Genetic diversity and macrolide resistance of Mycoplasma pneumoniae isolates from two consecutive epidemics in Slovenia.

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.

Ureaplasma parvum and Ureaplasma urealyticum detected with the same frequency among women with and without symptoms of urogenital tract infection.

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.

Design and validation of a qPCR assay for accurate detection and initial serogrouping of Legionella pneumophila in clinical specimens by the ESCMID Study Group for Legionella Infections (ESGLI).

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.

Outbreak of Legionnaires' disease in a nursing home, Slovenia, August 2010: preliminary report.

We report an outbreak of Legionnaires' disease ina nursing home in Slovenia in August 2010 affecting 15 of 234 residents. To date, Legionnaires' disease has been confirmed in four patients. Further serum analyses and genotyping of isolates are ongoing. The building's water distribution system with dead end sections has been identified as the probable source of infection.

Chlamydia trachomatis infections in heterosexuals attending sexually transmitted disease clinics in Slovenia.

This study assessed the age and gender distribution of Chlamydia trachomatis infections among patients attending two clinics for sexually transmitted diseases (STDs) in Slovenia. Between January 1999 and December 2003, 1714 heterosexual male and 892 heterosexual female patients were tested for C. trachomatis. The prevalence of C. trachomatis infection was 19.5% (n = 334) for male patients and 10.7% (n = 96) for female patients, with the highest prevalence in the group aged 15-30 years. The prevalence decreased between 2000 and 2003 among female patients. The results support the implementation of routine screening for C. trachomatis genital infection among male and female patients aged < 30 years attending STD clinics in Slovenia.

Presence of Chlamydia pneumoniae DNA in the artery wall--biomarker of coronary artery disease.

Many authors have shown an association between Chlamydia pneumoniae (CPn) infection and coronary artery disease (CAD). However, whether CPn infection demonstrated by CPn DNA presence in the artery wall plays an important role in pathogenesis of CAD and acute coronary events (i.e. unstable angina) remains to be elucidated. One hundred and fifteen consecutive patients with CAD (51 with unstable angina and 64 with stable angina) were compared with 52 control subjects with aortic valve disease without angiographic evidence of CAD. The presence of CPn DNA in the aortic wall was assessed with nested polymerase chain reaction (PCR), and the IgM, IgG and IgA anti-CPn titres were assessed with microimmunofluorescence test. CPn DNA presence in the artery (i.e. aortic) wall was associated with 3.7-fold increased risk of CAD (95% CI 1.2-11.3, P < 0.01); however, no statistically significant difference in CPn DNA presence was demonstrated between unstable and stable angina (17.6% vs. 25%). In the CPn DNA positive group more often than in the CPn DNA negative group, serological signs of chronic infection (55.2% vs. 27%, P = 0.004) were demonstrated, whereas no statistically significant differences were demonstrated in prevalence of either acute infection (9.3% vs. 0%) or reinfection (0% vs. 0%). In conclusion, CPn DNA presence in the artery (i.e. aortic) wall was associated with CAD, therefore may be used as a biomarker for CAD. Moreover, no statistically significant differences in CPn DNA presence in the artery wall and in serology were present between unstable and stable angina; therefore, CPn infection does not seem implicated in triggering an acute coronary event.

Long-term study of Chlamydophilosis in Slovenia.

Immune reactivity for Chlamydophila (C.) psittaci in Slovenia was monitored in parrots, canaries, finches and nine species of recently captured free-living birds (house sparrows, Eurasian goldfinches, tree sparrows, chaffinches, European greenfinches, European serines, Eurasian siskins, Eurasian linnets and Eurasian bullfinches) in the period from 1991 to 2001. In subsequent years, specific IgG antibodies were found using immunofluorescence in parrots (0.7-53.6%), canaries (0.0-3.5%), finches (0.0-5.7%) and in captured free-living birds (33.3% of Eurasian goldfinches in 1994). An experimental infection with C psittaci was performed in order to study clinical signs and pathological changes in canaries and finches. The C. psittaci strain used for experimental infection was isolated from a cockatiel (Nymphicus hollandicus). Chlamydial DNA was extracted from clinical material followed by RFLP-PCR analysis. Infection of canaries and finches was confirmed in organ smears by direct immunofluorescence and a modified Gimenez staining method. In addition, serological tests of indirect immunofluorescence and complement fixation were applied. However, in spite of positive immunological reaction there were no clinical signs three weeks after infection. The present study includes results of a serological survey of persons belonging to the most important risk groups (breeders, pet shopkeepers and veterinarians). The results of microimmunofluorescence to identify the presence of specific antibodies and correlation between appearance of infection in birds and important risk groups are presented. Out of 143 persons belonging to the high-risk group we found 10 (7%) persons who were immunologically positive. Testing of two successive samples was used to demonstrate an increase in IgG and IgA titres in human sera. However, IgM, which is indicative of acute infection, could not be detected.

Feasibility of testing for Chlamydia trachomatis in a general population sexual behaviour survey in Slovenia.

Our objective was to assess the feasibility of integrating first void urine (FVU) specimens testing for Chlamydia trachomatis genitourinary infection into a general population sexual behaviour survey. A total of 752 randomly selected respondents aged 18 to 54 were enrolled into the survey. Face to face interviewing with self-administered sensitive questions was used. Overall survey response rate was 77.4%. A convenience sub-sample of 83 respondents were invited to provide FVU specimens for confidential testing for C. trachomatis genitourinary infection. Fifty-five complied. This resulted in 66% FVU specimen participation rate among targeted respondents. Two specimens tested positive by Amplicor polymerase chain reaction. High feasibility study overall response rate indicated good acceptability of the survey. It proved feasible to collect FVU specimens for C. trachomatis testing in the small sub-sample. Consequently, we proceeded with integration of testing for C. trachomatis into the ongoing main survey.

Polymerase chain reaction for detection of Legionellae DNA in urine samples from patients with community-acquired pneumonia.

Polymerase chain reaction (PCR) was used for detecting Legionella DNA in water, sputum, tracheal aspirate and bronchoalveolar lavage fluid. There is paucity of data on the use of PCR for detection of Legionella in serum and urine samples. In 82 patients admitted with community-acquired pneumonia, urinary PCR was used in addition to urinary antigen assay for Legionella pneumophila serogroup 1 and serological tests (indirect immunofluorescence and ELISA) in paired sera. PCR was positive in urine samples from 21 patients (26%): in six of seven patients with acute legionellosis by CDC criteria, and 15 patients with negative urine antigen showing no fourfold rise in antibody titers in immunofluorescence test.

Effect of rat (beta)-interferon on intracellular levels of hydrolases in rat embryonal fibroblasts (Wistar strain).

The effect of rat beta-interferon (IFN) on the intracellular level of thiole and acid proteases and alkaline phosphatases was investigated. When nontransformed rat embryonal fibroblasts (REF) (Wistar strain) were treated with homologous IFN, a time independent decrease of hydrolase enzyme levels was observed. IFN treatment of transformed cells lead to a time dependent decrease of thiole proteases within 90 min. The values for acid proteases remained unchanged after the short term IFN treatment.

Comparison of serologic tests with urinary antigen detection for diagnosis of legionnaires' disease in patients with community-acquired pneumonia.

OBJECTIVE: To compare serologic methods and detection of urinary antigen in the diagnosis of community-acquired pneumonia. METHODS: Paired sera from 84 patients with community-acquired pneumonia were tested for Legionella pneumophila serogroup (LP SG) 1-7 and Legionella micdadei by use of the indirect immunofluorescence antibody test (IIF), enzyme-linked immunosorbent assay (ELISA) for LP SG 1-7 and complement-fixation (CF) test for LP SG 1. All patients were evaluated by ELISA urinary antigen detection for LP SG 1. RESULTS: Seven patients met the CDC criteria for acute Legionella infection, while in the rest of them we failed to detect urinary Legionella antigen. Thirty-three patients had non-diagnostic IIF antibody titers. Serum ELISA (IgG and/or IgM) was positive in 40 patients. Nine patients showed at least one CF titer of >/=1:32. The sensitivities of ELISA IgM for the first and the second serum samples compared with IIF were 42.8% and 46.6%, respectively, while the specificities were higher, i.e. 87% and 88.4%, respectively. The sensitivities of ELISA IgG for the first and the second samples were 42.8% and 53.3%, and the specificities were 77.9% and 76.8%, respectively. CONCLUSIONS: Although ELISA is simple to perform and easy to automate, we think that its advantages over indirect immunofluorescence and urinary antigen detection remain questionable.

Prevalence of genital Chlamydia trachomatis infection in the general population of Slovenia: serious gaps in control.

OBJECTIVES: One of the objectives of the first national survey of sexual lifestyles, attitudes, and health in Slovenia was to estimate the prevalence of and risk factors for genital Chlamydia trachomatis infection in Slovenian adults aged 18-49 years. METHODS: Data were collected over 1999-2001 from a probability sample of the general population by face to face interviews and anonymous self administered questionnaires. Respondents were invited to provide a first void urine (FVU) specimen for polymerase chain reaction testing for C trachomatis infection. We compared the results to the equivalent British survey. RESULTS: 1447 individuals contributed FVU specimens (82.6% of survey respondents, 55.3% of those eligible). C trachomatis infection was diagnosed in 3.0% of men and 1.6% of women. Prevalence was highest in men and women aged 18-24 years (4.1% for both). Individuals reporting first heterosexual intercourse before the age of 16, unprotected sexual intercourse with at least one heterosexual partner during the preceding year, concurrent heterosexual relationships during the preceding year, and five or more lifetime heterosexual partners had a higher prevalence. The association was statistically significant only for five or more lifetime partners (adjusted OR 3.0; 95% CI 1.3 to 6.9; p = 0.01). CONCLUSIONS: A relatively high prevalence of genital C trachomatis infection among 18-24 year old Slovenians, in the presence of relatively low risk sexual behaviour and low reported incidence rates of chlamydia infection, suggest serious gaps in the diagnosis and treatment of the condition. The results provide support for the introduction of chlamydia screening in Slovenia.

Aetiology and clinical presentation of mild community-acquired bacterial pneumonia.

A prospective study was initiated to analyse the bacterial aetiology and clinical picture of mild community-acquired pneumonia in Slovenia using the previously described Pneumonia Severity Index. Radiographically confirmed cases of pneumonia in patients treated with oral antibiotics in seven study centres were included. An aetiological diagnosis was attempted using culture of blood and sputum, urinary antigen testing for Streptococcus pneumoniae and Legionella pneumophila, and antibody testing for Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella pneumophila in paired serum samples. One hundred thirteen patients were evaluable for clinical presentation and 109 for aetiological diagnosis. At least one pathogen was detected in 62.4% patients. The most common causative agents were Mycoplasma pneumoniae in 24.8%, Chlamydia pneumoniae in 21.1%, and Streptococcus pneumoniae in 13.8% of patients. Dual infection was detected in 8.3% of patients. Most patients suffered from cough, fatigue, and fever. Patients with atypical aetiology of pneumonia differed from those with typical bacterial pneumonia or pneumonia of unknown aetiology in age, presence of dyspnea, and bronchial breathing on lung auscultation. Patients with pneumococcal, chlamydial, and mycoplasmal infections differed in age, risk class, presence of dyspnea, bronchial breathing, and proteinuria. There was an overlap of other clinical symptoms, underlying conditions, and laboratory and radiographic findings among the groups of patients classified by aetiology. Since patients with mild community-acquired pneumonia exhibit similar clinical characteristics and, moreover, since a substantial proportion of cases are attributable to atypical bacteria, broad-spectrum antibiotic treatment seems to be recommended.