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Detection of Mycoplasma genitalium in urogenital specimens by real-time PCR and by conventional PCR assay
Örebro University, Department of Clinical Medicine.
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2005 (English)In: Journal of Medical Microbiology, ISSN 0022-2615, E-ISSN 1473-5644, Vol. 54, no 1, p. 23-29Article in journal (Refereed) Published
Abstract [en]

A real-time LightCycler PCR (LC-PCR) with hybridization probesfor detection of Mycoplasma genitalium in endocervical and firstvoid urine specimens was developed and compared to a conventionalPCR. The primers for both assays were identical and designedto amplify a 427 bp fragment of the 16S rRNA gene of M. genitalium.The LC-PCR assay had a detection limit of < 5 bacterial genomesper reaction when dilutions of genomic DNA from a type strainof M. genitalium were tested. First void urine from 398 menand first void urine and endocervical specimens from 301 womenattending an STD clinic were analysed by LC-PCR and by the conventionalPCR. Using the conventional PCR as reference, the LC-PCR hada specificity of 99.7 % and a sensitivity of 72.2 % for thedetection of M. genitalium in first void urine samples frommen. There was no significant difference in the performanceof the LC-PCR assay compared to the conventional PCR when endocervicalswabs were considered (58 and 65 %, respectively) or with aset of endocervical swab/urine specimens for which the LC-PCRassay detected 73 % of the infections (specificity = 98.6 %and sensitivity = 68.2 %) while the conventional PCR detected85 % of the infections. With female urine specimens there wasa significant difference between the two assays (38 and 73 %,respectively; P = 0.01 McNemar's test). This illustrates theneed to analyse both endocervical and urine specimens, becauseM. genitalium DNA was detected in only one of the two specimensin a great number of the M. genitalium-infected women. The lowersensitivity of the LC-PCR assay was probably caused by a combinationof inhibition and limitations regarding the amount of templateDNA. The LC-PCR assay was easy to perform and the simultaneousamplification and detection eliminated the need for furtherhandling of PCR products. With improvement in sample preparationmethods and increased volumes of the template DNA, the LC-PCRassay could be a useful routine diagnostic method.

Place, publisher, year, edition, pages
2005. Vol. 54, no 1, p. 23-29
National Category
Medical and Health Sciences
Research subject
Medicine
Identifiers
URN: urn:nbn:se:oru:diva-3196DOI: 10.1099/jmm.0.45732-0OAI: oai:DiVA.org:oru-3196DiVA, id: diva2:137287
Available from: 2006-11-17 Created: 2006-11-17 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Detection of Chlamydia trachomatis and Mycoplasma genitalium by genetic and serological methods
Open this publication in new window or tab >>Detection of Chlamydia trachomatis and Mycoplasma genitalium by genetic and serological methods
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Chlamydia trachomatis infections are associated with a spectrum of clinical diseases including urethritis, prostatitis and epididymitis among men and cervicitis and pelvic inflammatory disease (PID), with an increased risk of infertility and ectopic pregnancy (EP), among women. In the search for other pathogens causing urethritis, Mycoplasma genitalium was isolated from urethral specimens from two men with acute urethritis (1980). Mycoplasma bacteria are extremely difficult to isolate by culture, and clinical studies have been possible only after the advent of the first PCR-based detection method. M. genitalium has been found to be associated with lower genital tract infections in both men and women. Finding evidence for a connection between M. genitalium and upper genital tract infections in women is still of major importance.

The aim in papers I and II was to develop a PCR method for genetic characterization of clinical C. trachomatis isolates by sequence analysis of the omp1 gene, and to study the distribution of genotypes within sexual networks and determine if genotyping would improve partner notification. The method was used to determine the genotypes of C. trachomatis in 237 positive urogenital and/or urine specimens from men and women attending the STDClinic in Örebro during one year. Sequence analysis of the omp1 gene revealed that the most prevalent genotypes corresponded to C. trachomatis serovar E (47%), followed by F (17%), and K (9%). There were 161 networks found and specimens were sequenced from at least two patients in 47 networks. In seven of these 47 networks there were discrepant genotypes. In the largest network comprising 26 individuals two different C. trachomatis genotypes were found, and one partner had urethritis due to a Mycoplasma genitalium infection but was C. trachomatis negative.

The need for a new method for M. genitalium DNA detection was one reason for study III. An existing conventional PCR protocol for detection of M. genitalium DNA was further developed into a real-time PCR (RT-PCR) with hybridisation probes. In order to evaluate the RT-PCR assay with clinical material, specimens from 398 men and 301 women attending the STD Clinic in Örebro were analysed, using the RT-PCR assay, and also by the well established conventional PCR in Copenhagen. Using the conventional PCR method as “gold standard”, the sensitivity for the RT-PCR assay was 72.2% and 68.2% and the specificity was 99.7% and 98.6%, respectively, in urogenital specimens from men and women.

The aim in paper IV was to adapt a Triton X-114 extracted Lipid-Associated Membrane Protein (LAMP) Enzyme Immuno Assays (EIA) method to detect antibodies against M. genitalium and to evaluate the association between M. genitalium and PID and EP, using sera sampled in Örebro during the 1980s, and also to compare the number of sera having M. genitalium antibodies against those having C. trachomatis antibodies, using a commercial anti- Chlamydia trachomatis EIA assay. No statistical significant association could be demonstrated between M. genitalium antibodies and PID or EP in our serum material. However, a slight trend toward association was found when focusing on younger individuals. Antibodies against C. trachomatis were found to be significantly associated with PID and EP.

Place, publisher, year, edition, pages
Örebro: Örebro universitetsbibliotek, 2006. p. 61
Series
Örebro Studies in Medicine, ISSN 1652-4063 ; 8
Keywords
Chlamydia trachomatis, genotyping, contact tracing, sexually transmitted diseases, Mycoplasma genitalium, PCR, serology
National Category
Medical and Health Sciences
Research subject
Medicine
Identifiers
urn:nbn:se:oru:diva-793 (URN)91-7668-506-3 (ISBN)
Public defence
2006-12-08, Wilandersalen, Universitetssjukhuset, Örebro, 13:00
Opponent
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Available from: 2006-11-17 Created: 2006-11-17 Last updated: 2017-10-18Bibliographically approved

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Jurstrand, Margaretha

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