The Relevance of a Novel Quantitative Assay to Detect up to 40 Major Streptococcus pneumoniae Serotypes Directly in Clinical Nasopharyngeal and Blood Specimens

International audienceFor epidemiological and surveillance purposes, it is relevant to monitor the distribution and dynamics of Streptococcus pneumoniae serotypes. Conventional serotyping methods do not provide rapid or quantitative information on serotype loads. Quantitative serotyping may enable prediction of the invasiveness of a specific serotype compared to other serotypes carried. Here, we describe a novel, rapid multiplex real-time PCR assay for identification and quantification of the 40 most prevalent pneumococcal serotypes and the assay impacts in pneumonia specimens from emerging and developing countries. Eleven multiplex PCR to detect 40 serotypes or serogroups were optimized. Quantification was enabled by reference to standard dilutions of known bacterial load. Performance of the assay was evaluated to specifically type and quantify S. pneumoniae in nasopharyngeal and blood samples from adult and pediatric patients hospitalized with pneumonia (n = 664) from five different countries. Serogroup 6 was widely represented in nasopharyngeal specimens from all five cohorts. The most frequent serotypes in the French, South African, and Brazilian cohorts were 1 and 7A/F, 3 and 19F, and 14, respectively. When both samples were available, the serotype in blood was always present as carriage with other serotypes in the nasopharynx. Moreover, the ability of a serotype to invade the bloodstream may be linked to its nasopharyngeal load. The mean nasopharyngeal concentration of the serotypes that moved to the blood was 3 log-fold higher than the ones only found in the nasopharynx. This novel, rapid, quantitative assay may potentially predict some of the S. pneumoniae serotypes invasiveness and assessment of pneumococcal serotype distribution

Primers/Probes Sequences and Concentration Used in Each Pool for the <i>S</i>. <i>pneumoniae</i> Serotype Identification and Quantification Assay.

<p>Primers/Probes Sequences and Concentration Used in Each Pool for the <i>S</i>. <i>pneumoniae</i> Serotype Identification and Quantification Assay.</p

Pneumococcal load distribution in NP samples from the South African cohort.

<p>Comparison between invasive (present in both NP and WB samples of the same patient; n = 36) and non-invasive serotypes (present in NP samples only; n = 145). Each plot represents a sample. The bold bars indicate the mean bacterial concentration for each group (Student's <i>t</i>-test, <i>P</i><0.001).</p

<i>S</i>. <i>pneumoniae</i> serotypes distribution determined by the 40 <i>S</i>. <i>pneumoniae</i> serotype real-time PCR typing assay (40-PCR).

<p>(A) in NP samples (n = 562) of patients from Cambodia (n = 149), France (n = 45), South Africa (n = 227), Mali (n = 86) and Brazil (n = 55), (B) in WB samples (n = 102) of patients from France (n = 5), Mali (n = 16) and South Africa (n = 81). Each column represents the cumulative number of patients for a given serotype.</p

Serotype-Dependent Detection Limits and Linear Equation Values.

<p>Serotype-Dependent Detection Limits and Linear Equation Values.</p