Asymptomatic Leishmania infantum infections in humans living in endemic and non-endemic areas of Croatia, 2007 to 2009.

The prevalence of asymptomatic leishmaniasis in the general population of Croatia has not been studied to date. To assess the prevalence of Leishmania infantum specific IgG antibodies among immunocompetent residents of Croatia, sera from 2,035 persons (eastern coast of Adriatic Sea, n=1,186; Adriatic islands, n=653; mainland, n=196), were tested by an enzyme immunoassay. A total of 231 (11.4%) persons had anti-Leishmania antibodies. Multivariate analysis revealed that seropositivity was associated with geographic location and age. Residents of coastal areas and islands were significantly more seropositive than mainland residents (odds ratios (OR) 20.37 to 28.51). Moderate to high anti-Leishmania seroprevalence was found throughout the eastern Adriatic coast and islands (4.0% to 22.2%) including the sites previously considered non-endemic. A highly endemic focus was identified in central coastal Dalmatia (seroprevalence 22.2%; OR: 1.72; 95% confidence interval (CI): 1.33-2.22). Regarding age, children aged 0-9 years were the most vulnerable group for asymptomatic Leishmania infection (OR: 2.19; 95% CI: 1.16-4.14).

Antibiotic resistance among enterococcal strains isolated from clinical specimens.

The distribution and resistance patterns of clinical isolates of enterococci from hospital patients were compared with those obtained from outpatients. Of 235 enterococcal isolates 212 (90.2%) were identified as Enterococcus faecalis and 23 (9.8%) as E. faecium. E. faecium occurred more frequently in specimens from hospitalized patients than from outpatients (P < 0.001). Over 90% of all E. faecalis isolates were susceptible to ampicillin. Resistance to ampicillin occurred in 66.7% of hospital strains of E. faecium. High-level resistance to gentamicin (MIC > 500 mg/l) was seen in 37.03% of inpatients' and in 11.5% of outpatients' E. faecalis isolates and in 76.2% of hospital isolates of E. faecium. High-level streptomycin resistance (MIC > 2000 mg/l) occurred in 52.8% of E. faecalis and 76.2% of E. faecium hospital isolates. There were no isolates resistant to vancomycin. The community acquired strains isolated from outpatients were more susceptible than isolates from hospitalized patients to all antimicrobial agents tested.

Combined anti-herpes virus activity of nucleoside analogs and interferon.

Addition of interferon (IFN) to nucleoside analog therapy for herpetic keratitis has been shown to significantly increase the efficacy of therapy compared to nucleoside alone. We have analysed several nucleoside analogs and recombinant IFN-alpha 2 to determine which combinations have increased anti-herpes simplex virus type 1 (HSV) activity. Synergistic anti-HSV activity between IFN-alpha 2 and the acyclic guanosine analogs, acyclovir (ACV) and ganciclovir (DHPG), was demonstrated in cytopathic effect reduction assay in human corneal cell cultures as well as in Vero cells. In this assay system IFN-alpha 2 alone had little detectable antiviral activity at titers of greater than or equal to 2,000 IU/ml, however, treatment of cells with about 100 IU/ml of IFN-alpha 2 for 24 hrs prior to infection decreased the ED50 of ACV approximately 2- to 3-fold and of DHPG approximately 5- to 6-fold in Vero cells. Combinations of IFN-alpha 2 with bromovinyldeoxyuridine (BVdU) in Vero cells or human corneal stromal cells did not increase the antiviral activity of BVdU. Combinations of IFN-alpha 2 with trifluorothymidine (TFT) also did not increase the effective antiviral activity of this nucleoside and resulted in decreased uptake of TFT from the medium. These studies document that combinations of acyclic nucleoside analogs, ACV and DHPG, with IFN-alpha 2 resulted in synergistic anti-HSV activities in both Vero and human corneal stromal cells, while the pyrimidine analogs, TFT and BVdU, were not synergistic with IFN-alpha 2. IFN-alpha 2 treatment of cells induced modifications of nucleoside (e.g., thymidine and TFT), but not nucleobase (e.g., ACV) uptake. These studies suggest that selective inhibition of nucleoside versus nucleobase uptake may contribute to the mechanism of IFN/nucleobase synergy in the inhibition of HSV replication.

[Rickettsiales--modern findings]. / Rickettsiales--suvremene spoznaje.

The term rickettsiae has as a rule encompassed the intracellular bacteria. Molecular studies brought new data to rickettsial taxonomy. Many new data on rickettsia have been accumulated over recent years, and a comparison of the newly discovered diseases with previously known rickettsioses, as well as with their agents is of interest. The number of "new" agents that have been discovered in the last few years is remarkable. This review discusses the current knowledge of bacterial species that historically belonged to the order Rickettsiales, as well as the role of these agents as human and animal pathogens.

[Determination of antibody kinetics in Mediterranean spotted fever using the Weil-Felix agglutination test, complement fixation and indirect immunofluorescence]. / Kretanje protutijela u osoba s mediteranskom pjegavom groznicom odredivano Weil-Felixovom aglutinacijom, reakcijom vezanja komplementa i neizravnom imunofluorescencijom.

The kinetics of antibodies in human cases of Mediterranean spotted fever was studied by applying Weil-Felix (WF) agglutination with Proteus OX antigens, complement fixation (CF) and indirect immunofluorescence (IF) tests with Rickettsia conorri antigen to 46 sera from 21 patients. The kinetics of specific (CF and IF) and nonspecific (WF) antibodies was similar. The percentage of the positive sera and geometric mean titers of antibodies reached the maximum after 14 days from the onset of illness.

[Monitoring bacterial resistance to antibiotics in the CroatianRepublic]. / Pracenje rezistencije bakterija na antibiotike u Republici Hrvatskoj.

In 1996 a Committee for antibiotic resistance surveillance in Croatia was founded by the Croatian Academy of Medical Sciences. In this study antibiotic surveillance results for the period June 1-December 31, 1997 from 12 microbiology laboratories throughout Croatia are presented. Sensitivity to antibiotics was determined by disk diffusion method for the following bacteria: Streptococcus pneumoniae, Staphylococcus aureus, Enterococcus spp., Escherichia coli, Klebsiella spp. and Pseudomonas aeruginosa. In general, high proportion of resistant isolates was recorded throughout Croatia, although some regional variations were noticed. Mean resistance of pneumococci to penicillin was 38%, in S. aureus resistance to methicillin was 47%, and 3rd generation cephalosporin-resistance in E. coli was 6% and in Klebsiella spp. 21%. In P. aeruginosa resistance to gentamicin averaged 50%, to imipenem 13% and to ceftazidim 8%. Future aims of the Committee are to continue routine antibiotic resistance surveillance during certain periods every year, and to estimate clinical significance of resistant bacteria, detect mechanisms of resistance and improve the quality of laboratory work through education and quality control projects.

Prevalence of Toscana virus antibodies in residents of Croatia.

To assess the prevalence of Toscana virus (TOSV) infection among healthy residents of Croatia we tested sera from 2016 persons, for IgG antibodies to TOSV, by an enzyme immunoassay. A total of 755 (37.5%) persons had IgG antibodies to TOSV: 53.9%, 33.6% and 6.1% among residents of the islands, coastal area and mainland of Croatia, respectively. Risk factors significantly associated with seropositivity to TOSV were: living on islands (OR, 11.10; 95% CI, 6.02-20.50; p <0.001) or in coastal areas (OR, 6.96; 95% CI, 3.81-12.71; p <0.001) and increase of age (OR, 1.03; 95% CI, 1.02-1.03; p <0.001).

Antimicrobial susceptibility and beta-lactamase production of selected gram-negative bacilli from two Croatian hospitals: MYSTIC study results.

The meropenem yearly Susceptibility Test Information Collection (MYSTIC) programme is a global, longitudinal resistance surveillance network that monitors the activity of meropenem and compares its activity with other broadspectrum antimicrobial agents. We now report the antimicrobial efficacy of meropenem compared to other broad-spectrum agents within the selective Gram-negative pathogen groups from two Croatian Hospitals investigated between 2002-2007. A total of 1510 Gram-negative pathogens were tested and the minimum-inhibitory concentrations (MICs) were determined by broth microdilution method according to CLSI.There was no resistance to either imipenem or meropenem observed for Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis in both medical centers. High resistance rates of K. pneumoniae to ceftazidime (18%), cefepime (17%) and gentamicin (39%) are raising concern. Acinetobacter baumannii turned out to be the most resistant Gram-negative bacteria with 81% resistant to ceftazidime, 73% to cefepime, 69% to gentamicin and 71% to ciprofloxacin. Almost 20% of Pseudomonas aeruginosa strains were resistant to imipenem, 13% to meropenem, 69% to gentamicin and 38% to ciprofloxacin.The prevalence of extended-spectrum beta-lactamases (ESBLs) in E. coli was 10% and in K. pneumoniae 49%. PCR and sequencing of the amplicons revealed the presence of SHV-5 in nine E. coli strains and additional tem-1 beta-lactamase five strains. Five K. pneumoniae strains were positive for bla(SHV-5 )gene. Eight ESBL positive Enterobacter spp. strains were found to produce tem and CtX-m beta-lactamases. Plasmid-mediated AmpC beta-lactamases were not found among K. pneumoniae, E. coli and Enterobacter spp. Three A. baumannii strains from Zagreb University Center were identified by multiplex PCR as OXA-58 like producers. Six A. baumannii strains from Split University Center were found to possess an ISAba1 insertion sequence upstream of bla(OXA-51 )gene. According to our results meropenem remains an appropriate antibiotic for the treatment of severe infections caused by Gram-negative bacteria. These data indicate that despite continued use of meropenem, carbapenem resistance is not increasing among species tested, except for A. Baumannii, in the two study hospitals and suggest that clinicians can still administer carbapenems as a reliable and effective choice in managing serious nosocomial infections.

Epidemiological features of Mediterranean spotted fever, murine typhus, and Q fever in Split-Dalmatia County (Croatia), 1982-2002.

We determined the epidemiological features of three zoonoses in hospitalized patients in southern Croatia. Patients were diagnosed by serological testing. Clinical and epidemiological data were also collected. Between 1982 and 2002, Mediterranean spotted fever (MSF) was diagnosed in 126 (incidence rate 1.27/100,000 per year), murine typhus (MT), in 57 (incidence rate 0.57/100,000 per year), and Q fever in 170 (incidence rate 1.7/100,000 per year) patients. MSF and Q fever were characterized by a marked seasonality. Incidences of Q fever and of MSF were higher for males than for females (P<0.0001 and P=0.0024, respectively). The most frequent of the three zoonoses in children was MSF. Q fever and MT cases were mostly seen in the 21-50 years age group. We found no statistically significant differences between season- and gender-specific incidence rates of MT. Whereas infections due to rickettsiae decreased, the incidence of Q fever increased over the last 12 years of the study.

Prevalence of antibodies to Toxoplasma gondii in the female population of the County of Split Dalmatia, Croatia.

The prevalence of IgG antibodies reactive with Toxoplasma gondii in the female population of the County of Split Dalmatia was investigated by enzyme linked immunosorbent assay. Of a total of 1109 serum samples collected from female subjects, 423 (38.1%) reacted with T. gondii. The frequency of positive sera increased with age. Theoretical incidence of congenital toxoplasmosis was calculated from the annual increase in cumulative prevalence of antibodies between different age groups among the women of childbearing age. The estimated theoretical incidence of congenital toxoplasmosis was 1.4 per 100 pregnancies of adolescents (16-20 years) and decreased to 0.1% in seronegative pregnant women aged 41-45.

Neutralizing antibodies for sandfly fever Naples virus in human sera on the island of Mljet.

Sera from apparently healthy residents of various age from different localities of the Adriatic island of Mljet were tested by the plaque reduction neutralization test (PRNT) for antibodies to sandfly fever Naples virus (SFN). An average of 51.4% of the sera examined was found to have neutralizing antibodies. Reactors to SFN virus were found in all the age groups examined and the antibody prevalence appeared to increase with age. The presence of SFN neutralizing antibodies in all age groups indicates that the virus must have been present endemically and that it is still active on the island of Mljet.

Geographical distribution of arboviruses in Yugoslavia.

Studies of arboviruses started in Yugoslavia in 1953 following the isolation of TBE virus which caused a severe epidemic that year. Until now the following viruses have been proven to circulate in the country: tick-borne encephalitis (TBE), Crimean-Congo hemorrhagic fever (CCHF), Bhanja (BHA), sandfly fever (SF), Tahyna (TAH), Calovo (CVO), West Nile (WN), dengue (DEN), Jug Bogdanovac (JB), and Hantaviruses. TBE virus is endemic in the north-west part of the country, causing also epidemics in cyclical intervals. Its typical clinical picture is aseptic meningitis, but severe cases with paralysis have also been described. The bite of ticks is confirmed in about 80% of cases. CCF caused a small epidemic with ten clinical cases in Macedonia in 1976. Bhanja virus was isolated on the Dalmatian island of Brac in 1977, the antibody rate there, determined by the HI method, being about 31%. The first human disease in the world was caused by the Yugoslav Bhanja virus strain. Sandfly fever is still active in the country. The Naples type is prevailing and has proved hazardous for newcomers. Hantaviruses have been studied since 1980. They caused severe epidemics (1967, 1980, 1989) and sporadic cases all over the country. Three different strains are in circulation. Further studies are needed for the rest of the above mentioned viruses to learn more about their significance in human pathology.

Prevalence of antibodies to rickettsiae in the north-western part of Bosnia and Herzegovina.

The prevalence of rickettsial antibodies in north-western part of Bosnia and Herzegovina was studied. Among 231 sera tested by complement fixation (CF) positive were: 61.5% for Rickettsia typhi, 4.3% for R. prowazekii, 1.7% for R. conorii and 19.0% for Coxiella burnetii. Of 183 sera tested by indirect immunofluorescence assay (IFA) 37.7% reacted with R. typhi, 1.6% with R. conorii and 22.4% with C. burnetii. The results show that at least R. typhi and C. burnetii are highly endemic in this area.

Synergistic anti-varicella-zoster virus activity of interferon-alpha 2a and acyclovir in corneal cells.

Human corneal stromal (HCS) cells in cultures established from donor corneas can serve as host cells for replication of varicella-zoster virus (VZV). Comparative infectious centers assays suggest that HCS cells are more restrictive hosts than MRC-5 cells, a line of human embryonic lung fibroblasts, commonly used for VZV isolation. VZV propagated in MRC-5 cells was infectious for both MRC-5 and HCS cells, but titers in HCS were one fifth of those in MRC-5 cells. Inhibition of VZV replication in HCS cells by acyclovir (ACV), recombinant human interferon-alpha 2a (IFN-alpha 2a), or the combination of these two antivirals was detected by both an infectious centers-plaque-reduction assay and an ELISA method using monoclonal anti-VZV antibody. In the infectious centers-plaque-reduction assay combinations of ACV with IFN-alpha 2a showed synergistic anti-VZV activity. VZV protein synthesis, as detected by the ELISA, was a less sensitive measure of the antiviral effects producing an ED50 value of 50 microM for ACV, five to ten times the ED50 determined in the plaque reduction assay. In the ELISA, high titers of IFN-alpha 2a (2000 IU/ml) decreased virus antigen expression only slightly, while combinations of ACV and IFN-alpha 2a were synergistic in their detected anti-VZV activity. These data document the replication of VZV in HCS cells and demonstrate that VZV is sensitive to the synergistic antiviral action of combinations of IFN-alpha 2a and ACV.

Antibodies to spotted fever group rickettsiae in dogs in Croatia.

The prevalence of IgG antibodies to spotted fever group rickettsiae was studied by the indirect immunofluorescent antibody assay in a sample of 194 canine sera from different parts of Croatia. Dogs (n = 141) from urban and suburban-semirural environs from the middle part of the eastern coast of Adriatic sea, and dogs (n = 53) from Zagreb, the capital of Croatia located in the northern continental part of Croatia, were tested. The total of 88 (62.4%) dogs from southern coastal Croatia had antibodies to Rickettsia conorii, with a significantly higher prevalence (69.9%) in suburban-semirural areas. The prevalence rate of antibodies to Rickettsia conorii in canine sera from urban environment in the north of Croatia was 20.7%. The results show that the members of spotted fever group rickettsiae are present in both coastal and continental Croatia.