The Dynamic Change in Testing Strategies in the Testing Times of COVID-19

Introduction

COVID-19 struck mankind as a pandemic like never before. The magnitude of infectivity was baffling and it crippled healthcare across the globe affecting the most affluent countries with robust and highest quality of healthcare delivery systems.

The basis of management rested on effective testing to identify the positive cases and isolate them in order to prevent the spread of infection. While some countries had strong molecular diagnostics infrastructure to support the ever-increasing demand for Reverse Transcription Polymerase Chain Reaction (RT-PCR) tests, most were caught unaware and found it difficult to meet the exponentially increasing demand of RT-PCR machines and kits.

In this article, we seek to embark upon the evolution of testing with resultant changes in policies right from the inception.

First Wave

The first case of COVID-19 in India was reported in January 2020 in Kerala, diagnosed at National Institute of Virology (NIV), Pune by testing of oropharyngeal swab from a 20-year-old woman who presented with a one-day history of sore throat and dry cough. It took three days for the case to be concluded as positive for COVID-19 disease. The patient was managed at the Government Hospital, Thrissur and was reported as Covid negative on the 19^th day of illness. The testing at that time composed predominantly of routine blood work up with special attention to total and differential leucocyte count and Erythrocyte Sedimentation Rate (ESR). The findings at presentation mostly comprised of leukopenia, lymphopenia and raised ESR.¹

By February 2020, India had 14 documented laboratories to detect COVID-19 infection through RT-PCR.

Another major change in the policy regarding testing took place in the form of scrapping of state surveillance approvals. In the initial days, for each case, it was mandatory to fill a form and send it to the Integrated Disease Surveillance Programme (IDSP) officials for approval. The idea behind this was to keep indiscriminate testing in check since the kits were in short supply. This was actually a hindrance, as many a times a clinically suspected case of COVID-19 was not tested due to lack of approvals which resulted in cluster outbreaks, as in certain cases these clinically suspected candidates were actually positive. At the same time, countries like South Korea had managed its COVID-19 cases effectively based on mass testing of nearly 4% of its entire population, thereby sending the message loud and clear that the basis of controlling this menace was adequate testing followed by tracking and tracing.^2,3

By mid-March 2020, the Ministry of Health and Family Welfare (MOHFW) approved testing by private accredited laboratories and conducted surveillance to test for community transmission.

As the number of cases increased, newer modalities of testing based on Nucleic acid amplification, with much shorter Turnaround time (TAT) were implemented effectively. These included the TrueNat and Cartridge based nucleic acid amplification test (CBNAAT) which brought down the TAT to 30-60 minutes. These were very effective in tier two and tier three cities as well and had a major advantage of portability with ease of performing the tests since they didn’t need the technically complicated steps of genetic material extraction involved in conventional RT-PCR testing.

By mid-June, the Rapid antigen test kits were also validated which served as a point-of-care testing especially in containment zones and hospital setups. These could provide a result in 30 minutes, in which a positive result was taken as confirmatory, but a negative result in symptomatic cases was subjected to confirmation by RT-PCR, given the low sensitivity of the test.

The phase also saw a rapid increase in the number of validated testing kits, making sure testing met the surge in demand and also that monopolization in kits was averted. This was a major success in lowering the costs of the kits and thereby the test price, resulting in wide spread testing. By mid-August, more than 700 kits were validated and an official portal was made available to expedite the validation process.

The total number of laboratories conducting RT-PCR testing significantly rose to 1596 by August 2020. This was mainly brought about by consolidation of resources, investing in developing infrastructure and training laboratory professionals along with publicprivate partnerships. The government also established mentoring institutes to guide in upscaling testing in hospitals and medical institutes.

Neutrophil to lymphocyte ratio (NLR), Platelet to lymphocyte ratio (PLR) and Lymphocyte to monocyte ratio (LMR) gave an idea of the severity of the disease. These biomarker ratios increasingly started aiding in prognostication of cases and helping clinicians to detect potentially severe cases early. Higher NLR and PLR and lower LMR are indicative of a more severe disease course.⁴

The major pathophysiology centred around the release of cytokines secondary to viral induced endothelial damage which played a pivotal role in the onset of dreaded and fatal cytokine storm. This necessitated the quantification of specific markers like C-Reactive Protein (CRP) and Interleukin-6 (IL-6) which were also identified as potential targets to impede the cytokine mediated damage.

Eventually, the impact of the virus on endothelial cells resulting in microthrombi, leading to coagulopathy was well established, which brought about the advent of testing for coagulation profile including Prothrombin Time (PT), activated Partial Thromboplastin Time (a-PTT) and D-Dimer in all the patients.

The October 2020 saw a major decline in the number of new cases and by February 2021, India reached its lowest number of new cases of COVID-19.

Second Wave

A major development around the advent of new year 2021 was the identification of different variants of the SARS-CoV-2 virus responsible for causing major outbreaks across the globe, especially in the United Kingdom (UK). Amongst these variants, the Alpha and Delta variants caused major outbreaks, morbidity and mortality. While the Alpha variant was detected in the UK, the Delta variant had its origin in India.

By mid-March, India saw a rapid spike in the number of cases, initially in Delhi and Maharashtra and later even in the villages which were earlier unaffected. This worsened with acute shortage of oxygen and fatal secondary infections which left many maimed and/or deceased. One such infection which stood out was Mucormycosis, popularly known by its misnomer as ‘Black Fungus’. Laboratories started getting increased load of tissue biopsies ranging from sino-nasal scrapings to orbital exenteration specimens. These required morphological identification of broad, aseptate hyphae, branching at obtuse angles, aided by special stains like Gomori’s Methenamine Silver (GMS) and Periodic acid-Schiff (PAS). Given the fatality involved, it was imperative that the diagnosis be made accurately and timely to prevent spread of infection. The altered glycaemic state and rampant use of corticosteroids only worsened the state of affairs which reached dreaded proportions in light of shortage of liposomal Amphotericin B.

The tissue diagnosis in these cases is supplemented and confirmed by microbiological studies which range from a simple KOH mount to culture, the gold-standard.

PCR based molecular identification and DNA sequencing based on bar codes along with matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) are available in very few laboratories but offer detection of fungal genetic material in serum and paraffin-embedded tissues along with species identification.⁵

The immense pressure on the national RT-PCR testing resources and massive delays in TAT prompted the development of newer modalities like Molecular point of care tests based on isothermal technology, using proprietary enzymes and constant temperature control to achieve immensely rapid RNA amplification. These devices are capable of detecting positive samples in less than 15 minutes and provide a fast and accurate detection of COVID-19.

Antibody Testing

Like any other infection, antibody testing in COVID-19 has a major role in ascertaining the infection rate, herd immunity, predicted humoral protection and in understanding the disease epidemiology. Detection of neutralizing antibodies also plays a pivotal role in determining vaccine efficacy.

Though IgM is the first to rise, its detection is less specific due to cross reactivity. Also, it has been observed that not in all cases it rises before IgG, thus raising questions on its credibility to be used as a marker for recent/acute infection. Thus, initial antibody testing revolved around testing of total and IgG antibodies.⁶

Antibodies that target the spike protein of SARS-CoV-2, with antibodies to S1 RBD have shown to be responsible for almost 90% of neutralizing activity.

The current kits detect various types of antibodies like Spike (IgG and/or IgM) and Nucleocapsid.

Conclusion

We hereby present in a nutshell the various modalities of laboratory testing for both diagnosis of COVID-19 and serosurveillance. In any infectious pandemic which threatens the existence of mankind at large, the first step is always to estimate the magnitude of threat and to detect the potential spreaders, thereby highlighting the importance of testing. In COVID-19, the limitation of time against the infectivity and lethality of the disease prompted development in testing, management and vaccination like never seen before, both in magnitude and in timelines. This also necessitated a dynamic state of policy making which needed a change at every step whenever a new finding was deciphered. We are still learning about the infection, changing with each new finding that comes to light and it might be sometime before we are certain about our victory in toto!

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Conflicts of interest

The authors declare no conflict of Interest, financial or otherwise