Selection of Ideal Reference Genes for Gene Expression Analysis in COVID-19 and Mucormycosis.

Selection of reference genes during real-time quantitative PCR (qRT-PCR) is critical to determine accurate and reliable mRNA expression. Nonetheless, not a single study has investigated the expression stability of candidate reference genes to determine their suitability as internal controls in SARS-CoV-2 infection or COVID-19-associated mucormycosis (CAM). Using qRT-PCR, we determined expression stability of the nine most commonly used housekeeping genes, namely, TATA-box binding protein (TBP), cyclophilin (CypA), ß-2-microglobulin (B2M), 18S rRNA (18S), peroxisome proliferator-activated receptor gamma (PPARG) coactivator 1 alpha (PGC-1α), glucuronidase beta (GUSB), hypoxanthine phosphoribosyltransferase 1 (HPRT-1), ß-ACTIN, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in patients with COVID-19 of various severities (asymptomatic, mild, moderate, and severe) and those with CAM. We used statistical algorithms (delta-CT [threshold cycle], NormFinder, BestKeeper, GeNorm, and RefFinder) to select the most appropriate reference gene and observed that clinical severity profoundly influences expression stability of reference genes. CypA demonstrated the most consistent expression irrespective of disease severity and emerged as the most suitable reference gene in COVID-19 and CAM. Incidentally, GAPDH, the most commonly used reference gene, showed the maximum variations in expression and emerged as the least suitable. Next, we determined expression of nuclear factor erythroid 2-related factor 2 (NRF2), interleukin-6 (IL-6), and IL-15 using CypA and GAPDH as internal controls and show that CypA-normalized expression matches well with the RNA sequencing-based expression of these genes. Further, IL-6 expression correlated well with the plasma levels of IL-6 and C-reactive protein, a marker of inflammation. In conclusion, GAPDH emerged as the least suitable and CypA as the most suitable reference gene in COVID-19 and CAM. The results highlight the expression variability of housekeeping genes due to disease severity and provide a strong rationale for identification of appropriate reference genes in other chronic conditions as well. IMPORTANCE Gene expression studies are critical to develop new diagnostics, therapeutics, and prognostic modalities. However, accurate determination of expression requires data normalization with a reference gene, whose expression does not vary across different disease stages. Misidentification of a reference gene can produce inaccurate results. Unfortunately, despite the global impact of COVID-19 and an urgent unmet need for better treatment, not a single study has investigated the expression stability of housekeeping genes across the disease spectrum to determine their suitability as internal controls. Our study identifies CypA and then TBP as the two most suitable reference genes for COVID-19 and CAM. Further, GAPDH, the most commonly used reference gene in COVID-19 studies, turned out to be the least suitable. This work fills an important gap in the field and promises to facilitate determination of an accurate expression of genes to catalyze development of novel molecular diagnostics and therapeutics for improved patient care.

A Cold Chain-Independent Specimen Collection and Transport Medium Improves Diagnostic Sensitivity and Minimizes Biosafety Challenges of COVID-19 Molecular Diagnosis.

Equitable and timely access to COVID-19-related care has emerged as a major challenge, especially in developing and low-income countries. In India, ∼65% of the population lives in villages where infrastructural constraints limit the access to molecular diagnostics of COVID-19 infection. Especially, the requirement of a cold chain transport for sustained sample integrity and associated biosafety challenges pose major bottlenecks to the equitable access. Here, we developed an innovative clinical specimen collection medium, named SupraSens microbial transport medium (SSTM). SSTM allowed a cold chain-independent transport at a wide temperature range (15°C to 40°C) and directly inactivated SARS-CoV-2 (<15 min). Evaluation of SSTM compared to commercial viral transport medium (VTM) in field studies (n = 181 patients) highlighted that, for the samples from same patients, SSTM could capture more symptomatic (∼26.67%, 4/15) and asymptomatic (52.63%, 10/19) COVID-19 patients. Compared to VTM, SSTM yielded significantly lower quantitative PCR (qPCR) threshold cycle (Ct) values (mean ΔCt > -3.50), thereby improving diagnostic sensitivity of SSTM (18.79% [34/181]) versus that of VTM (11.05% [20/181]). Overall, SSTM had detection of COVID-19 patients 70% higher than that of VTM. Since the logistical and infrastructural constraints are not unique to India, our study highlights the invaluable global utility of SSTM as a key to accurately identify those infected and control COVID-19 transmission. Taken together, our data provide a strong justification to the adoption of SSTM for sample collection and transport during the pandemic. IMPORTANCE Approximately forty-four percent of the global population lives in villages, including 59% in Africa (https://unhabitat.org/World%20Cities%20Report%202020). The fast-evolving nature of SARS-CoV-2 and its extremely contagious nature warrant early and accurate COVID-19 diagnostics across rural and urban population as a key to prevent viral transmission. Unfortunately, lack of adequate infrastructure, including the availability of biosafety-compliant facilities and an end-to-end cold chain availability for COVID-19 molecular diagnosis, limits the accessibility of testing in these countries. Here, we fulfill this urgent unmet need by developing a sample collection and transport medium, SSTM, that does not require cold chain, neutralizes the virus quickly, and maintains the sample integrity at broad temperature range without compromising sensitivity. Further, we observed that use of SSTM in field studies during pandemic improved the diagnostic sensitivity, thereby establishing the feasibility of molecular testing even in the infrastructural constraints of remote, hilly, or rural communities in India and elsewhere.

Exploring salivary diagnostics in COVID-19: a scoping review and research suggestions.

INTRODUCTION: Molecular diagnostics for SARS-CoV-2 infection characteristically involves the sampling of the throat or nasopharyngeal swab (NPS). However, these procedures are invasive, require necessary skills for sample collection, cause patient discomfort, and are non-conducive for extensive scale testing. Saliva is increasingly being suggested as an alternate diagnostic sample in SARS-CoV-2 infection. OBJECTIVES: This scoping review was done with the objective of exploring the evidence on the role of saliva as an alternate diagnostic sample in SARS-CoV-2 condition. METHODS: Thorough search of the literature in major databases was undertaken in June 2020 using free text and MESH terms, followed by PRISMA to identify 17 studies for data extraction. RESULTS AND CONCLUSIONS: Evidence was summarised for study characteristics, salivary sampling characteristics, viral load, and longevity of virus in saliva. The literature supports that saliva offers a simple sample collection method compared to technique-sensitive NPS and has the advantage of point-of-care testing for initial screening in community or hospital-based set-up. The additional highlights of this review are heterogeneity in the current literature and the gaps in methodology. Therefore, a robust study design to generate higher levels of evidence has been proposed.

Development of a highly effective low-cost vaporized hydrogen peroxide-based method for disinfection of personal protective equipment for their selective reuse during pandemics.

BACKGROUND: Personal Protective Equipment (PPE) is required to safely work with biological agents of bacterial (i.e. Mycobacterium tuberculosis) or viral origin (Ebola and SARS). COVID-19 pandemic especially has created unforeseen public health challenges including a global shortage of PPE needed for the safety of health care workers (HCWs). Although sufficient stocks of PPE are currently available, their critical shortage may develop soon due to increase in demand and depletion of existing supply lines. To empower our HCWs and ensure their continued protection, proactive measures are urgently required to develop procedures to safely decontaminate the PPEs to allow their "selective reuse" during contingency situations. METHODS: Herein, we have successfully developed a decontamination method based on vaporized hydrogen peroxide (VHP). We have used a range of concentration of hydrogen peroxide to disinfect PPE (coveralls, face-shields, and N-95 masks). To ensure a proper disinfection, we have evaluated three biological indicators namely Escherichia coli, Mycobacterium smegmatis and spores of Bacillus stearothermophilus, considered as the gold standard for disinfection processes. We next evaluated the impact of repeated VHP treatment on physical features, permeability, and fabric integrity of coveralls and N-95 masks. Next, we performed Scanning Electron Microscopy (SEM) to evaluate microscopic changes in fiber thickness of N-95 masks, melt blown layer or coverall body suits. Considering the fact that any disinfection procedure should be able to meet local requirements, our study included various regionally procured N-95 masks and coveralls available at our institute All India Institute of Medical Sciences (AIIMS), New Delhi, India. Lastly, the practical utility of VHP method developed herein was ascertained by operationalizing a dedicated research facility disinfecting used PPE during COVID-19. RESULTS: Our prototype studies show that a single VHP cycle (7-8% Hydrogen peroxide) could disinfect PPE and PPE housing room of about 1200 cubic feet (length10 ft × breadth 10 ft × height 12 ft) in less than 10 min, as noted by a complete loss of B. stearothermophilus spore revival. The results are consistent and reproducible as tested in over 10 cycles in our settings. Further, repeated VHP treatment did not result in any physical tear, deformity or other appreciable change in the coverall and N-95 masks. Our permeation tests evaluating droplet penetration did not reveal any change in permeability post-VHP treatments. Also, SEM analysis indeed revealed no significant change in fiber thickness or damage to fibers of coveralls or melt blown layer of N-95 masks essential for filtration. There was no change in user comfort and experience following VHP treatment of PPE. Based on results of these studies, and parameters developed and optimized, an institutional research facility to disinfect COVID-19 PPE is successfully established and operationalized with more than 80% recovery rate for used PPE post-disinfection. CONCLUSIONS: Our study, therefore, successfully establishes the utility of VHP to effectively disinfect PPE for a possible reuse as per the requirements. VHP treatment did not damage coveralls, cause physical deformity and also did not alter fabric architecture of melt blown layer. We observed that disinfection process was successful consistently and therefore believe that the VHP-based decontamination model will have a universal applicability and utility. This process can be easily and economically scaled up and can be instrumental in easing global PPE shortages in any biosafety facility or in health care settings during pandemic situation such as COVID-19.