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PCR detection and prevalence of Mycoplasma genitalium
Karlstad University, Faculty of Technology and Science, Department of Chemistry and Biomedical Sciences. (Biomedicinsk vetenskap)
2010 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Chlamydia and gonorrhea are major causes of sexually transmitted infections (STI) in adolescents worldwide. The infections are caused by Chlamydia trachomatis or Neisseria gonorrhoeae, bacteria with clinical manifestations such as urethritis, prostatitis and epididymitis among men, and urethritis, cervicitis and upper genital tract infection (i.e. pelvic inflammatory disease) among women. However, in many cases of genital tract infection, the etiology remains uncertain. In light of this, Mycoplasma genitalium was somewhat accidentally isolated in 1980 after prolonged incubation of urogenital specimens from men with non-gonococcal urethritis. Following the initial isolation in 1980, repeated attempts have been made to recover the extremely fastidious organism from clinical samples by culture techniques, but isolates have been rare and difficult to obtain. With the development of PCR methods in the early 1990s, detection of M. genitalium infection became more feasible.

The aim in paper I was to compare three different PCR assays (conventional and real-time 16S rRNA gene PCR as well as real-time Mycoplasma genitalium adhesin protein (MgPa) gene PCR) for detection of M. genitalium. The study also determined the prevalence of M. genitalium. Clinical specimens collected from STI attendees, 381 men and 298 women, were used to determine the prevalence of M. genitalium and 213 of these specimens were used in the PCR comparative study. The prevalence of M. genitalium infection in men and women was 27/381 (7.1 %) and 23/298 (7.7 %) respectively. In the PCR comparative study, M. genitalium DNA were detected in 61/76 (80.3 %) of true-positive specimen by conventional 16S rRNA gene PCR, in 52/76 (68.4 %) by real-time 16S rRNA gene PCR and in 74/76 (97.4 %) by real-time MgPa gene PCR. Hence, real-time MgPa gene PCR is well suited for clinical diagnosis of M. genitalium in urogenital specimens from men and women.

The aim in paper II was to determine whether a patients’ endocervical swab specimen can be transported in first void urine (FVU) as combined specimens in detection of Mycoplasma genitalium by real-time PCR. The study also compared two different DNA extraction methods (manual Chelex DNA extraction and automated BioRobot M48 DNA extraction) for observation of possible PCR inhibition. Clinical specimens collected from 329 women attending a STI clinic were used in the study. A total of 100 endocervical swab specimens transported in FVU was used in the PCR inhibition analysis. M. genitalium was detected in 25/329 (7.6 %) women. Endocervical swab specimens transported in FVU demonstrate higher sensitivity compared to both FVU alone and specimens transported in 2-SP medium detecting 24/25 (96 %), 22/25 (88 %) and 17/25 (68 %) of M. genitalium positive women, respectively. Automated BioRobot M48 DNA extraction was shown to be superior to manual Chelex extraction leaving no PCR inhibition and slightly higher DNA yield and/or better sensitivity. The results from these two studies are important knowledge in establishing the future diagnostic level of this STI in our county and also nationally.

Place, publisher, year, edition, pages
Karlstad: Karlstad University , 2010. , p. 38
Series
Karlstad University Studies, ISSN 1403-8099 ; 2010:11
Keywords [en]
PCR. Real-time PCR. Mycoplasma genitalium. Endocervical. Urogenital.
National Category
Microbiology in the medical area Microbiology in the medical area
Research subject
Biomedical Sciences
Identifiers
URN: urn:nbn:se:kau:diva-5481ISBN: 978-91-7063-297-6  (print)OAI: oai:DiVA.org:kau-5481DiVA, id: diva2:304724
Presentation
2010-05-21, 9C 203, Karlstads universitet, Karlstad, 10:30 (English)
Opponent
Supervisors
Available from: 2010-05-07 Created: 2010-03-19 Last updated: 2018-01-12Bibliographically approved
List of papers
1. A comparative study of three different PCR assays for detection of Mycoplasma genitalium in urogenital specimens from men and women
Open this publication in new window or tab >>A comparative study of three different PCR assays for detection of Mycoplasma genitalium in urogenital specimens from men and women
Show others...
2008 (English)In: Journal of Medical Microbiology, ISSN 0022-2615, E-ISSN 1473-5644, Vol. 57, no 3, p. 304-309Article in journal (Refereed) Published
Abstract [en]

The aim of this study was to compare conventional 16S rRNA gene PCR, real-time 16S rRNA gene PCR and real-time Mycoplasma genitalium adhesin protein (Mg gene PCR as detection methods for M. genitalium infection. The study also determined the prevalence of M. genitalium in male and female patients attending a sexually transmitted infections clinic in a rural area in the west of Sweden. First void urine (FVU) and/or urethral swabs were collected from 381 men, and FVU and/or cervical swabs and/or urethral swabs were collected from 298 women. A total of 213 specimens were used in the PCR comparative study: 98 consecutively sampled specimens from patients enrolled in the prevalence study, 36 consecutively sampled specimens from patients with symptoms of urethritis and 79 specimens from patients positive for M. genitalium by real-time MgPa gene PCR in the prevalence study. A true-positive M.genitalium DNA specimen was defined as either a specimen positive in any two PCR assays or a specimen whose PCR product was verified by DNA sequencing. The prevalence of M. genitalium infection in men and women was 27/381 (7.1 %) and 23/298 (7.7 %), respectively. In the PCR comparative study, M. genitalium DNA was detected in 61/76 (80.3%) of true-positive speciemens of conventional 16S rRNA gene PCR, in 52/76 (68.4 %) by real-time 16S rRNA gene PCR and in 74/76 (97.4 %) by real-time MgPa gene PCR. Real-time MgPa gene PCR thus had higher sensitivity compared with conventional 16S rRNA gene PCR and had considerably increased sensitivity compared with real-time 16S rRNA gene PCR for detection of M. genitalium DNA. Real-time MgPa gene PCR is well suited for the clinical diagnosis of M. genitalium.

Keywords
PCR. Mycoplasma genitalium. urogenital
National Category
Microbiology in the medical area
Research subject
Biomedical Sciences
Identifiers
urn:nbn:se:kau:diva-5474 (URN)10.1099/jmm.0.47498-0 (DOI)
Projects
biomedicin
Available from: 2010-03-17 Created: 2010-03-17 Last updated: 2018-01-12Bibliographically approved
2. Endocervical swabs transported in first void urine as combined specimens in the detection of Mycoplasma genitalium by real-time PCR
Open this publication in new window or tab >>Endocervical swabs transported in first void urine as combined specimens in the detection of Mycoplasma genitalium by real-time PCR
Show others...
2009 (English)In: Journal of Medical Microbiology, ISSN 0022-2615, E-ISSN 1473-5644, Vol. 58, p. 117-120Article in journal (Refereed) Published
Abstract [en]

The aim of this study was to determine whether a patient’s endocervical swab specimen can be transported in first void urine (FVU) as combined specimens for the detection of Mycoplasma genitalium by real-time PCR. The study also compared two different DNA extraction methods for observation of possible PCR inhibition. Three specimens, one endocervical swab specimen transported in 2-SP medium, one endocervical swab specimen transported in FVU and a FVU specimen, were collected from 329 women. All sample types underwent manual DNA extraction whereas in the DNA extraction study, 329 endocervical swab specimens transported in FVU were subjected to both manual Chelex and automated BioRobot M48 DNA extraction. A total of 100 endocervical swab specimens transported in FVU from patients PCR-negative for M. genitalium in the study were used in the PCR inhibition analysis. M. genitalium was detected in 25/329 (7.6 %) women. The endocervical swab specimens transported in 2-SP medium and transported in FVU were positive for M. genitalium in 17/25 (68 %) and 24/25 (96 %) women, respectively. The FVU specimens alone were positive for M. genitalium in 22/25 (88 %) women. In the DNA extraction study, M. genitalium DNA was detected in 24/329 (7.3 %) and 28/329 (8.5 %) of endocervical swab specimens transported in FVU subjected to manual Chelex extraction and automated BioRobot M48 extraction, respectively. Partial PCR inhibition was detected in 6% of samples subjected to manual Chelex extraction whereas no inhibition was detected with the automated BioRobot M48 extraction. Thus endocervical swab specimens transported in FVU demonstrate higher sensitivity than FVU specimens only and have considerably increased sensitivity compared with endocervical swab specimens transported in 2-SP medium for detection of M. genitalium DNA. Moreover, automated BioRobot M48 extraction was shown to be superior to a crude manual Chelex extraction, leaving no PCR inhibition and giving a slightly higher DNA yield and/or better sensitivity.

Keywords
real-time PCR. prevalence. mycoplasma genitalium.
National Category
Microbiology in the medical area
Research subject
Biomedical Sciences
Identifiers
urn:nbn:se:kau:diva-5473 (URN)10.1099/jmm.0.003681-0 (DOI)
Projects
biomedicinsk vetenskap
Available from: 2010-03-17 Created: 2010-03-17 Last updated: 2018-01-12Bibliographically approved

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