Rickettsiae and rickettsial diseases in Croatia: Implications for travel medicine.

AIM: To review the current state of knowledge concerning rickettsiae and rickettsioses in Croatia and to discuss their implications for travellers. METHODS: The PubMed database was searched from 1991 to 2015 by combining the words "rickettsia," "rickettsiosis", "travellers" and "Croatia". RESULTS: Since 1969, Croatia appears to be free of epidemic typhus (ET) caused by Rickettsia prowazekii and the last case of Brill-Zinsser disease was recorded in 2008. Mediterranean spotted fever (MSF) caused by Rickettsia conorii is the most frequent human rickettsial infection in Croatia, followed by murine typhus caused by Rickettsia typhi. Human cases of MSF and murine typhus have been predominantly observed along the eastern Adriatic coast from Zadar to Dubrovnik and between Zadar and Split, respectively. Rickettsia akari, etiologic agent of rickettsialpox, was isolated from blood of a patient diagnosed with MSF in Zadar, but no cases of rickettsialpox were reported. Several species of pathogenic (Rickettsia slovaca, Rickettsia aeschlimannii, Ricketsia helvetica, and Ricketsia raoultii) and species of undetermined pathogenicity (Ricketsia hoogstraalii sp. nov.) rickettsiae were identified in ticks collected in different ecological regions of Croatia. A search of the literature revealed no evidence of rickettsial infection in travellers visiting Croatia. Three imported cases of Rickettsia africae were observed in travellers returning from South Africa. CONCLUSION: Rickettsiae and rickettsial diseases continue to be present in Croatia. As they can be acquired while travelling, physicians should consider rickettsial infection in the differential diagnosis of patients returning from Croatia and presenting with febrile illness.

A case of Mediterranean spotted fever associated with severe respiratory distress syndrome.

Mediterranean spotted fever (MSF) is usually a mild endemic rickettsial disease occurring in southern Croatia. We have reported the clinical and epidemiological characteristics of an acute MSF case associated with severe respiratory distress syndrome and hemodynamical instability. The patient recovered completely after antimicrobial treatment. Indirect immunofluorescence assay (FOCUS Diagnostics Inc.) was performed to detect IgM and IgG antibodies to Rickettsia conorii. A significant increase of both IgM and IgG antibody titres found in paired acute- and convalescent-phase serum confirmed the diagnosis of acute MSF.

Association of Kaposi's sarcoma-associated herpesvirus (KSHV) with bladder cancer in Croatian patients.

As the seventh most common human malignancy, bladder cancer represents a global health problem. In addition to well-recognized risk factors such as smoking and exposure to chemicals, various infectious agents have been implicated as cofactors in the pathogenesis of urothelial malignancies. The aim of the present study was to assess the possible association of viral infection and bladder cancer in Croatian patients. Biopsy specimens were collected from a total of 55 patients diagnosed with different stages of bladder cancer. Initial screening of DNA extracts for the presence of viruses on Lawrence Livermore Microbial Detection Array revealed Kaposi's sarcoma-associated herpesvirus (KSHV) in each of three randomly chosen biopsy specimens. The prevalence of infection with KSHV among study population was then examined by KSHV-specific polymerase chain reaction (PCR) and immunoblotting. By nested PCR, KSHV DNA was detected in 55% of patients. KSHV, also known as human herpesvirus 8, is an infectious agent known to cause cancer. Its oncogenic potential is primarily recognized from its role in Kaposi's sarcoma, but it has also been involved in pathogenesis of two lymphoproliferative disorders. A high prevalence of KSHV infection in our study indicates that KSHV may play a role in tumorigenesis of bladder cancer and warrants further studies.

Antimicrobial susceptibility of gram-negative and gram-positive bacteria collected from countries in Eastern Europe: results from the Tigecycline Evaluation and Surveillance Trial (T.E.S.T.) 2004-2010.

The Tigecycline Evaluation and Surveillance Trial (T.E.S.T.) commenced in 2004 to longitudinally monitor global changes in bacterial susceptibility to a suite of antimicrobial agents. The current study examined the activity of tigecycline and comparators against isolates collected across Eastern Europe between 2004 and 2010. Minimum inhibitory concentrations were determined using Clinical and Laboratory Standards Institute (CLSI) broth microdilution methodologies. Antimicrobial susceptibility was determined using CLSI interpretive criteria, and tigecycline susceptibility was established using European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. This study included 10 295 Gram-negative and 4611 Gram-positive isolates from 42 centres. Extended-spectrum ß-lactamases (ESBLs) were reported among 15.3% of Escherichia coli and 39.3% of Klebsiella pneumoniae isolates; the highest rates were observed in Turkey (30.9%) and Bulgaria (53.8%), respectively. Imipenem-non-susceptible K. pneumoniae were identified only in Turkey. ESBL-positive E. coli were highly susceptible to imipenem (95.1%), meropenem (98.0%) and tigecycline (98.5%). Most antimicrobials showed poor activity against Acinetobacter baumannii and Pseudomonas aeruginosa. Vancomycin resistance was noted among 0.9% of Enterococcus faecalis and 11.7% of Enterococcus faecium isolates. High rates of susceptibility were reported for linezolid (99.7%) and tigecycline (100%) against E. faecium. One-quarter of Staphylococcus aureus isolates were meticillin-resistant S. aureus (MRSA), with the highest rate in Romania (51.5%); all MRSA were susceptible to linezolid, tigecycline and vancomycin. Antimicrobial resistance is high in much of Eastern Europe, with considerable variation seen among countries. Tigecycline and the carbapenems retain excellent activity against many pathogens from Eastern Europe; linezolid and vancomycin are active against most Gram-positive pathogens.

Evidence of an autochthonous Toscana virus strain in Croatia.

BACKGROUND: Phleboviruses are large and widespread group of viruses that are transmitted by arthropods and they have been reported to circulate in endemic regions of Mediterranean Basin, including Croatia. OBJECTIVES: To investigate the role of Toscana virus, as a cause of the aseptic meningitis, in summer months in Croatia. STUDY DESIGN: Samples from 30 patients with aseptic meningitis were retrospectively tested by serology and RT-PCR for TOSV. RESULTS: TOSV RNA was detected in 2/30 and TOSV IgM antibodies were found in 4/30 of patients. Phylogenetic analysis of partial L and S segments suggests that TOSV from Croatia represents an autochthonous strain. CONCLUSIONS: The study has confirmed the role of TOSV as an agent that causes aseptic meningitis in Croatia, therefore it should be considered by physicians when encountering meningitis or febrile illness among indigenous population or travellers during the summer months.

Antimicrobial effects of wine: separating the role of polyphenols, pH, ethanol, and other wine components.

While the antimicrobial effectiveness of wine is well documented, relative contributions of the wine components to its antimicrobial activity is controversial. To separate the role of wine phenolics, ethanol, and pH from other wine constituents, the antimicrobial effects of intact wine were compared to that of phenols-stripped wine, dealcoholized wine, ethanol, and low pH applied separately and in combination, against 2 common foodborne pathogens, Salmonella enterica serovar Enteritidis and Escherichia coli. All samples were biochemically characterized with respect to their total phenolics and resveratrol content, antioxidant capacity, ethanol content, and pH. Antioxidative activity of the samples corresponded to their total phenolics content. Except for respective controls, pH and ethanol content were similar in all samples. The order of antibacterial activity of the samples was: intact wine > phenols-stripped wine > dealcoholized wine > combination of ethanol and low pH > low pH > ethanol. Separate application of ethanol or low pH showed negligible antibacterial activity while their combination showed synergistic effect. Antibacterial activity of the samples could not be related to their total phenolics and resveratrol content, antioxidant capacity, ethanol content, or pH. Our study indicates that antimicrobial activity of complex solutions such as intact wine cannot be exclusively attributed to its phenolic or nonphenolic constituents, nor can the antimicrobial activity of wine be predicted on the basis of its particular components.

High prevalence and molecular characterization of extended-spectrum ß-lactamase-producing Proteus mirabilis strains in southern Croatia.

The aim of this study was to determine the prevalence and antibiotic resistance rates of extended-spectrum ß-lactamase (ESBL)-producing Proteus mirabilis strains isolated from inpatients at the Split University Hospital (southern Croatia) during a survey performed between 2005 and 2008. A total of 2152 consecutive isolates of P. mirabilis were isolated. The prevalence was 0.5 % in 2005 and increased significantly to 20.9 % by 2008. Strains were most frequently isolated from urine (36.5 %) and bronchial aspirates and wound swabs (11.3 %). ESBL-producing P. mirabilis isolates showed very high resistance rates to the majority of non-ß-lactam antibiotics and were susceptible to a ß-lactam/ß-lactamase inhibitor and carbapenems. The isolates were genotyped and their ESBLs were molecularly characterized. Strains originating from the intensive care unit and the surgery and neurosurgery wards were clonally related. All P. mirabilis isolates produced the TEM-52 type of ESBL. To the best of our knowledge, our work detailed here and summarized in an earlier communication is the first report of such isolates from southern Croatia. Increased monitoring and screening for ESBL production in this species at our hospital is mandatory.

Rickettsia hoogstraalii sp. nov., isolated from hard- and soft-bodied ticks.

A novel spotted fever group Rickettsia was found in Haemaphysalis sulcata ticks collected from sheep and goats in Croatia in 2006. At the same time, a genetically identical organism was co-isolated with the embryonic cell line CCE3 obtained from the soft tick Carios capensis in Georgia, USA. In this study, further phenotypic and genotypic characteristics of the novel rickettsial strain present in H. sulcata ticks were investigated. Based on the cultivation of bacteria in mosquito and Vero cell cultures, the presence of rickettsiae in tick tissues and cell cultures [confirmed by transmission electron microscopy (TEM)] and the amplification and sequencing of five rickettsial genes, it was demonstrated that the novel Rickettsia strain fulfils the criteria to be classified as a novel species. The name Rickettsia hoogstraalii sp. nov. is proposed for the new strain. Rickettsia hoogstraalii sp. nov., an obligately intracellular bacterium, was grown in Vero cells and arthropod CCE3, ISE6 and C6/36 cell lines. The morphology of the cells of the novel species was typical of SFG rickettsiae. The small coccobacillary appearance of the bacteria was apparent with light microscopy. A Gram-negative bacterial cell wall and a cytoplasmic membrane separated by a narrow periplasmic space were visible by TEM. To date, Rickettsia hoogstraalii sp. nov. has been isolated from two species of ticks, H. sulcata and C. capensis. The novel species appears to be geographically widely distributed, having been detected in Croatia, Spain and Georgia, USA. Although no information is available regarding the possible pathogenicity of the novel species for vertebrate hosts, R. hoogstraalii sp. nov. has a cytopathic effect in Vero, CCE3 and ISE6 cells. Sequence analyses of the 16S rRNA, 17 kDa, gltA, ompA and ompB genes indicated that even though R. hoogstraalii sp. nov. was closely related to Rickettsia felis, it represents a separate species within the spotted fever group. The type strain of R. hoogstraalii sp. nov. is strain Croatica(T) (=DSM 22243(T)=UTMB 00003(T)).

Occurrence of OXA-107 and ISAba1 in carbapenem-resistant isolates of Acinetobacter baumannii from Croatia.

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 bla(OXA-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.

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.

Molecular detection of Theileria sp. in ticks and naturally infected sheep.

Seven healthy sheep and 10 sheep diagnosed with piroplasmosis based on clinical signs were tested for the presence of babesiae and theileriae. Using the molecular techniques, two species of theileriae were detected and characterized. Theileria ovis was present mostly in healthy sheep and in Rhipicephalus ticks collected from infected sheep. Theileria sp. OT3 parasite was detected mostly in ill animals which represent additional evidence to the possible pathogenic nature of Theileria sp. OT3. The presence of babesiae in sheep or in ticks was not determined. The results of this study showed that ovine piroplasmosis due to Theileria is present in Southern Croatia. It was concluded that clinical diagnosis of ovine piroplasmosis should be confirmed by molecular analysis in order to identify the species of piroplasm, to select the appropriate treatment and to exclude the threat for public health.

War-associated cases of typhoid fever imported to Split-Dalmatia County (Croatia).

OBJECTIVE: The goal was to investigate the number and origin of typhoid fever cases in Split-Dalmatia County, Croatia, during the period of 1970-2005. METHODS: Data from the medical records of typhoid fever cases treated at the University Hospital during the 35-year period were analyzed. Cases were defined as those in which Salmonella typhi was isolated from blood and/or stool. All isolates were tested for susceptibility to antimicrobial agents. RESULTS: A total of 45 cases were identified. Forty-four patients originated from Bosnia and Herzegovina, and all of their isolates were susceptible to all antimicrobial agents tested. One patient from Pakistan was infected with multidrug-resistant S. typhi. No autochthonous case of typhoid fever was identified. CONCLUSION: All cases of typhoid fever were acquired in countries where typhoid fever is endemic, and their importation to Croatia was the result of war-associated population movements. Increased awareness of health care workers regarding persons who originated from or have traveled to a typhoid-endemic area is needed. To avoid treatment failure, particular attention should be given to persons coming from regions with a known presence of multidrug-resistant S. typhi.

Molecular identification of Rickettsia felis-like bacteria in Haemaphysalis sulcata ticks collected from domestic animals in southern Croatia.

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.

High prevalence of antibodies to lymphocytic choriomeningitis virus in a murine typhus endemic region in Croatia.

A retrospective serological survey was carried out by the indirect immunofluorescence assay (IFA) and complement fixation (CF) test in a sample of 425 healthy residents (240 females and 185 males) to investigate whether Lymphocytic choriomeningitis virus (LCMV) circulates in the rural area of the northern Croatian island of Vir, which is known to be endemic for murine typhus. The overall prevalence of LCMV antibodies detected by IFA was found to be 36% (155 out of 425) and 13% (54 out of 425) by CF. No significant difference in the LCMV seroprevalence was observed with respect to age and gender by any of the methods used. The results show that inhabitants in the area studied are clearly being exposed to LCMV and the infection of the population occurs in the first 10 years of life. Equal prevalence of IgG and especially of short-lived CF antibodies in the elderly in comparison to other age groups and children can be explained by reinfections and boosting of antibodies through permanent contact with the virus. To the best of our knowledge this study reports the first evidence of endemic LCMV in Croatia and one of the highest LCMV human prevalence reported worldwide to date. The epidemiological association between LCMV and other zoonoses which can be expected in this community as well as the etiology of summer influenza-like illness along the coastal area of Croatia has to be explored further.

Clinical and epidemiological features of hospitalized acute Q fever cases from Split-Dalmatia County (Croatia), 1985-2002.

BACKGROUND: Q fever shows a wide diversity of clinical manifestation. Q fever is endemic in northern Croatia, but the epidemiological and clinical characteristics of this disease in various ecological areas of southern Croatia are unclear. MATERIAL/METHODS: From January 1985 to December 2002, acute Q fever cases hospitalized at Split University Hospital were analyzed. Acute Q fever was defined as fever (>38 degrees C) with clinical findings in lung and/or liver verified by serologic testing with Coxiella burnetii phase II antigen. RESULTS: During the period of observation, 155 acute Q fever cases were hospitalized. The mean incidence of acute Q fever in the study region was 0.20/100,000/year (95%CI:0-0.78) in the coastal area and 4.64/100,000/year (95%CI:0.44-8.85) in the non-coastal areas, with a male predominance (chi2=60.0; p=0.0000) and a mean male to female ratio of 4.2:1. People of essentially all ages (4-76 years) were affected, the highest rate of infection being recorded in 20- to 49-year-old age groups. In contrast to adults, girls were more frequently affected than boys (2:1). No case of acute Q fever was recorded on any of the nearby islands. Clinically, acute Q fever most commonly presented with both pneumonia and hepatitis (60.0%), followed by pneumonia (25.8%), hepatitis (9.0%), and nonspecific febrile illnesses (5.2%). CONCLUSIONS: C. burnetii is endemic in rural, coastal, and non-coastal areas of southern Croatia and is associated with stock breeding. In these areas, Q fever occurs sporadically and epidemically. Males 20-49 years of age were the prevalent cases.

Prevalence and antimicrobial resistance of extended-spectrum beta-lactamases-producing Escherichia coli and Klebsiella pneumoniae strains isolated in a university hospital in Split, Croatia.

The prevalence of Escherichia coli and Klebsiella pneumoniae that produce extended-spectrum b-lactamases (ESBL) was investigated in patients of a university hospital in Split, Croatia. Patients were grouped according to age (pediatric vs. adult), antibiotic type, and hospital ward. From Jan. 2001 to Dec. 2002, the susceptibility of E. coli and K. pneumoniae isolates to antimicrobials was tested. ESBL production was assayed using the double-disk synergy test. ESBL-producing E. coli and K. pneumoniae were detected in all sites of infection sampled. The percentages of ESBL-positive isolates were higher in the pediatric wards than in the adult wards. The antibiotics most commonly prescribed to patients in all hospital wards belonged to the third-generation cephalosporin group. Among ESBL producers, E. coli isolates were more resistant to aminoglycosides, but less resistant to ciprofloxacin and cotrimoxazole. Resistance of E. coli and K. pneumoniae to ciprofloxacin was exclusively found in isolates from adult patients. None of the isolates, regardless of ESBL production, was resistant to carbapenemes. In addition, the prevalence and antimicrobial resistance of ESBL-producing E. coli and K. pneumoniae isolates differed between pediatric and adult patients.

Prevalence and antimicrobial resistance of extended-spectrum β-lactamases-producing Escherichia coli and Klebsiella pneumoniae strains isolated in a university hospital in Split, Croatia / Prevalencia y resistencia antimicrobiana de cepas de Escherichia coli y Klebsiella pneumoniae productoras de β-lactamasas de espectro extendido aisladas en un hospital universitario de Split (Croacia)

The prevalence of Escherichia coli and Klebsiella pneumoniae that produce extended-spectrum β-lactamases (ESBL) was investigated in patients of a university hospital in Split, Croatia. Patients were grouped according to age (pediatric vs. adult), antibiotic type, and hospital ward. From Jan. 2001 to Dec. 2002, the susceptibility of E. coli and K. pneumoniae isolates to antimicrobials was tested. ESBL production was assayed using the double-disk synergy test. ESBL-producing E. coli and K. pneumoniae were detected in all sites of infection sampled. The percentages of ESBL-positive isolates were higher in the pediatric wards than in the adult wards. The antibiotics most commonly prescribed to patients in all hospital wards belonged to the third-generation cephalosporin group. Among ESBL producers, E. coli isolates were more resistant to aminoglycosides, but less resistant to ciprofloxacin and cotrimoxazole. Resistance of E. coli and K. pneumoniae to ciprofloxacin was exclusively found in isolates from adult patients. None of the isolates, regardless of ESBL production, was resistant to carbapenemes. In addition, the prevalence and antimicrobial resistance of ESBL-producing E. coli and K. pneumoniae isolates differed between pediatric and adult patients (AU)

Se ha investigado la frecuencia de Escherichia coli y Klebsiella pneumoniae productoras de β-lactamasas de espectro extendido (ESBL) en pacientes de un hospital universitario de Split (Croacia). Los pacientes se agruparon en relación con la edad, el tipo de antibiótico recetado y la ubicación en el hospital. Desde enero de 2001 a diciembre de 2002 se realizaron ensayos de susceptibilidad a antimicrobianos en aislados de E. coli y K. pneumoniae. La producción de ESBL fue ensayada mediante tests de sinergia de disco doble. En todos los lugares de infección se detectaron aislados de E. coli y K. pneumoniae productores de ESBL. En las salas de pediatría se detectaron los porcentajes más elevados de ESBL-positivos. Los antibióticos más recetados en el hospital fueron las cefalosporinas de tercera generación. Entre los aislados productores de ESBL, los de E. coli fueron más resistentes a los aminoglicósidos, pero menos a ciprofloxacina y cotrimoxazol. Sólo se halló resistencia de E. coli y K. pneumoniae a la ciprofloxacina en aislados obtenidos de pacientes adultos. Ningún aislado, independientemente de la producción de ESBL, fue resistente a los carbapenemos. Se hallaron diferencias en la incidencia y resistencia antimicrobiana de los aislados de E. coli y K. pneumoniae productores de ESBL entre pacientes pediátricos y pacientes adultos (AU)

Q fever epidemic among employees in a factory in the suburb of Zadar, Croatia.

AIM: To examine the role of wind in the spread of Q fever (Coxiella burnetii) from the source of infection (sheep on the pastures) to the factory where there was an outbreak of Q fever among the employees. METHODS: We performed clinical (fever, coughing, myalgias, arthralgias), laboratory (complete blood test, aminotransferases, antibodies to Coxiella burnetii), radiographic (chest X-ray), and epidemiological (questionnaire) analysis on 47 of 110 employees of the plant in a suburb of Zadar. Sera of 182 sheep were tested for antibody to C. burnetii by complement fixation reaction. RESULTS: During the first half of March 2004, 14 of 110 employees of a factory in a suburb of Zadar were diagnosed with Q fever on the basis of clinical and laboratory findings. In three sections of the plant, directly exposed to the north wind, a diagnosis of Q fever was confirmed in 14 of 110 employees by clinical, laboratory, and X-ray analysis, whereas there were no sick employees in the other four sections. North of the plant there were pastures where many flocks of sheep grazed. Antibodies to C. burnetii were found in 20 out of 182 sheep sera. Employees who were exposed to the north wind, had a significantly higher possibility of acquiring Q fever than did those working in sections protected from the wind. CONCLUSION: North wind (bura) containing the aerosolized C. burnetii likely influenced the Q fever outbreak in persons far from the source of the infection.

First isolation of Rickettsia conorii from human blood in Croatia.

AIM: To detect and isolate rickettsial strains from blood samples of patients with presumptive diagnosis of Mediterranean spotted fever (MSF) in the coastal region of south Croatia, and to compare the results with routine serology. METHODS: A "suicide" polymerase chain reaction (PCR), and a shell vial culture were done on samples of ethylenediamine tetra-acetic acid (EDTA) and citrate-anticoagulated blood samples. Indirect immunofluorescence was performed on sera collected from 17 patients clinically diagnosed with MSF during summer in three consecutive years, from 1998 to 2000. RESULTS: The primers used in PCR amplified the expected part of the rickettsia genomic DNA and Rickettsia conorii grew from the shell vial-cultured blood of a single patient. In 13 patients, the diagnosis was confirmed serologically by paired sera, whereas in 4 patients the diagnosis remained presumptive, since no paired sera were available. Analyzing sequences of the ompA and citrate synthase gene, respectively, derived from the shell vial isolate, a 100% similarity with Rickettsia conorii, strain Seven (Malish), was found. CONCLUSION: To the best of our knowledge, this is the first isolation of Rickettsia conorii from a human sample in Croatia, and the first proof of a causative agent of MSF in the country. Beside PCR-based methods and isolation, correct diagnosis of MSF could be still routinely reached by serology.