An Overview of Tick-Borne Encephalitis Epidemiology in Endemic Regions of Continental Croatia, 2017-2023.

Tick-borne encephalitis (TBE) represents an important public health problem in Europe. We analyzed the epidemiology of TBE based on data from humans, animals, and Ixodes ricinus ticks in endemic regions of continental Croatia. In the period from 2017 to 2023, cerebrospinal fluid (CSF) and serum samples of 684 patients with neuroinvasive diseases, 2240 horse serum samples, and 300 sheep serum samples were tested for TBEV. In addition, 8751 I. ricinus ticks were collected. CSF samples were tested using RT-PCR. Serological tests (serum, CSF) were performed using commercial ELISA, with confirmation of cross-reactive samples by a virus neutralization test. Eighty-four autochthonous human TBEV cases were confirmed. The majority of patients were in the age group of 40-69 years (58.3%) with a male predominance (70.2%). TBE showed a bimodal seasonality with a large peak in April-August and a small one in October-November. In addition to humans, TBEV IgG antibodies were found in 12.2% of horses and 9.7% of sheep. Seasonal tick abundance corresponds to the reported number of human infections. Continental Croatia is still an active natural focus of TBE. Continuous monitoring of infections in humans, sentinel animals, and ticks is needed for the implementation of preventive measures.

An international outbreak of Salmonella enterica serotype Enteritidis linked to eggs from Poland: a microbiological and epidemiological study.

BACKGROUND: Salmonella spp are a major cause of food-borne outbreaks in Europe. We investigated a large multi-country outbreak of Salmonella enterica serotype Enteritidis in the EU and European Economic Area (EEA). METHODS: A confirmed case was defined as a laboratory-confirmed infection with the outbreak strains of S Enteritidis based on whole-genome sequencing (WGS), occurring between May 1, 2015, and Oct 31, 2018. A probable case was defined as laboratory-confirmed infection with S Enteritidis with the multiple-locus variable-number tandem repeat analysis outbreak profile. Multi-country epidemiological, trace-back, trace-forward, and environmental investigations were done. We did a case-control study including confirmed and probable cases and controls randomly sampled from the population registry (frequency matched by age, sex, and postal code). Odds ratios (ORs) for exposure rates between cases and controls were calculated with unmatched univariable and multivariable logistic regression. FINDINGS: 18 EU and EEA countries reported 838 confirmed and 371 probable cases. 509 (42%) cases were reported in 2016, after which the number of cases steadily increased. The case-control study results showed that cases more often ate in food establishments than did controls (OR 3·4 [95% CI 1·6-7·3]), but no specific food item was identified. Recipe-based food trace-back investigations among cases who ate in food establishments identified eggs from Poland as the vehicle of infection in October, 2016. Phylogenetic analysis identified two strains of S Enteritidis in human cases that were subsequently identified in salmonella-positive eggs and primary production premises in Poland, confirming the source of the outbreak. After control measures were implemented, the number of cases decreased, but increased again in March, 2017, and the increase continued into 2018. INTERPRETATION: This outbreak highlights the public health value of multi-country sharing of epidemiological, trace-back, and microbiological data. The re-emergence of cases suggests that outbreak strains have continued to enter the food chain, although changes in strain population dynamics and fewer cases indicate that control measures had some effect. Routine use of WGS in salmonella surveillance and outbreak response promises to identify and stop outbreaks in the future. FUNDING: European Centre for Disease Prevention and Control; Directorate General for Health and Food Safety, European Commission; and National Public Health and Food Safety Institutes of the authors' countries (see Acknowledgments for full list).

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.

Carbapenem-Resistant Acinetobacter baumannii Recovered from Swine Manure.

Recently, concerns have been raised about the possibility of Acinetobacter baumannii transmission between animals and humans. So far, A. baumannii has been reported in animals with which people can come into contact. The presence of this pathogen in animal manure presents an equally important public health risk. In this study, we report the finding of two A. baumannii isolates in swine manure from a Croatian pig farm. Both isolates shared features with the widespread human clinical isolates: affiliation to the international clonal lineage 2 (ST-195), carbapenem, and extensive drug resistance and the plasmid-located acquired blaOXA-23 gene. These two A. baumannii isolates survived anaerobic conditions, competition with other microorganisms, and elevated concentrations of heavy metals in the stagnant swine manure for at least 2 weeks. These findings call for bacteriological analysis and disinfection of liquid swine manure before its application as a fertilizer in traditional extensive agriculture.

Activity of fosfomycin against nosocomial multiresistant bacterial pathogens from Croatia: a multicentric study.

AIM: To determine in vitro susceptibility of multiresistant bacterial isolates to fosfomycin. METHODS: In this prospective in vitro study (local non-random sample, level of evidence 3), 288 consecutively collected multiresistant bacterial isolates from seven medical centers in Croatia were tested from February 2014 until October 2016 for susceptibility to fosfomycin and other antibiotics according to Clinical and Laboratory Standards Institute methodology. Susceptibility to fosfomycin was determined by agar dilution method, while disc diffusion was performed for in vitro testing of other antibiotics. Polymerase chain reaction and sequencing were performed for the majority of extended spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae (K. pneumoniae) and carbapenem-resistant isolates. RESULTS: The majority of 288 multiresistant bacterial isolates (82.6%) were susceptible to fosfomycin. The 236 multiresistant Gram-negative isolates showed excellent susceptibility to fosfomycin. Susceptibility rates were as follows: Escherichia coli ESBL 97%, K. pneumoniae ESBL 80%, Enterobacter species 85.7%, Citrobacter freundii 100%, Proteus mirabilis 93%, and Pseudomonas aeruginosa 60%. Of the 52 multiresistant Gram-positive isolates, methicillin-resistant Staphylococcus aureus showed excellent susceptibility to fosfomycin (94.4%) and vancomycin-resistant enterococcus showed low susceptibility to fosfomycin (31%). Polymerase chain reaction analysis of 36/50 ESBL-producing K. pneumoniae isolates showed that majority of isolates had CTX-M-15 beta lactamase (27/36) preceded by ISEcp insertion sequence. All carbapenem-resistant Enterobacter and Citrobacter isolates had blaVIM-1 metallo-beta-lactamase gene. CONCLUSION: With the best in vitro activity among the tested antibiotics, fosfomycin could be an effective treatment option for infections caused by multiresistant Gram-negative and Gram-positive bacterial strains in the hospital setting.

Ureaplasma urealyticum and Ureaplasma parvum in women of reproductive age.

OBJECTIVES: To determine the incidence of Ureaplasma urealyticum and Ureaplasma parvum (UP) in symptomatic and asymptomatic women of reproductive age and to estimate antibiotic susceptibility of ureaplasma isolates. MATERIALS AND METHODS: This study included 424 ureaplasma positive women of 1,370 tested women who visited gynecological practices during 2010. Cervicovaginal or urethral swab specimens from each patient were obtained for cultivation and molecular typing by RT-PCR. RESULTS: Ureaplasma spp. was identified by cultivation in 424 (34.4 %) cases, of which 79.0 % were from women with symptoms and 21.0 % from women without symptoms. Among ureaplasma positive women, 121 (28.5 %) were pregnant. Genotyping was successful in 244 strains, and the majority of samples were identified as UP (92.6 %). Among genotyped isolates, there were 79.5 % from symptomatic and 20.5 % from asymptomatic women; 29.9 % from pregnant and 70.1 % from non-pregnant women. There was no difference in the incidence of ureaplasma type regarding symptoms. Antibiotic susceptibility of 424 ureaplasma isolates identified by cultivation showed that all strains were susceptible to doxycycline, josamycin, erythromycin, tetracycline, clarithromycin and pristinamycin, but there was lower susceptibility to quinolone antibiotics, i.e., 42.9 and 24.5 % isolates were susceptible to ofloxacin and ciprofloxacin, respectively. CONCLUSION: This study shows that UP was the most frequent isolated ureaplasma species (92.6 %). Regarding antibiotic susceptibility, quinolones are not the best choice for the treatment of ureaplasma infections, while macrolides and tetracyclines are still effective.

Lower urinary tract infections and the effects of hormone therapy in postmenopausal women in the Zagreb region.

Urinary tract infections (UTI) remain one of the most common bacterial infections seen in adult women of all ages. In postmenopausal women, the aging process contributes to local complaints in the lower urogenital tissue, including UTI. Our study was conducted at gynecological practices of the health centers in the Zagreb region, Croatia, during 2009. The study included postmenopausal women with urinary symptoms divided into two groups: hormone therapy (HT) users and controls. The objectives were to estimate microbiologically proven lower UTIs (LUTIs) in postmenopausal women with urinary symptoms and the effect of regular HT use on microbiologically confirmed LUTIs. Out of 2338 postmenopausal patients, there was a significantly higher rate of women with urinary symptoms in HT users, namely 64.4% (143/221), compared to the control group at 4.8% (102/2116). Of the 245 patients with urinary symptoms, in 58.8% (144/245) the infection was microbiologically confirmed. Hormone therapy users showed a statistically significant lower rate of microbiologically proven LUTIs (46.9%, 67/143) compared to controls (75.5%, 77/102, p < 0.001). Data analysis also showed the efficacy of local as well as systemic HT treatment compared with the control group (p < 0.00, p = 0.049). But there was a significant difference in the frequency of LUTIs between patients who used local (30.3%, 20/66) and systemic (61.1%, 47/77) HT (p < 0.001). The patients who, regulary used therapy, in the local HT group as well as in the group on systemic HT showed a lower incidence of LUTIs compared to controls (p < 0.00, p = 0.006). In patients who did not regulary use therapy, there were no significant differences between either local (63.6%, 7/11)) or systemic (76.9%, 20/26) HT non- regular users and the control group (75. 5%, 77/102) (p = 0.917, p = 0.625). The high percentage of patients with non-microbiologically confirmed LUTIs (41.2%, 101/245) suggested the significant role microbiological testing has in LUTI diagnosis. Both local and systemic HT use was related to LUTI reduction.

Prevalence of Coxiella burnetii antibodies among febrile patients in Croatia, 2008-2010.

Despite the widespread distribution of Q fever, the prevalence in humans is not accurately known, because many infected people seroconvert without symptoms or with a mild febrile disease. The aim of this study was to determine the seroprevalence of Q fever in different regions of Croatia. During a 2-year period (2008-2010), serum samples from 552 febrile patients with prolonged cough aged 1-88 were tested for the presence of Coxiella burnetii antibodies by using indirect immunofluorescent assay. Sera from 27.5% patients showed IgG antibodies. Serological evidence of C. burnetii infection was found in patients from all parts of Croatia. Seroprevalence rates significantly differed among regions from 21.5% to 41.2% (p=0.001). Men were more often seropositive (31.6%) than women (22.2%; p=0.016). According to age, a progressive increase in the IgG seropositivity rates was observed as ranging from 6.7% in children less than 10 years of age to 39.2% in patients aged 40-49 (p=0.001). Above the age of 50, the IgG seroprevalence remained stable. Patients from rural areas were more often seropositive than patients from urban areas (40.8% vs. 19%), p<0.001). Acute Q fever was confirmed in 5.8% of patients. Cases occurred throughout the year. A majority of cases were reported during summer months.

[Evaluation of microbiological diagnostics in urogenital infections in postmenopausal women]. / Znacenje mikrobioloske dijagnostike u infekcijama urogenitalnog sustava zena u postmenopauzi.

OBJECTIVE: To establish the percentage of infections in postmenopausal women with urinary symptoms which can be confirmed by microbiological analysis, the most common causative agents and whether the urethra and vagina in patients with cystitis are concurrently colonized by pathogenic microorganisms. METHODS: Laboratories of the Croatian National Institute of Public Health in Zagreb, in the period of two years, analyzed 245 samples taken from patients with urinary symptoms who had been postmenopausal at least for a year. Urine samples, as well as urethral and vaginal swabs were taken from each patient and tested for causative agents of urogenital infections, genital mycoplasma and Chlamydia trachomatis. RESULTS: Cystitis was confirmed by microbiological analysis in 31.4% women, urethritis in 24.8%, and vaginitis in 15.1%. The most common causative agent of urethritis was Ureaplasma urealyticum, while Gardnerella vaginalis was the most common in vaginal infections. E. coli was concurrently isolated in urine, urethral and vaginal samples in 65.1% of patients with E. coli cystitis, while Streptococcus agalactiae was isolated in urethral and vaginal samples in each patient with Streptococcus agalactiae cystitis. CONCLUSION: Mucosal colonization of the urethra and vagina contributes to the incidence of cystitis in postmenopausal women. Microbiological diagnostics is necessary to distinguish between the symptoms of ageing of the urogenital system and infection, with a view to preventing unnecessary antibiotic therapy.

[Uropathogens and antimicrobial susceptibility in outpatients]. / Uzrocnici urinarnih infekcija i antimikrobna osjetljivost u vanbolnickoj populaciji.

In 2003, primary health care reported 230,356 (2.7% of diagnosed diseases and conditions) cases of urinary bladder infections in Croatia. Results of this study including 815 outpatient urine samples confirmed the diagnosis of urinary system infection in 27.4%. Positive urine cultures were recorded in 32.8% of female and 20.1% of male patients, Escherichia (E.) coli and Enterococcus spp. with 67.3% and 12.6%, respectively were the most common causative agents of urinary system infection. Considering the efficacy of antibiotic action on individual pathogenic isolates, sulphametoxazole-trimethroprim showed high efficacy (94.3%) against E. coli strains. Th diagnosis of cystitis is established more often than confirmed by study the findings. Urine culture makes a considerable contribution to the diagnosis, rationalizing the costs of treatment by preventing the unnecessary prescribing of antibiotics and the development of antibiotic resistance. Thus it is necessary to make it fully accessible. Application of the current state-of-the-art in patients attending primary health care clinics, should be enabled by proactive or interactive measurable use of (on-line) guidelines on the treatment of urinary tract infections.