The Coronavirus testing conundrum

The global pandemic of novel coronavirus disease 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV2) began in Wuhan, China in December 2019. Since then it has been declared a pandemic by World Health organization (WHO) in March 2020. Approximately 3 billion people are under lockdown, ~3,000,00 have been infected and 200,000 fatalities as on April 28th, 2020.

Origins of Covid-19

The 2019-nCoV belongs to the group of viruses, which include the 2003 Severe Acute `1`Respiratory Syndrome (SARS) and the 2012 Middle East Respiratory Syndrome (MERS) viruses. Since the SARS outbreak in 2003, scientists have discovered a large number of SARS-related coronaviruses from their natural hosts - bats. Previous studies have shown some of these bat coronaviruses to have the potential to infect humans.

General Biology

The Covid-19 virus is a single stranded RNA virus. It belongs to Betacoronavirus family and has ~ 80%, ~50%and ~95% similarities to genomes of severe acute respiratory syndrome virus (SARS-CoV2), Middle East respiratory syndrome virus (MERS-CoV) and bat corona virus RaTG13 respectively. The differences in their genomic and phenotypic structures determines their pathogenicity. Compared to SARS-CoV and MERS-CoV, Covid-19 exhibits faster human-to-human transmission. It is estimated that SARS-CoV2 infected person can infect approximately three new people (reproductive number is averaged to 3.28). However symptoms can vary with some patients remaining asymptomatic while others presenting with fever, cough, shortness of breath, fatigue and host of other symptoms.

SARS-CoV2 has four structural proteins which include spike surface glycoprotein (S), small envelope protein E, matrix protein (M) and nucleocapsid protein (N). The spike glycoprotein (S protein) on the virus’s surface mediates receptor recognition and membrane fusion. The SARS-CoV2 virus enters a person through nose and throat. There is a receptor called ACE2 which normally helps regulate blood pressure and is present in all human organs. This is also present in the respiratory tract of humans. 

SARS-CoV2 appears to gain entry into the cells by latching on to the angiotensin-converting enzyme 2 (ACE2) receptor through the altered spikes S protein. 

Image: A 2D image of COVID-19 virus showing all its proteins.Source: L. Mousavizadeh and S. Ghasemi, Genotype and Phenotype of Covid-19: their roles in Pathogenesis, Journal of Microbiology, Mar 2020.

Types of tests:

Once the SARS CoV2 is cleared from the body, viral RNA is no longer available for detection in the respiratory tract, leaving a short window during infection for the virus to be detected. The RT-PCR method can be used for detection of viral RNA during this phase, however it gives no indication if the patient’s infection history and his immune status or if they are still susceptible to infections. Unlike RNA, antibodies last longer in the body and persist in blood for several months to years depending upon the type of immune response elicited by the body when exposed to different viruses.

Source: Why we need antigen and antibody tests for Covid-19, The Native Antigen Co, accessed Mar 24, 2020

1.    Nucleic acid testing (RT-PCR, LAMP and CRISPR):

The RT-PCR technique is highly sensitive and specific and can deliver a reliable diagnosis as fast as three hours, though usually laboratories take on average between 6 to 8 hours. Compared to other available virus isolation methods, RT-PCR is real time, significantly faster and has a lower potential for contamination or errors as the entire process can be done within a closed tube. It continues to be the most accurate method available for detection of the coronavirus. A sample is collected from a suspect carrier’s nose or throat via nasopharyngeal swabs.

RT-PCR involves reverse transcription of SARS CoV2 RNA into complementary cDNA which involves amplification of specific regions of the cDNA. There are three conserved sequences in the SARS related viral genomes (1) RdRP gene (RNA dependent RNA polymerase gene) (2) the E gene (envelope protein gene) and (3) the N gene (nucleocapsid protein gene). Both RdRP and E gene have high analytical sensitivity for detection while the N gene has provided poorer analytical sensitivity. The assays can be designed as two-target system, where one primer detects numerous coronaviruses including SARS CoV2 and second set of primersare specific to SARS-CoV2. One of the drawbacks of RT-PCR is its throughput. In most of the RT-PCR testing systems, one can only run less than 20 samples an hour.

Globally dozens of manufacturers are able to develop the molecular based diagnostic tests and get approval in a short period of few months after the onset of the pandemic, testifying to the ingenuity ang global collaborations.

Several countries including Germany have successfully used ‘pooled samples’ from multiple patients to address shortage of test kits. A pooled sample test can be used instead of conducting individual tests on a wider population. If the ‘super sample’ tests positive, the group can be made into smaller pools again to test which one turns positive within the smaller pool of samples. If 1% of the population has SARS Cov-19, 19.5 test kits would suffice to test 100 individuals.

Isothermal amplification (Loop-mediated isothermal amplification (LAMP) amplifies nucleic acid under isothermal conditions without the use of a thermal cycler and is a fast, specific, and cost-effective technique. In LAMP, the target sequence is amplified at a constant temperature of 60–65 °C using either two or three sets of primers and a polymerase with high strand displacement activity in addition to a replication activity. Typically, 4 different primers are used to amplify 6 distinct regions on the target gene, which increases specificity. An additional pair of "loop primers" can further accelerate the reaction. The amount of DNA produced in LAMP is considerably higher than PCR-based amplification. LAMP represents a cheaper alternative amplification method. However, it has lower sensitivity than real-time RT-PCR. Not a single LAMP assay has been approved by FDA so far.

CRISPER-Based Covid 19 Paper based test kit: Scientists at Univ. of California San Francisco (UCSF), MIT, Broad Inst., Mammoth Biosciences, Cephid and Institute of Genomics and Integrative Biology (IGIB, India) have developed a relatively inexpensive rapid test which does not require specialized equipment. Samples from carriers are collected via nasal or throat swabs. The test uses CRISPR technology by using Cas protein, along with viral genetic material to test for Corona specific gene- Gene N (Specified by CDC) and gene E (specified by WHO). The process is faster and less resource intensive than traditional RT-PCR technology. The pitfall is that it requires high viral load, hence may miss out patients with initial or late stages of infections.

The new test is yet to receive formal approval from regulatory authorities and US FDA. However, the researchers plan to fast-track the approval process under emergency use authorization process.

2. Rapid diagnostic tests based on antigen detection

Rapid Antibody Tests detects the presence of viral proteins (antigens) expressed by the Covid-19 virus in samples from the respiratory tract of a person. Target antigen binds to specific antibodies (IgG and IgM) present on the paper strip enclosed in plastic casing and results can be detected in as early as 15min. RDT is typically a qualitative (positive or negative) lateral flow assay that is small, portable and can be used at point of care (POC).  RDTs are often similar to pregnancy tests, in that the test shows the user colored lines to indicate positive or negative results.

RDT tests accuracy depends on several factors such as timing of onset of symptoms, concentration of virus in the specimen, quality of specimen collected etc. There could be higher chance of false positive, by cross reaction to antigens produced by other human corona viruses such as common cold.

The gold standard frontline test for COVID-19 diagnosis continues to be real time PCR based molecular test, which is aimed at early virus detection. The rapid antibody test cannot replace the frontline test.

Based on current data, WHO does not currently recommend the use of antigen-detecting rapid diagnostic tests for patient care, although research into their performance and potential diagnostic utility is highly encouraged.

3. Rapid diagnostic tests based on host antibody detection

Serological tests measure the amount of antibodies present in the blood when the body is exposed to a specific infection, like COVID-19. In other words, the test detects the body’s immune response to the infection caused by the virus rather than detecting the virus itself. In the early days of an infection when the body’s immune response is still building, antibodies may not be detected. This limits the test’s effectiveness for diagnosing COVID-19 and why it should not be used as the sole basis to diagnose COVID-19.

Serological tests can play a critical role in the fight against COVID-19 by helping healthcare professionals to identify individuals who have overcome an infection in the past and have developed an immune response. In future, this may potentially be used to help determine, together with other clinical data, individuals that are no longer susceptible to infection and can return to work. In addition, these test results can aid in determining individuals who can donate blood plasma, that may serve as a possible treatment for those who are seriously ill from COVID-19. Serological tests can also be useful in the validation of vaccine candidates in clinical trials.

Based on current data, WHO does not recommend the use of antibody-detecting rapid diagnostic tests for patient care but encourages the continuation of work to establish their usefulness in disease surveillance and epidemiologic research. There is still a great deal about COVID-19 immunity that we don’t yet fully understand. For example, we don’t yet know that just because someone has developed antibodies, that they are fully protected from reinfection, or how long any immunity lasts.

Conclusions:

Countries like S. Korea where extensive testing has been done early on has been able to flatten the curve and hence have had lesser fatalities. South Korea has been able to manage the pandemic much better due to prior experience with MERS and SARS. There are incidences of second wave of infection hitting countries now in S. Korea, Japan, Singapore where reinfections are also being reported. Premature relaxation of social distancing, travel restrictions and other norms can have significant toll on containing the outbreak. Governments should act and plan for extensive testing, contact tracing, containment and treatment facilities to minimize mortality due to Covid-19 and to minimize the economic fallout of these pandemics This will not be the last pandemic the world will see. It is imperative that the world should step up global collaborations and funding to develop better strategies to mitigate similar pandemics in the future. The world is connected like never before. It’s like one large village. Disease can originate anywhere in the world and will spread to the rest of the world in no time. Also, it's imperative that the rich countries should devote resources to strengthen healthcare in countries who can't afford it in their own self-interest.

With one of the lowest numbers of positive cases and deaths per million in the world, Indian authorities, both central and state, through stringent laws and restrictions have done a phenomenal job so far. The ongoing battle against Covid-19 from the trenches has to continue until we develop a vaccine and can start immunizing the entire population.

Dr Aman Iqbal PhD, Dr Nita Sachan PhD, Dr Ramesh Byrapaneni MD DM

Opinions expressed solely are our own and do not express the views or opinions of my organizations we are associated with.