Single-plex quantitative assays for the detection and quantification of most pneumococcal serotypes

Abstract

Streptococcus pneumoniae globally kills more children than any other infectious disease every year. A prerequisite for pneumococcal disease and transmission is colonization of the nasopharynx. While the introduction of pneumococcal conjugate vaccines has reduced the burden of pneumococcal disease, understanding the impact of vaccination on nasopharyngeal colonization has been hampered by the lack of sensitive quantitative methods for the detection of >90 known S. pneumoniae serotypes. In this work, we developed 27 new quantitative (q)PCR reactions and optimized 26 for a total of 53 qPCR reactions targeting pneumococcal serotypes or serogroups, including all vaccine types. Reactions proved to be target-specific with a limit of detection of 2 genome equivalents per reaction. Given the number of probes required for these assays and their unknown shelf-life, the stability of cryopreserved reagents was evaluated. Our studies demonstrate that two-year cryopreserved probes had similar limit of detection as freshly-diluted probes. Moreover, efficiency and limit of detection of 1-month cryopreserved, ready-to-use, qPCR reaction mixtures were similar to those of freshly prepared mixtures. Using these reactions, our proof-of-concept studies utilizing nasopharyngeal samples (N=30) collected from young children detected samples containing ≥2 serotypes/serogroups. Samples colonized by multiple serotypes/serogroups always had a serotype that contributes at least 50% of the pneumococcal load. In addition, a molecular approach called S6-q(PCR)2 was developed and proven to individually detect and quantify epidemiologically-important serogroup 6 strains including 6A, 6B, 6C and 6D. This technology will be useful for epidemiological studies, diagnostic platforms and to study the pneumobiome.

Fig 1. Distribution and densities of S. pneumoniae serotypes in single or multiple colonized nasopharyngeal (NP) samples.

Density (CFU/ml as determined by lytA qPCR) of the specified serotype is presented on the x axis. Symbols for each serotype indicates the number of times they were detected as a unique serotype (•) or in samples with multiple colonization (◊).

Fig 2. Quantification and subtyping, by exclusion, of serotypes 6A, 6B, 6C, and 6D by S6-q(PCR)².

DNA from NP samples was utilized as template in qPCR and PCR reactions. (I) The first set of qPCR reactions quantified serogroup 6, including serotype 6A, 6B, 6C and 6D (top panel), whereas the second specifically quantified serotypes 6C and 6D (bottom panel). Serotype 6A and 6B or serotype 6C or 6D are differentiated by one single nucleotide polymorphisms (SNPs) in the wciP gene. To type individual serotypes, PCR reactions were performed targeting subtype SNPs within the wciP gene to identify (II) serotypes 6A and 6C or (III) serotypes 6B and 6D strain. A PCR targeting (IV) serotypes 6C and 6D specifically by amplifying the wciNβ gene was performed as an additional control.

Fig 3. Algorithm for the identification and quantification of serotype 6 types directly from NP samples by S6-q(PCR)².

DNA purified from NP samples is utilized as template in qPCR and PCR reactions. Arrows indicate reactions to be performed, qPCR in dark gray boxes or PCR in light gray boxes, and continuous lines indicate reaction results. The final serotype obtained is indicated in the black boxes.