9 For all patients, 500 µL of the liquid collected from the nasopharyngeal swab were passed through 0.22-µm pore sized centrifugal filter (Merck millipore, Darmstadt, Germany), then were inoculated in wells of 96-well culture microplates, of which 4 wells contained Vero E6 cells (ATCC CRL-1586) in Minimum Essential Medium culture medium with 4% fetal calf serum and 1% glutamine. After centrifigation at 4,000 g, microplates were incubated at 37°C. Plates were observed daily for evidence of cytopathogenic effect. Presumptive detection of virus in supernatant was done using SU5000 SEM (Hitachi) then confirmed by specific RT- PCR. Outcome The primary endpoint was virological clearance at day-6 post-inclusion. Secondary outcomes were virological clearance overtime during the study period, clinical follow-up (body temperature, respiratory rate, long of stay at hospital and mortality), and occurrence of side- effects. Statistics Assuming a 50% efficacy of hydroxychloroquine in reducing the viral load at day 7, a 85% power, a type I error rate of 5% and 10% loss to follow-up, we calculated that a total of 48 COVID-19 patients (ie, 24 cases in the hydroxychloroquine group and 24 in the control group) would be required for the analysis (Fleiss with CC). Statistical differences were evaluated by Pearson’s chi-square or Fisher’s exact tests as categorical variables, as appropriate. Means of quantitative data were compared using Student’s t-test. Analyses were performed in Stata version 14.2. 3. Results (detailed results are available in supplementary Table 1)
Hydroxychloroquine and Azithromycin as a Treatment of COVID-19 Page 8 Page 10