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High Levels of Detection of Nonpneumococcal Species of Streptococcus in Saliva from Adults in the United States

While the sensitivity of detection of pneumococcal carriage can be improved by testing respiratory tract samples with quantitative PCR (qPCR), concerns have been raised regarding the specificity of this approach. We therefore investigated the reliability of the widely used lytA qPCR assay when appli...

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Autores principales: Hislop, Maikel S., Allicock, Orchid M., Thammavongsa, Darani A., Mbodj, Sidiya, Nelson, Allison, Shaw, Albert C., Weinberger, Daniel M., Wyllie, Anne L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Microbiology 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10269540/
https://www.ncbi.nlm.nih.gov/pubmed/37067447
http://dx.doi.org/10.1128/spectrum.05207-22
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author Hislop, Maikel S.
Allicock, Orchid M.
Thammavongsa, Darani A.
Mbodj, Sidiya
Nelson, Allison
Shaw, Albert C.
Weinberger, Daniel M.
Wyllie, Anne L.
author_facet Hislop, Maikel S.
Allicock, Orchid M.
Thammavongsa, Darani A.
Mbodj, Sidiya
Nelson, Allison
Shaw, Albert C.
Weinberger, Daniel M.
Wyllie, Anne L.
author_sort Hislop, Maikel S.
collection PubMed
description While the sensitivity of detection of pneumococcal carriage can be improved by testing respiratory tract samples with quantitative PCR (qPCR), concerns have been raised regarding the specificity of this approach. We therefore investigated the reliability of the widely used lytA qPCR assay when applied to saliva samples from older adults in relation to a more specific qPCR assay (piaB). During the autumn/winter seasons of 2018/2019 and 2019/2020, saliva was collected at multiple time points from 103 healthy adults aged 21 to 39 (n = 34) and >64 (n = 69) years (n = 344 total samples). Following culture enrichment, extracted DNA was tested using qPCR for piaB and lytA. By sequencing the variable region of rpsB (S2 typing), we identified the species of bacteria isolated from samples testing lytA-positive only. While 30 of 344 (8.7%) saliva samples (16.5% individuals) tested qPCR-positive for both piaB and lytA, 52 (15.1%) samples tested lytA-positive only. No samples tested piaB-positive only. Through extensive reculture attempts of the lytA-positive samples collected in 2018/2019, we isolated 23 strains (in 8 samples from 5 individuals) that were also qPCR-positive for only lytA. Sequencing determined that Streptococcus mitis and Streptococcus infantis were predominantly responsible for this lytA-positive qPCR signal. We identified a comparatively large proportion of samples generating positive signals with the widely used lytA qPCR and identified nonpneumococcal Streptococcus species responsible for this signal. This highlights the importance of testing for the presence of multiple gene targets in tandem for reliable and specific detection of pneumococcus in polymicrobial respiratory tract samples. IMPORTANCE Testing saliva samples with quantitative PCR (qPCR) improves the sensitivity of detection of pneumococcal carriage. The qPCR assay targeting lytA, the gene encoding the major pneumococcal autolysin, has become widely accepted for the identification of pneumococcus and is even considered the “gold standard” by many. However, when applying this approach to investigate the prevalence of pneumococcal carriage in adults in New Haven, CT, USA, we identified nonpneumococcal Streptococcus spp. that generate positive signals in this widely used assay. By testing also for piaB (encoding the iron acquisition ABC transporter lipoprotein, PiaB), our findings demonstrate the importance of testing for the presence of multiple gene targets in tandem for reliable molecular detection of pneumococcus in respiratory tract samples; targeting only lytA may lead to an overestimation of true carriage rates.
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spelling pubmed-102695402023-06-16 High Levels of Detection of Nonpneumococcal Species of Streptococcus in Saliva from Adults in the United States Hislop, Maikel S. Allicock, Orchid M. Thammavongsa, Darani A. Mbodj, Sidiya Nelson, Allison Shaw, Albert C. Weinberger, Daniel M. Wyllie, Anne L. Microbiol Spectr Research Article While the sensitivity of detection of pneumococcal carriage can be improved by testing respiratory tract samples with quantitative PCR (qPCR), concerns have been raised regarding the specificity of this approach. We therefore investigated the reliability of the widely used lytA qPCR assay when applied to saliva samples from older adults in relation to a more specific qPCR assay (piaB). During the autumn/winter seasons of 2018/2019 and 2019/2020, saliva was collected at multiple time points from 103 healthy adults aged 21 to 39 (n = 34) and >64 (n = 69) years (n = 344 total samples). Following culture enrichment, extracted DNA was tested using qPCR for piaB and lytA. By sequencing the variable region of rpsB (S2 typing), we identified the species of bacteria isolated from samples testing lytA-positive only. While 30 of 344 (8.7%) saliva samples (16.5% individuals) tested qPCR-positive for both piaB and lytA, 52 (15.1%) samples tested lytA-positive only. No samples tested piaB-positive only. Through extensive reculture attempts of the lytA-positive samples collected in 2018/2019, we isolated 23 strains (in 8 samples from 5 individuals) that were also qPCR-positive for only lytA. Sequencing determined that Streptococcus mitis and Streptococcus infantis were predominantly responsible for this lytA-positive qPCR signal. We identified a comparatively large proportion of samples generating positive signals with the widely used lytA qPCR and identified nonpneumococcal Streptococcus species responsible for this signal. This highlights the importance of testing for the presence of multiple gene targets in tandem for reliable and specific detection of pneumococcus in polymicrobial respiratory tract samples. IMPORTANCE Testing saliva samples with quantitative PCR (qPCR) improves the sensitivity of detection of pneumococcal carriage. The qPCR assay targeting lytA, the gene encoding the major pneumococcal autolysin, has become widely accepted for the identification of pneumococcus and is even considered the “gold standard” by many. However, when applying this approach to investigate the prevalence of pneumococcal carriage in adults in New Haven, CT, USA, we identified nonpneumococcal Streptococcus spp. that generate positive signals in this widely used assay. By testing also for piaB (encoding the iron acquisition ABC transporter lipoprotein, PiaB), our findings demonstrate the importance of testing for the presence of multiple gene targets in tandem for reliable molecular detection of pneumococcus in respiratory tract samples; targeting only lytA may lead to an overestimation of true carriage rates. American Society for Microbiology 2023-04-17 /pmc/articles/PMC10269540/ /pubmed/37067447 http://dx.doi.org/10.1128/spectrum.05207-22 Text en Copyright © 2023 Hislop et al. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Research Article
Hislop, Maikel S.
Allicock, Orchid M.
Thammavongsa, Darani A.
Mbodj, Sidiya
Nelson, Allison
Shaw, Albert C.
Weinberger, Daniel M.
Wyllie, Anne L.
High Levels of Detection of Nonpneumococcal Species of Streptococcus in Saliva from Adults in the United States
title High Levels of Detection of Nonpneumococcal Species of Streptococcus in Saliva from Adults in the United States
title_full High Levels of Detection of Nonpneumococcal Species of Streptococcus in Saliva from Adults in the United States
title_fullStr High Levels of Detection of Nonpneumococcal Species of Streptococcus in Saliva from Adults in the United States
title_full_unstemmed High Levels of Detection of Nonpneumococcal Species of Streptococcus in Saliva from Adults in the United States
title_short High Levels of Detection of Nonpneumococcal Species of Streptococcus in Saliva from Adults in the United States
title_sort high levels of detection of nonpneumococcal species of streptococcus in saliva from adults in the united states
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10269540/
https://www.ncbi.nlm.nih.gov/pubmed/37067447
http://dx.doi.org/10.1128/spectrum.05207-22
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