Monday June 14, 2021

A new review published Monday, July 19, 2021, in Pathogens and Immunity looks at nucleic acid amplification testing and the essential factors influencing the performance of diagnostic tests. Authors also offer guidance on the benefits and limitations to antigen and RT-PCR testing related to the novel coronavirus.

Nucleic acid amplification and antigen-detection tests have played an important role in the defense against SARS-CoV-2. Results from nucleic acid sequencing of the novel coronavirus have led to the development of highly sensitive RT-PCR tests which can identify the presence of virus in the respiratory tract. As more information was collected and these tools evolved, these tests have been used to inform best practices for patient care and broader strategies for public health.

A nucleic acid amplification test (NAAT) is a diagnostic test that is used to detect genetic material related to a specific virus. A sample is taken from the nose or saliva of a person suspected to be infected. The test makes copies of the genetic material present in a person’s sample in order to detect even small amounts of virus.

While factors influencing efficacy of these tests include how the specimen is collected, stored and processed; the sensitivity and specificity of the test; and whether or not the individual being tested is symptomatic, it is important to acknowledge that response to the COVID-19 outbreak required a fluid application of these established diagnostic strategies while simultaneously developing controls for benchmarking diagnostic test performance, the authors write.

“As optimization of various diagnostic assays continues, a system for monitoring the resilience of the diagnostic assays in the face of SARS-CoV-2 evolution has not yet been developed. Given the rapid emergence of COVID-19, it has not been surprising that intense scrutiny has been focused on diagnostic test performance and validity, and that measures of these metrics vary across studies,” write Gary Procop, MD, Cleveland Clinic, Cleveland, Ohio, lead author of the review and colleagues.

According to the review, one should keep in mind the following things regarding molecular diagnostic testing, crossing threshold values, genome sequencing and antigen detection tests:

  • When assessing test performance, it is important to distinguish whether a test is being used as a diagnostic test or a screening test. Applied as a diagnostic test, there should be a high pre-test probability that the patient is infected.
  • It is not possible at this time to identify a crossing threshold value after which a patient can be designated as non-infectious. According to the authors, crossing threshold values have been most effectively used to clarify or confirm clinical findings.
  • As the virus continues to mutate, viral genome sequence variation may contribute to false negative outcomes where the RT-PCR creates a mismatch against the viral sequence.
  • Antigen tests for respiratory viral pathogens are less sensitive than RT-PCR assays. Antigen tests are most useful to confirm infections in a symptomatic patient. If the antigen test is negative on a symptomatic patient, the patient should be retested with an RT-PCR assay.

“As the genetic landscape becomes more complicated, we will need multiple tools to assist us in the implementation of appropriate diagnosis, the administration of therapy, and the execution of in­fection control,” the authors write. “We remain entirely optimistic that we will find the appropriate testing algorithms and technologies that will help us overcome this global threat.”

The authors report no financial support for the research, authorship, and/or publication related to this article.

The full paper is available freely at the Pathogens and Immunity website:

DOI: 10.20411/pai.v6i1.422

Media Contact:

Gary W. Procop, MD

[email protected]

About Pathogens and Immunity

Pathogens and Immunity is a novel open access journal published at Case Western Reserve University. We consider manuscripts describing original research that is relevant to human health and disease. We look for work that offers new perspectives or asks new questions about microbes and/or host defenses. Our underlying mission is to make the process of biomedical publication better. We charge no fees to authors and will review submissions in any format. The initial online submission process takes less than 5 minutes.