Marcus Pond

High Prevalence of Antibiotic-Resistant Mycoplasma genitalium in Nongonococcal Urethritis: The Need for Routine Testing and the Inadequacy of Current Treatment Options

Mycoplasma genitalium infections were as frequent a cause of nongonococcal urethritis as Chlamydia trachomatis, had high rates of macrolide-associated genotypic resistance, and were nonclonal, suggesting an established community infection. Detection of genotypic resistance to fluoroquinolones is cause for concern.

Clinical Application of Whole-Genome Sequencing To Inform Treatment for Multidrug-Resistant Tuberculosis Cases

ABSTRACT The treatment of drug-resistant tuberculosis cases is challenging, as drug options are limited, and the existing diagnostics are inadequate. Whole-genome sequencing (WGS) has been used in a clinical setting to investigate six cases of suspected extensively drug-resistant Mycobacterium tuberculosis (XDR-TB) encountered at a London teaching hospital between 2008 and 2014. Sixteen isolates from six suspected XDR-TB cases were sequenced; five cases were analyzed in a clinically relevant time frame, with one case sequenced retrospectively. WGS identified mutations in the M. tuberculosis genes associated with antibiotic resistance that are likely to be responsible for the phenotypic resistance. Thus, an evidence base was developed to inform the clinical decisions made around antibiotic treatment over prolonged periods. All strains in this study belonged to the East Asian (Beijing) lineage, and the strain relatedness was consistent with the expectations from the case histories, confirming one contact transmission event. We demonstrate that WGS data can be produced in a clinically relevant time scale some weeks before drug sensitivity testing (DST) data are available, and they actively help clinical decision-making through the assessment of whether an isolate (i) has a particular resistance mutation where there are absent or contradictory DST results, (ii) has no further resistance markers and therefore is unlikely to be XDR, or (iii) is identical to an isolate of known resistance (i.e., a likely transmission event). A small number of discrepancies between the genotypic predictions and phenotypic DST results are discussed in the wider context of the interpretation and reporting of WGS results.

Accurate detection of Neisseria gonorrhoeae ciprofloxacin susceptibility directly from genital and extragenital clinical samples: towards genotype-guided antimicrobial therapy

Introduction Increasing use of nucleic acid amplification tests (NAATs) as the primary means of diagnosing gonococcal infection has resulted in diminished availability of Neisseria gonorrhoeae antimicrobial susceptibility data. We conducted a prospective diagnostic assessment of a real-time PCR assay (NGSNP) enabling direct detection of gonococcal ciprofloxacin susceptibility from a range of clinical sample types. Methods NGSNP, designed to discriminate an SNP associated with ciprofloxacin resistance within the N. gonorrhoeae genome, was validated using a characterized panel of geographically diverse isolates (n = 90) and evaluated to predict ciprofloxacin susceptibility directly on N. gonorrhoeae-positive NAAT lysates derived from genital (n = 174) and non-genital (n = 116) samples (n = 290), from 222 culture-confirmed clinical episodes of gonococcal infection. Results NGSNP correctly genotyped all phenotypically susceptible (n = 49) and resistant (n = 41) panel isolates. Ciprofloxacin-resistant N. gonorrhoeae was responsible for infection in 29.7% (n = 66) of clinical episodes evaluated. Compared with phenotypic susceptibility testing, NGSNP demonstrated sensitivity and specificity of 95.8% (95% CI 91.5%–98.3%) and 100% (95% CI 94.7%–100%), respectively, for detecting ciprofloxacin-susceptible N. gonorrhoeae, with a positive predictive value of 100% (95% CI 97.7%–100%). Applied to urogenital (n = 164), rectal (n = 40) and pharyngeal samples alone (n = 30), positive predictive values were 100% (95% CI 96.8%–100%), 100% (95% CI 87.2%–100%) and 100% (95% CI 82.4%–100%), respectively. Conclusions Genotypic prediction of N. gonorrhoeae ciprofloxacin susceptibility directly from clinical samples was highly accurate and, in the absence of culture, will facilitate use of tailored therapy for gonococcal infection, sparing use of current empirical treatment regimens and enhancing acquisition of susceptibility data for surveillance.

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

Objectives Gram-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. Methods Receiver 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. Results An 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). Conclusions AUFC 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.

Impact of deploying multiple point-of-care tests with a ‘sample first’ approach on a sexual health clinical care pathway. A service evaluation

Objectives To assess clinical service value of STI point-of-care test (POCT) use in a ‘sample first’ clinical pathway (patients providing samples on arrival at clinic, before clinician consultation). Specific outcomes were: patient acceptability; whether a rapid nucleic acid amplification test (NAAT) for Chlamydia trachomatis/Neisseria gonorrhoeae (CT/NG) could be used as a POCT in practice; feasibility of non-NAAT POCT implementation for Trichomonas vaginalis (TV) and bacterial vaginosis (BV); impact on patient diagnosis and treatment. Methods Service evaluation in a south London sexual health clinic. Symptomatic female and male patients and sexual contacts of CT/NG-positive individuals provided samples for diagnostic testing on clinic arrival, prior to clinical consultation. Tests included routine culture and microscopy; CT/NG (GeneXpert) NAAT; non-NAAT POCTs for TV and BV. Results All 70 (35 males, 35 females) patients approached participated. The ‘sample first’ pathway was acceptable, with >90% reporting they were happy to give samples on arrival and receive results in the same visit. Non-NAAT POCT results were available for all patients prior to leaving clinic; rapid CT/NG results were available for only 21.4% (15/70; 5 males, 10 females) of patients prior to leaving clinic. Known negative CT/NG results led to two females avoiding presumptive treatment, and one male receiving treatment directed at possible Mycoplasma genitalium infection causing non-gonococcal urethritis. Non-NAAT POCTs detected more positives than routine microscopy (TV 3 vs 2; BV 24 vs 7), resulting in more patients receiving treatment. Conclusions A ‘sample first’ clinical pathway to enable multiple POCT use was acceptable to patients and feasible in a busy sexual health clinic, but rapid CT/NG processing time was too long to enable POCT use. There is need for further development to improve test processing times to enable POC use of rapid NAATs.

Tools for Detection of Mycoplasma amphoriforme: a Primary Respiratory Pathogen?

ABSTRACT Mycoplasma amphoriforme is a recently described organism isolated from the respiratory tracts of patients with immunodeficiency and evidence of chronic infection. Novel assays for the molecular detection of the organism by real-time quantitative PCRs (qPCRs) targeting the uracil DNA glycosylase gene (udg) or the 23S rRNA gene are described here. The analytical sensitivities are similar to the existing conventional M. amphoriforme 16S rRNA gene PCR, with the advantage of being species specific, rapid, and quantitative. By using these techniques, we demonstrate the presence of this organism in 17 (19.3%) primary antibody-deficient (PAD) patients, 4 (5%) adults with lower respiratory tract infection, 1 (2.6%) sputum sample from a patient attending a chest clinic, and 23 (0.21%) samples submitted for viral diagnosis of respiratory infection, but not in normal adult control subjects. These data show the presence of this microorganism in respiratory patients and suggest that M. amphoriforme may infect both immunocompetent and immunocompromised people. Further studies to characterize this organism are required, and this report provides the tools that may be used by other research groups to investigate its pathogenic potential.

Chlamydia testing: Reaching high-risk sexually active young people in the community

Falasinnu et al. found strong evidence highlighting the importance of age, number of sexual partners and ethnicity in identifying at-risk populations for chlamydia and gonorrhoea. We aimed to explore if offering on the spot, rapid sexually transmitted infections (STI) tests might be an acceptable way of accessing sexually experienced people aged <25. In September 2014, as part of improving clinical outreach in the community, we conducted the first UK one-day service evaluation pilot of the Cepheid GeneXpert rapidChlamydia trachomatis/Neisseria gonorrhoeae testing system in an inner London further education college. Consecutive students in public areas were invited to provide urine samples for ‘on-the-spot’ chlamydia/gonorrhoea testing, and to give telephone feedback in the next two weeks. The mean age of 52 eligible students was 19 years (range 16 to 24), 65% were female and 37% were smokers. Reported ethnicity was AfroCaribbean 67%, white 22% or other 11%. Mean number of sexual partners in the last 12 months was three (range 0–18), with most students (64%) never having been tested for STIs. Of 39 participants with a new sexual partner in the last six months only seven had been tested for chlamydia during this period. Seven urine samples (13%, 95% confidence interval 4% to 22%) were positive: six for chlamydia and one for gonorrhoea. Negative results were sent by text in a mean time of 2 h 12min after providing the sample (range 1 h 30min–5 hr 50min). Students with infections were telephoned and given advice about obtaining treatment. Six were given their result in a mean time of 3 h 18min (range 1 h 40min–5 h 40min). The final student was contacted 55 h later. Five students were confirmed treated by two weeks of whom four had notified partners. None of the seven infected students had planned to get tested. A total of 42 students (81%) provided feedback: all were happy to be tested and liked the rapid results. Comments included: ‘it is good to be safe’, ‘helps people – makes them aware’, ‘easy’, ‘reliable’, ‘less technical’, ‘in college so right there’, ‘good so you don’t get worried a lot’, ‘sooner you know the better’, ‘other clinics should do that’, ‘couldn’t believe I got the result the same day’. As this was the first STI test for most students, this level of engagement is encouraging. They also suggested advertising testing; making it less obvious the testing was for STIs; testing for a range of infections including HIV and providing same day treatment. We provided same day results to a group shown by Falasinnu et al. to be at high risk of STIs. Most students would not otherwise have been tested, including all those who had positive results. Our pilot could inform commissioning of future community-based service delivery of chlamydia/gonorrhoea testing in this high-risk group.

P1.32 Hand-held rapid whole genome nanopore sequencing to predict neisseria gonorrhoeae antibiotic susceptibility: steps towards clinic based tailored antimicrobial therapy

Introduction Next generation sequencing can accurately predict antibiotic susceptibility in Neisseria gonorrhoeae (NG) allowing preservation of first-line treatments in the face of widespread antimicrobial resistance (AMR). The rapid nature of novel hand-held nanopore sequencing (NPS) gives promise for utility at the point of care. We evaluated time to result post DNA extraction, and accuracy of MinION (Oxford Nanopore Technologies) NPS to predict phenotypic antimicrobial susceptibility (PAMS) of NG to ciprofloxacin and azithromycin. Methods One-directional (1-D) NPS using bar-coded DNA library preparations from 48 NG isolates, prospectively collected from a London clinic, were run on NPS flow cells (3 per R9.0 flow cell) and illumina MiSeq as a comparator. NPS raw sequences were transferred to the cloud for base-calling, alignment, and variant calling using standard tools. Results Mean time for 1-D library preparation was roughly 1 hour; NPS and alignment took <40 min per sample with single nucleotide polymorphism (SNP) calling adding little extra time. NPS genome coverage was >30X per isolate. Of 48 samples, PAMS to ciprofloxacin, and azithromycin was 74% and 87% respectively. Accuracy of NPS-based genotypic susceptibility, defined as absence of any known AMR-associated SNP’s, to predict PAMS for ciprofloxacin and azithromycin, was 34/34 (100%; 95% CI 89.8%–100%) and 35/40 (87.5%; 95% CI 73.9%–94.5%) respectively. Accuracies improved significantly for azithromycin when only high quality reads were included, and with Illumina sequencing. 30 of the 34 isolates susceptible to azithromycin were also susceptible to ciprofloxacin, and 3 of 6 isolates resistant to azithromycin were also resistant to Ciprofloxacin. Conclusion NPS accurately predicted ciprofloxacin PAMS but was less accurate for azithromycin. With new iterations of the technology, imminent rapid barcoded library preparation (10 min) and rapid DNA extraction from clinical samples, NPS may allow accurate ceftriaxone-adjunctive treatment combinations, for a substantial proportion of patients.

Diagnostic accuracy of a prototype rapid chlamydia and gonorrhoea recombinase polymerase amplification assay: a multicentre cross-sectional preclinical evaluation

Objectives Rapid and accurate sexually transmitted infection diagnosis can reduce onward transmission and improve treatment efficacy. We evaluated the accuracy of a 15-minute run-time recombinase polymerase amplification–based prototype point-of-care test (TwistDx) for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG). Methods Prospective, multicentre study of symptomatic and asymptomatic patients attending three English sexual health clinics. Research samples provided were additional self-collected vulvovaginal swab (SCVS) (female participants) and first-catch urine (FCU) aliquot (female and male participants). Samples were processed blind to the comparator (routine clinic CT/NG nucleic acid amplification test (NAAT)) results. Discrepancies were resolved using Cepheid CT/NG GeneXpert. Results Both recombinase polymerase amplification and routine clinic NAAT results were available for 392 male and 395 female participants. CT positivity was 8.9% (35/392) (male FCU), 7.3% (29/395) (female FCU) and 7.1% (28/395) (SCVS). Corresponding NG positivity was 3.1% (12/392), 0.8% (3/395) and 0.8% (3/395). Specificity and positive predictive values were 100% for all sample types and both organisms, except male CT FCU (99.7% specificity (95% confidence interval (CI) 98.4–100.0; 356/357), 97.1% positive predictive value (95% CI 84.7–99.9; 33/34)). For CT, sensitivity was ≥94.3% for FCU and SCVS. CT sensitivity for female FCU was higher (100%; 95% CI, 88.1–100; 29/29) than for SCVS (96.4%; 95% CI, 81.7–99.9; 27/28). NG sensitivity and negative predictive values were 100% in FCU (male and female). Conclusions This prototype test has excellent performance characteristics, comparable to currently used NAATs, and fulfils several World Health Organization ASSURED criteria. Its rapidity without loss of performance suggests that once further developed and commercialized, this test could positively affect clinical practice and public health.

P015 Receiving 1g azithromycin as part of mass drug administration (mda) for the control of trachoma is associated with reduced genital mycoplasma genitaliumprevalence

Introduction Mass Drug Administration (MDA) with 1g oral azithromycin for ocular Chlamydia trachomatis (CT) infection, a key component of trachoma control, can concomitantly reduce genital CT prevalence. However, this dose is known to be sub-optimal for the treatment of genital Mycoplasma genitalium (MG) infection. Here we investigate factors associated with MG infection in pre- and post-MDA sample sets. Methods Pre-MDA (T1) and 6 months post-MDA (T2) CT-negative self-collected vulvo-vaginal swabs from women attending three outpatient antenatal clinics (Honiara, Solomon Islands), were tested for MG infection using nucleic acid amplification. Logistic regression was used to determine factors associated with infection. Variables tested included: patient age, clinic attended, ethnicity, time spent in education, living in an urban or rural environment, marital status, living with spouse, presence of symptoms associated with a sexually transmitted infection (STI), having an STI in the last 12 months, current CT, Gonorrhoea or Trichomonas vaginalis infection, and at T2 only receipt of MDA dose. Results MG positivity was found in 11.9% (95%CI: 8.3–16.6; 28/236) of women at T1 and in 10.9% (95%CI: 7.7–15.4; 28/256) at T2 (p=0.7467). The only factor associated with having an MG infection was history of not having received MDA with azithromycin at T2 (odds ratio 0.19, 95%CI 0.07–0.53, p=0.001). Discussion Not having MG infection was associated with receiving 1g azithromycin as part of MDA for trachoma control six months previously. However there was no overall drop in population prevalence, indicating individual but not population benefits of MDA with regard to MG infection control.