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.

The adenoviral infections in children admitted to hospital with pneumonia, acute bronchiolitis or respiratory viral infections.

UNLABELLED: The objective of this study was to investigate the percent of infections with adenovirus (ADV) in children who had pneumonia, acute bronchiolitis or viral respiratory infections and were admitted to two pediatrics hospitals in Bucharest (Grigore Alexandrescu Hospital and Alfred Rusescu Hospital). SUBJECTS: 70 children aged one month - five years, admitted to the above mentioned pediatrics hospitals in Bucharest, who were negative for the Respiratory Syncytial Virus (RSV) and the human Metapneumovirus (hMPV) by Reverse Transcription -Polymerase Chain Reaction (RT-PCR). 48 of them presented pneumonia upon admission to hospital, 6--acute bronchiolitis and 16 respiratory viral infections. Samples (nasal swabs) were taken from patients and introduced in viral transport medium. DIAGNOSTIC METHODS: RT-PCR for RSV and hMPV, Multiplex PCR by seeplex multi-detection system with Seeplex RV/PB 18 ASE Detection for detection of 5 pneumonial bacteria and Real-Time PCR, Duplica Real Time Adenovirus Detection for ADV. RESULTS: Of the total 70 patients negative for RSV, hMPV and 5 pneumonial bacteria (Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila), 10 were ADV positive; none of the children < 6 months (N = 16) presented ADV infection. In the 6 months - 2 years group (N = 35), 6 were ADV positive. In the 2 - 5 years group (N = 19), 4 were ADV positive. CONCLUSIONS: The percent of ADV infections in children hospitalized with acute respiratory infections (ARI) caused by neither RSV or hMPV is 14.2%. ADV is most frequently encountered in the 6 months - 2 years and then 2 - 5 years groups, but the most severe pneumonia forms can be seen in the 6 months - 2 years group. In children < 6 months with acute bronchiolitis ADV was not found to be an etiologic agent.

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.

Etiology of viral pneumopathies in patients in intensive care unit under mechanical ventilation.

The objective of this work was to define the etiology of viral pneumopathies at the patients from reanimation section being under mechanical ventilation, making reference to viruses with respiratory tropism, and also to Chlamydia Pneumoniae and Mycoplasma pneumoniae. The subjects were 36 patients hospitalized into Service of Medical Reanimation from CHU Caen and who needed mechanical ventilation more than 48 hours. The samples from the patients were mostly nasal aspirate, 1 bronchial aspirate and 2 tracheal aspirates. The diagnosis tests were: the test of direct immunofluorescence (DIF) from the samples (for Influenza viruses A and B, Parainfluenza 1,2,3, Adenovirus and Respiratory Syncytial Virus (RSV), inoculation on the tissue culture of diploid cells MRC5, and at the appearance of cythopatic effect specific for Herpes Simplex Virus (HSV), it was made DIF for the detection of type 1 or 2, and also there were made 6 techniques of Polymerase Chain Reaction (PCR). The results of the tests were: at admission before installing the mechanical ventilation, 6 patients presented an infection with Rhinoviruses (RV), 3 with Influenza type A, 3 with HSV type 1 and 2 with Enterovirus. After a period of time from installing the mechanical ventilation, 8 patients presented an infection with HSV typel, among who 1 presented at admission an infection with RV, and 1 patient presented at 7 days from installing the mechanical ventilation an infection with RSV, and at 16 days an infection with HSV type 1. Thus, it could be concluded that in 25% from the cases of viral pneumopathies from patients being under mechanical ventilation it was an endogen reactivation of HSV type1 and only into a single case was diagnosed initially with an infection with RSV, after that it appeared also an infection with HSV typel.

First detection of human metapneumovirus in children with respiratory infections in Romania.

The human metapneumovirus (hMPV) was first isolated in 2001 in the Netherlands (Van der Hoogen and collaborators) from a nasopharyngeal aspirate sampled from an infant. Based on the morphological, biochemical and genetic characteristics, the hMPV was initially classified in the genus Metapneumovirus with the avian metapneumovirus (APV), the agent causing the respiratory infections of the upper tract in turkeys and other birds. Subsequently, together with the respiratory syncytial virus (RSV), it was classified in the Pneumovirus genus which is a part of the Pneumovirinae subfamily, the Paramyxoviridae family. The aim of the present study was to optimize hMPV molecular detection and to detect the virus in samples form children with respiratory infections in Romania. Two types of RTPCR commercial kits were evaluated for the detection of hMPV. Tests were performed on 28 pharyngeal exudates from children aged from 9 months to 6 years, which were negative for influenza viruses and for Respiratory Syncytial Virus (RSV). Among the tested samples 7 (25%) have been positive for hMPV by RT-PCR. These results document for the first time that hMPV is circulating in Romania and causes respiratory infections, especially in newborns and children under 6 years old.

The viral bronchiolites diagnosis in children by PCR multiplex.

The aim of the study was to determine the etiology of the viral bronchiolites in children by using direct immunofluorescence test and 3 RT-PCR Multiplex (S.Bellau-Pujol) The study was performed on 122 nasal inspirations collected from 3 weeks-6 month old children hospitalizated in the pediatrics service of CH Rouen. The results were that the majority (53%) of bronchiolites in children had like etiology RSV and a lot of these infections had double viral etiology (26% RSV+ Rhinovirus; 2,7% RSV+HMPV and 1% RSV+Coronavirus 229E). An important viral factor which gives bronchiolitis in children is HMPV (11%). We also find respiratory infections with triple viral etiology: RSV+Influenza A virus + Rhinovirus.