COVID-19: developing quantitative RT-PCR tests to assess new variants and viral loads

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Although the roll-out of the severe acute respiratory syndrome coronavirus 2 vaccination programme is proving effective at reducing levels of serious COVID-19 and associated deaths, the pandemic is by no means over. CLI caught up with Alina Merz (Senior Product Manager, Bruker Microbiology and Diagnostics) to find out more about why the need for new COVID-19 tests remains and what questions we want these tests to answer.

Introduction

The recent COVID-19 outbreak was officially declared a pandemic by the World Health Organization (WHO) on 11 March 2020. Over a year later, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to challenge healthcare systems worldwide. As at 10 August 2021, there have been 203 295 170 confirmed cases of COVID-19 globally, reported to the WHO, including more than 4 million deaths [1].

One of the key challenges is the continuing emergence of new variants, which highlights the need for rapid and accurate tests that can identify and differentiate them. RNA viruses like SARS-CoV-2 are known for high rates of mutations leading to variants that have the potential for different clinical properties. Therefore, researchers are focusing their work on better understanding how easily the new variants might be transmitted, how severely they affect patients and how effective currently authorized vaccines are.

Identifying and differentiating variants is key in mapping COVID-19 epidemiology and viral load testing is also important for optimizing patient treatment.

How are scientists currently detecting COVID-19 variants and can you tell us more about the testing methods?

Variant identification and differentiation are currently being met on two fronts: by extensive research and by new developments in the speed and accuracy of testing solutions from manufacturers. One of the most important focuses of the latter involves devising a rapid version of the reverse transcription polymerase chain reaction (RT-PCR) assay, which needs to be complemented by a greater emphasis on viral load testing.

Various technologies are currently being used for SARS-CoV-2 detection. The nucleic acid amplification (NAAT) test, in the form of an RT-PCR assay has become the gold standard of SARS-CoV-2 tests. It is a highly sensitive method of detecting viral RNA from respiratory samples (e.g. nasopharyngeal swabs or bronchial aspirate) [2]. However, RT-PCR tests are conducted in a laboratory and can take days to produce results, with bottlenecks easily occurring.

Alternatively, rapid antigen testing seeks to identify protein from the viral surface, again from a bronchial or nasal sample. This test is faster than the RT-PCR test but is widely reported to be less accurate [3]. The third approach is an antibody test, which looks for antibodies that have been produced in response to the presence of the virus.

Can you tell us more about the mutations we are seeing across the world and why this might be?

An additional complication that has arisen is the need to detect and track new emerging SARS-CoV-2 mutations. Changes are common in both DNA and RNA viruses, but RNA viruses are particularly known for high mutation rates, which can lead to variants that have different properties. For coronaviruses, mutations often occur in the crown-like spikes that give these viruses their names. Although most are not of great concern to clinicians, a few can increase the infection and transmission rate of the virus as well as its virulence.

Multiple mutations can lead to new variants. As the virus adapts, therefore, further research and more accurate and rapid means of detection of any variants are needed. It is vital that any significant changes can be detected and mapped so that the epidemiology of the virus can be tracked. This will also help to identify any impact on the efficacy of existing vaccines or the need for revisions to the vaccines or treatments.

The first two main variants to be identified were discovered in the UK (variant B.1.1.7, Alpha) and South Africa (B.1.351, Beta). Although their changes to the spike protein differ, they have the N501Y mutation in common.

What does this mean for viral load?

Both the Alpha and Beta variants are believed to lead to a higher viral load in infected patients and therefore are more likely to have greater transmissibility. Some research suggests that the Alpha variant might have a greater pathogenicity than the initial virus, although the Beta variant does not appear to be any more severe [4]. However, it may still lead to a decreased effectiveness of human antibodies, due to the presence of the E484K mutation. In terms of resistance to current vaccines, initial studies suggest that the BioNTech/Pfizer, AstraZeneca and Moderna vaccines still offer protection against the Alpha variant, whereas they might be less effective against the Beta variant (although still reducing the number of severe cases and deaths).

Two further variants have been detected in Brazil (P.1, Gamma, and P.2, Zeta), which also carry the E484K and N501Y mutations. The Gamma variant has been classified as a variant of concern and carries different unique mutations, including mutations that affect the spike (S) protein. It appears that current vaccines might have a lower level of efficacy against these two variants. The Gamma variant is seen as a particular danger, and is believed to be highly transmissible, especially as most diagnostic tests cannot easily identify it. This variant is largely responsible for the current | 18 chaos caused by the pandemic in Brazil. It is the dominant variant of the virus in the country, and has also been detected in more than 60 countries all over the world so far.

Meanwhile, other new variants are also being investigated, including one identified in India (B.1.617.2, Delta), which has mutations that include L452R, and a further variant first documented in Peru (C.37, Lambda).

How important are the tests we use in identifying different variants and how is this helping scientists to develop new ways to combat Covid-19?

The often unknown danger posed by new variants highlights the need for newer, even more effective tests. Researchers are focusing on gaining a greater understanding of how easily variants might be transmitted, how severely they affect patients and how they might reduce the effectiveness of currently authorized vaccines. Along with healthcare professionals, they need more information about the virological, epidemiological and clinical characteristics of these variants.

Part of the research has focused on the accuracy of quantitative PCR (RT-qPCR) assays in identifying variants. Standard commercial assays look at sequences chosen from a number of target genes in the virus’s genome (e.g. the E and N genes). However, mutations in these genes can affect the sensitivity and specificity of assays. In one study, a novel mutation of the N gene was found to affect the detection of the SARS-CoV-2 virus when using a commercial assay [5]. This suggests that some new variants could escape detection with the most commonly used tests.

Another study concluded that some new mutations might reduce or negate the impact of targeted therapies that are currently under evaluation. Mutations can cause changes in the amino acids in the S-protein that locks onto the ACE2 (angiotensin-converting enzyme 2) membrane protein of human cells. The study looked at two mutations that resulted in the virus being able to bind to the cell more securely, which could lead to an increase in its transmissibility. The researchers concluded that ACE2 and spike-S1 are not suitable targets for peptide drugs that could be used to treat COVID-19 [6].

To meet the need for more accurate, real-time tests, manufacturers are developing rapid RT-PCR assays that can identify variants of potential concern, based on their underlying mutations. The WHO recommends the use of NAAT tests for screening suspected virus infections, and a faster and more accurate quantitative test could mitigate many of the issues identified by the researchers, resulting in information that will help to protect patients as well as enabling the pandemic’s progression to be tracked more easily.

Can you tell us more about the need to evaluate viral load in a Covid-19 infected patient and the effect this has on research capabilities?

A further key aspect of ongoing research aimed at combating the virus is to find ways of evaluating the viral load – i.e. the quantity of the genetic material of a virus (RNA for SARS-CoV-2) that can be measured in the blood of an infected person. Quantitative assessments of the viral load can help clinicians to categorize patients according to risk; to monitor disease progression, to choose the most appropriate therapies to deliver at the right time; to make more informed decisions on any isolation measures; and to assess the transmission risks and the overall severity of the disease.

There is a growing body of research in these areas. A recent study has shown that the SARS-CoV-2 viral load could possibly be used as a way of predicting mortality [7]. In this study, the viral load was calculated using RT-qPCR technology in a large group of hospitalized patients, which enabled clinicians to more accurately assess patients and predict clinical outcomes.

Viremia has also been shown to correlate with an increase in severity of the disease [8]. In hospitalized patients, a higher plasma viral load of SARS-CoV-2 was associated with a higher level of respiratory disease, lower absolute lymphocyte counts and increased markers of inflammation.

Other research has shown that the viral load can provide a better understanding of SARS-CoV-2 transmissibility [9]. The viral loads of hospitalized patients who had transmitted the disease to at least one other person were compared with those who were not the cause of secondary transmission. The study concluded that “high nasopharyngeal viral loads around onset may contribute to secondary transmission of COVID-19”.

These investigations highlight the need for effective, reliable and accurate quantitative assays that can assess the viral load in line with the First WHO International Standard for SARS-CoV-2 RNA [10], such as the FluoroType® SARS-CoV-2 varID Q (Bruker). This CE-IVD kit specifically detects SARS-CoV-2 and the respective viral load in a multiplex, RT-PCR test for nasopharyngeal or oropharyngeal swab sample analysis.

Compared to a year ago, the research into SARS-CoV-2 is phenomenal and the advances made in such a short time have truly been a global effort to suppress the virus. Can you tell us why the development of the RT-qPCR assays is significant?

The global COVID-19 pandemic continues to present new challenges to scientists and clinicians alike. Despite the welcome success of the current crop of rapidly developed vaccines, the situation continues to evolve, with the emergence of new SARS-CoV-2 variants worldwide causing concern.

The wealth of new research on the virus and possible new variants has highlighted the need for more rapid and sensitive, RT-qPCR assays that are able to detect and differentiate between the various mutations that arise. These assays can also be used to quantify the viral load, providing vital data on the severity and transmissibility of the disease. This will enable clinicians to make well-informed decisions on patient treatment and will help manufacturers to modify their vaccines, where necessary, in response to any new variants. The new assays will therefore form a vital tool in the ongoing efforts against the spread of COVID-19.

The interviewee

Alina Merz BSc, MSc, Senior Product Manager, Bruker Microbiology and Diagnostics (M&D) Bruker-Hain, Hain Lifescience GmbH, 72147 Nehren, Germany

E-mail: alina.merz@bruker.com

For further information visit Bruker (www.bruker.com)

References

1. WHO Coronavirus (COVID-19) Dashboard [website]. 2021, 10 August (https://covid19.who.int/).
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3. Pollock NR, Savage TJ, Wardell H, Lee RA, Mathew A, et al. Correlation of SARS-CoV-2 nucleocapsid antigen and RNA concentrations in nasopharyngeal samples from children and adults using an ultrasensitive and quantitative antigen assay. J Clin Microbiol 2021; 59(4): e03077-20.
4. Covid variants: latest on the Indian, UK, South African and Brazilian variants [website]. Heart Matters; Journal of the British Heart Foundation 2021, May (https://www.bhf.org.uk/ informationsupport/heart-matters-magazine/news/coronavirusand-your-health/covid-variant#UKdeadly).
5. Hasan MR, Sundararaju S, Manickam C, Mirza F, Al-Hail H, et al. A novel point mutation in the N gene of SARS-CoV-2 may affect the detection of the virus by reverse transcription-quantitative PCR. J Clin Microbiol 2021; 59(4): e03278-20.
6. Shu CJ, Huang X, Tang HH, Mo DD, Zhou JW, Deng C. Mutations in spike protein and allele variations in ACE2 impact targeted therapy strategies against SARS-CoV-2. Zool Res 2021; 42(2): 170–181.
7. Pujadas E, Chaudhry F, McBride R, Richter F, Zhao S, et al. SARS-CoV-2 viral load predicts COVID-19 mortality. Lancet Respir Med 2020; 8(9): e70.
8. Fajnzylber J, Regan J, Coxen K, Corry H, Wong C, et al. SARS-CoV-2 viral load is associated with increased disease severity and mortality. Nat Commun 2020; 11(1): 5493.
9. Kawasuji H, Takegoshi Y, Kaneda M, Ueno A, Miyajima Y, et al. Transmissibility of COVID-19 depends on the viral load around onset in adult and symptomatic patients. PLoS One 2020; 15(12): e0243597