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

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

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

Immune responses to Sinopharm/BBIBP-CorV in individuals in Sri Lanka

As there are limited data of the immunogenicity of the Sinopharm/BBIBP-CorV in different populations, antibody responses against different SARS-CoV-2 variants of concern and T cell responses, we investigated the immunogenicity of the vaccine, in individuals in Sri Lanka. SARS-CoV-2-specific antibodies were measured in 282 individuals who were seronegative at baseline, and ACE2 receptor blocking antibodies, antibodies to the receptor-binding domain (RBD) of the wild-type (WT), alpha, beta and delta variants, ex vivo and cultured IFNγ ELISpot assays, intracellular cytokine secretion assays and B cell ELISpot assays were carried out in a sub cohort of the vaccinees at 4 and 6 weeks (2 weeks after the second dose). Ninety-five percent of the vaccinees seroconverted, although the seroconversion rates were significantly lower (p 60 years (93.3%) compared to those who were 20–39 years (98.9%); 81.25% had ACE2 receptor blocking antibodies at 6 weeks, and there was no difference in these antibody titres in vaccine sera compared to convalescent sera (p = 0.44). Vaccinees had significantly less (

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

Immune responses to a single dose of the AZD1222/Covishield vaccine in health care workers

Several COVID-19 vaccines have received emergency approval. Here we assess the immunogenicity of a single dose of the AZD1222 vaccine, at one month, in a cohort of health care workers (HCWs) (629 naïve and 26 previously infected). 93.4% of naïve HCWs seroconverted, irrespective of age and gender. Haemagglutination test for antibodies to the receptor binding domain (RBD), surrogate neutralization assay (sVNT) and ex vivo IFNγ ELISpot assays were carried out in a sub-cohort. ACE2 blocking antibodies (measured by sVNT) were detected in 67/69 (97.1%) of naïve HCWs. Antibody levels to the RBD of the wild-type virus were higher than to RBD of B.1.1.7, and titres to B.1.351 were very low. Ex vivo T cell responses were observed in 30.8% to 61.7% in naïve HCWs. Previously infected HCWs, developed significantly higher (p