Tatjana Vilibić-Čavlek

The biennial cycle of respiratory syncytial virus outbreaks in Croatia.

The paper analyses the epidemic pattern of respiratory syncytial virus (RSV) outbreaks in children in Croatia. Over a period of 11 consecutive winter seasons (1994–2005) 3,435 inpatients from Zagreb County aged from infancy to 10 years who were hospitalised with acute respiratory tract infections were tested for RSV-infection. RSV was identified in nasopharyngeal secretions of patients by virus isolation in cell culture and by detection of viral antigen with monoclonal antibodies.In the Zagreb area, RSV outbreaks were proven to vary in a two-year cycle, which was repeated every 23–25 months. This biennial cycle comprised one larger and one smaller season. Climate factors correlated significantly with the number of RSV cases identified only in the large seasons, which suggests that the biennial cycle is likely to continue regardless of meteorological conditions. Knowledge of this biennial pattern should be useful in predicting the onset of RSV outbreaks in Croatia, and would facilitate planning for the prevention and control of RSV infections in the region.

Seroprevalence of TORCH infections in women of childbearing age in Croatia

During 2005–2009, a seroepidemiological study was carried out in Croatia to define the population susceptible to common TORCH agents among pregnant and non-pregnant women of childbearing age. The IgG seroprevalence was 29.1% forT. gondii, 94.6% for rubella, 75.3% for cytomegalovirus (CMV), 78.7% for herpes simplex virus type 1 (HSV-1), and 6.8% for HSV-2. Acute toxoplasmosis and CMV infection (positive IgM antibodies with low IgG avidity) were documented in 0.25% and 0.09% women, respectively. IgM prevalence was 1.2% for both HSV-1 and HSV-2. None of the participants showed acute rubella infection. Seropositivity to T. gondii and HSV-2 varied significantly between age groups (p = 0.001 and p = 0.036, respectively). Women residing in rural regions showed a significantly higher seroprevalence rate for T. gondii, CMV, and HSV-1 than urban women (T. gondii: 44.0% vs. 25.4%, p < 0.001; CMV: 85.0% vs. 73.1%, p = 0.018; HSV-1: 86.0% vs. 76.4%, p = 0.041).

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.

Clinical and molecular characterization of a parechovirus type 1 outbreak in neonates in Croatia.

During July 2009 an outbreak in neonates represented with gastrointestinal and respiratory symptoms was observed at the Neonatal Postintensive Care Unit, Clinical Hospital Center, Zagreb. Human parechovirus type 1 (HPeV1) was isolated from seven patients, one of whom was asymptomatic. All but one were premature neonates with serious underlying conditions, and all recovered fully after several days. In order to characterize the HPeV1s, sequencing of the VP1/2A region was conducted on six isolates from the outbreak and four isolates detected in Croatia in 2008 and 2007. The analysis of sequence similarity showed that the nucleotide identity between the prototype strain (Harris) and HPeV1 isolated in Croatia was 76.5–77.5%. Croatian strains from 2007 and 2009 clustered together with strains from the Netherlands and Germany detected in 2003 and 2006, respectively, while strains from 2008 clustered with the strain from Finland detected in 2000. Change of the dominant strains each year may suggest antigenic variation as a result of viral response to specific immunity of the target population. J. Med. Virol. 83:137–141, 2011.

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.

Prevalence and dynamics of cytomegalovirus infection among patients undergoing chronic hemodialysis.

Cytomegalovirus (CMV) is an important pathogen in immunocompromised individuals. The aim of this study was to analyze prevalence and dynamics of CMV infection among patients undergoing chronic hemodialysis. From 2010 to 2012, a total of 162 patients and 160 control subjects were tested for the presence of CMV IgM and IgG antibodies using enzyme-linked immunosorbent assay. IgM/IgG reactive samples were further evaluated for IgG avidity to confirm or rule out recent primary CMV infection. The overall IgG seropositivity was higher in hemodialysis patients compared to controls (90.7% vs. 81.9%; crude odds ratio [OR] =2.02, 95% confidence interval [CI] =1.05–3.89; OR adjusted for age and gender = 2.18, 95% CI = 1.05–4.55). CMV IgG antibody titers were similar in both groups. There was no difference in CMV prevalence between males (87.9%) and females (96.3%). According to age, a progressive increase in seropositivity was observed in both hemodialysis patients and the control group. Three hemodialysis patients (1.9%) developed recurrent CMV infection (positive IgM with high avidity IgG antibodies). In one patient (2.9%), seroconversion was documented during the second year of the follow-up period indicating primary infection. In contrast, in the control group, recent primary CMV infection (positive IgM with low/borderline IgG avidity) was demonstrated in three subjects (1.9%), whereas one (0.6%) developed recurrent infection. On multivariate logistic regression, hemodialysis and older age were significant predictors for CMV seropositivity.

Epidemiology of hepatitis C in Croatia in the European context.

We analyzed prevalence, risk factors and hepatitis C virus (HCV) genotype distribution in different population groups in Croatia in the context of HCV epidemiology in Europe, with the aim to gather all existing information on HCV infection in Croatia which will be used to advise upon preventive measures. It is estimated that 35000-45000 of the Croatian population is chronically infected with HCV. Like in other European countries, there have been changes in the HCV epidemiology in Croatia over the past few decades. In some risk groups (polytransfused and hemodialysis patients), a significant decrease in the HCV prevalence was observed after the introduction of routine HCV screening of blood/blood products in 1992. Injecting drug users (IDUs) still represent a group with the highest risk for HCV infection with prevalence ranging from 29% to 65%. Compared to the prevalence in the Croatian general population (0.9%), higher prevalence rates were found in prison populations (8.3%-44%), human immunodeficiency virus-infected patients (15%), persons with high-risk sexual behavior (4.6%) and alcohol abusers (2.4%). Low/very low prevalence was reported in children and adolescents (0.3%) as well as in blood donors (0%-0.009%). In addition, distribution of HCV genotypes has changed due to different routes of transmission. In the general population, genotypes 1 and 3 are most widely distributed (60.4%-79.8% and 12.9%-47.9%, respectively). The similar genotype distribution is found in groups with high-risk sexual behavior. Genotype 3 is predominant in Croatian IDUs (60.5%-83.9%) while in the prison population genotypes 3 and 1 are equally distributed (52.4% and 47.6%). Data on HCV prevalence and risk factors for transmission are useful for implementation of preventive measures and HCV screening.

Seasonal occurrence of human metapneumovirus infections in Croatia.

Background: Human metapneumovirus (HMPV) is 1 of the commonest causes of viral ARI especially among pediatric patients. Its incidence varies from year to year in countries belonging to moderate climate zone. The aim of this study was to investigate epidemiologic characteristics of HMPV infections in Croatia. Methods: During a 4-year period (January 1, 2009, through December 31, 2012), nasopharyngeal aspirates were collected from 2610 children <10 years who were admitted to hospitals with acute respiratory infections. Direct immunofluorescence assay was used to detect the virus from clinical samples. Demographics and clinical data were also analyzed. Results: HMPV was detected in 8.4% of patients. While many of HMPV-infected children were 13–24 months of age (30.9% of all proven HMPV infections), the highest incidence of HMPV infection was recorded in 2- to 5-year-old children (11.4% of all children in this age group). HMPV caused 7.1% of upper respiratory tract infections and 11.7% of lower respiratory tract infections. Annual prevalence rates of HMPV infection varied significantly from year to year (P < 0.001). Peak incidence was detected in spring or winter months, depending on the year. Conclusions: This study indicates that HMPV infections in Croatia show a biennial outbreak pattern characterized by alternation of winter and spring activity. HMPV outbreaks alternate with respiratory syncytial virus outbreaks.

The role of IgG avidity determination in diagnosis of Epstein-Barr virus infection in immunocompetent and immunocompromised patients

There is a high degree of variability in the serologic response to Epstein-Barr virus (EBV) infection, especially in viral capsid antigen (VCA)-IgM antibodies. Therefore, additional tests are needed to confirm primary infection. We evaluated the value of IgG avidity determination in diagnosis of EBV infection in immunocompetent and immunocompromised patients. A total of 236 serum samples from immunocompetent patients with symptoms suggestive of EBV infection were tested for the presence of VCA-IgM/IgG antibodies and IgG avidity. Using IgG avidity, acute primary infection was confirmed in 56.7% of the immunocompetent patients with positive and in 1.8% of patients with negative VCA-IgM. Recent primary infection was documented in 8.9% of the IgM positive and 3.5% of the IgM negative patients. In patients with indeterminate serology (equivocal IgM), 6.7% were classified by avidity index (AI) as acute primary infection, 10.0% as post-acute and 83.3% as past infection cases. Concerning the 32 immunocompromised patients, recent primary infection was documented in 3 of the 14 IgM positive patients. High AI was detected in 11 of these patients, indicating an IgM response due to reactivation. Determination of IgG avidity in combination with classical serologic markers seems to be a reliable method to confirm primary infection both in immunocompetent and immunocompromised patients. It may be especially useful to differentiate cases of primary infection in patients with undetectable VCA-IgM antibodies or indeterminate routine EBV serology.

Herpes simplex virus infection in the Croatian population

Abstract Background: Herpes simplex virus (HSV) infections are caused by 2 types of virus, HSV-1 and HSV-2. Both viruses are endemic worldwide. There are marked variations in the seroprevalences of HSV-1 and HSV-2 in Europe. The aim of this study was to determine the seroprevalence of HSV infections in Croatia. Methods: During a 3-y period (2008–2010), a total of 1672 patients were tested for the presence of HSV-1 and HSV-2 antibodies using an enzyme-linked immunosorbent assay. Results: The overall immunoglobulin G (IgG) seroprevalence rates were 72.5% for HSV-1 and 9.9% for HSV-2. There was no significant difference in seropositivity between males and females for HSV-1 (72.0% vs 73.0%) or HSV-2 (8.9% vs 10.7%). HSV-1 seroprevalence increased from 26.4% in those aged 6 months–9 y to 89.9% in those aged 40–49 y, and remained stable thereafter, ranging from 87.4% to 91.5% (p < 0.001). HSV-2 IgG seropositivity increased progressively from 5.7% in participants aged 20–29 y to 26.5% in participants aged ≥ 60 y (p < 0.001). HSV-1 seroprevalence did not differ between participants residing in urban and rural areas (72.5% vs 72.6%). Urban place of residence was a significant factor for HSV-2 seroprevalence in univariate analysis, but after standardization for age, it was no longer significant.Conclusions: Multiple logistic regression showed that age was a significant predictor of both HSV-1 and HSV-2 seropositivity, while female gender was a significant predictor of HSV-2 seropositivity. In pregnant women, obstetric history was not a significant predictor of either HSV-1 or HSV-2 seroprevalence.