Single gene targeted nanopore sequencing enables simultaneous identification and antimicrobial resistance detection of sexually transmitted infections.

OBJECTIVES: To develop a simple DNA sequencing test for simultaneous identification and antimicrobial resistance (AMR) detection of multiple sexually transmitted infections (STIs). METHODS: Real-time PCR (qPCR) was initially performed to identify Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT), Mycoplasma genitalium (MG) and Trichomonas vaginalis (TV) infections among a total of 200 vulvo-vaginal swab samples from female sex workers in Ecuador. qPCR positive samples plus qPCR negative controls for these STIs were subjected to single gene targeted PCR MinION-nanopore sequencing using the smartphone operated MinIT. RESULTS: Among 200 vulvo-vaginal swab samples 43 were qPCR positive for at least one of the STIs. Single gene targeted nanopore sequencing generally yielded higher pathogen specific read counts in qPCR positive samples than qPCR negative controls. Of the 26 CT, NG or MG infections identified by qPCR, 25 were clearly distinguishable from qPCR negative controls by read count. Discrimination of TV qPCR positives from qPCR negative controls was poorer as many had low pathogen loads (qPCR cycle threshold >35) which produced few specific reads. Real-time AMR profiling revealed that 3/3 NG samples identified had gyrA mutations associated with fluoroquinolone resistance, 2/10 of TV had mutations related to metronidazole resistance, while none of the MG samples possessed 23S rRNA gene mutations contributing to macrolide resistance. CONCLUSIONS: Single gene targeted nanopore sequencing for diagnosing and simultaneously identifying key antimicrobial resistance markers for four common genital STIs shows promise. Further work to optimise accuracy, reduce costs and improve speed may allow sustainable approaches for managing STIs and emerging AMR in resource poor and laboratory limited settings.

Chlamydia trachomatis and Mycoplasma genitalium prevalence and associated factors among women presenting to a pregnancy termination and contraception clinic, 2009-2019.

BACKGROUND: Risk of pelvic inflammatory disease associated with Chlamydia trachomatis and Mycoplasma genitalium is increased after termination of pregnancy (TOP) and may be increased after insertion of intrauterine devices (IUDs). Screening prior to these procedures is recommended only for C. trachomatis. We examined C. trachomatis and M. genitalium prevalence and associated factors among women presenting to a pregnancy termination and contraception service over 10 years. METHODS: Retrospective analysis of clinical data collected from 17 573 women aged 15-45 years in 2009-2019 and for 266 M. genitalium positive women tested for macrolide resistance-associated mutations in 2016-2019. RESULTS: C. trachomatis and M. genitalium prevalence was 3.7% and 3.4%, respectively. In multivariable analyses, shared risk factors were younger age (p<0.001, for both C. trachomatis and M. genitalium), socioeconomic disadvantage (p=0.045 and p=0.008, respectively) and coinfection (p<0.001, for both sexually transmitted infections), with 10.1% of C. trachomatis positive women also positive for M. genitalium. Additional risk factors were earlier year of visit (p=0.001) for C. trachomatis and for M. genitalium residing outside a major city (p=0.013). The proportion of M. genitalium infections tested between 2016 and 2019 with macrolide resistance-associated mutations was 32.7%. CONCLUSIONS: Given the high level of antimicrobial resistance and the prevalence of coinfection, testing C. trachomatis positive women for M. genitalium could be considered in this setting to prevent further spread of resistant infections. Further research is required into the causal link between M. genitalium and pelvic inflammatory disease in women undergoing TOP and IUD insertion.

Antimicrobial resistance in Neisseria gonorrhoeae and Mycoplasma genitalium isolates from the private healthcare sector in South Africa: A pilot study.

BACKGROUND: Reports have emerged globally of antimicrobial resistance (AMR) in Neisseria gonorrhoeae and Mycoplasma genitalium infections. In South Africa (SA), there are substantial differences between private and public healthcare with regard to antimicrobial drug prescribing practice, which could affect AMR patterns of private and public healthcare patients. OBJECTIVES: To perform a pilot study to determine the frequency of AMR of N. gonorrhoeae and M. genitalium in patients accessing SA's private healthcare sector. METHODS: In this cross-sectional study, N. gonorrhoeae-positive cultures and M. genitalium DNA samples were collected from a private healthcare reference laboratory from August 2018 to August 2019. In N. gonorrhoeae-positive cultures, antimicrobial susceptibility testing was performed, followed by N. gonorrhoeae multiantigen sequence typing (NG-MAST) to determine genetic relatedness of the isolates. To determine macrolide and fluoroquinolone resistance rates, M. genitalium-positive samples were analysed by sequencing the 23S rRNA, gyrA and parC genes. RESULTS: Twenty-one N. gonorrhoeae- and 27 M. genitalium-positive specimens were included in this analysis. High rates of resistance were detected among gonococcal isolates, with 90% resistance to tetracycline, 86% to penicillin and 62% to ciprofloxacin, but no resistance to azithromycin, cefixime and ceftriaxone. NG-MAST revealed genetically diverse isolates with 83% novel NG-MAST sequence types. Macrolide and fluoroquinolone resistance-associated mutations were detected in 18.5% (n=5/27) and 7.4% (n=2/27) of M. genitalium strains, respectively. CONCLUSIONS: We observed high frequencies of ciprofloxacin, penicillin and tetracycline resistance in N. gonorrhoeae and macrolide resistance-associated mutations in M. genitalium in private healthcare sector patients in SA. This finding highlights the need to use diagnostics for sexually transmitted infections and to include the private healthcare sector in antimicrobial surveillance and stewardship programmes.

Mycoplasma genitalium prevalence and macrolide resistance-associated mutations and coinfection with Chlamydia trachomatis in Southern Jutland, Denmark.

This study aims to investigate prevalence of Mycoplasma genitalium and macrolide resistance-associated mutations and coinfection with other sexually transmitted bacteria in Southern Jutland, Denmark, where this information is very limited. Urinary samples from patients suspected of sexually transmitted bacterial infections collected at primary healthcare facilities in Southern Jutland are routinely tested for Chlamydia trachomatis and Neisseria gonorrhoeae. 601 of these samples were analysed with SpeeDx MG+23S reagents, which can detect M. genitalium and macrolide resistance-mediating mutations in the 23S rRNA gene. Moreover, 147 C. trachomatis positive urinary samples from routine test were also analysed with the PCR assay to detect M. genitalium. 72 out of 601 samples were detected positive for C. trachomatis (12%), five samples (0.83%) positive for N. gonorrhoeae and 25 samples positive for M. genitalium (4.2%). 14 of the 25 M. genitalium samples were detected having 23S rRNA gene mutations associated with macrolide resistance (56%). 25 of 147 C. trachomatis positive samples were tested positive for M. genitalium (17%) and two of them were positive for M. genitalium and N. gonorrhoeae (1.4%). The high prevalence of M. genitalium and macrolide resistance-associated mutation and the coinfection with C. trachomatis in the region suggesting that M. genitalium testing should be included in routine sexually transmitted infection screening.

Prevalence and risk factors of Mycoplasma genitalium infection in patients attending a sexually transmitted infection clinic in Reunion Island: a cross-sectional study (2017-2018).

BACKGROUND: Mycoplasma genitalium (MG) is an emerging sexually transmitted infection (STI) for whose management remains controversial. We aimed to assess the prevalence and risk factors of MG infection in patients attending an STI clinic in Reunion Island. METHODS: Between January 2017 and December 2018, all patients attending the Saint-Pierre STI clinic in Reunion Island were screened for MG, Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG). Urogenital, pharyngeal and/or anal samples were collected based on sexual behaviour and analysed by triplex PCR. Risk factors were identified using a Poisson regression for binary outcome. RESULTS: Among 2069 screened subjects, the overall prevalence of MG was 4.88% [95% Confidence Interval (CI) 3.98-5.93]. The prevalence of urogenital MG was 4.38%, with women being more affected than men (5.33% vs 3.22%, prevalence ratio (PR) 1.66, p = 0.02). The prevalence of anal MG was 3.06% and that of pharyngeal MG was 0.61%, with men being more affected in both cases. Infection with MG was independently associated with multiple partners (6-10 partners: adjusted prevalence ratio-aPR 2.55, p < 0.048; > 10 partners: aPR 4.33, p < 0.004), previous history of STI (aPR 1.89, p = 0.026), non-use of condoms (aPR 2.56, p < 0.003) and co-infection with CT (aPR 2.56, p < 0.017). CONCLUSION: Compared to other countries, the prevalence of MG is high in Reunion Island, especially in women aged under 25 years, and co-infection with CT is common. Routine MG screening and treatment should be performed in at-risk women and co-infection with MG should be considered when deciding on treatment for CT, particularly in regions where azithromycin is still in use.

Are Urogenital Symptoms Caused by Sexually Transmitted Infections and Colonizing Bacteria?

OBJECTIVE: This study aimed to investigate the prevalence of sexually transmitted infections (STIs) and colonizing bacteria in relation to urogenital symptoms. MATERIALS AND METHODS: In this cross-sectional study, patients visiting the STI clinic at Umeå University Hospital were asked for symptoms and condom use. Samples from 759 patients (465 male and 294 female) were analyzed for 4 STIs (Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, and Mycoplasma genitalium) and 3 colonizing bacteria (Mycoplasma hominis, Ureaplasma parvum, and Ureaplasma urealyticum). RESULTS: Chlamydia trachomatis prevalence was 11% among women and 9.5% among men. Neisseria gonorrhoeae prevalence was 0.7% among women and 0.9% among men. Mycoplasma genitalium was found in 11% and 5.6% of women and men, respectively. Asymptomatic men and women had similar distribution patterns of microorganisms as those with urogenital symptoms, with the exceptions of Neisseria gonorrhoeae- and Mycoplasma genitalium-infected men who declared symptoms more frequently. Of 158 men with urogenital symptoms, 55% were test-negative. Of 129 women with urogenital symptoms, 12% were test-negative. CONCLUSIONS: This study reveals a complex picture, where a large number of multi-positive tests made it complicated to correlate urogenital symptoms with microorganisms. A high number of test-negative but symptomatic patients indicate a need of searching for additional pathogens.

Comparison between Abbott m2000 RealTime and Alinity m STI systems for detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and Mycoplasma genitalium.

The new Abbott Alinity m STI Assay was compared with Abbott m2000 RealTime PCR. For Chlamydia trachomatis, 26 (7.5%) of 347 samples were positive in the Alinity assay and 24 (6.9%) in the m2000 assay. Corresponding figures for Neisseria gonorrhoeae were 23 (6.6%) and 17 (4.9%). For Mycoplasma genitalium, 22 (7.9%) of 279 samples were positive in the Alinity assay and 18 (6.5%) in the m2000 assay, for which DNA extraction was performed on an m2000sp instrument combined with in-house real-time PCR. The Alinity assay has at least the same sensitivity as the m2000 assay. The specificity was evaluated by discrepancy analysis.

Detection of parC gene mutations associated with quinolone resistance in Mycoplasma genitalium: evaluation of a multiplex real-time PCR assay.

Introduction. Increasing levels of antibiotic resistance are complicating treatment for the sexually transmitted pathogen Mycoplasma genitalium. Resistance to fluoroquinolones is associated with mutations in the parC gene. Although the precise mutations conferring resistance are not fully understood, the single nucleotide polymorphism (SNP) G248T/S83I is most implicated.Aim. To evaluate the performance of the MG+parC(beta2) assay (SpeeDx, Australia), which detects single nucleotide polymorphisms (SNPs) in the parC gene at amino acid position S83 (A247C/S83R, G248T/S83I, G248A/S83N) and D87 (G259A/D87N, G259T/D87Y, G259C/D87H).Methods. Clinical samples were analysed by MG+parC(beta2) assay and results compared to Sanger sequencing. Sensitivity, specificity, and predictive value for treatment failure were calculated.Results. From analysis of 205 samples, the MG+parC(beta2) assay performed with a high sensitivity 98.2% (95% CI:90.3-100) and specificity 99.3% (95% CI:96.3-100) for parC SNP detection with a kappa of 0.97 (95% CI:0.94-1.00). The predictive value of G248T/S83I detection (the most common SNP, prevalence of 13% in the study population) was analysed with respect to treatment failure (patients received sequential doxycycline-moxifloxacin). The positive-predictive-value for moxifloxacin failure after detection of S83I was only 44% (95% CI:24.4-65.1), while negative-predictive-value was high at 96.9% (95% CI:92.7-99.0), suggesting that other SNPs are contributing to resistance.Conclusion. MG+parC(beta2) performed with high concordance compared to Sanger sequencing. Such qPCR assays can assist in understanding causes of treatment failure, inform the development of diagnostic assays, and can be applied to surveillance of mutations in populations. Due to an incomplete understanding of the basis for fluoroquinolone resistance, such tests do not appear to be ready for clinical application.

Evaluation of 5 commercial assays for the detection of Mycoplasma genitalium and other Urogenital Mycoplasmas.

The focus on urogenital mycoplasmas as the possible etiologic agents of urogenital infections and syndromes, has increased in the last decade. Of these, Mycoplasma genitalium is proven to be pathogenic and sexually transmitted. We compared five commercially available assays for the detection of these organisms in urogenital mycoplasma culture specimen remnants. Stored specimen remnants were tested on Aptima Mycoplasma genitalium, Allplex™ STI Essential and CGMT, ResitancePlus®MG and Allplex™ MG & AziR Assays. All positive M. genitalium specimens and culture negative, nucleic acid positive Ureaplasmas were sent to the National Microbiology Laboratory for confirmation. The Aptima Mycoplasma genitalium assay detected 7 M. genitalium infections, the Allplex™ STI-EA and the Allplex™ CGMT detected 6 M. genitalium positives, and the Allplex™MG and AziR and SpeeDx ResistancePlus® MG detected 5 M. genitalium positives, four with macrolide resistant genes. The Allplex™ STI Essential assay was 100% sensitive and specific for Mycoplasma hominis and Ureaplasma targets. As seen in other studies, the Aptima Mycoplasma genitalium assay was 100% sensitive and specific for the detection of M. genitalium. The multiplex assays had lower sensitivities for M. genitalium detection (Allplex™ STI Essential and CGMT sensitivity of 85.71%; Allplex™ MG & AziR and SpeeDx ResistancePlus® MG sensitivity of 71.43%) with high specificities of 100%. Assays tested have high sensitivities and specificities for the detection of urogenital mycoplasmas especially M. genitalium macrolide resistance markers. All labs wanting to perform onsite detection of these organisms will find an assay to easily fit into their workflow.

Prevalence of urogenital, anal, and pharyngeal infections with Chlamydia trachomatis, Neisseria gonorrhoeae, and Mycoplasma genitalium: a cross-sectional study in Reunion island.

BACKGROUND: Recommendations for sexually transmitted infection (STI) screening vary significantly across countries. This study evaluated the prevalence of urogenital and extragenital infections with Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Mycoplasma genitalium (MG) in patients visiting a French STI clinic in the Indian Ocean region to determine whether current STI screening practices should be updated. METHODS: This cross-sectional study examined all patients who visited the STI clinic between 2014 and 2015. Triplex polymerase chain reaction screening for CT, NG, and MG was performed on urine, vaginal, pharyngeal, and anal specimens (FTD Urethritis Basic Kit, Fast Track Diagnostics, Luxembourg). RESULTS: Of the 851 patients enrolled in the study, 367 were women (367/851, 43.2%) and 484 were men (484/851, 56.0%). Overall, 826 urogenital specimens (826/851, 97.1%), 606 pharyngeal specimens (606/851, 71.2%), and 127 anal specimens (127/851, 14.9%) were taken from enrolled patients. The prevalence of urogenital CT and MG was high in women ≤25 years (19/186, 10.21%; 5/186, 2.69%) and in men who have sex with women ≤30 years (16/212, 7.54%; 5/212, 2.36%). Among patients with urogenital CT infection, 13.7% (7/51) had urethritis. All patients with urogenital MG infection were asymptomatic. Men who have sex with men had a high prevalence of pharyngeal CT (2/45, 4.44%) and NG (3/44, 6.81%) and a high prevalence of anal CT (2/27, 7.41%), NG (2/27, 7.40%), and MG (1/27, 3.70%). After excluding patients with concomitant urogenital infection, extragenital infections with at least 1 of the 3 pathogens were found in 20 swabs (20/91, 21.9%) taken from 16 patients (16/81, 19.7%), all of them asymptomatic. CONCLUSIONS: Routine multisite screening for CT, NG, and MG should be performed to mitigate the transmission of STIs in high-risk sexually active populations.

Comparison between Aptima® assays (Hologic) and the CoBAS® 6800 system (Roche) for the diagnosis of sexually transmitted infections caused by Chlamydia trachomatis, Neisseria gonorrhoeae, and Mycoplasma genitalium.

Nowadays, it is of utmost importance to use fully validated assays for molecular-based diagnosis. In the field of sexually transmitted disease (STD), Roche and Hologic provide assays for diagnosing Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Mycoplasma genitalium (MG), and Trichomonas vaginalis (TV). A total of 212 clinical samples were tested. Aptima® Combo 2 (detecting CT and NG), Aptima® M. genitalium and the Aptima® T. vaginalis on the Panther® system were compared to CoBAS® CT/NG and CoBAS® TV/MG running on the CoBAS® 6800 system. To solve the discrepancies, Allplex™ STI Essential assay (Seegene®) and/or Sanger DNA sequencing were used. The diagnostic performance was calculated by mean of the sensitivity and specificity parameters. Aptima® (sensitivity: 98.90%, specificity: 100%), CoBAS® (sensitivity 100%, specificity: 96.67%). The CoBAS® combo (CT/NG) failed detecting NG from an anal/rectum specimen, which is not included into the validated specimens of the assay. Aptima® combo 2 produced two false positives (CT and NG), not detected by the third tests. All the assays showed an optimal diagnostic capacity, meeting the requirements for IVD DNA-based assays. All products work optimally on automatic platforms, minimizing time and risk of contamination during handling.

The Associations of Genital Mycoplasmas with Female Infertility and Adverse Pregnancy Outcomes: a Systematic Review and Meta-analysis.

The roles of genital mycoplasmas including Mycoplasma genitalium (M. genitalium), Mycoplasma hominis (M. hominis), Ureaplasma urealyticum (U. urealyticum), and Ureaplasma parvum (U. parvum) in reproductive diseases are equivocal. To investigate whether genital mycoplasmas are risk factors of female infertility and adverse pregnancy outcomes, we performed a systematic review and meta-analysis. Electronic databases were searched for related studies. A random-effects model or fixed-effects model was employed to generate forest plots. Pooled odd ratios (ORs) with 95% confidence intervals (CIs) were applied to measure the strength of associations. Meanwhile, heterogeneity was evaluated by H statistic and I2 statistic, and publication bias was explored by funnel plots based on Egger's test and Begg's test. The search yielded 2054 relevant records, and 35 articles were ultimately included for meta-analysis. M. genitalium was a significant risk factor for female infertility (OR, 13.03 [95% CI, 3.46-48.98]) and preterm birth (PTB) (OR, 1.81 [95% CI, 1.17-2.80]), but not for spontaneous abortion (SA) (OR, 0.58 [95% CI, 0.25-1.35]). M. hominis can significantly increase the potential risk of female infertility (OR, 1.56 [95% CI, 1.02-2.38]), SA (OR, 9.14 [95% CI, 4.14-20.18]), stillbirth (OR, 3.98 [95% CI, 1.39-11.36]), and premature rupture of membranes (PROM) (OR, 1.79 [95% CI, 1.26-2.55]), but was not associated with PTB (OR, 1.29 [95% CI, 0.78-2.15]). U. urealyticum had no significant risk effect on female infertility (OR, 0.68 [95% CI, 0.42-1.11]). Coinfections of M. hominis and Ureaplasma were significantly associated with female infertility, SA, and stillbirth, but not with PROM. On the basis of current evidences, this meta-analysis supports that M. genitalium is a risk factor for female infertility and PTB; M. hominis is a potential risk factor for female infertility, SA, stillbirth, and PROM; U. urealyticum has no significant association with female infertility; and the relationship of U. parvum with female infertility and adverse pregnancy outcomes needs to be paid more attention to and remains to be further revealed.

Mycoplasma genitalium coinfection in men with symptomatic gonococcal urethritis.

OBJECTIVES: International guidelines recommend Mycoplasma genitalium testing, preferably using an assay to detect macrolide resistance-associated mutations, for men presenting with non-gonococcal urethritis, but there is no specific guidance on such testing for men with gonococcal urethritis. METHODS: This study aimed to estimate the proportion of men with gonococcal urethritis who have coinfection with M. genitalium through a retrospective analysis of cases of symptomatic urethral gonorrhoea at Western Sydney Sexual Health Centre in 2017 and 2018. RESULTS: Fourteen of 184 (7.6%, 95% CI 3.7 to 11.5) men with gonococcal urethritis had M. genitalium detected in the urine at the time of presentation. No demographic or behavioural factors predicted M. genitalium coinfection. Coinfection with urethral Chlamydia trachomatis was detected in 29 of 184 (15.8%, 95% CI 10.5 to 21.1). All five men with macrolide-resistant M. genitalium detected returned for treatment with moxifloxacin at a median of 8 days (range 5-16 days) after presentation and treatment of gonorrhoea; three of five were documented to remain symptomatic at this visit. CONCLUSION: Although M. genitalium coinfection is less common than chlamydia among men with symptomatic gonococcal urethritis, M. genitalium testing, using an assay to detect macrolide resistance, will potentially reduce symptom duration particularly for men with macrolide-resistant infections, but may not be justifiable on cost-benefit analysis.

Prevalence of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium and Trichomonas vaginalis including relevant resistance-associated mutations in a single center in the Netherlands.

PURPOSE: In this study, we report the prevalence of Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Mycoplasma genitalium (MG) and Trichomonas vaginalis (TV) amongst clinical specimens of patients suspected for sexually transmitted infections received at our laboratory and in addition report the prevalence of resistance-associated mutations (RAM) for ciprofloxacin in NG and azithromycin and moxifloxacin in MG. METHODS: All specimens received from December 2018 to May 2019 were tested for the four pathogens. In addition, the presence of RAM associated with resistance to ciprofloxacin in NG and to azithromycin and moxifloxacin in MG was determined by different real-time PCR assays on all NG- and MG-positive specimens. RESULTS: CT was detected most often (267/2613, 10.2%), followed by MG (106/2592, 4.1%), NG (41/2613, 1.6%) and TV (10/2592, 0.4%) amongst all specimens. The prevalence of ciprofloxacin RAM in NG was 21.2%, and the prevalence of RAM in MG was 40.6% for azithromycin and 8.1% for moxifloxacin. Nearly all specimens containing moxifloxacin-resistant MG also contained azithromycin-resistant MG. CONCLUSION: CT is found most often in our population followed by MG and NG. By using molecular assays to detect RAM supplementary to pathogen identification of NG and MG, optimal therapy can be advised.

Molecular epidemiological analysis of Mycoplasma genitalium shows low prevalence of azithromycin resistance and a well-established epidemic in South Africa.

OBJECTIVES: Macrolide resistance in Mycoplasma genitalium is emerging globally. There is paucity of data from sub-Saharan Africa where syndromic management is used to treat sexually transmitted infections (STIs). We conducted a molecular epidemiological study to determine the prevalence of azithromycin resistance and epidemic diversity of M. genitalium infections in South Africa. METHODS: We analysed 90 M. genitalium-positive specimens that had been collected consecutively from men and women (50% symptomatic) from geographically diverse communities across the northern part of South Africa between 2015 and 2019. Melting curve analysis followed by targeted sequencing of the 23S rRNA gene was performed to detect azithromycin resistance. Molecular typing was done through single nucleotide polymorphism (SNP) analysis of the MG191 gene and short tandem repeats (STR) assessment of the MG309 gene. An overview of all published M. genitalium sequence types was generated and novel sequence types identified in this study were allocated numbers accordingly. RESULTS: Azithromycin resistance was detected in 1/90 M. genitalium-positive specimens (1.1%; 95% CI 0% to 3.3%) as conferred by A2071G mutation; this strain also harboured a C234T mutation in the parC gene with wild type gyrA gene. SNP typing and STR assessment was successful in 38/90 specimens (42%) and showed a genetically diverse epidemic, without geographic clustering, with eight novel sequence types identified. CONCLUSION: This is the first study that determines resistance in M. genitalium infection since introduction of azithromycin in the syndromic management regimen for STIs in South Africa in 2015. Despite a well-established epidemic, azithromycin-resistant M. genitalium infection is still uncommon in the public healthcare sector. However, it has the potential to undermine the effectiveness of syndromic management. Introduction of molecular diagnostics and continuous surveillance are warranted for early detection emergence of resistance.

Homelessness, sex and a tale of two sexually transmitted infections.

The Young People's Health Service (YPHS) is a free, nurse-led Primary Health Care Clinic, in Melbourne, for young people aged 12-24 who are experiencing homelessness. Sexually transmitted infection (STI) screening is routinely offered as part of comprehensive psychosocial assessments. We wanted to determine the number of people positive for Chlamydia trachomatis (Ct) and Mycoplasma genitalium (Mg), amongst this asymptomatic high-risk population. We also wanted to review our screening practice. All asymptomatic sexually active clients seen by YPHS between 2014 and 2016 were offered a first pass urine polymerase chain reaction-based test for Ct and Mg. Urine samples were taken for men and women. Positivity for Ct and Mg out of those tested was determined and association with gender examined. Between 2014-2016, 272 males and 278 females (n = 550) were screened for Ct, and 72 infections were detected (13.1%. Chlamydia positivity did not differ between males (n = 35; 12.9%, 95% confidence interval [CI]: 8.8-16.8) and females (n = 37; 13.3%, 95%CI: 9.3-17.3). Over the same period 273 males and 284 females were screened for Mg (n = 557) and 55 infections were detected (9.9%). A higher proportion of females (n = 35; 12.3%, 95%CI: 8.5-16.1) tested positive compared to males (n = 20; 7.3%, 95%CI: 4.2-10.4), p = 0.048. Our study demonstrates both Ct and Mg are prevalent in the population, Mg being more common in young women than young men. Referral for specialist care for macrolide-resistant Mg increased and the updated Australian STI management guidelines led to a review of practice.

Evaluation of ResistancePlus MG FleXible, a 'near-patient' test for the detection of Mycoplasma genitalium and macrolide resistance mutations, using freshly collected clinical samples.

Introduction. Mycoplasma genitalium is a sexually transmitted pathogen with increasing resistance to first- and second-line antimicrobials. The 'near-patient test' ResistancePlus MG FleXible (SpeeDx) detects M. genitalium plus four macrolide resistance mutations (MRMs), facilitating same-day patient follow up.Hypothesis/Gap Statement. This assay has not been assessed on freshly collected samples.Aim. Our goal was to evaluate the performance of the ResistancePlus MG FleXible test against the standard of care open platform test.Methods. ResistancePlus MG FleXible (analysed on the Cepheid GeneXpert platform) was evaluated on freshly collected samples and compared to the standard of care open platform test ResistancePlus MG (SpeeDx) analysed on the LightCycler 480 II (Roche).Results. For 270 valid tests, ResistancePlus MG FleXible yielded a high positive per cent agreement (PPA) of 94.1% [96/102; 95 % confidence interval (CI): 87.6-97.8 %] and negative per cent agreement (NPA) of 95.2% (160/168; 95 % CI: 90.8-97.9%) for M. genitalium detection compared to the reference assay (kappa for test concordance of 0.89; 95 % CI: 0.83-0.95). Performance was similar across different sample types. For the detection of MRMs, ResistancePlus MG FleXible had a PPA of 97.1% (66/68; 95% CI: 89.8-99.6) and NPA of 78.6% (22/28; 95 % CI: 59.0-91.7), with test comparison kappa of 0.79 (95 % CI: 0.65-0.93). Notably, of six discordant results (i.e. determined to be wild type by the reference assay), five were positive for MRMs by Sanger sequencing, indicating that the ResistancePlus MG FleXible assay has an improved performance for mutation detection.Conclusion. ResistancePlus MG FleXible had comparable test performance for M. genitalium detection as the open platform assay, with improved detection of MRMs. The ResistancePlus MG FleXible 'near-patient' assay can deliver a rapid result to expedite appropriate treatment.

Macrolide and fluoroquinolone associated mutations in Mycoplasma genitalium in a retrospective study of male and female patients seeking care at a STI Clinic in Guangzhou, China, 2016-2018.

BACKGROUND: Antimicrobial resistance in M. genitalium is a growing clinical problem. We investigated the mutations associated with macrolide and fluoroquinolone resistance, two commonly used medical regimens for treatment in China. Our aim is to analyze the prevalence and diversity of mutations among M. genitalium-positive clinical specimens in Guangzhou, south China. METHODS: A total of 154 stored M. genitalium positive specimens from men and women attending a STI clinic were tested for macrolide and fluoroquinolone mutations. M. genitalium was detected via TaqMan MGB real-time PCR. Mutations associated with macrolide resistance were detected using primers targeting region V of the 23S rRNA gene. Fluoroquinolone resistant mutations were screened via primers targeting topoisomerase IV (parC) and DNA gyrase (gyrA). RESULTS: 98.7% (152/154), 95.5% (147/154) and 90.3% (139/154) of M. genitalium positive samples produced sufficient amplicon for detecting resistance mutations in 23S rRNA, gyrA and parC genes, respectively. 66.4% (101/152), 0.7% (1/147) and 77.7% (108/139) samples manifested mutations in 23S rRNA, gyrA and parC genes, respectively. A2072G (59/101, 58.4%) and S83I (79/108, 73.1%) were highly predominating in 23S rRNA and parC genes, respectively. Two samples had amino acid substitutions in gyrA (M95I and A96T, respectively). Two samples had two amino acid substitutions in parC (S83I + D87Y). 48.6% (67/138) of samples harbored both macrolide and fluoroquinolone resistance-associated mutations. The most common combination of mutations was A2072G (23S rRNA) and S83I (parC) (40/67, 59.7%). One sample had three amino acid changes in 23S rRNA, gyrA and parC genes (A2072G + A96T + S83I). CONCLUSIONS: The high antimicrobial resistance rate of M. genitalium in Guangzhou is a very worrying problem and suggests that antimicrobial resistance testing and the development of new antibiotic regimens are crucially needed.