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

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The Pneucarriage Project: a Multi-Centre Comparative Study to Identify the Best Serotyping Methods for Examining Pneumococcal Carriage in Vaccine Evaluation Studies RESEARCH ARTICLE The PneuCarriage Project: A Multi-Centre Comparative Study to Identify the Best Serotyping Methods for Examining Pneumococcal Carriage in Vaccine Evaluation Studies Catherine Satzke1,2*, Eileen M. Dunne1, Barbara D. Porter1, Keith P. Klugman3,E. a11111 Kim Mulholland1,4, PneuCarriage project group¶ 1 Pneumococcal Research Group, Murdoch Childrens Research Institute, Royal Children’s Hospital, Parkville, Victoria, Australia, 2 Department of Microbiology and Immunology, University of Melbourne, Peter Doherty Institute for Infection and Immunity, Parkville, Victoria, Australia, 3 Hubert Department of Global Health, Rollins School of Public Health, Emory University, Atlanta, Georgia, United States of America, 4 Department of Infectious Disease Epidemiology, London School of Hygiene & Tropical Medicine, London, United Kingdom OPEN ACCESS ¶ Membership of the PneuCarriage project group is provided in the Acknowledgments. Citation: Satzke C, Dunne EM, Porter BD, Klugman * [email protected] KP, Mulholland EK, PneuCarriage project group (2015) The PneuCarriage Project: A Multi-Centre Comparative Study to Identify the Best Serotyping Methods for Examining Pneumococcal Carriage in Abstract Vaccine Evaluation Studies. PLoS Med 12(11): e1001903. doi:10.1371/journal.pmed.1001903 Academic Editor: Derek Bell, Imperial College Background London, UNITED KINGDOM The pneumococcus is a diverse pathogen whose primary niche is the nasopharynx. Over Received: February 26, 2015 90 different serotypes exist, and nasopharyngeal carriage of multiple serotypes is common. Accepted: October 9, 2015 Understanding pneumococcal carriage is essential for evaluating the impact of pneumococ- cal vaccines. Traditional serotyping methods are cumbersome and insufficient for detecting Published: November 17, 2015 multiple serotype carriage, and there are few data comparing the new methods that have Copyright: © 2015 Satzke et al. This is an open been developed over the past decade. We established the PneuCarriage project, a large, access article distributed under the terms of the Creative Commons Attribution License, which permits international multi-centre study dedicated to the identification of the best pneumococcal ser- unrestricted use, distribution, and reproduction in any otyping methods for carriage studies. medium, provided the original author and source are credited. Methods and Findings Data Availability Statement: All relevant data are Reference sample sets were distributed to 15 research groups for blinded testing. Twenty within the paper and its Supporting Information files. pneumococcal serotyping methods were used to test 81 laboratory-prepared (spiked) sam- Funding: This project was initially funded through the ples. The five top-performing methods were used to test 260 nasopharyngeal (field) sam- PneumoCarr Consortium (Grant 37875 funded by the Grand Challenges in Global Health Initiative which ples collected from children in six high-burden countries. Sensitivity and positive predictive was supported by The Bill & Melinda Gates value (PPV) were determined for the test methods and the reference method (traditional Foundation, the Foundation for the National Institutes serotyping of >100 colonies from each sample). of Health, the Wellcome Trust, and the Canadian For the alternate serotyping methods, the overall sensitivity ranged from 1% to 99% (ref- Institutes of Health Research). The PneuCarriage project was subsequently directly funded by The Bill erence method 98%), and PPV from 8% to 100% (reference method 100%), when testing & Melinda Gates Foundation (www.gatesfoundation. the spiked samples. Fifteen methods had 70% sensitivity to detect the dominant (major) PLOS Medicine | DOI:10.1371/journal.pmed.1001903 November 17, 2015 1/30 Serotyping Methods for Pneumococcal Carriage Studies org), Grant 52099. This project was also supported serotype, whilst only eight methods had 70% sensitivity to detect minor serotypes. For the by the Murdoch Childrens Research Institute (www. field samples, the overall sensitivity ranged from 74.2% to 95.8% (reference method mcri.edu.au) and the Victorian Government's Operational Infrastructure Support Program. The 93.8%), and PPV from 82.2% to 96.4% (reference method 99.6%). The microarray had the funders had no role in the study design, data highest sensitivity (95.8%) and high PPV (93.7%). The major limitation of this study is that collection and analysis, decision to publish, or not all of the available alternative serotyping methods were included. preparation of the manuscript; except for the specific roles of DCHB, DH and KPK as described in the author contributions. All authors have read, and Conclusions confirm that they meet, ICMJE criteria for authorship. Most methods were able to detect the dominant serotype in a sample, but many performed Competing Interests: I have read the journal's policy poorly in detecting the minor serotype populations. Microarray with a culture amplification and the authors of the manuscript have the following step was the top-performing method. Results from this comprehensive evaluation will inform competing interests: DCHB and DH are previous employees of the Bill and Melinda Gates Foundation, future vaccine evaluation and impact studies, particularly in low-income settings, where which provided funding for this project. KPK was pneumococcal disease burden remains high. 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 Introduction boards for Merck (CS, KPK, EKM), GSK (KPK, EKM), and Pfizer (KPK). Research group authors Streptococcus pneumoniae (the pneumococcus) is a dominant cause of childhood illness and have a scientific and/or commercial interest in the death worldwide. It has been estimated to cause ~800,000 deaths in children aged under 5 y performance of their methods, so blinded testing was annually, most due to pneumonia and most in low-income countries [1]. Pneumococci com- conducted. monly colonise the nasopharynx of healthy children. Although carriage is usually asymptom- Abbreviations: CDC, US Centers for Disease atic, it can cause local inflammation and is considered a prerequisite for pneumococcal disease Control and Prevention; CFU, colony-forming units; [2]. Pneumococci are diverse, with 97 serotypes identified to date. Understanding carriage is ESI-MS, electrospray ionisation mass spectrometry; HBA, horse blood agar; IQR, interquartile range; MFI, important for understanding pneumococcal population biology and transmission, assessing mean fluorescence intensity; MLST, multilocus vaccine impact, and evaluating the performance of new vaccines. sequence typing; mPCR, multiplex PCR; PBS, Pneumococcal conjugate vaccines (PCVs) are effective at preventing pneumonia and inva- phosphate-buffered saline; PCV, pneumococcal sive pneumococcal disease, and are currently being introduced in many resource-poor coun- conjugate vaccine; PPV, positive predictive value; tries (e.g., by Gavi, the Vaccine Alliance). PCVs reduce nasopharyngeal carriage of the RFLP, restriction fragment length polymorphism; pneumococcal serotypes they contain (vaccine types). Although transmission and disease due RLB, reverse line blot; SSI, Statens Serum Institut; STGG, skim milk-tryptone-glucose-glycerol; ULT, to vaccine types is reduced, non-vaccine types fill the ecological niche, becoming more com- ultra-low temperature. mon in carriage and disease [3–5]. This phenomenon of serotype replacement, which may be more pronounced in low-income settings (because of higher rates, load, and diversity of pneu- mococcal carriage [6] and higher rates of disease), represents a significant risk to the global pneumococcal immunisation strategy. It is difficult to monitor the serotypes causing pneumo- nia or invasive disease because of the insensitivity of blood culture for detecting pneumococcal pneumonia, inconsistencies in clinical practice (e.g., performing of lumbar punctures), and limitations on diagnostic laboratory capacity, particularly in resource-poor settings [7–9]. Fur- thermore, any improvement in laboratory or clinical diagnostic practices over time in vacci- nated populations would lead to enhanced detection of non-vaccine types. This increase may not reflect true replacement, and may cause unnecessary concerns about vaccine effectiveness and inappropriate changes to vaccine policy. Carriage studies offer a practical approach for monitoring serotype replacement and can help in assessing the impact of PCVs and other pneumococcal vaccines [8,10]. Pneumococcal carriage is an important endpoint for efficacy trials of new pneumococcal vaccines such as common protein, combination (PCV + common protein), and whole-cell vaccines [11]. Tradi- tional serotyping methods involve typing a small number of colonies, frequently missing car- riage of multiple serotypes and providing no quantitative data. The gold-standard serotyping PLOS Medicine | DOI:10.1371/journal.pmed.1001903 November 17, 2015 2/30 Serotyping Methods for Pneumococcal Carriage Studies method (the Quellung reaction) was developed in the early 1900s (see [12]) and is performed by testing colonies with a set of antisera and visualising the bacteria with a microscope. It is laborious and requires a complete set of type-specific antisera, and is therefore mainly per-
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