Genomic variants in SARS-CoV-2 isolates from India and its impact on diagnostic assays - what we need to know

Coronavirus Disease 19 (COVID-19) originated from Wuhan in China and rapidly emerged as a global pandemic affecting millions of people worldwide. It is estimated that about 80% of individuals affected by COVID-19 are asymptomatic or have mild symptoms but act as a carrier and spread it to the other individuals[1]. This has necessitated the screening of the individuals even with low viral load with high sensitivity and specificity. Reverse transcription polymerase chain reaction (RT-PCR) has been considered as a gold standard and widely used for diagnosis and screening of COVID-19 patients [2,3]. This assay amplifies SARS-CoV-2 genomic region with the help of the complementary oligonucleotide primers whose readout is examined through specific fluorescence-labeled probes. 


Like all viruses, SARS-CoV-2 has been continuously evolving through accumulation of genetic mutations[4]. Our previous analysis has estimated the mutation rate around 1.64 x 10-3 variants per site per year [5]. While these variants could mean different things depending on where they occur, variations occurring at the primer or probe binding sites on the viral genome could potentially impact the efficiency of the test/assay[6]. Additionally analysis of the genome and genomic variants could also provide insights into developing newer primers & probes in regions relatively devoid of variants. 


In a recent study [7], the authors have analysed variants from 717 high quality SARS-CoV-2 genomes from India and deposited from multiple laboratories in the GISAID database (https://www.gisaid.org/) [8]. Through a crowdsourcing approach involving a number of students, we could catalogue a total of 132 primer or probe sequences that were widely used for RT-PCR testing. The SARS-CoV-2 genomic variants were mapped on these 132 primer or probe sequences. The mapping variants were further evaluated to understand the potential impact of the variants on the RT-PCR efficiency by calculating its melting temperature (Tm) and Gibbs free energy. 


In the analysis, a total of 125 unique genetic variants were found mapped to 80 primer or probe sequences. Out of which 13 variants had frequency ≥ 1% in Indian isolated SARS-CoV-2 genomes. A total of 15 primer/probes had variants encompassing ≥1% of genomes from India. One of these primers include the sequence “GTGARATGGTCATGTGTGGCGG” which had variants in > 3% of genomes in India and predicted to have an impact on the efficiency of the RT-PCR tests. This primer is part of the assay which is recommended by national agencies for use in India 

These findings could provide insights towards evidence based approvals of diagnostic assays. For example, regulatory agencies could monitor the frequency of the variants at the primer or probe sites that impacts the efficiency of the assays. Resources like IndiCoV (http://clingen.igib.res.in/indicov/) which systematically compiles genetic variants and annotations in Indian SARS-CoV-2 isolates could potentially aid this.

The findings could also potentially mean a lot to clinicians. In clinical settings, where a false negative RT-PCR test is suspected, revalidation on an alternate test primer/probe set could provide a solution. Alternatively more sensitive assays like next-generation sequencing could be used. Thirdly, this opens up a possibility to design better diagnostic assays targeting regions in the genome which are unlikely to have frequency variants opening up a new opportunity 

It is also imperative to note that more specific assays for detection of SARS-CoV-2 are in the anvil, including sequencing based approaches like COVIDseq which has recently been implemented in clinical samples by the CSIR Institute of Genomics and Integrative Biology with  an increase in diagnostic yield of between 8 and 10%  compared to PCR . While not recommended for general surveillance, these assays could be used to offer confirmation in cases of inconclusive results, could be used in surveillance of individuals who are at high risk of transmitting to a large number of people, like clinicians, public-facing personnel like police etc who if tested false negative could potentially infect large number of people, and also could be used in people who for example take to air-travel and are likely to remain in a confined space and therefore likely to infect a large number of people. 

References


2. Shen, M. et al. Recent advances and perspectives of nucleic acid detection for coronavirus. J Pharm Anal (2020) doi:10.1016/j.jpha.2020.02.010.

3. Noh, J. Y. et al. Correction to: Simultaneous detection of severe acute respiratory syndrome, Middle East respiratory syndrome, and related bat coronaviruses by real-time reverse transcription PCR. Arch. Virol. 163, 819 (2018).

4. Li, X. et al. Evolutionary history, potential intermediate animal host, and cross-species analyses of SARS-CoV-2. J. Med. Virol. 92, 602–611 (2020).

Please note that this article is a preprint and currently under peer review

6. Yang, J.-R. et al. Newly emerging mutations in the matrix genes of the human influenza A(H1N1)pdm09 and A(H3N2) viruses reduce the detection sensitivity of real-time reverse transcription-PCR. J. Clin. Microbiol. 52, 76–82 (2014).

Please note that this article is a preprint and currently under peer review

8. Shu, Y. & McCauley, J. GISAID: Global initiative on sharing all influenza data - from vision to reality. Euro Surveill. 22, (2017).


All opinions expressed are personal and do not reflect the opinion of their employers or organisations associated.

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