Detection of Putative Virulence Genes alr, goiB, and goiC in Mycoplasma hominis Isolates from Austrian Patients.

In Mycoplasma hominis, two genes (alr and goiB) have been found to be associated with the invasion of the amniotic cavity, and a single gene (goiC) to be associated with intra-amniotic infections and a high risk of preterm birth. The syntopic presence of Ureaplasma spp. in the same patient has been shown to correlate with the absence of goiC in M. hominis. The aim of our study was to investigate the presence of alr, goiB, and goiC genes in two groups of M. hominis isolates collected from symptomatic and asymptomatic male and non-pregnant female patients attending an Outpatients Centre. Group A consisted of 26 isolates from patients with only M. hominis confirmed; group B consisted of 24 isolates from patients with Ureaplasma spp. as the only co-infection. We extracted DNA from all M. hominis isolates and analysed the samples for the presence of alr, goiB, and goiC in a qPCR assay. Additionally, we determined their cytotoxicity against HeLa cells. We confirmed the presence of the alr gene in 85% of group A isolates and in 100% of group B isolates; goiB was detected in 46% of the samples in both groups, whereas goiC was found in 73% of group A and 79% of group B isolates, respectively. It was shown that co-colonisation with Ureaplasma spp. in the same patient had no effect on the presence of goiC in the respective M. hominis isolate. We did not observe any cytotoxic effect of the investigated isolates on human cells, regardless of the presence or absence of the investigated genes.

Characterisation of Trichomonas vaginalis Isolates Collected from Patients in Vienna between 2019 and 2021.

Trichomonas vaginalis (TV) is the causative agent of trichomoniasis, the most common nonviral sexually transmitted disease. TV can carry symbionts such as Trichomonas vaginalis virus (TVV) or Mycoplasma hominis. Four distinct strains of TV are known: TVV1, TVV2, TVV3, and TVV4. The aim of the current study was to characterise TV isolates from Austrian patients for the presence of symbionts, and to determine their effect on metronidazole susceptibility and cytotoxicity against HeLa cells. We collected 82 TV isolates and detected presence of TVV (TVV1, TVV2, or TVV3) in 29 of them (35%); no TVV4 was detected. M. hominis was detected in vaginal/urethral swabs by culture in 37% of the TV-positive patients; M. hominis DNA was found in 28% of the TV isolates by PCR. In 15% of the patients, M. hominis was detected in the clinical samples as well as within the respective TV isolates. In 22% of the patients, M. hominis was detected by culture only. In 11 patients, M. hominis was detected only within the respective cultured TV isolates (13%), while the swab samples were negative for M. hominis. Our results provide a first insight into the distribution of symbionts in TV isolates from Austrian patients. We did not observe significant effects of the symbionts on metronidazole susceptibility, cytotoxicity, or severity of symptoms.

Substantial underdiagnosis of lymphogranuloma venereum in men who have sex with men in Europe: preliminary findings from a multicentre surveillance pilot.

OBJECTIVES: Understanding the public health impact of lymphogranuloma venereum (LGV) in Europe is hampered by inadequate diagnostics and surveillance systems in many European countries. We developed and piloted LGV surveillance in three European countries without existing systems and performed a preliminary investigation of LGV epidemiology, where little evidence currently exists. METHODS: We recruited STI or dermatovenereology clinics and associated laboratories serving men who have sex with men (MSM) in Austria, Croatia and Slovenia, using the UK for comparison. We undertook centralised LGV testing of Chlamydia trachomatis (CT)-positive rectal swabs collected between October 2016 and May 2017 from MSM attending these clinics. Stored specimens from Austria (2015-2016) and Croatia (2014) were also tested. Clinical and sociodemographic data were collected using a standardised proforma. The ompA gene of LGV-positive specimens was sequenced. RESULTS: In total, 500 specimens from CT-positive MSM were tested, and LGV positivity was 25.6% (128/500; 95% CI 22.0% to 29.6%) overall, and 47.6% (79/166; 40.1% to 55.2%) in Austria, 20.0% (3/15; 7.1% to 45.2%) in Croatia, 16.7% (1/6; 3.0% to 56.4%) in Slovenia and 14.4% (45/313; 10.9% to 18.7 %) in the UK. Proformas were completed for cases in Croatia, Slovenia and in the UK; proformas could not be completed for Austrian cases, but limited data were available from line listings. Where recorded, 83.9% (78/93) of LGV-CT cases were HIV-positive compared with 65.4% (149/228) of non-LGV-CT cases; MSM with LGV-CT were more likely to have proctitis (Austria, 91.8% vs 40.5%, p<0.001; Croatia, 100% vs 25%, p=0.04; UK, 52.4% vs 11.7%, p<0.001) than those with non-LGV-CT. Six different ompA sequences were identified, including three new variants; the L2 ompA sequence predominated (58.6%, 51/87). CONCLUSIONS: LGV is substantially underdiagnosed in MSM across Europe. Unified efforts are needed to overcome barriers to testing, establish effective surveillance, and optimise diagnosis, treatment and prevention.

The Changing Spectrum of Sexually Transmitted Infections in Europe.

As long as 400 years ago, syphilitic ulcers and gonococcal discharge were observed in connection with sexual intercourse. War, poverty, and lack of efficient therapeutic options led to a high incidence of venereal diseases, many of which had devastating outcomes. This situation continued until the beginning of the 20th century, when the microbial aetiology of venereal diseases was discovered. The infection rate dropped with the availability of antibiotic therapy after the Second World War. However, since the beginning of the 21st century, a steady increase in sexually transmitted infections (STIs) has been recognized worldwide. The number of reported cases of syphilis is increasing in Europe, especially in men having sex with men (MSM). Antibiotic resistance in several genital pathogens, such as Neisseria gonorrhoeae and Mycoplasma genitalium, causes therapeutic problems. Viral genital infections have become a therapeutic challenge, especially for prevention of STIs. Due to better knowledge of the long-term consequences of STIs and the connection between genital cancer and papillomavirus infections, sexual health services with screening programmes have been established in many European countries. There is general awareness of the importance of human papilloma virus vaccination programmes for young adolescents as a preventive strategy for genital cancer.

Ten years of external quality assessment (EQA) of Neisseria gonorrhoeae antimicrobial susceptibility testing in Europe elucidate high reliability of data.

BACKGROUND: Confidence in any diagnostic and antimicrobial susceptibility testing data is provided by appropriate and regular quality assurance (QA) procedures. In Europe, the European Gonococcal Antimicrobial Susceptibility Programme (Euro-GASP) has been monitoring the antimicrobial susceptibility in Neisseria gonorrhoeae since 2004. Euro-GASP includes an external quality assessment (EQA) scheme as an essential component for a quality-assured laboratory-based surveillance programme. Participation in the EQA scheme enables any problems with the performed antimicrobial susceptibility testing to be identified and addressed, feeds into the curricula of laboratory training organised by the Euro-GASP network, and assesses the capacity of individual laboratories to detect emerging new, rare and increasing antimicrobial resistance phenotypes. Participant performance in the Euro-GASP EQA scheme over a 10 year period (2007 to 2016, no EQA in 2013) was evaluated. METHODS: Antimicrobial susceptibility category and MIC results from the first 5 years (2007-2011) of the Euro-GASP EQA were compared with the latter 5 years (2012-2016). These time periods were selected to assess the impact of the 2012 European Union case definitions for the reporting of antimicrobial susceptibility. RESULTS: Antimicrobial susceptibility category agreement in each year was ≥91%. Discrepancies in susceptibility categories were generally because the MICs for EQA panel isolates were on or very close to the susceptibility or resistance breakpoints. A high proportion of isolates tested over the 10 years were within one (≥90%) or two (≥97%) MIC log2 dilutions of the modal MIC, respectively. The most common method used was Etest on GC agar base. There was a shift to using breakpoints published by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) in the latter 5 years, however overall impact on the validity of results was limited, as the percentage categorical agreement and MIC concordance changed very little between the two five-year periods. CONCLUSIONS: The high level of comparability of results in this EQA scheme indicates that high quality data are produced by the Euro-GASP participants and gives confidence in susceptibility and resistance data generated by laboratories performing decentralised testing.

WGS analysis and molecular resistance mechanisms of azithromycin-resistant (MIC >2 mg/L) Neisseria gonorrhoeae isolates in Europe from 2009 to 2014.

OBJECTIVES: To elucidate the genome-based epidemiology and phylogenomics of azithromycin-resistant (MIC >2 mg/L) Neisseria gonorrhoeae strains collected in 2009-14 in Europe and clarify the azithromycin resistance mechanisms. METHODS: Seventy-five azithromycin-resistant (MIC 4 to >256 mg/L) N. gonorrhoeae isolates collected in 17 European countries during 2009-14 were examined using antimicrobial susceptibility testing and WGS. RESULTS: Thirty-six N. gonorrhoeae multi-antigen sequence typing STs and five phylogenomic clades, including 4-22 isolates from several countries per clade, were identified. The azithromycin target mutation A2059G (Escherichia coli numbering) was found in all four alleles of the 23S rRNA gene in all isolates with high-level azithromycin resistance (n = 4; MIC ≥256 mg/L). The C2611T mutation was identified in two to four alleles of the 23S rRNA gene in the remaining 71 isolates. Mutations in mtrR and its promoter were identified in 43 isolates, comprising isolates within the whole azithromycin MIC range. No mutations associated with azithromycin resistance were found in the rplD gene or the rplV gene and none of the macrolide resistance-associated genes [mef(A/E), ere(A), ere(B), erm(A), erm(B), erm(C) and erm(F)] were identified in any isolate. CONCLUSIONS: Clonal spread of relatively few N. gonorrhoeae strains accounts for the majority of the azithromycin resistance (MIC >2 mg/L) in Europe. The four isolates with high-level resistance to azithromycin (MIC ≥256 mg/L) were widely separated in the phylogenomic tree and did not belong to any of the main clades. The main azithromycin resistance mechanisms were the A2059G mutation (high-level resistance) and the C2611T mutation (low- and moderate-level resistance) in the 23S rRNA gene.

Gonococcal infections in Austria: a long-term observation of prevalence and resistance profiles from 1999 to 2014.

BACKGROUND AND OBJECTIVES: The increase in minimum inhibitory concentrations (MICs) of cephalosporins for Neisseria gonorrhoeae has given rise to concerns regarding potentially untreatable gonococcal infections. The goal was to ascertain the prevalence of gonorrhea in a Viennese patient group and determine resistance patterns. Another objective was to evaluate resistance profiles and MIC values of gonococcal isolates in an Austria-wide surveillance project. PATIENTS AND METHODS: From 1999 to 2014, 350,000 individuals were tested for gonococci at the Viennese Outpatient Clinic. In addition, from 2010 to 2014, the MICs of recommended antibiotics was determined in 3,584 gonococcal isolates, initially by agar dilution and breakpoint determination, and, from 2012 onwards, by Etest®. RESULTS: During the observation period, the prevalence of gonorrhea increased eightfold, with a significantly greater number of quinolone, penicillin, and tetracycline- resistant strains. In gonococcal strains isolated from across Austria, there was an increase in cefixime and ceftriaxone MICs toward breakpoints. Twenty-one isolates showed cefixime resistance, and while there was an increase in azithromycin resistance from 0.9 % (2013) to 3.2 % (2014), no resistance to ceftriaxone was observed. CONCLUSION: Currently, there is no imminent risk of untreatable gonorrhea in Austria. However, continuing the use of gonococcal cultures as a diagnostic tool for establishing resistance profiles is essential in order to monitor trends in the development of Neisseria (N.) gonorrhoeae resistance.

The Prevalence of Genital Mycoplasmas and Coinfection with Trichomonas vaginalis in Female Patients in Vienna, Austria.

Trichomonas vaginalis causes trichomoniasis, the most recurrent sexually transmitted infection (STI) worldwide. Genital mycoplasmas, not considered STI agents, are frequently isolated from the female genital tract. A symbiosis between Mycoplasma species and T. vaginalis has been described. The aim of this study was to conduct molecular-based analyses of vaginal specimens, thus assessing the prevalence of non-STI Mycoplasma infections. In total, 582 samples from female patients and an additional 20 T. vaginalis isolates were analyzed by PCR using Mycoplasma specific 16S rRNA primers, and the obtained PCR products were sequenced. Mycoplasma species were detected in 28.2% of the collected vaginal samples. Mycoplasma hominis was found in 21.5% of the specimens, Ureaplasma species were found in 7.5% of the samples. The molecular data of the newly described species, CandidatusMycoplasma girerdii, were obtained for the first time in Austria, in a sample also positive for T. vaginalis. Analyses of the cultivated T. vaginalis strains confirmed the presence of M. hominis in two out of 20 samples. A comparably high prevalence of genital mycoplasmas was revealed through advanced diagnostic assays, with M. hominis and U. parvum being the most prevalent species. The previously described symbiotic relationship between M. hominis and T. vaginalis was confirmed.

European surveillance of antimicrobial resistance in Neisseria gonorrhoeae.

OBJECTIVE: To perform a European sentinel surveillance study for antimicrobial resistance (AMR) in Neisseria gonorrhoeae as part of the European Surveillance of Sexually Transmitted Infections Programme. METHODS: From 2006 to 2008 17 countries participated in the AMR surveillance programme. The susceptibility of a total of 3528 consecutive isolates was tested using the agar dilution breakpoint technique or Etests for ciprofloxacin, penicillin, tetracycline, azithromycin, spectinomycin and ceftriaxone. Nitrocefin was used to detect ß-lactamase activity. RESULTS: Rates of resistance to ciprofloxacin, the previously recommended treatment, were high across Europe (42-52%), indicating that usage is no longer appropriate. Although resistance to the currently recommended treatment, ceftriaxone, was not demonstrated, a concerning upward drift in the minimal inhibitory concentration (MIC) distribution was identified since an earlier European study in 2004. No resistance to spectinomycin was seen, whereas azithromycin resistance varied from 2% to 7% and isolates from Scotland (n=4) and Ireland (n=1) showed high-level resistance (MIC >256 mg/l). High-level resistance to tetracycline and penicillin remained relatively constant at 16% and 12%, respectively. CONCLUSIONS: AMR is an ongoing problem in Europe, with high rates of resistance to many previously recommended therapeutic agents observed in many European countries. Continual European and global surveillance of AMR in N gonorrhoeae is essential to monitor for increasing, emerging and high-level resistance to therapeutically relevant agents and to inform treatment guidelines so optimum treatments are administered.

Lymphogranuloma venereum outbreak in Europe.

Lymphogranuloma venereum (LGV) is a venereal disease caused by Chlamydia trachomatis biovars L1 to L3. Unlike other anogenital C. trachomatis infections, LGV preferably affects lymphatic tissue after invasion through an epithelial surface. LGV has been considered an exotic tropical disease in Europe. This changed in 2003 as there was an outbreak of LGV in Rotterdam followed by additional reports from other European countries and North America. Most patients were HIV-positive men who presented with proctitis. Most of these patients were infected by C. trachomatis L2b biovar, a variant that was first identified in patients from Amsterdam. This review will address the recent developments of the LGV outbreak in Europe and discuss epidemiology, clinical manifestations, new subtypes of LGV genotypes and appropriate diagnostic measures.

The accuracy and efficacy of screening tests for Chlamydia trachomatis: a systematic review.

Screening women for lower genital tract infection with Chlamydia trachomatis is important in the prevention of pelvic inflammatory disease, ectopic pregnancy and infertility. This systematic review aims to state clearly which of the available diagnostic tests for the detection of C. trachomatis would be most effective in terms of clinical effectiveness. The review included all studies published from 1990 onward that evaluated diagnostic tests in asymptomatic, young, sexually active populations. Medline and Embase were searched electronically and key journals were hand-searched. Further studies were identified through the Internet and contact with experts in the field. All studies were reviewed by two reviewers and were scored by Irwig's assessment criteria. Additional quality assessment criteria included a documented sexual history and recording of previous chlamydial infection. The reviews were subjected to meta-analysis and meta-regression. The 30 studies that were included examined three types of DNA-based test--ligase chain reaction (LCR), PCR and gene probe--as well as enzyme immuno-assay (EIA). The results showed that while specificities were high, sensitivities varied widely across the tests and were also dependent on the specimen tested. Pooled sensitivities for LCR, PCR, gene probe and EIA on urine were 96.5%, 85.6%, 92% and 38%, respectively, while on cervical swabs the corresponding sensitivities of PCR, gene probe and EIA were 88.6%, 84% and 65%. Meta-analysis demonstrated that DNA amplification techniques performed best for both urine and swabs in low prevalence populations. We conclude that nucleic acid amplification tests used on non-invasive samples such as urine are more effective at detecting asymptomatic chlamydial infection than conventional tests, but there are few data to relate a positive result with clinical outcome.

New Chlamydia trachomatis L2 strains identified in a recent outbreak of lymphogranuloma venereum in Vienna, Austria.

BACKGROUND: Since 2003, an ongoing outbreak of lymphogranuloma venereum (LGV), caused by Chlamydia trachomatis biovar L2b, has been reported among men who have sex with men. METHODS: Twenty-four samples positive for C. trachomatis were analyzed for specific biovars and genovariants by genotyping of the variable segment (VS) 4, VS2 and VS1 regions of the outer membrane protein (omp) A. In addition we assessed the patients' sociodemographic background and clinical signs and symptoms. RESULTS: Twenty-four men who have sex with men presented with either anorectal or inguinal symptoms and tested positive for C. trachomatis DNA. Of these, the L2 genotype accounted for 15 patients, with a high coinfection rate with HIV (73.3%) and other sexually transmitted infections (53.4%). Analysis of the VS1, VS2, and VS4 regions of the ompA gene revealed the variant L2b in 8 patients. In 4 patients, 3 new L2 sequences were identified with nucleotide changes in the VS1, VS2, and VS4 region, respectively, defining new strains designated L2c, d, e. CONCLUSIONS: This outbreak of LGV represents the further spread of C. trachomatis L2 infection. Sequence analysis of ompA regions shows heterogeneity of L2 variants, suggesting more than 1 source of the LGV infections diagnosed in Vienna.

Detection of Trichomonas vaginalis on modified Columbia agar in the routine laboratory.

Broth culture of Trichomonas vaginalis is considered the "gold standard" for the diagnosis of trichomoniasis. Two studies were carried out to evaluate modified Columbia agar (MCA) for the isolation of T. vaginalis from clinical samples. Study I compared isolation on MCA to that on liquid medium with 889 vaginal samples. Out of 63 samples positive for T. vaginalis (7.1% of total), MCA identified 62 (98.4%) and broth identified 58 (92.1%). In study II, trichomoniasis was diagnosed within the scope of a screening program for a total of 39,585 men and women by culture on MCA and direct microscopy. Culture on MCA detected 199 (98.5%) and Gram staining detected 163 (80.7%) of 202 positive specimens. Wet-mount preparations used for symptomatic patients identified 103 (92.8%) of 111 cases. Culture of T. vaginalis from clinical samples on MCA is highly sensitive and reliable, as well as timesaving, and therefore suitable for screening of symptomatic and asymptomatic individuals.

Efficacy of povidone-iodine vaginal suppositories in the treatment of bacterial vaginosis.

A prospective, randomized clinical trial was performed to study the efficacy of povidone iodine (Betadine) suppositories for the treatment of bacterial vaginosis (BV) in comparison to capsules containing lactobacilli (Döderlein Med). Seventy patients with BV included in the study were randomly assigned to be either treated with povidone iodine suppositories or lactobacilli. Patients were treated once a day for 5 days. Initial examinations took place on the first day of the study with follow-up examinations on days 8 and 15. The examinations included clinical parameters, patient evaluation, secretion screens, and quantitative and qualitative microbiological tests of vaginal flora. Both treatment groups showed improvement of clinical parameters, condition of secretions and subjective state of health. At day 15 there was a trend towards a better efficacy of the treatment with povidone iodine but this was not significant. However, patients with acute BV treated with povidone iodine had significantly better scores after 15 days. Both treatments were well tolerated. The microbiological examinations showed an increase of the mean number of lactobacilli in the vagina on day 8 after initiation of treatment with lactobacilli, but a decrease on day 15. Contrary to that the lactobacilli counts from patients treated with povidone iodine suppositories decreased after the first week but increased in the second one. Potentially pathogenic germs, e.g. Gardnerella, Bacteroides and Enterobacteria were reduced in a higher extent and with a longer lasting effect after treatment with povidone iodine suppositories than with capsules containing lactobacilli. The results of this study show that native lactobacilli rapidly re-colonize after the antiseptic treatment with povidone iodine. Therefore, there is no need to use lactobacilli in addition.