I ABSTRACT
Respiratory tract infections (RTI), that have high cause of mortality and morbidity worldwide, are caused by infectious pathogens are able to replicate in the respiratory tract, in pulmonary parenchyma or pleuric sierosa. RTI can be classified into infections of the upper respiratory tract (URI), that affect the nose and throat and infections of the lower respiratory tract (LRTI), that affect the lower Airways and lungs. The infectious causative agents of RTI can be: bacteria, fungi and protozoa, but most frequently viruses. The viruses most frequently associated with RTI are, Adenovirus (ADV), Bocavirus (BOV), Coronavirus (CoV), Enterovirus (ENTV), influenza (A, B, C), Metapneumovirus (MetV), Parainfluenzavirus (IPV 1, 2, 3, 4a and b), Rhinoviruses (RV), Respiratory Syncytial Virus(RSV). Respiratory viruses belong to different families and often show ovelapping symptom, thaw identifying the single causative agent can be compromised by wide variety of potential pathogens involved and frequent co-infections, that make difficult for clinicians to formulate a diagnostic question to the laboratory. For these reasons diagnostic methods must be rapid, sensitive and specific in order to identify all possible viruses that may be present in clinical specimens collected from patients affected from respiratory syndromes. The techniques most commonly used today to diagnose infectionsare often viral low sensitive and specific and require highly specific skills. In recent years the technology used for the laboratory diagnosis of viral RTI has led to the development of techniques based nucleic acid amplification for the simultaneous detection of viruses, most commonly associated with RTI. Arrays are a technology developed for the detection of multiple targets at the same time; there are various platforms, which recently has been applied to the virological diagnosis; clinical array (CLART) which are based on labelling and amplification of RNA and DNA from different viruses simultaneously and their detection through hybridization with specific probes. To evaluate the use of the CLART technology in the diagnosis of respiratory infections, in this study we analysed 8 quality control samples for Molecular Diagnostics (QCMD) with know viral load,
II separately and then mixing to test the sensitivity and specificity of the method. 10 leftover respiratory specimens (throat swabs) collected from patients with symptoms related to respiratory tract infections in the Laboratory of Virology of Pisa hospital and university from April to may 2009 during the influenza virus H1N1v pandemia. Clinicians had asked only the search of H1N1; all samples resulted negative for H1N1v infection. The samples were analyzed with CLART PneumoVir ® kit (Genomicas) technology which allows the contemporary rilevation of 17 respiratory viruses: INF A, B and C; IPV 1, 2, 3 and 4 (subtypes A and B); A and B, RSV RSV, MPV (A and B); ENTV (Echovirus), ADV, CoV and BoV. The method involves the use of arrays positioned or at the bottom of a eppendorf tube and the amplification and biotin labelling with two multiplex PCR of specific viral genome fragments of about 120-330 BP, extracted and purified by different types of samples (nasal pharyngeal wash, pharyngeal exudates, naso-pharyngeal exudates), which are then hybridized with capture specific probes. For the resolution of hybridization and the interpretation of results the image processing software, optical scanner (SAICLART ®), has been used, that the automatic detection of the viral genotypes. All samples were analysed in three times and all viruses detected were confirmed by Real Time PCR (Light Cycler2.0, Roche). By CLART Pneumovir all viral genomes were detected in QCMD samples. In clinical specimens single infections were detected with: RSV, MPV, CoV 229 a co-infection of MPV to RSV, A and B, a co-infection of CoV 229 Ipv3 and RSV and a co-infection of BoV and MPV. Only in the sample with the co-infection of Cov 229, Ipv3, RSV, the Real Time PCR did not confirm the presence of the Ipv3 genome. The obtained data in demonstrate that CLART can detect properly respiratory viruses most frequently associated with respiratory syndromes with high sensitivity and specificity.
