P-BEST: Fast efficient COVID-19 virus testing before symptoms appear

By Dr. Liji Thomas, MDApr 22 2020

A highly promising new study published on the preprint server medRxiv in April 2020 reports an efficient new method to conduct high-throughput testing of blood samples for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which is causing the rapidly spreading pandemic of COVID-19 disease throughout the world. The method could revolutionize the diagnosis of new cases before they become symptomatic.

Why was the new test developed?

The spread of SARS-CoV-2 appears to have resisted efforts in most democratic countries to contain it. Recent studies report that up to a third of infected people remain asymptomatic, and many more shed large amounts of the virus before the onset of symptoms.

Study: Efficient high throughput SARS-CoV-2 testing to detect asymptomatic carriers. Image Credit: David Herraez Calzada / Shutterstock

In view of the transmission of the virus from both asymptomatic and presymptomatic individuals, it is necessary to routinely identify all infected individuals to stop the march of the pandemic. However, there are significant bottlenecks inhibiting the ability to carry out such diagnostic testing.

These include not only inadequate laboratory facilities but the limited availability of the reagents needed to carry out the real-time polymerase chain reaction (rtPCR) tests that directly detect the virus.

How does the new test work?

The principle of the new testing method, called P-BEST - a method for Pooling-Based Efficient SARS-CoV-2 Testing, is based on a low percentage of carriers in the tested population, below 1%. In such a case, the pooled sample can yield a correct identification of all positive individuals with a much lower requirement of diagnostic tests compared to individual sample testing.

The group-testing method is based on pooling samples into groups, each of which is tested separately for the virus by the rtPCR assay that is the current standard of diagnostic testing. Using a combinatorial pooling strategy maximizes the odds of identifying all positives within a large pool.

The current study

The present study was a proof-of-concept study where 384 patient samples were pooled into 48 pools of 48 unique samples each. Each pool was designed on the basis of a Reed-Solomon error-correcting code. Liquid consisting of samples diluted in a lysis buffer was added to each pool by an automated robot.

After carrying out PCR on each of the 48 pools within each set of 384 samples, the results were broken down by a special decoding algorithm to identify the carriers. Each such carrier was then identified separately.

The pooling time took less than 5 hours, and a standard biosafety level 2 laboratory was used in view of the inactivation of all viral particles by the lysis buffer used to dilute the individual samples.

The samples were from already tested patients. The testing set consisted of four sets of 384 samples each. Each of these had an increased number of positive carriers, from 2 in the first to 5 in the fourth.

What did the study find?

The investigators found that using P-BEST, they could correctly pick up all the positive carriers from the four sets of 384 samples, pooled into 48-sample pools. The significant advantage was that only 48 PCR tests needed to be carried out for each set. In other words, the use of reagents jumped eight-fold in efficiency.

When they simulated the method, it was found that P-BEST can be used to rightly identify all the carriers, even up to 5/384 samples in the final set, which comes to only 1.3% of the whole set. The number of false positives is less than 2.75, with less than 0.33 negatives.

What does the study show?

The study shows that inactivated virus samples can be used, with the samples diluted in lysis buffer. This allows automated pooling techniques to be carried out in laboratories conforming to lower levels of biosafety (BSL-2) due to the non-infectious nature of the pooled samples. The only additional equipment needed is a basic and readily available automated dispensing robot for laboratory use.

The increase in efficiency of reagent use by a factor of 8 is not the maximum that can be achieved; by increasing the number of samples pooled within a set, even greater savings of reagent can be accomplished with the same equipment. This can boost the number of tests that can be done with the same resources in terms of not just reagent, but staff and laboratory equipment.

Early follow-up experiments show that a single positive sample can be picked out even from a pool of 128 subjects, allowing for more than 1000 tests to be done. Thus the present pooling technique, P-BEST, is optimally designed for the fast and efficient screening of low-risk asymptomatic populations with a low carrier frequency. It should not be used to test people who are at high risk of being infected, for instance, those who came into contact with COVID-19 patients.

With a carrier frequency above 1.3%, the P-BEST method is less efficient. This has a side benefit: it can help to identify hotspots for viral transmission. The scientists conclude, “P-BEST provides an efficient and easy-to-implement solution for increasing testing capacity that will work with any clinically approved genome-extraction and PCR-based diagnostic methodologies.”

Important Notice

medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.