by Biotech Express Bureau | Updated on April 20, 2020
Current molecular methods to detect infections of the novel coronavirus rely on identifying particular genetic sequences (Nucleic acid amplification tests) of SARS-CoV2, but new assays are being developed to meet the growing demand for rapid answers.
The quick sequencing of the SARS-CoV-2 genome and distribution of the data early on in the COVID-19 outbreak has enabled the development of a variety of assays to diagnose patients based on snippets of the virus’s genetic code. But as the number of potential cases increases, and concerns rise about the possibility of a global pandemic, the pressure is on to enable even faster, more-accessible testing.
The full genome of the novel coronavirus was published on January 10 of this year, just two weeks after the disease was first identified in Wuhan, China. A week later, a group of researchers led by German scientists released the first diagnostic protocol for COVID-19 using swabbed samples from a patient’s nose and throat; this PCR-based protocol has since been selected by the World Health Organization (WHO). This test detects the presence of SARS-CoV-2’s E gene, which codes for the envelope that surrounds the viral shell, and the gene for the enzyme RNA-dependent RNA polymerase, although to prevent false positives lab technicians may also sequence full genome of all samples. This method of RT-PCR takes 24–48 hours to get a result.
The US CDC has developed an assay that looks for three sequences in the N gene, which codes for the nucleocapsid phosphoprotein found in the virus’s shell, also known as the capsid. The assay also contains primers for the RNA-dependent RNA polymerase gene. The kit contains PCR primer–probe sets for 2 regions of the viral nucleocapsid gene (N1 and N2), and for the human RNase P gene to ensure the RNA extraction was successful.
RT-PCR is gold standard but there are now numerous scientists are taking a different approach for the development of commercial test kits in response to the rising diagnostic demands of the epidemic. For instance, the researchers are using CRISPR technology to tag the target SARS-CoV-2 sequences with a fluorescent probe.
Another CRISPR-based diagnostic protocol developed by researchers at the McGovern Institute at MIT uses paper strips to detect the presence of a target virus, and claims to take around one hour to deliver the result. The test still needs to be developed and validated for clinical use, for COVID-19 or any other viral disease.
A different diagnostics approach is through serology i.e. blood tests for antibodies detect IgM, IgA, IgG, or total antibodies against the SARS-CoV-2 virus. The test has major drawback because peak Abs generation takes around 21 days, as a result of this natural delay, antibody testing is not useful in the setting of an acute illness.
Radiographic Tests: Many centers have evaluated the utility of chest imaging for diagnosis. On chest radiography, bilateral pneumonia is the most frequently reported feature.
The most common laboratory biomarkers reported in patients with COVID-19 include decreased albumin (75.8% [95% CI, 30.5% to 100%]), elevated C-reactive protein (58.3% [CI, 21.8% to 94.7%]), and elevated lactate dehydrogenase levels (57.0% [CI, 38.0% to 76.0%]), and lymphopenia (43.1% [CI, 18.9% to 67.3%]).
Other biomarkers that have been reported include increased erythrocyte sedimentation rates; elevated aspartate aminotransferase, alanine aminotransferase, and creatinine kinase levels; leukopenia; leukocytosis; and increased bilirubin and creatinine levels. Such findings are not surprising, because these biomarkers represent an inflammatory host response to SARS–CoV-2 or are early markers of end-organ dysfunction, similar to that seen in patients with sepsis.
No biomarker or combination of biomarkers currently exists that is sensitive or specific enough to establish a diagnosis of COVID-19
How India is doing CoV diagnostic testing
India contains world’s second largest population where most of the people lives in congested atmosphere and thus chance of infection is more among them. The decision of early lockdown has proved useful in this way but still diagnosis of this large population has become a hercules task for the govt. officials.
Diagnosis depend upon population, the more any country have the more number of tests require to rule out the possibility of infection.
“We should be not be panicking, we should be preparing,” Luv Agarwal joint secretary in the Indian health ministry said.”We have followed a pro-active policy from the beginning. Our focus has been on surveillance and contact tracing. Instead of the virus chasing us, we have been chasing the virus.”
India’s top medical body, the Indian Council of Medical Research (ICMR), has granted approvals for two types of testing kits. One is the real-time polymerase chain reaction (RT-PCR) test, which takes six to eight hours for results to be processed. The second type of test is a rapid antibody test, which gives results in 30 minutes. Though it is not conclusive, ICMR has said that it could be used for cluster-based testing in government-designated hotspots.
The Indian Council of Medical Research on 27th March floated a guidance for rapid testing kits which are based on blood, serum or plasma, and can generate results for exposure to the novel Coronavirus, or SARS-CoV2, within 30 minutes. As per the guidance document, while the kits can indicate exposure to the virus it cannot test for the infection. “Positive test indicates exposure to SARS-CoV-2. Negative test does not rule out COVID-19 infection,” the government’s nodal bio-research agency said. This is because ‘Rapid testing kits’ are considered less reliable compared to real-time polymerase chain reaction (RT-PCR), bit help in giving quick result for positive cases. Both tests usually come positive after 7-10 days of infection.
The authority had listed 12 approved rapid testing kits, with 11 having certification from the European regulator. The test kits certified by the European regulator include those by one Singapore-based firm Sensing Self Ltd, two US-based firms BioMedomics and CTK Biotech Inc, and at least five China-based firms like Getein Biotech, Hangzhou Biotest Biotech Co Ltd and Beijing Tigsun Diagnostics Co Ltd. Another Chinese firm Wondfo got its test kit validated by National Institute of Virology in Pune.
India is likely to start manufacturing new serological diagnostic testing kits within the next two months as many advances have been made after the virus was isolated by research agencies, the Indian Council of Medical Research (ICMR) said on April 1st.
Meanwhile, to take speedy decisions on R&D for SARS-CoV2 and Covid-19, the government formed a science and technology committee co-chaired by V K Paul and principal scientific adviser K Vijayraghavan. The committee will work in coordination with Council of Scientific and Industrial Research (CSIR), department of biotechnology(DBT), department of science and technology (DST) and other science agencies, scientists, industries and regulatory bodies for taking quick decisions on solutions to the disease.
“We are hopeful that at least a serological diagnostic testing will be made in India in next one to two months,” Dr Gangakhedkar said. Serological tests are tests that look for antibodies in blood and help in diagnosing diseases.
In India, testing for COVID-19 is being done by a range of accredited laboratories across the country. Medsource Ozone is gearing up to roll out 300,000 tests a week. It will have the first set of kits in two weeks. It is the only Indian company to have received approvals for both type of test kits—RT-PCR-based and the antibody tests. The 17-year-old biotech company makes a range of test kits, for everything from malaria to HIV. Its test costs slightly more than competitors at 1,600 rupees ($21) a pop.
According to a speech by PM Modi India has more than 600 Diagnostic centres across India.
Mylab Discovery, in the western city of Pune, became the first Indian firm to get full approval to make and sell testing kits. It shipped the first batch of 150 to diagnostic labs in Pune, Mumbai, Delhi, Goa and Bengaluru (Bangalore) this week. The molecular diagnostic company, which also makes testing kits for HIV and Hepatitis B and C, and other diseases, says it can supply up to 100,000 Covid-19 testing kits a week and can produce up to 200,000 if needed. Each Mylab kit can test 100 samples and costs 1,200 rupees ($16; £13) – that’s about a quarter of the 4,500 rupees that India pays to import Covid-19 testing kits from abroad.
Ms Bhosale, who headed the team that designed the coronavirus testing kit called Patho Detect, said it was done “in record time” – six weeks instead of three or four months. She submitted the kit for evaluation by the National Institute of Virology (NIV) on 18 March.
Before submitting the kits for evaluation, the team had to check and re-check all the parameters to ensure its results that were precise, and accurate. “If you carry out 10 tests on the same sample, all 10 results should be same,” said Ms Bhosale. “And we achieved that. Our kit was perfect.” The government-run Indian Council for Medical Research (ICMR), under which NIV operates, agreed. It said Mylab was the only Indian company to achieve 100% results.
In the past few days, India has scaled up testing. Initially, only the state labs were allowed to test for coronavirus, but permission has now been extended to several private labs too. And on 28th March, India also gave approvals to 15 private companies to commercially sell diagnostic kits based on licences they have obtained in the US, European Union and some other countries.
According to update on ICMR website, Total Operational (initiated independent testing) Government Laboratories reporting to ICMR was 197 including 3 collection sites. 82 Private Laboratories were designated to test COVID-19 and 6 new COVID-19 Govt. & Private testing laboratories were approved as per request of the State Govt.
In India, National Institute of Virology, ICMR, Pune has been assigned as WHO reference laboratories providing confirmatory testing for COVID-19. As per recent update on 18 April 2020, a total of 3,72,123 samples from 3,54,969 individuals have been tested and 16,365 individuals have been confirmed positive among suspected cases and contacts of
References:
1.https://www.fda.gov/medical-devices/emergency-situations-medical-devices/faqs-diagnostic-testing-sars-cov-2
2.https://mbio.asm.org/content/11/2/e00722-20
3.https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(20)30788-1/fulltext
4.https://annals.org/aim/fullarticle/2764737/diagnostic-testing-severe-acute-respiratory-syndrome-related-coronavirus-2-narrative
5.https://www.who.int/docs/default-source/coronaviruse/who-reference-laboratories-providing-confirmatory-testing-for-covid-19.pdf?sfvrsn=a03a01e6_6
6.https://icmr.nic.in/content/covid-19
7.https://icmr.nic.in/sites/default/files/upload_documents/Govt_Labs_18042020.pdf
