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Molecular Diagnostics Technologies for Covid-19: Limitations and Challenges

Dr Shivaji K

This pandemic has drawn several parallels to Spanish Flu but the former could be characterized with the progress of molecular technology, although there remain several challenges to overcome for swift containment of the novel coronavirus, writes Dr Shivaji K Jadhav, Head of Molecular Biology (Covid Laboratory) at SRL Diagnostics, Fortis Hospital, Bengaluru.

As we all know that in December 2019, the health officials in Wuhan, reported a disease outbreak involving a cluster of unknown type cases of pneumonia of unknown cause, type and unidentified, Since then, coronavirus disease (COVID-19 or SARS-CoV-2) outbreak has been characterized as a pandemic, spread to 188 countries worldwide, It is estimated globally, as of February 14, 2021, there have been 108,153,741 confirmed cases of Covid-19, including, 2,381,295 deaths, reported to WHO.

The pandemic has proven to be a significant challenge to our ability to reduce the global spread of the SARS-CoV-2. Given the global scale of infections due to the novel virus and the lack of approved therapeutics and vaccines, the Covid-19 pandemic has drawn comparisons to the deadly 1918 Spanish Flu pandemic. A key difference is current advances in molecular diagnostic technology that has enabled us to rapidly characterize the novel virus, identify infectious (including asymptomatic) patients, and potentially isolate them to control the disease spread.

However, initial delays in assay design and supply chain bottlenecks prevented the deployment of accurate diagnostic tests at scale globally. This was found to be a critical gap in arresting the spread of this devastating disease worldwide. The unprecedented global pandemic known as SARS-CoV-2 has exercised to its limits nearly all aspects of modern viral diagnostics. In doing so, it has illuminated both the advantages and limitations of current technologies. Tremendous effort has been put forth to expand our capacity to diagnose this deadly virus.

Current methods for SARS-CoV-2 diagnostic testing include nucleic acid amplification, CBNAAT, CRISPR, Genome sequencing, antigen and antibody-based detection methods. Additionally, it is important to include analysis of equally critical aspects of Covid-19 diagnostics, including sample collection and preparation, transport medium, testing models, and commercial response. The overall emphasize the integrated nature of assays, wherein issues in sample collection procedures and expertise to collect the swab and preparation of the sample for processing could impact the overall performance in a clinical setting.

Molecular diagnosis of Covid-19 is primarily relying on the detection of RNA of the SARS-CoV-2 virus, the causative infectious agent of the pandemic. Reverse transcription polymerase chain reaction (RTPCR) enables sensitive detection of specific sequences of genes that encode the RNA dependent RNA polymerase (RdRP), nucleocapsid (N), envelope (E), and spike (S) proteins of the virus. Although RT-PCR tests have been widely used and many alternative assays have been developed, the current testing capacity and availability cannot meet the unprecedented global demands for rapid, reliable, and widely accessible molecular diagnosis. Challenges remain throughout the entire analytical process, from the collection and treatment of specimens to the amplification and detection of viral RNA and the validation of clinical sensitivity and specificity.

We highlight the main issues surrounding the molecular diagnosis of Covid-19, including false negatives from the detection of viral RNA, temporal variations of viral loads, selection and treatment of specimens, and limiting factors in detecting viral proteins. Real-time RT-PCR is the major workhorse in the field of molecular diagnostics. It has been extensively used for high-throughput screening and early diagnosis of Covid-19, and other infectious diseases in the past. RT-PCR can amplify and detect a single copy of the specific genomic sequence and therefore, it is extremely sensitive. Furthermore, real-time RT-PCT is a quantitative technique as the number of copies of RNA generated in a PCR increases exponentially and is proportional to the amount of starting material, i.e. viral load. At the moment, a vast majority of the commercially available tests for early diagnosis of Covid-19 are based on real-time RT-PCR assays.

The RT-PCR is the most widely used method for the detection of viral pathogens, including the SARS-CoV-2 virus. However, given the current challenges with RT-PCR, alternative techniques involving CRISPR-Cas and isothermal amplification are being explored. For instance, there are severe limitations associated with the availability, costs, and the need for trained personnel to run RT-PCR tests. Diagnostic capabilities with lower cost, faster turnaround times, and portability are critical, given the global scope and magnitude of the pandemic. There is a critical research need such as improvements in RTPCR, development of alternative nucleic acid amplification techniques, incorporating CRISPR technology for point-of-care (POC) applications, validation of POC tests, and sequencing of viral RNA and its mutations. Further advances in analytical technology and research through multidisciplinary collaboration will contribute to the development of mitigation strategies, therapeutics, and vaccines. Lessons learned from molecular diagnosis of Covid-19 are valuable for better preparedness in response to other infectious diseases.

CBNAAT assays played an important role in the diagnosis of Covid-19. The limitation or concerns associated with assay includes high Ct values with false positivity. This is very crucial in the interpretation of clinical cases and emergency cases which are on an urgent basis due to operation or critical surgeries. We have also observed some of the lytic transport medium is not suitable for the CBNAAT assay and it will show false positivity.

In general performance of commercial and pre-commercial molecular diagnostics tests depends on clinical performance in terms of time-to-result, clinical sensitivity, clinical specificity, and limit of detection (LoD). The most important criteria to diagnose Covid-19 accurately at an early stage of infection and to avoid false negative results. Understandably, during a pandemic of this scale, the standards for authorization of these diagnostics are relaxed to afford the availability of test kits and large-scale screening of the samples. Also, due to the lack of time to pre-screen and test a significant number of samples, it is difficult to accumulate reliable performance data. Thus, 100% clinical sensitivity was reported by several manufacturers, while others directly submitted kits for evaluation without an appropriate number of clinical tests. The tested sensitivity values for these diagnostics are satisfactory in most cases due to the inherent sensitivity of the real-time RTPCR method. However, sensitivity values drop well below 95% in some cases, which may be attributed to the deficiencies in the test kits or sampling and handling errors.

Many biomedical companies are rising to the challenge and developing COVID-19 diagnostics. In the last few months, some of these diagnostics have become commercially available for healthcare workers and clinical laboratories. However, diagnostic technologies have specific limitations and reported several false-positive and false-negative cases, especially during the early stages of infection. We need to strengthen future research and development challenges to enable timely, rapid, low-cost, and accurate diagnosis of emerging infectious diseases.

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