Genotype replacement of the human parainfluenza virus type 2 in Croatia between 2011 and 2017 - the role of neutralising antibodies.

Previously we reported on the HPIV2 genotype distribution in Croatia 2011-2014. Here we expand this period up to 2017 and confirm that G1a genotype has replaced G3 genotype from the period 2011-2014. Our hypothesis was that the G1a-to-G3 genotype replacement is an antibody-driven event. A cross-neutralisation with anti-HPIV2 sera specific for either G1a or G3 genotype revealed the presence of genotype-specific antigenic determinants. By the profound, in silico analyses three potential B cell epitopic regions were identified in the hemagglutinin neuraminidase (regions 314-361 and 474-490) and fusion protein (region 440-484). The region identified in the fusion protein does not show any unique site between the G1a and G3 isolates, five differentially glycosylated sites in the G1a and G3 genotype isolates were identified in epitopic regions of hemagglutinin neuraminidase. All positively selected codons were found to be located either in the region 314-316 or in the region 474-490 what indicates a strong positive selection in this region and reveals that these regions are susceptible to evolutionary pressure possibly caused by antibodies what gives a strong verification to our hypothesis that neutralising antibodies are a key determinant in the inherently complex adaptive evolution of HPIV2 in the region.

Seroepidemiology of Hepatitis E in Selected Population Groups in Croatia: A Prospective Pilot Study.

Hepatitis E has become an emerging infection in many European countries. We analysed the prevalence of hepatitis E virus (HEV) infection in selected population groups in Croatia. Overall HEV IgG seropositivity was 5.6%, while 1.9% participants showed IgM antibodies suggestive of recent infection. No IgM-positive sample was positive for HEV RNA. HEV IgG antibodies were most prevalent in alcohol abusers (8.9%) and war veterans (8.6%), compared with 6.1% among injecting drug users and 2.7% in healthcare professionals. No individual with high-risk sexual behaviour tested HEV seropositive. HEV IgG positivity increased significantly with age from 1.8% to 2.3% in individuals younger than 40 years to 11.3% in individuals older than 50 years (P = 0.023). The mean age of HEV-positive participants was significantly higher than that of HEV-negative participants (50.9 ± 11.8 years versus 41.2 ± 11.8 years, P = 0.008). Seroprevalence rates were significantly higher in residents of suburban and rural areas compared with residents of urban areas (14.5% versus 2.5%, P = 0.003). Additionally, an increasing prevalence of HEV IgG antibodies was observed from 1.8% in participants living in families with two household members to 12.1% in those living with more than four members (P = 0.046). Gender, marital status, educational level, sexual orientation, source of drinking water, history of blood transfusions, surgical procedures, tattooing and travelling were not associated with HEV seroprevalence. Logistic regression showed that living in suburban/rural areas was the main risk factor for HEV seropositivity (OR = 6.67; 95%CI = 1.89-25.0; AOR = 7.14, 95%CI = 1.89-25.0).

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.

Seroprevalence of Taenia solium infections in Croatian patients presenting with epilepsy.

Epilepsy is one of the most common neurological disorders, while neurocysticercosis caused by Taenia solium infection of the central nervous system currently represents the leading cause of secondary epilepsy in Central and South America, East and South Asia, and sub-Saharan Africa. As a result of increased migration from these endemic regions, neurocysticercosis and subsequent epilepsy are becoming a growing public health problem in developed countries as well. In order to determine the prevalence of T. solium infection in patients with epilepsy in Croatia, a retrospective serological study was conducted. A total of 770 serum samples were tested for the presence of T. solium IgG antibodies using a commercial qualitative enzyme immunoassay. The Western blot technique was used as a confirmatory test for the diagnosis. The overall seroprevalence rate of T. solium infection in patients with clinically proven epilepsy was 1.5%. Although the results have shown that infection with this tapeworm is rare in Croatia, this study hopes to increase awareness about the importance of preventive measures and benefits of accurate and timely diagnosis. Intervention measures for infection control are crucial, namely sanitation improvement, control of domestic pig-breeding, detailed meat inspection, detection and treatment of tapeworm carriers, hand washing and health education.

Autochthonous dengue fever in Croatia, August-September 2010.

After information about a dengue case in Germany acquired in Croatia, health professionals and the public in Croatia were alerted to assess the situation and to enhance mosquito control, resulting in the diagnosis of a second case of autochthonous dengue fever in the same area and the detection of 15 persons with evidence of recent dengue infection. Mosquito control measures were introduced. The circumstances of dengue virus introduction to Croatia remain unresolved.

Seroprevalence of Echinococcus granulosus infection in Croatian patients with cystic liver disease.

Cystic liver disease (CLD), presenting with solitary or multiple cysts in the liver, is a common diagnosis today, primarily due to the frequent application of modern radiological methods. There is a wide range of possible causes. CLD of infective origin is usually caused by an echinococcal species. During the past three decades a number of cystic echinococcosis (CE) control programmes have led to a significant decrease in the incidence of human hydatidosis in some endemic areas. The aim of this study was to determine the seroprevalence of E. granulosus infection in Croatian patients with CLD. A total of 540 serum samples from patients with hepatic cysts detected by imaging methods were screened for the presence of E. granulosus IgG antibodies using semiquantitative enzyme-linked immunosorbent assay. The Western blot technique was used as a confirmatory test for the CE diagnosis. The overall E. granulosus seroprevalence rate in patients with CLD was 3.9%. There was no significant difference in seroprevalence rate between male and female patients (P = 0.541). According to age groups, there was a significant difference in seropositivity among age groups (P = 0.002). The highest seroprevalence rate was detected in the youngest age group (up to 18 years), both in males and females (20% and 13%, respectively). This study indicates that CE still represents a public health problem in Croatia. Preventive measures should be used to control Echinococcus infections, including avoidance of contact with infected dogs, egg-contaminated soil or plants; control and treatment of dogs with antihelmintics; hand washing, improved sanitation and health education.

Enumeration of haemagglutinin-specific CD8+ T cells after influenza vaccination using MHC class I peptide tetramers.

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.

Outcome of influenza vaccination in combat-related post-traumatic stress disorder (PTSD) patients.

Post-traumatic stress disorder (PTSD) is an anxiety disorder that can occur after exposure to extreme traumatic experience such as war trauma, and is accompanied by fear, helplessness or horror. Exposure to trauma can result in immune dysregulation and influence susceptibility to infectious disease as well as vaccine efficacy. The aim of the study was to determine the relation of psychological stress and the immune response to influenza vaccination in combat-related PTSD patients (n = 28). Detection of anti-viral antibody titre was performed by inhibition of haemagglutination assay. Ex vivo tetramer staining of CD8(+) T lymphocytes was used to monitor T cells specific for human leucocyte antigen (HLA)-A*0201-restricted influenza A haemagglutinin antigens before and after vaccination. Twenty patients showed a fourfold antibody titre increase to one or both influenza A viral strains, and 18 of them showed the same response for both influenza B viral strains. Ten of 15 healthy controls showed a fourfold rise in antibody titre to both influenza A viral strains and eight of them showed the same response for both influenza B viral strains. HLA-A*0201(+) PTSD patients (n = 10) showed a significant increase of influenza-specific CD8 T cells after vaccination. Although those PTSD patients had a lower number of influenza-specific CD8(+) T cells before vaccination compared to HLA-A*0201(+) healthy controls (n = 6), there was no difference in influenza A antibody titre between PTSD patients and control subjects before vaccination. The generated humoral and cellular immune response in PTSD patients argues against the hypothesis that combat-related PTSD in war veterans might affect protection following influenza vaccination.

Increased Toll-like receptor 4 expression in infants with respiratory syncytial virus bronchiolitis.

The fusion protein of the respiratory syncytial virus (RSV) binds to the pattern recognition receptors, TLR4 and CD14, and initiates innate immunity response to the virus. The aim of the study was to investigate the expression of TLR4 on peripheral blood lymphocytes and monocytes in peripheral blood of infants in both acute and convalescent phase of RSV bronchiolitis (n = 26). In addition, TNF-alpha expression in lipopolysaccharide-stimulated monocytes was also assessed. The results showed TLR4 to be expressed predominantly by monocytes in both sick infants and controls. During the acute phase of infection monocytes up-regulated TLR4 in eight infants, which returned to the levels recorded in controls 4-6 weeks from infection. There was no difference in the percentage of TNF-alpha secreting monocytes. Of the clinical parameters tested, minimal oxygen saturation was found to correlate negatively with this expression in the group of infants with increased TLR4. Additional studies are under way to correlate this finding with the outcome of the immune response to RSV.

Non-radioactive digoxigenin DNA labeling and immunologic detection of HSV PCR products.

BACKGROUND: Herpes simplex virus (HSV) is a common cause of human skin and mucous membrane infections, and also causes sporadic meningoencephalitis. As a new method for rapid HSV diagnostics, polymerase chain reaction (PCR) has been introduced in clinical laboratories. Radioactive labeling of DNA probes has become a common practice in experimental laboratories. To avoid radioactive labeling of HSV oligonucleotide probes or PCR products, non-radioactive compounds, which are easily detected by enzyme or immunoassay techniques, are introduced. OBJECTIVES: The aim of our study was (1) to introduce non-radioactive labeling of HSV DNA probe by digoxigenin-labeled dUTP; (2) to establish a rapid and reliable laboratory method for rapid HSV diagnostics; (3) to compare the PCR method with the standard virology techniques, such as cell culture virus isolation and HSV direct fluorescent antibody test (DFA). STUDY DESIGN: We have tested the efficiency of PCR method and non-radioactive labeling of HSV DNA probe for detection of HSV from 30 clinical specimens (skin and mucous membrane swabs). HSV was detected in the specimens by standard virology techniques and PCR. Replicated HSV DNA was non-radioactively labeled by random incorporation of digoxigenin-labeled deoxyuridine triphosphate (DIG-dUTP), and the hybrids were detected by the antibody conjugates and the appropriate enzyme-mediated staining reaction (DIG DNA labeling and detection kit non-radioactive, Boehringer Mannheim GmbH). RESULTS: Non-radioactive labeling of hybridization DNA probes with digoxigenin-dUTP was obtained. HSV DNA was successfully multiplied and detected in the HSV-infected cell culture supernatant; however, it was not detected in the clinical specimen supernatant or sediment. HSV DNA was detected by direct PCR method in non-centrifugated clinical specimens. CONCLUSIONS: The PCR method could be successfully used for diagnoses of HSV infections. Since the sensitivity of this method is partly limited by the virus quantity in the specimen, we recommend cultivating the virus in the cell culture at least 24 h prior to PCR. The use of non-radioactive labeling of hybridization DNA probes, such as random primed DNA labeling with digoxigenin-dUTP, has proven both sensitive and specific, and more appropriate for diagnostic purposes than radioactive DNA labeling to be used until standardized commercial tests appear.

[Herpes simplex viruses: biological characteristics, immunopathogenesis, diagnosis and treatment]. / Herpes simpleks-virusi: bioloske osobine, imunopatogeneza, dijagnostika i lijecenje.

Herpes simplex virus (HSV) is the most common cause of sporadic encephalitis in developed countries. Herpes simplex virus type 1 (HSV-1) causes oropharyngeal infections, keratoconjunctivitis and infections of the central nervous system, while herpes simplex virus type 2 (HSV-2) in the immunocompetent most frequently causes genital infections. HSV-1 primary infection usually occurs in the early childhood but is also possible at adolescent age. HSV-2 primary infection is usually postponed till the adult age and coincides with the sexual activity. Common characteristics of these two viruses are a relatively rapid reproductive cycle, an efficient elimination of the infected cells, and the ability of causing a latent infection in the sensory ganglia. Since nowadays there is a specific therapy, the prognoses of severe HSV infections are much better. However, it is necessary that the antiviral therapy be applied shortly after the first symptoms of the disease have appeared. Therefore, the application of a rapid and safe method for detection of HSV from clinical materials is the first step in the treatment of severe and lethal infections like meningoencephalitis. In that light, the method called polymerase chain reaction (PCR) represents a new in vitro technique of DNA replication which enables exponential replication of a well defined DNA fragment. The advantages of this diagnostic method are its rapidity and sensitivity, and it does not require live cells for virus detection.

Nosocomial respiratory syncytial virus infections in children's wards.

During community outbreak, nosocomial infections caused by both groups (A and B) of respiratory syncytial virus (RSV) occur as the most common nosocomial infections at pediatric wards. RSV cross-infection is considered to have taken place when a child acquires an infection after being in the ward longer than 7 days, and its frequency at the ward could be calculated in several ways. That frequency ranges worldwide between 30% and 70% in neonatal units, and between 20% and 40% at pediatric wards. The infections are manifested as lower respiratory tract infections (LRTI) in 20-60% and 30-40% of cases, respectively. These infections could be early diagnosed by an RSV rapid detection method. In RSV-positive children who develop LRTI and belong to the category with a high risk of developing severe RSV disease, a specific therapy is recommended. The frequency of RSV nosocomial infections at children's wards could be considerably reduced (to only a few per cent) by providing education to hospital personnel in the etiology and transmission of respiratory viruses and by compliance with pediatric droplet precautions (cohort nursing, and gown and glove wearing/handwashing in any contact with infected children).

Predominant type-2 response in infants with respiratory syncytial virus (RSV) infection demonstrated by cytokine flow cytometry.

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.

[Subtypes of respiratory syncytial viruses]. / Podtipovi respiratornoga sincicijskog virusa.

Antigenically the respiratory syncytial virus (RSV) is of two types: A and B. Five structural proteins express virus type differences. Antigenic and genetic differences among individual strains of the same virus type are classed accordingly by monoclonal antibody reactivity into antigenic subtypes; for nucleotide sequence and restriction maps of individual gene polymerase chain reaction products they have different genetic categories: SHL 1-6, NP 1-6. Still unproved is the association between the virus-caused clinical picture severity and virus type. One type or both may be the causative agents of an RSV epidemic. Between 1988 and 1994, both types of RSV strains circulated in Central Europe, as well as the different subtypes within each type; type A, particularly subtype A1, was absolutely dominant. RSV isolates were of genotypes SHL 2, SHL 1/3/4, SHL 5, NP 1. They occurred in the same order as the genotypes shown in the rest of Europe and the world.

Clinical and epidemiological aspects of respiratory syncytial virus lower respiratory tract infections.

This paper has analyzed respiratory syncytial virus lower respiratory tract infections in 201 hospitalized children. In children with wheezing, erythrocyte sedimentation rate (ESR) was significantly higher in those with pneumonia than with syndroma pertussis, while the white blood cell (WBC) count was significantly lower in patients with bronchitis than in those with bronchiolitis and syndroma pertussis. Bronchodilatators were applied in 75.6% and corticosteroids in 20% of patients. Ten patients were ventilated. Fatal disease outcome was observed in one infant. Twelve consecutive-year study of respiratory syncytial virus (RSV) infections showed that 27.3% of these diseases were bronchiolitis and pneumonia.

Antigenic and genomic diversity of central European respiratory syncytial virus strains.

Thirty-two RSV strains recovered during the winter months of 1987/88 to 1993/94 from hospitalized children in Vienna, Austria and Zagreb, Croatia were analysed for antigenic and genetic variations. Twenty-nine of the 32 isolates investigated belonged to group A and 3 to group B, with the majority of infections caused by subgroup A1 (21 of 29). Restriction endonuclease mapping of PCR products derived from parts of the N and G gene of 18 group A strains identified 3 distinct lineages, very similar to those defined by analysis of recurrent epidemics in Birmingham, United Kingdom during the same period. Results of this study provide further information on the global pattern of RSV and show that very similar viruses are present simultaneously in widely separated areas.

Increase in CD23+ B cells in infants with bronchiolitis is accompanied by appearance of IgE and IgG4 antibodies specific for respiratory syncytial virus.

Infection with respiratory syncytial virus (RSV) may induce asthma-like symptoms and RSV-specific IgE in infected infants as a result of Th2-like response to RSV. The effect of RSV infection on the expression of B cell antigens CD21 and CD23, putative participants in Th2 responses, was investigated. Samples from bronchiolitic infants (n = 19) were tested by three-color immunofluorescence flow cytometry during the acute phase of infection and 4-6 weeks later. In 6 of 10 RSV-positive infants, the percentage of CD23+ B cells was higher than in 9 RSV-negative children and in controls. Both CD21+ and CD21- B cells exhibited a higher percentage of CD23. The group with increased expression of CD23 antigen had RSV-specific IgE and IgG4 antibodies. These findings corroborate the hypothesis that RSV could provoke a Th2-type response, but the relationship between CD23 antigen and RSV infection must be determined.

Respiratory syncytial virus infection in the elderly.

Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infections in infants and children throughout the world. Respiratory syncytial virus infections in the elderly represent reinfections in the hosts who have had many prior episodes. Thus, RSV infections are usually not considered serious in adults, since reinfections are generally known to result in mild disease. Nevertheless, in adults, as in children, the infection has been reported to cause altered airway resistance and exacerbation of chronic obstructive lung disease. In people over 60 years of age, RSV usually causes mild nasal congestion, but can also result in fever, anorexia, pneumonia, bronchitis, and even death. Diagnosis of RSV infection in the elderly by the standard methods used in children is not as successful as in the latter group. This may be due to a combination of factors such as shorter shedding phase, lower viral titers, and dry mucosa. An alternative, rapid, and direct viral diagnostic method, the polymerase chain reaction, has recently been introduced in the diagnosis of RSV infections.

Antigenic diversity of respiratory syncytial virus subgroup B strains circulating during a community outbreak of infection.

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.

Preparation of respiratory syncytial virus subgroup A and B antigens for enzyme immunoassay antibody detection.

A simplified method was described for purification of respiratory syncytial virus (RSV) subgroup A and B aimed to be used as antigens in enzyme immunoassay (EIA). The titer of each RSV subgroup and the amount of protein was determined from the visible band in 45% sucrose gradient. The quality of prepared RSV subgroup antigens for EIA was described in terms of the achievable final titer, the amount of protein, and EIA criss-cross titration. The RSV subgroup A and B antigens, diluted as 1:100 (low opalescent band in 45% sucrose layer) or 1:800 (high opalescent band in 45% sucrose layer) produced a positive reaction in EIA criss-cross titration with IgG antibodies from the patient's serum (convalescent phase) diluted as 1:25,600 (for RSV A) and 1:6,400 (for RSV B). This method offers shorter and more simplified steps of viral antigen purification, and provides acceptable quantity and quality of viral antigens appropriate for use in EIA.