Non-pneumococcal mitis-group streptococci confound detection of pneumococcal capsular serotype-specific loci in upper respiratory tract
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-...
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PeerJ Inc.
2013-06-01
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| Online Access: | https://peerj.com/articles/97.pdf |
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| author | Maria da Gloria Carvalho Fabiana C. Pimenta Iaci Moura Alexis Roundtree Robert E. Gertz Jr Zhongya Li Geofrey Jagero Godfrey Bigogo Muthoni Junghae Laura Conklin Daniel R. Feikin Robert F. Breiman Cynthia G. Whitney Bernard W. Beall |
| author_facet | Maria da Gloria Carvalho Fabiana C. Pimenta Iaci Moura Alexis Roundtree Robert E. Gertz Jr Zhongya Li Geofrey Jagero Godfrey Bigogo Muthoni Junghae Laura Conklin Daniel R. Feikin Robert F. Breiman Cynthia G. Whitney Bernard W. Beall |
| author_sort | Maria da Gloria Carvalho |
| collection | DOAJ |
| description | 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. |
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| issn | 2167-8359 |
| language | English |
| last_indexed | 2024-03-09T07:24:24Z |
| publishDate | 2013-06-01 |
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| spelling | doaj.art-cca2f5f35a834bbeadab36ff722959a02023-12-03T07:09:20ZengPeerJ Inc.PeerJ2167-83592013-06-011e9710.7717/peerj.9797Non-pneumococcal mitis-group streptococci confound detection of pneumococcal capsular serotype-specific loci in upper respiratory tractMaria da Gloria Carvalho0Fabiana C. Pimenta1Iaci Moura2Alexis Roundtree3Robert E. Gertz Jr4Zhongya Li5Geofrey Jagero6Godfrey Bigogo7Muthoni Junghae8Laura Conklin9Daniel R. Feikin10Robert F. Breiman11Cynthia G. Whitney12Bernard W. Beall13National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, USANational Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, USANational Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, USANational Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, USANational Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, USANational Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, USAKenya Medical Research Institute, KenyaKenya Medical Research Institute, KenyaKenya Medical Research Institute, KenyaNational Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, USAKenya Medical Research Institute, KenyaKenya Medical Research Institute, KenyaNational Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, USANational Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, USAWe 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.https://peerj.com/articles/97.pdfPneumococcal serotype-specific lociMitis group streptococciOropharyngeal and nasopharyngeal floraPCR for serotype deduction |
| spellingShingle | Maria da Gloria Carvalho Fabiana C. Pimenta Iaci Moura Alexis Roundtree Robert E. Gertz Jr Zhongya Li Geofrey Jagero Godfrey Bigogo Muthoni Junghae Laura Conklin Daniel R. Feikin Robert F. Breiman Cynthia G. Whitney Bernard W. Beall Non-pneumococcal mitis-group streptococci confound detection of pneumococcal capsular serotype-specific loci in upper respiratory tract PeerJ Pneumococcal serotype-specific loci Mitis group streptococci Oropharyngeal and nasopharyngeal flora PCR for serotype deduction |
| title | Non-pneumococcal mitis-group streptococci confound detection of pneumococcal capsular serotype-specific loci in upper respiratory tract |
| title_full | Non-pneumococcal mitis-group streptococci confound detection of pneumococcal capsular serotype-specific loci in upper respiratory tract |
| title_fullStr | Non-pneumococcal mitis-group streptococci confound detection of pneumococcal capsular serotype-specific loci in upper respiratory tract |
| title_full_unstemmed | Non-pneumococcal mitis-group streptococci confound detection of pneumococcal capsular serotype-specific loci in upper respiratory tract |
| title_short | Non-pneumococcal mitis-group streptococci confound detection of pneumococcal capsular serotype-specific loci in upper respiratory tract |
| title_sort | non pneumococcal mitis group streptococci confound detection of pneumococcal capsular serotype specific loci in upper respiratory tract |
| topic | Pneumococcal serotype-specific loci Mitis group streptococci Oropharyngeal and nasopharyngeal flora PCR for serotype deduction |
| url | https://peerj.com/articles/97.pdf |
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