Integration of RT-LAMP and Microfluidic Technology for Detection of SARS-CoV-2 in Wastewater as an Advanced Point-of-care Platform

Development of lab-on-a-chip (LOC) system based on integration of reverse transcription loop-mediated isothermal amplification (RT-LAMP) and microfluidic technology is expected to speed up SARS-CoV-2 diagnostics allowing early intervention. In the current work, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and RT-LAMP assays were performed on extracted RNA of 7 wastewater samples from COVID-19 hotspots. RT{square}LAMP assay was also performed on wastewater samples without RNA extraction. Current detection of SARS-CoV-2 is mainly by RT-qPCR of ORF (ORF1ab) and N genes so we targeted both to find the best surrogate marker for SARS-CoV-2 detection. We also performed RT-LAMP with/without RNA extraction inside microfluidic device to target both genes. Positivity rates of RT-qPCR and RT-LAMP performed on extracted RNA were 100.0% (7/7) and 85.7% (6/7), respectively. RT-qPCR results revealed that all 7 wastewater samples were positive for N gene (Ct range 37-39), and negative for ORF1ab, suggesting that N gene could be used as a surrogate marker for detection of SARS-CoV-2. RT-LAMP of N and ORF (ORF1a) genes performed on wastewater samples without RNA extraction indicated that all 7 samples remains pink (negative). The color remains pink in all microchannels except microchannels which subjected to RT-LAMP for targeting N region after RNA extraction (yellow color) in 6 out of 7 samples. This study shows that SARS-CoV-2 was successfully detected from wastewater samples using RT-LAMP in microfluidic chips.

An assessment of efficacy of Iodine complex (Renessans) against SARS-CoV-2 in non-human primates (Rhesus macaque)

Renessans is an iodine complex which has proven in vitro antiviral activity including Anti-SARS-CoV-2 activity. The present study was designed to determine its efficacy against SARS-CoV-2 in monkeys (Rhesus macaque). A total of 14 monkeys were divided into four groups: A) Prophylactic group (n=03), (B) Treatment group (n=03), (C) infection control group (n=04) and (D) negative control group (n=04) and were housed in BSL-3 Animal facility while group D was housed at another animal house. Group A was administered with Renessans @ 2.85 mg/7 kg from 5 days prior to the infection to 08 days post infections (DPI). Group B was administered with Renessans from 03-08 DPI @ 2.85 mg/7 kg. Group C was administered with WIF only. The infection @ 2 x 106 TCID of SARS-CoV-2 was given to all group monkeys through intranasal and oral route under anesthesia. Nasal swab samples (at different times) and fecal matter on daily basis were collected for the detection of SARS-CoV-2 through real-time quantitative PCR. Three monkeys (one from each of group A, B and C) were euthanized at 07 DPI to determine the gross pathological lesions and SARS-CoV-2 detection from internal tissues. Nasal swabs from all the monkeys from group A, B and C were positive for SARS-CoV-2 at 02 and 07 DPI (Day 05 of treatment). At 14 DPI, all (100%) nasal swabs from group A were negative for SARS-CoV-2 while 50% and 100% were positive from group B and C, respectively. At 21 DPI, monkeys from group B were negative and all in group C were still positive for SARS-CoV-2. Similarly, fecal matter of monkeys in group A and B was returned negative in significantly lesser time as compared to monkeys from infection control group. Based on these research findings it is concluded that the Renessans has in-vivo SARS-CoV-2 activity and may result in early clearance of SARS-CoV-2. Therefore, a clinical trial of the drug in COVID-19 patients may reveal its anti-COVID-19 potential.

A longitudinal survey for genome-based identification of SARS-CoV-2 in sewage water in selected lockdown areas of Lahore city, Pakistan; a potential approach for future smart lockdown strategy

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections has affected more than 15 million people and, as of 22 July 2019, caused deaths of more than 0.6 million individuals globally. With the excretion of SARS-CoV-2 in the stool of symptomatic and asymptomatic COVID-19 patients, its genome detection in the sewage water can be used as a powerful epidemiological tool to predict the number of positive cases in a population. This study was conducted to detect SARS-CoV-2 genome in sewage water during the lockdown. Sewage samples, from 28 pre-selected sites, were collected on alternate days from 13-25 July, 2020 from two selected areas [Johar Town (n = 05) and Township (n = 23)], where smart lockdown were implemented by the government authorities on 9th July, 2020. Genomic RNA was extracted and the SARS-CoV-2 was detected and quantified using commercially available kit through Real-Time PCR. Out of 28, sixteen samples were positive on day one while 19, 17, 23, 17, 05 and 09 samples were positive on day 3, 5, 7, 9, 11, and 13, respectively. Results revealed a decreased positivity rate and SARS CoV-2 genome copies in sewage towards the end of lockdown however few sampling sites did not follow a clear pattern indicating the complexities in sewage water based surveillance i.e time of sampling etc. Hourly sampling from two sites for 24 hours also revealed the impact of sampling time on detection of SARS-CoV-2 genome in sewage. Results of current study insinuate a possible role of sewage-based COVID-19 surveillance in monitoring and execution of smart lockdowns.

An in vitro assessment of anti-SARS-CoV-2 activity of oral preparations of iodine complexes (RENESSANS)

Since the emergence of CoVID-19 pandemic in China in late 2019, scientists are striving hard to explore non-toxic, viable anti-SARS-CoV-2 compounds or medicines. We determined In Vitro anti-SARS-CoV-2 activity of oral formulations (syrup and capsule) of an Iodine-complex (Renessans). A monolayer of vero cells were exposed to SARS-CoV-2 in the presence and absence of different concentrations (equivalent to 50, 05 and 0.5 g/ml of I2) of Renessans. Anti-SARS-CoV-2 activity of each of the formulation was assessed in the form of cell survival, SARS-CoV-2-specific cytopathic effect (CPE) and genome quantization. With varying concentrations of syrup and capsule, a varying rate of inhibition of CPE, cells survival and virus replication was observed. Compared to 0.5 g/ml concentration of Renessans syrup, 5 and 50 g/ml showed comparable results where there was a 100% cell survival, no CPEs and a negligible viral replication ({Delta}CT= 0.11 and 0.13, respectively). This study indicates that Renessans, containing iodine, may have potential activity against SARS-CoV-2 which needs to be further investigated in human clinical trials.