Seroprevalence and entomological study on Chikungunya virus at the Croatian littoral.

During 2011-2012, a total of 1008 serum samples from randomly selected inhabitants of seven Croatian counties located on the Adriatic Coast were tested for the presence of chikungunya virus (CHIKV) IgG antibodies using indirect immunofluorescence assay. Nine participants (0.9%) from four counties were found to be seropositive to CHIKV. Seroprevalence varied from 0.5% to 1.8% between counties. Additionally, a total of 3,699 mosquitoes were captured in 126 localities from August 16 to September 24, 2011. Three mosquito species were found: Ae. albopictus (3010/81.4%), Cx. pipiens (688/18.6%) and only one specimen of the Cs. longiareolata. Female mosquitoes (N = 1,748) were pooled. All pools tested negative for CHIKV RNA using a real-time RT-PCR.

Dengue virus infection in Croatia: seroprevalence and entomological study.

During 2011-2012, a total of 1,180 sera samples were collected from residents of seven Croatian counties located on the Adriatic Coast and four counties in northeastern Croatia and tested for the presence of dengue virus (DENV) IgG antibodies using ELISA. Reactive samples were further tested by IFA. Seven samples (0.59%) tested positive for DENV antibodies. Seroprevalence rates by county varied from 0-2.21%. The highest seropositivity rate (2.21%) was found in the Dubrovnik-Neretva County where autochthonous dengue cases were recorded in 2010. Additionally,3,699 mosquitoes were collected from 126 localities along the Adriatic coast in August-September, 2011. Aedes albopictus was the most prevalent species (81.37%). No evidence of DENV RNA was detected by RT-PCR among 1,748 female mosquitoes.

Sexually Transmitted Diseases as a Risk for Acquiring HIV Infection among the Population of Men Who Have Sex with Men--A Case-Control Study.

At the beginning of the 1980-ies, HIV infection and AIDS were described for the first time, this among the population of men who have sex with other men. Nearly thirty years later, the MSM population is still a population under heightened risk for acquiring HIV infection and other sexually transmitted diseases. This study investigates sexually transmitted diseases as a risk for HIV infection. A total of 296 men who have sex with men (MSM) were included in this case control study. Differences among the frequencies of sexually transmitted diseases among the MSM of HIV positive and HIV negative status were tested. The history of HIV positive more often states falling ill with sexually transmitted diseases than this was the case before they became HIV positive, unlike those MSM who are not HIV infected (45.9%:11.1% that is OR 6.79, 95% CI 3.49-13.19). Hepatitis B infection is more frequent in HIV positive MSM (11.5%:1.9%; OR 6.58, 95% CI 1.86-23.3). The frequency of gonorrhea in case history of HIV positive MSM is significantly higher than in the HIV negative group (11.5%:3.8%, OR 3.24, 95% CI 1.13-9.34). In the group of HIV positive MSM, unlike the HIV negative group, syphilis (14.8:1.0%, OR 1774, 95% CI 3.43-122.87) and genital herpes (8.2%:0.5%, OR 18.39, 95% CI 2.03-424.7) are more frequent. The results of this study will be used in future preventive activities focused on the population of MSM, as a population under particular risk for acquiring sexually transmitted infections.

First outbreak of West Nile virus neuroinvasive disease in humans, Croatia, 2012.

Between September 6 and 21, 2012, seven human cases of West Nile virus (WNV) neuroinvasive infection were laboratory confirmed in Croatia. The median patient age was 62 years (range 48-77). Five patients presented with meningoencephalitis and two patients with meningoencephalitis followed by acute flaccid paralysis. Four of them had an underlying disease (hypertension). Using enzyme-linked immunosorbent assay (ELISA), WNV-specific immunoglobulin M (IgM) and IgG antibodies of low avidity were detected in six patients, whereas one showed only IgM antibodies. All samples were confirmed using plaque-reduction neutralization and microneutralization tests. Five patients recovered fully. Before human cases were reported, acute asymptomatic WNV infection was demonstrated by detection of IgM antibodies in sentinel horses. Moreover, an increased WNV IgG seropositivity in horses was detected in counties where human cases occurred. Adulticidal and larvicidal treatments were administered immediately in the respective places of residence. The end of the warm season contributed to the fact that there were no new cases of WNV disease recorded.

[West nile virus infection: re-emergent disease in Croatia]. / Infekcija virusom zapadnog nila: re-emergentna bolest u hrvatskoj.

West Nile virus (WNV) is a small, enveloped, spherical virus that belongs to the family Flaviviridae, genus Flavivirus, Japanese encephalitis serocomplex. Natural reservoirs of WNV are birds, and the main vectors are mosquitoes of the genus Culex. There are seven genetic lineages of WNV. Lineages 1 and 2 are the most widely distributed (Africa, North and South America, Europe, Asia and Australia). About 80% of infections are asymptomatic. In 20% of patients nonspecific febrile disease occurs (West Nile fever). Less than 1% of infected persons will develop neuroinvasive WNV disease (meningitis, encephalitis, and poliomyelitis). In Croatia, antibodies to WNV were demonstrated in humans,bears and horses. In August-September 2012 clinical cases of human WNV neuroinvasive disease and asymptomatic acute infection in horses were reported for the first time in three eastern Croatian counties. The diagnosis was confirmed by serologic tests (enzyme immunoassay, IgG avidity, plaque-reduction neutralization test).

Demonstration of Usutu virus antibodies in horses, Croatia.

We report the first serological evidence of Usutu virus (USUV) infection in horses in Croatia. During 2011, 1380 horse serum samples from healthy animals were collected from six northern Croatian counties. All samples were first screened for West Nile virus (WNV) immunoglobulin G (IgG) antibodies using an enzyme-linked immunosorbent assay (ELISA). Sixty-nine WNV ELISA-reactive samples were further tested for WNV antibodies by a virus neutralization assay (VN assay) and plaque reduction neutralization test (PRNT), and USUV by a VN assay and tick-borne encephalitis virus (TBEV) antibodies by PRNT. During the same period, 306 human serum samples from patients coming for routine testing with no symptoms of acute febrile disease were tested for USUV IgG using ELISA. Reactive samples were tested for both USUV and WNV using a VN assay. USUV-specific neutralizing antibodies were detected in two of 69 WNV ELISA-reactive horse serum samples. Seropositive animals were found in two different regions of Croatia. One additional sample showed specific WNV-neutralizing antibodies that cross-neutralized USUV. Only one human sample (0.3%) was reactive to USUV antibodies in an ELISA test. In a confirmatory test, WNV-neutralizing antibodies were detected, indicating cross-reactive antibodies with USUV in ELISA. The exposure to USUV was documented in two WNV ELISA-reactive horses at distant locations. These results indicate the presence of USUV in northern Croatia.

[Epidemiology of viral hepatitis]. / Epidemiologija virusnih hepatitisa.

Understanding the country-specific epidemiology of disease, which may vary greatly among countries, is crucial for identifying the most appropriate preventive and control measures. An overview of the local epidemiology of viral hepatitis in Croatia is given in this paper. The overall prevalence of hepatitis B in Croatia is low (less than 2% HBsAg carriers in the general population). Hepatitis B incidence and prevalence began to decline significantly following the introduction of universal hepatitis B vaccination in 1999. Information on HBsAg seroprevalence is derived from routine testing of certain subpopulations (pregnant women, blood donors) and seroprevalence studies mostly targeted at high-risk populations. Universal childhood vaccination against hepatitis B remains the main preventive measure. We recommend testing for immunity one to two months after the third dose of hepatitis B vaccine for health-care workers. The incidence and prevalence of hepatitis C have also been declining in the general population. The main preventive measures are ensuring safety of blood products, prevention of drug abuse, and harm reduction programs for intravenous drug users. Hepatitis A incidence has declined dramatically since fifty years ago, when thousands of cases were reported annually. In the last five years, an average of twenty cases have been reported per year. The reduction of hepatitis A is a consequence of improved personal and community hygiene and sanitation. Hepatitis D has not been reported in Croatia. The risk of hepatitis D will get to be even smaller as the proportion of population vaccinated against hepatitis B builds up. Hepatitis E is reported only sporadically in Croatia, mostly in persons occupationally in contact with pigs and in travelers to endemic countries. In conclusion, Croatia is a low prevalence country for hepatitides A, B and C. Hepatitis D has not been reported to occur in Croatia and there are only sporadic cases of hepatitis E. Since hepatitis A is a rare disease occurring sporadically, which is a consequence of improved sanitation and hygiene, hepatitides B and C are the main causes of viral hepatitis in Croatia. The introduction of universal mandatory hepatitis B vaccination of schoolchildren in 1999 resulted in a decrease in the incidence of hepatitis B, which is most pronounced in adolescents and young adults, and further decrease in the incidence and prevalence is expected as the pool of susceptible individuals decreases through vaccination. The incidence of hepatitis C is decreasing as well. In spite of a relatively favorable epidemiological situation, hepatitis B and C are still a significant public health burden with an estimated 25,000 persons chronically infected with HBV and about 40,000 persons chronically infected with HCV in Croatia.

Epidemiological patterns of tuberculosis in Croatia in the period 1996-2005.

The last comprehensive publication on tuberculosis in Croatia and the earliest impact of war, besides the yearly routine reports, was done in 1996 in Croatian. We were, therefore, interested to explore incidence trends and to highlight the early post-war tuberculosis epidemiological patterns in the next ten years period (1996-2005). A retrospective analysis of epidemiological data on all registered tuberculosis cases in Croatia searching the databases of 21 Croatian Public Health Institutes and the National Tuberculosis Registry was made. During the study period, the total tuberculosis incidence rates in Croatia dropped from 45 to 25.8/100 000 inhabitants. The average highest age-specific rates were recorded in the age group > or = 65 years being in decrease in all age groups. Paediatric cases (0-14 years) represented 4.5% of all cases. Tuberculosis cases among males were recorded in 64% cases, and 83.6% were indigenous population. Tuberculosis was bacteriologically confirmed in 67.7% cases. A low proportion of drug resistance (3.3%) was recorded. During 1985-2005, 56 tuberculosis cases among 242 AIDS cases were reported. Tuberculosis mortality showed a decreasing trend (p < 0.001). However, tuberculosis has still had the highest mortality rates among infectious diseases in Croatia. Despite the War chain of events and tuberculosis programmatic changes, tuberculosis incidence rates in Croatia have been decreasing but they are still far away from national target, incidence rate of 10/100 000 declared in 1998 and much higher than in European Union and Western Europe. Tuberculosis among children, resistance to tuberculosis drugs and HIV prevalence, significant problems in many European countries, have not caused problems in tuberculosis control in Croatia. This favourable epidemiological situation must be kept and improved through strengthened tuberculosis control measures.

Risk behaviors for getting HIV infection among the Croatian men who have sex with men.

This study was conducted with the aim of obtaining the very first information on the sexual risk behavior of Croatian men who have sex with men (MSM). There were 1127 respondents recruited at four venues: three physical meeting places of the Croatian MSM population (disco club, bar and sauna) in Zagreb and one virtual (gay oriented web-site) meeting place of the Croatian MSM population. The overall response rate was only 19%. The rate of condom use during last anal intercourse was 59% and 56% of those who used a lubricant had chosen an incorrect product to use with latex condoms. There was no connection between drug-use and unprotected anal intercourse (UAI). Only a fifth of those who had also had sex with women (MSM/MSW) during last 12 months regularly used protection. The obtained findings will serve for focused and effective prevention activities and a basis for comparison in future research.

[An epidemic of trichinellosis in autumn 2004 in Slavonski Brod]. / Epidemija trihineloze na podrucju Slavonskog Broda u jesen 2004. Godine.

Trichinellosis is a worldwide zoonotic disease caused by a nematode Trichinella sp. Cases of Trichinella spiralis infection in Slavonski Brod were investigated. A total of 64 trichinellosis cases were notified to the Institute of Public Health, Brod-Posavska County between October and November 2004. There were 64 patients, 32 of them female, age range 3-67, mean 35 years. The mean incubation period was 3 (range 1-5) weeks. The epidemic started in October and terminated 39 days later. The patients ate smoked sausages. Trichinella spiralis was identified in sausages by artificial digestion. Among 64 people infected, 65% had facial and/or periorbital edema, 56% diarrhea, 53% myalgia, 48% fever, 26% headache, 23% weakness, 6% conjunctivitis, 6% cough and 3% vomitus, 6% of patients were free from symptoms and 17% were hospitalized. Eosinophils were elevated in 92%, creatinine kinase in 60%, and lactate dehydrogenase in 59% of patients; 97% of patients were positive for Trichinella spiralis on indirect immunofluorescence.

Vaccine regulations in Croatia.

In this paper legal prerequisites for vaccine licensure in Croatia are discussed. The Croatian legislation concerning vaccine licensing, marketing authorisation and utilization is reviewed. The procedures for including a vaccine into the Mandatory Childhood Vaccination Programme are also discussed with focus on Human papillomavirus (HPV) vaccines. Non-obligatory vaccination recommendations are given when according to professional opinion; vaccination is beneficial for the vaccinee. There is little doubt that HPV vaccines should be recommended for preadolescent girls in Croatia. However, reaching a decision on its possible introduction into the Childhood Vaccination Programme will require careful consideration of the larger picture and a comparison of the cost-effectiveness of a mandatory vaccination against other competing public health priorities.

Mumps virus strains isolated in Croatia in 1998 and 2005: Genotyping and putative antigenic relatedness to vaccine strains.

Two mumps virus strains 9218/Zg98 and Du/CRO05 were isolated in two locations in Croatia in 1998 and 2005, respectively. Genetic characterization of these temporally distinct mumps virus isolates was carried out in order to determine their genotype and putative antigenic relatedness to mumps virus vaccine strains. Sequence analysis of the small hydrophobic (SH) gene revealed that isolate 9218/Zg98 shows less than 95% of similarity to any reference strain, thus representing a potential reference strain for a new genotype. Isolate Du/CRO05 clearly belongs to genotype G with the 97% of homology to the reference strain Glouc1/UK96. When compared to each other, the two Croatian strains have extremely low level of homology of only 89% indicating no relatedness between them. Putative antigenic properties of the HN protein of these two isolates were compared to different vaccine strains. The results reveal a higher level of homology of antigenic determinants to non-A genotype vaccine strains than to A genotype vaccine strain.

Epidemiology of HIV infection and AIDS in Croatia--an overview.

This article presents an overview of HIV/AIDS epidemiology and surveillance in Croatia 20 years after the first documented case of AIDS in the country. Here we describe strategies employed for HIV/AIDS surveillance in Croatia as well as preliminary results of HIV seroprevalence among most-at-risk populations (MARPs) research conducted by the Infectious Diseases Epidemiology Service at the Croatian National Institute of Public Health (CNIPH). Croatia has a low incidence and prevalence of HIV and AIDS. At the end of 2005, there were 553 documented cases of HIV infection, 239 of which progressed to AIDS. In Croatia, AIDS is being registered within MARPs only and dominantly among men who have sex with men (MSM). AIDS patients and HIV infected persons are found in all parts of the country. Crude prevalence of HIV among MARPs was found to be 0.9%. It is necessary to continue with current prevention and control measures in the country, and to create a culture of awareness and precaution, a strategy that has proven effective in reducing risk of HIV infection.

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.

[MHC tetramers: tracking specific immunity]. / MHC-tetrameri: na tragu specificnoj imunosti.

In an adaptive immune response, antigen is recognized by two distinct sets of highly variable receptor molecules: (1) immunoglobulins, that serve as antigen receptors on B cells and (2) the antigen-specific receptors on T cells. T cells play important role in the control of infection and in the development of protective immunity. These cells can also mediate anti-tumor effects and, in case of autoimmune syndromes, contribute to the development and pathology of disease. The specificity of T cells is determined by T cell receptors (TCR). Understanding of the success of immune responses requires the direct measurement of antigen-specific T lymphocytes. Cell with major histocompatibility complex (MHC) class I molecules are able to present antigens to antigen-specific CD8+ cytotoxic T lymphocytes. MHC class I molecules present small peptides (epitopes) processed from intracellular antigens such as viruses and intracellular bacteria. MHC class I molecules in humans are designated as human leukocyte antigen (HLA) class I and divided into HLA-A, -B and -C. CD8+ T cells recognize MHC class I molecules and after activation produce proteins that destroy infected cells. MHC class II molecules receive their peptides mainly from extracellular and soluble antigens and present them to the CD4+ T helper cells. A recently described technique that can be used in flow cytometry enables us to quantify ex vivo antigen-specific T cells by binding of soluble tetramer MHC-peptide complexes attached to fluorochrome. Quantitative analyses of antigen-specific T cell populations provide important information on the natural course of immune responses. The interaction of T cell receptors on T lymphocytes with tetrameric MHC-peptide complexes mimics the situation on the cell surface, and allows for reliable binding. Tetramers consist of four biotinylated HLA-peptide epitope complexes bound to streptavidin conjugated with fluorescent dye. Tetramer technology has sensitivity of detection as little as 0.02% of total cytotoxic T cell pool or T helper cell pool (i.e. approximately 1 in 50.000 lymphocytes). The combination of this technology with intracellular cytokine staining methods opens up significantly better ways of studying these cells than previously possible, allowing immunologists to look at their life cycle (activation and proliferation), manner of death (aging and apoptosis) and effector function (cytotoxic potential and cytokine production). MHC tetramers class I have yielded useful insights into in vivo dynamic and function of antigen-specific CD8+ T cells in viral infections, parasitic infections, cancer, autoimmune disease and transplantation. This knowledge is of special interest for immunotherapy, diagnostic monitoring of T cell mediated immunity, and the development of new vaccination strategies. There is some possibility for cell therapy with antigen-specific CD8+ T cells for various diseases including cancer and viral infections. Targeted immunotherapy of selective deletion of auto--or alloreactive T cells with MHC tetramers may be important for the treatment of autoimmune disease, or to prevent the rejection of transplanted organs. The utility of this technique for the immunotherapy in vivo needs to be confirmed and modified in further research. Understanding how antigen-specific cells develop and function in different circumstances and pathologies will be the key to unravelling the secrets of cellular immune system.

Role of epidemiologic information system in sudden death prevention among Croatia's haemodialysis patients.

The main aim of Croatia's epidemiological information system is to enable the monitoring of infectious diseases as well as to enable necessary interventions. Its 24-hour epidemiologic alert mechanism made it possible for initial reports of sudden death clusters in haemodialysis units to reach epidemiologists on the night of 12 October 2001. The alert about the sudden deaths among haemodialysis patients in two hospitals has prompted us to do an epidemiological inquiry during that night and early the next morning. It looked at suspicions of a possible link between Baxter Plivadial P-15 lot 2001F075 dialyser and these deaths. As the suspicion was based on a small amount of data, it needed confirmation from a countrywide inquiry involving the other 40 haemodialysis units. A phone inquiry showed that 23 haemodialysis deaths occurred on 8-13 October 2001. It suggested that also dialyser of a different type and lot (P-18 2001B17R) should be incriminated. During the inquiry (October 13), all haemodialysis units were informed of the suspicions regarding incriminated dialysers, and of the necessity of temporary withdrawal of these from use. The withdrawal on 13 October of incriminated dialysers stopped the deaths among haemodialysis patients. No more such sudden deaths have been recorded since that could be linked either to the described period or to this clinical picture.

[The variola virus as a biological weapon]. / Virus variole kao biolosko oruzje.

In view of the threat of use of the variola virus as a biological weapon, the interest of medical and other public in this causative agent that was eradicated in the wild at the end of the 1970s has increased. The paper gives an outline of the current knowledge on biological properties of the variola virus, and on the epidemiology, pathogenesis, clinical picture and prophylaxis of the disease caused by this virus. Descriptions of two sudden smallpox epidemics (Germany in 1970 and former Yugoslavia in 1972) could illustrate the potential of the smallpox virus as a biological weapon in bioterrorism and biological warfare. In fact, this virus can spread very readily through aerosol, which may lead to explosive epidemics. Not having been immunised, our population aged less than 25 years totally lacks the immunity. Older individuals are likely to have a low residual specific immunity to the agent. The only way to prevent a smallpox epidemic is by vaccination and patient isolation. A rapid smallpox diagnostics and prompt vaccination of all contacts is of utmost importance in stopping the outbreak.

Epidemiological analysis of tuberculosis in the Kingdom of Croatia and Slavonia during 1901-1910.

AIM: To analyze data on epidemiology and treatment of tuberculosis from the sources related to the history and epidemiology of tuberculosis in the Kingdom of Croatia and Slavonia in the 1900-1910 period. METHODS: Epidemiological data were obtained from the statistical yearbooks of the Kingdom of Croatia and Slavonia for years 1905 and 1910. A number of sources and publications on the history of medicine, especially on the history of tuberculosis in the studied period were analyzed. Mortality ratio, birth rate ratio, and population growth were calculated. Tuberculosis mortality per 100,000 inhabitants was calculated for four major cities and eight counties of the Kingdom of Croatia and Slavonia. The difference in tuberculosis mortality rates between urban and rural areas and socio-economic situation and organization of the health service were analyzed. The incidence, prevalence, and morbidity from tuberculosis were not analyzed because of the lack of data. RESULTS: The tuberculosis mortality ratio per 100,000 inhabitants in the Kingdom of Croatia and Slavonia were constantly high (above 400 on average), with higher mortality ratio in urban than in rural areas. According to the opinion of the Croatian physicians and statistical data of the period, this was due to the constant growth of population, insufficient health care system, and bad living conditions. The highest tuberculosis mortality rates were in the cities of Zemun (600-800) and Zagreb (500-700), and in the counties of Srijem (600-400), Virovitica (600-400), and Lika- Krbava (500-400). CONCLUSION: There was a concordance between the statistical data and the publications, reports, and articles written in the 19th and 20th century on tuberculosis situation in the Kingdom of Croatia and Slavonia. The analysis showed that tuberculosis was one of the major causes of death in the Kingdom of Croatia and Slavonia from 1901 to 1910. This fact had a significant influence on tuberculosis prevention and eradication efforts in the first half of the 20th century in Croatia and laid basis for further and more elaborate measures against tuberculosis epidemics.