Introduction
The COVID-19 pandemic which started from Wuhan city of China now has spread globally to more than 180 countries with 5,819,962 confirmed cases as on 31’st May.1 The critical importance of diagnostic services in tackling this pandemic was emphatically proven because diagnostics are fundamental for Identification & control of an Outbreak. WHO also emphasized that diagnostic testing, isolation, and contact tracing should be the backbone of the global response to COVID-19 as social distancing measures and hand washing alone will not stop the epidemic.2 Eventhough currently there are legion of tests available globally for COVID-19, what tests to be used in COVID-19 response and what strategy is to be used to scale up the testing capacity is critical for correct identification followed by isolation of confirmed cases & contact tracing.Timeline of key dates of diagnostic response globally & in India are mentioned.
Key dates in the diagnostic response (global) timeline3, 4 include
7 January 2020: The full genetic sequence of the novel coronavirus was published. This enabled the development of real-time PCR diagnostic (RT-PCR) tests by different countries and diagnostic companies.
4 February 2020: The US Centers for Disease Control and Prevention’s (CDC’s) testing protocol [CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel] received Emergency Use Authorization from the Food and Drug Administration (FDA).
4 February 2020: The Republic of Korea’s Ministry of Food and Drug Safety approved the emergency use of RT- PCR kits for COVID-19.
19 February 2020: FIND launched an expression of interest for test developers of in vitro diagnostics that detect SARS-CoV-2 nucleic acid (molecular tests) to undergo an independent evaluation, in collaboration with WHO.
12 March 2020: FDA issues Emergency Use Authorization for first commercial molecular test for SARS-CoV-2.
13 March 2020: FIND launched an EOI for test developers of immunoassays (manual ELISA, machine-based or lateral flow, rapid tests specific for SARS-CoV-2 antigen or antibodies) to be evaluated using a standardized, independent protocol, in collaboration with WHO.
2 April 2020: FDA authorizes the first antibody-based test for COVID-19.
7 April 2020: WHO lists two tests for emergency use: one RT-PCR test and one qualitative assay.
12 April 2020: Close to 14 million SARS-CoV-2 tests have been performed worldwide (data from 184/195 countries) with around 1.8 million positive results reported on FIND’s Test Tracker.
Key dates in the diagnostic response (India) timeline5, 6, 7, 8 includes
A Total of 3126119 samples were tested for SARS CoV-2 (COVID-19) in India as on 26Th May 2020.
First case for COVID-19 in India was reported on 31’st December.
By 13 April 2020, there had been nearly 9,000 confirmed cases and over 300 deaths.
Rapid scaling up of PCR testing from mid-March, from 19 to over 669 (Government laboratories: 466 & Private laboratories: 203)9 total Operational Laboratories reporting to ICMR as on 31/5/20 which includes 466 Real-Time (RT PCR) testing labs, 134 TrueNat testing labs & 55 CBNAAT Testing labs for COVID-19:
17/03/2020: Advisory Strategy of COVID 19 testing in India (Version 2).
04/04/2020: Advisory to start rapid antibody based blood test for COVID-19 17/04/2020: Protocol for using 'Rapid antibody test' in Hot area - epidemiological studies and surveillance for the State/UT.
27/04/2020: Revised Advisory to state on Rapid Antibody Blood Test.
09/05/2020: Advisory for use of Cartridge Based Nucleic Acid Amplification Test (CBNAAT) using Cepheid Xpert Xpress SARS-CoV2.
18/05/2020: Revised Strategy for COVID19 testing in India (Version 5).
19/05/2020: Revised Guidelines for TrueNat testing for COVID-19.
WHO recommends a strategy for COVID-19 testing and has defined four transmission scenarios.10, 11 Each scenario gives the aim, how the testing will be done and what will be the actions taken based on the results.
Overview of Diagnostics for Covid-19
When we consider the role of diagnostics in the detection of infection, there are many important goals to consider like right clinical sample taken for the right test & right patient at the right time in the right setting. And of course right interpretation also matters. Basic diagnostic methods for infectious diseases typically fall into two different categories-Pathogen detection & Host biomarker detection as shown in Figure 1. Main differences between molecular (or PCR) assays and immunoassays, and advantages of each technology is shown in Table 1.
Table 1
Difference between molecular assay & immuno assay
Specimen ollection and Lab Biosafety
The World Health Organization (WHO)12 and Centers for Disease Control and Prevention (CDC)13 recommend specimens from the lower respiratory tract, including sputum, bronchoalveolar lavage and tracheal aspirate, for the diagnosis of COVID-19 using the approved molecular testing methods. Specimen collection have to be done donning appropriate PPE14 and the same has to be doffed carefully & disposed as per bio medical waste management guidelines. Table 2 describes specimen type recommended and collection material & storage for the same. Laboratory biosafety for COVID-19 testing is shown in Table 3.
Table 2
Specimen types and storage for COVID-19
Table 3
Lab biosafety for COVID -19 Testing14
Molecular assay -RT PCR
The targets for molecular assay foe SARS COV-2 is shown in Figure 2
Current testing modalities for laboratory diagnosis of COVID-19 by Real Time PCR
Principles of good molecular biology testing practice15, 16
Molecular labs should follow best practices to minimize nucleic acid contamination of the laboratory environment & best practices to minimize and detect contamination of samples in an analytical run. This could be achieved by proper design of a molecular lab. Contamination is a major concern because, they cause false positive results.
To reduce the likelihood of contamination and to protect the integrity of specimens, proper design of laboratory, and following general laboratory practices are important. Separation of pre- and post-amplification work areas and reagent preparation areas to prevent contamination is mandatory. Ideally, molecular laboratory should have three work areas that are physically separated and preferably at a distance from each other:
Area 1: reagent preparation room (positive air pressure relative to outside);
Area 2: specimen control/preparation, PCR set-up room (negative air pressure relative to outside); and
Area 3: nucleic acid amplification and amplification product analysis room (negative air pressure relative to outside).
These work areas must be arranged so that there is a unidirectional flow of personnel and specimens from “clean” areas to “dirty” areas, i.e. from Area 1 to Area 3 (this includes cleaning and maintenance personnel). See theFigure 2 for an example of work area layout.
Molecular testing on open platforms:17, 18, 19
Test kits and protocols that are used on open PCR platforms require the end user to extract the viral RNA in a separate step to the PCR reaction. As these manual PCR kits and protocols are compatible with multiple PCR machines (thermocyclers), the end user may also choose which thermocycler to use. There are two types of manual PCR protocols that can be performed on open PCR machines: those that follow laboratory-developed (or in-house) protocols and those that follow commercial kit protocols.
Advantages of tests for use on open platforms
Tests may be well supported in-country by the manufacturer’s distributor network and service staff (if using commercial kits but does not apply to Local Developed Tests).
It may be easier to scale testing using multiple kits that are compatible with the same open system, as long as consistent quality can be guaranteed by the laboratory, which may mitigate any supply chain concerns.
Laboratory staff may already have been trained on testing, troubleshooting and maintaining open platforms.
Tests are usually less expensive than tests for use on closed platforms.
Disadvantages of open platforms
Strong quality control, quality assurance procedures and validation of the methods required in every laboratory to ensure high test performance & Quality.
Sample processing methods may not be specific for the specimen types recommended for COVID-19 diagnosis.
Well-trained staff are needed to perform the many manual steps and interpret results.
Open platforms require additional consumables, equipment or quality controls that may not be supplied with the machine, test or test component.
Molecular testing using closed (proprietary) diagnostic platforms17, 18, 20
Use of closed/proprietary systems generally enables greater automation of workflow, and frequently includes extraction, amplification and output of results all on the same machine. A number of manufacturers have rapidly developed SARS CoV-2 tests to run on their existing closed platforms to take advantage of available machines and associated resources in laboratories. Example of some COVID-19 closed platform tests authorized for use are shown in Figure 4.
Advantages of closed platforms
Closed platform instruments are often already available in many countries, where they have been deployed for TB, HIV and other testing.
Closed platforms are well-supported by manufacturers or their representatives (e.g. manufacturers may have in-country service staff supporting the platforms).
Some closed platforms allow for high-throughput testing, which can help to rapidly scale up COVID-19 testing, whereas others may be more amendable to more decentralized testing.
Laboratory staff will be trained on testing, troubleshooting and maintaining closed platforms already in use.
Most, if not all, required reagents or consumables for closed platforms are directly provided by the test supplier.
Closed platforms require limited hands-on time and have largely automated result reporting and interpretation.
Internal quality controls for closed platforms are included in the platform or test.
What should one consider when selecting an open/Closed platform test?
When implementing molecular tests for use on open/closed platforms, one should consider performing an assessment of the available platform tests at their setting including assessment of infrastructure/resources. Once the best combination of tests or test components is matched to the correct PCR and result visualization machine, then procurement and training of the staff needs to be considered.
Molecular Assay- Loop-Mediated Isothermal Amplification (LAMP)
Although real‐time RT‐PCR is sensitive and reliable, it is time‐consuming (~2 h) and requires a specific detection device or instrument, which limits its broad application to current huge demand for the global pandemic of COVID‐19.
LAMP It is a very sensitive, easy and time efficient method. (LAMP) is a rapid technology of DNA amplification which has been applied to pathogen detection such as virus, bacteria and malaria. The LAMP reaction generally needs one constant temperature, and the target DNA can be amplified in 30 min. COVID‐19 diagnosis kit for the rapid detection of SARS‐CoV‐2, using one‐step reverse transcription and loop‐mediated isothermal amplification (RT‐LAMP) has been developed.21, 22, 23 But commercial kits based on this principle is not yet available in India
Immunoassays
{Current WHO recommendations do not support their use in clinical management, but encourage their inclusion in research projects to assess their performance and clinical utility}.
Immunoassays are used in detection or quantification of viral proteins, antigens or antiviral antibodies produced by the immune response against a pathogen from clinical specimens. All immunoassays rely on the binding of an antibody to an antigen, and the ability to produce a measurable signal in response to the binding, either via chemically linking antibodies or antigens with some kind of detectable label. A variety of different labels and detection methods used are either emission of radiation, prediction of a color change in solution, or fluorescence.
Targets of immunoassays for SARS-CoV-224, 25, 26
Antigen tests for SARS-CoV-2 detect and/or quantify the nucleocapsid (N) or spike (S1 & S2) proteins of the virus, Spike protein S1 is pecific. Antibody tests detect the antibodies (IgG, IgM or IgA) produced against the N or S viral proteins.
Spike protein (S) is the most exposed viral protein of SARS-CoV-2, which attaches to a specific receptor on human cells, allowing the virus to infect the cell. The S protein consists of S1 and S2 subunits.7
Nucleoprotein (N) is the most abundant viral protein produced and shed during infection with SARS-CoV-2. It is highly immunogenic and can be found in blood and urine.8
Matrix protein (M is the most abundant protein inside a SARS-CoV-2 virus
In SARS-CoV-2, published evidence shows that viral load peaks at 3-5 days and that both IgM and IgG start to increase around 10 days after the onset of symptoms. Most patients produce antibodies in response to the virus (known as seroconversion) within the first three weeks.
Types of SARS-CoV-2 immunoassays24, 25, 26
The most common types of immunoassays developed for COVID-19 are either enzyme-linked immunosorbent assays (ELISAs) or lateral flow immunoassays (LFIs), also known as rapid diagnostic tests (RDTs).
ELISAs are one of the most frequently used types of immunoassays. Some key features of ELISAs mentioned in Table 1. Labs can use commercial reagent kits or in-house protocols; and have high throughput – run on 96 or 324-well plates;
All laboratory procedures for ELISA must be performed in a facility using procedures equivalent to a BSL-2, be based on a risk assessment, and conducted by trained personnel. Initial processing of samples from patients suspected of having COVID-19 infection, as well as any aerosol-generating procedures such as vortexing and ELISA plate washing, must be done in a biological safety cabinet. Refer to TABLE on Laboratory biosafety for COVID-19 testing.
LFIs or RDTs allow for the rapid detection of SARS-CoV-2 antigens or antibodies using a test strip. RDTs use the same principles of antigen-antibody binding as ELISAs but are generally less sensitive. When a sample is added to the test strip, the antigen or antibody migrates up the test strip, where it is captured by a detector antibody. The antibody for detection can be conjugated to gold nanoparticles or latex beads. An example of RDT in use is shown in Figure 5. Some key features of RDTs in use are mentioned in Table 1. One can perform the test with limited technical expertise. They can be run on different sample types: nasopharyngeal swab/respiratory specimens for rapid antigen detection and serum/plasma/whole blood, and other fluids for antibody detection.
RDT antibody tests could be very helpful in research studies to understand, at a community level, the population attack rate, the case fatality rate, and to understand transmission patterns in specific subgroups, such as children, teachers, and frontline medical staff. It is not known how long the antibodies detected by these RTD's will persist, nor whether they offer immunity to reinfection. As molecular testing is not always available, especially outside large urban areas, there is substantial pressure to use these tests.
Testing of samples using RDTs outside a laboratory setting should follow standard precautions to provide a barrier between the specimen and personnel during specimen manipulation. Although the use of immunoassays is not currently recommended for routine diagnosis, Table 4 shows where immunoassays for COVID-19, especially RDTs, can potentially be used.
Table 4
Potential applications for COVID-19 immunoassays26
Other potential diagnostic applications for immunoassays include aiding in the estimation of the disease stage (acute, early, convalescent);assessing the immune status of healthcare and other essential personnel;assessing convalescent plasma and vaccine efficacy (more suitable for ELISAs versus RDTs); and Guiding identification of low-risk people to exit quarantine (e.g. lifting lockdown measures).
The following Figure 6 27 adapted from the Institute of Tropical Medicine, Antwerp, describes general performance requirements for antigen and antibody tests according to different testing scenarios:
Sars-Cov-2 Diagnostics: Performance Data
Test performance is an important component that should be considered when selecting specific diagnostic products and determining the most appropriate implementation approaches. Some important performance characteristics of molecular assays & Immunoassay include:
Analytical sensitivity – also known as limit of detection, the lowest concentration of viral RNA that the test can detect.
Analytical specificity - or cross-reactivity – describes whether the test will detect other viruses or other infections that are not SARS-CoV-2, which can lead to false positive results.
Clinical sensitivity - the ability of a test to correctly identify COVID-19 positive individuals.
Clinical specificity - the ability of a test to correctly identify COVID-19 negative individuals.
A note on test utility.
The clinical utility of any test is affected by the prevalence or the probability of disease, in a population (even where the sensitivity and specificity of the test remain the same). Given a positive or negative result, the probability of having or not having COVID-19 (known as positive predictive value [PPV] and negative predictive value [NPV] respectively), will differ depending on the population tested.
Where to find data on Molecular/immunoassay Test Performance
FIND (Foundation for innovative new diagnostics) gives performance data on molecular & Immunoassay regarding detection of COVID-1928 and is in the process of performing an independent evaluation of a large number of different SARS-CoV-2 tests including immunoassays. The website will make the data publicly available when the evaluations are complete. FIND is working closely with WHO on this. List of validated test kits for both Molecular assay and antibody assay are published by ICMR.29, 30
List of molecular and immuno assay test kits available from FIND website
The complete list of US-FDA and/or CE-approved SARS-CoV-2 real time PCR kits and immunoassays are available at FIND COVID-19 website.31, 32 The status of the Tests if they are commercialized, in development or withdrawn, type of the test if it’s a Manual /automated/POC molecular test, Antigen/Antibody RDT, which regulatory is performing the tests are published in the FIND COVID-19 diagnostic pipeline website.
Conclusion
Diagnostic tests play an important role in the COVID-19 pandemic response. We need to understand the attributes and limitations of each type of test and use the right test for the right patient at the right time in the right setting. This is a rapidly evolving area as many countries are trying to scale up community testing for COVID-19. Many tests are now commercially available but their performance is not clear. FIND the Foundation for Innovative New Diagnostics, is evaluating their test performance and WHO will soon issue recommendations on the desired test performance characteristics for each use case.
Summary of the links from where information collected
Indian Council of Medical Research. COVID-19.
Ministry of Health and Family Welfare Government of India. https://www.mohfw.gov.in/#.
Foundation for Innovative New Diagnostics. COVID-19 diagnostics resource centre.
World Health Organization. Coronavirus disease (COVID-19) technical guidance: Laboratory testing for 2019-nCoV in humans.
World Health Organization. Country and technical guidance - coronavirus disease (COVID-19).
World Health Organization. Coronavirus disease (COVID-2019) daily situation reports.
Africa Centres for Disease Control and Prevention. Latest updates on the COVID-19 crisis from Africa CDC.
US Centers for Disease Control and Prevention. Coronavirus (COVID-19) updates.
London School of Hygiene & Tropical Medicine. COVID-19 updates.
European Centre for Disease Prevention and Control. COVID-19 updates.
National Institute for Communicable Diseases. Division of the National Health Laboratory Service South Africa. COVID-19.
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