PARIS – Stilla Technologies SAS, of Villejuif, France, is supplying a new, cost-effective approach for COVID-19 testing by combining its digital polymerase chain reaction (PCR) technology with the group testing method. “This approach greatly increases testing capacity and meets the highest quality standards,” Rémi Dangla, co-founder and CEO of Stilla Technologies, told BioWorld.
Real-time-PCR (RT-PCR) represents the standard method of testing for the presence of COVID-19 in a sample. However, the technique is reaching its limits as demand for SARS-CoV-2 RT-PCR testing increases worldwide.
For example, testing individual samples using RT-PCR is not scalable. Indeed, facilities face a global shortage of reagents, while demand is reaching millions of tests. Laboratories experienced a severe shortage of reagents at the start of the pandemic and remain under pressure as they stock up on supplies. In addition, the cost of testing is not scalable to the world population.
“Group testing, or sample pooling, could be offered as a solution to expand testing capability,” said Dangla. The idea is to pool samples together before moving on to do the PCR test. If the test is negative, it means that all members of the group being tested are healthy individuals. On the other hand, if the test is positive, at least one [member] of the group is carrying the virus. The infected patient then can be pinpointed by undergoing an individual test. The advantage of this method is a saving in the overall number of tests required to screen a given population, and thereby an increase in testing capability for fixed reagent and instrumentation resources. Savings depend on key parameters, such as disease prevalence and group size.
Digital PCR, technology compatible with group testing
Group testing is not new; Robert Dorfman used it in 1943 for a program to detect syphilis in the U.S. military. It also has been used regularly for cases of hepatitis B, avian pneumovirus and, in 2010, HIV. Group testing protocols using RT-PCR have been evaluated and implemented for COVID-19 screening around the world in several experiments using RT-PCR detection techniques, notably in Israel, Germany, California, Nebraska, New York state and Italy.
Although these studies have shown that positive individuals can be detected in pooled samples, the number of amplification cycles needed to detect those often is increased by dilution, and perhaps by inhibition effects. This can prevent a weak positive specimen from being detected in group samples. Concerns about the sensitivity of group testing have been raised by French medical authorities, leading to a recommendation against their use in that country.
“Digital PCR (RT-dPCR), and especially our Naica system platform, is known for its higher sensitivity and precision over classical RT-PCR,” said Dangla. Its three-color PCR platform, Crystal digital PCR, uses microfluidic technology to integrate the whole digital PCR process onto a single chip. Recent studies have confirmed the high sensitivity of Crystal digital PCR in detecting SARS-CoV-2 – up to 10 times more than RT-PCR. Thus, associating digital PCR with the group testing method would address the main problem of sensitivity.
Largest comparative study performed to date
The company has wrapped up a study that systematically compared group testing for SARS-CoV-2 to individual reference tests using the Cobras SARS-CoV-2 from Roche Holding AG. This study was conducted in May by the Virology Department at Bichat Hospital in conjunction with the Center for Research in Economics and Statistics.
“We tested the protocol in a direct comparison with reference RT-PCR testing on 448 samples, split into three groups for RT-dPCR analysis: 56 groups of eight samples, 28 groups of 16 samples and 14 groups of 32 samples,” said Dangla. This was the first study to evaluate group testing combined with digital PCR.
Of the 448 samples tested, 26 and 25 tested positive using the digital PCR group test, for group sizes of eight and 16 samples. Against this, 25 tested positive with individual reference tests in RT-PCR. Thus, the study demonstrates a comparable to better sensitivity for group testing using digital PCR vs. individual testing using RT-PCR.
Individual RT-PCR testing identified 25 positive samples. For groups of eight, testing by RT-dPCR identified 23 groups as positive, corresponding to 26 true positive samples, including two samples not initially detected by individual RT-PCR, but confirmed positive by further RT-PCR and RT-dPCR testing. For groups of 16, 15 groups tested positive, corresponding to 25 true positive samples identified. “One hundred percent concordance is found for groups of 32 but with limited data points,” said Dangla.
The French study showed that the group testing approach using digital PCR with the Naica system displays a similar or even better diagnostic sensitivity compared with individual RT-PCR testing for group sizes up to 16 samples. “The main benefit of this method is that it reduces the amount of reagent required to test a population by about 80%, while reducing costs … and increasing test capacity by up to 10 times,” said Dangla. Moreover, this approach multiplies the number of individuals tested for a given amount of reagents by a factor of five.