Viral Profile of COPD Exacerbations According to Patients§

Dimopoulos, G; Tsiodras, S; Lerikou, M; Chranioti, Aik; Perros, E; Anagnostopoulou, U; Karakitsos, P; Armaganidis, A

Following first evaluation and before the initiation of any treatment, sputum and oropharyngeal samples (gargle) were collected and submitted for viral detection by clinical microarrays technique i.e. CLART ® PneumoVir kit (GENOMICA, Spain). This method is able to detect and characterize the 17 most frequent types of human viruses responsible for respiratory infections, by identifying very small quantities of viral genomic material. It uses a sequence which corresponds to a highly preserved region within the viral genome and binding probes specific to each respiratory virus type. Viruses analyzed include, human Respiratory Syncytial Virus (hRSV) type A and B, human Metapneumovirus (hMPV) type A and B Influenza virus (all types A, B, C), Rhinovirus, Parainfluenza virus (hPIV) 1, 2, 3 and 4 (subtypes A and B), Enterovirus (Echovirus), Adenovirus, Coronavirus and Bocavirus. The samples were collected in Thin Prep CytoLyt ® solution and centrifuged at 2000g. The molecular procedure was performed according to the manufacturer's instructions. In brief, viral DNA/RNA was extracted by using 200µl of clinical sample mixed with lysis buffer and then allowed to stand in room temperature for 15 minutes. Isopropanol was added and centrifugation was performed at 13000 rpm for 20 min followed by removal of the supernatant. Then the precipitate was resuspended with 1000µl 70% ethanol followed by centrifugation at 13000 rpm for 15 min. The supernatant was removed again and the sample was left to dry for 15 min (until there were no ethanol residues left). At the end the pellet was resuspended in 20 µl of dilution solution. The viral DNA/RNA extracts were stored at -20°C until amplification. Virus amplification was performed via two RT (reverse transcriptase) Multiplex PCR reactions of a specific 120-330 bp fragment of the viral genome. The PCR employed the following thermal cycler settings: 1 cycle of 45 min at 45°C and 15 min at 95°C, followed by 45 cycles of 30 sec at 95°C, 1,5 min at 50°C and 1 min at 68°C and 1 final cycle of 10 min at 68°C. Visualization of the amplified product was performed on a platform based on low-density micro-arrays, which is called Array Tube (AT). This detection system is based on the precipitation of an insoluble product at those sites of the AT where the hybridization of the products amplified by specific molecular probes is produced. The amplified products are labeled with biotin during the PCR. After the amplification, they hybridize with their respective specific probes that are immobilized in specific and known sites of the AT, after which they are incubated with a streptavidin-peroxidase conjugate. The conjugate binds via streptavidin with the biotin present in the amplified products (which are also bound to their specific probes), while in the presence of o-dianisidine, the peroxidase activity of the conjugate induces the appearance of an insoluble product which precipitates at the hybridization sites of the AT" [10] .