Frequently Asked Questions
What are the components of the real-time RT-PCR diagnostic tests for the COVID-19 virus?
Polymerase Chain Reaction (PCR)– Is a means of amplifying targeted DNA fragments billions of times. The DNA polymerase enzyme synthesizes DNA sequences that are complementary to the targeted sequence of interest. This “targeting” is directed by primers defined to flank both ends of the DNA sequence of interest.
Reverse Transcription PCR (RT)– In this process, the enzyme reverse transcriptase uses mRNA in lieu of DNA to synthesize a single stranded DNA sequence.
RT-PCR– Is the combined process in which initially, mRNA (isolated from the tissue of interest) is the template utilized by reverse transcriptase to synthesize a single stranded DNA chain. Subsequently, DNA polymerase is used to convert the single stranded DNA chain into double-stranded DNA. A fragment of the DNA can then be amplified to indirectly identify the mRNA of interest present in the original source sample.
Real-Time Quantitative-RT-PCR– This procedure results in the measurement of products of interest generated during a PCR cycle. An identifiable oligonucleotide probe (which can contain a fluorophore source) that can emit a flourescent signal of a specific frequency is integrated into the desired target sequence region. During the PCR process, cleaving of the probe and degradation leads to the emitting of a fluorescent signal. Upon detection of the fluorescence, the quantified amount of mRNA in the source sample is determined.
Are these tests FDA approved?
On June 18, 2020, the FDA granted an EUA for BioHit Healthcare’s SARS-CoV-2 lgM/lgG antibody test (Download FDA EUA letter of authorization here).
As per the FDA’s guidance on March 16th, 2020, the FDA does not intend to object to the development and distribution by commercial manufacturers or development and use by laboratories of serology tests to identify antibodies to SARS-CoV-2, where the test has been validated, notification is provided to the FDA, and information along the lines of the following is included in the test reports:
“Negative results do not rule out SARS-CoV-2 infection, particularly in those who have been in contact with the virus. Following-up with a molecular diagnostic testing should be considered to rule out infection in these individuals. Results from antibody testing should not be used as the sole basis to diagnose or exclude SARS-CoV-2 infection or to inform infection status. Positive results may be due to past or present infection with non-SARS-CoV-2 coronavirus strains, such as coronavirus HKU1, NL63, OC43, or 229E.”
Can these tests be used at home or with remote supervision?
No. COVID-19 IgM/IgG Test Kits are not intended for at-home use, nor are they intended to be used under remote supervision. These are “Point-of-Care” kits and are meant to be administered by trained healthcare professionals. If you are not a healthcare professional, and think that you need to be tested, please reach out to a local clinic or physician and ask them about offering this test.
What are the prominent antibodies detected in serologic diagnostic CoVID-19 testing?
IgM– Is the first antibody synthesized as part of an immune response possibly because synthesis occurs without B cell isotope switching. Although IgM molecules synthesized initially manifest low affinity, they form pentamers with 10 antigen binding sites which in essence is consistent with a state of high avidity. Additionally, the IgM pentamers are important in complement activation that in turn promotes phagocytic cell uptake and removal of immune complexes.
IgG– B cell synthesis of IgG antibodies as part of an immune response occurs later, after isotope switching. Furthermore, it circulates unlike IgM as high affinity antibody monomers. IgG efficiently activates the complement system and plays an integral role in pathogen opsonization.
Timing of antibody synthesis during an immune response (e.g. response to an infection)– Is initiated with an early IgM response that begins during the first week of an illness and usually maximizes during the second week. In contrast, IgG response initiates after the IgM response begins and maximizes by three to four weeks after the infection began. IgM detection, with regards to the BioHit SARS-CoV-2 antibody test kit, is detectable by the seventh day after infection or on the third day of symptoms.
What are the advantages and disadvantages to the two most common forms of COVID-19 viral detection?
RT-PCR viral detection– In general, accuracy is best during the first several days of infection and decreases over time. This is noted and exemplified in a study by Zhao et al. ( Zhao J et al. Clin Infect Dis 2020 Mar 28 ) in which viral RNA detectability diminished from 66.7% of samples collected before seven days of illness to 54% of samples tested during the second week of illness. In contrast, 89.6% of samples tested serologically for the presence of virus-related IgM, during the second week of illness, were reported as positive. In another study by Gua et al., it was demonstrated that the serologic IgM antibody test results were more accurate than results attained from PCR methods for samples collected on or after five-and-a-half days of illness (Guo L et al. Clin Infect Dis March 21, 2020).
The advantages of newer serologic antibody kits, such as the BioHit SARS-CoV-2 IgM/IgG antibody test kit include simplicity, quick attainment of results, accuracy and reproducibility. This point-of-care test kit is simple to use and is fully contained aside from the patient finger-stick blood sample that is added. The test results (both IgM and IgG) are processed and readily available within 15 minutes. The IgM and IgG test results are very accurate as manifested by the excellent sensitivity (IgM-97.5%, IgG-97.5%) and specificity (IgM-99.5%, IgG-100%) which are discussed more fully in the answer to question four below. In contrast, RT-PCR viral detection is a more complex method requiring processing at a relatively sophisticated laboratory utilizing molecular methodology. Processing time typically takes six hours with results often reported 24 hours later.
What is the meaning of the terms sensitivity, specificity, positive predictive value and negative predictive value and how do they apply to disease detection methodology?
Sensitivity is the performance of a test to accurately identify a patient as having a disease. This is determined using the following formula:
Sensitivity = # of true positive/ # of true positive + # of false negative
Specificity is the performance of a test to accurately identify a patient as being disease free. This is determined by using the following formula:
Specificity =# of true negative/ # of true negative plus # of false positive
Examples of Sensitivity and Specificity for a new diagnostic serologic test for disease Z.
|Blood Test||Patients with Disease Z||Patients without Disease Z|
|Test Positive||90 (True Positives)||8 (False positive)|
|Test Negative||10 (False Negatives)||222 (True Negative)|
Sensitivity = True positives/ True Positives + False Negatives = 90 / 90 +10
Thus, Sensitivity for this test is 90%
Specificity = True Negatives / True Negatives =+ False Positives = 222/ 222 + 8
Thus, the Specificity for this test is 96.5%
Now we will determine the sensitivity and specificity of the IgM portion of the BioHit SARS-CoV-2 IgM/IgG antibody test kit. The test results and patient sample numbers were from a retrospective study of 226 patient samples from the First Affiliated Hospital of Anhui Medical University including 108 samples from normal individuals, 40 samples from those diagnosed with COVID-19 and 78 samples from individuals with other respiratory tract infections.
|Colloidal Gold IgM Results||
C L I N I C A L
|D I A G N O S I S Negative||Total Results|
Sensitivity = True positives/ True Positives + False Negatives = 39 / 39 + 1 = 39/40 = 97.5%
Specificity = True Negatives / True Negatives + False Positives = 185/ 185 + 1 = 185/186 = 99.5%
Thus, based on these results, the BioHit SARS-CoV-2 IgM test kit has both an excellent sensitivity and specificity. In a similar manner, the BioHit SARS-CoV-2 IgG test kit also has excellent associated sensitivity and specificity, respectively 97.5% and 100% (data not shown).
Predictive Values (PV): PV vary with the prevalence of a particular disease within the population under study. In contrast, sensitivity and specificity do not directly depend upon disease prevalence.
The positive predictive value (PPV) is the correlation of the proportion of positive diagnostic tests that are truly positive and represent the presence of disease. Whereas, the negative predictive value (NPV) is the proportion of negative tests that are truly negative and represent the lack of disease.
The table below demonstrates the affects of disease prevalence on PPV and NPV for a diagnostic test with a sensitivity and specificity of 90%. This table shows that as the disease prevalence increases the PPV also increases. In contrast, as the disease prevalence increases, the NPV diminishes.
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