Abundance of the nasopharyngeal microbiome effects pertussis diagnosis and explains the sensitivity difference between bacterial culture and real-time PCR
Quantitative real time PCR (qPCR)is used for pertussis diagnosis. The positive rate of qPCR is generally much higher than that of bacterial culture, which may cause confusion. The current study utilized the 16S ribosomal RNA (16S rRNA) sequencing to assess the correlation between conventional culture and qPCR and to explore the value of 16S rRNA in diagnosing pertussis. Nasopharyngeal swabs, collected from 102 children meeting clinical diagnostic criteria for pertussis, were subjected to Bordetella pertussis culture and qPCR. Bioinformatic microbiota analysis was based on 16S rRNA V3-V4 gene sequencing. Among 102 samples, 14 (13.7%) were culture-positive for Bordetella pertussis, while 61 (59.8%) were qPCR positive. Genus Bordetella was identified in 68 (66.7%) samples via 16S rRNA sequencing. When the relative abundance of Bordetella genus exceeded 0.70%, both qPCR and culture results were positive. Samples with a relative abundance of less than 0.20% exhibited positive qPCR and negative culture results. Samples with a low Bordetella abundance are the key factors underlying poor correlation between culture and qPCR results in laboratory tests.
KeywordsBordetella pertussis Diagnosis Sensitivity Culture Quantitative real-time PCR
We are grateful to the Department of Microbiology and Immunology at the Beijing Children’s hospitals for provision of laboratory space and technical assistance.
Yijun Ding, Kaihu Yao, and Tianyou Wang contribute to the conception and the design of the work. Qing Wang and Dongfang Li contribute to the acquisition, the analysis, and the interpretation of the data. The first draft of the manuscript was written by Yijun Ding and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
This work was supported by the National Major Scientific and Technological Special Project for “Significant New Drugs Development” (grant number 2017ZX09304029) and Beijing Hospitals Authority Youth Programme (grant number QML 20181207).
Compliance with ethical standards
Conflicts of interest
The authors declare that they have no conflict of interest.
This study was approved by the Ethics Committee of Beijing Children’s Hospital Affiliated to Capital Medical University. The committee exempted request for informed consent because this study only focused on bacteria and did not affect patients. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
- 2.Cimolai N, Trombley C, O'Neill D (1996) Diagnosis of whooping cough: a new era with rapid molecular diagnostics. Pediatric emergency care 12 (2):91-93. Doi:org/ https://doi.org/10.1097/00006565-199604000-00006
- 3.Edelman K, Nikkari S, Ruuskanen O, He Q, Viljanen M, Mertsola J (1996) Detection of Bordetella pertussis by polymerase chain reaction and culture in the nasopharynx of erythromycin-treated infants with pertussis. The pediatric infectious disease journal 15 (1):54-57. Doi:org/ https://doi.org/10.1097/00006454-199601000-00012
- 7.Chinese Medical Association Pediatrics Infectious Group. Chinese children’s pertussis diagnosis and treatment recommendations (2017). Chin J Pediatr 55 (8):568–572. doi: https://doi.org/10.3760/cma.j.issn.0578-1310.2017.08.004
- 14.Srinivasan R, Karaoz U, Volegova M, MacKichan J, Kato-Maeda M, Miller S, Nadarajan R, Brodie EL, Lynch SV (2015) Use of 16S rRNA gene for identification of a broad range of clinically relevant bacterial pathogens. PLoS One 10(2):e0117617. https://doi.org/10.1371/journal.pone.0117617 CrossRefPubMedPubMedCentralGoogle Scholar
- 15.CDC Best practices for healthcare professionals on the use of polymerase chain reaction (PCR) for diagnosing pertussis 2019[updated 2/21/2019. https://www.cdc.gov/pertussis/clinical/diagnostic-testing/ diagnosis-pcr-bestpractices.html. Accessed 21 February 2019
- 16.Lee AD, Cassiday PK, Pawloski LC, Tatti KM, Martin MD, Briere EC, Tondella ML, Martin SW, Clinical Validation Study G (2018) Clinical evaluation and validation of laboratory methods for the diagnosis of Bordetella pertussis infection: culture, polymerase chain reaction (PCR) and anti-pertussis toxin IgG serology (IgG-PT). PLoS One 13(4):e0195979. https://doi.org/10.1371/journal.pone.0195979 CrossRefPubMedPubMedCentralGoogle Scholar
- 17.Zhang Q, Li M, Wang L, Xin T, He Q (2013) High-resolution melting analysis for the detection of two erythromycin-resistant Bordetella pertussis strains carried by healthy schoolchildren in China. Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases 19(6):E260–E262. https://doi.org/10.1111/1469-0691.12161 CrossRefGoogle Scholar
- 19.Bourgeois N, Ghnassia JC, Doucet-Populaire F (2003) In vitro activity of fluoroquinolones against erythromycin-susceptible and -resistant Bordetella pertussis. The journal of antimicrobial chemotherapy 51 (3):742-743. Doi:org/ https://doi.org/10.1093/jac/dkg145
- 20.Guillot S, Descours G, Gillet Y, Etienne J, Floret D, Guiso N (2012) Macrolide-resistant Bordetella pertussis infection in newborn girl, France. Emerg Infect Dis 18 (6):966–968. doi: https://doi.org/10.3201/eid1806.120091
- 21.Fry NK, Duncan J, Vaghji L, George RC, Harrison TG (2010) Antimicrobial susceptibility testing of historical and recent clinical isolates of Bordetella pertussis in the United Kingdom using the Etest method. European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology 29(9):1183–1185. https://doi.org/10.1007/s10096-010-0976-1 CrossRefGoogle Scholar
- 23.Wang Z, Cui Z, Li Y, Hou T, Liu X, Xi Y, Liu Y, Li H, He Q (2014) High prevalence of erythromycin-resistant Bordetella pertussis in Xi’an, China. Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases 20(11):O825–O830. https://doi.org/10.1111/1469-0691.12671 CrossRefGoogle Scholar
- 24.Mattoo S, Cherry JD (2005) Molecular pathogenesis, epidemiology, and clinical manifestations of respiratory infections due to Bordetella pertussis and other Bordetella subspecies. Clin Microbiol Rev 18(2):326–382. https://doi.org/10.1128/CMR.18.2.326-382.2005 CrossRefPubMedPubMedCentralGoogle Scholar