Page 48 - IMCJ19s1

• Antibody production begins soon after symptoms viral RNA. SARS-CoV-2 has an RNA genome; thus,
(5-14 days). determining if RNA sequences specific to the known
• Timing of antibody development (IgM, IgG, & IgA) SARS-CoV-2 genome are present can determine if viral
varies across individuals. particles are or were recently present in the sample. This
• Median antibody production is at 15 days after test is not a direct measure of infective virus, which would
exposure. have to be evaluated with viral culture. Viral RNA detection
is most often achieved via nucleic acid amplification
Accuracy methods (NAA) using a polymerase chain reaction (PCR).
Before assessing the specific tests in more detail, it is Samples are collected from nasopharyngeal (NP) swab,
important to understand the accuracy of a test; in other oropharyngeal (OP) swab, bronchoalveolar lavage, sputum
words, how likely is a test to return a true result? We (research), or saliva and then placed in medium that
measure the accuracy of a diagnostic test via the following preserves the RNA.
characteristics: At the lab, RNA is isolated from the sample and then
subjected to a reverse transcription reaction to convert the
• First, sensitivity, which is the percentage of truly RNA sequence to DNA. Specific part(s) of the sequence
infected individuals who will test positive on a test. are then amplified using real-time reverse transcription
• Second, specificity, which is the percentage of truly PCR (RT-PCR). The RT-PCR technique uses primers,
uninfected individuals who test negative. which are short fragments of DNA that are complementary
to specific parts of the transcribed viral RNA. These
The sensitivity of a test shows how well a test detects the primers are added to the reaction with appropriate
proportion of true positives (TP); the probability that the test enzymes that amplify the target sequence hundreds to
will capture a positive result and avoid a false negative (FN) thousands of times through successive cycles of heating
result: (TP/TP+FN). Clinically, using RNA nucleic acid and cooling, making it easier to identify whether the
amplification (NAA) tests with high sensitivity ensures that specific sequence is present in a large amount of other
clinicians do not falsely miss the presence of the virus. genetic information such as host DNA. The RT-PCR
The specificity of a test shows how well a test detects technique uses fluorescently labeled DNA strands to
the proportion of true negatives (TN); the probability that monitor the amplification reaction and identify the
the test will not give a false positive (FP) result: presence of the specific strand in “real-time” as the
(TN/TN+FP). Clinically, using serology tests with high reaction proceeds. It has the advantage over conventional
specificity ensures that clinicians do not falsely assume the PCR in that it takes place in a single reaction, resulting in
presence of antibodies to the virus (e.g. a false positive from less chance for error and a shorter completion time. It has
antibodies to SARS-CoV-1 or other viruses). the additional advantage of being quantitative, thus giving
Additional characteristics of diagnostic tests that are an estimate of viral load (i.e., quantity). Simultaneously
important to consider are positive predictive value, or PPV, performing multiplex RT-PCR, which tests for multiple
and negative predictive value, or NPV. Positive predictive target sequences, increases both sensitivity and specificity. 11
value is defined as the percentage of those testing positive Individual laboratory assays have different cut-offs
on a test who are truly infected. Put another way, if you have for the amount of RNA that defines a positive result. This
someone who tests positive, the positive predictive value is will affect the sensitivity and specificity of the assay. Thus,
the percent chance that that person actually does have the test performance characteristics and cut-offs are
infection. Negative predictive value is defined as the laboratory-dependent and will affect clinical and
percentage of those testing negative on a test who are truly epidemiological interpretation. Finally, despite the ability
uninfected. Both PPV and NPV depend not only on the to quantify the viral RNA in the initial sample, most
sensitivity and specificity of the test but also on the results are reported as a qualitative presence or absence of
prevalence of infection in the population studied. viral RNA. Quantitative results are primarily used in the
Issues of sampling error with antigen testing (insufficient research setting at this time.
or incorrect sample procurement) are noted below and are Novel amplification techniques such as isothermal
independent of the sensitivity and specificity of the assay. amplification and loop-mediated isothermal amplification
(LAMP) which utilizes unique primers and DNA
Understanding Testing polymerases that allow rapid amplification of the targeted
Viral Testing sequences at a constant temperature are also available.
Viral RNA
RT-PCR Techniques CRISPR-Cas12
Tests that look for the presence of SARS-CoV-2 in the In addition to the widely adopted RT-PCR discussed
respiratory tract are used to determine if an individual has above, new methods also include the use of CRISPR-
an active infection and/or is contagious. Most of the tests Cas12 gene targeting technology to test for the presence of
currently available for this purpose detect the presence of SARS-CoV-2 RNA in OP & NP swabs. CRISPR-Cas12 can

46 Integrative Medicine • Vol. 19, No. S1 • Epub Ahead of Print Messier—Primer on SARS-CoV-2 Testing