Dataset Information

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

ABSTRACT: Rapid and sensitive detection of macrolide resistance in Mycoplasma genitalium is required for the guidance of adequate antimicrobial treatment. Previous studies have confirmed that single-base mutations at position 2058 or 2059 in domain V of the 23S rRNA gene of M. genitalium result in high-level macrolide resistance. Sequencing of PCR products remains the gold standard for the identification of mutations conferring resistance to macrolides but is laborious and time-consuming. The aim of the present study was to develop a 5' nuclease genotyping assay to detect single nucleotide polymorphisms in the 23S rRNA gene of Mycoplasma genitalium that are associated with macrolide resistance by combining PCR with hydrolysis probes and subsequent endpoint genotyping analysis. The 5' nuclease genotyping assay was used as a referral test to be used on M. genitalium-positive samples and was validated on 259 positive samples, of which 253 (97.7%) were successfully sequenced. With the newly developed assay, 237/259 (91.5%) investigated M. genitalium-positive samples were genotyped. The positive and the negative predictive values were 100% when evaluated on successfully genotyped samples. The newly developed assay discriminated macrolide-resistant M. genitalium in clinical specimens possessing A2058G, A2058C, A2058T, and A2059G mutations with a sensitivity of 94.4% (95% confidence interval [CI], 90.7% to 98.2%) and a specificity of 92.7% (95% CI, 87.8% to 97.6%) when evaluated on successfully sequenced samples. The assay can correctly guide antimicrobial treatment of M. genitalium infections.

SUBMITTER: Kristiansen GQ

PROVIDER: S-EPMC4879279 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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

Kristiansen Gitte Qvist GQ Lisby Jan Gorm JG Schønning Kristian K

Journal of clinical microbiology 20160406 6

Rapid and sensitive detection of macrolide resistance in Mycoplasma genitalium is required for the guidance of adequate antimicrobial treatment. Previous studies have confirmed that single-base mutations at position 2058 or 2059 in domain V of the 23S rRNA gene of M. genitalium result in high-level macrolide resistance. Sequencing of PCR products remains the gold standard for the identification of mutations conferring resistance to macrolides but is laborious and time-consuming. The aim of the p ...[more]

PMID: 27053672

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Project description:BackgroundThis study evaluated the distribution of macrolide-resistant Mycoplasma genitalium in multiple urogenital specimens collected from women enrolled in a prospective multicenter US clinical study.MethodsFour female urogenital specimens (vaginal swab, urine, endocervical swab, ectocervical brush/spatula) collected from each subject were tested using a transcription-mediated amplification (TMA) assay for M. genitalium. TMA-positive specimens were evaluated by reverse transcription-polymerase chain reaction and bidirectional Sanger sequencing of M. genitalium 23S rRNA to identify the presence of macrolide-resistance-mediating mutations (MRMs) at base positions 2058/2059.ResultsOf 140 women with ≥1 TMA-positive specimens, 128 (91.4%) yielded M. genitalium 23S rRNA sequence. MRMs were found in 52% of vaginal specimens, 46.3% of urine specimens, 37.8% of endocervical specimens, and 46% of ectocervical specimens. There were 44 unique specimen type/sequence phenotype combinations of M. genitalium infection. Most (81; 63.3%) women had single specimen-sequence phenotype (macrolide-susceptible, MRM, or both) infections, while 24 (18.8%) women had multiple specimen-sequence phenotype concordant infections, and 23 (17.9%) women had multiple specimen-sequence phenotype discordant infections. The sensitivity for any single specimen type to detect overall urogenital tract macrolide-resistant M. genitalium infection status was 96.3% for vaginal swab samples, 82.6% for urine samples, 70.8% for endocervical swab samples, and 82.1% for ectocervical brush/spatula liquid Pap samples.ConclusionsThe distribution of M. genitalium infections in female urogenital tract specimens is highly complex, with multiple phenotypic combinations of the organism infecting a significant proportion of women at different anatomic specimen collection sites. Vaginal swab sampling yielded the highest sensitivity for identifying women with macrolide-resistant M. genitalium urogenital tract infections.

Project description:BackgroundMycoplasma genitalium (MG) causes urethritis, cervicitis and pelvic inflammatory disease. The MG treatment failure rate using 1 g azithromycin at an Australian Sexual Health clinic in 2007-9 was 31% (95%CI 23-40%). We developed a rapid high resolution melt analysis (HRMA) assay targeting resistance mutations in the MG 23S rRNA gene, and validated it against DNA sequencing by examining pre- and post-treatment archived samples from MG-infected patients.Methodology/principal findingsAvailable MG-positive pre-treatment (n = 82) and post-treatment samples from individuals with clinical treatment failure (n = 20) were screened for 23S rRNA gene mutations. Sixteen (20%) pre-treatment samples possessed resistance mutations (A2058G, A2059G, A2059C), which were significantly more common in patients with symptomatic azithromycin-treatment failure (12/26; 44%) than in those clinically cured (4/56; 7%), p<0.001. All 20 patients experiencing azithromycin-failure had detectable mutations in their post-treatment samples. In 9 of these cases, the same mutational types were present in both pre- and post-treatment samples indicating transmitted resistance, whilst in 11 of these cases (55%), mutations were absent in pre-treatment samples indicating likely selection of resistant isolates have occurred. HRMA was able to detect all mutational changes determined in this study by DNA sequencing. An additional HRMA assay incorporating an unlabelled probe was also developed to detect type 4 single-nucleotide polymorphisms found in other populations, with a slightly lower sensitivity of 90%.Conclusions/significanceTreatment failure is associated with the detection of macrolide resistance mutations, which appear to be almost equally due to selection of resistant isolates following exposure to 1 g azithromycin and pre-existing transmitted resistance. The application of a rapid molecular assay to detect resistance at the time of initial detection of infection allows clinicians to shorten the time to initiate effective second line treatment. This has the potential to reduce transmission of resistant strains and to avoid sequelae associated with persistent untreated infection.

Dataset Information

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

Publications

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

Similar Datasets

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