introduction
At the end of 2019, a novel coronavirus was identified as the cause of a cluster of pneumonia cases in Wuhan. It rapidly spread, resulting in an epidemic throughout China, followed by an increasing number of cases in other countries throughout the world. In February 2020, the World Health Organization designated the disease COVID-19, which stands for coronavirus disease.
In symptomatic patients, the possibility of COVID-19 should be considered primarily in those with new-onset fever and/or respiratory tract symptoms (e.g., cough, dyspnea). Other consistent symptoms include smell or taste disturbances, myalgias, and diarrhea, Headache, Sore throat, Nausea/vomiting. As SARS-CoV-2 shows no symptom in almost 33% of patients, clinicians should have a low threshold for suspicion of COVID-19.
SEVERITY OF SYMPTOMATIC INFECTION
The spectrum of symptomatic infection ranges from mild to critical.
- Mild disease (no or mild pneumonia) was reported in 81 percent.
- Severe disease (e.g., with dyspnea, hypoxia, or >50 percent lung involvement on imaging within 24 to 48 hours) was reported in 14 percent.
- Critical disease (e.g., with respiratory failure, shock, or multiorgan dysfunction) was reported in 5 percent.
The overall case fatality rate (CFR) that was reported in this study was 2.3%, and there was a CFR of 49% among those patients with critical disease. Among children, disease severity was lower with 94% having asymptomatic, mild or moderate disease, 5% having severe disease, and <1% having critical disease
Whom to test
Symptomatic patients — If possible, all symptomatic patients with suspected infection should undergo testing; the diagnosis cannot be definitively made without microbiologic testing.
asymptomatic individuals — Testing certain asymptomatic individuals may also be important for public health or infection control purposes. Indications for testing asymptomatic individuals include
- Following close contact with an individual with COVID-19 (this includes neonates born to mothers with COVID-19). The time to detectable RNA following exposure is unknown, so the optimal time to test for COVID-19 following exposure is uncertain. The CDC recommends testing immediately after the exposure is identified to quickly identify infection and if the test is negative, retesting five to seven days after the last exposure. In some cases, testing can be used to help determine the length of quarantine (e.g., reduce the quarantine period to seven days if an individual remains asymptomatic and has a negative viral test within 48 hours of the planned end of quarantine)
- Early identification of infection in congregate living facilities that house individuals at risk for severe disease (e.g., long-term care facilities, correctional and detention facilities, homeless shelters). This includes testing in response to identified COVID-19 cases within the facility as well as intermittent screening of employees and residents.
- Screening hospitalized patients at locations where prevalence is high (e.g., ≥10 percent PCR positivity in the community).
- Prior to surgical procedures or aerosol-generating procedures.
- Prior to receiving immunosuppressive therapy (including prior to transplantation)
Initial testing and specimen collection
Nucleic acid amplification testing (NAAT), most commonly with a reverse-transcription polymerase chain reaction (RT-PCR) assay, to detect SARS-CoV-2 RNA from the upper respiratory tract is the preferred initial diagnostic test for COVID-19. In some settings, antigen testing may be the initial test used, but the sensitivity of antigen tests is lower than that of NAATs, and negative antigen tests should usually be confirmed with NAAT.
The CDC recommends collection of one of the following specimens
- Nasopharyngeal swab specimen
- Nasal swab specimen from both anterior nares
- Nasal mid-turbinate swab
- Nasal or nasopharyngeal wash/aspirate
- Oropharyngeal swab specimen
- Saliva specimen (1 to 5 mL)
Test interpretation and additional testing
Positive NAAT result — A positive nucleic acid amplification test (NAAT; eg, RT-PCR) for SARS-CoV-2 generally confirms the diagnosis of COVID-19. No additional diagnostic testing is necessary. However, additional testing may be warranted for management in hospitalized patients.
Patients with COVID-19 can have detectable SARS-CoV-2 RNA in upper respiratory tract specimens for weeks after the onset of symptoms. However, prolonged viral RNA detection does not necessarily indicate ongoing infectiousness.
Negative NAAT result — For many individuals, a single negative NAAT result is sufficient to exclude the diagnosis of COVID-19. However, false-negative NAAT tests (eg, RT-PCR) from upper respiratory specimens have been well documented. If initial testing is negative but the suspicion for COVID-19 remains (eg, suggestive symptoms without evident alternative cause) and confirming the presence of infection is important for management or infection control, we suggest repeating the test. The optimal timing for repeat testing is not known; it is generally performed 24 to 48 hours after the initial test. Repeat testing within 24 hours is not recommended.
It is recommended to reserve lower respiratory tract specimen NAAT testing for hospitalized patients who have an initial negative test on an upper respiratory tract specimen but for whom suspicion for lower respiratory tract SARS-CoV-2 infection remains.
In hospitalized patients suspected of having COVID-19 who have a negative SARS-CoV-2 NAAT, characteristic laboratory or imaging findings can further support the clinical diagnosis of COVID-19 and be reasons to maintain infection control precautions. It is also important to consider other potential causes of symptoms in patients with negative SARS-CoV-2 NAATs.
Impact of SARS-CoV-2 mutations on test accuracy
Several SARS-CoV-2 variants that have mutations in the spike protein have been identified globally.
Some of these mutations (in particular, a deletion at amino acids 69-70) impact the ability of SARS-CoV-2 NAAT to detect the S gene that encodes the spike protein. Most NAAT will still detect SARS-CoV-2 RNA even if they fail to detect the mutated S gene because they have been designed to detect more than one gene target. S gene target failure can be used a marker for variants that contain spike protein mutations for surveillance purposes.
Most antigen tests target nucleocapsid protein, so mutations in the spike protein would not impact the accuracy of such antigen tests.
SPECIFIC DIAGNOSTIC TECHNIQUES
- NAAT (including RT-PCR) to diagnose current infection
Types of NAATs — The diagnosis of COVID-19 is made primarily by direct detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA by nucleic acid amplification tests (NAATs), most commonly reverse-transcription polymerase chain reaction (RT-PCR) from the upper respiratory tract. Various RT-PCR assays are used around the world; different assays amplify and detect different regions of the SARS-CoV-2 genome. Some target two or more genes, including the nucleocapsid (N), envelope (E), and spike (S) genes, and regions in the first open reading frame, including the RNA-dependent RNA polymerase (RdRp) gene. Other, less common types of NAAT include isothermal amplification, CRISPR-based assays, and next-generation sequencing.
Accuracy — The accuracy and predictive values of SARS-CoV-2 NAATs have not been systematically evaluated. They are highly specific tests. Although NAATs have high analytic sensitivity in ideal settings (i.e., they are able to accurately detect low levels of viral RNA in test samples known to contain viral RNA), clinical performance is more variable.
False-positive results are rare but have been reported with certain platforms.
Reported false-negative rates have ranged from less than 5 to 40 percent.
Sensitivity of testing likely depends on the type and quality of the specimen obtained, the duration of illness at the time of testing, and the specific of assay.
- Antigen testing as an alternative to NAAT
Tests that detect SARS-CoV-2 antigen can be performed rapidly and at the point of care and thus may be more accessible with a faster time to results than some NAATs. Antigen tests are typically less sensitive than NAATs. Nevertheless, they can be useful when NAATs are not available or where NAAT turnaround times are too long to be clinically useful, provided that clinicians are aware of the possibility of false negatives and the results are interpreted based on the pretest probability of COVID-19. Antigen test is useful in symptomatic individuals within 5 to 7 days of symptom onset. Its sensitivity is ≥80% and have very high specificity (97%).
3.Antibody testing
Serologic tests detect antibodies to SARS-CoV-2 in the blood and can be used to determine different aspects of the adaptive immune response and functionality of antibodies. The tests can be broadly classified to detect either binding or neutralizing antibodies.
Binding antibody detection: These tests use purified proteins of SARS-CoV-2, not live virus. Individual antibody types, like IgG, IgM, and IgA, can be determined. Both IgM and IgG may be detected around the same time after infection. While IgM is most useful for determining recent infection as it usually becomes undetectable weeks to months following infection, IgG may remain detectable for longer periods. IgA is important for mucosal immunity and can be detected in mucous secretions like saliva in addition to blood.
- Laboratory tests use lateral flow, ELISA or chemiluminescent immunoassay (CIA) methods for antibody detection in serum, plasma, whole blood and dried blood spots, which for some assays may require trained laboratorians and specialized instruments. Based on the reagents, total antibody (Ig) can be detected, or IgG and IgM, can be detected separately. While most tests detect antibodies against either S or N proteins, some tests can detect antibodies against both immunodominant proteins (multiplex assays).
Neutralizing antibody detection tests determine the functional ability of antibodies to prevent infection by virus in vitro. These tests monitor inhibition of viral growth in cell culture when incubated with serum or plasma. Three types of neutralization tests are conducted:
- Virus neutralization tests (VNT),such as the plaque-reduction neutralization test (PRNT) and microneutralization, use a SARS-CoV-2 virus from a clinical isolate or recombinant SARS-CoV-2 virus expressing reporter proteins. This testing may take up to 5 days to complete. There are currently no EUA authorized VNTs.
- Pseudovirus neutralization tests (pVNT)use recombinant pseudoviruses (like vesicular stomatitis virus, VSV or lentiviruses) that incorporate the S protein of SARS-CoV-2. These reporter-based tests can be performed in BSL-2 laboratories depending on the virus strain used. There are currently no EUA authorized pVNTs.
- Competitive neutralization tests (cVNT) have also been developed and one has been authorized by the FDA. These are binding antibody tests designed to qualitatively detect potentially neutralizing antibodies, often those that prevent interaction of RBD with the ACE-2 receptor. The test mimics the interaction of RBD with ACE-2 in an ELISA format (similar to RBD on a virus particle binding to a cell surface ACE-2 receptor) and the ability of RBD specific antibodies to interfere with the interaction detected using a decrease in signal based on the reporter fused RBD. These tests can be conducted in BSL-2 laboratories because they do not require live virus.
- Other tests
- Viral culture– For safety reasons, specimens from a patient with suspected or documented COVID-19 should not be submitted to clinical laboratories for viral culture. Viral culture is mainly reserved for research purposes.
- Other tests of SARS-CoV-2 immunity– The clinical utility of tests that can detect cell-mediated immune responses to SARS-CoV-2, such as with an interferon-gamma release assay, is also being explored.
At-home tests of SARS-CoV-2
So far, the FDA has given what’s called emergency use authorization (EUA) to several at-home tests. That means the tests haven’t gone through the full rigorous FDA approval process, but the agency wanted to get them on the market quickly due to the severity of the pandemic.
Generally, these tests are authorized that can be done without a prescription. And can be considered as a key tool for schools, workplaces and communities to use to quickly screen people for COVID-19 during the pandemic.
- Molecular test
The test is the first molecular test authorized for at-home use without a prescription. Molecular tests are generally more accurate than those that use antigen technology, especially in patients who are asymptomatic. Although overall results for diagnosing and ruling out COVID-19 were good (95.1% of infections correctly diagnosed and 99% correctly ruled out), 69% of the studies used the tests in laboratories instead of at the point-of-care and few studies followed test manufacturer instructions.
These Tests utilizes RT-LAMP [1] technology to detect RNA of the N gene for SARS-CoV-2. This technology can create a signal from a few copies of RNA in less than 30 minutes. The RT-LAMP amplification reaction occurs in two phases, a non-cyclic phase followed by a cyclic phase. During the non-cyclic phase, reverse transcriptase, with RNase H activity, converts the RNA target into cDNA. A DNA polymerase with strand displacement activity then amplifies the cDNA. A successful amplification reaction creates a pH change and subsequently a color change of the halochromic agents within the reaction mixture.
- Antigen test
These tests are used for the qualitative detection of SARS‑CoV‑2 nucleocapsid antigen present in human nasal samples. In people with confirmed COVID-19, antigen tests correctly identified COVID-19 infection in an average of 72% of people with symptoms, compared to 58% of people without symptoms. Tests were most accurate when used in the first week after symptoms first developed (an average of 78% of confirmed cases had positive antigen tests). This is likely to be because people have the most virus in their system in the first days after they are infected.
Tests Detect Novel Coronavirus and Influenza in a Single Sample
As influenza (flu) season nears and COVID-19 continues to spread in many parts of the United States, the Food and Drug Administration (FDA) has authorized new molecular-based tests that use a single sample and can distinguish between influenza, SARS-CoV-2 (the virus that causes COVID-19), and other respiratory pathogens.
The Tests
As of August 18, 2020, the FDA has granted emergency use authorization (EUA) to combination tests manufactured by the CDC and two commercial companies. All three tests detect genetic material from infectious pathogens, and the preferred sample is a swab of the back of the nose (nasopharyngeal or NP swab). Additionally, this test can use other respiratory samples, such as nasal and throat swabs.
The CDC’s test detects two types of influenza viruses—influenza A and B—and differentiates them from SARS-CoV-2, the virus that causes COVID-19. The test takes four hours to perform.
The combination tests detect numerous respiratory pathogens. In addition to SARS-CoV-2, influenza B and three distinct strains of the influenza A virus, the tests detect four other coronaviruses, human metapneumovirus, human rhinovirus/enterovirus, parainfluenza virus and respiratory syncytial virus (RSV). Besides viruses, these tests can also identify a few types of bacteria that cause respiratory illnesses, such as Bordetella pertussis, which causes whooping cough, Chlamydia pneumoniae, and Mycoplasma pneumoniae. These commercial tests take about one hour to complete.
Although the FDA has granted EUA for these combination tests, they may not be immediately available for patients in all areas. Before offering the new combination tests or other existing influenza and COVID-19 tests, labs must consider several factors. Their decisions may be influenced by local prevalence of influenza and COVID-19, availability of reagents and other supplies needed to run specific tests, their turnaround times, and whether lab staff has appropriate expertise.
Likewise, healthcare professionals also take several considerations into account when choosing testing for their patients. Not everyone with symptoms of a respiratory infection is tested with a comprehensive panel. Healthcare practitioners consider how quickly they need a diagnosis, whether an individual test is more appropriate based on signs and symptoms and medical history, and whether specific treatment is available.
Some healthcare practitioners or health facilities may reserve the new combination tests for people who need a quick diagnosis and appropriate treatment because they are seriously ill and/or hospitalized, have an increased risk for a severe infection with complications, have multiple infections, compromised immune systems, or other pre-existing chronic diseases.
COVID-19 Vaccines
The aim of COVID-19 vaccination program is to protect people who are at risk for acute illness or death from COVID-19, or people who are at high risk of transmitting the infection to vulnerable ones. So far, more than 280 cases of corona vaccine are in various stages of development. A person is considered fully vaccinated against COVID-19 about 2 or 3 weeks after receipt of the second dose in a 2-dose series. Although individual care should be taken to prevent Covid-19 infection after receiving the vaccine.
So far, Sputnik v, Astrazeneca, Sinopharm and Covaxin, have been imported under the supervision of the Food and Drug Administration of Iran and are recommended for the prevention of Covid-19 in people over 18 years of age.
A brief description of covid 19 vaccines is demonstrated in the table below.
[1] Reverse transcription loop-mediated isothermal amplification
