Superiority of trans-oral over trans-nasal sampling in detecting Streptococcus pneumoniae colonization in adults

Abstract

The human nasopharynx is the main reservoir for Streptococcus pneumoniae. We applied conventional and molecular methods to determine the prevalence of S. pneumoniae nasopharyngeal colonization in adults. Paired trans-orally and trans-nasally obtained nasopharyngeal samples from 268 parents of 24-month-old children were assessed for pneumococcal presence. Parents were classified as colonized when live pneumococci were recovered from either sample cultured on medium selective for S. pneumoniae. Of the 52 (19%) colonized parents 49 (18%) were culture-positive in trans-nasal and 10 (4%) in trans-oral samples. Bacterial growth was harvested from these cultures, DNA isolated and tested by quantitative-PCR (qPCR) targeting lytA and piaA genes specific for S. pneumoniae. A sample was considered positive if signals for both genes were detected. Altogether 105 (39%) individuals were classified as positive for pneumococcus by qPCR including 50 (19%) in trans-nasal and 94 (35%) in trans-oral settings. Although significantly more trans-nasal compared to trans-oral samples were culture-positive for S. pneumoniae at the primary diagnostic step (p<0.001) the opposite was observed in qPCR results (p<0.001). To confirm the presence of live pneumococcus in samples positive by qPCR but negative at the initial diagnostic step, we serially-diluted cell harvests, re-cultured and carefully examined for S. pneumoniae presence. Live pneumococci were recovered from an additional 43 parents including 42 positive in trans-oral and 4 in trans-nasal samples increasing the number of individuals culture- and qPCR-positive to 93 (35%) and positive by either of two methods to 107 (40%). There were significantly more trans-oral than trans-nasal samples positive for pneumococcus by both culture and qPCR (n = 71; 27%; vs. n = 50; 19%; p<0.05). Our data suggest that pneumococcal colonization is more common in adults than previously estimated and point towards the superiority of a trans-oral over a trans-nasal approach when testing adults for colonization with S. pneumoniae.

Figure 1. Molecular quantification of bacterial presence in nasopharyngeal samples from children and their parents.

Quantity of bacterial DNA in femtograms per microliter of template recovered directly from Amies medium of nasopharyngeal trans-nasal (TN) and trans-oral (TO) samples collected from nine randomly selected parents, plus trans-nasal samples from their children. Horizontal lines represent median concentrations per niche sampled.

Figure 2. PCR based detection of Streptococcus pneumoniae versus recovery of live pneumococci from nasopharyngeal cultures.

Figure depicts results of qPCR-based detection of S. pneumoniae-specific genes lytA and piaA and results of live Streptococcus pneumoniae isolation from trans-nasal (upper graphs) and trans-oral (lower graphs) nasopharyngeal cultures from 268 parents of 24-month-old children. Samples are divided into culture-positive (graphs on the left) versus culture-negative (graphs on the right) according to the results of conventional culture at the diagnostic step (n  =  number of samples per category). Each dot represents an individual sample. Position of the dot corresponds to C_T values for lytA- and piaA-specific signals as marked on corresponding axes. Red dots represent samples from which live pneumococci were isolated either from a clinical specimen at the primary diagnostic step (graphs on the left) or from re-processed gentamicin plate culture harvests (graphs on the right). Dotted lines mark the threshold arbitrarily assigned to discriminate between positive (C_T<35) and negative samples, and the total number of 45 cycles in each qPCR reaction. Number next to a dot depicts number of samples with a negative C_T score of 45 for both genes targeted or with limited or no bacterial growth and no growth of alpha-hemolytic colonies in gentamicin plate cultures. Numbers in square brackets depict the number (in black) of samples with C_T values below 35 for both targeted genes/number (in red) of cultures from which live S. pneumoniae was isolated at recovery culture step.