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COVID-19 testing

Diagnostic testing for the respiratory illness COVID-19 and the underlying pathogen SARS-CoV-2 coronavirus

Top 10 COVID-19 testing related articles

The US CDC's COVID-19 laboratory test kit

COVID-19 testing involves analyzing samples to assess the current or past presence of SARS-CoV-2. The two main branches detect either the presence of the virus or of antibodies produced in response to infection.[1][2] Tests for viral presence are used to diagnose individual cases and to allow public health authorities to trace and contain outbreaks. Antibody tests instead show whether someone once had the disease. They are less useful for diagnosing current infections because antibodies may not develop for weeks after infection.[3] It is used to assess disease prevalence, which aids the estimation of the infection fatality rate.[4]

Individual jurisdictions have adopted varied testing protocols, including whom to test, how often to test, analysis protocols, sample collection and the uses of test results.[5][6][7] This variation has likely significantly impacted reported statistics, including case and test numbers, case fatality rates and case demographics.[8][9][10][11] Because SARS-CoV-2 transmission occurs days after exposure (and before onset of symptoms) there is an urgent need for frequent surveillance and rapid availability of results.[12]

Test analysis is often performed in automated, high-throughput, medical laboratories by medical laboratory scientists. Alternatively, point-of-care testing can be done in physician's offices and parking lots, workplaces, institutional settings or transit hubs.

COVID-19 testing Intro articles: 127

Methods

Explanation of the underlying pathophysiology pertaining to diagnosis of COVID-19[13]

Positive viral tests indicate a current infection, while positive antibody tests indicate a prior infection.[14] Other techniques include a CT scan, checking for elevated body temperature, checking for low blood oxygen level, and the deployment of detection dogs at airports.[15][16][17]

Detection of the virus

Reverse transcription polymerase chain reaction

Polymerase chain reaction (PCR) is a process that amplifies (replicates) a small, well-defined segment of DNA many hundreds of thousands of times, creating enough of it for analysis. Test samples are treated with certain chemicals[18][19] that allow DNA to be extracted. Reverse transcription converts RNA into DNA.

Reverse transcription polymerase chain reaction (RT-PCR) first uses reverse transcription to obtain DNA, followed by PCR to amplify that DNA, creating enough to be analyzed.[19] RT-PCR can thereby detect SARS-CoV-2, which contains only RNA. The RT-PCR process generally requires a few hours.[20]

Real-time PCR (qPCR)[21] provides advantages including automation, higher-throughput and more reliable instrumentation. It has become the preferred method.[22][23]

The combined technique has been described as real-time RT-PCR[24] or quantitative RT-PCR[25] and is sometimes abbreviated qRT-PCR,[26] rRT-PCR[27] or RT-qPCR,[28] although sometimes RT-PCR or PCR are used. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines propose the term RT-qPCR,[29] but not all authors adhere to this.

Average sensitivity for rapid molecular tests were 95.2% (ranging from 68% to 100%) and average specificity was 98.9% (ranging from 92% to 100%) between test results of different company brands and sampling methods.[30]

Samples can be obtained by various methods, including a nasopharyngeal swab, sputum (coughed up material),[31] throat swabs,[32] deep airway material collected via suction catheter[32] or saliva.[33][34] Drosten et al. remarked that for 2003 SARS, "from a diagnostic point of view, it is important to note that nasal and throat swabs seem less suitable for diagnosis, since these materials contain considerably less viral RNA than sputum, and the virus may escape detection if only these materials are tested."[35]

Sensitivity of clinical samples by RT-PCR is 63% for nasal swab, 32% for pharyngeal swab, 48% for feces, 72–75% for sputum, and 93–95% for bronchoalveolar lavage.[36]

The likelihood of detecting the virus depends on collection method and how much time has passed since infection. According to Drosten tests performed with throat swabs are reliable only in the first week. Thereafter the virus may abandon the throat and multiply in the lungs. In the second week, sputum or deep airways collection is preferred.[32]

Collecting saliva may be as effective as nasal and throat swabs,[33] although this is not certain.[37][34] Sampling saliva may reduce the risk for health care professionals by eliminating close physical interaction.[38] It is also more comfortable for the patient.[39] Quarantined people can collect their own samples.[38] A saliva test's diagnostic value depends on sample site (deep throat, oral cavity, or salivary glands).[34] Some studies have found that saliva yielded greater sensitivity and consistency when compared with swab samples.[40][41][42]

On 15 August 2020, the US FDA granted an emergency use authorization for a saliva test developed at Yale University that gives results in hours.[43][44]

On 4 January 2021, the US FDA issued an alert about the risk of false results, particularly false negative results, with the Curative SARS-Cov-2 Assay real-time RT-PCR test.[45]

Viral burden measured in upper respiratory specimens declines after symptom onset.[46]

Isothermal amplification assays

Isothermal nucleic acid amplification tests also amplify the virus's genome. They are faster than PCR because they don't involve repeated heating and cooling cycles. These tests typically detect DNA using fluorescent tags, which are read out with specialized machines. CRISPR gene editing technology was modified to perform the detection: if the CRISPR enzyme attaches to the sequence, it colors a paper strip. The researchers expect the resulting test to be cheap and easy to use in point-of-care settings.[47][48] The test amplifies RNA directly, without the RNA-to-DNA conversion step of RT-PCR.[49]

Antigen

An antigen is the part of a pathogen that elicits an immune response. Antigen tests look for antigen proteins from the viral surface. In the case of a coronavirus, these are usually proteins from the surface spikes.[50] SARS-CoV-2 antigens can be detected before onset of COVID-19 symptoms (as soon as SARS-CoV-2 virus particles) with more rapid test results, but with less sensitivity than PCR tests for the virus.[51]

Antigen tests may be one way to scale up testing to much greater levels.[50] Isothermal nucleic acid amplification tests can process only one sample at a time per machine. RT-PCR tests are accurate but require too much time, energy and trained personnel to run the tests.[50] "There will never be the ability on a [PCR] test to do 300 million tests a day or to test everybody before they go to work or to school," Deborah Birx, head of the White House Coronavirus Task Force, said on 17 April 2020. "But there might be with the antigen test."[52]

Samples may be collected via nasopharyngeal swab, a swab of the anterior nares, or from saliva. The sample is then exposed to paper strips containing artificial antibodies designed to bind to coronavirus antigens. Antigens bind to the strips and give a visual readout. The process takes less than 30 minutes, can deliver results at point of care, and does not require expensive equipment or extensive training.[50]

Swabs of respiratory viruses often lack enough antigen material to be detectable.[53] This is especially true for asymptomatic patients who have little if any nasal discharge. Viral proteins are not amplified in an antigen test.[50][54] According to the WHO the sensitivity of similar antigen tests for respiratory diseases like the flu ranges between 34% and 80%. "Based on this information, half or more of COVID-19 infected patients might be missed by such tests, depending on the group of patients tested," the WHO said. While some scientists doubt whether an antigen test can be useful against COVID-19,[54] others have argued that antigen tests are highly sensitive when viral load is high and people are contagious, making them suitable for public health screening.[55][56] Routine antigen tests can quickly identify when asymptomatic people are contagious, while follow-up PCR can be used if confirmatory diagnosis is needed.[57]

Imaging

Typical visible features on CT initially include bilateral multilobar ground-glass opacities with a peripheral or posterior distribution.[58] COVID-19 can be identified with higher precision using CT than with RT-PCR.[59]

Subpleural dominance, crazy paving, and consolidation may develop as the disease evolves.[58][60] Chest CT scans and chest x-rays are not recommended for diagnosing COVID-19. Radiologic findings in COVID-19 lack specificity.[61][58]

Antibody tests

Left: Automated analyzer for immunoassays, used, for example, to find SARS-CoV-2 antibodies. Right: Example of quantitative results for SARS-CoV-2 antibody test.

The body responds to a viral infection by producing antibodies that help neutralize the virus. Blood tests (serology tests) can detect the presence of such antibodies.[62] Antibody tests can be used to assess what fraction of a population has once been infected, which can then be used to calculate the disease's mortality rate.[4]

SARS-CoV-2 antibodies' potency and protective period have not been established.[4][63] Therefore, a positive antibody test may not imply immunity to a future infection. Further, whether mild or asymptomatic infections produce sufficient antibodies for a test to detect has not been established.[64] Antibodies for some diseases persist in the bloodstream for many years, while others fade away.[50]

The most notable antibodies are IgM and IgG. IgM antibodies are generally detectable several days after initial infection, although levels over the course of infection and beyond are not well characterized.[65] IgG antibodies generally become detectable 10–14 days after infection and normally peak around 28 days after infection.[66][67] This pattern of antibody development seen with other infections, often does not apply to SARS-CoV-2, however, with IgM sometimes occurring after IgG, together with IgG or not occurring at all.[68] Generally, however, median IgM detection occurs 5 days after symptom onset, whereas IgG is detected a median 14 days after symptom onset.[69] IgG levels significantly decline after two or three months.[70]

Average specificity of antigen tests is 99.5%, and average sensitivity is 56.8%, but there is extreme variation in sensitivity results (ranging from 0 to 94%) between test results of different company brands.[30]

Genetic tests verify infection earlier than antibody tests. Only 30% of those with a positive genetic test produced a positive antibody test on day 7 of their infection.[64]

Types

Rapid diagnostic test (RDT)

RDTs typically use a small, portable, positive/negative lateral flow assay that can be executed at point of care. RDTs may process blood samples, saliva samples, or nasal swab fluids. RDTs produce colored lines to indicate positive or negative results.[71]

COVID-19 Antigen Rapid Test Kit
Mucus from nose or throat in a test liquid is placed onto a COVID-19 rapid antigen diagnostic test device
Enzyme-linked immunosorbent assay (ELISA)

ELISAs can be qualitative or quantitative and generally require a lab. These tests usually use whole blood, plasma, or serum samples. A plate is coated with a viral protein, such as a SARS-CoV-2 spike protein. Samples are incubated with the protein, allowing any antibodies to bind to it. The antibody-protein complex can then be detected with another wash of antibodies that produce a color/fluorescent readout.[71]

Neutralization assay

Neutralization assays assess whether sample antibodies prevent viral infection in test cells. These tests sample blood, plasma or serum. The test cultures cells that allow viral reproduction (e.g., VeroE6 cells). By varying antibody concentrations, researchers can visualize and quantify how many test antibodies block virus replication.[71]

Chemiluminescent immunoassay

Chemiluminescent immunoassays are quantitative lab tests. They sample blood, plasma, or serum. Samples are mixed with a known viral protein, buffer reagents and specific, enzyme-labeled antibodies. The result is luminescent. A chemiluminescent microparticle immunoassay uses magnetic, protein-coated microparticles. Antibodies react to the viral protein, forming a complex. Secondary enzyme-labeled antibodies are added and bind to these complexes. The resulting chemical reaction produces light. The radiance is used to calculate the number of antibodies. This test can identify multiple types of antibodies, including IgG, IgM, and IgA.[71]

Neutralizing vis-à-vis binding antibodies

Most if not all large scale COVID-19 antibody testing looks for binding antibodies only and does not measure the more important neutralizing antibodies (NAb).[72][73][74] A NAb is an antibody that defends a cell from an infectious particle by neutralizing its biological effects. Neutralization renders the particle no longer infectious or pathogenic.[75] A binding antibody binds to the pathogen but the pathogen remains infective; the purpose can be to flag the pathogen for destruction by the immune system.[76] It may even enhance infectivity by interacting with receptors on macrophages.[77] Since most COVID-19 antibody tests return a positive result if they find only binding antibodies, these tests cannot indicate that the subject has generated protective NAbs that protect against re-infection.[73][74]

It is expected that binding antibodies imply the presence of NAbs[74] and for many viral diseases total antibody responses correlate somewhat with NAb responses[78] but this is not established for COVID-19. A study of 175 recovered patients in China who experienced mild symptoms reported that 10 individuals had no detectable NAbs at discharge, or thereafter. How these patients recovered without the help of NAbs and whether they were at risk of re-infection was not addressed.[73] An additional source of uncertainty is that even if NAbs are present, viruses such as HIV can evade NAb responses.[72]

Studies have indicated that NAbs to the original SARS virus (the predecessor to the current SARS-CoV-2) can remain active for two years[79] and are gone after six years.[80] Nevertheless, memory cells including Memory B cells and Memory T cells[81] can last much longer and may have the ability to reduce reinfection severity.[80]

Other tests

Following recovery, many patients no longer have detectable viral RNA in upper respiratory specimens. Among those who do, RNA concentrations three days following recovery are generally below the range in which replication-competent virus has been reliably isolated.[82]

No clear correlation has been described between length of illness and duration of post-recovery shedding of viral RNA in upper respiratory specimens.[83]

COVID-19 testing Methods articles: 58

Infectivity

Infectivity is indicated by the basic reproduction number (R0, pronounced "R naught") of the disease.[84] SARS-CoV-2 is estimated to have an R0 of 2.2 to 2.5.[85][86] This means that in a population where all individuals are susceptible to infection, each infected person is expected to infect 2.2 to 2.5 others in the absence of interventions.[87] R0 can vary according factors such as geography, population demographics and density.[88] In New York state R0 was estimated to be 3.4 to 3.8.[89]

On average, an infected person begins showing symptoms five days after infection (the "incubation period") and can infect others beginning two to three days before that.[85][90] One study reported that 44% of viral transmissions occur within this period.[85][91] According to the CDC, a significant number of infected people who never show symptoms are nevertheless contagious.[91][86] In vitro studies have not found replication-competent virus after 9 days from infection.[92] The statistically estimated likelihood of recovering replication-competent virus approaches zero by 10 days.[93]

Infectious virus has not been cultured from urine or reliably cultured from feces;[94] these potential sources pose minimal if any risk of transmitting infection and any risk can be sufficiently mitigated by good hand hygiene.

Patterns and duration of illness and infectivity have not been fully described. However, available data indicate that SARS-CoV-2 RNA shedding in upper respiratory specimens declines after symptom onset. At 10 days recovery of replication-competent virus in viral culture (as a proxy of the presence of infectious virus) approaches zero. Although patients may produce PCR-positive specimens for up to six weeks,[95] it remains unknown whether these samples hold infectious virus. After clinical recovery, many patients do not continue to shed. Among recovered patients with detectable RNA in upper respiratory specimens, concentrations after three days are generally below levels where virus has been reliably cultured. These data were generated from adults across a variety of age groups and with varying severity of illness. Data from children and infants were not available.[92]

COVID-19 testing Infectivity articles: 3

History

Timeline of total number of tests in different countries[96]

2020

January

Public Health England announced a test on the 10th,[97] using a real-time RT-PCR (RdRp gene) assay based on oral swabs.[98] The test detected the presence of any type of coronavirus, including specifically identifying SARS-CoV-2. It was rolled out to twelve laboratories across the United Kingdom on 10 February.[99]

Scientists from China first released information on the viral genome on 11 January 2020,[100][101] sending multiple genomic sequences to GISAID, an indispensable mechanism for sharing influenza genetic sequence data.[102] That day the Malaysian Institute for Medical Research (IMR) produced "primers and probes" specific to a SARS-CoV-2 RT-PCR test.[103] The IMR's materials were used to diagnose Malaysia's first patient on 24 January.[104] BGI Group was one of the first companies to receive emergency use approval from China's National Medical Products Administration for a nucleic acid test.[105]

The German nucleic acid testing protocol was published on the 17th. Another early PCR test was developed by Charité University hospital in Berlin, working with academic collaborators in Europe and Hong Kong, and published on the 23rd. It used rtRT-PCR, and formed the basis of 250,000 kits distributed by the World Health Organization (WHO).[106]

The first case in South Korea was confirmed on 19 January.[107]

In Russia, the first COVID‑19 test was developed by the State Research Center of Virology and Biotechnology VECTOR. Production began on 24 January.[108]

In the US, the Centers for Disease Control and Prevention (CDC) developed its SARS-CoV-2 Real Time PCR Diagnostic Panel.[109] The protocol became available on the 28th.[110] One of three tests in early kits failed due to faulty reagents.

February

South Korean company Kogenebiotech's clinical grade, nucleic acid test (PowerChek Coronavirus) was approved by Korea Centers for Disease Control and Prevention (KCDC) on 4 February.[111]

In Wuhan, BGI opened a makeshift 2000-sq-meter emergency detection laboratory named "Huo-Yan" (Chinese: 火眼, "Fire Eye") on the 5th.[112][113] It processed more than 10,000 samples/day.[114][113] Construction required 5 days.[115] The Wuhan Laboratory was followed by Huo-Yan labs in Shenzhen, Tianjin, Beijing, and Shanghai, in a total of 12 cities across China.

On 11 February, the test was approved by the Federal Service for Surveillance in Healthcare in Russia.[116]

In the United States, the CDC refused to let other labs process tests that month, allowing an average of fewer than 100 samples/day to be processed. Tests using two components were not determined to be reliable until the 28th, and only then were state and local laboratories permitted to begin testing.[117] The test was approved by the FDA under an EUA.

March

Due to limited testing, no countries had reliable data on the prevalence of the virus in their population.[118] Testing variability distorts reported case fatality rates, which were probably overestimated in many countries due to sampling bias.[8][119] Shortages of reagent and other supplies became a bottleneck for mass testing in the EU and UK[120] and the US.[121][122]

By 4 March, China reached 50,000 tests per day.[123] Early in March, China reported accuracy problems with its PCR tests.[124] A study examined 1070 samples from 205 Wuhan patients and reported varied sensitivity according to the methods and location of sample collection. Samples from bronchoalveolar lavage fluid specimens returned the highest sensitivity.[125] The authors argued that CT scans showed even higher sensitivity.[126]

US commercial labs began testing in early March. As of the 5th, LabCorp announced nationwide availability of COVID‑19 testing based on RT-PCR.[127] Quest Diagnostics made nationwide testing available as of 9 March.[128] US testing demand grew rapidly, causing backlogs of hundreds of thousands of tests at private US labs. Supplies of swabs and chemical reagents continued strained.[129] On 25 May, the US required each state to take responsibility for meeting its testing needs.[130] In March, the FDA issued EUAs for nucleic acid tests to Hologic (3/16),[131] Abbott Laboratories (3/18),[132] Thermo Fisher Scientific (3/19)[133] Cepheid (3/21)[134][135] and LabCorp (4/30).[132]

On 12 March, Mayo Clinic announced a nucleic acid test.[136]

On 16 March, the WHO called for ramping up testing programmes as the best way to slow the spread.[137][138] Several European countries initially conducted more tests than the US.[139][140] By 19 March, drive-in tests were offered in several large cities.[141]

As of 22 March, according to the president of the Robert Koch Institute, Germany had capacity for 160,000 tests per week.[142] As of 26 March, German Health Minister Jens Spahn estimated that Germany was conducting 200,000 tests per week.[143] Germany has a large medical diagnostics industry, with more than a hundred testing labs that provided the technology and infrastructure to enable rapid increases in testing. Costs are borne by insurance when the test is ordered by a physician.[144] As of the end of March at least 483,295 samples were tested and 33,491 (6.9%) had tested positive.[145]

On 26 March, it was reported that 80% of test kits that Czechia purchased from China gave inaccurate results.[146][147] Slovakia purchased 1.2 million antibody-based test kits from China that were found to be inaccurate.[148] China accused Czechia and Slovakia of incorrect use of those tests.[149] Ateş Kara of the Turkish Health Ministry said the test kits Turkey purchased from China had a "high error rate".[150][151]

Spain purchased test kits from Chinese firm Shenzhen Bioeasy Biotechnology Co Ltd, but found that results were unacceptable. The maker explained that the incorrect results may stem from failure to collect samples or use the kits correctly. On 27 March, the Spanish ministry switched to another vendor, Shenzhen Bioeasy.[152]

By 31 March, the United Arab Emirates was testing more of its population per head than any other country.[153] UAE implemented a combination of drive-through sample collection, and a mass-throughput laboratory from Group 42 and BGI. The lab conduced tens of thousands RT-PCR tests per day and was the first to be operational at that scale other than China.[154]

By the month's end, testing had surpassed 200k/week.[155]

April

The FDA gave an EUA for the US' first antibody test on the 2nd.[156][63]

On 5 April, the U.S. subsidiary of China's BGI Group sent a proposal to the state of California offering to build in California, at cost ($10 million), the world's largest COVID-19 testing site, in two weeks, and train Americans to operate it. California's consultants recommended against it, because of the risk of security and commercial competition.[157]

As of 7 April, the World Health Organization (WHO) had accepted two diagnostic tests for procurement under the Emergency Use Listing procedure (EUL).[158]

On 13 April,