By Deborah Borfitz
August 30, 2021 | The pandemic ushered in a climate of uncertainty and high-stakes decision-making that has inspired a lot of ingenuity and innovation in clinical laboratories, according to Esther Babady, Ph.D., medical director of the clinical microbiology service at Memorial Sloan Kettering (MSK) Cancer Center, speaking last week at the Next Generation Dx Summit. After COVID-19 was declared a public health emergency early last year, it quickly became clear that labs couldn’t simply sit on the sidelines.
The question initially was whether developing a diagnostic test for the virus, which would require Emergency Use Authorization (EUA) from the Food and Drug Administration (FDA), was even a good idea, Babady says. That was before the first test to be so authorized—a RT-PCR diagnostic panel developed by the Centers for Disease Control and Prevention—roused concerns about accuracy and missteps in deploying the test into public health labs.
Still, clinical labs were hesitant to step up given the complexities of the regulatory process, she continues. The FDA’s Feb. 29 release of guidance on the EUA process was the “game changer” and substantially modified the way assays get validated relative to the traditional 510(k) process.
One day later, New York State reported its first case of COVID-19 and within weeks the governor issued a “pause” executive order closing non-essential businesses statewide, Babady says. Meanwhile, Vice President Mike Pence announced everyone can get tested, and “labs had to figure out how to make it happen.”
Supply Chain Struggles
At MSK Cancer Center, the Feb. 29 decision to start working on a test for SARS-CoV-2 resulted in the creation of a new molecular diagnostic in nine days despite a seemingly endless succession of supply chain issues, she says. “We never thought we would test a virus in saliva,” she adds, noting that saliva had previously been the sample material only on occasion for detecting the flu. But nasopharyngeal swabs, at the time, were nowhere to be found.
Encouragingly, a handful of published studies out of Asia found saliva provided good samples for the SARS-CoV-2 virus, Babady says. The team also wanted to look at the oral rinse technique.
Saliva-based testing won out when its performance was compared to that of swab-based and oral rinse methods, as reported last November in the Journal of Molecular Diagnostics (DOI: 10.1016/j.jmoldx.2020.10.018). While the oral wash approach had good specificity (96%), its sensitivity relative to nasopharyngeal swabs was unacceptable (63%). Agreement between saliva and the nasopharyngeal swabs was 97.7% with a sensitivity of 94.1% (93% and 96.7%, respectively, when using oropharyngeal swab), and the viral load in the nasopharyngeal samples was only slightly higher than in the saliva samples.
Subsequently, researchers elsewhere published a study in the Journal of Clinical Microbiology (DOI: 10.1128/JCM.00876-20) where 160 swab designs were evaluated, and four prototypes ultimately validated, in a clinical trial where volunteers were swabbed with a prototype and reference swab and results were compared with RT-PCR. Cycle threshold values were “pretty equivalent” between each prototype and the control, Babady says.
Viral transport media was also a scarce resource, for which saline became the viable alternative, she continues. Using two different RT-PCR tests, a Mayo Clinic study found RNA integrity was maintained when saline was used to preserve specimens. To deal with a shortage of extraction kits, researchers in Spain ingeniously made do without them by heating patient samples to 65 degrees C. for half an hour without compromising specificity.
The need to expand testing capacity from 100 to 1,000 and then 5,000 per day was another challenge faced by clinical labs, which responded by looking at pooling strategies. Published research showed it was possible to pool between nine and 11 patient samples and achieve sensitivity greater than 95% relative to individual tests, says Babady, and a MSK study on the practice and available on the open science platform Europe PMC (DOI: 10.1016/j.jcv.2021.104790) further found it to be a promising option.
The latter study used the Roche Cobar SARS-CoV-2 test platform, which received an EUA from the FDA for pooling of up to six nasopharyngeal swab samples. Babady and her colleagues evaluated the approach on both nasopharyngeal swab and saliva samples and found pooling in both cases to be a “great option” with, respectively, 100% and 90% sensitivity.
News of the Indian, Brazilian, and South African variants emerged in June 2020 and by the fall discussion about variants (now focused squarely on Delta) became the predominant topic of conversation, she says. To establish the risk of transmission within the hospital, MSK last October began tracking circulating variants of concern by sequencing patients who test positive for SARS-CoV-2.
Most recently, researchers with The Ohio State University have reported on the development of a PCR test specific to variants of concern by screening for spike protein deletions and mutations (Journal of Clinical Microbiology, DOI: 10.1128/JCM.00926-21), Babady notes. A multiplex, RT-PCR test developed at Emory University that detects specific mutations in the spike receptor binding domain also demonstrated 100% sensitivity in detecting variants of concern when compared with SARS-CoV-2 genome sequencing (Journal of Clinical Microbiology, DOI: 10.1128/JCM.01446-21).
Babady says sequencing done at MSK Cancer Center is supported by philanthropic funding and cost-free to patients. “We sequence every single positive [case] if there is enough viral load,” which describes 80%-85% of the positive samples. Nearly 2,000 samples have been sequenced to date.