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Performance of Bordetella pertussis IS481 real-time PCR in a vaccine trial setting
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
2007 (English)In: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 115, no 12, p. 1370-1375Article in journal (Refereed) Published
Description
Abstract [en]

A real-time PCR method targeting the Bordetella pertussis IS481 gene fragment was evaluated in a vaccine trial setting in which real-time PCR results could be validated against culture and serology results. Two commonly used DNA extraction methods, Amplicor((R)) Respiratory Preparation kit and the QIAamp((R)) DNA Mini Kit, were compared. An approximately 50-fold higher sensitivity was achieved using the Amplicor kit. 89 of 276 aspirates analysed with the IS481 real-time PCR were positive. Interestingly, six of these were culture negative and came from serology-negative patients. Defining true positive cases either as culture-positive or as PCR-positive cases that had been confirmed with a serology-positive result or verified with a newly constructed recA PCR, the sensitivity and specificity of the IS481 real-time PCR were 89% and 98%, respectively. This study confirms the specificity and high diagnostic sensitivity of IS481-based PCR methods for diagnosis of B. pertussis.

Place, publisher, year, edition, pages
2007. Vol. 115, no 12, p. 1370-1375
Keywords [en]
Bordetella pertussis, IS481, real-time PCR
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-113051DOI: 10.1111/j.1600-0463.2007.00774.xISI: 000251859600007PubMedID: 18184407OAI: oai:DiVA.org:uu-113051DiVA, id: diva2:289814
Available from: 2010-01-25 Created: 2010-01-25 Last updated: 2019-04-06Bibliographically approved
In thesis
1. Molecular detection and epidemiological studies of atypical bacteria causing respiratory tract infections
Open this publication in new window or tab >>Molecular detection and epidemiological studies of atypical bacteria causing respiratory tract infections
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Respiratory infections are common causes of morbidity and mortality. Chlamydia pneumoniae, Mycoplasma pneumoniae and Bordetella pertussis cause respiratory infection, often with similar symptoms. Molecular diagnostic methods are preferred since these bacteria are difficult to culture. The aim of this thesis was to evaluate and improve the diagnostics and knowledge of the epidemiology of these bacteria.

A real-time polymerase chain reaction (PCR) method targeting the IS481 element present in the genome of B. pertussis was compared to culture and serology results, and a duplex real-time PCR method was constructed for detecting C. pneumoniae and M. pneumoniae, which was compared to two endpoint PCR methods. Both real-time PCR methods showed high sensitivity and specificity.

Typing of 624 M. pneumoniae samples, collected from 1996 to 2017 from four counties, was performed by P1 typing and multiple-locus variable number tandem repeat analysis (MLVA). A polyclonal distribution of strains was seen over all epidemic periods, but strains of P1 type 2/variant 2 and MLVA types 3-5-6-2 and 4-5-7-2 predominated in 2010−2013. A shift from type 2 strains to different variant 2 strains was seen and a new variant, 2e, was detected in 2016−2017. An A2063G mutation associated with macrolide resistance was detected by a fluorescence resonance energy transfer (FRET) PCR method in one (0.16%) of 608 M. pneumoniae strains.

Molecular characterisation using whole-genome sequencing of 93 B. pertussis isolates, collected between 1986 and 2016 from three counties showed that there were polyclonal strains in the county of Dalarna, Gävleborg and Uppsala in the years 2014−2016. Changes in virulence-related genes were detected: a shift from isolates harbouring the ptxP3 allele in favour of ptxP1 was seen, and almost all isolates had a disrupted prn gene. No detection of macrolide resistance in B. pertussis was detected.

In conclusion, the validated real-time PCR methods for detection of B. pertussis, C. pneumoniae and M. pneumoniae have led to improved diagnostic methods for use in clinical laboratories. The molecular characterisation of M. pneumoniae and B. pertussis strains has contributed to the wider understanding of the genetic changes that has occurred over the epidemic periods, but further studies is needed.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. p. 63
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1564
Keywords
Chlamydia pneumoniae, Mycoplasma pneumoniae, Bordetella pertussis, real-time PCR, P1 typing, MLVA, whole-genome sequencing, macrolide resistance, molecular diagnostics, molecular epidemiology
National Category
Infectious Medicine
Identifiers
urn:nbn:se:uu:diva-381158 (URN)978-91-513-0632-2 (ISBN)
Public defence
2019-05-29, Rudbeckssalen, Rudbeckslaboratoriet, Dag Hammarskjölds v 20, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2019-05-06 Created: 2019-04-06 Last updated: 2019-06-17

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