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With emergence of MHC class I tetramers loaded with CD8+ T-cell viral epitopes, it is possible to study virus-specific CD8 cells in humans during infection and after vaccination. MHC class I tetramers was used to detect the frequency of haemagglutinin (HA)-specific T cells in 26 healthy influenza-vaccinated humans. Peripheral blood was collected before, and 7, 14 and 28 days after vaccination. Four-colour flow cytometry was used for monitoring of vaccine induced T-cell response. In 15 donors, two- to fivefold increase in frequency of HA-specific T cells was observed 7 days after vaccination. In addition, in 12 of these donors, this increase was accompanied with fourfold increase of H1N1 antibody titre. The increase in frequency of HA-specific CD8+/IFN-γ+ cells was low and peaked 28 days after vaccination in three of the six donors tested. Frequencies of HA-specific CD8+ T cells and antibody titre returned to prevaccination values 1 year after vaccination. Subunit influenza vaccines have the ability to induce HA-specific CD8+ cells. As the immune response to this vaccine decreased significantly after 1 year, our results confirm the importance of annual immunization for adequate protection. [ABSTRACT FROM AUTHOR]

Abstract Background Toll-like receptors (TLRs) are part of the innate immune system, able to recognize pathogen-associated molecular patterns and activate immune system upon pathogen challenge. Respiratory syncytial virus (RSV) is a RNA virus particularly detrimental in infancy. It could cause severe lower respiratory tract disease and recurrent infections related to inadequate development of anti-viral immunity. The reason could be inadequate multiple TLRs engagement, including TLR8 in recognition of single-stranded viral RNA and diminished synthesis of inflammatory mediators due to a lower expression. Methods Intracellular TLR8 expression in peripheral blood monocytes from RSV-infected infants was profiled and compared to healthy adults and age matched controls. Whether the observed difference in TLR8 expression is a transitory effect, infants in convalescent phase (4-6 weeks later) were retested. Specific TLR8-mediated TNF-α production in monocytes during an acute and convalescent phase was analyzed. Results RSV-infected and healthy infants had lower percentage of TLR8-expressing monocytes than healthy adults whereas decreased of TLR8 protein levels were detected only for RSV-infected infant group. Lower protein levels of TLR8 in monocytes from RSV-infected infants, compared to healthy infants, negatively correlated with respiratory frequency and resulted in lower TNF-α synthesis upon a specific TLR8 stimulation. In the convalescent phase, levels of TLR8 increased, accompanied by increased TNF-α synthesis compared to acute infection. Conclusions Lower TLR8 expression observed in monocytes, during an acute RSV infection, might have a dampening impact on early anti-viral cytokine production necessary to control RSV replication, and subsequently initiate an adaptive Th1 type immune response leading to severe disease in infected infants.

Abstract Background The epidemic pattern of respiratory syncytial virus (RSV) is quite different in regions of Europe (biennial epidemics in alternating cycles of approximately 9 and 15 months) than in the Western Hemisphere (annual epidemics). In order to determine if these differences are accounted for by the circulation of different RSV subtypes, we studied the prevalence of RSV subtype A and B strains in Zagreb County from 1 January 2006 to 31 December 2007. Results RSV was identified in the nasopharyngeal secretions of 368 inpatients using direct fluorescence assays and/or by virus isolation in cell culture. The subtype of recovered strains was determined by real-time PCR. Of 368 RSV infections identified in children during this interval, subtype A virus caused 94 infections, and subtype B 270. Four patients had a dual RSV infection (subtypes A and B). The period of study was characterized by two epidemic waves of RSV infections-one, smaller, in the spring of 2006 (peaking in March), the second, larger, in December 2006/January 2007 (peaking in January). The predominant subtype in both outbreaks was RSV subtype B. Not until November 2007 did RSV subtype A predominate, while initiating a new outbreak continuing into the following calendar year. Conclusion Though only two calendar years were monitored, we believe that the biennial RSV cycle in Croatia occurs independently of the dominant viral subtype.

Abstract 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.

Background: Cytomegalovirus (CMV) is endemic worldwide, with marked differences in the seroprevalence rates between countries. The aim of this study was to analyze the seroprevalence of CMV infections in Croatia., Methods: During a 3-year period (2013-2015) 2438 consecutive serum samples collected from Croatian residents were tested for the presence of CMV IgM and IgG antibodies using enzyme-linked immunoassay. The IgM/IgG positive samples were further tested for IgG avidity., Results: The overall seroprevalence rates for CMV IgG and IgM antibodies were 74.4 % and 4.3 %, respectively. The IgG seroprevalence showed significant differences between population groups: children/adolescents 54.6 %, general adult population 77.2 %, hemodialysis patients 91.4 % (p < 0.001). Seropositivity of CMV was strongly age-dependent with prevalences ranging from 53.0 % in children less than 10 years old to 93.8 % in persons above 60 years (p < 0.001). There was no difference in the prevalence rate between women with normal pregnancy and women with poor obstetric history. Gender and place of residence were not associated with CMV seropositivity. Using IgG avidity, current/recent primary CMV infection was confirmed by a low/borderline avidity index (AI) in 46.7 % participants, while in 53.3 % a high AI indicated CMV reactivation or reinfection. Primary infections were detected mainly in children and adolescents (83.2 % and 70.5 %, respectively), while reactivation/reinfection was common in persons older than 40 (77.0-100 %). Reactivation/reinfection was most commonly detected in hemodialysis patients (92.3 %). Logistic regression showed that older age and being on hemodialysis were significant predictors of CMV seropositivity., Conclusion: Infections with CMV are widespread in the Croatian population. Older age and being on hemodialysis appear to be the main risk factors for CMV infection.

Introduction: Croatia is endemic for hemorrhagic fever with renal syndrome (HFRS), with both Puumala (PUUV) and Dobrava virus (DOBV) documented. Several large outbreaks were recorded in 1995, 2002, and 2012. We analyzed demographic, clinical, laboratory, and virological characteristics of HFRS cases detected in three geographically close natural foci (Ogulin, Slunj, and the Plitvice Lakes surroundings) during the 2014 outbreak., Methodology: From January to December 2014, 122 patients with suspected HFRS were tested for hantavirus IgM/IgG antibodies using an indirect immunofluorescence assay (IFA). Cross-reactive samples were further tested using a western blot (WB). For hospitalized patients from Ogulin area, clinical and laboratory data were analyzed., Results: Acute infection was documented in 57 (46.7%) patients, of whom 75.4% were hospitalized. Ten (8.2%) patients were found to be IgG seropositive. Patients were 15-69 years of age and predominantly male (74.5%). The outbreak started in winter months, with most cases recorded from May to July (80.7%). The most frequently reported symptoms were fever (96.3%), chills/shivering (62.9%), and lumbar pain (48.1%). Mild clinical form was found in 66.7% patients, moderate in 18.5%, and severe in 14.8% patients (all but one infected with PUUV). One patient died. Using IFA, 48.8% patients showed monotypic antibody response, while in 51.2%, cross-reactive antibodies were found. PUUV was confirmed in 94.7% and DOBV in 5.3% HFRS cases by WB., Conclusions: Central mountainous Croatian regions are still highly endemic areas for HFRS. A higher percentage of severe PUUV infections could be at least partly associated with a patient's immune status.

A total of 52 serum samples from patients with symptoms suggestive of tick-borne encephalitis virus (TBEV) infection and positive IgM and/or IgG antibodies were tested for IgG avidity. Acute/recent TBEV infection was confirmed by low/borderline avidity index (AI) in 94.8% IgM positive/IgG positive samples, while in 5.2% high AI was found indicating persisting IgM antibodies. Majority of IgM negative/IgG positive samples (78.6%) showed high AI consistent with past TBEV infection. However, in 21.3% patients without measurable IgM antibodies current/recent infection was confirmed by AI. IgG avidity represents an additional serologic marker that improves diagnosis of TBEV, especially in cases of atypical antibody response.

Objective: Characterization of the phylogeny and diversity of human respiratory syncytial virus (HRSV) genotype ON1 that occurred during its early evolution (within the first 3.5 years since the detection of the first ON1 strains). ON1 strains have a 72-nucleotide-long in-frame duplication within the second hypervariable domain of the glycoprotein gene (HVR2)., Methods: All available HVR2 sequences of strains belonging to the ON1 genotype published prior to June 20, 2014 were collected. Multiple sequence alignments, phylogeny, phylogeography, sequence clustering and putative protein analyses were performed., Results: The worldwide spread and diversification of ON1 strains are presented. Only in a minority of ON1 strains do the two replicas remain identical, and various ON1 strains possess common differences between the first and the second copy (segments A and B). Mutations of the progenitor sequence were more frequent in segment B, a higher overall diversity on the protein level and more putative glycosylation sites exist in segment B, and, unlike in segment A, positive selection acts on that protein region., Conclusions: The fast spread of the novel HRSV genotype ON1 has been accompanied by its rapid concurrent diversification. Differences in variability of the two replicas within HVR2 were detected, with C-terminal replica being more variable.

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.

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.

Although Chlamydia trachomatis resistance is not of great concern due to its excellent sensitivity to the currently recommended first-line antibiotics (azithromycin and doxycycline), clinical treatment failures have been reported and some of them were linked to laboratory proved resistance. The aim of this study was to determine in vitro susceptibility to azithromycin and doxycycline for 24 urogenital chlamydial strains isolated in Croatia-a country with the highest consumption of azithromycin in Europe and with very high antibiotic prescription rates. Fourteen isolates from cervical swabs, nine from male urethral swabs, and one isolate from expressed prostatic secretion were tested in McCoy cell culture system. All strains were susceptible to azithromycin and doxycycline with minimal inhibitory concentration for azithromycin and doxycycline ranging from 0.064 to 0.125 μg/mL and 0.016 to 0.064 μg/mL, and minimal chlamydicidal concentration ranging from 0.064 to 2.0 μg/mL and 0.032 to 1.0 μg/mL, respectively. Since we still lack information on whether C. trachomatis is evolving in vivo in response to antibiotic selection pressure, this kind of surveillance for resistance is essential in detecting shifts in antimicrobial susceptibilities.

Human respiratory syncytial virus (HRSV) is a common etiological agent of acute lower respiratory tract disease in infants. The molecular epidemiology of HRSV in Croatia over four consecutive seasons (from 2006 to 2008) was investigated. A total of 72 HRSV samples were chosen from 696 screened cases in a pediatric clinic in Zagreb. Molecular characterization of HRSV revealed the predominance of HRSV group B viruses in the first two epidemic seasons and HRSV group A viruses in the next two seasons. According to the phylogenetic analysis, NA1 and BA9 were the predominant circulating HRSV genotypes detected during the study. Overall, 82.9% of all HRSV A strains belonged to the NA1 genotype. The HRSV B genotype BA9, detected in two consecutive seasons (2006 and 2007), was the predominant circulating HRSV B genotype, accounting for 80.6% of all HRSV B strains. This study provides data on the circulation pattern of HRSV genotypes in Croatia and their molecular characterization., (Copyright © 2012 Wiley Periodicals, Inc.)

Objective: The aim of this study was to determine the immunity to varicella-zoster virus (VZV) in Croatian pregnant and non-pregnant women of reproductive age., Methods: During 2007-2011, a total of 638 women aged 16-45 years were tested for the presence of VZV IgM and IgG antibodies using commercial enzyme-linked immunosorbent assay. Samples positive for IgG antibodies with positive or equivocal IgM antibodies were tested for IgG avidity., Results: The overall IgG seroprevalence was 84.3 %. There was a significant increase in IgG seropositivity with age (OR = 1.04 for 1-year increase in age; 95 % CI 1.01-1.08). The lowest seroprevalence rate was reported in the 16-20 age groups (78.6 %), and the highest was in the 41-45 age groups (94.3 %). There was no significant difference in seroprevalence among women residing in urban and rural areas (83.6 vs. 87.0 %, OR 0.76, 95 % CI 0.43-1.34)., Conclusions: The results of this study have shown that a high proportion of Croatian childbearing-aged women (15.7 %) who were referred to the laboratory for VZV serology testing are susceptible to VZV and, thus, at risk for contracting varicella during pregnancy. Serology testing of adolescent girls and adult women who do not have a documented history of varicella is encouraged with the aim of vaccinating seronegative girls and women against VZV before pregnancy. In addition, testing of pregnant women is advised to identify susceptible women and vaccinate them after delivery.

Background: The epidemic pattern of respiratory syncytial virus (RSV) in Croatia is biennial. In order to determine if the circulation of different RSV subtypes affects the outbreak cycle, the aim of the present study was to analyze the epidemic pattern of RSV in children in Croatia (Zagreb region) over a period of 3 consecutive years., Methods: The study group consisted of 696 inpatients, aged 0-5 years, who were hospitalized with acute respiratory tract infections caused by RSV, in Zagreb, in the period 1 January 2006-31 December 2008. The virus was identified in nasopharyngeal secretions using direct immunofluorescence. The virus subtype was determined on real-time polymerase chain reaction., Results: Of 696 RSV infections identified in children, subtype A virus caused 374 infections, and subtype B, 318. Four patients had a dual RSV infection (subtypes A and B). The period of study was characterized by four epidemic waves of RSV infections: the first, smaller, in the spring of 2006; the second, larger, in December 2006/January 2007; the third in spring 2008, followed by a fourth outbreak beginning in November of 2008. The biennial virus cycles were persistent although the predominant RSV subtype in the first two epidemic waves was subtype B, and in the second two it was subtype A., Conclusion: Over a 3 year period of observation, the biennial RSV cycle in Croatia cannot be explained by a difference in the predominant circulating subtype of RSV. Other unknown factors account for the biennial cycle of RSV epidemics in Croatia., (© 2011 The Authors. Pediatrics International © 2011 Japan Pediatric Society.)

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.

Between 2007 and 2010, a total of 268 Croatian patients with lymphadenopathy were tested for IgM/IgG antibodies to Bartonella (B.) henselae and B. quintana. Samples from 44.4% patients showed positive IgG antibodies: 35.8% to B. henselae, 6.7% to B. quintana and 1.9% to both Bartonella species. There was no difference in seropositivity between males and females (47.4% vs. 41.5%). Seroprevalence was high in all age groups (40.4-60.9%). Patients from urban and rural areas showed a similar seroprevalence rate (44.1% vs. 44.8%). Positive IgM antibodies were found in 28.3% patients varying from 17.5% and 37.5% among age groups. Most cases were reported from August to March.

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.

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.

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).

Background: Toll-like receptors (TLRs) are part of the innate immune system, able to recognize pathogen-associated molecular patterns and activate immune system upon pathogen challenge. Respiratory syncytial virus (RSV) is a RNA virus particularly detrimental in infancy. It could cause severe lower respiratory tract disease and recurrent infections related to inadequate development of anti-viral immunity. The reason could be inadequate multiple TLRs engagement, including TLR8 in recognition of single-stranded viral RNA and diminished synthesis of inflammatory mediators due to a lower expression., Methods: Intracellular TLR8 expression in peripheral blood monocytes from RSV-infected infants was profiled and compared to healthy adults and age matched controls. Whether the observed difference in TLR8 expression is a transitory effect, infants in convalescent phase (4-6 weeks later) were retested. Specific TLR8-mediated TNF-α production in monocytes during an acute and convalescent phase was analyzed., Results: RSV-infected and healthy infants had lower percentage of TLR8-expressing monocytes than healthy adults whereas decreased of TLR8 protein levels were detected only for RSV-infected infant group. Lower protein levels of TLR8 in monocytes from RSV-infected infants, compared to healthy infants, negatively correlated with respiratory frequency and resulted in lower TNF-α synthesis upon a specific TLR8 stimulation. In the convalescent phase, levels of TLR8 increased, accompanied by increased TNF-α synthesis compared to acute infection., Conclusions: Lower TLR8 expression observed in monocytes, during an acute RSV infection, might have a dampening impact on early anti-viral cytokine production necessary to control RSV replication, and subsequently initiate an adaptive Th1 type immune response leading to severe disease in infected infants.

Background: The epidemic pattern of respiratory syncytial virus (RSV) is quite different in regions of Europe (biennial epidemics in alternating cycles of approximately 9 and 15 months) than in the Western Hemisphere (annual epidemics). In order to determine if these differences are accounted for by the circulation of different RSV subtypes, we studied the prevalence of RSV subtype A and B strains in Zagreb County from 1 January 2006 to 31 December 2007., Results: RSV was identified in the nasopharyngeal secretions of 368 inpatients using direct fluorescence assays and/or by virus isolation in cell culture. The subtype of recovered strains was determined by real-time PCR. Of 368 RSV infections identified in children during this interval, subtype A virus caused 94 infections, and subtype B 270. Four patients had a dual RSV infection (subtypes A and B). The period of study was characterized by two epidemic waves of RSV infections-one, smaller, in the spring of 2006 (peaking in March), the second, larger, in December 2006/January 2007 (peaking in January). The predominant subtype in both outbreaks was RSV subtype B. Not until November 2007 did RSV subtype A predominate, while initiating a new outbreak continuing into the following calendar year., Conclusion: Though only two calendar years were monitored, we believe that the biennial RSV cycle in Croatia occurs independently of the dominant viral subtype.

Background: Respiratory syncytial virus (RSV) is the most common cause of severe lower respiratory tract infections (LRTI) in infants. The aim of the present study was to analyze the epidemiologic characteristics of RSV outbreaks in Croatian children., Methods: Over a period of 11 consecutive years (1994-2005), 3435 inpatients with acute respiratory infections (ARI) aged from birth to 10 years and were residing in Zagreb County were tested for infection with RSV and other respiratory viruses at the Virology Department, Croatian National Institute of Public Health. RSV was identified in nasopharyngeal secretions by isolation on cell culture and/or detection with monoclonal antibodies using a direct fluorescence assay., Results: RSV was the most common causative agent of ARI (42.2%; 658/1559) for the infants 0-6 months of age. It was also the etiologic agent of LRTI in 49% (495/1010) of infants of similar age. RSV was demonstrated in 56.5% (382/676) of infants with bronchiolitis, and in 36.5% (49/134) of those with pneumonia in this age group., Conclusion: The overall prevalence of RSV infection in Croatian children with acute respiratory illness, and its occurrence in various age groups, has remained stable over the past decade. RSV was found to be the most common cause of bronchiolitis occurring throughout childhood (52.7%; 482/913).

Respiratory syncytial virus (RSV) glycoprotein G mimics fractalkine, a CX(3)C chemokine, which mediates chemotaxis of leukocytes expressing its receptor, CX(3)CR1. The aim of this study was to examine the relationship between RSV infection and expression of perforin and IFN-gamma in CX(3)CR1-expressing peripheral blood CD8(+) T cells. Samples were collected from infants with RSV bronchiolitis, both in the acute and convalescence phase (n = 12), and from their age- and sex-matched healthy controls (n = 15). Perforin expression and IFN-gamma secretion in CX(3)CR1(+) CD8(+) T cells were assessed by four-color flow cytometry. The NF-kappaB p50 and p65 subunit levels were also determined as markers of RSV-induced inflammation. Study results showed perforin and CX(3)CR1 expression to be significantly lower in the convalescent phase of infected infants than in healthy controls. There was no significant difference in IFN-gamma secretion and NF-kappaB binding activity between two time-points in RSV-infected infants, or when compared with healthy controls. Infants with prolonged wheezing had lower acute-phase CX(3)CR1 levels in peripheral blood. These data indicate existence of an event persisting after acute RSV infection that is able to modulate effector functions of cytotoxic T cells, and also link disease severity with CX(3)CR1 expression.

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.

With emergence of MHC class I tetramers loaded with CD8+ T-cell viral epitopes, it is possible to study virus-specific CD8 cells in humans during infection and after vaccination. MHC class I tetramers was used to detect the frequency of haemagglutinin (HA)-specific T cells in 26 healthy influenza-vaccinated humans. Peripheral blood was collected before, and 7, 14 and 28 days after vaccination. Four-colour flow cytometry was used for monitoring of vaccine induced T-cell response. In 15 donors, two- to fivefold increase in frequency of HA-specific T cells was observed 7 days after vaccination. In addition, in 12 of these donors, this increase was accompanied with fourfold increase of H1N1 antibody titre. The increase in frequency of HA-specific CD8+/IFN-gamma+ cells was low and peaked 28 days after vaccination in three of the six donors tested. Frequencies of HA-specific CD8+ T cells and antibody titre returned to prevaccination values 1 year after vaccination. Subunit influenza vaccines have the ability to induce HA-specific CD8+ cells. As the immune response to this vaccine decreased significantly after 1 year, our results confirm the importance of annual immunization for adequate protection.

Acute RSV infection in infancy may produce some asthma-like symptoms and may be followed by a recurrent wheeze later in childhood. It has been proposed that RSV infection stimulates type-2 cytokine responses, resembling those found in atopy and asthma. Peripheral blood cells were obtained from RSV-infected infants (n = 30) and healthy controls (n = 10). After in vitro restimulation of the cells, intracellular IL-4 and interferon-gamma (IFN-gamma) were measured by flow cytometry. The cells from RSV-infected infants produced more IL-4 and less IFN-gamma than those from healthy controls. IL-4 production was more frequent in CD8 than in CD4 cells, and the bias toward IL-4 production was greatest in infants with mild infections, whereas IFN-gamma production increased with disease severity. Our conclusions are that RSV infection is associated with IL-4 production in peripheral T cells, and that peripheral blood in infants with severe disease may be depleted of cytokine-producing cells.

The epidemiological characteristics and relationship between respiratory syncytial virus (RSV) subgroup and virulence during an outbreak of RSV infection occurring in Southeast Texas in the winter season 1991/92 are described. Fifty-two infants and children were diagnosed with RSV infection by rapid viral antigen detection and/or viral isolation. Subgrouping of the isolates was carried out using 11-monoclonal anti-bodies. Ten isolates were found to be subgroup B, and 8 isolates were subgroup A. The subgroup B strains showed 3 different patterns of reaction with monoclonal antibodies; one of these subgroups was examined further by restriction analysis of parts of its nucleocapsid and attachment protein genes. The peak of RSV outbreak was in December 1991. Both subtypes A and B circulated simultaneously in the same territory, and caused lower respiratory tract infections in similar proportions. The more frequent occurrence of the B subgroup and the diversity of its simultaneously circulated RSV strains have made this outbreak unusual.