Capping the price of coronavirus tests at ₹800 has forced some private labs to use inexpensive and less sensitive kits with lower limit of detection to keep the cost of testing low, which raises the risk of false negatives and compromises data coming out of India, say experts. The issue has come into focus now because it has emerged that RT-PCR tests of one kind can be used as a proxy for identifying the new variant of the Sars-CoV-2 virus, which is raging though the UK, and has panicked the rest of the world.
The ORF-1a, RdRp, E, N, and S genes are most frequently targeted for Sars-CoV-2 detection by reverse transcription-polymerase chain reaction (RT-PCR) testing, which is the standard test for Covid-19 diagnosis in India. The test that can successfully identify the variant is a three-gene test, where one of the genes doesn’t show up in the result because of a mutation in the virus.
“The four coronavirus-specific genes include a screening gene for (E) protein, which is found in all bat coronaviruses, and four confirmatory genes specific to Sars-CoV2, which include ORF-1a, RdRp, nucleocapsid (N), and spike (S) proteins. Various test kits approved by Indian Council of Medical Research (ICMR) test for the E protein, along with one or more confirmatory genes to confirm Covid-19 diagnosis. While we use three-gene PCR test kits, a few labs use two-gene kits,” said Dr Navin Dang, consultant microbiologist and founder of Dr Dang’s Labsin New Delhi.
The new UK variant Sars-CoV-2 VOC 202012/01 has a mutation in the receptor binding domain (RBD) of the spike protein (S) at position 501, where amino acid asparagine (N) has been replaced with tyrosine (Y). This variant carries many other mutations, including a double deletion at positions 69 and 70.
“With standard kits, testing for two genes is usually enough. Sequencing, however, is most conclusive,” said Dr Anurag Agrawal, director, Council for Scientific and Industrial Research-Institute of Genomics and Integrative BioIogy (CSIR-IGIB). His reference is to the fact that sequencing the viral genome is the surest way to identify the variant.
“Most labs use three gene test in India that test for E, RdRp and N gene, but with mutations found in the S protein in Sars-CoV2 in the UK, RT-PCR tests that rely on the S gene alone for confirmatory diagnosis can fail. All kits testing for other confirmatory genes will still give accurate results, but kits that test only for E and S genes will need to be reconfirmed against a kit testing for other confirmatory genes,” said Dr Vidur Mahajan, head of research at the Delhi-based Centre for Advanced Research in Imaging, Neurosciences and Genomics (Caring), which collaborated with CSIR-IGIB for the world’s first successful run of Covid-Seq, a sequencing-based test for Covid-19 in India that can also diagnose the UK strain.
Beyond RT-PCR testing
A positive RT-PCR test for Covid-19 has more weight than a negative test because of the test’s high specificity but moderate sensitivity of around 70%.
“Unfortunately, with the price capping, some very substandard kits that cost as little as ₹100 are available, which can give misleading results,” said Dang.
And, of course, many of these tests do not check for three genes.
On March 21, 2020, ministry of health and family welfare capped for RT-PCR Covid-19 tests at ₹4,500 per test, which Delhi government lowered to ₹2,400 per test on June 18 for samples collected by government, on November 11, lowered it further to ₹800 per test.
Karnataka, which allow differential pricing depending on the type test being used, began using the Abbott ID Now express RT-PCR method, which gives positive in less than five minutes and negative within 13 minutes at Bengaluru’s Kempegowda International Airport (KIA).
The RT-PCR Express test will cost ₹2,400, compared to the commonly used open-system RT-PCR test that cost ₹800 and gives results in around six hours.
Karnataka also has differential pricing for samples collected by Government and sent to private labs and those collected by private labs themselves at their collection centres.
In Delhi, prices for home collection are ₹1,200 at home collection compared to ₹800 at labs.
Scaling up sequencing
“While there is obvious concern over a mutated strain that spreads faster at a time when things are looking better, there is no reason to be afraid. As far as we know, it (the infection caused by the new strain) is not more severe. In some ways, the situation is similar to when D 614G mutation first spread. Right now, with high community spread followed by recovery, the only thing to be concerned about is whether it will escape immunity. At this point, there is no reason to believe that, so I would advise caution but not worry,” said Dr Agrawal.
What we need is more sequencing, he said. “We need to sequence at scale to truly know that it (the new strain) has not entered India. In any case, it may appear on its own as well. IGIB had identified this site earlier for likely mutation,” said Agrawal.
Tony Cox, CEO, UK’s National Biosample Centre and Milton Keynes “Lighthouse” Coronavirus Testing Mega-Lab tweeted recently: “MK LHL testing data showing increasing prevalence of H69/V70 variant in positive test data — which is detected incidentally by the commonly used 3-gene PCR test. Numerous other mutations are circulating but are invisible to this PCR test. So genome sequencing of viral samples is very important.”
India has sequenced around 5,000 Sars-CoV2 genomes since March, according to Agrawal, and shared at least 4,338 in the public domain for analysis.
India has also stepped up sequencing following the possibility of the faster-spreading UK variant reaching India.
Over the past 24 hours, the government has stepped up surveillance and reached out to public and private labs for samples of extracted RNA of patients who tested positive for Covid-19 and have a travel history to the UK, for sequencing to identify if the UK variant was already present in India.Delhi government has asked all labs to store and not discard all positive samples collected since November 25.
“The government has stepped up analysis of past positive cases who have had a history of travel to UK for genomic sequencing since December 18. Today, we have collected 70 samples for people who have arrived from the UK on Wednesday,” said Dr Mahajan.