Comparison of traditional culture and molecular qPCR for detection of simultaneous carriage of multiple pneumococcal serotypes in African children

Abstract

S. pneumoniae is a common colonizer of the human nasopharynx in high income and low-middle income countries. Due to limitations of standard culture methods, the prevalence of concurrent colonization with multiple serotypes is unclear. We evaluated the use of multiplex quantitative PCR (qPCR) to detect multiple pneumococcal serotypes/group colonization in archived nasopharyngeal swabs of pneumococcal conjugate vaccine naive children who had previously been investigated by traditional culture methods. Overall the detection of pneumococcal colonization was higher by qPCR (82%) compared to standard culture (71%; p < 0.001), with a high concordance (kappa = 0.73) of serotypes/groups identified by culture also being identified by qPCR. Also, qPCR was more sensitive in detecting multiple serotype/groups among colonized cases (28.7%) compared to culture (4.5%; p < 0.001). Of the additional serotypes detected only by qPCR, the majority were of lower density (<104 CFU/ml) than the dominant colonizing serotype, with serotype/group 6A/B, 19B/F and 23F being the highest density colonizers, followed by serotype 5 and serogroup 9A/L/N/V being the most common second and third colonizers respectively. The ability of qPCR to detect multiple pneumococcal serotypes at a low carriage density might provide better insight into underlying mechanism for changes in serotype colonization in PCV vaccinated children.

Hands are vehicles for transmission of Streptococcus pneumoniae in novel controlled human infection study

“…Our results support other studies in suggesting that determining pneumococcal colonisation by both culture and qPCR are complementary [11,12]. Higher rates of detection using molecular methods likely represent superior sensitivity at low-density colonisation.…”

Use of Multiplex Quantitative PCR To Evaluate the Impact of Pneumococcal Conjugate Vaccine on Nasopharyngeal Pneumococcal Colonization in African Children

“…Although we have shown the value of molecular assays for surveillance of pneumococcal colonization, limitations of our study include the fact that the qPCR assays could not discriminate between all vaccine serotypes within their respective serogroups due to the high genotypic similarities within each group as described previously ( 4 ); however, due to the high concordance between serotypes identified by culture and qPCR, we can assume from colonization data using traditional culture methods that 93.3%, 88.9%, and 91.4% of all 9A/L/N/V, 18A/B/C, and 19B/F serogroups identified by qPCR to be vaccine serotypes 9V, 18C, and 19F, respectively. The qPCR assay did not detect all pneumococcal serotypes and nontypeable pneumococci could not be identified.…”

“…These serotypes that have a high invasive disease potential in our settings, were thus considered nonvaccine serotypes at the time of our studies. Further, qPCR increases the detection of serotypes 5 and 19A previously missed by traditional culture methods ( 4 ) and highlights the importance of continued surveillance using sensitive molecular methods able to detect serotypes at a low carriage density to more accurately measure the effectiveness of these vaccines in reducing/eliminating carriage or change in the circulating pneumococcal serotypes. For example, after the introduction of PCV13, serotype 12F has emerged as a leading “replacement” serotype, causing IPD in many settings ( 16 – 18 ).…”

“…Briefly, stored nasopharyngeal swabs were thawed, and total nucleic acids were extracted using the automated NucliSens easyMAG extraction system (BioMérieux, Marcy l’Etoile, France) according to the manufacturer’s instructions; extracted nucleic acids were stored at −20°C. The quantitative PCR (qPCR) method used in this study has previously been described and validated ( 4 ). Briefly, target DNAs were prescreened for the Streptococcus lytA gene ( 26 ).…”

“…Increased NVT colonization in vaccinees could involve either replacement colonization through reduced VT acquisition or unmasking of previously prevailing NVT that were not identified by traditional culture methods that detect only the dominant colonizing serotype or both mechanisms ( 3 ). Molecular detection of pneumococci in the nasopharynx has several advantages over traditional culture-based methods, including the detection of multiple serotypes from a single sample with high sensitivity, as well as quantitative PCR (qPCR) methods being able to measure the density of colonization and relative proportion of colonizing serotypes ( 4 ).…”