Detection and prediction of Streptococcus pneumoniae serotypes directly from nasopharyngeal swabs using PCR

Abstract

Monitoring Streptococcus pneumoniae serotype distribution is important to assess the impact and effectiveness of pneumococcal vaccine programs. With the challenges of Quellung serotyping, PCR-based serotype prediction is increasingly being used for large-scale epidemiological studies. This study used real-time (RT)-PCR targeting the genes encoding autolysin (lytA) and capsular biosynthesis gene A (cpsA) of S. pneumoniae in nucleic acids extracted directly from nasopharyngeal (NP) swabs submitted for viral studies. If the specimen was lytA or cpsA PCR-positive, then serotype prediction was performed on the same nucleic acid using eight conventional multiplex PCRs (cmPCRs) and seven real-time multiplex PCRs (rmPCRs). Of 1770 NP swabs, 132 (7.5 %) were lytA-positive and 122 (6.9 %) were positive for both targets (lytA and cpsA). Of the 122 lytA + cpsA + specimens, a serotype could be assigned in 52 (41.8 %) using cmPCR alone and the yield was increased to 70 (57.4 %) with the addition of rmPCR. Based on sensitivity, incremental yield and more efficient workflow, an algorithm was proposed where lytA and cpsA RT-PCR screening was followed by serotype deduction using rmPCR and a modified set of four instead of eight cmPCRs. This algorithm was validated for use on NP swabs, and the distribution of S. pneumoniae serotypes deduced from this approach showed good concordance with those obtained with cultured isolates serotyped by Quellung and PCR. Overall, molecular detection and serotyping of S. pneumoniae from NP swabs was found to be a valuable tool to assess S. pneumoniae colonization and monitor trends in serotype distribution.

Assessing the diagnostic accuracy of PCR-based detection ofStreptococcus pneumoniaefrom nasopharyngeal swabs collected for viral studies in Canadian adults hospitalised with community-acquired pneumonia: a Serious Outcomes Surveillance (SOS) Network of the Canadian Immunization Research (CIRN) study

“…[4][5][6][7][8] While traditional methods of serotyping (Quellung reaction) require live organism, PCR-based detection and serotyping of S. pneumoniae can be performed on a variety of clinical specimens without culture. [9][10][11][12][13][14][15] Our laboratory previously demonstrated the feasibility of these PCR methods on nasopharyngeal (NP) swabs routinely collected for respiratory virus studies. 9 The serotype distribution mirrored trends obtained with traditional Quellung serotyping, but the PCR methods were not thoroughly validated with specimens collected from patients with pneumococcal disease.…”

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

“…[19,[34][35][36][37][38][39] For example, while serotype 7F is covered in all three pneumococcal vaccines, 7F cannot be discriminated from serotype 7A using these molecular methods, and therefore the serotype is assigned 7F/7A. Other vaccinepreventable serotypes identified by PCR-based serotyping cannot discriminate: 6A from 6B; 9V from 9A; 9N from 9L; 11A from 11D; 12F from serotypes 12A, 12B, 44, and 46; 15B from 15C; 18C from serotypes 18F, 18B, and 18A; 22F from 22A; 33F from serotypes 33A and 37.…”

“…As previously reported, confounding amplicons (as a result of non-specific amplification) sometimes occurred with cmPCR and cmPCRmod reactions. [19] If present, these could readily be resolved with repeat reactions using individual primer pairs targeting the suspected serotype. Normalization of the S. pneumoniae culture to a McFarland value of 1.0 prior to extraction provided sufficient template that gave strong and reliable amplicons for all detectable serotypes.…”

“…Nucleic acids and cmPCR reactions were performed under conditions previously described (Lang et al, 2015). Nucleic acids were isolated from 200 µl bacterial suspension using a MagNA Pure Total Nucleic Acid Isolation kit (Roche, Laval, QC) on a MagNA Pure LC instrument, as recommended by the manufacturer.…”

“…[15] More recently, molecular methods for serotype deduction like conventional multiplex PCRs (cmPCR) have been developed, and are widely used from DNA extracted from S. pneumoniae isolates or clinical specimens. [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.…”