ObjectivesUnderstanding 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.MethodsWe 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.ResultsIn 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).ConclusionsLGV 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.

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.

There is mounting evidence stating that Ureaplasma urealyticum causes non-gonococcal urethritis in males, whereas Ureaplasma parvum does not seem to be of clinical significance. However, the clinical role of U. parvum and U. urealyticum in lower urogenital tract infections in females remains unclear. The aim of the study was to determine the frequency of U. parvum and U. urealyticum among 145 Ureaplasma spp. culture-positive women with symptoms of lower urogenital tract infection (n = 75) and those without (n = 70), and to determine possible associations between the detection of U. parvum and U. urealyticum with selected characteristics. Endocervical, urethral, and vaginal swabs, and first voided urine were obtained. Polymerase chain reaction (PCR) was performed to differentiate ureaplasmas. No significant association between the detection of U. parvum or U. urealyticum and symptom status was found. Significantly more women aged 25 years and younger were infected with U. urealyticum (23.4 %) compared to those aged above 25 years (9.2 %) [odds ratio (OR) 3.0 (1.1; 8.1); p = 0.03] and significantly less women aged 25 years and younger (83.5 %) were infected with U. parvum compared to those aged above 25 years (95.5 %) [OR 0.2 (0.1; 0.9); p = 0.03]. The detection of Chlamydia trachomatis was significantly associated to both U. parvum and U. urealyticum (p = 0.021), and to U. parvum alone with borderline significance (p = 0.063). Although neither U. parvum nor U. urealyticum seem to cause symptoms in females, their role in the female urogenital tract remains unknown, taking into account their ubiquity, possible augmentation of the urogenital microenvironment, and ascending capability to the sterile upper reproductive tract.

Two nationwide Mycoplasma pneumoniae epidemics occurred in Slovenia between 2006 and 2016. The aim of this study was to assess which M. pneumoniae genotypes were present in our area during the selected timeframe, whether the origin of the epidemics was monoclonal or polyclonal and whether the proportion between detected genotypes changed over time. We were also interested in the presence of macrolide resistance (MR) and whether it could be linked to specific genotypes. We performed pyrosequencing of the P1 gene and multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) typing from 872 M. pneumoniae isolates obtained from respiratory tract infections (RTI)-suffering patients. Additionally, isolates were tested for the presence of MR implicated mutations in the 23S rRNA gene. The MLVA typing results revealed that three main genotypes, MLVA-3,5,6,2, MLVA-3,6,6,2 and MLVA-4,5,7,2, were constantly present and occasionally joined by less abundant, short-lived genotypes, which were detected mostly, but not exclusively, during epidemics. We also noticed a switch in abundance from MLVA-3,5,6,2 and MLVA-3,6,6,2, which dominated in the first epidemic (77.0%; 97/126), to MLVA-4,5,7,2 (71.6%; 428/598), which dominated in the second. Similar to this finding, the dominant P1 type also shifted from type 2 to type 1, although a complete P1 type shift was not observed, since both types remained in circulation. MR was detected in 0.8% (7/872) of M. pneumoniae isolates. Our results seem to suggest that MR remains sporadic in Slovenia at this point in time and that both recent epidemics were polyclonal in nature and, possibly, to some extent, fuelled by the P1 type dominance change.

In this retrospective study we employed real-time polymerase chain reaction (PCR) to analyse the occurrence of Mycoplasma pneumoniae among upper and lower respiratory tract infections (RTI) in the Central Region of Slovenia between January 2006 and December 2014. We also used a culture and pyrosequencing approach to genotype strains and infer their potential macrolide resistance. Of a total 9,431 tested samples from in- and out-patient with RTI, 1,255 (13%) were found to be positive by M. pneumoniae PCR. The proportion of positive samples was 19% (947/5,092) among children (?16 years-old) and 7% (308/4,339) among adults (>16 years-old). Overall, among those PCR tested, the highest proportions of M. pneumoniae infections during the study period were observed in 2010 and 2014. In these two years, 18% (218/1,237) and 25% (721/2,844) of samples were positive respectively, indicating epidemic periods. From the 1,255 M. pneumoniae PCR-positive samples, 783 (614 from paediatric and 169 from adult patients) were successfully cultured. Of these, 40% (312/783) were constituted of strains belonging to the P1 type II genomic group, while 60% (469/783) contained strains of the P1 type I group. Two isolates comprised both P1 type I and II strains. Results of a genotype analysis by year, showed that the dominant M. pneumoniae P1 type during the 2010 epidemic was P1 type II (82% of isolates; 81/99), which was replaced by P1 type I in the 2014 epidemic (75%; 384/510). This observation could indicate that the two epidemics may have been driven by a type shift phenomenon, although both types remained present in the studied population during the assessed period of time. Only 1% of strains (7/783) were found to harbour an A2063G mutation in the 23S rRNA gene, which confers macrolide resistance, suggesting that the occurrence of M. pneumoniae macrolide resistance still seems to be sporadic in our geographic area.

Prompt detection of Legionella pneumophila is essential for rapid investigation of legionellosis. Furthermore, as the majority of L. pneumophila infections are caused by serogroup 1 (sg1) strains, rapid identification of such strains can be critical in both routine and outbreak scenarios. The ESCMID Study Group for Legionella Infections (ESGLI) was established in 2012 and immediately identified as a priority the validation of a reliable, easy to perform and interpret, cost-effective qPCR assay to standardise the detection of L. pneumophila DNA amongst members. A novel L. pneumophila assay targeting the mip gene was designed and combined with previously published methodologies amplifying the sg1 marker (wzm) and the green fluorescent protein gene (gfp) internal process control. The resulting triplex assay was validated internationally on the three qPCR platforms used by the majority of European Legionella reference laboratories: ABI 7500 (Life Technologies), LightCycler 480 Instrument II (Roche) and Rotor-Gene Q (Qiagen). Clinical and EQA specimens were tested together with a large panel of strains (251 in total) to validate the assay. The assay proved to be 100% specific for L. pneumophila and sg1 DNA both in silico and in vitro. Efficiency values for mip and wzm assays ranged between 91.97 and 97.69%. Limit of detection values estimated with 95% confidence were adopted for mip and wzm assays on all three qPCR platforms. Inhibition was not observed. This study describes a robust assay that could be widely implemented to standardise the molecular detection of L. pneumophila among ESGLI laboratories and beyond.

A laboratory-confirmed lymphogranuloma venereum (LGV) case in Slovenia was reported in 2015, in a human immunodeficiency virus (HIV)-negative man presenting with inguinal lymphadenopathy. He reported unprotected insertive anal intercourse with two male partners in Croatia. Variant L2c of Chlamydia trachomatis was detected in clinical samples. Although the patient was eventually cured, the recommended treatment regimen with doxycycline had to be prolonged.

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