Knowledge of diagnostic tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, and a clear understanding of the nature of the tests and the interpretation of their findings is important.
This viewpoint describes how to interpret 2 types of diagnostic tests commonly in use for SARS-CoV-2 infections: reverse transcriptase polymerase chain reaction (RT-PCR) and IgM and IgG enzyme-linked immunosorbent assay (ELISA). — and how results may vary over time.
Detection of viral RNA by RT-PCR
Until now, the most commonly used and reliable test for the diagnosis of COVID-19 has been the RT-PCR test performed using nasopharyngeal swabs or other upper respiratory tract samples, including throat swabs or, more recently, saliva.
Different manufacturers use a variety of RNA gene targets, with most tests targeting 1 or more of the genes: envelope (ENV), nucleocapsid (N), spike (S), RNA-dependent RNA polymerase (RdRp), and ORF1.
The sensitivity of the tests to individual genes is comparable according to comparison studies, except for the RdRp-SARSr priming probe (Institut Charité), which has a slightly lower sensitivity probably due to a mismatch in the reverse priming. 1
In most people with symptomatic COVID-19 infection, viral RNA measured by cycle threshold (Ct) is detected as early as the first day of symptoms and peaks in the first week of symptom onset. . The Ct is the number of replication cycles necessary to produce a fluorescent signal; lower Ct values represent higher viral RNA loads. A Ct value less than 40 is clinically reported as PCR positive.
This positivity begins to decrease in week 3 and subsequently becomes undetectable. However, Ct values obtained in severely ill hospitalized patients are lower than Ct values in mild cases, and PCR positivity may persist beyond 3 weeks after illness onset, when most Mild cases will produce a negative result.2 However, a “positive” PCR result reflects only the detection of viral RNA and does not necessarily indicate the presence of viable viruses.3
In some cases, viral RNA has been detected by RT-PCR even beyond week 6 after the first positive test. Some cases have also been reported positive after 2 consecutive negative PCR tests performed 24 hours apart. It is unclear whether this is a test error, reinfection, or reactivation.
In a study of 9 patients, attempts to isolate the virus in culture were not successful beyond day 8 of illness onset, which correlates with decline in infectivity beyond the first week.3
This is partly why the CDC’s "symptom-based strategy" indicates that healthcare workers can return to work, "if at least 3 days (72 hours) have passed since recovery, defined as resolution." of fever without the use of medications and improvement of respiratory symptoms (e.g., cough, shortness of breath); and at least 10 days have passed since the onset of symptoms."4
The chronology of PCR positivity is different in samples other than the nasopharyngeal swab. PCR positivity decreases more slowly in sputum and can still be positive after nasopharyngeal samples are negative. 3
In one study, stool PCR positivity was observed in 55 of 96 (57%) infected patients and remained positive in stool beyond the nasopharyngeal swab for 4 to 11 days, but was not related to clinical severity. 2 The persistence of PCR positivity in sputum and feces was found to be similar, as assessed by Wölfel et al.3
In a study of 205 patients with confirmed COVID-19 infection, RT-PCR positivity was highest in bronchoalveolar lavage samples (93%), followed by sputum (72%), nasal swab (63%), and pharyngeal swab ( 32%).5 False negative results occurred mainly due to inappropriate timing of sample collection in relation to disease onset and deficiency in sampling technique, especially nasopharyngeal swabs.
The specificity of most RT-PCR tests is 100% because the primer design is specific to the SARS-CoV-2 genome sequence. Occasional false positive results may occur due to technical errors and reagent contamination.
Detection of antibodies against SARS-CoV-2
COVID-19 infection can also be detected indirectly by measuring the host’s immune response to SARS-CoV-2 infection.
Serological diagnosis is especially important for patients with mild to moderate disease who may present late, beyond the first 2 weeks of disease onset.
Serological diagnosis is also becoming an important tool to understand the extent of COVID-19 in the community and identify people who are immune and potentially "protected" from becoming infected.
The most sensitive and earliest serological marker are total antibodies, whose levels begin to increase from the second week after the onset of symptoms. 6
Although IgM and IgG by ELISA have been found to be positive as early as the fourth day after the onset of symptoms, the highest levels occur in the second and third weeks of illness.
For example, IgM and IgG seroconversion occurred in all patients between the third and fourth week of onset of clinical disease as measured in 23 patients by To et al.7 and 85 by Xiang et al.8 Thereafter. IgM begins to decline and reaches lower levels at week 5 and almost disappears at week 7, while IgG persists beyond 7 weeks. 9
In a study of 140 patients, the combined sensitivity of PCR and IgM by ELISA targeting nucleocapsid (N) antigen was 98.6% versus 51.9% with a single PCR test. During the first 5.5 days, quantitative PCR had a higher positivity rate than IgM, while IgM (ELISA) had a higher positivity rate after day 5.5 of illness. 10
ELISA-based IgM and IgG antibody tests have a specificity greater than 95% for the diagnosis of COVID-19. Testing serum samples paired with the initial PCR and the second two weeks later may further increase the accuracy of diagnosis. Typically, most antibodies are produced against the most abundant protein of the virus, which is N.
Therefore, tests that detect antibodies against N would be the most sensitive. However, receptor binding domain protein S (RBD-S) is the host binding protein, and antibodies against RBD-S would be more specific and are expected to be neutralizing. Therefore, the use of one or both antigens to detect IgG and IgM would result in high sensitivity. 7 However, antibodies may be cross-reactive with SARS-CoV and possibly other coronaviruses.
Rapid tests for the detection of antibodies have been widely developed and marketed and are of variable quality. Many manufacturers do not disclose the nature of the antigens used. These tests are purely qualitative in nature and can only indicate the presence or absence of antibodies to SARS-CoV-2.
The presence of neutralizing antibodies can only be confirmed by a plaque reduction neutralization test. However, it has been shown that high titers of IgG antibodies detected by ELISA correlate positively with neutralizing antibodies. 7
The long-term persistence and duration of protection conferred by neutralizing antibodies is unknown.
Conclusions
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