal density (qPCR cycle threshold and standard curve) and relative abundance (microarray) and varied widely. Compared to all serotype densities observed, the strongest evidence of differences was seen for serotypes 21 and 35B (higher) and 3, 38 and non-typeables (lower) (p<0.05) with a similar hierarchy when only a single serotype carriage was assessed. There was no evidence of any overall density differences between children with single or multiple serotypes detected but serotypes with mid-range densities were more prevalent. The hierarchy of distinct pneumococcal serotype carriage densities described here for the first time, may help explain the dynamics of transmission between children

Impact of a quadrivalent meningococcal ACWY glycoconjugate or a serogroup B meningococcal vaccine on meningococcal carriage: an observer-blind, phase 3 randomised clinical trial

Background: Meningococcal conjugate vaccines protect individuals directly, but also confer herd protection by interrupting carriage transmission. This Phase III observer-blind, randomised, controlled study evaluated the effects of meningococcal quadrivalent (ACWY) glycoconjugate (MenACWY-CRM) or serogroup B (4CMenB) vaccination on meningococcal carriage rates in young adults. Methods: University students (aged 18–24 years) from ten sites in England were randomised to receive two vaccinations one month apart: two doses of Japanese Encephalitis vaccine (controls), two doses of 4CMenB (4CMenB), or one dose of MenACWY-CRM then placebo (MenACWY-CRM). Meningococci were isolated from oropharyngeal swabs collected before vaccination and at five scheduled intervals over one year. Primary analysis was cross-sectional carriage one month after the vaccine course; secondary analyses included comparison of carriage at any time point after primary analysis until study termination. Findings: 2954 subjects were randomised (control, n=987; 4CMenB, n=988; MenACWY-CRM, n=979); approximately one-third of each group was positive for meningococcal carriage at study entry. By one month, there was no significant difference in carriage between controls and 4CMenB (Odds Ratios (OR) [95% CI]; 1·2 [0·8−1·7]) or MenACWY-CRM (OR [95% CI], 0·9 [0·6–1·3]) groups. From three months after dose two, 4CMenB vaccination resulted in significantly lower carriage of any meningococcal strain (calculated efficacy 18·2% [95% CI: 3·4–30·8]) and capsular groups BCWY (calculated efficacy 26·6% [95% CI: 10·5–39·9]) compared to control vaccination. Significantly lower carriage rates were also observed in the MenACWY-CRM group compared with controls: calculated efficacies 39·0% [95%CI: 17·3-55·0] and 36.2% [95%CI: 15·6-51·7] for serogroups Y and CWY, respectively. Interpretation: MenACWY-CRM and 4CMenB vaccines reduced meningococcal carriage rates over 12 months post-vaccination and, therefore, may affect transmission where widely implemented

Effect of a quadrivalent meningococcal ACWY glycoconjugate or a serogroup B meningococcal vaccine on meningococcal carriage: an observer-blind, phase 3 randomised clinical trial

Background: Meningococcal conjugate vaccines protect individuals directly, but also confer herd protection by interrupting carriage transmission. This Phase III observer-blind, randomised, controlled study evaluated the effects of meningococcal quadrivalent (ACWY) glycoconjugate (MenACWY-CRM) or serogroup B (4CMenB) vaccination on meningococcal carriage rates in young adults.Methods: University students (aged 18–24 years) from ten sites in England were randomised to receive two vaccinations one month apart: two doses of Japanese Encephalitis vaccine (controls), two doses of 4CMenB (4CMenB), or one dose of MenACWY-CRM then placebo (MenACWY-CRM). Meningococci were isolated from oropharyngeal swabs collected before vaccination and at five scheduled intervals over one year. Primary analysis was cross-sectional carriage one month after the vaccine course; secondary analyses included comparison of carriage at any time point after primary analysis until study termination.Findings: 2954 subjects were randomised (control, n=987; 4CMenB, n=988; MenACWY-CRM, n=979); approximately one-third of each group was positive for meningococcal carriage at study entry. By one month, there was no significant difference in carriage between controls and 4CMenB (Odds Ratios (OR) [95% CI]; 1·2 [0·8−1·7]) or MenACWY-CRM (OR [95% CI], 0·9 [0·6–1·3]) groups. From three months after dose two, 4CMenB vaccination resulted in significantly lower carriage of any meningococcal strain (calculated efficacy 18·2% [95% CI: 3·4–30·8]) and capsular groups BCWY (calculated efficacy 26·6% [95% CI: 10·5–39·9]) compared to control vaccination. Significantly lower carriage rates were also observed in the MenACWY-CRM group compared with controls: calculated efficacies 39·0% [95%CI: 17·3-55·0] and 36.2% [95%CI: 15·6-51·7] for serogroups Y and CWY, respectively.Interpretation: MenACWY-CRM and 4CMenB vaccines reduced meningococcal carriage rates over 12 months post-vaccination and, therefore, may affect transmission where widely implemented