Faced with an ongoing lack of protective equipment and testing supplies, medical professionals have been seeking alternatives to accurately diagnose cases of COVID-19, a pandemic that has caused more than 11 million cases and more than 530,000 deaths worldwide. Supplies of nasopharyngeal swabs were some of the first testing materials to run low in mid-March, prompting a pivot to nasal swabs. More recently, saliva-based testing has come forward as an attractive, low-cost alternative.

The first spit tests are already being sold to consumers, with more poised to apply for emergency use authorization from the US Food and Drug Administration soon. While saliva can be a crude sample for diagnosing disease using traditional PCR, it pairs well with a cheap PCR alternative known as loop-mediated isothermal amplification (LAMP), previously used to detect outbreaks of Zika and Ebola in resource-poor countries. Propelled by a global pandemic, researchers in the US and the UK are now modifying LAMP and assessing its utility as a diagnostic tool for COVID-19.

“By diversifying the possible choice of assay, you diversify the supply chain as well,” says Robert Meagher, a chemical engineer at the Sandia National Laboratories who develops tools for diagnosing emerging diseases. Making additional testing options available to healthcare workers will help mitigate backlogs if one reagent or material runs low. 

Saliva-based testing also offers an improvement over the standard nasopharyngeal swab because people can collect their own samples with minimal discomfort—simply spit into a sterile tube and mail it to a lab for processing. And due to an emergency use authorization given to Rutgers University in May, some tests can now be carried out by patients in their own homes, allowing personnel and protective equipment to be saved for when they are most needed. Andrew Brooks, the director of technology development at RUCDR Infinite Biologics, the Rutgers-affiliated biorepository that developed the test, says the use of in-home testing “completely mitigates the risk of contracting the disease while you’re getting a test,” and requires only gloves. 

Several companies are selling Rutgers’s kits directly to consumers through online orders, although all tests must be sent back to its lab in New Jersey for processing. Positive results are reported to local health officials, and individuals are advised on how to respond.

Universities and clinicians alike are now planning to incorporate saliva-based sampling into their workflow for diagnosing COVID-19. The UK government recently partnered with the molecular diagnostics company Optigene to develop a pilot study involving more than 14,000 people to test the efficacy of its saliva test. In the US, the University of Chicago will use spit tests to clear patients prior to elective surgeries in its hospital for the next several years, Evgeny Izumchenko, a UChicago oncologist who helped develop the university’s test, tells The Scientist.

In another screening application, University of Illinois chemist Martin Burke laid out plans to use a new test designed by himself and colleagues on more than 50,000 students as they return to campus in the fall. “We imagine this will be just part of their orientation,” Burke said during a webinar on June 16. “So you get your housing information, your dining card, your ID card, and you also submit your saliva sample.” 

Despite the ease of sampling, saliva is not without its challenges. For PCR, the virus’s RNA must first be reverse transcribed into DNA, and saliva contains enzymes that chew up nucleic acids and inhibitors that interfere with the DNA amplification process used to detect the virus. As a result, saliva must often be purified before the DNA can be amplified. “It’s kind of a dogma . . . that you have to start with absolutely pure DNA or RNA,” Meagher says.

LAMP in the spotlight

While quantitative reverse transcription PCR remains the most-used method to diagnose COVID-19 regardless of how the sample is collected, many of the bottlenecks in the pipeline stem from the high cost and low scalability of the approach, says Meagher. A single benchtop thermal cycler, the machine that controls the temperature changes during the PCR, typically costs around $25,000 and can usually only run between 96 and 384 samples at a time.

To address these challenges, a team of researchers at Columbia University recently modified a LAMP protocol used by their fertility clinic to identify abnormalities in chromosome numbers in human embryos. Rather than count chromosomes, the tool now detects the presence of SARS-CoV-2 RNA in saliva in as little as half an hour, changing the color of the sample from red to yellow when the virus is present.The team’s new test is one of several using LAMP. Unlike PCR, which creates new copies of DNA through cyclical temperature changes, LAMP reactions take place at a consistent 63°C, eliminating the need for complex machinery. The chemicals used in the reaction are also more robust against the enzymes and inhibitors in saliva, doing away with the need to purify each sample.LAMP has traditionally been deployed in resource poor countries because it requires less power and equipment than PCR. But under the current pandemic, the whole world has become somewhat resource limited, and Meagher stresses that LAMP can be just as useful in diagnosing emerging diseases in first-world nations such as the US.

Author: nupurmisra