Volga Punda-Polić

Rickettsia helvetica in Dermacentor reticulatus Ticks

We report on the molecular evidence that Dermacentor reticulatus ticks in Croatia are infected with Rickettsia helvetica (10%) or Rickettsia slovaca (2%) or co-infected with both species (1%). These findings expand the knowledge of the geographic distribution of R. helvetica and D. reticulatus ticks.

Detection and identification of spotted fever group rickettsiae in ticks collected in southern Croatia

A total of 197 ticks belonging to four species (Haemaphysalis punctata, Hyalomma marginatum, Rhipicephalus bursa and Dermacentor marginatus) collected in October 2000 from domestic animals in southern Croatia were examined for the presence of rickettsiae by molecular techniques. Specific sequences of the rickettsiae were detected in 25 (12.7%) of ticks tested. The prevalence of infection in D. marginatus and H. marginatum ticks was 36.8 and 64.7%, respectively. None of the ticks belonging to the species H. punctata or Rh. bursa were infected. Sequence analysis of amplified products revealed that D. marginatus ticks are infected with Rickettsia slovaca, whereas H. marginatum are infected with R. aeschlimannii. The results of this study extend the knowledge of the geographic distribution of SFG rickettsiae and indicate that at least two of them, with yet uncertain pathogenicity to humans, are present in ticks in southern Croatia.

Molecular Identification of Rickettsia felis‐like Bacteria in Haemaphysalis sulcata Ticks Collected from Domestic Animals in Southern Croatia

Abstract:  Haemaphysalis sulcata ticks collected from sheep and goats in southern Croatia were found infected with rickettsiae. Molecular analysis of the complete gltA gene and portion of 17 kDa and ompB genes revealed the presence of Rickettsia felis‐like bacteria in up to 26% of tested ticks.

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.

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.

First report of molecular characterization of carbapenem-resistant Acinetobacter baumannii in different intensive care units in University Hospital Split, Croatia.

Acinetobacter baumannii is an important cause of nosocomial infections such as pneumonia, septicaemia, urinary tract infections, and wound infections 1. It may become resistant to a wide range of antibiotics, thus complicating the treatment of nosocomial infections 1. The emergence of carbapenem resistance in Acinetobacter baumannii has become a global concern since these β -lactams are often the only effective treatment left against many multiresistant strains . According to a recent report, the overall resistance rate to imipenem in A. baumannii isolated from 17 representative laboratories in Croatia was 1 % (range 0%-8%) . However, molecular basis of carbapenem resistance was not investigated yet. We would like to report the molecular epidemiology of carbapenem resistant strains obtained from clinical samples of several patients hospitalised on different intensive care units inside University Hospital Split, Croatia. Eleven isolates of A. baumannii with an unusual resistance profile were isolated during 2003 and 2004, from patients hospitalised in three different intensive care units (two adults and one children ICU) on different locations inside UHS. Routine susceptibility testing was done by standard disc diffusion method according to the National Committee for Clinical Laboratory Standards guidelines. All collected isolates of A. baumannii displayed intermediate (MICs>8 mg/L) or resistant (MICs>16 mg/L) profile to imipenem and/or meropenem. Minimum inhibitory concentrations were also determined for ceftazidime, cefepime, ceftriaxone, amikacin, gentamicin, ciprofloxacin and piperacillin-tazobactam by broth microdilution method according to CLSI recommendation.. All isolates were multidrug-resistant exhibiting highly resistance to ceftazidime, cefepime, piperacillin/tazobactam, amikacin, gentamicin, netilmicin and ciprofloxacin. No resistance to ampicillin/sulbactam was observed which is not in use in our hospital. The isolates were genetically characterized with pulsed-field gel electrophoresis (PFGE). The preparation of genomic DNA of A. baumannii isolates was performed as previously described. Oxacillinases were detected by using a standard PCR technique with primers specific for blaOXA-23, blaOXA-40, blaOXA-58, and blaOXA-69 genes. Production of metallo-β -lactamases was screened by E test-MBL and PCR with primers specific for VIM and IMP β -lactamases. Β -lactamases were prepared from overnight broth cultures, released by sonication and cell debris was removed by centrifugation. PCRs were positive for the OXA-69 like enzyme in ten Acinetobacter strains belonging to unique PFGE profile characterised to pulsotype A, while strain characterised to pusotype B was the only strain isolated from children intensive care unit and carbapenemase gene was not found with tested primers. No significant hydrolysis of imipenem was detected. Acccording to the bibliographical data OXA-69 is a naturally occurring oxacillinase in A. baumannii and it does not confer on the producing isolates significant resistance to carbapenems, thus it can be concluded that resistance to imipenem and meropenem in our isolates is due to other mechanisms such as porin loss or efflux. Spread of the pulsotype A with OXA-69-type oxacillinases was due to clonal dissemination of multi-drug resistant A. baumannii between two different adult Intensive Care Units, probably by hospital staff during medical procedures. The infection control team of the hospital implemented restriction of carbapenem usage and strict antiseptic techniques, which included the rigorous use of alcohol-clorhexidine solutions before and between patient and equipment contact and before leaving the unit. To our knowledge, this is the first report on the molecular characterization carbapenem resistance in A. baumannii in Croatia. A similar finding underscores the progressive emergence of these determinants in different geographic areas. Consequently, incidence and spread of multidrug-resistant A. baumannii nosocomial infections suggest the necessity of a surveillance program and enforcing adequate control measures in different hospital settings

Occurrence of OXA-107 and ISAba1 in Carbapenem-Resistant Isolates of Acinetobacter baumannii from Croatia

ABSTRACT Carbapenem-resistant isolates of Acinetobacter baumannii from intensive care units at Split University Hospital, Split, Croatia, were studied. Most (100 of 106) had ISAba1 inserted upstream of a blaOXA-107 gene, encoding an unusual OXA-51-type oxacillinase. Pulsed-field gel electrophoresis revealed that the isolates formed three clusters belonging to the sequence group 2 (European clone 1) lineage.

Prevalence of antibodies to spotted fever group rickettsiae in the region of Split (southern Croatia)

The prevalence of IgG antibodies to spotted fever group rickettsiae (SFG) was investigated by the indirect immunofluorescence assay in a sample of 1207 healthy residents of the central part of southern Croatia (eastern coast of Adriatic Sea). Sera obtained from subjects living in three different places of residency (urban, suburban and rural environs of the region) were tested. Overall 528 (43.7%) persons had significant antibody titers (1:40 or higher), with a significantly higher prevalence in suburban and rural areas. The results show that inhabitants in the area are clearly being exposed to SFG rickettsiae. Risk factors significantly associated with seropositivity to SFG rickettsiae were: living in suburban and rural settlements (odds ratio [OR] = 1.81; 95% confidence interval [CI] = 1.35–2.4), contact with animals (OR: 1.52; 95% CI: 1.15–1.99), and increase of age (OR: 1.18; 95% CI: 1.09–1.26).

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.

Demographic and Epidemiologic Features of Mediterranean Spotted Fever Cases in the Region of Split, Croatia

Mediterranean spotted fever (MSF) is an acute, febrile, tick-transmitted rickettsiosis caused by Rickettsia conorii , an obligate intracellular bacterium endemic in Mediterranean countries, Africa, and Asia.The major vector of the disease is the dog brown tick Rhipicephalus sanguineus . 1,2 The first clinical description of spotted fever disease in Croatia was reported in the city of Split (central part of southern Croatia, eastern coast of Adriatic Sea) by Tartaglia in 1935. 3 The first well documented, serologically confirmed cases of MSF in Croatia were reported among inhabitants and tourists in Split and its adjacent suburban-semirural area in 1982. 4 Ever since a prospective study on MSF in the area of Split was carried out at University Hospital Split, the only hospital in the region, where cases of MSF are observed every year. This article presents an overview of data accumulated from 1982 through 1999 and describes demographic and epidemiological features of patients with MSF in the region of Split, Croatia.