Development of a rotor-gene real-time PCR assay for the detection and quantification of Mycoplasma genitalium

Müller, Etienne; Venter, Johanna M. E.; Magooa, Mahlape Precious; Morrison, Charles; Lewis, David A.; Napierala, Sue

doi:10.1016/j.mimet.2011.12.017

Cited by 31 publications

(25 citation statements)

References 28 publications

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“…In order to further investigate this finding, we assessed the relationship between UWCC and pathogen load. Bacterial load range of M. genitalium was comparable with other studies 24 25 and there was no difference in pathogen loads between M. genitalium and C. trachomatis , although this analysis was a secondary outcome of the study and may not have been sufficiently powered to demonstrate this. However, UWCC were higher in C. trachomatis infection compared with M. genitalium , suggesting different interactions or effects of inflammation on bacterial replication in the male genital tract between the two organisms.…”

Section: Discussionsupporting

confidence: 74%

Performance evaluation of automated urine microscopy as a rapid, non-invasive approach for the diagnosis of non-gonococcal urethritis

et al. 2015

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ObjectivesGram-stained urethral smear (GSUS), the standard point-of-care test for non-gonococcal urethritis (NGU) is operator dependent and poorly specific. The performance of rapid automated urine flow cytometry (AUFC) of first void urine (FVU) white cell counts (UWCC) for predicting Mycoplasma genitalium and Chlamydia trachomatis urethral infections was assessed and its application to asymptomatic infection was evaluated.MethodsReceiver operating characteristic curve analysis, determining FVU-UWCC threshold for predicting M. genitalium or C. trachomatis infection was performed on 208 ‘training’ samples from symptomatic patients and subsequently validated using 228 additional FVUs obtained from prospective unselected patients.ResultsAn optimal diagnostic threshold of >29 UWC/µL gave sensitivities and specificities for either infection of 81.5% (95% CI 65.1% to 91.6%) and 85.8% (79.5% to 90.4%), respectively, compared with 86.8% (71.1% to 95%) and 64.7% (56.9% to 71.7%), respectively, for GSUS, using the training set samples. FVU-UWCC demonstrated sensitivities and specificities of 69.2% (95% CI 48.1% to 84.9%) and 92% (87.2% to 95.2%), respectively, when using validation samples. In asymptomatic patients where GSUS was not used, AUFC would have enabled more infections to be detected compared with clinical considerations only (71.4% vs 28.6%; p=0.03). The correlation between UWCC and bacterial load was stronger for M. genitalium compared with C. trachomatis (τ=0.426, p≤0.001 vs τ=0.295, p=0.022, respectively).ConclusionsAUFC offers improved specificity over microscopy for predicting C. trachomatis or M. genitalium infection. Universal AUFC may enable non-invasive diagnosis of asymptomatic NGU at the PoC. The degree of urethral inflammation exhibits a stronger association with pathogen load for M. genitalium compared with C. trachomatis.

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Section: Discussionsupporting

confidence: 74%

Performance evaluation of automated urine microscopy as a rapid, non-invasive approach for the diagnosis of non-gonococcal urethritis

et al. 2015

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“…Testing was done using a laboratory-developed quantitative PCR (qPCR) using hydrolysis probes targeting the pdhD and mgpB genes of M. genitalium and an additional sampleprocessing control. The primers and probes of this laboratory-developed test have previously been published (14)(15)(16). In the multiplex qPCR, the final concentrations of M. genitalium-specific primers and of probes were 500 nM and 100 nM, respectively.…”

Section: Methodsmentioning

confidence: 99%

A 5′ Nuclease Genotyping Assay for Identification of Macrolide-Resistant Mycoplasma genitalium in Clinical Specimens

2016

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M ycoplasma genitalium is a sexually transmitted bacterium that causes nongonococcal urethritis in men and has been associated with cervicitis and pelvic inflammatory disease in women (1). The current recommended treatment of M. genitalium infection in Scandinavia is an extended course of oral macrolide. However, if empirical treatment of nongonococcal urethritis is initiated, usually only a single dose of azithromycin is given as treatment for suspected Chlamydia infection. Single-dose azithromycin treatment of M. genitalium infection is associated with a suboptimal treatment response and, in the case of treatment failure, with the development of M. genitalium macrolide resistance (2, 3). Macrolide-resistant M. genitalium strains are consequently prevalent, and resistance rates of 41% in a cohort of men with urethritis in London (4) and 38% in a recent population-based Danish survey (5) have been reported. In proven M. genitalium infection, macrolide susceptibility reporting is therefore necessary to guide therapy (6).Macrolide resistance in M. genitalium is strongly associated with the mutations A2058G and A2059G in the gene encoding 23S rRNA (3,7,8). In a recent Dutch study, a high proportion of the A2058T mutation causing resistance was observed (9). Resistance-associated mutations can be identified by PCR amplification and sequencing. This method is best suited to process samples in bulk and may result in unacceptable reporting times. Highresolution melting (HRM) analysis is another powerful tool to detect mutations in a target sequence, and PCR assays using HRM analysis for detecting mutations associated with macrolide resistance have recently been reported (10, 11). An alternative method for identifying known mutations is genotyping assays using differentially labeled hydrolysis probes targeting wild-type and mutated alleles (12,13). In the present study, we report such a 5= nuclease genotyping assay for the detection of the macrolide resistance-associated mutations in M. genitalium. The assay was used to determine macrolide resistance in 259 samples that had previously tested positive for the presence of M. genitalium, and the results obtained using this assay were compared to sequencing of the region of interest in the 23S rRNA gene. The 5= nuclease genotyping assay was highly accurate compared to sequencing. MATERIALS AND METHODSM. genitalium detection. Between 8 October 2013 and 12 September 2014, the Department of Clinical Microbiology received 3,147 samples to test for the presence of M. genitalium. Testing was done using a laboratory-developed quantitative PCR (qPCR) using hydrolysis probes targeting the pdhD and mgpB genes of M. genitalium and an additional sampleprocessing control. The primers and probes of this laboratory-developed test have previously been published (14-16). In the multiplex qPCR, the final concentrations of M. genitalium-specific primers and of probes were 500 nM and 100 nM, respectively. Reactions were done in a total volume of 20 l with LightCycler 480 probes master (Roche A...

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“…Initially, samples were submitted for β‐globin amplification by qPCR to evaluate DNA integrity [Lindh et al, ]. β‐globin positive samples were tested with specific primers previously validated by qPCR for DNA detection of CT, GV, MG, and UP [Ling et al, ; Muller et al, ; Hajikhani et al, ; Jalal et al, ]. Ureaplasma infection was detected with a single pair of oligonucleotides which amplified the following biovars: (1) U. Parvum and (2) Urealyticum [Jalal et al, ].…”

Section: Methodsmentioning

confidence: 99%

Prevalence of sexually transmitted pathogens associated with HPV infection in cervical samples in a Mexican population

et al. 2015

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Cervical cancer development has been mainly associated with persistent human papillomavirus (HPV) infections. However, HPV infection is unlikely to be sufficient to cause cervical cancer, and the contribution of other sexually transmitted infections (STIs) could be the determining factor for cervical lesion-progression. The aim of this study was to estimate the prevalence of STIs associated with HPV-positivity in 201 cervical samples from patients who underwent annual routine gynecological exams. The overall prevalence of STIs was 57.7%, and the most frequent infection was Ureaplasma spp (UP) (39.8%), followed by Gardnerella vaginalis (GV) (25.9%), α-HPV (18.4%), Chlamydia trachomatis (CT) (1.5%), and Mycoplasma genitalium (MG) (0.5%). The highest prevalence rate of multiple non-HPV infections was observed for the age-range 31-40; for papillomavirus infection, the age-range was 21-30. In normal cervical samples, HPV16 was the most prevalent genotype (24.3%), followed by genotypes 58 (13.5%) and 52 (10.8%). Intriguingly, HPV18 was not detected in the study population, and genotypes 52 and 58 were found exclusively in samples with abnormal cytology. Papillomavirus infection with oncogenic types was significantly associated with GV (P = 0.025) and strongly associated with multiple non-HPV pathogens (P = 0.002). The following variables correlated significantly with cytological diagnosis of low-grade squamous intraepithelial lesion (LSIL): GV (P = 0.028), multiple non-HPV infections (P = 0.001), and high-risk HPV positivity (P = 0.001). Epidemiological data from this study will contribute to the molecular detection of sexually transmitted pathogens from screening programs to identify those women who are at risk for developing cervical lesions.

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Development of a rotor-gene real-time PCR assay for the detection and quantification of Mycoplasma genitalium

Cited by 31 publications

References 28 publications

Performance evaluation of automated urine microscopy as a rapid, non-invasive approach for the diagnosis of non-gonococcal urethritis

Performance evaluation of automated urine microscopy as a rapid, non-invasive approach for the diagnosis of non-gonococcal urethritis

A 5′ Nuclease Genotyping Assay for Identification of Macrolide-Resistant Mycoplasma genitalium in Clinical Specimens

Prevalence of sexually transmitted pathogens associated with HPV infection in cervical samples in a Mexican population

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