Timely Diagnosis of Incubating Syphilis Infections Using Treponema pallidum Transcription-Mediated Amplification Assay.

BACKGROUND: Syphilis is a complex, multistage, sexually transmitted infection (STI) caused by the bacterium Treponema pallidum subspecies pallidum (TP). New diagnostic tools are needed to minimize transmission. In this study, we aimed to assess the additional value of an investigational transcription-mediated amplification test for TP (TP-TMA) for routine diagnostics. METHODS: Between September 2021 and August 2022, visits by all participants of the national preexposure prophylaxis (PrEP) program at the sexual health center (SHC) in Amsterdam were included. Anal, pharyngeal, vaginal, and urine samples collected for Chlamydia trachomatis and Neisseria gonorrhoeae screening were additionally tested with the TP-TMA assay based on detection of 23S rRNA of TP. RESULTS: In total, 9974 SHC visits by 3283 participants were included. There were 191 infectious syphilis cases diagnosed: 26 (14%) primary syphilis, 54 (29%) secondary syphilis, and 111 (58%) early latent syphilis. In 79 of the 191 (41%) syphilis cases, at least 1 sample was TP-TMA-positive. For 16 participants, the positive TP-TMA result was not concordant with routine diagnostics. Of those, 2 participants were treated for syphilis within a week before the visit. Eight participants were treated for a syphilis notification at the visit or for another STI. Five participants were diagnosed with syphilis at the following visit, and 1 participant was lost to follow-up. CONCLUSIONS: By adding the TP-TMA assay to routine diagnostics, we identified 14 of 191 (7%) additional syphilis infections among participants of the national PrEP program. The TP-TMA assay is a useful diagnostic tool to increase syphilis case finding and thus limit the transmission of syphilis.

Treponema pallidum Strains Among Women and Men Who Have Sex With Women in Amsterdam, the Netherlands and Antwerp, Belgium Between 2014 and 2020.

ABSTRACT: The Treponema pallidum strain distribution among men who have sex with women were similar to the strain distribution men who have sex with men. The most prevalent strains and percentage of strains belonging to the Nichols lineage are similar to previous studies in Amsterdam focusing on men who have sex with men.

Enhanced detection rate of Mycoplasma genitalium in urine overtime by transcription-mediated amplification in comparison to real-time PCR.

BACKGROUND: Diagnosis of infected individuals with Mycoplasma genitalium (MG) is often performed by real-time PCR or transcription-mediated amplification (TMA). A limitation of the MG-TMA assay is the relatively short time span of 24 h in which the collected urine is required to be transferred into a Urine Specimen Transport Tube, according to the manufacturer's guidelines. If not transferred within 24 h, the manufacturer's claimed sensitivity cannot be guaranteed anymore, and samples may instead be tested with an in-house validated real-time PCR, despite its recognized lower sensitivity. This study aimed to validate an exception to the sample transport and storage conditions of the MG-TMA assay as set by the manufacturer, being the prolongation of the acceptable testing time limit of 24 h. METHODS: From June to December 2022, first-void urines were collected from clients attending the clinic for sexual health in Amsterdam, the Netherlands. Urine samples that tested positive for MG by TMA assay at the day of collection were concomitantly stored at room (18-24 °C) and refrigerator temperature (4-8 °C) for 15 days. The stored urine samples were tested with both an in-house validated real-time PCR and MG-TMA assay after transfer of the original urine samples to the respective test tubes at 3, 7, 12 and 15 days post collection. RESULTS: In total, 47 MG-positive urine samples were collected, stored and tested for MG by real-time PCR and TMA assays. After storage at room temperature, the MG-detection rate by TMA was significantly higher compared to real-time PCR, at days 0 (p ≤ 0.001), 7 (p ≤ 0.001) and 12 (p < 0.05). After storage at refrigerator temperature, the MG-detection rate determined by TMA assay was significantly enhanced in comparison with real-time PCR at days 3 (p < 0.01), 7 (p ≤ 0.001) and 15 (p < 0.01). CONCLUSIONS: This validation study showed that the MG-TMA assay has a superior detection rate in urine compared to real-time PCR, up to 15 days post sample collection and irrespective of storage temperature. Accepting urines older than 24 h to be tested by TMA will improve clinical diagnosis of MG infections.

Epidemiologic and Genomic Analysis of SARS-CoV-2 Delta Variant Superspreading Event in Nightclub, the Netherlands, June 2021.

We report a severe acute respiratory syndrome coronavirus 2 superspreading event in the Netherlands after distancing rules were lifted in nightclubs, despite requiring a negative test or vaccination. This occurrence illustrates the potential for rapid dissemination of variants in largely unvaccinated populations under such conditions. We detected subsequent community transmission of this strain.

Shigella is common in symptomatic and asymptomatic men who have sex with men visiting a sexual health clinic in Amsterdam.

INTRODUCTION: Shigellosis is a reportable infectious disease. It can present as a severe bloody diarrhoea but is often asymptomatic. Shigella can be sexually transmissible. We performed a study among symptomatic and asymptomatic men who have sex with men (MSM) to assess the prevalence of Shigella, Salmonella and Campylobacter. METHODS: From March to June 2020, MSM attending the Amsterdam centre for sexual health were consecutively included. Predefined minimal numbers of inclusion of 150, 100 and 50 were determined, respectively, for MSM who reported no diarrhoea, diarrhoea during last month or diarrhoea on the day of visit to clinic. Anal samples were tested for the presence of Shigella, Salmonella and Campylobacter. During the same period, the frequency of these bacteria was assessed in routinely tested samples requested by general physicians or nursing home physicians. Characteristics of included MSM were compared between the men with different diarrhoea anamnesis, and the prevalence of shigellosis was estimated in each group. RESULTS: We included 212 MSM without diarrhoea, 109 MSM who recently had diarrhoea and 68 MSM who reported diarrhoea on the day of clinic visit. Thirteen (3.3%, 95% CI 1.7% to 5.6%) MSM were infected with Shigella, none with Salmonella and 7 (1.8%, 95% CI 0.7% to 3.7%) with Campylobacter. Shigella prevalence was 2.8% (95% CI 1.0% to 6.1%) in asymptomatic men, 3.7% (95% CI 1.0% to 9.1%) in men who recently had diarrhoea and 4.4% (95% CI 0.9% to 12.4%) in men with current diarrhoea (p=0.799). Shigella was more frequently found in MSM who had used pre-exposure prophylaxis (PrEP) in the preceding 3 months (10/151), compared with those not having used PrEP (2/146) or being HIV positive (1/75) (p=0.038). Shigella was significantly more often detected among MSM compared with routinely obtained faecal samples being 11/770 (1.4%) (p=0.031). CONCLUSION: Shigella infections are relatively common in both symptomatic and asymptomatic MSM. Future studies should focus on the risk of onward transmission via asymptomatic persons. Samenvatting Introductie Shigellose is een meldingsplichtige infectieziekte. Het kan zich presenteren als een ernstige bloederige diarree, maar is vaak asymptomatisch. Shigella kan seksueel overdraagbaar zijn. We hebben een onderzoek uitgevoerd onder symptomatische en asymptomatische mannen die seks hebben met mannen (MSM) om de prevalentie van Shigella, Salmonella en Campylobacter te bepalen. Methoden Van maart tot juni 2020 werden achtereenvolgens MSM van het Amsterdamse centrum voor seksuele gezondheid opgenomen. Vooraf gedefinieerde minimale aantallen van inclusie van respectievelijk 150, 100 en 50 waren bepaald voor MSM die geen diarree, diarree in de afgelopen maand of diarree op de dag van bezoek aan de kliniek meldden. Anale monsters werden getest op de aanwezigheid van Shigella, Salmonella en Campylobacter. In dezelfde periode werd de frequentie van deze bacteriën bepaald in routinematig geteste monsters aangevraagd door huisartsen of verpleeghuisartsen. Kenmerken van geïncludeerde MSM werden vergeleken tussen mannen met verschillende diarree anamnese, en de prevalentie van shigellose werd in elke groep geschat. Resultaten We includeerden 212 MSM zonder diarree, 109 MSM die onlangs diarree hadden en 68 MSM die diarree meldden op de dag van het bezoek aan de kliniek. Dertien (3,3%, 95% CI 1,7-5,6%) MSM waren geïnfecteerd met Shigella, geen enkele met Salmonella, en 7 (1,8%, 95% CI 0,7-3,7%) met Campylobacter. De prevalentie van Shigella was 2,8% (95%CI 1,0-6,1%) bij asymptomatische mannen, 3,7% (95%CI 1,0-9,1%) bij mannen die recent diarree hadden en 4,4% (95%CI 0,9-12,4%) bij mannen met huidige diarree (P=0,799). Shigella werd vaker gevonden bij MSM die in de voorgaande drie maanden (10/151) PrEP hadden gebruikt dan bij mensen die geen PrEP hadden gebruikt (2/146) of hiv-positief waren (1/75) (p=0,038). Shigella werd significant vaker gedetecteerd bij MSM in vergelijking met routinematig verkregen fecale monsters, namelijk 11/770 (1,4%) (p=0,031). Conclusie Shigella infecties komen relatief vaak voor bij zowel symptomatische als asymptomatische MSM. Toekomstige studies moeten zich richten op het risico van verdere overdracht via asymptomatische personen.

Incident urogenital and anorectal Chlamydia trachomatis in women: the role of sexual exposure and autoinoculation: a multicentre observational study (FemCure).

BACKGROUND: Anorectal infections with Chlamydia trachomatis (CT) are common in women visiting STI outpatient clinics. We here evaluated the risk posed by sexual exposure and by alternate anatomical site infection for incident anorectal and urogenital CT. METHODS: Prospective multicentre cohort study, FemCure. Participants were treated for CT, and after 4, 6, 8, 10 and 12 weeks, they self-collected anorectal and urogenital samples (swabs) for CT-DNA testing. We calculated the proportion with incident CT, that is, CT incidence (at weeks 6-12) by 2-week time-periods. Compared with no exposure (A), we estimated the risk of incident CT for (B) sexual exposure, (C) alternate site anatomic site infection and (D) both, adjusted for confounders and expressed as adjusted ORs with 95% CIs. RESULTS: We analysed data of 385 participants contributing 1540 2-week periods. The anorectal CT incidence was 2.9% (39/1343) (95 CI 1.8 to 3.6); 1.3% (A), 1.3% (B), 27.8% (C) and 36.7% (D). The ORs were: 0.91 (95% CI 0.32 to 2.60) (B), 26.0 (95% CI 7.16 to 94.34) (C), 44.26 (95% CI 14.38 to 136.21) (D).The urogenital CT incidence was 3.3% (47/1428) (95% CI 2.4 to 4.4); 0.7% (A), 1.9% (B), 13.9% (C) and 25.4% (D). The ORs were: 2.73 (95% CI 0.87 to 8.61) (B), 21.77 (95% CI 6.70 to 70 71) (C) and 49.66 (95% CI 15.37 to 160.41) (D). CONCLUSIONS: After initial treatment, an alternate anatomical site CT infection increased the risk for an incident CT in women, especially when also sex was reported. This may suggest a key role for autoinoculation in the re-establishment or persistence of urogenital and anorectal chlamydia infections.

Determinants associated with viable genital or rectal Chlamydia trachomatis bacterial load (FemCure).

BACKGROUND: Chlamydia trachomatis (CT) is routinely diagnosed by nucleic acid amplification tests (NAATs), which are unable to distinguish between nucleic acids from viable and non-viable CT organisms. OBJECTIVES: We applied our recently developed sensitive PCR (viability PCR) technique to measure viable bacterial CT load and explore associated determinants in 524 women attending Dutch sexual health centres (STI clinics), and who had genital or rectal CT. METHODS: We included women participating in the FemCure study (Netherlands, 2016-2017). At the enrolment visit (pre-treatment), 524 were NAAT positive (n=411 had genital and rectal CT, n=88 had genital CT only and n=25 had rectal CT only). We assessed viable rectal and viable genital load using V-PCR. We presented mean load (range 0 (non-viable) to 6.5 log10 CT/mL) and explored potential associations with urogenital symptoms (coital lower abdominal pain, coital blood loss, intermenstrual bleeding, altered vaginal discharge, painful or frequent micturition), rectal symptoms (discharge, pain, blood loss), other anatomical site infection and sociodemographics using multivariable regression analyses. RESULTS: In genital (n=499) CT NAAT-positive women, the mean viable load was 3.5 log10 CT/mL (SD 1.6). Genital viable load was independently associated with urogenital symptoms-especially altered vaginal discharge (Beta=0.35, p=0.012) and with concurrent rectal CT (aBeta=1.79; p<0.001). Urogenital symptoms were reported by 50.3% of women; their mean genital viable load was 3.6 log10 CT/mL (vs 3.3 in women without symptoms). Of 436 rectal CT NAAT-positive women, the mean rectal viable load was 2.2 log10 CT/mL (SD 2.0); rectal symptoms were reported by 2.5% (n=11) and not associated with rectal viable load. CONCLUSION: Among women diagnosed with CT in an outpatient clinical setting, viable genital CT load was higher in those reporting urogenital symptoms, but the difference was small. Viable genital load was substantially higher when women also had a concurrent rectal CT. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov NCT02694497.

Macrolide-Resistant Mycoplasma genitalium Impairs Clinical Improvement of Male Urethritis After Empirical Treatment.

BACKGROUND: Mycoplasma genitalium (MG) is associated with urethritis in men and could play a role in clinical outcome. We examined clinical improvement of symptoms in men receiving empirical treatment for urethritis and correlated the outcome with Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT), MG, and MG macrolide resistance-associated mutations (MRAM) status. METHODS: At the sexually transmitted infection clinic in Amsterdam, the Netherlands, empirical treatment for gonococcal urethritis is 1 g ceftriaxone and for nongonococcal urethritis 1 g azithromycin. In 2018 to 2019, we tested urine samples of men with urethritis for CT, NG, and MG using transcription-mediated amplification assays. Mycoplasma genitalium-positive samples were tested for MRAM using quantitative polymerase chain reaction. Two weeks after receiving therapy, men were sent a text message inquiring after clinical improvement. RESULTS: We evaluated 2505 cases of urethritis. The positivity rates of NG, CT, and MG were 26% (648 of 2489), 29% (726 of 2489), and 23% (522 of 2288), respectively. In 768 of 2288 of the cases (34%), no causative agent was detected. Most cases were infected with a single pathogen: NG, 417 of 2288 (18%); CT, 486 of 2288 (21%); and MG, 320 of 2288 (14%). The prevalence of MRAM among MG-positives was 74% (327 of 439). For 642 (25.6%) cases, we could evaluate clinical improvement after treatment of whom 127 (20%) indicated no improvement; 9% (15 of 174) in NG cases, 18% (35 of 195) in CT cases, 14% (4 of 28) in MG wild-type cases, and 40% (38 of 94) in MG-MRAM cases. Clinical improvement in MG-MRAM cases was significantly lower compared with all other groups (P < 0.001). CONCLUSIONS: Presence of MG-MRAM is associated with lack of clinical improvement in azithromycin-treated nongonococcal urethritis.

Emergence of a Neisseria gonorrhoeae clone with reduced cephalosporin susceptibility between 2014 and 2019 in Amsterdam, The Netherlands, revealed by genomic population analysis.

BACKGROUND: Emerging resistance to cephalosporins in Neisseria gonorrhoeae (Ng) is a major public health threat, since these are considered antibiotics of last resort. Continuous surveillance is needed to monitor the circulation of resistant strains and those with reduced susceptibility. OBJECTIVES: For the purpose of epidemiological surveillance, genomic population analysis was performed on Ng isolates from Amsterdam with a focus on isolates with reduced susceptibility to ceftriaxone. METHODS: WGS data were obtained from 318 isolates from Amsterdam, the Netherlands between 2014 and 2019. Isolates were typed according to MLST, Ng Multi-Antigen Sequence Typing (NG-MAST) and Ng Sequence Typing for Antimicrobial Resistance (NG-STAR) schemes and additional resistance markers were identified. Phylogenetic trees were created to identify genetic clusters and to compare Dutch and non-Dutch MLST7827 isolates. RESULTS: MLST7363 and MLST1901 were the predominant strains having reduced susceptibility to ceftriaxone during 2014-16; MLST7827 emerged and dominated during 2017-19. NG-STAR38 and NG-MAST2318/10386 were predominant among MLST7827 isolates. MLST7827 reduced susceptibility isolates carried a non-mosaic 13.001 penA allele with an A501V mutation and porB1b G120K/A121D mutations, which were lacking in susceptible MLST7827 isolates. Phylogenetic analysis of all publicly available MLST7827 isolates showed strong genetic clustering of Dutch and other European MLST7827 isolates. CONCLUSIONS: MLST7827 isolates with reduced ceftriaxone susceptibility have emerged during recent years in Amsterdam. Co-occurrence of penA A501V and porB1b G120K/A121D mutations was strongly associated with reduced susceptibility to ceftriaxone. Genetic clustering of Dutch and other European MLST7827 isolates indicates extensive circulation of this strain in Europe. Close monitoring of the spread of this strain having an alarming susceptibility profile is needed.

Oropharyngeal Chlamydia trachomatis in women; spontaneous clearance and cure after treatment (FemCure).

OBJECTIVES: Women attending STI clinics are not routinely tested for oropharyngeal Chlamydia trachomatis (CT) infections. We aimed to assess spontaneous clearance of oropharyngeal CT and cure after antibiotic treatment in women. METHODS: Women with vaginal or rectal CT (n=560) were recruited at STI clinics in 2016-2017, as part of the FemCure study (prospective cohort study). We included participants' data from week -1, that is, the diagnosis at initial visit, when clinics applied selective oropharyngeal testing. At week -1, a total of 241 women were oropharyngeally tested (30 positive) and 319 were untested. All FemCure participants provided nurse-collected oropharyngeal samples at study enrolment, that is, week 0, just prior to treatment (n=560), and after treatment at weeks 4 (n=449), 8 (n=433) and 12 (n=427). Samples were tested by nucleic acid amplification test, and at week 0 also by viability testing by viability PCR. Proportions of oropharyngeal CT test results were presented to represent spontaneous clearance and cure. RESULTS: Of 30 women diagnosed with oropharyngeal CT at week -1, fifteen (50%) were negative at week 0 after a median of 9 days, that is, 'spontaneous clearance'. At week 0, a total of 560 participants were tested, and 46 (8.8%) were oropharyngeal CT positive; 12 of them (26.1%) had viable CT. Of the 46 positive, 36 women had an oropharyngeal test after treatment; 97.2% (35/36) were negative at week 4, that is, 'cure'. Of all women with follow-up visits, the proportion of oropharyngeal CT positive was between 0.5% and 1.6% between weeks 4 and 12. Of those not tested at week -1 (n=319), 8.5% (n=27) were oropharyngeal positive at week 0. CONCLUSIONS: The clinical importance of oropharyngeal CT in women is debated. We demonstrated that spontaneous clearance of oropharyngeal CT among women is common; of those who did not clear for CT, three-quarters had non-viable CT. After regular treatment with azithromycin or doxycycline, cure rate (97%) of oropharyngeal CT is excellent. TRIAL REGISTRATION NUMBER: NCT02694497.

Molecular diversity of Treponema pallidum subspecies pallidum isolates in Amsterdam, the Netherlands.

OBJECTIVES: The prevalence of syphilis, caused by the spirochete Treponema pallidum subsp. pallidum (TPA), remains high despite the availability of effective antibiotics. In the Netherlands, most syphilis cases are found among men who have sex with men (MSM). We studied the distribution of TPA strain types by molecular characterisation and related this to available characteristics. In addition, resistance to macrolides was assessed. METHODS: TPA DNA was extracted from 136 genital ulcer swab or skin lesions samples deriving from 135 patients diagnosed with syphilis in 2016 and 2017 at the Public Health Service in Amsterdam, the Netherlands. Molecular typing was done according to the enhanced CDC method (E-CDC), in which three genetic regions of the arp, tpr and tp0548 genes are analysed by gel electrophoresis of the arp and tpr regions and by sequence analysis for the tp0548 region. Part of the 23S rDNA locus was sequenced to determine the presence of macrolide resistance-associated mutations. RESULTS: Full E-CDC strain types could be determined for 99/136 (73%) DNA samples, which tested positive in a diagnostic PCR targeting the polA gene. Types differed within one patient of whom two samples were available. No association was found between the demographic and clinical characteristics and the TPA types. The most prevalent type was 14d/g, found in 23 of the 99 (23%) fully typed samples. Part of the 23S rDNA locus was successfully sequenced for 93/136 (68%) samples and 83 (88%) contained the A2058G mutation. No A2059G mutation was found. CONCLUSIONS: A broad strain distribution was found. Few subtypes were clonally expanded, and most other subtypes were rare. Detection of the most prevalent strain type, 14d/g, is in concordance with other TPA typing studies. The high prevalence of genetic macrolide resistance indicates that azithromycin is not an alternative treatment option.

Spontaneous clearance of Chlamydia trachomatis accounting for bacterial viability in vaginally or rectally infected women (FemCure).

OBJECTIVES: Spontaneous clearance of Chlamydia trachomatis (CT) infections can occur between diagnosis and treatment. We followed CT patients to assess clearance using a conventional definition (no total CT-DNA, assessed by routine quantitative PCR methods) and a definition accounting for viability, assessed by viability PCR testing. METHODS: Three outpatient STI clinics included CT-diagnosed women (The Netherlands, 2016-2017, FemCure study); participants had vaginal CT (vCT) and rectal CT (rCT) (group A: n=155), vCT and were rectally untested (group B: n=351), single vCT (group C: n=25) or single rCT (group D: n=29). Follow-up (median interval 9 days) vaginal and rectal samples underwent quantitative PCR testing (detecting total CT-DNA). When PCR positive, samples underwent V-PCR testing to detect 'viable CT' (CT-DNA from intact CT organisms; V-PCR positive). 'Clearance' was the proportion PCR-negative patients and 'clearance of viable CT' was the proportion of patients testing PCR negative or PCR positive but V-PCR negative. We used multivariable logistic regression analyses to assess diagnosis group (A-D), age, days since initial CT test (diagnosis) and study site (STI clinic) in relation to clearance and clearance of viable CT. RESULTS: Clearance and clearance of viable CT at both anatomic sites were for (A) 0.6% and 3.9%; (B) 5.4% and 9.4%; (C) 32.0% and 52.0% and (D) 27.6% and 41.4%, respectively. In multivariate analyses, women with single infections (groups C and D) had higher likelihood of clearance than women concurrently infected with vCT and rCT (p<0.001).Of rectally untested women (group B), 76.9% had total CT-DNA and 46.7% had viable CT (V-PCR positive) at the rectal site. CONCLUSIONS: Of untreated female vCT patients who had CT also at the rectal site, or who were rectally untested, only a small proportion cleared CT (in fact many had viable CT) at their follow-up visit (median 9 days). Among single site infected women clearance was much higher. TRIAL REGISTRATION NUMBER: NCT02694497.

Simultaneous Detection of Neisseria gonorrhoeae and Fluoroquinolone Resistance Mutations to Enable Rapid Prescription of Oral Antibiotics.

BACKGROUND: Absence of rapid antimicrobial resistance testing of Neisseria gonorrhoeae (Ng) hinders personalized antibiotic treatment. To enable rapid ciprofloxacin prescription, a real-time polymerase chain reaction (PCR) for simultaneous detection of Ng and fluoroquinolone resistance-associated gyrA-S91F mutation was evaluated. METHODS: Analytical NG quantitative PCR kit (NYtor BV) performance was assessed on 50 Ng transcription-mediated amplification (TMA)-negative and 100 Ng TMA-positive samples. To assess clinical use, 200 samples were prospectively analyzed, in parallel to routine diagnostic tests. Also, 50 urine, 50 anal, 50 pharyngeal, and 50 vaginal Ng TMA-positive samples were retrospectively analyzed. To assess if patients carried strains with different ciprofloxacin sensitivity at different anatomical locations, 50 urine/anal or vaginal/anal sample pairs collected during a single visit were analyzed. RESULTS: The NG quantitative PCR kit showed 97% sensitivity and 100% specificity for Ng detection and 92% sensitivity and 99% specificity for gyrA-S91F detection. Relative to TMA results, 85% Ng detection sensitivity and 99% specificity were found. Regarding the 200 prospectively analyzed clinical samples, 13 were Ng positive, of which 10 were also tested for antibiotic susceptibility by culture. The kit showed concordance for GyrA-S91F detection in 9 of 10 samples. Ng was detected in 96% and 94% of vaginal and urine TMA-positive samples, in 84% of anal samples and only in 22% of pharyngeal samples. Discordant ciprofloxacin sensitivity was found for 2 of 26 characterized urine/anal sample pairs. CONCLUSION: The NG quantitative polymerase chain reaction (qPCR) kit can be implemented in diagnostic testing for vaginal, urine, and anal Ng TMA-positive samples to enable rapid prescription of oral ciprofloxacin.

Incidence and Clearance of Anal High-risk Human Papillomavirus Infections and Their Determinants Over 5 Years Among Human Immunodeficiency Virus-negative Men Who Have Sex With Men.

BACKGROUND: We aimed to assess the incidence and clearance of anal high-risk human papillomavirus (hrHPV) infections and determinants thereof among human immunodeficiency virus (HIV)-negative men who have sex with men (MSM) over a period of up to 5 years. METHODS: From 2010 to 2015, HIV-negative MSM were followed every 6 months. Anal self-swabs were collected at inclusion and every 6 months thereafter, and were HPV genotyped using the SPF10-PCR DEIA/LiPA25-system-v1. Incidence rates (IRs) and clearance rates (CRs) of incident anal hrHPV infections were assessed by hrHPV type (types 16, 18, 31, 33, 45, 52, and 58). Determinants of transitions between uninfected and infected states were assessed by hrHPV type using a time-homogenous multi-state Markov model. RESULTS: This study included 713 HIV-negative MSM, with a median age of 37 years (interquartile range [IQR] 31-43) and a median number of study visits of 6 (IQR 2-7). The IRs of anal infections had a median of 5.2 per 100 person-years (range: 2.2-7.9) across types, with HPV16 having the highest IR. The CRs of incident anal hrHPV infections had a median of 53.7 per 100 person-years (range: 33.4-65.3) across types, with HPV16 having the lowest CR. Having had over 100 lifetime sex partners was significantly associated with incident anal hrHPV infections in multivariable analyses. CONCLUSIONS: The high incidence and low clearance rates of anal HPV16 infection, compared to other hrHPV types, is consistent with HPV16 being implicated in the large majority of anal cancer cases.

National prevalence estimates of chlamydia and gonorrhoea in the Netherlands.

OBJECTIVES: National prevalence estimates of Chlamydia trachomatis (chlamydia) and Neisseria gonorrhoeae (gonorrhoea) are important for providing insights in the occurrence and control of these STIs. The aim was to obtain national prevalence estimates for chlamydia and gonorrhoea and to investigate risk factors associated with infection. METHODS: Between November 2016 and January 2017, we performed a national population-based cross-sectional probability sample survey among men and women aged 18-34 years in the Netherlands. Individuals were invited to complete a questionnaire about sexual health. At the end of the questionnaire, sexually active individuals could request a home-based sampling kit. Samples were tested for chlamydia and gonorrhoea using nucleic acid amplification test (NAAT). Logistic regression analyses were performed for predictors of participation and chlamydia infection. RESULTS: Of the 17 222 invited individuals, 4447 (26%) participated. Of these, 3255 were eligible for prevalence survey participation and 550 (17%) returned a sample. Participation in the prevalence survey was associated with age (20+) and risk factors for STI. We did not detect any gonorrhoea. The overall weighted prevalence of chlamydia was 2.8% (95% CI 1.5% to 5.2%); 1.1% (0.1% to 7.2%) in men and 5.6% (3.3% to 9.5%) in women. Risk factors for chlamydia infections in women aged 18-24 years were low/medium education level, not having a relationship with the person you had most recent sex with and age at first sex older than 16. CONCLUSIONS: Chlamydia and gonorrhoea prevalence were low in the general Dutch population, as was the participation rate. Repeated prevalence surveys are needed to analyse trends in STI prevalences and to evaluate control policies.

Ceftriaxone Reduced Susceptible Neisseria gonorrhoeae in the Netherlands, 2009 to 2017: From PenA Mosaicism to A501T/V Nonmosaicism.

OBJECTIVES: To compare molecular and epidemiological differences between ceftriaxone-reduced susceptible (CRO-RS) and ceftriaxone-susceptible (CRO-S) N. gonorrhoeae (Ng) and to study the genetic relatedness of CRO-RS isolates. METHODS: Demographic and clinical data and samples for cultures were routinely collected from gonorrhoea patients visiting the Amsterdam STI clinic in 2009 to 2017. Ng multiantigen sequence typing (NG-MAST) and penA types were compared between CRO-RS and CRO-S Ng (frequency matched on year of isolation and sexual risk group). Minimum spanning trees were produced based on multilocus variable number of tandem repeats analysis for Ng (NG-MLVA) genotypes. RESULTS: We selected 174 CRO-RS isolates (minimum inhibitory concentration, ≥0.064 mg/L) and 174 CRO-S isolates (minimum inhibitory concentration, ≤0.016 mg/L). Demographic and clinical characteristics of patients were overall comparable between those infected with CRO-RS Ng and CRO-S Ng. However, CRO-RS isolates were more often collected from the pharyngeal site (odds ratios [OR], 3.64; P < 0.001), and patients with CRO-RS Ng were less often human immunodeficiency virus (HIV) and syphilis positive (OR, 0.63; P = 0.041 and OR, 0.58; P = 0.028, respectively). We identified 12 clusters based on NG-MLVA genotypes, including 3 large (>25 isolates) clusters predominantly containing CRO-RS isolates. Those from cluster 1 (n = 32) were mostly from 2009 to 2012 (n = 24; 75.0%), with a mosaic penA XXXIV pattern (n = 27; 84.4%) and belonging to NG-MAST genogroup G1407 (n = 24; 75.0%). Isolates from cluster 2 (n = 29) were mostly from 2013 to 2015 (n = 24; 82.7%), had a nonmosaic penA IX + A501T mutation (n = 22; 75.9%) and NG-MAST G2400 (n = 14; 48.3%). Most isolates from cluster 3 (n = 37) were from 2015 to 2017 (n = 26; 70.2%), had a nonmosaic penA IV + A501V mutation (n = 24; 64.9%) and NG-MAST G2318 (n = 22; 59.5%). CONCLUSIONS: We observed a shift in the predominant penA (from mosaic toward nonmosaic plus A501T/V mutation), NG-MAST and NG-MLVA types among CRO-RS Ng over time. This indicates a successive spread of different CRO-RS Ng clones.

Genomic analyses of the Chlamydia trachomatis core genome show an association between chromosomal genome, plasmid type and disease.

BACKGROUND: Chlamydia trachomatis (Ct) plasmid has been shown to encode genes essential for infection. We evaluated the population structure of Ct using whole-genome sequence data (WGS). In particular, the relationship between the Ct genome, plasmid and disease was investigated. RESULTS: WGS data from 157 Ct isolates deposited in the Chlamydiales pubMLST database ( http://pubMLST.org/chlamydiales/ ) were annotated with 902 genes including the core and accessory genome. Plasmid associated genes were annotated and a plasmid MLST scheme was defined allowing plasmid sequence types to be determined. Plasmid allelic variation was investigated. Phylogenetic relationships were examined using the Genome Comparator tool available in pubMLST. Phylogenetic analyses identified four distinct Ct core genome clusters and six plasmid clusters, with a strong association between the chromosomal genotype and plasmid. This in turn was linked to ompA genovars and disease phenotype. Horizontal genetic transfer of plasmids was observed for three urogenital-associated isolates, which possessed plasmids more commonly found in isolates resulting from ocular infections. The pgp3 gene was identified as the most polymorphic plasmid gene and pgp4 was the most conserved. CONCLUSION: A strong association between chromosomal genome, plasmid type and disease was observed, consistent with previous studies. This suggests co-evolution of the Ct chromosome and their plasmids, but we confirmed that plasmid transfer can occur between isolates. These data provide a better understanding of the genetic diversity occurring across the Ct genome in association with the plasmid content.

Lactobacillus iners-dominated vaginal microbiota is associated with increased susceptibility to Chlamydia trachomatis infection in Dutch women: a case-control study.

INTRODUCTION: This prospective study aimed to study the composition and structure of the vaginal microbiota prior to Chlamydia trachomatis infection. METHODS: A nested case-control study was performed in 122 women, half of which acquired C. trachomatis within a year after their first visit. At the first visit, the composition and structure of vaginal microbial communities were analysed using 16S rRNA sequencing in the context of the sociodemographic and sexual risk behaviour information using logistic regression. RESULTS: Five vaginal community state types (CSTs) were identified. Four CSTs were dominated by Lactobacillus spp., of which L. crispatus (37%) and L. iners (33%) were the most common. One CST was characterised by the absence of Lactobacillus spp. (25%) and the presence of an array of strict and facultative anaerobes. Multivariate logistic regression analysis revealed that women with a L. iners-dominated CST had an increased risk of C. trachomatis infection (p=0.04; OR: 2.6, 95% CI 1.0 to 6.6). CONCLUSIONS: The distribution of CSTs dominated by Lactobacillus spp. agreed with previous studies. However, the frequency of dysbiosis among Caucasian women was relatively high (24%). Having vaginal microbiota dominated by L. iners was associated with an increased risk for C. trachomatis infection. Therefore, we hypothesise that specific signatures of vaginal microbiota are indicative of increased host predisposition to acquiring STIs.

Stable Low Hepatitis C Virus Antibody Prevalence Among HIV-Negative Men Who Have Sex With Men Attending the Sexually Transmitted Infection Outpatient Clinic in Amsterdam, 2007 to 2017.

BACKGROUND: In response to the increased hepatitis C virus (HCV) prevalence recently found among participants of the Amsterdam preexposure prophylaxis demonstration project, we evaluated HCV prevalence over time and the performance of the HCV-MOSAIC risk score for detection of HCV infection in HIV-negative men who have sex with men (MSM) attending the Amsterdam sexually transmitted infection (STI) clinic. METHODS: In October 2016, HIV-negative MSM were tested for anti-HCV and HCV RNA and completed the HCV-MOSAIC risk score. Anti-HCV prevalence was compared with that found in cross-sectional studies at the Amsterdam STI clinic (2007-2017). The time trend in HCV prevalence was modeled via logistic regression. The performance of the HCV-MOSAIC risk score, adjusted to identify prevalent HCV infection, was evaluated by calculating sensitivity and specificity. RESULTS: Of 504 HIV-negative MSM tested in October 2016, 5 were anti-HCV positive (1.0%, 95% confidence interval [CI], 0.4%-2.3%) and all were HCV RNA negative. Sensitivity and specificity of the adjusted HCV-MOSAIC risk score for prevalent infection were 80.0% (95% CI, 37.6%-96.4%) and 56.1% (95% CI, 51.7%-60.4%), respectively. The overall anti-HCV prevalence among 3264 HIV-negative MSM participating in cross-sectional studies at the Amsterdam STI clinic (2007-2017) was 0.8% (95% CI, 0.5%-1.2%) and did not change over time (P = 0.55). CONCLUSIONS: Anti-HCV prevalence among HIV-negative MSM attending the Amsterdam STI clinic in October 2016 was 1.0% and remained stable over time. We would therefore not recommend routine HCV screening of HIV-negative MSM at the STI clinic. However, given the increased prevalence among MSM using preexposure prophylaxis, periodic monitoring of HCV prevalence remains important.

The Cervicovaginal Microbiota in Women Notified for Chlamydia trachomatis Infection: A Case-Control Study at the Sexually Transmitted Infection Outpatient Clinic in Amsterdam, The Netherlands.

BACKGROUND: Increasing evidence suggests that the cervicovaginal microbiota (CVM) plays an important role in acquiring sexually transmitted infections (STIs). Here we study the CVM in a population of women notified by a sex partner for Chlamydia trachomatis infection. METHODS: We included 98 women who were contact-traced by C. trachomatis-positive sex partners at the STI outpatient clinic in Amsterdam, the Netherlands, and analyzed their cervicovaginal samples and clinical data. CVMs were characterized by sequencing the V3/V4 region of the 16S ribosomal RNA gene and by hierarchical clustering. Characteristics associating with C. trachomatis infection were examined using bivariable and multivariable logistic regression analysis. RESULTS: The CVM was characterized for 93 women, of whom 52 tested C. trachomatis positive and 41 C. trachomatis negative. We identified 3 major CVM clusters. Clustered CVM predominantly comprised either diverse anaerobic bacteria (n = 39 [42%]), Lactobacillus iners (n = 32 [34%]), or Lactobacillus crispatus (n = 22 [24%]). In multivariable analysis, we found that CVM was significantly associated with C. trachomatis infection (odds ratio [OR], 4.2 [95% confidence interval {CI}, 1.2-15.4] for women with diverse anaerobic CVM and OR, 4.4 [95% CI, 1.3-15.6], for women with L. iners-dominated CVM, compared with women with L. crispatus-dominated CVM), as was younger age (OR, 3.1 [95% CI, 1.1-8.7] for those ≤21 years old) and reporting a steady sex partner (OR, 3.6 [95% CI, 1.4-9.4]). CONCLUSIONS: Women who tested positive for Chlamydia trachomatis infection after having been contact-traced by a chlamydia-positive partner were more likely to have CVM dominated by L. iners or by diverse anaerobic bacteria, than by L. crispatus.