Revisiting Pneumococcal Carriage by Use of Broth Enrichment and PCR Techniques for Enhanced Detection of Carriage and Serotypes

Abstract

The measurement of pneumococcal carriage in the nasopharyngeal reservoir is subject to potential confounders that include low-density and multiple-strain colonization. To compare different methodologies, we picked a random sampling of 100 nasopharyngeal specimens recovered from infants less than 2 years of age who were previously assessed for pneumococcal carriage and serotypes by a conventional method that used direct plating from the transport/storage medium (50 specimens were culture negative and 50 specimens were culture positive for pneumococci). We used a broth enrichment approach and a conventional PCR approach (with and without broth enrichment) to determine pneumococcal carriage and serotypes, and the results were compared to the initial conventional culture-based results. Additionally, we used a lytA-targeted real-time PCR for pneumococcal detection. Broth enrichment for both the culture-based and the PCR-based methods enhanced the isolation of pneumococci and detection of serotype diversity, with the most effective serotype deduction method being one that used broth enrichment prior to sequential multiplex PCR. Similarly, we also found that broth enrichment followed by the lytA-specific real-time PCR was the most sensitive for the detection of apparent pneumococcal carriage. The broth enrichment, conventional multiplex PCR, and real-time PCR approaches used in this study were effective in detecting pneumococcal carriage in the 50 specimens that were negative by conventional direct plating from transport medium (range of numbers of positive specimens, 8/50 to 22/50 [16 to 44%]), and the three different serotyping approaches that used broth enrichment increased the number of serotype identifications from the 100 specimens (12 to 29 additional serotype identifications to be positive). A PCR-based approach that employed a broth enrichment step appeared to best enhance the detection of mixed serotypes and low-density pneumococcal carriage.The principal habitat of the opportunistic pathogen Streptococcus pneumoniae is the nasopharynx, where it normally resides commensally. Current multivalent vaccines against pneumococci target subsets of the 91 known capsular serotypes known to be expressed by this organism (12). Although the biology of pneumococcal carriage is not well understood, it appears that pneumococcal disease occurs as a sequela of nasopharyngeal (NP) carriage (7, 15). Accurate assessment of the serotype distribution associated with pneumococcal colonization, together with knowledge of the serotype distribution associated with pneumococcal disease, could be of great value in the evaluation and formulation of pneumococcal vaccines.Detection of the NP carriage of pneumococci and serotyping have traditionally relied on conventional culture methods of direct plating of NP specimens from transport or storage media. For example, a recent major study used classical culture isolation and Quellung reaction-based serotyping to reveal significant effects of vaccination on the carriage of vaccine serotype str...

Refining PCR-based serotyping for detection of vaccine-preventable Streptococcus pneumoniae

“…While other studies have used oligonucleotide permutations to account for geographical differences in S. pneumoniae serotype distribution (USA, Latin America, Africa, and Asia), [17,24,40] this study showed that oligonucleotides permutations did not affect the performance characteristics of PCR-based serotyping. This study does not preclude additional validation if different permutations are used, or use of ongoing quality assurance controls.…”

“…[16][17][18][19][20][21][22][23][24][25][26][27][28] Since cmPCR have a limited number of serotypes in each PCR reaction, they have been designed to target the most prevalent serotypes causing IPD, often in sequential reactions. [21][22][23][24][25][26] However, sequential PCR may not be the most cost effective strategy to identify S. pneumo-niae serotypes that are vaccine-preventable. This study used oligonucleotide permutations in a modified set of cmPCR reactions (termed cmPCRmod) to reduce the amount of testing required to identify S. pneumoniae serotypes covered by the PCV7, PCV13, or PPV23 vaccines.…”

Direct Detection and Prediction of All Pneumococcal Serogroups by Target Enrichment-Based Next-Generation Sequencing

“…Multiplex PCRs, microarrays, and latex agglutination assays have been used to detect multiple serotypes in colonization, with different success rates (23)(24)(25). The use of culture for detection of multiple serotypes is limited by the number of colonies that need to be identified in order to have a realistic probability of identifying a minor serotype (26,27).…”

PCR deduction of invasive and colonizing pneumococcal serotypes from Venezuela: a critical appraisal

“…After boiling for 10 minutes, cell debris was pelleted by centrifugation (13.000 x g for 2 minutes) and the supernatant was used for the PCR. The multiplex PCR was performed using the primers of the published protocol of Pai et al [7], adapting the primer set as proposed by Carvalho et al [8]. Because the CDC protocol detects 16 serotypes/groups which have never been reported from patients with invasive pneumococcal disease or in carriage studies in Venezuela, these serotypes were excluded from the typing scheme.…”

“…Ongoing research, especially nasopharynx carriage studies, carried out in the last 10 years in our country to increase the knowledge of pneumococcal epidemiology, has demonstrated a need for a faster and cheaper technique for serotyping. Here, we evaluate for future investigational use a serial multiplex PCR (mPCR) approach, based on one originally developed by Pai et al [7] [8]). For the PCR detection of strains from Venezuela, we adapted this multiplex PCR scheme by modifying the order of the reactions and moving serotypes into different reaction mixtures from where they were previously, based on the prevalence data of invasive pneumococcal serotypes/groups in Venezuela.…”