Human metapneumovirus in Pediatric Patients with Acute Respiratory Tract Infections in the Aseer Region of Saudi Arabia

Alsuheel, Ali Mohammed; Ali, Abdelwahid Saeed; Al-Hakami, Ahmed Musa; Shati, Ayed Abdullah; Chandramoorthy, Harish C.; Al-Qahtani, Saleh Mohammed

A volume of 25 µl of the resuspended cell deposit was placed in slides with 6-mm-diameter wells. The specimens were then allowed to air dry thoroughly and fixed with fresh acetone at room temperature (15°C-30°C) for 10 min. The slide was air-dried after fixation. A volume of 25 µl of IMAGEN™ hMPV reagent (Oxoid Ltd., Cambridge, UK), which contains monoclonal antibodies against hMPV conjugated to fluorescein isothiocyanate, was added to the fixed cell preparation on the slide to cover the wells. The same amount was also added to the positive control slide. The slides were then incubated with the reagent in a moist chamber for 15 min at 37°C. Following incubation, excess reagent was washed off with PBS, and the slide was gently washed in an agitating bath containing PBS for 5 min. The PBS was drained off, and slide was allowed to air dry at room temperature (15°C-30°C). One drop of IMAGEN™ hMPV mounting fluid was added to the center of each well, and cover-slip was placed over the mounting fluid and specimen to ensure that there are no trapped air bubbles. The stained slides were immediately examined under epifluorescence microscope at ×400 and then ×1000. Apple-green fluorescence was observed in the cells infected with hMPV, whereas non-infected cells appeared as red color because they were stained with the Evans blue counterstain. Images of these cells were captured using a microscopic camera (Nikon-DS-Fi1, Nikon Corp., Tokyo, Japan) and archived.