The Challenges of Using Oropharyngeal Samples To Measure Pneumococcal Carriage in Adults

Abstract

Streptococcus pneumoniae (the pneumococcus) carriage is commonly used to measure effects of pneumococcal vaccines. Based on findings from culture-based studies, the World Health Organization recommends both nasopharyngeal (NP) and oropharyngeal (OP) sampling for detecting adult carriage. Given evidence of potential confounding by other streptococci, we evaluated molecular methods for pneumococcal identification and serotyping from 250 OP samples collected from adults in Fiji, using paired NP samples for comparison. Samples were screened using lytA quantitative PCR (qPCR), as well as pneumococcal identification and serotyping conducted by DNA microarray. A subset of OP samples were characterized by latex sweep agglutination and multiplex PCR. Alternate qPCR assays (piaB and bguR) for pneumococcal identification were evaluated. The lytA qPCR was less specific and had poor positive predictive value (PPV) in OP samples (88% and 26%, respectively) compared with NP samples (95% and 64%, respectively). Using additional targets piaB and/or bguR improved qPCR specificity in OP, although the PPV (42 to 53%) was still poor. Using microarray, we found that 102/107 (95%) of OP samples contained nonpneumococcal streptococci with partial or divergent complements of pneumococcal capsule genes. We explored 91 colonies isolated from 11 OP samples using various techniques, including multiplex PCR, latex agglutination, and microarray. We found that nonpneumococcal streptococci contribute to false positives in pneumococcal serotyping and may also contribute to spurious identification by qPCR. Our results highlight that molecular approaches should include multiple loci to minimize false-positive results when testing OP samples. Regardless of method, pneumococcal identification and serotyping results from OP samples should be interpreted with caution. IMPORTANCE Streptococcus pneumoniae (the pneumococcus) is a significant global pathogen. Accurate identification and serotyping are vital. In contrast with World Health Organization recommendations based on culture methods, we demonstrate that pneumococcal identification and serotyping with molecular methods are affected by sample type. Results from oropharyngeal samples from adults were often inaccurate. This is particularly important for assessment of vaccine impact using carriage studies, particularly in low- and middle-income countries where there are significant barriers for disease surveillance.

Pneumococcal carriage in unvaccinated children at the time of vaccine implementation into the national immunization program in Poland

“…Despite qPCR having more than an eight-fold increase in the sensitivity of detection; from 6.2% for culture (95% CI 2.3–13.0) to 53.6% for qPCR (95% CI 43.2–63.8), the accuracy of carriage detection with qPCR was only marginally higher than for saliva culture, with 78.2% (95% CI 73.8–82.2) and 76.9% (95% CI 72.4–80.1), respectively. We attributed the poor accuracy of saliva compared to NPS testing to a high density of non-pneumococcal species on saliva culture plates, either masking the detection of pneumococcal colonies or making pneumococci non-viable, as it has been described by Boelsen et al 32 for the oropharyngeal samples from adults.…”

“…Pneumococcal DNA was detected with qPCR by testing on a 7500 Real Time PCR System (Life Technologies, USA) a 0.625 μl of eluted sample in 25 μl reaction volume, using TaqMan Universal MasterMix (Life Technologies, USA), and primers and probes targeting sequences within piaB and lytA genes 29 , 31 . A sample was considered positive for pneumococcus when both signals were < 40 C T 21 , 23 , 32 .…”

It Takes Two to Tango: Combining Conventional Culture With Molecular Diagnostics Enhances Accuracy of Streptococcus pneumoniae Detection and Pneumococcal Serogroup/Serotype Determination in Carriage

“…Using assays developed by our groups and others, we have tested the serotype composition of respiratory samples from children and adults and have demonstrated an under-detection of S. pneumoniae and of individual pneumococcal serotypes by culture when compared with molecular methods ( Trzcinski et al, 2013 ; Wyllie et al, 2014 , 2016a , b ; Krone et al, 2015 ; Miellet et al, 2020 ). However, some caution that molecular methods exhibit poor specificity due to the presence of pneumococcal genes among commensal streptococci ( Carvalho Mda et al, 2012 , 2013 ; Boelsen et al, 2020 ). It could also be argued that molecular detection is unable to discriminate between live bacteria and presence of relic DNA ( Lennon et al, 2018 ).…”

It Takes Two to Tango: Combining Conventional Culture with Molecular Diagnostics Enhances Accuracy of Streptococcus pneumoniae Detection and Pneumococcal Serogroup/Serotype determination in Carriage

“…Some studies have cautioned against the use of molecular methods due to the presence of pneumococcal genes among commensal streptococci [25–27]. This phenomenon is likely to be common among bacterial species co-existing in a shared niche [50–52] and of particular concern for highly poly-microbial samples, hence careful selection of targeted genes is important [53].…”

“…Using assays developed by our groups and others, we have tested the serotype composition of respiratory samples from children and adults and have demonstrated an under-detection of S. pneumoniae and of individual pneumococcal serotypes by culture when compared with molecular methods [7, 18, 21–24]. However, some caution that molecular methods exhibit poor specificity due to the presence of pneumococcal genes among commensal streptococci [25–27]. It could also be argued that molecular detection is unable to discriminate between live bacteria and presence of relic DNA [28].…”