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.

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.

Low prevalence of current and past SARS-CoV-2 infections among visitors and staff members of homelessness services in Amsterdam at the end of the second wave of infections in the Netherlands.

BACKGROUND: People experiencing homelessness (PEH) may be at increased risk of SARS-CoV-2 infection and severe COVID-19. The Dutch government established emergency shelters and introduced preventive measures for homelessness services. There were no major SARS-CoV-2 outbreaks noticed among PEH during the first two waves of infections. This study aimed to assess the prevalence of current and past infections among PEH and staff by conducting an on-site COVID-19 screening project at homelessness services in Amsterdam, the Netherlands. METHODS: We assessed the proportion of visitors and staff members of four homelessness services at two locations in Amsterdam with positive SARS-CoV-2 qPCR and antibody results (IgG/IgM Rapid Test/Biozek) in May 2021. We also assessed sociodemographic, clinical and lifestyle characteristics, compliance with basic prevention measures and intention to vaccinate against COVID-19 among PEH and staff. RESULTS: A total of 138 visitors and 53 staff members filled out a questionnaire and were tested. Among PEH, the SARS-CoV-2 positivity rate was 0% (0/133;95%CI = 0-1.9) and the antibody positivity rate was 1.6% (2/131;95%CI = 0.8-7.5) among those without prior COVID-19 vaccination. Among staff, these percentages were 3% (1/32;95%CI = 0.1-16.2) and 11% (5/53;95%CI = 3.6-23.6), respectively. Most participants were often compliant with the basic preventive measures 'not shaking hands', 'wearing a face mask' and 'washing hands', but not with 'physical distancing'. High vaccination intent was more common among staff members (55%) than among visitors (42%), while high trust in the governmental COVID-19 policies was more common among visitors (41%) than among staff (30%). CONCLUSIONS: We observed a low prevalence of past and current SARS-CoV-2 infections among PEH, which may be explained by instated shelter policies, limited daily activities of PEH and compliance with prevention measures. Vaccine hesitancy and mistrust among visitors and staff could hinder vaccination uptake, suggesting that interventions towards homelessness services are needed.

Putative transmission of extended-spectrum ß-lactamase-producing Escherichia coli among men who have sex with men in Amsterdam, the Netherlands.

OBJECTIVES: A previous study showed higher prevalence of Escherichia coli (E. coli) expressing extended-spectrum ß-lactamases (ESBL-Ec) among men who have sex with men (MSM) in Amsterdam, the Netherlands, compared with the general Dutch population. This study genetically characterised the ESBL-Ec isolates and investigated whether the increased prevalence could be explained by transmission between participants. METHODS: Whole-genome sequences were obtained from 93 unique ESBL-Ec isolates that were cultured from rectal swabs of 79 participants. Isolates were typed according to the Achtman MLST scheme and ESBL and virulence genes were identified. Pairwise SNP distances were determined between isolates. Isolate pairs with ≤ 25 SNPs were considered part of a putative transmission event, and events between multiple participants formed putative transmission clusters. To investigate whether putatively transmitted isolates belonged to globally expanded lineages, the level of hierarchical clustering with international isolates was assessed using core genome MLST (cgMLST) implemented on the Enterobase platform. RESULTS: The most frequently detected E. coli types were ST131:blaCTX-M-15 (16 of 117, 13.5%), ST131:blaCTX-M-27, ST3075:blaCTX-M-15 and ST14:blaSHV-12 (all six of 117, 6.5%). Fourteen putative transmission events were identified, forming four putative transmission clusters. The largest putative transmission cluster contained ST131 isolates, which clustered with multiple international isolates in SNP and cgMLST analysis. One other transmission cluster (ST14:blaSHV-12) and two transmission events (ST14:blaSHV-12 and ST394:blaCTX-M-15) contained very rarely reported strains. CONCLUSIONS: The identification of unique ESBL-Ec strains involved in putative transmission and carried by multiple participants demonstrated a high probability of ESBL-Ec transmission between MSM in Amsterdam; therefore, ESBL-Ec infection should be considered in cases of sexually active MSM having associated symptoms.

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.

Reptile-associated Salmonella urinary tract infection: a case report.

We present an 18-year-old woman with a urinary tract infection caused by Salmonella Oranienburg. S. Oranienburg was isolated from her pet snake and confirmed as the source of infection using whole genome sequencing. Our case demonstrates the risk of acquiring reptile-associated salmonellosis and stretches the need for awareness to prevent these infections.

The Neisseria gonorrhoeae Accessory Genome and Its Association with the Core Genome and Antimicrobial Resistance.

The bacterial accessory genome provides the genetic flexibility needed to facilitate environment and host adaptation. In Neisseria gonorrhoeae, known accessory elements include plasmids which can transfer and mediate antimicrobial resistance (AMR); however, chromosomal accessory genes could also play a role in AMR. Here, the gonococcal accessory genome was characterized using gene-by-gene approaches and its association with the core genome and AMR were assessed. The gonococcal accessory gene pool consisted of 247 genes, which were mainly genes located on large mobile genetic elements, phage associated genes, or genes encoding putative secretion systems. Accessory elements showed similar synteny across genomes, indicating either a predisposition for particular genomic locations or ancestral inheritance that are conserved during strain expansion. Significant associations were found between the prevalence of accessory elements and core genome multi-locus sequence types (cgMLST), consistent with a structured gonococcal population despite frequent horizontal gene transfer (HGT). Increased prevalence of putative DNA exchange regulators was significantly associated with AMR, which included a putative secretion system, methyltransferases and a toxin-antitoxin system. Although frequent HGT results in high genetic diversity in the gonococcus, we found that this is mediated by a small gene pool. In fact, a highly organized genome composition was identified with a strong association between the accessory and core genome. Increased prevalence of DNA exchange regulators in antimicrobial resistant isolates suggests that genetic material exchange plays a role in the development or maintenance of AMR. These findings enhance our understanding of gonococcal genome architecture and have important implications for gonococcal population biology. IMPORTANCE The emergence of antimicrobial resistance (AMR) against third generation cephalosporins in Neisseria gonorrhoeae is a major public health concern, as these are antibiotics of last resort for the effective treatment of gonorrhea. Although the resistance mechanisms against this class of antibiotics have not been entirely resolved, resistance against other classes of antibiotics, such as tetracyclines, is known to be mediated through plasmids, which are known gonococcal extra-chromosomal accessory elements. A complete assessment of the chromosomal accessory genome content and its role in AMR has not yet been undertaken. Here, we comprehensively characterize the gonococcal accessory genome to better understand genome architecture as well as the evolution and mechanisms of AMR in this species.

Within-Host Genetic Variation in Neisseria gonorrhoeae over the Course of Infection.

Knowledge of within-host genetic variation informs studies on transmission dynamics. We studied within-host genetic variation in Neisseria gonorrhoeae over the course of infection and across different anatomical locations. Isolates were obtained during a clinical trial, and isolates from consecutive time points reflected persistent infections after treatment failure. We compared sequence types (STs) and recombination unfiltered- and filtered core genome single nucleotide polymorphism (SNP) distances in 65 within-host isolate pairs from the same anatomical location over time-obtained with a median interval of 7 days-and 65 isolate pairs across different anatomical locations at one time point. Isolates with different Multi-Locus Sequence Types (MLST), NG-Sequence Types for Antimicrobial Resistance (NG-STAR) and NG-Multi Antigen Sequence Types (NG-MAST) had a median of 1466 recombination filtered SNPs, whereas a median of 1 SNP was found between isolates with identical STs or a different NG-MAST only. The threshold for differentiating between strains was set at 10 recombination filtered SNPs, showing that isolates from persistent infections could have different NG-MASTs. Antibiotic pressure applied through treatment did not lead to an increase in genetic variation in specific genes or in overall extent of variation, compared to variation across anatomical locations. Instead, within-host genetic variation was proposedly driven by the host immune response, as it was concentrated in genomic regions encoding surface exposed proteins involved in host-microbe interaction. Ultimately, 15/228 (6.5%) between-host pairs contained a single strain, suggesting between-host transmission. However, patient reported data are needed to differentiate within-host persistence from between-host transmission. IMPORTANCE Understanding transmission dynamics of Neisseria gonorrhoeae (Ng) is based on the identification of transmission events. These can be identified by assessing genetic relatedness between Ng isolates, expressed as core genome SNP distances. However, a SNP threshold to differentiate between strains needs to be defined, using knowledge on within- and between-host genetic variation. Here, we assessed within-host genetic variation, using a unique set of within-host Ng isolates from the same anatomical location over time or across different anatomical locations at one time point. The insights in genetic variation that occurred during the infection period contribute to the understanding of infection dynamics. In addition, the obtained knowledge can be used for future research on transmission dynamics and development of public health interventions based on bacterial genomic data.

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.

Self-reported symptoms as predictors of SARS-CoV-2 infection in the general population living in the Amsterdam region, the Netherlands.

INTRODUCTION: Most COVID-19 symptoms are non-specific and also common in other respiratory infections. We aimed to assess which symptoms are most predictive of a positive test for SARS-CoV-2 in symptomatic people of the general population who were tested. METHODS: We used anonymised data of all SARS-CoV-2 test results from the Public Health Service of Amsterdam from June 1,2020 through August 31, 2021. Symptoms were self-reported at time of requesting a test. Multivariable logistic regression models with generalized estimating equations were used to identify predictors of a positive test. Included symptoms were: cough, fever, loss of smell or taste, muscle ache, runny nose, shortness of breath, and throat ache; adjustments were made for age and gender, and stratification by month. RESULTS: Overall, 12.0% of 773,680 tests in 432,213 unique individuals were positive. All symptoms were significantly associated with a positive test result, the strongest positive associations were: cough (aOR = 1.78, 95%CI = 1.75-1.80), fever (aOR = 2.11, 95%CI = 2.07-2.14), loss of smell or taste (aOR = 2.55, 95%CI = 2.50-2.61), and muscle ache (aOR = 2.38, 95%CI = 2.34-2.43). The adjusted odds ratios for loss of smell or taste slightly declined over time, while that for cough increased. CONCLUSION: Cough, fever, loss of smell or taste, and muscle ache appear to be most strongly associated with a positive SARS-CoV-2 test in symptomatic people of the general population who were tested.

Enhanced IgA coating of bacteria in women with Lactobacillus crispatus-dominated vaginal microbiota.

BACKGROUND: Immunoglobulin A (IgA) plays an important role in maintaining a healthy intestinal microbiome, but little is known about the interaction between local immunoglobulins and the vaginal microbiome. We assessed immunoglobulins (unbound and bound to bacteria), their association with vaginal microbiota composition and the changes over time in 25 healthy women of reproductive age. RESULTS: In both Lactobacillus crispatus-dominated and non-L. crispatus-dominated microbiota, IgA and IgG (unbound and bound to bacteria) were higher during menses (T = 1) compared to day 7­11 (T = 2) and day 17­25 (T = 3) after menses onset. The majority of vaginal bacteria are coated with IgA and/or IgG. Women with L. crispatus-dominated microbiota have increased IgA coating of vaginal bacteria compared to women with other microbiota compositions, but contained less IgA per bacterium. Presence of a dominantly IgA-coated population at T = 2 and/or T = 3 was also strongly associated with L. crispatus-dominated microbiota. In women with non-L. crispatus-dominated microbiota, more bacteria were uncoated. Unbound IgA, unbound IgG, and bound IgG levels were not associated with microbiota composition. CONCLUSIONS: In conclusion, L. crispatus-dominated vaginal microbiota have higher levels of bacterial IgA coating compared to non-L. crispatus-dominated vaginal microbiota. Similar to its regulating function in the intestinal tract, we hypothesize that IgA is involved in maintaining L. crispatus-dominated microbiota in the female genital tract. This may play a role in L. crispatus-associated health benefits. Video abstract.

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.

Prevalence of fluoroquinolone resistance-associated mutations in Mycoplasma genitalium among clients of two sexual health centres in the Netherlands: a cross-sectional study.

OBJECTIVE: This study aimed to determine the prevalence of fluoroquinolone resistance-associated mutations (QRAMs) in Mycoplasma genitalium (MG) among clients of two sexual health centres (SHCs) in the Netherlands. DESIGN: A cross-sectional study. SETTING AND PARTICIPANTS: Between 2018 and 2019, 669 clients with MG were included from two previous studies: 375 male clients with urethritis from the SHC in Amsterdam; and 294 clients (male and female) from the SHC in Amsterdam and The Hague. Urogenital and anal samples (705 in total) that tested positive for MG by nucleic acid amplification tests were selected. OUTCOME MEASURES: The presence of QRAM was detected by an MG-QRAM PCR targeting four mutations in the parC gene and investigated by sequence analysis of relevant regions of the gyrA and parC genes. Possible risk factors for the presence of QRAM were investigated. RESULTS: We found QRAM in 58 of 669 (9%) clients with an MG infection: 36 of 375 (10%) in the study population of men with urethritis and 22 of 294 (7%) in the study population of other clients (including both men and women; p=0.334). Most prevalent mutations in the parC gene were S83I and D87N, occurring in 31 of 60 (52%) and 20 of 60 (33%) samples, respectively. Factors associated with the presence of QRAM were: men who have sex with men (adjusted OR (aOR) 3.4, 95% CI 1.7 to 6.9) and Asian origin (aOR 2.5, 95% CI 1.2 to 5.6). Multidrug resistance (QRAM plus macrolide resistance-associated mutations) was found in 46 of 669 (7%) clients. CONCLUSIONS: Nine per cent of MG-positive clients from two Dutch SHCs had QRAM. New treatment strategies and antibiotics are needed to treat symptomatic patients with multidrug-resistant MG.

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.

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.

Ongoing evolution of Chlamydia trachomatis lymphogranuloma venereum: exploring the genomic diversity of circulating strains.

Lymphogranuloma venereum (LGV), the invasive infection of the sexually transmissible infection (STI) Chlamydia trachomatis, is caused by strains from the LGV biovar, most commonly represented by ompA-genotypes L2b and L2. We investigated the diversity in LGV samples across an international collection over seven years using typing and genome sequencing. LGV-positive samples (n=321) from eight countries collected between 2011 and 2017 (Spain n=97, Netherlands n=67, Switzerland n=64, Australia n=53, Sweden n=37, Hungary n=31, Czechia n=30, Slovenia n=10) were genotyped for pmpH and ompA variants. All were found to contain the 9 bp insertion in the pmpH gene, previously associated with ompA-genotype L2b. However, analysis of the ompA gene shows ompA-genotype L2b (n=83), ompA-genotype L2 (n=180) and several variants of these (n=52; 12 variant types), as well as other/mixed ompA-genotypes (n=6). To elucidate the genomic diversity, whole genome sequencing (WGS) was performed from selected samples using SureSelect target enrichment, resulting in 42 genomes, covering a diversity of ompA-genotypes and representing most of the countries sampled. A phylogeny of these data clearly shows that these ompA-genotypes derive from an ompA-genotype L2b ancestor, carrying up to eight SNPs per isolate. SNPs within ompA are overrepresented among genomic changes in these samples, each of which results in an amino acid change in the variable domains of OmpA (major outer membrane protein, MOMP). A reversion to ompA-genotype L2 with the L2b genomic backbone is commonly seen. The wide diversity of ompA-genotypes found in these recent LGV samples indicates that this gene is under immunological selection. Our results suggest that the ompA-genotype L2b genomic backbone is the dominant strain circulating and evolving particularly in men who have sex with men (MSM) populations.

High prevalence and resistance rates of Mycoplasma genitalium among patients visiting two sexually transmitted infection clinics in the Netherlands.

Mycoplasma genitalium is a well-known cause of urethritis in men and has been associated with cervicitis, pelvic inflammatory disease, and adverse obstetric outcomes in women. In this cross-sectional study, we determined the current prevalence of M. genitalium infection and the rate of macrolide resistance in M. genitalium isolates, in patients visiting two large Dutch sexually transmitted infection (STI) clinics, to evaluate whether the recommendations in Dutch guidelines should be revised. In addition, risk factors for M. genitalium were identified. In total, 3225 patients were included. M. genitalium prevalence rates were 13.8% for all patients; 20.1% for men who have sex with men, 8.2% for men who have sex with women, and 12.6% for women. Macrolide resistance-associated mutations were detected in 66% of the patients infected with M. genitalium. Age, educational level, country of origin, number of sexual partners, HIV-positivity, infection with Neisseria gonorrhoeae, and urethral symptoms in men were independently associated with M. genitalium infection. In conclusion, we found very high prevalence rates and macrolide resistance rates of M. genitalium in patients visiting STI clinics.

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.