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Molecular detection and epidemiological studies of atypical bacteria causing respiratory tract infections
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology.
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 [en]
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: urn:nbn:se:uu:diva-381158ISBN: 978-91-513-0632-2 (print)OAI: oai:DiVA.org:uu-381158DiVA, id: diva2:1302859
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
List of papers
1. Performance of Bordetella pertussis IS481 real-time PCR in a vaccine trial setting
Open this publication in new window or tab >>Performance of Bordetella pertussis IS481 real-time PCR in a vaccine trial setting
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
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.

Keywords
Bordetella pertussis, IS481, real-time PCR
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-113051 (URN)10.1111/j.1600-0463.2007.00774.x (DOI)000251859600007 ()18184407 (PubMedID)
Available from: 2010-01-25 Created: 2010-01-25 Last updated: 2019-04-06Bibliographically approved
2. Simultaneous detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae by use of molecular beacons in a duplex real-time PCR
Open this publication in new window or tab >>Simultaneous detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae by use of molecular beacons in a duplex real-time PCR
2008 (English)In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 46, no 2, p. 727-31Article in journal (Refereed) Published
Abstract [en]

A real-time PCR was designed for detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae such that each pathogen could be detected in a single tube and differentiated using molecular beacons marked with different fluorochromes. This duplex PCR, targeting the P1 adhesion gene for M. pneumoniae and the ompA gene for C. pneumoniae, was compared with two conventional PCR assays targeting the 16S rRNA gene and the ompA gene. A total of 120 clinical throat and nasopharyngeal swab samples were tested. DNA extraction was performed using an alkali denaturation/neutralization method, and real-time amplification, detection, and data analysis were performed using a Rotor-Gene 2000 real-time rotary analyzer (Corbett Life Science, Sydney, Australia). Using conventional PCR as a reference in an analysis of 120 samples, 13 of 14 samples positive for C. pneumoniae were detected by the novel real-time PCR. In an analysis of M. pneumoniae, 22 samples were positive in the conventional PCR and the novel assay detected 24 positive samples. When using the conventional PCR as a reference, sensitivity and specificity were 93% and 100%, respectively, for C. pneumoniae and 100% and 98%, respectively, for M. pneumoniae. With an overall agreement of 98.8%, this suggests that performance of the new duplex real-time PCR is comparable to that of conventional PCR.

Keywords
Chlamydophila pneumoniae
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-103950 (URN)10.1128/JCM.01540-07 (DOI)000253100300047 ()18094125 (PubMedID)
Available from: 2009-05-26 Created: 2009-05-26 Last updated: 2019-04-06
3. No detection of macrolide-resistant Mycoplasma pneumoniae from Swedish patients, 1996-2013.
Open this publication in new window or tab >>No detection of macrolide-resistant Mycoplasma pneumoniae from Swedish patients, 1996-2013.
2016 (English)In: Infection Ecology & Epidemiology, ISSN 2000-8686, E-ISSN 2000-8686, Infection ecology & epidemiology, Vol. 6, no 1, article id 31374Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Mycoplasma pneumoniae is a common cause of respiratory infections which can cause life-threatening pneumonia and serious extrapulmonary manifestations. Since the year 2000, the emergence of macrolide-resistant M. pneumoniae strains has increased with varying incidences across countries. In China more than 90% of the strains are resistant. M. pneumoniae diagnostics is mostly done with molecular methods, and in Sweden antibiotic resistance surveillance is not routinely performed. The prevalence of macrolide-resistant M. pneumoniae has not previously been studied in Sweden.

MATERIAL AND METHODS: A total of 563 M. pneumoniae-positive respiratory samples, collected from four counties in Sweden between 1996 and 2013, were screened for mutations associated with macrolide resistance using a duplex FRET real-time PCR method. The real-time PCR targets the 23S rRNA gene, and differentiation between wild-type and resistant strains was achieved with a melting curve analysis.

RESULTS: Of the 563 samples included, 548 were analyzed for mutations associated with macrolide resistance. No mutations were found. The detection rate of macrolide-resistant M. pneumoniae in this study was 0% [0.00-0.84%].

CONCLUSION: No macrolide-resistant M. pneumoniae has been detected in Sweden. However, the emergence and spread of macrolide-resistant M. pneumoniae strains in many countries commands continuous epidemiological surveillance.

Keywords
Mycoplasma pneumoniae, antibiotic resistance, diagnostics, macrolide, treatment
National Category
Infectious Medicine
Identifiers
urn:nbn:se:uu:diva-319205 (URN)10.3402/iee.v6.31374 (DOI)27258207 (PubMedID)
Available from: 2017-03-31 Created: 2017-03-31 Last updated: 2019-04-06Bibliographically approved
4. Molecular typing of Mycoplasma pneumoniae strains in Sweden, 1996–2017, and the emergence of a new P1 cytadhesin gene, Variant 2e
Open this publication in new window or tab >>Molecular typing of Mycoplasma pneumoniae strains in Sweden, 1996–2017, and the emergence of a new P1 cytadhesin gene, Variant 2e
2019 (English)In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 57, no 6, article id e00049-19Article in journal (Refereed) Published
Abstract [en]

Mycoplasma pneumoniae causes respiratory infections, such as community-acquired pneumonia (CAP), with epidemics recurring every 3 to 7 years. In 2010 and 2011, many countries experienced an extraordinary epidemic peak. The cause of these recurring epidemics is not understood, but decreasing herd immunity and shifts in the strains' antigenic properties have been suggested as contributing factors. M. pneumoniae PCR-positive samples were collected between 1996 and 2017 from four neighboring counties inhabited by 12% of Sweden's population. A total of 578 isolates were characterized directly from 624 clinical samples using P1 typing by sequencing and multilocus variable number tandem repeat analysis (MLVA). A fluorescence resonance energy transfer (FRET)-PCR approach was also used to detect mutations associated with macrolide resistance in the 23S rRNA gene. Through P1 typing, the strains were classified into type 1 and type 2, as well as variants 2a, 2b, 2c, and a new variant found in nine of the strains, denoted variant 2e. Twelve MLVA types were distinguished, and 3-5-6-2 (42.4%), 4-5-7-2 (37.4%), and 3-6-6-2 (14.9%) predominated. Several P1 and MLVA types cocirculated each year, but type 2/variant 2 strains and MLVA types 3-5-6-2 and 4-5-7-2 predominated during the epidemic period comprising the peak of 2010 and 2011. In 2016 and 2017, type 1 became more common, and MLVA type 4-5-7-2 predominated. We also found that 0.2% (1/578) of the strains carried a macrolide resistance-associated mutation, indicating a very low prevalence of macrolide resistance in this region of Sweden.

Place, publisher, year, edition, pages
American Society for Microbiology, 2019
Keywords
MLVA, Mycoplasma pneumoniae, P1 typing, molecular typing
National Category
Infectious Medicine
Identifiers
urn:nbn:se:uu:diva-381150 (URN)10.1128/JCM.00049-19 (DOI)000469251500002 ()30918047 (PubMedID)
Available from: 2019-04-04 Created: 2019-04-04 Last updated: 2019-06-24Bibliographically approved
5. Epidemiologic characterisation with whole-genome sequencing of Bordetella pertussis isolates during an endemic in three counties in Sweden, 2014-2016
Open this publication in new window or tab >>Epidemiologic characterisation with whole-genome sequencing of Bordetella pertussis isolates during an endemic in three counties in Sweden, 2014-2016
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Infectious Medicine
Identifiers
urn:nbn:se:uu:diva-381151 (URN)
Available from: 2019-04-04 Created: 2019-04-04 Last updated: 2019-04-15

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