COVID-19 Pandemic: lessons learnt and the way forward

Although the scientific community had been predicting and preparing for a pandemic for the last 10 years, many policy makers did not envision that a virus could cause such devastation to human life, economies and to the social structure. COVID-19 has taught us many bitter lessons and while moving forward it is important to understand that this current pandemic is yet to end. However, COVID-19 is unlikely to be the last pandemic that we face, Due to certain human activities such as urbanization, deforestation, increased human and animal interactions and climate change, we will see more pandemics emerging in the coming years. Preparedness and anticipation of such an event is the only way forward

Comparison of different sequencing techniques for identification of SARS-CoV-2 variants of concern with multiplex real-time PCR

As different SARS-CoV-2 variants emerge and with the continuous evolvement of sub lineages of the delta variant, it is crucial that all countries carry out sequencing of at least >1% of their infections, in order to detect emergence of variants with higher transmissibility and with ability to evade immunity. However, due to limited resources as many resource poor countries are unable to sequence adequate number of viruses, we compared to usefulness of a two-step commercially available multiplex real-time PCR assay to detect important single nucleotide polymorphisms (SNPs) associated with the variants and compared the sensitivity, accuracy and cost effectiveness of the Illumina sequencing platform and the Oxford Nanopore Technologies’ (ONT) platform. 138/143 (96.5%) identified as the alpha and 36/39 (92.3%) samples identified as the delta variants due to the presence of lineage defining SNPs by the multiplex real time PCR, were assigned to the same lineage by either of the two sequencing platforms. 34/37 of the samples sequenced by ONT had <5% ambiguous bases, while 21/37 samples sequenced using Illumina generated <5%. However, the mean PHRED scores averaged at 32.35 by Illumina reads but 10.78 in ONT. This difference results in a base error probability of 1 in 10 by the ONT and 1 in 1000 for Illumina sequencing platform. Sub-consensus single nucleotide variations (SNV) are highly correlated between both platforms (R2 = 0.79) while indels appear to have a weaker correlation (R2 = 0.13). Although the ONT had a slightly higher error rate compared to the Illumina technology, it achieved higher coverage with a lower number or reads, generated less ambiguous bases and was significantly less expensive than Illumina sequencing technology

SARS-CoV-2 neutralizing antibodies in patients with varying severity of acute COVID-19 illness

In order to support vaccine development, and to aid convalescent plasma therapy, it would be important to understand the kinetics, timing and persistence of SARS-CoV-2 neutralizing antibodies (NAbs), and their association with clinical disease severity. Therefore, we used a surrogate viral neutralization test to evaluate their levels in patients with varying severity of illness, in those with prolonged shedding and those with mild/asymptomatic illness at various time points. Patients with severe or moderate COVID-19 illness had earlier appearance of NAbs at higher levels compared to those with mild or asymptomatic illness. Furthermore, those who had prolonged shedding of the virus, had NAbs appearing faster and at higher levels than those who cleared the virus earlier. During the first week of illness the NAb levels of those with mild illness was significantly less (p = 0.01), compared to those with moderate and severe illness. At the end of 4 weeks (28 days), although 89% had NAbs, 38/76 (50%) in those with > 90 days had a negative result for the presence of NAbs. The Ab levels significantly declined during convalescence (> 90 days since onset of illness), compared to 4 to 8 weeks since onset of illness. Our data show that high levels of NAbs during early illness associated with clinical disease severity and that these antibodies declined in 50% of individuals after 3 months since onset of illness

Transmission dynamics, clinical characteristics and sero-surveillance in the COVID-19 outbreak in a population dense area of Colombo, Sri Lanka April- May 2020

Background The transmission dynamics of SARS-CoV-2 varies depending on social distancing measures, circulating SARS-CoV-2 variants, host factors and other environmental factors. We sought to investigate the clinical and epidemiological characteristics of a SARS-CoV-2 outbreak that occurred in a highly dense population area in Colombo, Sri Lanka from April to May 2020. Methodology/principal findings We carried out RT-qPCR for SARS-CoV2, assessed the SARS-CoV-2 specific total and neutralizing antibodies (Nabs) in a densely packed, underserved settlement (n = 2722) after identification of the index case on 15th April 2020. 89/2722 individuals were detected as infected by RT-qPCR with a secondary attack rate among close contacts being 0.077 (95% CI 0.063–0.095). Another 30 asymptomatic individuals were found to have had COVID-19 based on the presence of SARS-CoV-2 specific antibodies. However, only 61.5% of those who were initially seropositive for SARS-CoV-2 had detectable total antibodies at 120 to 160 days, while only 40.6% had detectable Nabs. 74/89 (83.1%) of RT-qPCR positive individuals were completely asymptomatic and all 15 (16.9%) who experienced symptoms were classified as having a mild illness. 18 (20.2%) were between the ages of 61 to 80. 11/89 (12.4%) had diabetes, 8/89 (9%) had cardiovascular disease and 4 (4.5%) had asthma. Of the two viruses that were sequenced and were of the B.1 and B.4 lineages with one carrying the D614G mutation. Discussion/conclusion Almost all infected individuals developed mild or asymptomatic illness despite the presence of comorbid illnesses. Since the majority of those who were in this underserved settlement were not infected despite circulation of the D614G variant, it would be important to further study environmental and host factors that lead to disease severity and transmission

Molecular epidemiology of AY.28 and AY.104 delta sub-lineages in Sri Lanka

Background: The worst SARS-CoV-2 outbreak in Sri Lanka was due to the two Sri Lankan delta sub-lineages AY.28 and AY.104. We proceeded to further characterize the mutations and clinical disease severity of these two sub-lineages. Methods: 705 delta SARS-CoV-2 genomes sequenced by our laboratory from mid-May to November 2021 using Illumina and Oxford Nanopore were included in the analysis. The clinical disease severity of 440/705 individuals were further analyzed to determine if infection with either AY.28 or AY.104 was associated with more severe disease. Sub-genomic RNA (sg-RNA) expression was analyzed using periscope. Results: AY.28 was the dominant variant throughout the outbreak, accounting for 67.7% of infections during the peak of the outbreak. AY.28 had three lineage defining mutations in the spike protein: A222V (92.80%), A701S (88.06%), and A1078S (92.04%) and seven in the ORF1a: R24C, K634N, P1640L, A2994V, A3209V, V3718A, and T3750I. AY.104 was characterized by the high prevalence of T95I (90.81%) and T572L (65.01%) mutations in the spike protein and by the absence of P1640L (94.28%) in ORF1a with the presence of A1918V (98.58%) mutation. The mean sgRNA expression levels of ORF6 in AY.28 were significantly higher compared to AY.104 (p < 0.0001) and B.1.617.2 (p < 0.01). Also, ORF3a showed significantly higher sgRNA expression in AY.28 compared to AY.104 (p < 0.0001). There was no difference in the clinical disease severity or duration of hospitalization in individuals infected with these sub lineages. Conclusions: Therefore, AY.28 and AY.104 appear to have a fitness advantage over the parental delta variant (B.1.617.2), while AY.28 also had a higher expression of sg-RNA compared to other sub-lineages. The clinical implications of these should be further investigated

Genomic and Epidemiological Analysis of SARS-CoV-2 Viruses in Sri Lanka.

Background: In order to understand the molecular epidemiology of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in Sri Lanka, since March 2020, we carried out genomic sequencing overlaid on available epidemiological data until April 2021. Methods: Whole genome sequencing was carried out on diagnostic sputum or nasopharyngeal swabs from 373 patients with COVID-19. Molecular clock phylogenetic analysis was undertaken to further explore dominant lineages. Results: The B.1.411 lineage was most prevalent, which was established in Sri Lanka and caused outbreaks throughout the country until March 2021. The estimated time of the most recent common ancestor (tMRCA) of this lineage was June 1, 2020 (with 95% lower and upper bounds March 30 to July 27) suggesting cryptic transmission may have occurred, prior to a large epidemic starting in October 2020. Returning travellers were identified with infections caused by lineage B.1.258, as well as the more transmissible B.1.1.7 lineage, which has replaced B.1.411 to fuel the ongoing large outbreak in the country. Conclusions: The large outbreak that started in early October, is due to spread of a single virus lineage, B.1.411 until the end of March 2021, when B.1.1.7 emerged and became the dominant lineage

Sensitivity and specificity of two WHO approved SARS-CoV2 antigen assays in detecting patients with SARS-CoV2 infection

Background: SARS-CoV-2 rapid antigen (Ag) detection kits are widely used in addition to quantitative reverse transcription PCR PCR (RT-qPCR), as they are cheaper with a rapid turnaround time. As there are many concerns regarding their sensitivity and specificity, in different settings, we evaluated two WHO approved rapid Ag kits in a large cohort of Sri Lankan individuals. Methods: Paired nasopharangeal swabs were obtained from 4786 participants for validation of the SD-Biosensor rapid Ag assay and 3325 for the Abbott rapid Ag assay, in comparison to RT-qPCR. A short questionnaire was used to record symptoms at the time of testing, and blood samples were obtained from 2721 of them for detection of SARS-CoV-2 specific antibodies. Results: The overall sensitivity of the SD-Biosensor Ag kit was 36.5% and the Abbott Ag test was 50.76%. The Abbott Ag test showed specificity of 99.4% and the SD-Biosensor Ag test 97.5%. At Ct values  30 (46.1 to 82.9%). 32.1% of those who gave a positive result with the SD-Biosensor Ag test and 26.3% of those who gave positive results with the Abbott Ag test had SARS-CoV-2 antibodies at the time of detection. Conclusions: Both rapid Ag tests appeared to be highly sensitive in detecting individuals at lower Ct values, in a community setting in Sri Lanka, but it will be important to further establish the relationship to infectivity

Kinetics of immune responses to SARS-CoV-2 proteins in individuals with varying severity of infection and following a single dose of the AZD1222

To characterize the IgG and IgA responses to different SARS-CoV-2 proteins, we investigated the antibody responses to SARS-CoV-2 following natural infection and following a single dose of AZD1222 (Covishield), in Sri Lankan individuals. The IgG and IgA responses were assessed to S1, S2, RBD, and N proteins in patients at 4 weeks and 12 weeks since the onset of illness or following vaccination. Antibodies to the receptor-binding domain of SARS-CoV-2 wild type (WT), α, β, and λ and ACE2 (Angiotensin Converting Enzyme 2) receptor blocking antibodies were also assessed in these cohorts. For those with mild illness and in vaccines, the IgG responses to S1, S2, RBD, and N protein increased from 4 weeks to 12 weeks, while it remained unchanged in those with moderate/severe illness. In the vaccines, IgG antibodies to the S2 subunit had the highest significant rise (P < 0.0001). Vaccines had several-fold lower IgA antibodies to all the SARS-CoV-2 proteins tested than those with natural infection. At 12 weeks, the haemagglutination test (HAT) titres were significantly lower to the α in vaccines and significantly lower in those with mild illness and in vaccines to β and for λ. No such difference was seen in those with moderate/severe illness. Vaccines had significantly less IgA to SARS-CoV-2, but comparable IgG responses those with natural infection. However, following a single dose vaccines had reduced antibody levels to the VOCs, which further declined with time, suggesting the need to reduce the gap between the two doses, in countries experiencing outbreaks due to VOCs

Seroprevalence of SARS-CoV-2 infection in the Colombo Municipality region, Sri Lanka

Background: As the Municipality Council area in Colombo (CMC) experienced the highest number of cases until the end of January 2021, in Sri Lanka, we carried out a serosurvey prior to initiation of the vaccination program to understand the extent of the SARS-CoV-2 outbreak. Methods: SARS-CoV-2 seropositivity was determined in 2,547 individuals between the ages of 10–86 years, by the Wantai total antibody ELISA. We also compared seroprevalence using the haemagglutination test (HAT) to evaluate its usefulness in carrying out serosurveys. Results: The overall seropositivity rate was 24.46%, while seropositivity by HAT was 18.90%. Although The SARS-CoV-2 infection detection rates by PCR were highest in the population between the ages of 20–60 years of age, there was no statistically significant difference in the seropositivity rates in different age groups. For instance, although the seropositivity rate was highest in the 10–20 age group (34.03%), the PCR positivity rate was 9.80%. Differences in the PCR positivity rates and seropositivity rates were also seen in 60–70-year-olds (8.90 vs. 30.4%) and in individuals >70 years (4.10 vs. 1.20%). The seropositivity rate of the females was 29.70% (290/976), which was significantly higher (p < 0.002) than in males 21.2% (333/1,571). Conclusions: A high seroprevalence rate (24.5%) was seen in all age groups in the CMC suggesting that a high level of transmission was seen during this time. The higher PCR positivity rates between the ages of 20–60 are likely to be due to increased testing carried out in the working population. Therefore, the PCR positivity rates, appear to underestimate the true extent of the outbreak and the age groups which were infected