DNA methylation at individual CpG-sites of EPB41L3, HTERT and FAM19A4 are useful for detection of cervical high-grade squamous intraepithelial lesions (HSIL) or worse: Analysis of individual CpG-sites outperforms averaging.

Global methylation analysis of gene promoters is promising for detection of high-grade squamous intraepithelial lesions or worse (HSIL+) in high-risk human papillomavirus (hrHPV)-positive women. However, diagnostic performance of methylation data at individual CpG-sites is limited. We explored methylation for predicting HSIL+ in self- and clinician-collected samples from Papua New Guinea. Methylation of EPB41L3 (1-6 CpG-sites), hTERT (1-10 CpG-sites) and FAM19A4 (1-5 CpG-sites) was assessed through pyrosequencing from 44 HPV+ samples (4 cancers, 19 HSIL, 4 low-grade squamous intraepithelial lesions (LSIL), 17 normal). New primers were designed for FAM19A4 directed to the first exon region not explored previously. In clinician-collected samples, methylation at CpG-sites 4 and 5 of EPB41L3 were the best HSIL predictors (AUC >0.83) and CpG-site 4 for cancer (0.925). Combination of EPB41L3 sites 2/4 plus FAM19A4 site 1 were the best HSIL+ markers [100% sensitivity, 63.2% specificity]. Methylation at CpG-site 5 of FAM19A4 was the best HSIL predictor (0.67) in self-collected samples, and CpG-sites 1 and 3 of FAM19A4 for cancer (0.77). Combined, FAM19A4 site 1 plus HPV 16/18 detection yielded sensitivity of 82.6% and specificity of 61.9%. In conclusion, methylation at individual CpG-sites of EPB41L3 and FAM19A4 outperformed global analysis and improved HSIL+ detection, warranting further investigation.

Performance of CADM1, MAL and miR124-2 methylation as triage markers for early detection of cervical cancer in self-collected and clinician-collected samples: an exploratory observational study in Papua New Guinea.

OBJECTIVE: WHO recommends human papillomavirus (HPV) testing for cervical screening, with triage of high-risk HPV (hrHPV) positive women. However, there are limitations to effective triage for low-resource, high-burden settings, such as Papua New Guinea. In this exploratory study, we assessed the performance of host methylation as triage tools for predicting high-grade squamous intraepithelial lesions (HSIL) in self-collected and clinician-collected samples. DESIGN: Exploratory observational study. SETTING: Provincial hospital, same-day cervical screen-and-treat trial, Papua New Guinea. PARTICIPANTS: 44 hrHPV+women, with paired self/clinician-collected samples (4 squamous cell carcinomas (SCC), 19 HSIL, 4 low-grade squamous intraepithelial lesions, 17 normal). PRIMARY AND SECONDARY OUTCOME MEASURES: Methylation levels of CADM1, MAL and miR124-2 analysed by methylation-specific PCRs against the clinical endpoint of HSIL or SCC (HSIL+) measured using liquid-based-cytology/p16-Ki67 stain. RESULTS: In clinician-collected samples, MAL and miR124-2 methylation levels were significantly higher with increasing grade of disease (p=0.0046 and p<0.0015, respectively). miR124-2 was the best predictor of HSIL (area under the curve, AUC 0.819) while MAL of SCC (AUC 0.856). In self-collected samples, MAL best predicted HSIL (AUC 0.595) while miR124-2 SCC (AUC 0.812). Combined miR124-2/MAL methylation yielded sensitivity and specificity for HSIL+ of 90.5% (95% CI 69.6% to 98.8%) and 70% (95% CI 45.7% to 88.1%), respectively, in clinician-collected samples, and 81.8% (95% CI 59.7% to 94.8%) and 47.6% (95% CI 25.7% to 70.2%), respectively, in self-collected samples. miR124-2/MAL plus HPV16/HPV18 improved sensitivity for HSIL+ (95.2%, 95% CI 76.2% to 99.9%) but decreased specificity (55.0%, 95% CI 31.5% to 76.9%). CONCLUSION: miR124-2/MAL methylation is a potential triage strategy for the detection of HSIL/SCC in low-income and middle-income country.

Vaginal anaerobes are associated with cervicitis: A case-control study.

OBJECTIVES: Cervicitis is associated with important reproductive sequelae. Primary causes include chlamydia and gonorrhoea, but a known sexually transmitted infection (STI) is not identified in >50% of cases (i.e. STI-negative cervicitis). Bacterial vaginosis (BV) and specific BV-associated bacteria have also been associated with cervicitis, but data are limited. We investigated the association between STI-negative cervicitis and vaginal microbiota composition. METHODS: This was a case-control sub-study of the OhMG study conducted at the Melbourne Sexual Health Centre. Cases were women with cervicitis who tested negative for STIs (STI-negative cervicitis, n = 64). Controls were STI-negative asymptomatic women attending for STI-screening (n = 128). The vaginal microbiota was characterised using 16S rRNA gene sequencing. Vaginal community state types were compared between cases and controls using logistic regression. Differential abundance analysis was performed to identify taxa associated with STI-negative cervicitis. RESULTS: STI-negative cervicitis cases were more likely than controls to have a Lactobacillus-deficient non-optimal microbiota (adjusted-odds-ratio 2.55, 95% CI 1.18-5.50). Compared to controls, cases had increased abundance of four BV-associated bacteria (Gardnerella, Fannyhessea vaginae, Prevotella bivia, Dialister micraerophilus) and decreased abundance of optimal lactobacilli. CONCLUSIONS: We report a positive association between non-optimal vaginal microbiota composition and STI-negative cervicitis. Specific anaerobic BV-associated bacteria may represent infectious causes of cervicitis.

Economic evaluation alongside a clinical trial of near-to-patient testing for sexually transmitted infections.

BACKGROUND: Current clinical care for common bacterial STIs (Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Mycoplasma genitalium (MG)) involves empiric antimicrobial therapy when clients are symptomatic, or if asymptomatic, waiting for laboratory testing and recall if indicated. Near-to-patient testing (NPT) can improve pathogen-specific prescribing and reduce unnecessary or inappropriate antibiotic use in treating sexually transmitted infections (STI) by providing same-day delivery of results and treatment. METHODS: We compared the economic cost of NPT to current clinic practice for managing clients with suspected proctitis, non-gonococcal urethritis (NGU), or as an STI contact, from a health provider's perspective. With a microsimulation of 1000 clients, we calculated the cost per client tested and per STI- and pathogen- detected for each testing strategy. Sensitivity analyses were conducted to assess the robustness of the main outcomes. Costs are reported as Australian dollars (2023). RESULTS: In the standard care arm, cost per client tested for proctitis, NGU in men who have sex with men (MSM) and heterosexual men were the highest at $247.96 (95% Prediction Interval (PI): 246.77-249.15), $204.23 (95% PI: 202.70-205.75) and $195.01 (95% PI: 193.81-196.21) respectively. Comparatively, in the NPT arm, it costs $162.36 (95% PI: 161.43-163.28), $158.39 (95% PI: 157.62-159.15) and $149.17 (95% PI: 148.62-149.73), respectively. Using NPT resulted in cost savings of 34.52%, 22.45% and 23.51%, respectively. Among all the testing strategies, substantial difference in cost per client tested between the standard care arm and the NPT arm was observed for contacts of CT or NG, varying from 27.37% to 35.28%. CONCLUSION: We found that NPT is cost-saving compared with standard clinical care for individuals with STI symptoms and sexual contacts of CT, NG, and MG.

Lactate metabolism promotes in vivo fitness during Acinetobacter baumannii infection.

Acinetobacter baumannii is one of the most prevalent causes of nosocomial infections worldwide. However, a paucity of information exists regarding the connection between metabolic capacity and in vivo bacterial fitness. Elevated lactate is a key marker of severe sepsis. We have previously shown that the putative A. baumannii lactate permease gene, lldP, is upregulated during in vivo infection. Here, we confirm that lldP expression is upregulated in three A. baumannii strains during a mammalian systemic infection. Utilising a transposon mutant disrupted for lldP in the contemporary clinical strain AB5075-UW, and a complemented strain, we confirmed its role in the in vitro utilisation of l-(+)-lactate. Furthermore, disruption of the lactate metabolism pathway resulted in reduced bacterial fitness during an in vivo systemic murine competition assay. The disruption of lldP had no impact on the susceptibility of this strain to complement mediated killing by healthy human serum. However, growth in biologically relevant concentrations of lactate observed during severe sepsis, led to bacterial tolerance to killing by healthy human blood, a phenotype that was abolished in the lldP mutant. This study highlights the importance of the lactate metabolism pathway for survival and growth of A. baumannii during infection.

Seegene Anyplex II assays detect HPV consistently using DNA extracts from different extraction methods.

The efficiency of PCR-based diagnostic assays can be impacted by the quality of DNA template, and anal samples can be particularly problematic due to the presence of faecal contaminants. Here, we compared the Quick-DNA Viral Kit (Zymo, Zymo Research, CA) and MagNA Pure 96 DNA and Viral NA Small Volume Kit (MP96, Roche) for use of the Seegene Anyplex II HPV28 assay (Anyplex28, Seegene) with anal samples. A total of 94 anal samples extracted using the MP96 and Zymo kits were tested via the Anyplex28, which detects high-risk human papillomavirus (HR-HPV, Panel A) and low-risk (LR-HPV, Panel B) HPV types. Testing the HR-HPV types (Panel A), 86 (91.5%) MP96 and 84 (89.4%) Zymo samples were deemed assessable. Overall agreement between the two methods was 87/94 (92.6%, 95% CI: 85.3-97.0) with the Kappa value of 0.678 (0.5-0.9). Of the 87 assessable samples, 50 (57.5%) were concordant, 34 (39.1%) partially concordant, and 10 (11.5%)discordant. In conclusion, the Anyplex28 produces comparable HPV genotyping results when using DNA extracts from either of these two methods.

Human papillomavirus negative high grade cervical lesions and cancers: Suggested guidance for HPV testing quality assurance.

BACKGROUND: Some high-grade cervical lesions and cervical cancers (HSIL+) test negative for human papillomavirus (HPV). The HPV-negative fraction varies between 0.03 % and 15 % between different laboratories. Monitoring and extended re-analysis of HPV-negative HSIL+ could thus be helpful to monitor performance of HPV testing services. We aimed to a) provide a real-life example of a quality assurance (QA) program based on re-analysis of HPV-negative HSIL+ and b) develop international guidance for QA of HPV testing services based on standardized identification of apparently HPV-negative HSIL+ and extended re-analysis, either by the primary laboratory or by a national HPV reference laboratory (NRL). METHODS: There were 116 initially HPV-negative cervical specimens (31 histopathology specimens and 85 liquid-based cytology samples) sent to the Swedish HPV Reference Laboratory for re-testing. Based on the results, an international QA guidance was developed through an iterative consensus process. RESULT: Standard PCR testing detected HPV in 55.2 % (64/116) of initially "HPV-negative" samples. Whole genome sequencing of PCR-negative samples identified HPV in an additional 7 samples (overall 61.2 % HPV positivity). Reasons for failure to detect HPV in an HSIL+ lesion are listed and guidance to identify cases for extended re-testing, including which information should be included when referring samples to an NRL are presented. CONCLUSION: Monitoring the proportion of and reasons for failure to detect HPV in HSIL+ will help support high performance and quality improvement of HPV testing services. We encourage implementation of QA strategies based on re-analysis of "HPV negative" HSIL+ samples.

Near-to-patient-testing to inform targeted antibiotic use for sexually transmitted infections in a public sexual health clinic: the NEPTUNE cohort study.

Background: Empiric treatment of sexually transmitted infections can cause unnecessary antibiotic use. We determined if near-to-patient-testing (NPT) for Neisseria gonorrhoeae, Chlamydia trachomatis and Mycoplasma genitalium (MG) improved antibiotic-use for a range of clinical presentations. Methods: Clients attending with non-gonococcal urethritis (NGU), proctitis, as STI-contacts, or for an MG-test-of-cure (MG-TOC) between March and December 2021 were recruited. Participants received near-to-patient-testing (NPT-group) for the three STIs using the GeneXpert® System (Cepheid), and concurrent routine-testing by transcription-mediated-amplification (TMA; Aptima, Hologic). Antibiotic-use among NGU or proctitis cases in the NPT-group was compared to clinic-controls undergoing routine-testing only. The proportion in the NPT-group who notified partners <24 hrs of their STI-specific result was calculated. Findings: Among 904 consults by 808 NPT-participants, ≥1 STI was detected in 63/252 (25.0%) with NGU, 22/51 (43.1%) with proctitis, and 167/527 (31.7%) STI-contacts. MG was detected among 35/157 (22.3%) MG-TOC consults. Among NGU and proctitis cases, fewer in the NPT-group received empiric treatment compared to clinic-controls (29.4% [95% CI: 24.3-34.9%] vs 83.8% [95% CI: 79.2-87.8%], p < 0.001), resulting in more NPT-group cases appropriately treated (STI-specific drug/no drug appropriately; 80.9% [95% CI: 76.0-85.1%] vs 33.0% [95% CI: 27.7-38.6%], p < 0.001) and fewer mistreated (incorrect drug/treated but pathogen-negative; 17.8% [13.7-22.6%] vs 61.4% [55.6-66.9%], p < 0.001). Of 167/264 in the NPT-group with an STI who responded regarding partner-notification, 95.2% notified all/some partners; 85.9% notified them <24 hrs of the STI-specific result. Interpretation: Near-to-patient-testing significantly improved antibiotic use and a high proportion of individuals rapidly notified partners of STI-specific results, highlighting the broad benefits of timely diagnostic strategies for STIs in clinical decision making and partner notification. Funding: ARC ITRP Hub-grant; NHMRC.

Evaluation of Seegene Anyplex II Performance for Detection of Human Papillomavirus Genotypes in Formalin-Fixed, Paraffin-Embedded Cervical Cancer Specimens.

CONTEXT.­: Detection of human papillomavirus (HPV) in formalin-fixed, paraffin-embedded (FFPE) tissues may identify the cause of lesions and has value for the development of new diagnostic assays and epidemiologic studies. Seegene Anyplex II assays are widely used for HPV screening, but their performance using FFPE samples has not been fully explored. OBJECTIVE.­: To validate Anyplex II HPV HR Detection (Anyplex II, Seegene) using FFPE samples. DESIGN.­: We used 248 stored DNA extracts from cervical cancer FFPE samples collected during 2005-2015 that tested HPV positive using the RHA kit HPV SPF10-LiPA25, v1 (SPF10, Labo Biomedical Products) HPV genotyping assay, manufacturer-validated for FFPE samples. RESULTS.­: Of the selected 248 samples, 243 were used in our analysis. Consistent with SPF10 genotyping results, Anyplex II detected all 12 oncogenic types and had an overall HPV detection rate of 86.4% (210 of 243 samples). Anyplex II and SPF10 showed very high agreement for the detection of the 2 most important oncogenic genotypes: HPV 16 (219 of 226; 96.9%; 95% CI, 93.7-98.75) and HPV 18 (221 of 226; 97.8%; 95% CI, 94.9-99.3). CONCLUSIONS.­: Overall results showed that both platforms produced comparable HPV genotyping results, indicating the suitability of Anyplex II for FFPE samples. The Anyplex II assay has the added convenience of being an efficient, single-well semiquantitative polymerase chain reaction assay. Further optimization of Anyplex II may enhance its performance using FFPE samples by improving the detection limit.

Development of a touchdown droplet digital PCR assay for the detection and quantitation of human papillomavirus 16 and 18 from self-collected anal samples.

IMPORTANCE: The quantity of the human papillomavirus (HPV) is associated with disease outcome. We designed an accurate and precise digital PCR assay for quantitating HPV in anal samples, a sample type that is typically problematic due to the presence of PCR inhibitors.

Evaluation of TIB Molbiol LightMix® assays for detection of Mycoplasma genitalium and key resistance mutations for macrolides and fluoroquinolones.

The LightMix® Modular Mycoplasma Macrolide and LightMix® Modular parC Fluoroquinolone Resistance assays (TIB Molbiol) were evaluated using sequential Mycoplasma genitalium positive (n = 125) and negative (n = 93) clinical samples. Results were compared to the results of an established commercial assay (ResistancePlus MG assay, SpeeDx Pty Ltd) or Sanger sequencing (for parC). Detection of M. genitalium by the TIB Molbiol assay had a high agreement with the reference assay, with a positive percent agreement (PPA) of 97.6 [95% confidence interval (CI): 93.1-99.5] and negative percent agreement (NPA) of 95.7 (95% CI: 89.5-98.8). From 105 positive samples, macrolide resistance detection had a PPA of 100% (95% CI: 93.7-100) and NPA of 81.3% (95% CI: 67.4-91.1). For the detection of fluroquinolone resistance mutation G248T/S83I or "other mutation" in the quinolone resistance determinant region, from 95 samples there was 100% (95% CI: 86.3-100) sensitivity and 100% (95% CI: 94.5-100) specificity. The understanding of the basis for fluoroquinolone treatment failure is still developing; it is therefore important to use the output of parC-based resistance assays with caution to avoid the inappropriate use of antibiotic therapies, especially considering the limited number of alternative treatments.

Prevotella and Gardnerella Are Associated With Treatment Failure Following First-line Antibiotics for Bacterial Vaginosis.

BACKGROUND: Bacterial vaginosis (BV) is a common vaginal dysbiosis that often recurs following first-line antibiotics. We investigated if vaginal microbiota composition was associated with BV recurrence. METHODS: We analyzed samples and data from 121 women who participated in 3 published trials evaluating novel interventions for improving BV cure, including concurrent antibiotic treatment of regular sexual partners (RSPs). Women diagnosed with BV received first-line antibiotics and self-collected vaginal swabs pretreatment and the day after finishing antibiotics (immediately posttreatment). 16S rRNA gene sequencing was performed on vaginal samples. Logistic regression explored associations between BV recurrence and features of the vaginal microbiota pre- and posttreatment. RESULTS: Sixteen women (13% [95% confidence interval {CI}, 8%-21%]) experienced BV recurrence within 1 month of treatment. Women with an untreated RSP were more likely to experience recurrence than women with no RSP (P = .008) or an RSP who received treatment (P = .011). A higher abundance of Prevotella pretreatment (adjusted odds ratio [AOR], 1.35 [95% CI, 1.05-1.91]) and Gardnerella immediately posttreatment (AOR, 1.23 [95% CI, 1.03-1.49]) were associated with increased odds of BV recurrence. CONCLUSIONS: Having specific Prevotella spp prior to recommended treatment and persistence of Gardnerella immediately posttreatment may contribute to the high rates of BV recurrence. Interventions that target these taxa are likely required to achieve sustained BV cure.

A single dose of quadrivalent HPV vaccine is highly effective against HPV genotypes 16 and 18 detection in young pregnant women eight years following vaccination: an retrospective cohort study in Fiji.

Background: In 2008/9, Fiji vaccinated >30,000 girls aged 9-12 years with the quadrivalent human papillomavirus (4vHPV) vaccine coverage for at least one dose was >60% (one dose only was 14%, two dose only was 13%, three doses was 35%). We calculated vaccine effectiveness (VE) of one, two and three doses of 4vHPV against oncogenic HPV genotypes 16/18, eight years following vaccination. Methods: A retrospective cohort study was undertaken (2015-2019) in pregnant women ≤23 years old, eligible to receive 4vHPV in 2008/9, with confirmed vaccination status. The study was restricted to pregnant women due to the cultural sensitivity of asking about sexual behavior in Fiji. For each participant a clinician collected a questionnaire, vaginal swab and genital warts examination, a median eight (range 6-11) years post vaccination. HPV DNA was detected by molecular methods. Adjusted VE (aVE) against the detection of vaccine HPV genotypes (16/18), the comparison group of non-vaccine genotypes (31/33/35/39/45/51/52/56/58/59/66/68), and genital warts were calculated. Covariates included in the adjusted model were: age, ethnicity and smoking, according to univariate association with any HPV detection. Findings: Among 822 participants the prevalence of HPV 16/18 in the unvaccinated, one, two and three-dose groups were 13.3% (50/376), 2.5% (4/158), 0% (0/99) and 1.6% (3/189), respectively; and for the non-vaccine high-risk genotypes, the detection rate was similar across dosage groups (33.2%-40.4%, p = 0.321). The aVE against HPV 16/18 for one, two and three doses were 81% (95% CI; 48-93%), 100% (95% CI; 100-100%), and 89% (95% CI; 64-96%), respectively. Prevalence of HPV 16/18 was lower among women with longer time since vaccination. Interpretations: A single dose 4vHPV vaccine is highly effective against HPV genotypes 16 and 18 eight years following vaccination. Our results provide the longest duration of protection for reduced dose 4vHPV schedule in a low- or middle-income country in the Western Pacific region. Funding: This study was supported by the Bill & Melinda Gates Foundation and the Department of Foreign Affairs and Trade of the Australian Government and Fiji Health Sector Support Program (FHSSP). FHSSP is implemented by Abt JTA on behalf of the Australian Government.

Quantitation of Mycoplasma genitalium using droplet digital PCR.

Mycoplasma genitalium is a sexually transmitted infection with increasing concerns around antimicrobial resistance. Droplet digital PCR (ddPCR) is a rapid quantification method with high precision that may be useful for absolute quantitation of bacteria in samples. This study aimed to develop a ddPCR assay for the quantification of M. genitalium. ddPCR targeting the gene mgpB was established and analysed using the QX100 ddPCR system. The assay was evaluated against quantitated DNA standards, and then in comparison to an established quantitative PCR performed on the Lightcycler 480 II. DNA template of increasing complexity was used, including synthetic double stranded DNA, DNA extracts from laboratory-cultured M. genitalium strains (n = 17) and DNA from M. genitalium-positive clinical samples (n = 21). There was a strong correlation between ddPCR concentration estimates and measured DNA standards (r2 = 0.997), and between ddPCR and qPCR quantitation for different templates (r2 ranging from 0.953 to 0.997). ddPCR reliably detected template in a range from <10 copies per reaction to >104 copies per reaction and demonstrated linearity over dilution series. Concentration estimates by ddPCR were reproducibly less than those determine by qPCR. ddPCR demonstrated precise and reproducible quantitation of M. genitalium with a variety of templates.

gyrA Mutations in Mycoplasma genitalium and Their Contribution to Moxifloxacin Failure: Time for the Next Generation of Resistance-Guided Therapy.

BACKGROUND: Although single nucleotide polymorphisms (SNPs) in Mycoplasma genitalium parC contribute to fluoroquinolone treatment failure, data are limited for the homologous gene, gyrA. This study investigated the prevalence of gyrA SNPs and their contribution to fluoroquinolone failure. METHODS: Samples from 411 patients (male and female) undergoing treatment for M. genitalium infection (Melbourne Sexual Health Centre, March 2019-February 2020) were analyzed by Sanger sequencing (gyrA and parC). For patients treated with moxifloxacin (n = 194), the association between SNPs and microbiologic treatment outcome was analyzed. RESULTS: The most common parC SNP was G248T/S83I (21.1% of samples), followed by D87N (2.3%). The most common gyrA SNP was G285A/M95I (7.1%). Dual parC/gyrA SNPs were found in 8.6% of cases. One third of infections harboring parC G248T/S83I SNP had a concurrent SNP in gyrA conferring M95I. SNPs in gyrA cooccurred with parC S83I variations. Treatment failure was higher in patients with parC S83I/gyrA dual SNPs when compared with infections with single S83I SNP alone from analysis of (1) 194 cases in this study (81.2% vs 45.8%, P = .047), and (2) pooled analysis of a larger population of 535 cases (80.6% vs 43.2%; P = .0027), indicating a strong additive effect. CONCLUSIONS: Compared with parC S83I SNP alone, M. genitalium infections with dual mutations affecting parC/gyrA had twice the likelihood of failing moxifloxacin. Although antimicrobial resistance varies by region globally, these data indicate that gyrA should be considered as a target for future resistance assays in Australasia. We propose a strategy for the next generation of resistance-guided therapy incorporating parC and gyrA testing.

Performance of Human Papillomavirus Attribution Algorithms to Predict Causative Genotypes in Anal High-Grade Lesions.

BACKGROUND: Gay and bisexual men (GBM) are at increased risk of human papillomavirus (HPV)-associated anal high-grade squamous intraepithelial lesions (HSILs). Understanding the fractions of HSILs attributable to HPV genotypes is important to inform potential impacts of screening and vaccination strategies. However, multiple infections are common, making attribution of causative types difficult. Algorithms developed for predicting HSIL-causative genotype fractions have never been compared with a reference standard in GBM. METHOD: Samples were from the Study of the Prevention of Anal Cancer. Baseline HPV genotypes detected in anal swab samples (160 participants) were compared with HPV genotypes in anal HSILs (222 lesions) determined by laser capture microdissection (LCM). Five algorithms were compared: proportional, hierarchical, maximum, minimum, and maximum likelihood estimation. RESULTS: All algorithms predicted HPV-16 as the most common HSIL-causative genotype, and proportions differed from LCM detection (37.8%) by algorithm (with differences of -6.1%, +20.9%, -20.4%, +2.9%, and +2.2% respectively). Fractions predicted using the proportional method showed a strong positive correlation with LCM, overall (R = 0.73 and P = .002), and by human immunodeficiency virus (HIV) status (HIV positive, R = 0.74 and P = .001; HIV-negative, R = 0.68 and P = .005). CONCLUSIONS: Algorithms produced a range of inaccurate estimates of HSIL attribution, with the proportional algorithm performing best. The high occurrence of multiple HPV infections means that these algorithms may be of limited use in GBM.

Comparison of Seegene AnyPlexTM II STI-7e with standard-of-care diagnostic methods for the detection of Mycoplasma genitalium, Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis.

The AnyPlexTM II STI-7e panel assay (Seegene) detects seven sexually transmitted organisms (Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, M. hominis, Ureaplasma urealyticum, U. parvum, and Trichomonas vaginalis). This study compared the performance of AnyPlexTM II STI-7e with standard-of-care diagnostic methods. Samples (cervical or vaginal swabs, or urine) from 1330 women were tested on standard-of-care assays; 83/1318 (6.3%) tested positive for M. genitalium (ResistancePlus® MG), 99/1317 (7.5%) positive for C. trachomatis and 11/1316 (0.8%) positive for N. gonorrhoeae (Hologic® Aptima Combo 2®), and 6/689 (0.9%) positive for T. vaginalis (wet mount microscopy). AnyPlexTM II STI-7e had good agreement for the detection of M. genitalium [Cohen's kappa of 0.80, 95% confidence intervals (CI) 0.74-0.87] and C. trachomatis (kappa of 0.87, 95% CI 0.82-0.92), with positive and negative % agreement >96% for both infections. There was lower agreement for the detection of N. gonorrhoeae (kappa of 0.37, 95%CI 0.19-0.55) and T. vaginalis (kappa of 0.521, 95%CI 0.25-0.80). In summary, the test performed well in this comparison for M. genitalium and C. trachomatis detection, but results were less conclusive for N. gonorrhoeae and T. vaginalis due to low prevalence in the population.

The Urethral Microbiota of Men with and without Idiopathic Urethritis.

Nongonococcal urethritis (NGU) is a common genital tract syndrome in men, and up to 50% of cases are considered idiopathic, i.e., no etiological agent is identified. This poses challenges for clinicians in the diagnosis and treatment of NGU and often results in antibiotic misuse and overuse. Therefore, to identify potential infectious causes of urethritis and inform clinical management of urethritis cases, we characterized and compared the urethral microbiota of men with and without idiopathic urethritis. Participants were derived from a case-control study that examined viral and bacterial pathogens and sexual practices associated with NGU. Men with NGU who tested negative for established causes of NGU (Chlamydia trachomatis, Mycoplasma genitalium, Trichomonas vaginalis, adenoviruses, herpes simplex virus [HSV]-1, and/or HSV-2) were classified as idiopathic cases, and the controls were men reporting no current urethral symptoms. Men provided a urine sample that was used to characterize the urethral microbiota using 16S rRNA gene sequencing. Bacterial taxa associated with idiopathic urethritis were identified using analysis of compositions of microbiomes with bias correction. When stratified by sex of sexual partner, we found that the abundance of Haemophilus influenzae was significantly increased in men who have sex with men with idiopathic urethritis, and the abundance of Corynebacterium was significantly increased in men who have sex with women with idiopathic urethritis. Other taxa, including Ureaplasma, Staphylococcus haemolyticus, Streptococcus pyogenes, Escherichia, and Streptococcus pneumoniae/pseudopneumoniae, dominated the urethral microbiota of idiopathic urethritis cases but not controls, suggesting that these organisms may also contribute to urethritis. Importantly, the taxa we identified represent biologically plausible causes of urethritis and should be prioritized for future study. IMPORTANCE Nongonococcal urethritis (NGU) is the commonest genital tract syndrome in men and is nearly universally presumptively treated with an antibiotic. Common causes of NGU include Chlamydia trachomatis and Mycoplasma genitalium, but in more than 50% of cases, an infectious cause is not identified. In this case-control study, we found that the urethral microbiota composition differed between men with and without idiopathic urethritis and differed by sex of sexual partner. We identified specific bacterial taxa that were associated with idiopathic urethritis, including Haemophilus influenzae and Corynebacterium. These data, together with the finding that key bacterial taxa were found to dominate the urethral microbiota of cases but not controls, suggest that a range of bacteria contribute to urethritis and that these organisms may be influenced by sexual practices. Through identifying the infectious causes of urethritis, we can inform appropriate targeted diagnostic and treatment practices and importantly reduce misuse and overuse of antibiotics.