Development of a TaqMan Array Card for Pneumococcal Serotyping on Isolates and Nasopharyngeal Samples

Abstract

Streptococcus pneumoniae is both a commensal and a major pathogen that causes invasive disease in people of all ages. The introduction of serotype-specific pneumococcal vaccines has reduced the burden of disease but has also led to replacement with new strains; thus, serotyping remains important for vaccine-related disease surveillance. Conventional serotyping methods are laborious and expensive. We developed an easy-to-perform genotypic TaqMan array card (TAC) to identify S. pneumoniae strains, including lytA-based sequences, and 53 sequence-specific PCRs to identify 74 serotypes/serogroups covering all current vaccine types as well as prevalent nonvaccine types. The TAC method was evaluated on 146 clinical S. pneumoniae isolates and 13 nonpneumococcal species that naturally inhabit the upper respiratory tract and yielded 97% (142/146) sensitivity and 100% (13/13) specificity versus results of standard Quellung serotyping. The calculated limit of detection was 20 to 200 fg (∼8 to 84 genome equivalents) per reaction. On 23 blinded nasopharyngeal specimens that were pneumococcus culture positive, the TAC pan-pneumococcus lytA assay was positive in 21 (91% sensitivity versus culture). On TAC lytA-positive specimens, a serotype result was obtained on 86%, and the result was 95% accurate versus the subsequent culture's Quellung result. TAC also detected mixed serotypes in two specimens where Quellung detected only the predominant serotype. This TAC method yields fast and comprehensive serotyping compared to the standard method and may be useful on direct specimens.

Development of an Extended-Specificity Multiplex Immunoassay for Detection of Streptococcus pneumoniae Serotype-Specific Antigen in Urine by Use of Human Monoclonal Antibodies

“…Furthermore, this method of typing requires the recovery of a viable pneumococcal culture and thus precludes cases where an isolate is not obtained-for example, when antimicrobial treatment has been administered prior to specimen collection (13,14), or in cases of noninvasive disease. The development of pneumococcal capsular typing methods using a number of techniques, such as specific enzyme-linked immunosorbent assay (ELISA), competitive enzyme immunoassay (EIA), multiplex immunoassay (MIA), single and multiplex PCRs, and capsular sequence typing (CST) (15)(16)(17)(18)(19)(20)(21), has contributed to serogroup/serotype surveillance of invasive and noninvasive PD, particularly when an isolate is unavailable, by their direct application to clinical specimens.…”

Etiology of Pediatric Meningitis in West Africa Using Molecular Methods in the Era of Conjugate Vaccines against Pneumococcus, Meningococcus, and Haemophilus influenzae Type b

“… 24 Implementation of the TAC assay in routine surveillance would enhance the monitoring of pneumococcal meningitis in the post-PCV era across the subregion. To further enhance such surveillance, it would be useful to examine serotypes using direct molecular tools, 33 and these can be incorporated into a future TAC version. Worth noting is that TAC is easy to use and modular for customized use, 8 with a reagent cost of ∼$60/sample and 3-hour running time from sample to results.…”

Development and characterization of a synthetic DNA, NUversa, to be used as a standard in quantitative polymerase chain reactions for molecular pneumococcal serotyping

“… 2013 ) or a recently developed TAC for pneumococcal serotyping (Pholwat et al. 2016 ). The TAC, for example, detects 78 different pneumococcal serotypes.…”

“… 2016 ; Pholwat et al. 2016 ). Altogether, the reactions detect all 13 PCV types and 66 NVT strains, demonstrating high sensitivity and providing the absolute densities of individual serotypes in each sample.…”