A reverse-hybridization test for the identification of 76 pneumococcal serotypes, 42 individually and 34 in pairs

Highlights

• S. PneumoStrip is a reverse hybridization strip assay for pneumococcal serotyping.

• It allows for the identification of 76 pneumococcal serotypes.

• 42 pneumococcal serotypes are identified individually and 34 in pairs.

• 60/64 (93.8%) pneumococcal invasive isolates collected in 2016–2017 were serotyped.

Abstract

The S. PneumoStrip test is a recently developed reverse hybridization strip-based commercial assay that allows for the identification of 76 pneumococcal serotypes, 42 individually and 34 in pairs, according to their specific gene sequences. The test was validated with reference strains of 92 different pneumococcal serotypes and with a selection of 75 clinical isolates representing 55 serotypes, showing 100% sensitivity and specificity. The test was also applied to 64 pneumococcal invasive isolates (23 different serotypes) consecutively collected between June 2016 and March 2017, with 60 (93.8%) being serotyped. Four isolates belonging to serotypes 13, 29, and 35B (2 isolates), which are not included in the test, did not produce a hybridization signal with serotype specific probes. The identification of most serotypes causing invasive pneumococcal disease together with the simplicity of performance and results interpretation, and the use of routine laboratory equipment make this test very suitable for most clinical and research laboratories.

Introduction

Streptococcus pneumoniae (pneumococcus) is a human pathogen that frequently causes respiratory diseases such as community acquired pneumonia or otitis media, and less frequently invasive disease as meningitis or bacteremia (Centers for Disease Control and Prevention, n.d.). The external polysaccharide capsule constitutes the pneumococcal major virulence factor, being a highly immunogenic component (Griffith, 1928). Pneumococcal capsule is composed of complex polysaccharides (Kamerling, 2000) and more than 90 different capsular polysaccharide types (serotypes) have been described to date according to their reaction with specific antisera in the Quellung reaction.

Pneumococcal conjugate vaccines (PCV) commercialized since 2000 contain the polysaccharides of the serotypes most frequently causing invasive pneumococcal disease (IPD) in children, as the polysaccharide of all serotypes cannot be included in a unique vaccine. Although PCV have been very successful decreasing the incidence of IPD due to vaccine serotypes in vaccinated and unvaccinated populations due to herd protection, a replacement of serotypes causing disease has been observed (Flasche et al., 2011, Weinberger et al., 2011, Weil-Olivier et al., 2012). This serotype replacement necessitates continuous surveillance of serotypes responsible for IPD, not only to monitor the replacement events but to determine current vaccines´ efficacy, to design future vaccines and to control the possible emergence of particularly virulent serotypes. To achieve this goal, robust capsular typing tools capable of determining a wide range of serotypes in an easy way are needed. Ideally, these tools should be quick and simple and with a high sensitivity and specificity in order to be implemented in the daily work of clinical laboratories.

Genes for pneumococcal capsular synthesis are located in the capsular polysaccharide synthesis (cps) locus, which shows enough genetic divergence between serotypes to enable the determination of serotypes on the basis of molecular techniques (Kong et al., 2005, Tarragó et al., 2008, Tomita et al., 2011, Leung et al., 2012, Marimón et al., 2016). In general, a very good agreement between results obtained by molecular methods and the Quellung reaction has been demonstrated. Of the molecular techniques described, multiplex-PCR is one of the most simple and frequently used, identifying different number of serotypes on the basis of the different length of the amplicons obtained (Marimón et al., 2016, Brito et al., 2003, Pai et al., 2006, Coskun-Ari et al., 2012, Richter et al., 2013). However, the different multiplex-PCR designed showed the same problem that the similarity of capsular genes between related serotypes gave amplification products of the same length, making impossible to identify individually some serotypes. Amplicon hybridization with serotype-specific probes could distinguish between amplicons of the same size but with different sequences without the need of using more sophisticated techniques as sequencing. The objective of this work was to develop and assess the performance of the “S. PneumoStrip test”, a commercial strip reverse-hybridization assay for easy and robust pneumococcal typing from culture, based on multiplex-PCR followed by semi-automated strip hybridization.