3.5 Detection of Viral load
Using standard DNA, the qPCR assay detection limit was estimated to be 9.1 copies per reaction and the slope and correlation coefficient of the standard curve were −2·557 and 0·981 respectively (Figure 3). HAdV genome was detected by qPCR in all 61 PCR positive samples with Ct values ranging from 12 to 39.9 (median Ct value: 28.8) (Figure 3). Fifty-five specimens with Ct values < 35 found to contain DNA copies between 3.3 X 102 and 5.7 X 1010. Among them, 46 specimens were negative for RVA and NoV and of which 71.7% were with severe and very severe disease outcome. Analysis of the mean Ct values and viral load in specimens with different subgroups showed no significant difference (Table 5). However, the viral load in patients infected with HAdV-F strains was significantly high (n=32; 5.24± 2.38) as compared to non HAdV-F strains (n=29; 4.11± 1.61, p=0.036).
Among HAdV positive patients, 9, 28, 18 and 6 specimens showed Ct values between 10-20, 20-30, 30-35 and 35-40 respectively (Table 6). The distribution of patients with moderate, severe and very severe disease outcome in different groups of Ct values was not significantly different from each other (p=0.571, Table 6). Comparative analysis of the mean ± SD values obtained for disease severity score among these groups was without any significant difference. Analysis of moderate, severe and very severe disease outcome in different groups of Ct values in HAdV-F and non HAdV-F strains showed no significant difference (p= 0.299, Table 6)
Six specimens with Ct values >35 showed DNA copies between 2.7X102 and 0.69 per reaction and belong to genotype HAdVF-40, HAdVF-41, HAdVA-31, HAdVA-12, HAdVC-5 and untypeable hexon strain. Out of these six specimens, five belonged to patients with severe disease outcome displaying dual infection with Norovirus in two and RVA in one.