Gordana Mlinarić-Galinović

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‐γ) were measured by flow cytometry. The cells from RSV‐infected infants produced more IL‐4 and less IFN‐γ 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‐γ 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.

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‐α 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‐α 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.

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 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.

Adenovirus respiratory infections in hospitalized children: clinical findings in relation to species and serotypes.

Background: There are >50 adenovirus (ADV) serotypes that are divided into 7 species (A–G). The aim of this study was to characterize ADV serotypes and species in hospitalized infants and children in the City of Zagreb and Zagreb County and to describe clinical features and laboratory findings of ADV infections according to the causative ADV serotype. Methods: During the 3-year period from January 2006 to November 2008, 135 children (<10 years of age) with ADV respiratory infection, based on virus isolation, were treated at 2 hospitals in Zagreb. Demographics, clinical presentations and laboratory findings were evaluated. Results: Of the 135 ADV isolates, 77 (57.0%) were type 2, followed by 26 (19.3%) of type 1, 15 (11.1%) isolates of type 3, 2 (1.5%) of type 6 and only 1 (0.7%) was type 7. Male-to-female ratio was 3.2:1 (103 boys and 32 girls). The mean age was 22.9 months. The most common symptoms were fever (98%), rhinorrhea (89%) and cough (71%). The mean peak body temperature was 39.8°C. Tonsillitis was present in 79 (59%) and acute otitis media in 37 (28%) patients. Leukocytosis (>15.0 × 109/L) was noted in 103 (77%) patients. Serum C-reactive protein was >40 mg/L in 74 patients (56%). The erythrocyte sedimentation rate was ≥30 mm/h in 91 (71%) of the 127 patients tested. Conclusions: In this study, the most common isolated serotype was ADV type 2. Most affected children were younger than 3 years. ADV infections in young children can present with prolonged fever, leukocytosis and significantly elevated C-reactive protein and erythrocyte sedimentation rate, mimicking bacterial infections.

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 childrens 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).

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.

Decreased Toll-like receptor 8 expression and lower TNF-alpha synthesis in infants with acute RSV infection

BackgroundToll-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.MethodsIntracellular 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.ResultsRSV-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.ConclusionsLower 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.