Open-Source COVID-19 Test May Offer Greater Specificity, Acceptable Sensitivity

By Laurie Gelb, MPH, BCPA

Current Covid-19 tests are reverse transcription–quantitative polymerase chain reaction (RT-qPCR) assays that first extract RNA from patient swab samples, then detect viral RNA through RT-qPCR amplification of the extracted RNA.

These tests detect the presence of an antigen, rather than antibodies as do serological tests. Thus, they are highly sensitive, but not as specific; false negatives are common (up to 30% in some studies) [2] and have led to the conflicting results, even over a few days, experienced by some patients. Moreover, the reagents and other items used in the RNA extraction step have been in short supply, as has the time and labor this step entails.

To date, no extraction-free tests that work with the World Health Organization (WHO)’s open-source RT-qPCR assay have been developed. Accordingly, University of Vermont and University of Washington researchers have developed and tested the performance of a RT-qPCR approach, while compatible with WHO’s assay, eliminates the RNA extraction step [1]. Instead, patient swab material is loaded directly into the RT-qPCR mix.

Recently, the group reported that this truncated approach (“direct RT-qPCR”) correctly identified 92% of samples (n = 155) identified as positive for SARS-CoV-2 RNA by conventional RT-qPCR. In testing with 60 additional low-load samples, this approach had a sensitivity of 95% on samples with a clinical cycle threshold (CT) at or below 32. This CT level corresponds to a viral load of 1.7 × 104 copies/ml, below the threshold (106–107 copies/ml) at which samples are thought to contain sufficient live virus to signify infection.

In addition, when tested against 30 negative samples, the new method correctly identified each as negative, thus demonstrating 100% specificity, a significant improvement over current PCR tests.

In the next phase of testing, an international network of centers will run similar tests on their own samples.

The advantage of an open-source, more specific test is most readily appreciated when considering developing countries. However, even in more resource-rich environments, there are clearly populations for which greater test specificity would be desirable.

Stratified PCR Testing: A New Option for PCPs

As direct RT-qPCR tests become more widely distributed, primary care physicians will be better able to “triage” patients to the appropriate tests based on symptoms and the testing context.

Patients such as child care workers, students, and other populations that require screening for continued work or program participation might be best suited to the newer test, because trading off some low-viral load sensitivity for specificity could be optimal. On the other hand, health care workers, with broader interaction with vulnerable patients and viral loads that increase over time, might continue to require more sensitive, albeit less specific tests.

References
Bruce EA, Huang ML, Perchetti GA, Tighe S, Laaguiby P, et al. (2020) Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step. PLOS Biology 18(10): e3000896. https://doi.org/10.1371/journal.pbio.3000896

Woloshin S, Patel N, Kesselheim A. (2020) False negative tests for SARS-CoV-2 infection — challenges and implications. N Engl J Med 83:e38 DOI: 10.1056/NEJMp2015897