Optimization and validation of RT-LAMP assay for diagnosis of SARS-CoV2 including the globally dominant Delta variant.

BACKGROUND: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19 pandemic, has infected more than 179 million people worldwide. Testing of infected individuals is crucial for identification and isolation, thereby preventing further spread of the disease. Presently, Taqman™ Reverse Transcription Real Time PCR is considered gold standard, and is the most common technique used for molecular testing of COVID-19, though it requires sophisticated equipments, expertise and is also relatively expensive. OBJECTIVE: Development and optimization of an alternate molecular testing method for the diagnosis of COVID-19, through a two step Reverse Transcription Loop-mediated isothermal AMPlification (RT-LAMP). RESULTS: Primers for LAMP were carefully designed for discrimination from other closely related human pathogenic coronaviruses. Care was also taken that primer binding sites are present in conserved regions of SARS-CoV2. Our analysis shows that the primer binding sites are well conserved in all the variants of concern (VOC) and variants of interest (VOI), notified by World Health Organization (WHO). These lineages include B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.427/B.1.429, P.2, B.1.525, P.3, B.1.526 and B.1.617.1. Various DNA polymerases with strand displacement activity were evaluated and conditions were optimized for LAMP amplification and visualization. Different LAMP primer sets were also evaluated using synthetic templates as well as patient samples. CONCLUSION: In a double blind study, the RT-LAMP assay was validated on more than 150 patient samples at two different sites. The RT-LAMP assay appeared to be 89.2% accurate when compared to the Taqman™ rt-RT-PCR assay.

Potential of natural astaxanthin in alleviating the risk of cytokine storm in COVID-19.

Host excessive inflammatory immune response to SARS-CoV-2 infection is thought to underpin the pathogenesis of COVID-19 associated severe pneumonitis and acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). Once an immunological complication like cytokine storm occurs, anti-viral based monotherapy alone is not enough. Additional anti-inflammatory treatment is recommended. It must be noted that anti-inflammatory drugs such as JAK inhibitors, IL-6 inhibitors, TNF-α inhibitors, colchicine, etc., have been either suggested or are under trials for managing cytokine storm in COVID-19 infections. Natural astaxanthin (ASX) has a clinically proven safety profile and has antioxidant, anti-inflammatory, and immunomodulatory properties. There is evidence from preclinical studies that supports its preventive actions against ALI/ARDS. Moreover, ASX has a potent PPARs activity. Therefore, it is plausible to speculate that ASX could be considered as a potential adjunctive supplement. Here, we summarize the mounting evidence where ASX is shown to exert protective effect by regulating the expression of pro-inflammatory factors IL-1ß, IL-6, IL-8 and TNF-α. We present reports where ASX is shown to prevent against oxidative damage and attenuate exacerbation of the inflammatory responses by regulating signaling pathways like NF-ĸB, NLRP3 and JAK/STAT. These evidences provide a rationale for considering natural astaxanthin as a therapeutic agent against inflammatory cytokine storm and associated risks in COVID-19 infection and this suggestion requires further validation with clinical studies.

CRISPR-based assays for rapid detection of SARS-CoV-2.

COVID-19 pandemic posed an unprecedented threat to global public health and economies. There is no effective treatment of the disease, hence, scaling up testing for rapid diagnosis of SARS-CoV-2 infected patients and quarantine them from healthy individuals is one the best strategies to curb the pandemic. Establishing globally accepted easy-to-access diagnostic tests is extremely important to understanding the epidemiology of the present pandemic. While nucleic acid based tests are considered to be more sensitive with respect to serological tests but present gold standard qRT-PCR-based assays possess limitations such as low sample throughput, requirement for sophisticated reagents and instrumentation. To overcome these shortcomings, recent efforts of incorporating LAMP-based isothermal detection, and minimizing the number of reagents required are on rise. CRISPR based novel techniques, when merge with isothermal and allied technologies, promises to provide sensitive and rapid detection of SARS-CoV-2 nucleic acids. Here, we discuss and present compilation of state-of-the-art detection techniques for COVID-19 using CRISPR technology which has tremendous potential to transform diagnostics and epidemiology.

A flow virometry process proposed for detection of SARS-CoV-2 and large-scale screening of COVID-19 cases

The viral pneumonia COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread rapidly over 210 countries and declared as pandemic by WHO. WHO has emphasized on the scale-up of testing capacity, followed by isolation of infected individuals, and contact tracing, as the 'backbone' of managing the pandemic. Globally, the detection of SARS-CoV-2 in patients is done by real-time PCR (RT-PCR) and blood antibody-based testing. Here, a flow cytometry-based high-throughput screening system is proposed for testing of COVID-19 cases where the virus particle binds to specific primary antibodies and the resultant virus-antibody complex then binds to fluorescent-tagged secondary antibodies. The fluorescence signal could be measured in a flow channel for qualitative detection of virus in the test sample.