Sample collection and Diagnosis of COVID-19 (SARS-CoV-2)

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COVID-19 is a novel coronavirus that has caused a global pandemic and has affected millions of people worldwide. The symptoms of COVID-19 can range from mild to severe and can include fever, cough, shortness of breath, loss of taste or smell, headache, sore throat, muscle or body aches, fatigue, nausea, vomiting, diarrhea, and more. Some people may develop more serious complications such as pneumonia, acute respiratory distress syndrome, septic shock, blood clots, organ failure, or death.

The diagnosis of COVID-19 is based on the detection of specific viral genetic material or antigens in respiratory specimens or blood samples using laboratory tests. However, these tests are not 100% accurate and may have false negative or false positive results. Therefore, the diagnosis of COVID-19 should always be confirmed by a qualified health professional who can evaluate your medical history, symptoms, exposure history, and other relevant factors.

The information on this website is intended to provide general knowledge and guidance on the sample collection and diagnosis of COVID-19. It is not a substitute for professional medical advice, diagnosis, or treatment. The website does not endorse any specific products, services, tests, or treatments mentioned on this website. The website also does not guarantee the completeness, accuracy, timeliness, or reliability of the information on this website.

If you have any questions or concerns about your health or the health of someone else, please contact your physician or doctor immediately. Do not disregard professional medical advice or delay seeking it because of something you have read on this website. If you think you may have a medical emergency, call your doctor or local emergency service right away.

Since COVID-19 is an infectious respiratory tract infection, respiratory material should be collected, at a minimum:

• Upper respiratory specimens: nasopharyngeal and oropharyngeal swabs or wash in patients still capable of movement.
• Lower respiratory specimens: sputum (if produced) or endotracheal aspirate or bronchoalveolar lavage in patients with more severe respiratory disease.

Additional clinical specimens can be collected as the COVID-19 virus has been detected in blood and stool. In the case of deceased patients, the collection of autopsy material including lung tissue should be considered. In recovered patients, paired serum (acute and convalescent) can be favorable to define cases as serological assays become available retrospectively.

The type of specimen collected when testing for current or past infection with SARS-CoV-2 is based on the test being performed and its manufacturer’s instructions. Some of the specimen types listed above will not be appropriate for all tests. For diagnostic testing for current SARS-CoV-2 infections, CDC recommends collecting and testing an upper respiratory specimen.

Proper specimen collection is the most important step in the laboratory diagnosis of infectious diseases. A specimen that is not collected correctly may lead to false or inconclusive test results. The following specimen collection guidelines follow standard recommended procedures:

• Nasopharyngeal swabs: Insert a swab into the nostril parallel to the palate. Leave the swab in place for a few seconds to absorb secretions. Slowly remove the swab while rotating it. Swabs with synthetic fibres and plastic shafts are preferred.
• Nasal mid-turbinate swabs: Tilt patient’s head back 70 degrees. While gently rotating the swab, insert swab less than one inch (about 2 cm) into nostril (until resistance is met at turbinates). Rotate the swab several times against nasal wall and repeat in other nostril using the same swab.
• Anterior nares specimen collection: Insert a round-tipped swab into one nostril until resistance is met at turbinates (less than one inch into the nostril). Rotate the swab several times against nasal wall and repeat in other nostril using same swab.
• Saliva: Ask patient to spit into a sterile container without touching their mouth to the container. Alternatively, ask patient to cough deeply and spit into a sterile container without touching their mouth to the container.
• Sputum: Have patient rinse their mouth with water and then expectorate deep cough sputum directly into a sterile screw-cap collection cup or sterile dry container.
• Endotracheal aspirate or bronchoalveolar lavage: Collect 2-3 mL into a sterile, leak-proof, screw-cap sputum collection cup or sterile dry container.
• Blood: Collect 4 mL of whole blood in a serum separator tube. Store and transport specimen at 2-8°C up to 72 hours after collection. If a delay in testing or shipping is expected, store specimen at -20°C or below.
• Stool: Collect stool specimen in sterile, leak-proof, screw-cap container. Store and transport specimen at 2-8°C up to 72 hours after collection. If a delay in testing or shipping is expected, store specimen at -20°C or below.

Specimens for virus detection should reach the laboratory as soon as possible after collection. Specimens that can be delivered shortly to the laboratory can be stored and shipped at 2-8°C while if a delay is likely to happen, the viral transport medium (VTM) should be used. Specimens can be frozen to – 20°C or ideally -70°C and shipped on dry ice if further delays are expected. However, it is essential to avoid repeated freezing and thawing of specimens. The transport of potentially COVID-19 virus-containing samples to other countries should follow the UN Model Regulations, and any other essential regulations depending on the mode of transport being used.

For healthcare providers collecting specimens or working within 6 feet of patients suspected to be infected with SARS-CoV-2, maintain proper infection control and use recommended personal protective equipment (PPE), which includes an N95 or higher-level respirator (or face mask if a respirator is not available), eye protection, gloves, and a gown. For healthcare providers who are handling specimens, but are not directly involved in collection (e.g. handling self-collected specimens) and not working within 6 feet of the patient, follow Standard Precautions. Healthcare providers should wear a form of source control (face mask) at all times while in the healthcare facility. Healthcare providers can minimize PPE use if patients collect their own specimens while maintaining at least 6 feet of separation.

Clinical specimens for COVID-19 testing should be packed and shipped according to the international and national regulations for the transport of infectious substances. The following steps should be followed to ensure safe and proper packaging and shipping of specimens:

• Label the primary receptacle (such as a tube or vial) that contains the specimen with the patient`s name, identification number, specimen type, and date of collection.
• Place the primary receptacle in a watertight and leak-proof secondary container (such as a ziplock bag or a screw-cap container) with enough absorbent material to absorb all fluid in case of leakage or breakage.
• Place the secondary container in a rigid outer packaging (such as a cardboard box or a plastic container) that is strong enough to withstand shocks and pressure changes during transport. The outer packaging should have at least one surface with a minimum dimension of 100 mm x 100 mm.
• Fill any empty space in the outer packaging with cushioning material (such as bubble wrap or newspaper) to prevent movement of the secondary container during transport.
• Attach a completed laboratory request form or equivalent document to the outer packaging. The form should include the following information: sender`s name, address, and phone number; receiver`s name, address, and phone number; patient`s name, identification number, date of birth, sex, clinical symptoms, date of onset, date of specimen collection, specimen type, and test requested.
• Mark the outer packaging with the UN3373 label for biological substance, category B. The label should be affixed on the same surface as the address label and should not obscure any other markings or labels.
• Mark the outer packaging with the name and phone number of a responsible person who can provide additional information on the contents of the package in case of an emergency or a spill.
• Seal the outer packaging with adhesive tape or other appropriate material to prevent tampering or accidental opening during transport.
• Ship the package by the fastest possible means to minimize transit time and exposure to temperature extremes. Use a commercial carrier that complies with the International Air Transport Association (IATA) and U.S. Department of Transportation (USDOT) regulations for transporting infectious substances. Notify the receiving laboratory of the shipment details and expected arrival time.

For more information on packaging and shipping of clinical specimens for COVID-19 testing, please refer to:

• Interim Guidelines for Clinical Specimens for COVID-19 | CDC
• Interim Guidelines for Biosafety and COVID-19 | CDC
• Diagnostic Specimen Collection, Packaging and Shipping Guidance for COVID-19 Testing
• Best Diagnostic Packaging for Coronavirus Test Kit

Diagnosis of COVID-19 is the process of confirming or ruling out the presence of the virus that causes COVID-19 in a person who has symptoms or has been exposed to the virus. Diagnosis can help guide treatment, isolation and contact tracing measures to prevent further spread of the disease.

There are different types of tests that can be used to diagnose COVID-19, depending on the availability, accuracy and purpose of the test. The main types of tests are:

• RT-PCR test. This is the most common and reliable test for COVID-19. It detects the genetic material of the virus using a laboratory technique called reverse transcription polymerase chain reaction (RT-PCR). A sample is taken from the nose, throat or saliva of the person and sent to a lab for analysis. The result can take from a few hours to a few days, depending on the capacity of the lab. A positive RT-PCR test confirms the diagnosis of COVID-19, while a negative test may need to be repeated if the person has symptoms or high-risk exposure .

• Rapid antigen test. This is a faster and cheaper test that detects specific proteins on the surface of the virus using a device similar to a pregnancy test. A sample is taken from the nose or throat of the person and inserted into the device, which gives a result within minutes. A positive rapid antigen test indicates a high likelihood of COVID-19, while a negative test may need to be confirmed by a RT-PCR test if the person has symptoms or high-risk exposure .

• Serological test. This is a blood test that detects antibodies produced by the immune system in response to the virus. Antibodies usually appear after a few days or weeks of infection and may remain for months or longer. A positive serological test indicates a past or recent infection with COVID-19, while a negative test may mean that the person has not been exposed to the virus or has not developed antibodies yet . Serological tests are not routinely used for diagnosis of COVID-19, but they can be useful for epidemiological studies and vaccine evaluation.

The choice of which test to use for diagnosis of COVID-19 depends on several factors, such as:

• The availability and accessibility of testing resources and facilities
• The stage and severity of symptoms and disease
• The risk of exposure and transmission
• The clinical and public health objectives

If you develop symptoms of COVID-19 or you have been exposed to someone with COVID-19, you should contact your health care provider or local health authority for guidance on testing and isolation. You should also follow preventive measures such as wearing a mask, washing your hands and keeping physical distance from others .

A nucleic acid amplification test (NAAT) is a type of viral diagnostic test for SARS-CoV-2, the virus that causes COVID-19. NAATs detect genetic material (nucleic acids) of the virus, specifically the RNA (ribonucleic acid) sequences that comprise its genome. NAATs can use different methods to amplify and detect the viral RNA, such as:

• Reverse transcription polymerase chain reaction (RT-PCR)
• Isothermal amplification (e.g., nicking endonuclease amplification reaction , transcription mediated amplification , loop-mediated isothermal amplification , helicase-dependent amplification )
• Clustered regularly interspaced short palindromic repeats (CRISPR)
• Strand displacement amplification (SDA)

The advantage of NAATs is that they are highly sensitive for diagnosing COVID-19, meaning that they can reliably detect small amounts of SARS-CoV-2 RNA in a specimen and are unlikely to return a false-negative result . NAATs can also provide confirmation of the presence of the virus and inform molecular epidemiology studies by sequencing partial or whole genome of the virus.

NAATs can be performed on specimens collected from either the upper or lower respiratory tract, depending on the test being used and the manufacturer`s instructions . The type of specimen collected for testing SARS-CoV-2 is based on the test being performed and the manufacturer’s instructions. See CDC’s Collecting and Handling of Clinical Specimens for COVID-19 Testing. The most common specimens are nasopharyngeal, nasal mid-turbinate, anterior nasal, or throat swabs. Lower respiratory tract specimens, such as sputum, endotracheal aspirate, or bronchoalveolar lavage, have a higher yield than upper respiratory tract specimens, but they are often not obtained because of concerns about aerosolization of the virus during sample collection procedures.

NAATs have been authorized for use in different settings, such as in laboratory facilities by trained personnel (laboratory-based) or in point-of-care (POC) settings by trained personnel or by self-administration at home or in other non-healthcare locations . Some NAATs are considered rapid tests that can provide results within minutes, whereas others may take longer to complete. The level of sensitivity for the detection of SARS-CoV-2 genetic material in a specimen also varies depending on the methods and application of the NAAT. Laboratory-based NAATs generally have higher sensitivity than POC tests or self-administered tests. Therefore, laboratory-based NAATs can also be used to confirm the results of lower sensitivity tests, such as POC NAATs or antigen tests.

NAATs are recommended for everyone who has symptoms that are consistent with COVID-19 and people with known high-risk exposures to SARS-CoV-2. A NAAT should not be repeated in an asymptomatic person (with the exception of health care workers) within 90 days of a previous SARS-CoV-2 infection, even if the person has had a significant exposure to SARS-CoV-2. SARS-CoV-2 reinfection has been reported in people after an initial diagnosis of the infection; therefore, clinicians should consider using a NAAT for those who have recovered from a previous infection and who present with symptoms that are compatible with SARS-CoV-2 infection if there is no alternative diagnosis.

Serological testing is a method of detecting antibodies that are produced by the immune system in response to an infection or a vaccination. Antibodies are proteins that bind to specific antigens (such as viral proteins) and help fight off infections. Serological testing can help identify people who may have been exposed to SARS-CoV-2, the virus that causes COVID-19, or have recovered from it .

Serological testing is not routinely used for diagnosing COVID-19, as it cannot detect the presence of the virus itself. It can also take days to weeks after the infection for the body to produce detectable antibodies . Therefore, serological testing should not be used to determine if a person has an active COVID-19 infection or needs isolation or quarantine .

However, serological testing can be useful for other purposes, such as:

• Estimating the prevalence and spread of COVID-19 in a population, especially among people who may have had mild or asymptomatic infections .
• Evaluating the effectiveness of COVID-19 vaccines and the duration of immunity they provide .
• Supporting the development of treatments and therapies for COVID-19 by identifying potential donors of convalescent plasma (blood plasma from recovered patients that contains antibodies) .
• Understanding the immune response and pathogenesis of COVID-19 and its variants .

There are various types of serological tests available for COVID-19, such as:

• Enzyme-linked immunosorbent assay (ELISA): A laboratory-based test that uses an enzyme reaction to measure the amount and type of antibodies in a blood sample .
• Lateral flow immunoassay (LFIA): A rapid test that uses a paper strip with embedded antigens and color indicators to detect the presence or absence of antibodies in a blood sample .
• Chemiluminescence immunoassay (CLIA): A laboratory-based test that uses a chemical reaction to produce light signals that indicate the amount and type of antibodies in a blood sample .
• Neutralization assay: A laboratory-based test that measures the ability of antibodies in a blood sample to prevent the virus from infecting cells in culture .

Serological tests can detect different types of antibodies, such as:

• Immunoglobulin M (IgM): The first type of antibody that appears after an infection or vaccination, usually within a week. It indicates a recent or ongoing immune response .
• Immunoglobulin G (IgG): The most common and long-lasting type of antibody that appears after an infection or vaccination, usually within two to three weeks. It indicates a past or resolved immune response and may confer some level of protection against reinfection [^5

Viral sequencing is a process of determining the order of nucleotides (the building blocks of genetic material) in a virus`s genome. Viral sequencing can provide confirmation of the presence of SARS-CoV-2, the virus that causes COVID-19, and reveal its genetic variations. Viral sequencing can also inform molecular epidemiology studies, which can help track the origin, transmission, and evolution of the virus.

Viral sequencing can be done by using different methods, such as Sanger sequencing, next-generation sequencing (NGS), or nanopore sequencing. Sanger sequencing is a traditional method that reads one fragment of DNA at a time. NGS is a newer method that reads millions of fragments of DNA in parallel. Nanopore sequencing is an emerging method that reads long strands of DNA by passing them through tiny pores.

One of the challenges of viral sequencing is to obtain enough viral RNA from clinical specimens, such as nasopharyngeal or oropharyngeal swabs. Viral RNA may be degraded or contaminated by human or bacterial RNA, which can reduce the quality and quantity of the sequence data. To overcome this challenge, some researchers have developed methods to directly amplify and sequence viral RNA from clinical specimens without prior extraction .

Another challenge of viral sequencing is to analyze and interpret the sequence data. Viral genomes are constantly changing due to mutations, which may result in new variants with different characteristics. Some variants may have a competitive advantage over others and become more prevalent and circulate in a population. Some variants may also affect the virus`s ability to spread, cause disease, or evade immune responses.

To monitor the emergence and spread of SARS-CoV-2 variants, CDC and its partners use genomic surveillance, which involves collecting and analyzing sequence data from representative populations. CDC also classifies variants based on their attributes and characteristics into variants of interest, variants of concern, or variants of high consequence. For more information on current variants, visit CDC’s COVID Data Tracker.

Viral sequencing is an important tool for understanding and responding to the COVID-19 pandemic. By comparing the genomes of different viruses, scientists can identify their similarities and differences, trace their origins and movements, and monitor their changes and impacts on public health.

Viral culture is a method of growing viruses in cell lines or animal hosts to detect and identify them. It is not recommended as a routine diagnostic procedure for COVID-19 due to the lack of permissive cell lines, time to results, labour and expertise requirements, and the lack of commercial antisera for culture confirmation . SARS-CoV-2, the virus that causes COVID-19, can grow in primary monkey cells and cell lines such as Vero and LLC-MK2, but cell culture should not be performed for suspect cases in routine diagnostic laboratories for biosafety reasons .

However, virus isolation in cell culture is important to obtain isolates for characterization and to support the development of vaccines and therapeutic agents. It can also provide confirmation of the presence of the virus and inform molecular epidemiology studies . Viral sequencing can be used to confirm the identity and variant of the virus isolated in culture .

The duration of shedding of culturable virus in COVID-19 patients is variable and depends on several factors, such as the severity of illness, the type of specimen, and the viral variant. A study from South Korea reported that the latest positive viral culture was 12 days after symptom onset in hospitalized patients with mild-to-moderate COVID-19 . Another study from the United States reported that the median time from symptom onset or initial positive PCR test to culture conversion was 8 days for patients with omicron infection and 6 days for patients with delta infection . These findings suggest that the risk of transmission may decrease over time, but it is not eliminated.

Viral culture is a valuable tool for research and development purposes, but it has limited utility for clinical diagnosis of COVID-19. Other methods, such as nucleic acid amplification tests (NAATs) and antigen tests, are more widely available, faster, and easier to perform. However, viral culture can provide information on the viability and infectivity of the virus, which cannot be obtained by molecular or serological tests.

Rapid antigen tests are a type of COVID-19 test that detect certain proteins in the virus. They are also called lateral flow tests or LFTs. They can produce results in minutes and are usually performed at the point of care, such as a clinic, pharmacy, school or workplace. They are less expensive and easier to use than PCR tests, but they are also less sensitive and less specific. This means that they may miss some cases of COVID-19 infection or give false positive results.

Rapid antigen tests work by using a nasal swab to collect a sample from the front part of the nose. The swab is then inserted into a test device that contains antibodies that bind to the SARS-CoV-2 antigen. If the antigen is present, a colored line will appear on the test strip, indicating a positive result. If the antigen is not present, no line will appear, indicating a negative result.

Rapid antigen tests are most accurate when used within the first five days of symptom onset, when the viral load is highest. They are also more reliable for people who have symptoms than for people who do not have symptoms. A positive rapid antigen test result is considered accurate and does not need to be confirmed by a PCR test. However, a negative rapid antigen test result may not be accurate and may need to be confirmed by a PCR test, especially if the person has symptoms or has been exposed to someone with COVID-19.

Rapid antigen tests can be useful for screening large numbers of people quickly and cheaply, especially in settings where PCR testing is not readily available or feasible. They can also help identify people who are infectious and need to isolate themselves to prevent further transmission. However, rapid antigen tests are not a substitute for PCR testing and should not be used as the sole basis for diagnosis or treatment decisions. People who use rapid antigen tests should still follow public health guidelines on wearing masks, social distancing and hand hygiene.

A COVID-19 IgG/IgM rapid test is a lateral flow immunoassay that can be used to detect antibodies to SARS-CoV-2 in human blood samples. It is intended for use as an aid in identifying individuals with an adaptive immune response to SARS-CoV-2, indicating recent or prior infection .

The test consists of a cassette that contains a test strip with anti-human IgM and IgG antibodies immobilized on separate lines. The test also contains a control line with goat anti-mouse IgG antibodies. The test requires a small amount of blood sample (either from venipuncture or fingerstick) that is added to a buffer solution and applied to the sample well of the cassette. The blood sample migrates along the test strip by capillary action and reacts with the antibodies on the test lines. If IgM or IgG antibodies to SARS-CoV-2 are present in the sample, they will bind to the corresponding test line and produce a colored band. The control line will also produce a colored band if the test is performed correctly .

The test can provide results within 15 minutes and does not require any specialized equipment or laboratory personnel. The test can be performed at the point of care settings, such as clinics, hospitals, or pharmacies, under a CLIA certificate of waiver, compliance, or accreditation .

The advantages of the COVID-19 IgG/IgM rapid test are:

• It is easy to use and interpret
• It is fast and convenient
• It can detect both recent and past infections
• It can help monitor the seroprevalence and immune status of populations

The limitations of the COVID-19 IgG/IgM rapid test are:

• It is not intended for diagnosis or exclusion of acute SARS-CoV-2 infection
• It has low sensitivity and specificity compared to molecular tests
• It may produce false positive results due to cross-reactivity with other antibodies or causes
• It may produce false negative results due to low antibody levels or timing of testing
• It does not indicate the level or duration of immunity or protection against reinfection

Therefore, the COVID-19 IgG/IgM rapid test should be used with caution and in conjunction with other clinical and epidemiological information. The results should be confirmed by a second, different IgG or IgM assay if possible. The test should not be used to make decisions about infection control, treatment, vaccination, or return to work or school .

Electron microscopy (EM) is a technique that uses a beam of electrons to create an image of a specimen. EM can reveal the ultrastructure of cells and viruses, as well as the interactions between them. EM can be classified into two main types: scanning electron microscopy (SEM) and transmission electron microscopy (TEM).

SEM produces a three-dimensional image of the surface of a specimen by scanning it with a focused electron beam and detecting the secondary electrons emitted from the specimen. SEM can show the morphology and distribution of viral particles on the cell surface or in the extracellular environment.

TEM produces a two-dimensional image of a thin section of a specimen by transmitting a beam of electrons through it and detecting the electrons that pass through. TEM can show the internal structure and composition of viral particles and cells, as well as the intracellular location and replication of viruses.

EM has been used to visualize SARS-CoV-2, the causative agent of COVID-19, in various specimens, such as respiratory swabs, cell cultures, tissues, and fluids. EM can provide direct evidence of viral infection and shed light on the pathogenesis and transmission of COVID-19.

However, EM also has some limitations and challenges for the diagnosis of COVID-19. EM requires specialized equipment, expertise, and biosafety measures that are not widely available or accessible. EM is also time-consuming and labor-intensive, and it cannot provide quantitative or specific information about viral load or identity. Therefore, EM is not recommended as a routine diagnostic procedure for COVID-19, but rather as a complementary tool for research and confirmation purposes.

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