Determining specificity of the sVNT in measuring SARS-CoV2 Nabs in the Sri Lankan population
In order to determine the specificity of the sVNT in Sri Lankan individuals, we initially assessed the % of inhibition in 81 serum samples obtained from individuals who presented with a febrile illness to the outpatient department of the National Institute of Infectious Diseases (NIID), Sri Lanka in 2018. All these individuals had a % of inhibition less than the cut of value of ≤25%. We then assessed the specificity of the assay in 285 non-exposed individuals recruited from the Colombo Municipality area during the month of April. The percentage of inhibition in this population was also less than the cut-off value. Therefore , the specificity of this assay was found to be 100% as previously described 10.
Longitudinal changes in Nabs in patients with varying severity of clinical disease
In order to determine the longitudinal changes of SARS-CoV2 NAbs in patients with varying clinical disease severity, we assessed their levels in those with severe (n=6), moderate (n=5) and mild/asymptomatic illness (n=13) and also those who had mild illness but with prolonged shedding of the virus (n=21). The median duration of virus shedding in this whole cohort was 25 days (IQR 15 to 38 days) and therefore, those who had virus shedding for over 25 days and hospitalized for over 25 days were considered to have prolonged shedding of the virus. There were 10 individuals who had shedding for over 50 days. The patterns of virus shedding in those with mild but prolonged illness is shown in Fig 1A.
Three blood samples were obtained from these individuals during the course of illness and in the case of those with mild infection, 4 to 6 weeks after they were discharged from hospital.
In the longitudinal analysis of NAbs, they appeared earlier and faster, at higher levels in those who had severe and moderate pneumonia, followed by those who had prolonged shedding, while they appeared later, at lower levels in those who had mild/asymptomatic disease (Fig 1B). 4 individuals with mild illness had no detectable NAbs even 40 days since onset of illness, whereas all but 1/21 individuals had NAbs below this level.
The timing of the appearance of Nabs and their levels in relation to clinical disease severity
In order to further evaluate the appearance and the quantity of NAbs in relation to clinical disease severity, we assessed antibodies in blood samples at different time points from patients with severe pneumonia (n=10), moderate illness (n=19), mild illness (n=150) and prolonged shedding (n=82). The Nabs levels were determined in this larger cohort of individuals during the first 3 weeks and during week 4 to 8 of illness.
Again, those with moderate and severe illness had higher NAbs levels (median 63.16 and 48.9% of inhibition) during the 1st week, and in all subsequent time points compared to those with mild and prolonged shedding (Fig 2A). After the 3rd week (4th to 8th week), although all patients with severe, moderate and prolonged shedding had a positive test result, 23/69 (33.3%) of those with mild/asymptomatic illness were negative (% of inhibition <25). Those with prolonged shedding, who had mild or asymptomatic illness had significantly higher (p<0.0001) NeutAb levels (median 76.4%, IQR 52.32 to 89.5% of inhibition) than those with mild/asymptomatic illness (median 47.9%, IQR 18.9 to 77.1% of inhibition) during week 4 to 8. This data further reinforces the longitudinal analysis of NAbs as shown in Fig 1B.
SARS-CoV2 Nab positivity at different time points and persistence
We then proceeded to assess the detection of NAbs at various time points in illness, irrespective of clinical disease severity and also to assess if NAbs persisted over 90 days since onset of illness. NAbs were measured by the sVTN on day 14 to 21 (n=98), day 22 to 28 (n=100), day 29 to 36 (n=132), day 37 to 42 (n=32), day 43 to 49 (n=16), day 50 to 70 (n=29) and >90 days (n=15). The positivity rates during day 14 to 21 was 79.8%, day 22 to 28 was 88.9%, day 29 to 36 was 100Therefore, all patients tested positive by the end of 5th week of illness (Fig 2B). However, the positivity rates declined thereafter, as the positivity was 90.6% at 37 to 42 days, 65.5% between 50 to 70 days of illness, and 53.3% in those with >90 days since onset of illness suggesting that the NAbs could be declining with time. All those who had Nabs below the cut-off value were those who had mild/asymptomatic illness, while those with moderate/severe illness and individuals with prolonged shedding were positive for Nabs after day 90 of illness.