Most COVID-19 testing (such as qPCR) is currently performed in a laboratory environment that is quite laborious and time-consuming. Accurate and scalable point-of-care (POC) tests for the diagnosis of COVID-19 would increase the scope for diagnosis to be made in the temporary screening locations, doctor's offices, labs, and nursing homes at a larger scale, and outside the laboratory setting by
1) reducing the time to obtaining an actionable result (will identify whether to quarantine or not)
2) could support early identification of those with COVID-19 and
3) could also support appropriate use of isolation resources and infection control measures
4) identify those individuals who have previously been infected and recovered from the initial infection. This scenario may inform public health strategies by relaxing social distancing restrictions at or during the lockdown for these workers and
5) recruitment into clinical trials of treatments.
In this blog, we will consider the point-of-care assays that could run within few minutes to hours near the patient despite those typically placed within a laboratory and time-consuming. Many of these POC tests are either molecular-based PCR-type tests or serological assays, which detect the presence of virus in a blood sample. The main difference lies in the 1) sample used for identifying the target viral agents and the 2) tests' complexity, 3) speed, 4) scalability, and availability for mass screening.
Molecular diagnostics assays are primarily nucleic acid amplification tests built immediately upon identifying the pathogen or viral agent's sequence. The common targets for detecting SARS-CoV-2 in the specimen are:
• The envelope gene (E gene).
• Nucleocapsid gene (N gene).
• Spike gene (S gene).
• RNA-dependent RNA polymerase (RdRp).
A variety of samples have been studied using these assays, such as samples from the nares and oropharynx, salivary samples, tears, anal swabs, fecal and urine samples, and exhaled breath.
Most of the molecular POC tests have either gained CE marking or emergency FDA approval.
The average sensitivity of the molecular tests is 98%, and the average specificity is 99.8%. The specificity of POC tests depends on various parameters such as:
1. Availability of adequate specimen.
2. Quality of sample.
3. The time point of sample collection.
4. The handling and storage of the sample before analysis.
Recently, Tata Medical and Diagnostics Ltd. (TATA MD) in collaboration with CSIR-IGIB team developed end-to-end ‘Tata MD’s CHECK-Feluda’ diagnostics solution, which is the CRISPR (gene-editing technology) based test for coronavirus disease (Covid-19). Feluda is the world’s first COVID-19 detection test that employs Cas9. It was tested on 2,000 patients and achieved 96% sensitivity and 98% specificity – comparable to that of the RT-PCR.
Earlier, Cas9’s cousins – Cas12 and Cas13 – were used to develop COVID-19 detection kits in the US, and they were named ‘Detectr’ and ‘Sherlock’. Cas12 and 13 binds and cleaves RNA rather than DNA substrates and adopts an enzymatically “active” state. Both the enzymes then bind and cleaves additional RNAs regardless of homology. This is referred to as “collateral cleavage” that destroys DNA probes. DNA probes are designed similarly to conventional TaqMan probes, in which one end of the reporter is bound to a fluorophore and the opposite is linked to a quencher. Degradation of the DNA probes releases fluorophores and results in stable and strong fluorescent signal detected by a fluorimeter.
Current serological point of care tests uses ELISA, Chemiluminescence immunoassay, Electrochemiluminescence immunoassay, Time-resolved fluorescence immunoassay, Colloidal gold immune-chromatography, LFA to detect SARS-CoV-2 antibodies in body fluids such as whole blood, finger-prick blood, plasma, or serum. Due to the non-availability of validated assays, serological tests are not considered a part of the definitive or standard diagnosis of SARS-CoV-2. The significance of these tests lies in early detection and screening for immunity to disease.
Potential targets for SARS-CoV-2 antigen detection include the glycoprotein spikes on the surface of the viral capsid, N antigen, S antigen, and spike receptor-binding domain. Currently, available kits allow rapid quantification of the N and S antigens.
The reference standard used for comparison in these studies was RT-PCR testing which is subject to false negative results or misclassification. Any error in the reference standard may affect the performance, sensitivity and specificity of the POC tests being evaluated and is likely to have serious consequences. Besides, the RT-PCR test is complex and requires a centralized laboratory with expensive equipment and well-trained staffing.
There is an evident gap in current serological test development of POC
1) sample volume
2) time to prepare sample
4) flexibility to use with a quick turnaround time to results.
There is abundant opportunity to develop rapid and accurate serological tests for diagnosing COVID-19-related biomarkers. We believe that one of the key aspects that would enhance their widespread utility in clinics will be to equip sensing platforms in face masks etc. with wireless capability via Bluetooth and Wi-Fi so that the sensor information is transmitted safely and securely for clinicians.
Future research in POCT development lies in evidence-based clinical management and design of assays which are scalable during pandemic responses.
At Premas Biotech, we have optimized the parameters for the expression and purification of spike S1, E, M and N proteins. The proteins are produced in large amounts in single batch, which reduces batch-to-batch variation and COGs. SARS-CoV-2 purified proteins are commercially available for COVID-19-related research. Visit the supplier page for more protein information.
Contact us today if you would like to learn more.
10 December 2021
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