COVID-19: An insight into the developments in diagnostics and therapeutics in India

Diagnostic tests development in India

COVID KAVACH ELISA was developed by National Institute of Virology, Pune, India, by isolating virus from tested positive patients. The kit is manufactured by Zydus- Cadila which is the leading global pharmaceutical company in India. The Indian Council of Medical Research (ICMR) has approved this kit. The kit is reported to have 98.7% sensitivity and 100% specificity. As per the ICMR officials, the kit will be used in a national wide serosurvey conducted by the Health Ministry.^[11] GCC Biotech India Pvt. Ltd. has developed an indigenous low-cost COVID-19 test kit after rigorous clinical trial. It has high accuracy and can deliver results in <90 min. The firm has received the approval of ICMR.^[12] Quanitplus COVID-19 is an ICMR approved testing kit developed by Huwel Lifesciences from Hyderabad. The kit can deliver results within 2 h. It is a single formulation which does not require any addition of separate component while setting up reactions.^[13]

The local companies authorized by the Central Drugs Standard Control Organization to develop diagnostics kits are Mylab, Medsource Ozone Biomedicals, Voxtur Bio, and Alpine Biomedicals.^[14] Apart from these, many start-ups in different parts of the company are developing rapid, accurate, affordable diagnostics kits. Noida-based DNA Xperts have developed RT-PCR which can deliver the results in 58 min, cut the current testing time of 2 to 3 h. This kit is awaiting the ICMR approval.^[15] Sree Chitra Tirunal Institute for Medical Sciences and Technology in Thiruvananthapuram has developed an innovative diagnostic test kit named Chitra Gene LAMP-N for the diagnosis of SARS-CoV-2. This detects the N-Gene of virus using reverse transcriptase loop-mediated amplification of viral nucleic acid or RT- LAMP technique. This kit is considered as one of the first few confirmatory diagnostic tests for N-gene of SARS-CoV-2 virus using the RT-LAMP technique in the world. The test is highly specific that it can detect two regions of the gene which will ensure that the test does not fail even if one region of the viral gene undergoes mutation during its current spread. The detection time is only 10 min. The sample to result time is <2 hours.^[16]

Challenges and future directions

Although there are ample types of diagnostic kits in the market, their affordability and accessibility remain a major challenge, especially in countries like India. A few reasons for this include – non-availability of required reagents, lack of technical expertise to perform the test and operate the equipment, and limited accessibility to research facilities to acquire access to the virus samples. There are also significant constraints in meeting the medical requirements in terms of speed, volume, and cost of the diagnostic tools. One way to address this issue would be to enforce the “ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free and Deliverable to end-users) Criteria” as a benchmark for developing and identifying the most appropriate diagnostic tests for resource-constrained settings.^[17]

Cytokine therapy: Mild-moderate cases

On infection by SARS-CoV2, the innate arm of the immune system is triggered as the first line of defense, and this induces the expression and release of Type I interferons (IFNs) and other pro-inflammatory cytokines.^[19] Among these, IFNs exhibit potent antiviral activity by acting as signaling molecules to activate the required cellular/genetic machinery (e.g., immunomodulatory genes) to result in the production of antiviral proteins, which go to elicit effector functions such as the inhibition of viral replication.^[20]

However, these innate responses are not always successful in eliminating the virus. In such cases, the adaptive arm of the immune system takes over, and this is usually around the 4^th–7^th day post-infection. A successful transition to an adaptive immune response relies on: (a) The presence of required cytokine (IFNs and ILs) cues from innate immune cells and (b) the presence of an adequately large T-cell repertoire.^[21] A compromise in either of these results in immune evasion and associated pathophysiology. SARS- CoV2, through the action of several of its virulence factors, eventually leads to the inhibition of IFN-I production and functioning. Moreover, age-related degeneration of the thymic epithelium results in loss of thymic function due to a reduction in size and diversity of the naïve T-cell repertoire, which leads to weakened immune surveillance mechanisms in the elderly, increasing their susceptibility to and mortality from the infection.^[22] Thus, by stimulating the innate immune responses to trigger IFN production and release at early stages of the disease, we could effectively bridge the gap between the innate and adaptive immunity, and this would significantly improve prognosis and survival. This can be achieved by administration of IFNs.

A team of doctors at HCG, Bengaluru, has developed a novel cytokine cocktail containing INF-gamma (1000– 2000 pg/ml) and interleukin-6 (IL-6) 500–750 pg/ml. We propose that this cocktail, when administered to an infected patient, can result in a surge of cytokines in the body of the infected person and will boost his ability to fight the virus. They are currently evaluating the safety and efficacy of this cytokine cocktail through Phase I clinical trials where 1 ml of the study cytokine preparation will be administered as an intramuscular injection once a day for up to 10 days to patients with mild/moderate disease.

Convalescent plasma (CP) therapy: Severe cases

Severe pneumonia caused by SARS-CoV2 is characterized by rapid viral replication, massive inflammatory cell infiltration, and elevated pro-inflammatory cytokines, cytokine storm culminating in virtually irreversible lung injury.^[23] This severe form of the SARS-CoV2 infection is known to lead to lymphopenia with elevated cytokines, including IL-6, IL-10, TNF-α, and granulocyte-macrophage colony-stimulating factor.

Drawing on current data and literature on the use of CP in SARS-CoV2, it is clear that (a) CP therapy helps reduce mortality in severely ill patients, (b) an increase in neutralizing antibody titers and disappearance of SARS-CoV-2 was observed in almost all the patients after CPT therapy, and (c) there was a clear improvement of clinical symptoms post CP therapy.^[24] Thus, based on available yet limited scientific data, the administration of CP to severely ill-COVID-19 patients appears safe, clinically effective, and reduces mortality. The US-FDA has approved the use of CP in patients with COVID-19 on account of its beneficial effects, and the ICMR has approved several CP therapy trials in India.^[25]

In SARS-CoV2, viremia peaks in the 1^st week of infection. This generates a primary immune response within days which is followed by virus clearance. CP therapy relies on the administration of plasma (rich in specific IgG and IgM anti-SARS-CoV-19 antibodies) isolated from recovered individuals to severely ill patients. This suppresses viremia and blocks new infection by accelerating infected cell clearance.^[24] CP therapy would thus be particularly beneficial to severely ill patients who have not responded to other drugs/treatments, have developed (ARDS), and pose an increased risk organ failure. It might also help keep who are moderately ill patients and individuals comorbid chronic medical conditions, from becoming more ill and experiencing further complications. Current research is also evaluating the potential of CP therapy as a preventive intervention that could be administered to health care workers exposed to someone with COVID-19 to potentially prevent them from getting COVID-19.^[26]

Considering the current research and proven efficacy of CP in the past epidemics, a team of doctors at HCG, Bengaluru, has proposed a clinical trial to use CP therapy in patients diagnosed with COVID-19. Patients who have recovered from SARS-COV2 and who have been discharged from SARS-CoV2 treatment centers or units could be potential donors for CP which would be rich in immunoglobulins against SARS CoV2. The objective of their current study/ clinical trial is to investigate the potential use of CP in patients with coronavirus SARS-CoV-2 infection compared to standard care.

Mesenchymal stem cells: Critical cases

In critically ill COVID-19 patients, a lot of the symptoms are caused due to an overactive innate immune response (e.g., cytokine storms) that comes into play due to a dysfunctional adaptive immune response.^[27] Thus, the immunomodulatory effect of mesenchymal stem cells could potentially be implemented as a therapeutic intervention in critically ill or likely to be critically ill patients to help manage their symptoms and reduce risk of disease progression. There have been a few recent studies that have served to understand the therapeutic role of MSCs in respiratory infections similar to SARS-CoV2 and have displayed promising results.^[28]

Mesenchymal stem cells derived from umbilical cord, dental pulp, bone marrow, etc., have been used for immune modulation in various autoimmune disorders.^[29] The characteristic property of MSCs is that they aid tissue regeneration and repair. On account of low MHC expression, MSCs are a relatively non-immunogenic population of cells, making them ideal for therapeutic interventions using allogenic transfers without HLA matching.^[30] Interestingly, the pro-inflammatory cytokines released during this hyperactive immune response elicit anti-inflammatory effects on the MSCs.^[31] This results in the MSCs secreting several soluble factors such as nitric oxide, transforming growth factor beta, prostaglandin E2, indoleamine 2,3-dioxygenase, HLA-G5, and soluble IL-6, which function to inhibit the proliferation and activation of pro-inflammatory cells such as TH1 and TH17, thereby decreasing the production of pro-inflammatory cytokines such as IFN-gamma and IL- 17. Mesenchymal stem cells also inhibit the activation of cytotoxic T-cells and dendritic cell subsets, thereby reducing direct injury to lung parenchyma by increased regulation of T-Reg activity and production and release of anti-inflammatory cytokines such as IL-10.^[32] The lifespan, potential, and efficacy of MSCs can be controlled by priming the MSCs with IFN-gamma before transfusion.^[33]

Thus, to explore the use of mesenchymal stem cells in the treatment of COVID-positive patients with severe disease, a team of doctors at HCG, Bengaluru, has initiated a clinical trial to transfuse allogeneic mesenchymal stem cells derived from donated bone marrow or umbilical cord administered through intrapulmonary implantation where possible or administered through the intravenous route. To offset the neutralization of MSCs, we have proposed priming of MSC with a cytokine cocktail derived from Th1 cells of healthy donors to suppress the hyperactive immune response and promote tissue repair.