THE INDIRECT IMMUNOFLUORESCENCE ASSAY IN THE DIAGNOSIS OF HUMAN ROTAVIRUS GASTROENTERITIS

Rotavirus is the most frequently implicated etiological agent in infectious diarrhea in children. Several laboratory techniques were used to identify this virus in fecal material. This indirect immunofluorescence assay described by BRIDEN et alii (2) was used in the work, with some modifications, to study the frequency of rotavirus in infeccious diarrhea. The results were compared with those obtained with the enzyme immunoassay (EIARA) and the polyacrilamid gel electrophoresis (PAGE). The SA-11 strain of simian rotavirus was cultivated in MA-104 cells, in Eagle's MEM, without serum, with 10-mu-g/ml of trypsin (1:250 Difco). The cultures with total cytophatic effect were clarified with Freon TF, DuPont. The virus was concentrated by ultracentrifugation at 100.000 g/15 min (Sorwall centrifuge OTD-75B with AH 627 rotor) and then purified in a 20-40% Cesium-chloride gradient in Tris 0,05M, NaCl 0,15M buffer at 100.000 g/24h in a Beckmann L5-74 ultracentriguge with SW 65 rotor. This purified virus was used to prepare, in guinea-pigs, rotavirus antisera. Purified guinea-pig globulin was inoculated by subcutaneous route in rabbits to obtain antiguinea pig gamma - globulin antiserum which was conjugated with fluorescein-isotiocianate (6). Fecal species were collected from 268 children, under one year of age, with acute diarrhea. Approximately 20% suspensions were made in Tris/HCl 0,01M pH 7,4 buffer, containing 15 mM CaCl2, 1.000 U/ml penicilin and 1.000 ug/ml of streptomicim. The suspensions were clarified by centrifugation at 12.350g/20min after homogenization with Freon TF, DuPont. The supernates were examined as described below, immediatly or after storage at -20-degrees-C. The immunoflorescence reaction was performed in polystyrene microtitration plates (NUNC), with 96 wells, coated with MA-104 cells monolayer. An 25-mu-l amount of 20% fecal suspensions was inoculated in 2 wells, diluted at 1:4, and 25-mu-l in other 2 wells diluted at 1:16. The wells were filled with 75-mu-l of Eagle's MEM with 10-mu-g/ml of trypsin. The plates were centrifuged for 1 h at 1.200g in Beckmann TJ-6R centrifuge, with TH-4 rotor. The inoculum was pofur out, the wells refilled with fresh medium, and the plates incubated at 37-degrees-C for 18h. After incubation the plates were washed in PBS 0,01M pH 7,2, fixed with frozen metanol and allowed to dry. The infected monolayers were then treated with guinea-pig antirotavirus serum, diluted 1 in 40 (50-mu-l/well), at 37-degrees-C. After 60 min, plates were washed in PBS and stained for 60 min at 37-degrees-C with the fluorescencein-conjugated rabbit antiguinea-pig serum, diluted 1 in 20 (50-mu-l/well). After this period plates were washed in PBS, and allowed to dry. Readings of the fluorescence were performed in a Reichert-Jung, Microstar epifluorescent microscope. Samples with more than 3 cells with cythoplasmic granular fluorescence were regarded as positive. Enzyme-immunoassays were performed by double antibody sandwich tecnique with the EIARA/FIOCRUZ kit by technique described by PEREIRA et alii (15). Electrophoresis was carried out by LAEMLI's technique with modifications described by PEREIRA et alii (14). Slab gels were stained by silver impregnation. Rotavirus antigen could be detected in 40 out the 268 fecal samples (14,93%) by IF and PAGE, and in 46 (17,16%) by EIARA. A comparison between the three assays revealed a large percentage of agreement (72%). The results obtained were analysed using statistic kappa and the values obtained were between 0,72 and 0,76.