Macrolide Resistance-mediating Mutations Research Articles

Distribution of Macrolide Resistant Mycoplasma genitalium in Urogenital Tract Specimens From Women Enrolled in a US Clinical Study Cohort.

This study evaluated the distribution of macrolide-resistant Mycoplasma genitalium in multiple urogenital specimens collected from women enrolled in a prospective multicenter US clinical study. Four 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. Of 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. The 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.

Evaluation of Clinical, Gram Stain, and Microbiological Cure Outcomes in Men Receiving Azithromycin for Acute Nongonococcal Urethritis: Discordant Cures Are Associated With Mycoplasma genitalium Infection.

In men with nongonococcal urethritis (NGU), clinicians and patients rely on clinical cure to guide the need for additional testing/treatment and when to resume sex, respectively; however, discordant clinical and microbiological cure outcomes do occur. How accurately clinical cure reflects microbiological cure in specific sexually transmitted infections (STIs) is unclear. Men with NGU were tested for Neisseria gonorrhoeae, Chlamydia trachomatis (CT), Mycoplasma genitalium (MG), Trichomonas vaginalis, urethrotropic Neisseria meningitidis ST-11 clade strains, and Ureaplasma urealyticum (UU). Men received azithromycin 1 g and returned for a 1-month test-of-cure visit. In MG infections, we evaluated for the presence of macrolide resistance-mediating mutations (MRMs) and investigated alternate hypotheses for microbiological treatment failure using in situ shotgun metagenomic sequencing, phylogenetic analysis, multilocus sequence typing analyses, and quantitative PCR. Of 280 men with NGU, 121 were included in this analysis. In the monoinfection group, 52 had CT, 16 had MG, 7 had UU, 10 had mixed infection, and 36 men had idiopathic NGU. Clinical cure rates were 85% for CT, 100% for UU, 50% for MG, and 67% for idiopathic NGU. Clinical cure accurately predicted microbiological cure for all STIs, except MG. Discordant results were significantly associated with MG-NGU and predominantly reflected microbiological failure in men with clinical cure. Mycoplasma genitalium MRMs, but not MG load or strain, were strongly associated with microbiological failure. In azithromycin-treated NGU, clinical cure predicts microbiological cure for all STIs, except MG. Nongonococcal urethritis management should include MG testing and confirmation of microbiological cure in azithromycin-treated MG-NGU when MRM testing is unavailable.

Workflow and Throughput of Commercial Assays to Detect Mycoplasma genitalium and Macrolide Resistance-Mediating Mutations.

Aptima Mycoplasma genitalium (MG) required the shortest and STD6 the longest time to detect MG in clinical samples. ResistancePlus MG detected MG and macrolide resistance-mediating mutations simultaneously. Times were influenced by specimen numbers. M. genitalium positives from the other 2 assays required increased time for macrolide resistance-mediating mutation sequencing.

Resistance-guided treatment of Mycoplasma genitalium infection at a UK sexual health centre.

We evaluated the ResistancePlus® MG assay in providing macrolide resistance-guided treatment (RGT) for Mycoplasma genitalium infection at a UK sexual health centre. M. genitalium-positive samples from men with urethritis and women with pelvic inflammatory disease (PID) were tested for macrolide resistance-mediating mutations (MRMMs). MRMM-positive infections were given moxifloxacin 400mg; otherwise 2g azithromycin (1g single dose and then 500mg OD) was given. Among 57M. genitalium-positive patients (32 men and 25 women), MRMMs were detected in 41/57 (72% [95% confidence interval (95% CI) 58-83%). Thirty-two of 43 patients given RGT attended for test of cure. Treatment failure rate was significantly lower at 1/32 (3%) than 10/37 (27%) before RGT (n = 37 [men = 23 and women = 17]; p = 0.008). Treatment failure was lower in male urethritis (0/15 vs. 7/21 p = 0.027) but not in female PID. There was a trend of a shorter time to negative test of cure (TOC) in male urethritis (55.1 [95% 43.7-66.4] vs. 85.1 [95% CI CI 64.1-106.0] days, p = 0.077) but not in female PID. Macrolide resistance is higher than previous UK reports and higher than expected. RGT reduces overall treatment failure and is particularly beneficial in M. genitalium urethritis. Fluoroquinolone resistance will continue to rise with increasing fluoroquinolone use, and RGT is critical to direct appropriate azithromycin use and prevent overuse of moxifloxacin.

Molecular detection of Mycoplasma genitalium in endocervical swabs and associated rates of macrolide and fluoroquinolone resistance in Hong Kong.

There is a global trend of increasing macrolide and fluoroquinolone resistance in Mycoplasma genitalium (MG), such that international guidelines recommend molecular detection of resistance if a patient has MG-positive test results. Tests for MG are not routinely performed in Hong Kong. This study examined the detection of MG in endocervical swabs and the associated macrolide and fluoroquinolone resistance rates. Endocervical swabs received from two sexual health clinics in Hong Kong for routine assessments of Chlamydia trachomatis and Neisseria gonorrhoeae were also subjected to detection of MG. All MG-positive samples were tested for resistance-mediating mutations in 23S rRNA, parC, and gyrA genes. Laboratory records and past results for each patient were analysed. In total, endocervical swabs from 285 patients were included in this study. Mycoplasma genitalium was detected in swabs from 21 patients (7.4%) by real-time polymerase chain reaction with a commercial kit. Among MG-positive samples which were successfully analysed further, macrolide resistance-mediating mutations in 23S rRNA were found in 42.1% (8/19); fluoroquinolone resistance-related mutations in parC and gyrA were found in 65% (13/20) and 0% (0/20), respectively. All macrolide-resistant MG strains were also fluoroquinolone-resistant (42.1%, 8/19). No assessed factors were associated with the detection of MG or resistance-related mutations. In Hong Kong, MG was detected in endocervical swabs from 7.4% of patients in sexual health clinics, with high rates of macrolide and fluoroquinolone resistance. These findings warrant careful review of testing, clinical correlation, and treatment strategies for MG in the context of increasing antibiotic resistance.

High Prevalence of Vaginal and Rectal Mycoplasma genitalium Macrolide Resistance Among Female Sexually Transmitted Disease Clinic Patients in Seattle, Washington.

Rectal Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) are increasingly recognized as common infections among women. Little is known about the prevalence of rectal Mycoplasma genitalium (MG), rectal MG/CT/GC coinfection, or MG antimicrobial resistance patterns among women. In 2017 to 2018, we recruited women at high risk for CT from Seattle's municipal sexually transmitted disease clinic. Participants self-collected vaginal and rectal specimens for CT/GC nucleic acid amplification testing. We retrospectively tested samples for vaginal and rectal MG using nucleic acid amplification testing and tested MG-positive specimens for macrolide resistance-mediating mutations (MRM) and ParC quinolone resistance-associated mutations (QRAMs). Of 50 enrolled women, 13 (26%) tested positive for MG, including 10 (20%) with vaginal MG and 11 (22%) with rectal MG; 8 (62%) had concurrent vaginal/rectal MG. Five (38%) were coinfected with CT, none with GC. Only 2 of 11 women with rectal MG reported anal sex in the prior year. Of MG-positive specimens, 100% of rectal and 89% of vaginal specimens had an MRM. There were no vaginal or rectal MG-positive specimens with ParC QRAMs previously associated with quinolone failure. Five MG-infected women received azithromycin for vaginal CT, 4 of whom had a MG MRM detected in their vaginal and/or rectal specimens. We observed a high prevalence of macrolide-resistant vaginal and rectal MG among a population of women at high risk for CT. This study highlights how the use of antimicrobials designed to treat an identified infection-in this case, CT-could influence treatment outcomes and antimicrobial susceptibility in other unidentified infections.

Clinical evaluation of the ResistancePlus MG FleXible test on the GeneXpert Infinity-48s instrument: a near-patient assay for simultaneous detection of Mycoplasma genitalium and macrolide resistance

Antibiotic resistance in Mycoplasma genitalium (MG) is rising globally, especially to macrolides. In response to this challenge, assays reporting both the detection of MG and macrolide resistance-mediating mutations (MRMM) allow therapy to be tailored to the individual. The study evaluated the performance of the ResistancePlus® MG FleXible assay for the detection of MG and MRMM. Overall, the test performed well for the detection of MG compared to the AllplexTM STI Essential assay, used as a reference, with a kappa value of 0.926 (95% CI, 0.863–0.990). The kit also performed well for the detection of MRMM when compared with Sanger sequencing of the 23S rRNA gene, with a kappa value of 0.901 (95% CI, 0.807–0.996). The rate of MRMM in MG among the study population was 41.8%. In conclusion, the ResistancePlus® MG FleXible is a rapid, simple, and accurate cartridge-based assay for simultaneous detection of MG and MRMM in clinical settings.

Prevalence of Mycoplasma genitalium infection in women with bacterial vaginosis

BackgroundBacterial vaginosis (BV) is a common condition in reproductive-age women and is known to be positively associated with risk of acquisition of sexually transmitted infections (STI) such as chlamydia and gonorrhea. Mycoplasma genitalium is an emerging STI that has been linked to increased risk of pelvic inflammatory disease, adverse pregnancy outcomes and infertility. In the present study we sought to examine whether women diagnosed with symptomatic BV were at increased risk of having concurrent infection with Mycoplasma genitalium.MethodsWe used a novel PCR-based assay (ResistancePlus MG; SpeeDx Pty. Ltd., Sydney, Australia) to determine the prevalence of Mycoplasma genitalium infection and 23S rRNA macrolide-resistance mediating mutations (MRMM) in a cohort of 1532 women presenting with symptoms of vaginitis.ResultsM. genitalium was detected in 4.0% (62/1532) of samples with 37.1% (23/62) harboring MRMMs. The prevalence of M. genitalium infection in subjects with BV was significantly higher than in subjects with non-BV vaginitis (7.0% v 3.6%; OR = 1.97 (95% CI: 1.14–3.39).ConclusionsPrevalence of M. genitalium infection is associated with BV in women with symptomatic vaginitis. Improved management of BV is needed as a component of STI prevention strategies.

Evaluation of the ResistancePlus MG FleXible Assay for Detection of Wild-Type and 23S rRNA-Mutated Mycoplasma genitalium Strains.

Antibiotic resistance in Mycoplasma genitalium is rising globally, and resistance-guided diagnostics can facilitate targeted and timely treatment. The ResistancePlus MG FleXible (RPMG Flex) assay for the detection of M. genitalium and macrolide resistance-mediating mutations (MRMM) was evaluated for analytical sensitivity, specificity, reproducibility, and inhibition in the presence of interfering substances by simulating M. genitalium-negative pooled urine and swab matrices with M. genitalium cultures. Furthermore, the clinical sensitivity of the assay was evaluated and compared with a reference real-time PCR assay. The analytical sensitivity of the RPMG Flex assay was 157 genomes/ml for wild-type (WT) and 387 genomes/ml for MRMM strains in both matrices. For clinical specimens, the RPMG assay had an overall sensitivity of 96.1% (95% urine: 10/10 WT, 9/10 MRMM; 96.5% swab: 25/26 WT, 26/29 MRMM) compared to 85.7% for the MgPa/MagNAPure24 assay (95% urine: 19/20; 87% swab: 48/57). Clinical specificity was 100% for urine and 98.5% for swab specimens, respectively. No inhibition due to the presence of any of the tested interfering substances was observed. The RPMG Flex assay was more sensitive than the reference MgPa assay, in particular, for swab specimens. The implementation of this assay may increase ease of use and considerably decrease hands-on time for sample preparation compared to a standard block-based assay. The RPMG Flex assay for the GeneXpert Dx system provides a much-needed platform for the simultaneous detection of MG and MRMM and may thereby facilitate resistance-guided therapy for M. genitalium infections.

Macrolide-Resistant Mycoplasma genitalium in Southeastern Region of the Netherlands, 2014-2017.

Mycoplasma genitalium infections of the urogenital tract are usually treated with azithromycin; however, for the past several years, rates of azithromycin treatment failure have increased. To document the occurrence and frequency of macrolide resistance-mediating mutations (MRMMs) in M. genitalium infections, we collected 894 M. genitalium-positive samples during April 2014-December 2017 and retrospectively tested them for MRMMs. We designated 67 samples collected within 6 weeks after a positive result as test-of-cure samples; of these, 60 were MRMM positive. Among the remaining 827 samples, the rate of MRMM positivity rose from 22.7% in 2014 and 22.3% in 2015 to 44.4% in 2016 but decreased to 39.7% in 2017. Because of these high rates of MRMMs in M. genitalium infections, we recommend that clinicians perform tests of cure after treatment and that researchers further explore the clinical consequences of this infection.

424. Use of the ResistancePlus® MG Multiplex PCR Assay to Determine the Prevalence of Mycoplasma genitalium and Macrolide Resistance in a High-Risk US Population

Background Mycoplasma genitalium is a significant agent of sexually transmitted infection (STI). Cure rates have declined as rates of macrolide resistance has become increasingly prevalent. Diagnosis of M. genitalium infection and macrolide resistance detection is possible using nucleic-acid amplification tests (NAAT); use of such assays could improve patient management and antimicrobial stewardship. In this study we used one such assay, ResistancePlus MG (RPMG) to determine the prevalence of M. genitalium infection and macrolide resistance in a cohort of patients attending 3 public sexual health clinics in mid-Atlantic US states.MethodsDe-identified urogenital samples submitted to the LabCorp facility in Burlington, NC for routine Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) NAAT testing from 3 public sexual health clinics were analyzed in the study. All samples had been collected in the Aptima Specimen Collection system and tested with the Aptima Combo 2 CT/NG NAAT. A total of 1,261 samples (770 male, 491 female) from this cohort were successfully tested for M. genitalium and macrolide-resistance mediating mutations (MRMM) using the RPMG multiplexed PCR assay.ResultsThe prevalence of M. genitalium in this patient cohort was 10.4% (131/1,261), not significantly different to the prevalence of C. trachomatis (12.0%; P = 0.202) but significantly higher than the prevalence of Neisseria gonorrhoeae (6.7%; P = 0.0009). Sixty of the 131 M. genitalium positives were also positive for MRMM and thus azithromycin resistant.Conclusion M. genitalium infections were common amongst unselected individuals evaluated for treatable STI in the eastern United States and the rate of macrolide resistance in this population was significant. In addition, the RPMG assay was shown to be a simple and accurate method for simultaneously diagnosing M. genitalium infections and detecting MRMM and could be used to inform therapeutic decisions. Disclosures All authors: No reported disclosures.

Macrolide-Resistant Mycoplasma genitalium in Southeastern Region of the Netherlands, 2014-2017.

Mycoplasma genitalium infections of the urogenital tract are usually treated with azithromycin; however, for the past several years, rates of azithromycin treatment failure have increased. To document the occurrence and frequency of macrolide resistance–mediating mutations (MRMMs) in M. genitalium infections, we collected 894 M. genitalium–positive samples during April 2014–December 2017 and retrospectively tested them for MRMMs. We designated 67 samples collected within 6 weeks after a positive result as test-of-cure samples; of these, 60 were MRMM positive. Among the remaining 827 samples, the rate of MRMM positivity rose from 22.7% in 2014 and 22.3% in 2015 to 44.4% in 2016 but decreased to 39.7% in 2017. Because of these high rates of MRMMs in M. genitalium infections, we recommend that clinicians perform tests of cure after treatment and that researchers further explore the clinical consequences of this infection.

Macrolide-resistant Mycoplasma genitalium infections in Cuban patients: an underestimated health problem

BackgroundThe increasing prevalence of macrolide resistant Mycoplasma genitalium is a major concern worldwide. In Cuba, several cases of clinical treatment failure with 1 g single dose and extended azithromycin regimen have been detected and the aim of the present investigation was to retrospectively determine the prevalence of macrolide-resistance mediating mutations (MRMM) in M. genitalium-positive samples conserved at the Cuban National Reference Laboratory of Mycoplasma Research between 2009 and 2016.MethodsA total of 280 positive DNA extracts were analysed by a 5’ nuclease assay for detection of M. genitalium MRMM. Ten urogenital specimens from patients with azithromycin treatment failure and MRMM were inoculated in Vero cell to obtain the isolates for subsequent determination of antimicrobial susceptibility.ResultsThe overall prevalence of MRMM was 32%. No MRMM was detected in samples collected between 2009 and 2013 but since 2014 a dramatic increase to 90% (95% CI, 76–96%) in 2016 was seen. Three new M. genitalium isolates were isolated in Vero cell cultures and confirmed phenotypic resistance to macrolides in a cell-culture assisted susceptibility test. Preliminary observations suggest that combination therapy with levofloxacin and doxycycline may represent an affordable option for treatment of macrolide resistant M. genitalium infections.ConclusionsThis investigation showed the rapid emergence and high prevalence of MRMM in M. genitalium-infected patients in Cuba and confirmed the phenotypic resistance in isolates carrying MRMM. We suggest that Cuban guidelines for sexually transmitted infections are modified to include testing for M. genitalium and detection of MRMM in patients with failure of syndromic treatment, to ensure that in these cases, the treatment will be guided by etiologic diagnosis.

Long Duration of Asymptomatic Mycoplasma genitalium Infection After Syndromic Treatment for Nongonococcal Urethritis.

Although Mycoplasma genitalium (MG) is an acknowledged cause of nongonococcal urethritis (NGU), access to diagnostic testing is limited. Syndromic management is common, yet little is known about natural history. Between August 2014 and April 2016, 13 heterosexual men aged ≥16 years with MG were identified within a cohort study of men with and without NGU attending an urban sexually transmitted diseases clinic. Men had 6-7 monthly visits. NGU was defined as ≥5 polymorphonuclear leukocytes per high-power field on urethral Gram stain plus either visible urethral discharge or urethral symptoms. Men with NGU received 1 g of azithromycin. Men with persistent NGU received moxifloxacin 400 mg for 14 days. First-void urine was retrospectively tested for MG using transcription-mediated amplification. Resistance-associated mutations were detected by polymerase chain reaction (PCR) and sequencing. Organism load was determined by quantitative PCR. Sixty-two percent of MG-positive men had macrolide resistance-mediating mutations (MRMM) at enrollment; 31% had parC mutations (all outside the quinolone resistance-determining region). MG persisted after azithromycin in 7 men, 6 of whom had MRMM. The median duration of persistence in the absence of curative therapy was 143 days (range, 21-228). Five men experienced symptom resolution after azithromycin, but MG persisted for another 89-186 days before moxifloxacin. Organism load was somewhat lower in MRMM than wild-type infections (P = .16). The high prevalence of macrolide resistance and long duration of infection after symptom resolution highlights the need for diagnostic MG testing of men with NGU to direct therapy.

Mycoplasma genitalium Infection in Kenyan and US Women.

Little is known about the natural history of Mycoplasma genitalium (MG) infection in women. We retrospectively tested archived vaginal fluid samples to assess MG prevalence, incidence, persistence, recurrence and antimicrobial resistance markers among women participating in the Preventing Vaginal Infections trial, a randomized trial of monthly presumptive treatment to reduce vaginal infections. High-risk, nonpregnant, HIV-negative women aged 18 to 45 years from Kenya and the United States were randomized to receive metronidazole 750 mg + miconazole 200 mg intravaginal suppositories or placebo for 5 consecutive nights each month for 12 months. Clinician-collected swabs containing cervicovaginal fluid were tested for MG using Hologic nucleic acid amplification testing at enrollment and every other month thereafter. Specimens that were MG+ underwent additional testing for macrolide resistance-mediating mutations by DNA sequencing. Of 234 women enrolled, 221 had available specimens and 25 (11.3%) had MG at enrollment. Among 196 women without MG at enrollment, there were 52 incident MG infections (incidence, 33.4 per 100 person-years). Smoking was independently associated with incident MG infection (adjusted hazard ratio, 3.02; 95% confidence interval, 1.32-6.93), and age less than 25 years trended toward an association (adjusted hazard ratio, 1.70; 95% confidence interval, 0.95-3.06). Median time to clearance of incident MG infections was 1.5 months (interquartile range, 1.4-3.0 months). Of the 120 MG+ specimens, 16 specimens from 15 different women were macrolide resistance-mediating mutation positive (13.3%), with no difference by country. M. genitalium infection is common among sexually active women in Kenya and the Southern United States. Given associations between MG and adverse reproductive health outcomes, this high burden of MG in reproductive-aged women could contribute to substantial morbidity.

Evaluation of a real-time PCR assay for detection of Mycoplasma genitalium and macrolide resistance-mediating mutations from clinical specimens

Mycoplasma genitalium (MG) is a sexually transmitted pathogen for which there is no FDA-approved diagnostic test available in the United States. A modified real-time polymerase chain reaction assay for detecting MG and simultaneously identifying macrolide resistance mutations from clinical specimens was evaluated and proved to be sensitive and accurate for diagnostic purposes.

High levels of macrolide-resistant Mycoplasma genitalium in Queensland, Australia.

The macrolide azithromycin is recommended for treatment of Mycoplasma genitalium infection; however, M. genitalium strains possessing macrolide resistance-mediating mutations (MRMMs) are increasingly being reported. Here, we used the SpeeDx ResistancePlus MG kit, which provides simultaneous detection of M. genitalium and MRMMs, to assess MRMM carriage among M. genitalium infections in Queensland, Australia. Performance characteristics of the ResistancePlus MG kit for M. genitalium detection were compared to in-house PCR. Available M. genitalium PCR-positive (n=67) and negative (n=281) samples from the years 2011 to 2017 were tested using the SpeeDx ResistancePlus MG kit. In total, 63.6 % M. genitalium-positive samples were indicated to harbour MRMMs. The ResistancePlus MG method provided sensitivity and specificity of 97 and 99.6 % respectively compared to in-house PCR for M. genitalium detection. Such high levels of macrolide-resistant M. genitalium raise further concerns over future use of azithromycin for treatment of M. genitalium infection.

3: Detection of macrolide resistance-mediating mutations among women with mycoplasma genitalium infection in the preventing vaginal infections trial

3: Detection of macrolide resistance-mediating mutations among women with mycoplasma genitalium infection in the preventing vaginal infections trial