Abstract
We performed culture-based and PCR-based tests for pneumococcal identification and serotyping from carriage specimens collected in rural and urban Kenya. Nasopharyngeal specimens from 237 healthy children <5 years old (C-NPs) and combined nasopharyngeal/oropharyngeal specimens from 158 adults (A-NP/OPs, 118 HIV-positive) were assessed using pneumococcal isolation (following broth culture enrichment) with Quellung-based serotyping, real-time lytA-PCR, and conventional multiplexed PCR-serotyping (cmPCR). Culture-based testing from C-NPs, HIV-positive A-NP/OPs, and HIV-negative A-NP/OPs revealed 85.2%, 40.7%, and 12.5% pneumococcal carriage, respectively. In contrast, cmPCR serotypes were found in 93.2%, 98.3%, and 95.0% of these sets, respectively. Two of 16 lytA-negative C-NPs and 26 of 28 lytA-negative A-NP/OPs were cmPCR-positive for 1–10 serotypes (sts) or serogroups (sgs). A-NP/OPs averaged 5.5 cmPCR serotypes/serogroups (5.2 in HIV-positive, 7.1 in HIV-negative) and C-NPs averaged 1.5 cmPCR serotypes/serogroups. cmPCR serotypes/serogroups from lytA-negative A-NP/OPs included st2, st4, sg7F/7A, sg9N/9L, st10A, sg10F/10C/33C, st13, st17F, sg18C/18A/18B/18F, sg22F/22A, and st39. Nine strains of three non-pneumococcal species (S. oralis, S. mitis, and S. parasanguinis) (7 from A-OP, 1 from both A-NP and A-OP, and 1 from C-NP) were each cmPCR-positive for one of 7 serotypes/serogroups (st5, st13, sg15A/15F, sg10F/10C/33C, sg33F/33A/37, sg18C/18A/18B/18F, sg12F/12A/12B/ 44/46) with amplicons revealing 83.6–99.7% sequence identity to pneumococcal references. In total, 150 cmPCR amplicons from carriage specimens were sequenced, including 25 from lytA-negative specimens. Amplicon sequences derived from specimens yielding a pneumococcal isolate with the corresponding serotype were identical or highly conserved (>98.7%) with the reference cmPCR amplicon for the st, while cmPCR amplicons from lytA-negative specimens were generally more divergent. Separate testing of 56 A-OPs and 56 A-NPs revealed that ∼94% of the positive cmPCR results from A-NP/OPs were from OP microbiota. In contrast, A-NPs yielded >2-fold more pneumococcal isolates than A-OPs. Verified and suspected non-pneumococcal cmPCR serotypes/serogroups appeared to be relatively rare in C-NPs and A-NPs compared to A-OPs. Our findings indicate that non-pneumococcal species can confound serotype-specific PCR and other sequence-based assays due to evolutionarily conserved genes most likely involved in biosynthesis of surface polysaccharide structures.
Highlights
The primary reservoir for the opportunistic pathogen Streptococcus pneumoniae is the upper respiratory tract, where it coexists in varying proportions with other microbial species
Nineteen of the 20 serotypes recovered from A-nasopharyngeal specimens (NPs)/oropharyngeal swab specimens (OPs) were found in children, with the exception of 7A, recovered from a single human immunodeficiency virus (HIV)-positive A-NP/OP
Among A-NP/OPs there were 39 instances involving 21 different conventional multiplexed PCR-serotyping (cmPCR) types where we found a lack of any corresponding pneumococcal isolation (Quellung) -based results (Tables 3 and 4); among children, there was only 1 such instance
Summary
The primary reservoir for the opportunistic pathogen Streptococcus pneumoniae is the upper respiratory tract, where it coexists in varying proportions with other microbial species. Consistent with data indicating that children constitute a major pneumococcal reservoir (Hendley et al, 1975) is the fact that vaccinating young children with conjugate vaccines significantly decreases disease caused by vaccine serotypes in adults (Whitney et al, 2003). Studies of pneumococcal carriage serotype distributions, especially in young children, have revealed a great deal regarding the potential usefulness and impact of current multivalent conjugate vaccines that target small subsets of the >92 known pneumococcal capsular serotypes (Weinberger, Malley & Lipsitch, 2011), the complex biology of pneumococcal carriage is still poorly understood, especially as it pertains to culture-based detection rates that differ markedly between different disease-causing serotypes. Most available respiratory tract pneumococcal serotype distribution data have been obtained from studies of young children. Few studies of carriage have employed pneumococcal isolation independent, PCR-based detection of pneumococcal serotypes, especially in adults
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