Neurologic Complications of Influenza B Virus Infection in Adults, Romania.

We characterized influenza B virus-related neurologic manifestations in an unusually high number of hospitalized adults at a tertiary care facility in Romania during the 2014-15 influenza epidemic season. Of 32 patients with a confirmed laboratory diagnosis of influenza B virus infection, neurologic complications developed in 7 adults (median age 31 years). These complications were clinically diagnosed as confirmed encephalitis (4 patients), possible encephalitis (2 patients), and cerebellar ataxia (1 patient). Two of the patients died. Virus sequencing identified influenza virus B (Yam)-lineage clade 3, which is representative of the B/Phuket/3073/2013 strain, in 4 patients. None of the patients had been vaccinated against influenza. These results suggest that influenza B virus can cause a severe clinical course and should be considered as an etiologic factor for encephalitis.

Epidemiological and molecular investigation of a rubella outbreak, Romania, 2011 to 2012.

We describe a rubella outbreak that occurred in Romania between September 2011 and December 2012. During this period 24,627 rubella cases, 41.1% (n=10,134) of which female, were notified based on clinical criteria, and a total of 6,182 individuals were found serologically positive for IgM-specific rubella antibody. The median age of notified cases was 18 years (range: <1-65) and the most affected age group 15 to 19 years (n=16,245 cases). Of all notified cases, 24,067 cases (97.7%) reported no history of vaccination. Phylogenetic analysis of 19 sequences (739 nucleotides each), from 10 districts of the country revealed that the outbreak was caused by two distinct rubella virus strains of genotype 2B, which co-circulated with both temporal and geographical overlap. In addition to the 6,182 IgM-positive rubella cases, 28 cases of congenital rubella syndrome (CRS) were identified, including 11 neonatal deaths and one stillbirth. The outbreak underscores the need to encourage higher vaccination uptake in the population, particularly in women of reproductive age, and to strengthen epidemiological and laboratory investigations of suspected rubella cases. Genetic characterisation of wild-type rubella virus is an essential component to enhance surveillance and here we report rubella virus sequences from Romania.

I-MOVE multicentre case-control study 2010/11 to 2014/15: Is there within-season waning of influenza type/subtype vaccine effectiveness with increasing time since vaccination?

Since the 2008/9 influenza season, the I-MOVE multicentre case-control study measures influenza vaccine effectiveness (VE) against medically-attended influenza-like-illness (ILI) laboratory confirmed as influenza. In 2011/12, European studies reported a decline in VE against influenza A(H3N2) within the season. Using combined I-MOVE data from 2010/11 to 2014/15 we studied the effects of time since vaccination on influenza type/subtype-specific VE. We modelled influenza type/subtype-specific VE by time since vaccination using a restricted cubic spline, controlling for potential confounders (age, sex, time of onset, chronic conditions). Over 10,000 ILI cases were included in each analysis of influenza A(H3N2), A(H1N1)pdm09 and B; with 4,759, 3,152 and 3,617 influenza positive cases respectively. VE against influenza A(H3N2) reached 50.6% (95% CI: 30.0-65.1) 38 days after vaccination, declined to 0% (95% CI: -18.1-15.2) from 111 days onwards. At day 54 VE against influenza A(H1N1)pdm09 reached 55.3% (95% CI: 37.9-67.9) and remained between this value and 50.3% (95% CI: 34.8-62.1) until season end. VE against influenza B declined from 70.7% (95% CI: 51.3-82.4) 44 days after vaccination to 21.4% (95% CI: -57.4-60.8) at season end. To assess if vaccination campaign strategies need revising more evidence on VE by time since vaccination is urgently needed.

Surveillance for severe acute respiratory infections (SARI) in hospitals in the WHO European region - an exploratory analysis of risk factors for a severe outcome in influenza-positive SARI cases.

BACKGROUND: The 2009 H1N1 pandemic highlighted the need to routinely monitor severe influenza, which lead to the establishment of sentinel hospital-based surveillance of severe acute respiratory infections (SARI) in several countries in Europe. The objective of this study is to describe characteristics of SARI patients and to explore risk factors for a severe outcome in influenza-positive SARI patients. METHODS: Data on hospitalised patients meeting a syndromic SARI case definition between 2009 and 2012 from nine countries in Eastern Europe (Albania, Armenia, Belarus, Georgia, Kazakhstan, Kyrgyzstan, Romania, Russian Federation and Ukraine) were included in this study. An exploratory analysis was performed to assess the association between risk factors and a severe (ICU, fatal) outcome in influenza-positive SARI patients using a multivariate logistic regression analysis. RESULTS: Nine countries reported a total of 13,275 SARI patients. The majority of SARI patients reported in these countries were young children. A total of 12,673 SARI cases (95%) were tested for influenza virus and 3377 (27%) were laboratory confirmed. The majority of tested SARI cases were from Georgia, the Russian Federation and Ukraine and the least were from Kyrgyzstan. The proportion positive varied by country, season and age group, with a tendency to a higher proportion positive in the 15+ yrs age group in six of the countries. ICU admission and fatal outcome were most often recorded for influenza-positive SARI cases aged > 15 yrs. An exploratory analysis using pooled data from influenza-positive SARI cases in three countries showed that age > 15 yrs, having lung, heart, kidney or liver disease, and being pregnant were independently associated with a fatal outcome. CONCLUSIONS: Countries in Eastern Europe have been able to collect data through routine monitoring of severe influenza and results on risk factors for a severe outcome in influenza-positive SARI cases have identified several risk groups. This is especially relevant in the light of an overall low vaccination uptake and antiviral use in Eastern Europe, since information on risk factors will help in targeting and prioritising vulnerable populations.

Differentiation of influenza B lineages from clinical samples by one-step real-time RT-PCR.

BACKGROUND: Influenza viruses type A and type B are a leading cause of annual epidemics in human populations. Since the 1970s, influenza B viruses have diverged into two antigenically distinct virus lineages called the Yamagata and Victoria lineages. We describe the validation and implementation of a one-step real-time RT-PCR (rRT-PCR) assay that can differentiate between the two genetic lineages of type B. METHODS: Validation of rRT-PCR method was carried out using quantified positive control and reference influenza viruses with specific minor groove binder (MGB) probes. The assay was applied on 102 clinical specimens detected positive for influenza type B. RESULTS: Detection limit was found to be as low as 7.95 RNA copies per reaction. The interassay variability and intra-assay variability were found to be low, and comparable for Yamagata and Victoria lineages. No cross-reactivity with the tested subtypes of influenza type A, known to cause human infections, was noticed. Differentiation of influenza B lineages by rRT-PCR was successfully achieved on all of the known positive type B samples. From the total number of clinical specimens tested, 85 samples belonged to B/Yamagata and 17 samples to B/Victoria lineage. CONCLUSION: Differentiation of genetic lineage B influenza virus circulating in Romania in the next seasons by one-step real-time RT-PCR method will supplement the classical test, haemagglutination inhibition (HI), which requires growing of the virus. This method can be advantageous for a balanced selection of samples, in case of lineages co-circulation, for genetic and antigenic characterization.

Single and multipathogen viral infections in hospitalized children with acute respiratory infections.

We aimed to describe the viral etiology of acute respiratory tract infections in children aged 0-8 years admitted to Grigore Alexandrescu Hospital, the largest pediatric hospital in Romania. The patients had clinical diagnosis of pneumonia, bronchiolitis or viral respiratory infections and had been hospitalized between September 2010 and September 2011. The study was part of the "Molecular investigations of acute respiratory infections caused by non-influenza viruses, to assess the implications of infant and young child pathology" (2008-2011), a National Project II--42-164 (MIRVI). We included in the study 241 children that were swabbed in the first 8 days of the onset with the following symptoms during the previous 7 days: fever > 38 degrees C, AND cough or sore throat, and shortness of breath or difficulty breathing .We identified by RT-PCR 131 (54.4%) positive samples: 112 (85.5%) for a single pathogen, 18 (13.7%) for coinfection with two pathogens and 1(0.8%) for coinfection with three pathogens. The most frequent pathogen identified was respiratory syncytial virus (RSV) (40.18%), followed by Rhinovirus (RhV) (20.54%) and human Metapneumovirus (hMPV) (12.50%). We extrapolated our data to the National program of surveillance of SARI (severe acute respiratory infections). In this program, 191 children aged one month-8 years, were hospitalized in the same period, in which the highest percentage of positivity was due to Influenza viruses (62.65%), but RSV was identified with almost the same percent like in MIRVI (32.53%). It should be noted that among patients with pneumonia, bronchiolitis or respiratory viral infections were identified as the causal agent RhV.

Estimates of pandemic influenza vaccine effectiveness in Europe, 2009-2010: results of Influenza Monitoring Vaccine Effectiveness in Europe (I-MOVE) multicentre case-control study.

BACKGROUND: A multicentre case-control study based on sentinel practitioner surveillance networks from seven European countries was undertaken to estimate the effectiveness of 2009-2010 pandemic and seasonal influenza vaccines against medically attended influenza-like illness (ILI) laboratory-confirmed as pandemic influenza A (H1N1) (pH1N1). METHODS AND FINDINGS: Sentinel practitioners swabbed ILI patients using systematic sampling. We included in the study patients meeting the European ILI case definition with onset of symptoms >14 days after the start of national pandemic vaccination campaigns. We compared pH1N1 cases to influenza laboratory-negative controls. A valid vaccination corresponded to >14 days between receiving a dose of vaccine and symptom onset. We estimated pooled vaccine effectiveness (VE) as 1 minus the odds ratio with the study site as a fixed effect. Using logistic regression, we adjusted VE for potential confounding factors (age group, sex, month of onset, chronic diseases and related hospitalizations, smoking history, seasonal influenza vaccinations, practitioner visits in previous year). We conducted a complete case analysis excluding individuals with missing values and a multiple multivariate imputation to estimate missing values. The multivariate imputation (n = 2902) adjusted pandemic VE (PIVE) estimates were 71.9% (95% confidence interval [CI] 45.6-85.5) overall; 78.4% (95% CI 54.4-89.8) in patients <65 years; and 72.9% (95% CI 39.8-87.8) in individuals without chronic disease. The complete case (n = 1,502) adjusted PIVE were 66.0% (95% CI 23.9-84.8), 71.3% (95% CI 29.1-88.4), and 70.2% (95% CI 19.4-89.0), respectively. The adjusted PIVE was 66.0% (95% CI -69.9 to 93.2) if vaccinated 8-14 days before ILI onset. The adjusted 2009-2010 seasonal influenza VE was 9.9% (95% CI -65.2 to 50.9). CONCLUSIONS: Our results suggest good protection of the pandemic monovalent vaccine against medically attended pH1N1 and no effect of the 2009-2010 seasonal influenza vaccine. However, the late availability of the pandemic vaccine and subsequent limited coverage with this vaccine hampered our ability to study vaccine benefits during the outbreak period. Future studies should include estimation of the effectiveness of the new trivalent vaccine in the upcoming 2010-2011 season, when vaccination will occur before the influenza season starts.

Survival of H5N1 influenza virus in water and its inactivation by chemical methods.

The ability of H5N1 Avian Influenza Virus (AIV) to survive in surface water has been assessed in experimental laboratory conditions, based on non-pathogenic avian reassortant model, by titration of infectivity (TCID50) at different time intervals, in three different types of water. The effect of different chemicals on AIV's survival was assessed using the same type of experimental model. After exposure to the chemical, followed by growth on a suitable substrate, the AIV was quantified by a real-time quantitative reverse transcriptase PCR (qRT-PCR). The reassortant virus persisted, and remained infective in aquatic environments, for 12 days at 22-35 degrees C and up to 20 days at 4 degrees C, irrespective of the type of water, supporting the hypothesis of a potential risk for transmitting the virus among birds and contaminating the household water via common sources of water. A significant decrease for AIV persistence models was recorded for sea water, after 12 days, at 35 degrees C. An effective inactivation has been shown when using commercially available products based on glutaraldehyde and penta potassium bis (peroxy mono sulphate) bis(sulphate), respectively. This rapid and safe method for decontamination, developed in this study, might be helpful in implementation of biosafety measures in laboratory and farms against AIV.

Evaluation of the efficacy of a specific hyperimmune serum in experimental influenza infection in mice.

Serotherapy still remains a way of treatment in some diseases, and it could be consider superior to any other mode of action because the protecting substances of the body are the products of the organism itself. The aim of the study was to establish an "in vivo" method for testing the efficacy of therapeutic serum. Hyperimmune serum for influenza A/PR8/34 viral strain, was prepared in sheep, and tested for inhibition of haemagglutination and microneutralisation. Seroprotection was evaluated in mice one day after being challenged with a lethal dose of the same virus. Our study shows that protection occurred in all mice treated with undiluted hyperimmune serum one day post infection (no clinical signs, faster recovery of the body weight after the first three days of the infection, all mice survived).

[Efficiency of seasonal influenza vaccine in persons older than 65 in Romania. Pilot case-control study I-MOVE, 2008-2009]. / Eficacitatea vaccinului gripal sezonier la persoanele cu vârsta peste 65 de ani din România. Studiul pilot caz-martor I-MOVE, 2008-2009.

Cantacuzino Institute conducted between September 2008 and June 2009, a pilot case-control study to monitor the influenza vaccine effectiveness on people over 65 years of age from Romania. This study is part of the I-MOVE project "Monitoring the vaccine effectiveness during seasonal and pandemic influenza in EU/EEA member states, 2008-2009", coordinated by EpiConcept, France and financed by European Centre for Disease Prevention and Control (ECDC), Stockholm, Sweden. Forty seven family doctors and epidemiologists from Bucharest and seven influenza sentinel districts were selected to participate in this project, based on epidemiological and geographical criteria. Family doctors swabbed all people over 65-years-old consulting for influenza like illness (ILI). Influenza confirmed cases (classified as cases) were compared to influenza negative controls. Influenza vaccine effectiveness (IVE) was obtained using the formula: 1 - odds ratio, with 95% confidence interval (CI). One hundred and three ILI patients were enrolled in the study. Ninety nine from them (96.1%) were swabbed in the first 7 days after the onset, met the inclusion criteria and case definition and were included in analysis. Thirty (30.3%) were ILI flu positive and were classified as cases, sixty nine (69.7%) were ILI flu negative and classified as controls. Influenza vaccine effectiveness adjusted for the predefined set of confounders (age, sex, chronic diseases, smoking, previous influenza vaccination, functional status) was 86.8% (95% CI, 38.0, 97.2); influenza vaccine coverage in people older than 65 years was 49.4%. The result of the study showed a high influenza vaccine effectiveness in the elderly. In order to achieve a greater precision, the national and also the European samples should be extended for the next season.

Determinants of Fatal Outcome in Patients Admitted to Intensive Care Units With Influenza, European Union 2009-2017.

BACKGROUND: Morbidity, severity, and mortality associated with annual influenza epidemics are of public health concern. We analyzed surveillance data on hospitalized laboratory-confirmed influenza cases admitted to intensive care units to identify common determinants for fatal outcome and inform and target public health prevention strategies, including risk communication. METHODS: We performed a descriptive analysis and used Poisson regression models with robust variance to estimate the association of age, sex, virus (sub)type, and underlying medical condition with fatal outcome using European Union data from 2009 to 2017. RESULTS: Of 13 368 cases included in the basic dataset, 2806 (21%) were fatal. Age ≥40 years and infection with influenza A virus were associated with fatal outcome. Of 5886 cases with known underlying medical conditions and virus A subtype included in a more detailed analysis, 1349 (23%) were fatal. Influenza virus A(H1N1)pdm09 or A(H3N2) infection, age ≥60 years, cancer, human immunodeficiency virus infection and/or other immune deficiency, and heart, kidney, and liver disease were associated with fatal outcome; the risk of death was lower for patients with chronic lung disease and for pregnant women. CONCLUSIONS: This study re-emphasises the importance of preventing influenza in the elderly and tailoring strategies to risk groups with underlying medical conditions.

Influenza Vaccine Manufacturing: Effect of Inactivation, Splitting and Site of Manufacturing. Comparison of Influenza Vaccine Production Processes.

The aim of this study was to evaluate the impact of different inactivation and splitting procedures on influenza vaccine product composition, stability and recovery to support transfer of process technology. Four split and two whole inactivated virus (WIV) influenza vaccine bulks were produced and compared with respect to release criteria, stability of the bulk and haemagglutinin recovery. One clarified harvest of influenza H3N2 A/Uruguay virus prepared on 25.000 fertilized eggs was divided equally over six downstream processes. The main unit operation for purification was sucrose gradient zonal ultracentrifugation. The inactivation of the virus was performed with either formaldehyde in phosphate buffer or with beta-propiolactone in citrate buffer. For splitting of the viral products in presence of Tween®, either Triton™ X-100 or di-ethyl-ether was used. Removal of ether was established by centrifugation and evaporation, whereas removal of Triton-X100 was performed by hydrophobic interaction chromatography. All products were sterile filtered and subjected to a 5 months real time stability study. In all processes, major product losses were measured after sterile filtration; with larger losses for split virus than for WIV. The beta-propiolactone inactivation on average resulted in higher recoveries compared to processes using formaldehyde inactivation. Especially ether split formaldehyde product showed low recovery and least stability over a period of five months.

Immunolocalization and Distribution of Rubella Antigen in Fatal Congenital Rubella Syndrome.

BACKGROUND: An estimated 100,000 cases of congenital rubella syndrome (CRS) occur worldwide each year. The reported mortality rate for infants with CRS is up to 33%. The cellular mechanisms responsible for the multiple congenital defects in CRS are presently unknown. Here we identify cell types positive for rubella virus (RV) in CRS infants. METHODS: Cells and organs involved in RV replication were identified in paraffin-embedded autopsy tissues from three fatal case-patients by histopathologic examination and immunohistochemical (IHC) staining using a rabbit polyclonal RV antibody. Normal rabbit antisera and RV antisera preabsorbed with highly purified RV served as negative controls. RESULTS: RV antigen was found in interstitial fibroblasts in the heart, adventitial fibroblasts of large blood vessels, alveolar macrophages, progenitor cells of the outer granular layer of the brain, and in capillary endothelium and basal plate in the placenta. The antibody specificity was verified by IHC staining of multiple tissue sections from other infectious disease cases. RV infection of each cell type is consistent with abnormalities which have been identified in patients with CRS, in the heart, large blood vessels, and brain. Antigen distribution was consistent with inflammatory response to vascular injury and systemic spread of RV. CONCLUSIONS: The identification of RV positive cell types in CRS is important to better understand the pathology and pathogenesis of CRS.

Technology transfer of oil-in-water emulsion adjuvant manufacturing for pandemic influenza vaccine production in Romania: Preclinical evaluation of split virion inactivated H5N1 vaccine with adjuvant.

Millions of seasonal and pandemic influenza vaccine doses containing oil-in-water emulsion adjuvant have been administered in order to enhance and broaden immune responses and to facilitate antigen sparing. Despite the enactment of a Global Action Plan for Influenza Vaccines and a multi-fold increase in production capabilities over the past 10 years, worldwide capacity for pandemic influenza vaccine production is still limited. In developing countries, where routine influenza vaccination is not fully established, additional measures are needed to ensure adequate supply of pandemic influenza vaccines without dependence on the shipment of aid from other, potentially impacted first-world countries. Adaptation of influenza vaccine and adjuvant technologies by developing country influenza vaccine manufacturers may enable antigen sparing and corresponding increases in global influenza vaccine coverage capacity. Following on previously described work involving the technology transfer of oil-in-water emulsion adjuvant manufacturing to a Romanian vaccine manufacturing institute, we herein describe the preclinical evaluation of inactivated split virion H5N1 influenza vaccine with emulsion adjuvant, including immunogenicity, protection from virus challenge, antigen sparing capacity, and safety. In parallel with the evaluation of the bioactivity of the tech-transferred adjuvant, we also describe the impact of concurrent antigen manufacturing optimization activities. Depending on the vaccine antigen source and manufacturing process, inclusion of adjuvant was shown to enhance and broaden functional antibody titers in mouse and rabbit models, promote protection from homologous virus challenge in ferrets, and facilitate antigen sparing. Besides scientific findings, the operational lessons learned are delineated in order to facilitate adaptation of adjuvant technologies by other developing country institutes to enhance global pandemic influenza preparedness.

Severe acute respiratory infections surveillance in Romania - is it any gain to public health?

UNLABELLED: This article aims to present the operation and usefulness of severe acute respiratory infection (SARI) surveillance in Romania. Material and methods : The information obtained from surveillance data analysis using Epilnfo program revealed a greater severity of influenza in the second post-pandemic season (2011/2012) compared to the previous one (2010/2011). Detected risk factors were evidence-based arguments for recommending preventive measures in certain population groups. CONCLUSIONS: This surveillance system has proven useful, being able to provide information on the severity of influenza and other etiologies involved in severe acute respiratory infections.

High genetic diversity of measles virus, World Health Organization European Region, 2005-2006.

During 2005-2006, nine measles virus (MV) genotypes were identified throughout the World Health Organization European Region. All major epidemics were associated with genotypes D4, D6, and B3. Other genotypes (B2, D5, D8, D9, G2, and H1) were only found in limited numbers of cases after importation from other continents. The genetic diversity of endemic D6 strains was low; genotypes C2 and D7, circulating in Europe until recent years, were no longer identified. The transmission chains of several indigenous MV strains may thus have been interrupted by enhanced vaccination. However, multiple importations from Africa and Asia and virus introduction into highly mobile and unvaccinated communities caused a massive spread of D4 and B3 strains throughout much of the region. Thus, despite the reduction of endemic MV circulation, importation of MV from other continents caused prolonged circulation and large outbreaks after their introduction into unvaccinated and highly mobile communities.

Progress toward measles elimination in Romania after a mass vaccination campaign and implementation of enhanced measles surveillance.

In response to an outbreak of >33,000 measles cases in 1996-1998 and to prevent an outbreak predicted for 2002, Romania conducted a nationwide measles-rubella vaccination campaign in October 1998. Some 2.1 million children aged 7-18 years were vaccinated. Data from national surveillance and seroprevalence studies conducted in three districts were used to assess the campaign and status of measles control. Surveillance data showed a dramatic drop in measles despite enhanced surveillance starting in October 1999. From October 1999 to December 2001, 400 suspected measles cases were reported, down from about 5000 cases annually in non-outbreak years. Only 29 (8%) of 386 cases with specimens were laboratory confirmed; 14 were clinically confirmed. Seroprevalence estimates showed high measles antibody levels before (92.9%) and after (94.4%) the campaign. The low number of laboratory-confirmed cases and high population immunity suggest that interruption of indigenous measles virus transmission is a real possibility for Romania.