The PneuCarriage Project: A Multi-Centre Comparative Study to Identify the Best Serotyping Methods for Examining Pneumococcal Carriage in Vaccine Evaluation Studies

Abstract

Background: The pneumococcus is a diverse pathogen whose primary niche is the nasopharynx. Over 90 different serotypes exist, and nasopharyngeal carriage of multiple serotypes is common. Understanding pneumococcal carriage is essential for evaluating the impact of pneumococcal vaccines. Traditional serotyping methods are cumbersome and insufficient for detecting multiple serotype carriage, and there are few data comparing the new methods that have been developed over the past decade. We established the PneuCarriage project, a large, international multi-centre study dedicated to the identification of the best pneumococcal serotyping methods for carriage studies.

Methods and findings: Reference sample sets were distributed to 15 research groups for blinded testing. Twenty pneumococcal serotyping methods were used to test 81 laboratory-prepared (spiked) samples. The five top-performing methods were used to test 260 nasopharyngeal (field) samples collected from children in six high-burden countries. Sensitivity and positive predictive value (PPV) were determined for the test methods and the reference method (traditional serotyping of >100 colonies from each sample). For the alternate serotyping methods, the overall sensitivity ranged from 1% to 99% (reference method 98%), and PPV from 8% to 100% (reference method 100%), when testing the spiked samples. Fifteen methods had ≥70% sensitivity to detect the dominant (major) serotype, whilst only eight methods had ≥70% sensitivity to detect minor serotypes. For the field samples, the overall sensitivity ranged from 74.2% to 95.8% (reference method 93.8%), and PPV from 82.2% to 96.4% (reference method 99.6%). The microarray had the highest sensitivity (95.8%) and high PPV (93.7%). The major limitation of this study is that not all of the available alternative serotyping methods were included.

Conclusions: Most methods were able to detect the dominant serotype in a sample, but many performed poorly in detecting the minor serotype populations. Microarray with a culture amplification step was the top-performing method. Results from this comprehensive evaluation will inform future vaccine evaluation and impact studies, particularly in low-income settings, where pneumococcal disease burden remains high.

Conflict of interest statement

I have read the journal's policy and the authors of the manuscript have the following competing interests: DCHB and DH are previous employees of the Bill and Melinda Gates Foundation, which provided funding for this project. KPK was employed by the Bill and Melinda Gates Foundation towards the completion of the project, but was not involved in funding the project. KPK is a member of the Editorial Board of PLOS Medicine. CS and EMD were awarded the Robert Austrian Award which is funded by Pfizer. Authors have served on advisory boards for Merck (CS, KPK, EKM), GSK (KPK, EKM), and Pfizer (KPK). Research group authors have a scientific and/or commercial interest in the performance of their methods, so blinded testing was conducted.

Figures

Fig 1. Spiked sample testing results.

For each method (labelled m1–m22), the sensitivity of detection of the major serotypes (x-axis) and minor serotypes (y-axis) is plotted on the graph, with the PPV shown in colour according to the colour bar on the right. Methods that directly tested the sample or included a culture amplification step are represented by triangles and circles, respectively.

Fig 2. Serotype distribution in field samples.

A total of 307 serotypeable pneumococci (representing 49 serotypes) were identified in 260 nasopharyngeal swab samples collected from children in six countries. The 26 most common serotypes are shown here, with the remaining 23 serotypes identified combined as “other”.

Fig 3. Sensitivity and PPV of the five methods testing the 260 field samples.

The point estimates and 95% CIs for sensitivity (A) and PPV (B) are depicted. The sensitivity of method 4 is higher than those of the other methods.

Fig 4. Performance of microarray in determining percent abundance of serotypes in spiked and field samples.

The percent relative abundance reported by method 4 (culture microarray) compared with the inocula for 174 serotypeable pneumococci within 70 spiked samples with multiple serotypes (filled circles) and compared with results obtained by conventional serotyping according to the reference method for 61 serotypeable pneumococci within 27 field samples with multiple serotypes (open circles). For the spiked samples, the correlation of relative abundance results between the inocula and microarray was significant (p < 0.001): Spearman’s r = 0.863 (95% CI: 0.818, 0.897). Similarly, the correlation between actual relative abundance and microarray results was significant for the field samples (p < 0.001): Spearman’s r = 0.907 (95% CI: 0.847, 0.944).