Can we possibly improve the tools used for the testing of COVID-19?
What is Lab-on-a-chip? Can we further improve the diagnosis tools for COVID-19 and what is the science behind ID NOW test?
Since the outbreak of COVID-19, which is caused by (SARS)–CoV-2, there has been a staggering demand for affordable tools to diagnose COVID19. However, the conventional way of using RT-PCR and antibody testing to diagnose the patients needs special instruments, time-consuming and costly. Therefore, coping with the demand for these tools is causing havoc in rich countries and it is inevitable that COVID-19 will devastate poor countries. The map below shows how the pandemic COVID-19 is widespread across Africa and other poor countries (updated in 10–4–20).
However, these numbers are not accurate and it is very likely that there are larger outbreaks going on unnoticed in countries that don’t have access to tools in order to diagnose their patients.
The outbreak of the pandemic COVID-19 made us realize the shortcoming of the current diagnostic platforms. With the advances of our knowledge, it is possible to optimize the current tools to create more sensitive, affordable and easy to perform tools.
The conventional way to spot any virus (including HIV, hepatitis C, enterovirus, HSV, VZV, …) is by extracting the genetic materials (RNA in our case) then amplify it and read it so we would know if it belongs to the virus in question (e.g SARS-CoV-2) using a method called Polymerase chain reaction (PCR). The other way is less sensitive as it doesn’t look into the virus itself but detect the antibodies which were generated in our body to fight the virus. The tools are usually based on an immunoassay (e.g. Enzyme-Linked Immunosorbent Assay (ELISA)). This method was extensively discussed in my previous article. Since PCR is not a unique tool for (SARS)–CoV-2, is it possible to be developed further?
What is polymerase chain reaction (PCR)?
Briefly, this method needs a special instrument and can last between 4–6 hours. At first, RNA should be extracted from the sample (this step itself needs a minimum of 2 hours using typical kits). After RNA extraction, we need to amplify the virus RNA. This is done by two primers and an enzyme called Taq polymerase.
The primers are only short template sequences that correspond to part of the virus RNA sequence. They bind to the template at their complementary sites and serve as the starting point for copying whereas Taq polymerase does the building by adding on free nucleotides. The reaction is repeatedly cycled through a series of temperature changes, which allow many copies of the target region to be produced.
The disadvantage of this method:
- Unprocessed clinical samples of blood, sputum, and mucous swabs can’t be applied immediately. RNA should be extracted using other kits. The extraction steps itself takes an average of 2 hours.
- It needs temperature cycling. It relies on three thermal cycling steps to amplify a target sequence (e.g.RNA). In other words, regulation of temperature multiple times is needed which makes PCR unappealing for point-of-care (POC) testing.
- It usually relies on special instruments for reading the results as seen in the CDC PCR kit.
Can we automate the RNA extraction and PCR in one step?
On March 20, 2020, FDA has approved a point-of-care COVID-19 diagnostic for the Cepheid Xpert Xpress SARS-CoV-2 test. However, this test has to operate on one of Cepheid’s automated GeneXpert systems. Therefore, rural areas in poor countries might find these systems unaffordable.
Can we develop the diagnostic tool further and reduce both time-to-result and cost in order to be used in poor countries? Yes
1) Isothermal exponential amplification techniques:
Since PCR was invented in 1985, scientists developed other methods which are referred to as “Isothermal amplification techniques”. These include strand-displacement amplification (SDA), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), helicase-dependent amplification (HDA), and recombinase polymerase amplification (RPA), nicking enzyme amplification reaction (NEAR) which have great potential for on-site and point-of-care (POC) applications. Detailed schematic and animation of the complex amplification scheme can be found on-line.
Most of these techniques can use unprocessed samples and don’t need an extra step of RNA or DNA extraction and they outpreform PCR in terms of speed and sensitivity.
The ID NOW™ COVID-19 from Abbott which takes a maximum of 14 minutes to produce results directly from nose or throat swab samples (unprocessed) is based on isothermal exponential amplification technique called nicking enzyme amplification reaction (NEAR).
Nicking enzyme amplification reaction (NEAR) is simply relies on the synergy of two enzymes which are nicking endonuclease and DNA polymerase. These two enzymes help each other to produce a continuous cleavage and extension of a specific part of the DNA or RNA. Although ID NOW™ COVID-19 is extremely fast but it is not an instrument-free tool and might not be very affordable to be used in very poor countries. So, is there a better way? Yes, we can use lateral flow assays (LFA) in combination with isothermal exponential amplification techniques to produce low-cost instrument-free diagnostic tools.
2) Lateral flow assays (LFA):
There is a growing need for lab-on-a-chip devices. This term referred to the miniaturization of devices into a single chip on a very small scale. The most common example is lateral flow assays (LFA), where analytes wick through a paper strip, with the help of labeling reagents that eventually produce a visible band in a readout (e.g. pregnancy test). Combining a type of isothermal exponential and lateral flow assays (LFA) was used in tools to diagnose HIV and other diseases caused by parasites which are common in Africa such as Trypanosoma brucei. For example, Coris Bioconcept has used NASBA amplification and LFA to produce assay for the diagnosis of 80 different species.
Not just that this method offers instrument-free testing and but also displayed analytical sensitivity, far exceeding that of microscopy and PCR, and largely benefiting developing countries where the cost and bulk of the more sensitive PCR assay are prohibitive. It is highly likely that isothermal amplification strategies will become commonplace in the next generation of point-of-care diagnostic devices.
In these unprecedented and uncertain times, the burden of COVID-19 takes place in low-income and middle-income countries, there is an unmet need for reliable diagnostic methods to identify COVID-19 rapidly.
We can’t overlook the damage COVID-19 will cause to poor countries.
Therefore, we will need to develop a similar sample-to-answer system with minimum user intervention for COVID-19.
