By Dr. Peter Ikhianosimhe Imoesi
The use of Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) in pool testing is not new in molecular diagnostic, but of common use is the individual testing method.
Also, there is nothing wrong with the individual testing approach in use by the Nigeria Centre for Disease Control (NCDC). I was initially an advocate for individual testing; however, this approach is not feasible for a country like Nigeria with a population of over 200 million people.
Again, with the shortfall in supplies of required consumables to run the RT-qPCR, it is clear we will continue to struggle in ramping up our testing capacity. Worth mentioning is that individual testing requires the use of more consumables and by extension, capital intensive.
This is not just limited to supplies, but also the required human capacity in terms of competence and skilled experts. These limitations are evident in the total number of tests (23,835, NCDC, 7TH May 2020) we’ve done so far, with over 2,500 backlogs of samples according to Dr Ifeanyi Casmir – National Publicity Secretary, Nigeria Medical Laboratory Scientists – Channels Television program, 07 May 2020, 9 pm, left undone.
In the case of a small outbreak e.g. endemic disease, RT-qPCR individual testing is feasible. However, in a pandemic such as the one at hand – COVID-19 disease this may not be sustainable. And ramping up testing with this approach in many developing countries – Nigeria may require months if not years. The resultant effect of under-testing will potentiate the continuous transmission of the disease among the entire population owing to the slow pace at which we identify, and isolate COVID-19 positive cases.
Apparently, if we continue with this individual testing approach, we may have more people infected with the COVID-19 disease, and since we are under-testing, the virus will spread further. Little wonder Dr Chikwe Ihekweazu said, we may likely live with the virus for a little longer, why, because we are not testing enough to quickly isolate infected individuals. Science is exploratory, shouldn’t we explore other testing approaches like sample pool testing? At this point in time, and as a trained Molecular Biologist and Molecular Neuroscientist, I am of the opinion we adopt sample pool testing if truly we intend to halt the spread of the virus quickly. Except there is a form of political undertone.
Pool sample testing was first proposed for syphilis screening and the same testing approach has been employed in screening blood donors for antibodies to HIV, hepatitis B and C virus respectively. This sort of testing can obviate 90% of individual tests, with significant resources save and yet maintaining high specificity (86%) and sensitivity (95%) without necessarily sacrificing diagnostic certainty.
First, we need to establish if the current primers in used by the NCDC is highly specific without necessary generating primer-dimers and by extension false-positive results. To effectively establish this, we need to run a standard thermal cycler PCR test with the said primers and subsequently, run an agarose gel electrophoresis to establish the specificity of the primers (single band). Also, to boost the sensitivity of the pool testing RT-qPCR assay, we could increase the final reaction mix to 20 or 25ul per well and of this volume, 8ul should come from the sample pool cDNA. There is evidence that the molecular pool testing can detect DNA concentration of about 0.001ng/ul; this further justified the high level of RT-qPCR sensitivity.
Since asymptomatic populations may typically show low levels of the virus, pool testing could easily damp the sensitivity of the assay and by extension false-negative results. However, this can be addressed by using a few sample pool, larger amount of cDNA from each sample to make the sample pool and increasing the reaction mix as previously explained.
Pool Testing Procedure
What is sample pool? a sample mix made up from a number of individual samples pull together into a tube to form one whole sample. For instance, you could mix ten individual samples to produce a single sample tube. To increase the level of sensitivity of the test, one could increase the required volume from each sample. However, since RT-qPCR is highly sensitive, 10ul of cDNA from the individual samples may work just fine. The sample pool is then mix thoroughly before use. In any case, this may require careful optimisation to ascertain the actual volume required to generate precise results.
A layman explanation of pool testing is a cup of salty water (COVID19 positive – this is unknown to the scientist), and nine other cups with normal water only (COVID19 negative equally unknown to the scientist). To achieve pool testing, you withdraw 4 teaspoons from the salty water and also from the nine normal water into an empty cup and mix thoroughly.
If you have a good taste bud, the final mixture should taste differently. The taste bud here is the same as the highly sensitive RT-qPCR. If by chance you presume you’ve got poor taste bud, you could increase the number of teaspoons.
The same approach is employed in pool testing with a virus were many are asymptomatic. The flipside of this is that, if the final cup mixture tastes salty, one is then required to taste the individual cups to ascertain the cup with the salty water (COVID19 positive).
However, when the final mixture is tasteless, all ten cups are declared clean (COVID19 negative); this is the speed side of pool testing. When you have 100 samples for instance, and you batch the samples into a pool of ten samples each, for every pool of test samples that comes out negative, that is 10 individual samples in 2 hours run as against one sample run in 2 hours. Based on the above explanation, let’s integrate this into actual molecular testing.
The cups I mentioned initially is the same as a 96 or 384 well plate in RT-qPCR experiment. A 96 or 384 well plate means the plate can hold 384 samples and if you intend to run the samples in duplicates, that is 192 samples per plate and run.
Depending on the protocol, RT-qPCR could run for 1h:30min or 2hours. A 384 well plate is numbered from left to right (1 – 24 wells) and from top to bottom A – P respectively. In pool testing, each well represents 10
individual sample and in duplicates across 24 wells divided by 2 that is 10 samples into 12 wells and a
total of 120 samples across the plate row.
Leaving out the bottom wells i.e. P1 – P24 for positive or negative control samples; 2 hours run will produce 1,800 samples in the entire plate. Assuming only 10 wells produce COVID19 positive result, i.e. 10 samples in a well multiply by 10 wells = 100 samples, this would mean running 100 individual samples separately. However, we have achieved 1,700 (COVID-19 negative cases) sample run in 2hours. Again, if for instance, from sample
preparation to RT-qPCR takes about 4 hours, it means we could achieve 10,800 tests in 24 hours per
So far, the NCDC has done a total of 23,835 tests and of these numbers, only 3,912 are confirmed positive cases. This simply means the percentage of positive case as of 9th May 2020 is 0.16%; this into 10,800 tests per machine (as previously explained) would mean 1,728 positive COVID19 cases to be rerun from the pool testing. However, we’ve successfully run 9,072 COVID-19 confirmed negative cases and 1,728 COVID-19 confirmed positive cases respectively.
Individual COVID-19 RT-qPCR testing is highly expensive for low- and middle-income countries, and with the current deficit in supply chain, sourcing for needed reagents will limit these countries in conducting a large number of tests. However, a number of countries (Ghana, South Africa, Andaman and Nicobar Island, and German Red Cross Blood Donor Service and the Institute of Medical Virology at the University Hospital Frankfurt at Goethe University have equally developed pool testing procedure) have adopted the pool testing strategy in the fight against COVID19. If we must catch up with this coronavirus, we need to change our strategy and that strategy is migrating from individual testing to carefully optimised sample pool testing.
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