The COVID-19 Pandemic: a Summary

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Curious about COVID-19? Expert pathologist Fred Plapp summarizes the current state of play… 2  Special Feature

The COVID-19 Pandemic: A Summary

Curious about COVID-19? Expert pathologist Fred Plapp summarizes the current knowledge…

By Fred Plapp

What is a coronavirus? Coronaviruses are a large family of enveloped, non-segmented, single- stranded, positive-sense RNA viruses that circulate among animals including camels, cats, and bats. Coronaviruses Figure 1. A coronavirus viewed under an electron microscope. Credit: CDC/Fred Murphy. derive their name from their electron microscopic image, which resembles a CoV has a R0 of 4, meaning that each glycoprotein to dipeptidyl peptidase 4, crown – or corona (see Figure 1). infected person spreads the disease to an which is present in the lower respiratory Six strains of coronavirus have average of four others, and a case fatality tract, gastrointestinal tract, and kidney. infected humans, four of which are rate of 9.5 percent. Although the virus Like SARS, health professionals are together responsible for about one- infected 8,069 persons and caused 774 at high risk of contracting MERS. The third of common colds. In the past two deaths, the last known case of SARS was disease is still circulating and, to date, decades, there have been three global detected in September 2003. has infected approximately 2,500 people coronavirus outbreaks (1). Severe Nine years later, MERS-CoV – which and caused 850 deaths. The main factor Acute Respiratory Syndrome (SARS), causes Middle Eastern Respiratory that controls the spread of MERS-CoV caused by a coronavirus termed SARS- Syndrome (MERS) – emerged in is its very low R0 of 1. However, the case CoV, started in 2003 in Guangdong, Saudi Arabia. MERS is characterized fatality rate is very high at 35 percent. China, and spread to many countries in by sporadic zoonotic transmission from southeast Asia, North America, Europe, camels and limited episodes of person- What is SARS-CoV-2? and South Africa. Bats are the natural to-person transmission. Explosive On December 30, 2019, a cluster of hosts of SARS-CoV; its intermediate nosocomial transmission has been linked patients with pneumonia of unknown hosts are palm civets and raccoon dogs. to single super-spreaders of infection. etiology was observed in Wuhan, Early cases of SARS were linked to Almost all cases have been linked to China, and reported to the World human and animal contact at live game people in or near the Arabian Peninsula. Health Organization (WHO)’s China markets. Transmission occurred person- The symptoms of MERS are bureau in Beijing. By January 2, 2020, to-person through droplets produced nonspecific, but many patients develop the full genome of a new coronavirus by coughing or sneezing, via personal atypical pneumonia and severe acute (SARS-CoV-2) had been sequenced contact, and by touching contaminated respiratory distress. Up to 80 percent of by Shi Zhengli, a coronavirus expert surfaces. In SARS, peak viral shedding patients with MERS require mechanical at the Wuhan Institute of Virology; occurs approximately 10 days after the ventilation. Additionally, patients just over a week later, the sequence onset of illness, when many patients are often have prominent gastrointestinal had been published and the Chinese hospitalized, which explains why health symptoms and acute kidney failure. National Health Commission warned care professionals have a particularly This constellation of symptoms is due of its potential danger. The virus was high risk of becoming infected. SARS- to the binding of the MERS-CoV S initially referred to as “novel coronavirus Special Feature  3

2019” (2019-nCoV) by the WHO – but, 16 nonstructural proteins (NSP) that proteins to form new virions. Infected on February 11, 2020, was given the assist in replicating and proofreading cells can produce 100 to 1,000 virions official name of SARS-CoV-2 by the the viral genome (see Figure 3). per day. International Committee on Taxonomy SARS-CoV-2 virions attach to human of Viruses (2). cells with their densely glycosylated Where did SARS-CoV-2 come from? SARS-CoV-2 is a betacoronavirus (an spike protein and bind with high affinity How the virus evolved to become enveloped, single-stranded RNA virus) to the angiotensin-converting enzyme transmissible to humans is not known, but that shares 79 percent of its genetic 2 (ACE2) receptor on human cells. The two theories predominate: either natural sequence with SARS-CoV and has 96 spike protein is functionally divided into selection in an animal host before zoonotic percent homology with the RATG13 the S1 domain, responsible for receptor transfer to humans, or natural selection coronavirus strain in bats. However, binding, and the S2 domain, responsible in a human host after zoonotic transfer. unlike bat coronaviruses, SARS- for cell membrane fusion. Specifically, The first scenario is possible because CoV-2 has a spike protein optimized for the RBD of the spike protein mediates different coronaviruses infecting the high-affinity binding to human ACE2 recognition of the ACE2 receptor. These same host can exchange gene segments. receptors and a functional polybasic receptors are present on many types of A bat virus like RATG13 coinfecting cleavage site at the junction of the spike cells throughout the body – including an animal with another coronavirus protein’s S1 and S2 subunits (a feature lungs, heart, liver, intestines, kidneys, could have acquired a receptor-binding that enhances spike protein cleavage and testes, and blood vessels. These cells domain (RBD) more adept at infecting increases viral infectivity). also possess the TMPRSS2 serine humans, leading to SARS-CoV-2. In The virion contains four structural protease, which is needed to cleave the this scenario, the pandemic would have proteins (spike, envelope, membrane, spike protein and facilitate cell entry by emerged rapidly as soon as humans were and nucleocapsid) and single-stranded SARS-CoV-2. infected, because the virus had already RNA (see Figure 2). Once the virus has attached to the evolved to become highly infectious. The RNA genome consists of 29,903 ACE2 receptors, the TMPRSS2 In the second scenario, a non- nucleotides – larger than most other protease cleaves the spike protein to pathogenic version of the virus jumped RNA viruses. One-third of the genome expose a fusion peptide. Virions are from an animal host into humans and consists of genes for the four structural then able to enter and release their RNA then evolved to its current pathogenic proteins and eight genes for accessory into infected cells, where it is replicated state. For instance, some pangolin proteins that inhibit host defenses. Most and translated into new viral proteins. coronaviruses have an RBD structure of the remainder of the genome consists Nucleocapsid proteins bind to RNA nearly identical to that of SARS-CoV-2. of the replicase gene, which encodes two molecules and are then encapsulated A pangolin coronavirus could have been large polyproteins that are cleaved into by the envelope, spike, and membrane transmitted to a human; these animals are highly valued in traditional Chinese medicine and sold in markets such as the Wuhan Seafood and Wildlife Market, where many early human cases occurred. Another, more provocative theory suggests that SARS-CoV-2 was created (accidentally or intentionally) at The Wuhan Institute of Virology, a facility with a long history of bat coronavirus research. These theories suggest that the virus was either intentionally or accidentally released into the surrounding community. Although the lab has researched recombining the genomes of coronaviruses from different species to determine their potential to Figure 2. The structure of SARS-CoV-2. Credit: Scientific Animations™. infect human cells, prominent virologists

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Figure 3. The organization of the SARS-CoV-2 genome. Credit: Wikimedia user Furfur.

Figure 4. Global COVID-19 cases as recorded by the Johns Hopkins Center for Systems Science and Engineering. in the US consider it highly unlikely that have accidentally released both Ebola 2019 – or COVID-19. SARS-CoV-2 could have acquired both and a deadly strain of avian influenza; According to the Johns Hopkins Center of its unique features (a highly infectious and Chinese laboratory workers have for Systems Science and Engineering, as RBD and a polybasic cleavage site) in been infected with SARS-CoV and of July 6, 2020, there have been over 11.4 tissue culture. transmitted it to outside contacts on million confirmed cases of COVID-19 This type of research is performed in at least two occasions. Today, there are and 534,460 deaths worldwide (3) – but Biosafety Level 4 (BSL-4) laboratories, approximately 70 BSL-4 laboratories these numbers are still growing steadily which provide the highest level of in 30 countries, with more planned. (see Figure 4). Globally, the confirmed biocontainment and follow the most Many scientists fear that, with so case fatality rate is above 5 percent – stringent biosafety protocols. However, many biologists actively hunting for bat that is, one in every 20 people with a pathogen leakage from BSL-4 labs has viruses and performing gain-of-function confirmed positive COVID-19 test has been documented on several occasions. experiments, the world is at increasing died of the disease. The world’s last known case of smallpox risk of a laboratory-derived pandemic. The first US COVID-19 patient was was caused by a leak from a British diagnosed in late January. As of July 6, 2020, laboratory in 1978; an outbreak of foot What is COVID-19? there have been 2,888,729 confirmed cases and mouth disease in 2007 had a similar The disease caused by the SARS-CoV-2 of COVID-19 in the country and 129,947 origin; laboratories in the United States virus is known as coronavirus disease deaths. The average number of new cases Special Feature  5

per day in the US peaked at 31,000 on and acute respiratory distress syndrome April 10, 2020, and then slowly declined (ARDS). Vital signs predictive of a severe to a plateau of approximately 22,000 per course include respiratory rate over 24 day. A few weeks after reopening the breaths per minute, heart rate over 125 a b economy, however, the number of new beats per minute, and oxygen saturation cases per day has increased steadily up over 90 percent on room air. to 33,000. Current models estimate that The most common abnormal laboratory between 3 and 10 percent of Americans findings include lymphopenia (70 percent), (between 10 and 33 million people) have increased CRP (61–86 percent), mildly been infected so far. prolonged prothrombin time (58 percent), a b Fortunately, the number of deaths per elevated lactate dehydrogenase (40 day in the US has decreased from over percent), elevated AST and ALT (4–22 Figure 5. CT imaging of COVID-19 (5). a) 2,000 per day in April to approximately percent). Results that predict severe disease Thin layer CT and b) high-resolution CT 600 per day in mid-June. The decrease include D-dimer >1000 ng/mL, ferritin showing multiple patchy and light consolidation in deaths may be explained by a shift to >300 μg/L, lactate dehydrogenase >245 in both lungs of a 50-year-old woman with infections of younger people, continued IU/L, absolute lymphocyte count <800, COVID-19. c) Thin layer CT and d) high- protection of older people, more testing neutrophil-to-lymphocyte ratio >3, platelet resolution CT showing multiple patches and of people who are asymptomatic or have count <35,000/μL, CRP >100 mg/L, and “crazy paving” in both lungs of a 38-year-old mild symptoms, and better treatment. elevated high-sensitivity troponin. male with COVID-19. Other countries have not experienced this Cytokine release syndrome (“cytokine disconnect between the increase in new storm”) is caused by the overproduction People with underlying health cases per day and the number of deaths per of early-response proinflammatory conditions are six times more likely to be day – but, because deaths lag behind new cytokines, including tumor necrosis hospitalized and 12 times more likely to die cases by approximately three to four weeks, factor, IL-1β, and IL-6 (one of the best from the disease compared with patients deaths in the US are expected to rise again. biomarkers of cytokine storm severity who had no pre-existing conditions. The incubation period before the onset and mortality risk). Approximately 30 percent of of COVID-19 symptoms ranges from Chest radiographs are abnormal in 60 hospitalized COVID-19 patients one to 14 days, with a median of 5–7 percent of cases (77 percent if severe). develop progressive pulmonary disease. days. Patients, who have a median age of Chest CT is abnormal in 86 percent of The major cause of COVID-19 mortality 59 years, present with fever, dry cough, cases (95 percent if severe). Chest X-rays is respiratory failure secondary to ARDS loss of smell or taste, shortness of breath are characterized by bilateral patchy and thrombosis. ARDS is characterized by chills, rigor, fatigue, myalgia, headache, infiltrates and chest CT scans demonstrate leakage of fibrin-rich fluid from pulmonary sore throat, and diarrhea. ground-glass opacities in 86 percent of capillaries into alveoli. It may be caused by COVID-19 has a broad clinical cases. There is a peripheral distribution in direct binding of SARS-CoV-2 to ACE2 spectrum, ranging from asymptomatic over 50 percent of cases. “Crazy paving” receptors, which regulate the production infection or mild upper respiratory tract and consolidation become the dominant of angiotensin, on endothelial cells. illness to multifocal pneumonia, respiratory CT findings (see Figure 5), peaking 9 Impairment of ACE2 activity may lead failure, and death. Approximately 80 to 13 days, followed by slow clearing (4). to activation of the kallikrein-bradykinin percent of patients experience mild to pathway, which in turn increases vascular moderate disease, 15 percent have a Risk factors for COVID-19 include: permeability. Infected endothelial cells severe course requiring intensive care, and • older age also express leukocyte adhesion molecules 5 percent require mechanical ventilation. • ethnicity that recruit activated neutrophils and Patients may develop pneumonia towards • male gender lymphocytes to the site of injury. The the end of the first week of infection. The • comorbidities (including accumulation of cytokines, neutrophils, mean interval from onset of symptoms to hypertension, diabetes, coronary and lymphocytes causes inflammation, hospitalization is between 9 and 12 days; artery disease, chronic lung/ loosens endothelial cell junctions, mean duration from symptom onset to kidney/liver disease, cancer, increases vascular permeability, promotes discharge from the hospital is 25 days. hematologic malignancy, organ alveolar fluid retention, and enhances The most severe cases develop pneumonia transplant, or immunosuppression) pulmonary tissue damage.

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Figure 6. A transmission electron microscopic image of an isolate from the first US case of COVID-19. Credit: CDC.

A recent autopsy report compared the admission to intensive care units with who were symptom-free when they histologic patterns of lungs from patients Pediatric Inflammatory Multisystem tested positive was consistently high. who died from influenza with patients Syndrome (PIMS) associated with SARS- Because these studies tested circumscribed who died from COVID-19. Both groups CoV-2 infection. The syndrome has many populations, the percent of people who are had diffuse alveolar damage with hyaline overlapping features with Kawasaki asymptomatic and test positive is likely membranes and perivascular T-lymphocyte disease. Thus far, children have been given overestimated. Some experts suggest that infiltrates. The lungs from COVID-19 anti-inflammatory treatments, including the asymptomatic rate is 40 to 45 percent. patients had distinctive vascular features parenteral immunoglobulin and steroids. Asymptomatic patients have the same due to SARS-CoV-2 invasion of viral load as many symptomatic ones and endothelial cells, including disruption of What about asymptomatic disease? can transmit the virus for at least 14 days. cell membranes and severe endothelial Early identification and testing of And the absence of symptoms in people injury. This caused microangiopathy individuals with COVID19 symptoms infected with SARS-CoV-2 does not and widespread thrombosis in the small have been the primary focus of public mean that they are free from harm; of the vessels and capillaries of the lungs. health mitigation. However, many asymptomatic individuals who had lung Alveolar capillary microthrombi were studies have shown that a significant CT scans, 33–48 percent had ground nine times more prevalent in patients who proportion of individuals infected with glass opacities. died from COVID-19 than in those who SARS-CoV-2 do not have any symptoms died from influenza. at the time of testing. What about coagulopathy? Recent reports from Europe and Infection rates vary widely between The risks of COVID-19 are highest North America have described clusters populations (see Table 1). However, in in older patients with pre-existing of children and adolescents requiring all studies, the proportion of individuals conditions such as hypertension, diabetes, Special Feature  7

Population PCR tested Positive (%) Positive asymptomatic (%)

Iceland residents 13,080 0.8 43.0

Vo, Italy residents 5,155 2.0 42.2

Diamond Princess 3,711 19.2 46.5

Boston homeless shelter 408 36.0 87.8

New York City obstetric patients 214 15.4 87.9

USS Theodore Roosevelt 4,954 17.3 58.4

Japanese evacuated from Wuhan 565 2.3 30.8

Greeks evacuated from Europe 783 2.3 87.5

Charles de Gaulle aircraft carrier 1,760 59.4 47.8

Los Angeles homeless shelter 178 24.2 62.8

King County, Washington nursing home 76 63.2 6.3

US prisoners (AR, NC, OH, VA) 4,693 69.8 96.0

Rutgers University 829 4.9 65.9

Indiana residents 4,611 1.7 44.8

Table 1. A summary of studies on COVID-19 testing results, highlighting the percentage of positive test results obtained from asymptomatic patients. cardiovascular disease, and obesity. The of tissue factor. Together, these events lead cleaved (due to decreased secretion common theme? Their association with to thrombosis and tissue ischemia. of ADAMTS13), which can lead to vascular inflammation and endothelial An imbalance of VWF and thrombosis and thrombocytopenia. dysfunction. Multiple studies have ADAMTS13 may also play a role in In response to blood clot formation, noted a higher incidence of pulmonary COVID-19-associated coagulopathy. the fibrinolytic pathway is activated embolism and venous thromboembolism VWF is an acute phase response protein (which explains the high levels of in COVID-19 patients admitted to the released by activated endothelial cells in D-dimer and its close correlation with intensive care unit. response to inflammatory stimuli. In the poor patient outcome). SARS-CoV-2 may directly infect event of vascular injury, VWF facilitates SARS-CoV-2-induced thrombosis endothelial cells by binding their ACE2 binding of platelets to sub-endothelium may be responsible for the very low receptors. Endothelialitis – infection through its interactions with collagen, oxygen saturation levels in some patients. of endothelial cells and subsequent inducing thrombus formation. VWF also COVID-19 reduces tissue oxygenation not perivascular inflammation – disrupts binds to neutrophil extracellular traps and only by causing pneumonia and ARDS, but vascular integrity, exposes capillary recruits platelets and leukocytes to promote also by promoting thrombosis. basement membranes, and activates the thrombosis. In contrast, ADAMTS13 is a coagulation cascade. Activated endothelial negative acute response protein; its activity How is COVID-19 transmitted? cells express P-selectin, von Willebrand decreases in response to inflammation. Two factors facilitated the initial rapid factor (VWF), and fibrinogen, which Release of proinflammatory mediators spread of COVID-19 in Wuhan: i) a enhances platelet adhesion. Activated during the severe phase of COVID-19 population of 11 million inhabitants platelets release VEGF, which triggers may increase the secretion of ultra-large that increased the chance of person-to- endothelial cells to upregulate the expression VWF multimers that are not completely person contact, and ii) the city’s busy

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transportation hub, which increased the traveling up to 200 miles per hour, most of COVID-19 deaths that occur at home are likelihood of exporting cases to other which are small and travel great distances. underreported compared with those that locations. Despite Chinese containment A single cough or a sneeze emitted by an occur in a hospital. measures, COVID-19 has grown into a infected person may spread as many as 200 The infection fatality rate is the full-blown pandemic. million virus particles. proportion of infected people who will The R factor, a virus’ basic reproductive In contrast, a single breath releases only die from COVID-19, including those who number, is referred to as R0 – the average 50 to 5,000 droplets, most of which travel do not get tested or become symptomatic. number of people someone carrying the at low velocity and drop quickly. Because The infection fatality rate is estimated to be virus will infect. The higher the R0, the breath is expelled at low force, viral particles between 0.5 and 1 percent. Even at this rate, faster an epidemic can spread. At the start residing in the lower respiratory areas are COVID-19 is a serious public health threat. of the pandemic, R0 for SARS-CoV-2 not expelled – meaning that breathing may For comparison, the infection fatality rate was estimated at 2.0 to 2.5, indicating release as few as 20 to 30 viral particles of seasonal influenza is approximately 0.1 that one patient could transmit the virus per minute. Speaking increases the release percent – and it nevertheless kills hundreds to two (or slightly more) other people. about tenfold (200 virus particles per of thousands of people each year. The doubling time for COVID-19 cases minute), so five or more minutes of face- If one assumes that the number of is estimated at three to six days. to-face conversation could lead to infection. asymptomatic or minimally symptomatic The virus (see Figure 6) is transmitted But infection with SARS-CoV-2 cases is several times as high as the number primarily through droplets 5–10 μm depends not only on dose, but also of reported cases, the case fatality rate in diameter, released when an infected exposure time. If an infected person coughs may be less than 1 percent. Even though person coughs, sneezes, talks, or even or sneezes directly toward someone, they its case fatality rate is lower than MERS- exhales. These airborne droplets can can inhale 1,000 viral particles in a few CoV, SARS-CoV-2 will cause many more attach to the respiratory tract mucosa minutes. If someone enters a room shortly deaths, because there have been – and will or conjunctiva of another person. They after an infected person coughs or sneezes, continue to be – so many more cases. As can also settle on surfaces or fomites and it may take only a few breaths – whereas with other coronaviruses, health care- be transferred to another person upon if they simply occupied a room where an associated transmission appears to be a contact. SARS-CoV-2 is more stable on infected person was breathing, it might major mode of infection. plastic and steel (up to three days) than take 50 minutes or longer to inhale an One histopathological study of the lungs on cardboard (up to one day) or copper. infectious dose. of a deceased patient reported the presence Viral transmission is possible if someone of hyaline membrane formation (see Figure touches their face, eyes, nose, or mouth How deadly is COVID-19? 7), interstitial mononuclear inflammatory following contact with contaminated The mean duration from symptom onset infiltrates, and multinucleated giant cells. surfaces or fomites. to death is 18 days. Case fatality rate These findings were consistent with acute Transmission may also occur through (CFR), which is calculated by dividing respiratory distress syndrome. aerosols, which are particles smaller than 5 the number of deaths by the number of SARS-CoV-2 RNA has been detected μm. SARS-CoV-2 remains viable in these known cases, has been reported at 6.4 in several body fluids including 93 percent particles for up to three hours. Aerosol percent worldwide – significantly higher in of bronchoalveolar lavage fluid, 72 percent transmission is a serious risk to health older patients. But CFR almost certainly of sputum, 63 percent of nasal swabs, 32 care workers during procedures such as overestimates the true lethality of the virus. percent of pharyngeal swabs, 29 percent of intubation, bronchoscopy, suctioning, The number of confirmed cases usually feces, and 1 percent of blood samples. No turning a patient to the prone position, or includes only people whose symptoms urine specimens have tested positive. The disconnecting a patient from the ventilator. were severe enough to be tested, resulting average cycle threshold for all specimens Some experts estimate that exposure to in a severity bias. Epidemiologists estimate was 31, which corresponded to a viral as few as 1,000 SARS-CoV-2 particles can there are five to 10 times more people with load of less than 2.6x104 copies/mL. cause infection. One releases about 3,000 asymptomatic infections. Additionally, Nasal swabs had a cycle threshold of 24, respiratory droplets that travel at 50 miles the number of deaths may be inaccurate indicating a much higher viral load of per hour; most are large and quickly fall to at the time of calculation because deaths 1.4x106 copies/mL. the ground, but many remain airborne and typically occur one to two months after About 12 percent of COVID-19 can travel across a room in a few seconds. a person becomes infected and not all patients have gastrointestinal symptoms A sneeze releases about 30,000 droplets deaths are apparent at the same time. and 41 percent shed viral RNA in their Special Feature  9

Figure 7. Hyaline membrane formation in diffuse alveolar damage, the histological correlate of acute respiratory distress syndrome. Credit: Wikimedia user Nephron. feces. The presence of viral RNA does and airborne precautions, including the use deemed unusable. Although the problems not necessarily indicate the presence of of masks and eye protection. Health care were raised on February 7, more than 50 live virus, but raises the possibility of workers should maintain social distancing days passed before the CDC developed human-to-human transmission by the in the workplace. an alternative test. Even after kits became fecal-oral route. SARS-CoV-2 has also available, testing was hampered by a been detected in human breast milk from How does COVID-19 RT-PCR shortage of RNA extraction reagents and a single mother on days 10, 12, and 13 after testing work? nasopharyngeal swabs. birth. Detection of viral RNA coincided The sequence of SARS-CoV-2 was Eventually, the CDC published with mild COVID-19 symptoms and a published by Chinese scientists on January primers, probes, and protocols. The US positive PCR in the newborn. 11, 2020; the following week, virologists Food and Drug Administration (FDA) CDC guidelines state that routine in Berlin, Germany, produced the first issued new guidance on February 29, BSL-2 laboratory practices are adequate reverse transcriptase real-time polymerase 2020, so that labs could develop and for specimens from patients that may have chain reaction (RT-PCR) diagnostic test use COVID-19 molecular diagnostic SARS-CoV-2 infection, with the exception for COVID-19. This test was supplied to tests (but had to apply for Emergency that potentially infectious specimens from the WHO and many countries adopted it. Use Authorization, or EUA, within 15 these patients should be manipulated only Unfortunately, the US Centers for Disease business days of clinical use). Although in a biological safety cabinet. The CDC Control and Prevention (CDC) refused to clinical labs could purchase primers and explicitly recommends against viral culture employ this test and prevented laboratories probes for the CDC assay from Integrated from specimens that may contain SARS- from producing their own assays. On DNA Technologies (IDT), other reagents CoV-2. However, clinical laboratory staff February 5, the CDC began shipping its had to be procured elsewhere. To remain should wear personal protective equipment own SARS-CoV-2 RT-PCR kit – but in FDA compliance, labs had to follow the (PPE) and implement standard, contact, it produced unreliable results and was exact specifications under which the EUA

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was granted. If they ran the IDT kit on an intervals between specimens increases the Worldwide, concerns have been alternative platform, new EUA approval risk of missed diagnosis because viral load expressed about the reliability of tests was required – an ordeal too onerous for decreases with time. Repeat testing of a that have been rapidly developed and most hospital clinical laboratories. lower respiratory sample (bronchoalveolar marketed without rigorous oversight. The WHO’s RT-PCR assay targets lavage) might be necessary for diagnosis Some companies claim high sensitivity the SARS-CoV-2 envelope gene and the of a patient with severe or progressive and specificity without accompanying RNA-dependent RNA polymerase gene. disease and repeat negative results with data, and the FDA has warned that some If both targets are detected, the result is nasopharyngeal swabs. companies have falsely claimed FDA reported as positive; if only one is detected, Nasopharyngeal swabs, not throat approval. SARS-CoV-2 antibody tests the result is reported as inconclusive. The swabs, should be submitted for RT- marketed prior to or without an EUA CDC’s original assay included three PCR testing. If a nasopharyngeal (NP) are not FDA-authorized and have not different amplification regions of the swab is not inserted properly, there is a received a CLIA categorization. These N gene; NS3 was designed to detect all higher likelihood of a false negative result. tests are considered high-complexity by SARS-like coronaviruses, whereas the NP swabs should be transported to the default until they receive an approval N1 and N2 regions were specific for laboratory in universal transport media that permits them to be considered SARS-CoV-2. The NS3 target produced or saline, not sent by pneumatic tube, and moderate-complexity or CLIA-waived. too many false positive results and had to should be stored at 2–8°C. Currently available tests target be eliminated. Viral loads measured on NP specimens antibodies to one of two SARS-CoV-2 Other laboratories and diagnostic are much higher in patients with severe proteins, either the nucleocapsid companies designed RT-PCR assays that disease than in patients with mild disease, phosphoprotein or the spike protein. Most targeted various combinations of the open although RT-PCR testing may return lateral flow assays detect IgG and IgM reading frame, envelope, nucleocapsid, positive results even in some asymptomatic antibodies separately. Enzyme-linked and RNA-dependent RNA polymerase patients. Most patients with mild disease and chemiluminescent immunoassays genes. The limit of detection of most test negative by 10 days after symptom detect either total antibody, IgG alone, such assays was 100 viral copies/mL or onset. In contrast, patients with severe or IgG and IgM separately. There is higher. Several commercial reference disease shed virus for a median of 20 no substantive advantage to assays that labs began testing during the second days post-symptom onset. Virus may be detect IgG over total antibody. week of March, courtesy of major in continuously detectable until death in The Infectious Disease Society of vitro diagnostic vendors with EUAs for some non-survivors. America (IDSA) recently published their assays, but validations for these their guidelines on COVID-19 serologic assays used synthetic RNA sequences How does COVID-19 antibody testing (5). They state that antibody spiked into respiratory samples and data testing work? testing has not been clinically verified documenting their clinical diagnostic Serologic tests detect antibodies that and should not be used as the sole test performance was limited. form in blood after SARS-CoV-2 for diagnostic decisions. Antibody test Recent studies indicate that SARS- infection. To increase availability, the results should not be used to make CoV-2 viral load peaks in the first five FDA permits companies to develop staffing decisions or decisions regarding to six days of disease onset. Viral RNA and distribute serology tests if they the need for PPE until more evidence can be detected during the second validate the tests with specimens from about protective immunity is available. week of disease onset, but viral load confirmed COVID-19 patients and According to the IDSA, SARS- is lower. Despite high sensitivity, a notify the FDA of their intent. Results CoV-2 serology may: negative PCR is insufficient to exclude must be accompanied by a statement: SARS-CoV-2 infection in patients with “This test has not been reviewed by • support the diagnosis of a high pretest probability of infection; the FDA.” COVID-19 in patients who present timing of specimen collection, specimen Over 200 manufacturers have begun late and have a negative PCR source, specimen quality, and method marketing serologic tests in the US. result, or when lower respiratory performance affect the accuracy of results. Most of the tests flooding the market tract sampling is not possible If repeat PCR testing is warranted, the are lateral flow assays; laboratory-based • identify people with an antibody second specimen should be performed at antibody tests are either enzyme-linked response to serve as convalescent 24 hours after the first collection; longer or chemiluminescent immunoassays. plasma donors Special Feature  11

• allow epidemiologic studies of disease prevalence • verify vaccine response once antibody correlate(s) of protection are identified

Antibody tests should not be used to diagnose acute COVID-19 infections. Individuals with symptomatic COVID-19 generally do not have detectable antibodies to SARS-CoV-2 within 10 days of symptom onset. Most hospitalized patients with confirmed viral RNA have detectable IgG antibodies 14 days after symptom onset; IgM antibodies become detectable only one to two days earlier – so these tests Figure 8. “Flattening the curve,” a mitigation approach to lower and delay the epidemic peak. miss infectious patients in the early Credit: Esther Kim and Carl T. Bergstrom. stages of disease and patients with mild symptoms (who may produce antibodies that neutralized virus in other viral pneumonias. It consists lower antibody titers). They can also plaque growth assays (the standard test for primarily of supportive care and fail in elderly or immunocompromised antibody effectiveness). The best way to oxygen supplementation when needed. patients, who may not develop detectable investigate immunity is to follow people Dexamethasone has been reported to levels of antibodies after infection. Even with and without antibodies to determine decrease the mortality rate of patients more worrisome, some patients continue whether they become reinfected. with severe respiratory illness (6). to shed viral RNA after seroconversion. SARS-CoV-2 seroprevalence remains Remdesivir, a nucleoside prodrug that A negative serologic test might give low even in the most severely affected inhibits transcription of many RNA patients a false sense of security, leading communities. When overall disease viruses, may shorten COVID-19-related to reckless behavior. prevalence is low, maximizing specificity hospital stays by an average of three days Antibody tests may play a role in and positive predictive value is preferred. (7). Tocilizumab, a monoclonal antibody detecting unrecognized past infection An antibody test with specificity of 99 to IL-6, is being trialed in patients with and immunity, but that role must be percent or greater will yield the highest cytokine storm and severe respiratory rigorously evaluated. Currently, no one positive predictive value in populations disease. Additionally, lopinavir/ritonavir knows how long antibodies to SARS- with a prevalence of 5 percent or higher. (Kaletra), a mixture of two HIV protease CoV-2 persist. Seasonal coronavirus This test has an even greater chance of inhibitors, is under investigation. antibodies decline only a few weeks being accurate when testing a high- Recently, China approved the use of after infection and some people are risk group, such as exposed healthcare favilavir (Favipiravir), an antiviral drug susceptible to reinfection within one workers or family members. used for influenza, as an investigational year. More encouragingly, SARS-CoV When should serologic test results therapy for COVID-19 (8). antibodies peak approximately four not be used? They should not contribute Hydroxychloroquine, much touted for its months after infection and protect to decisions about grouping people in potential therapeutic effect, was shown in patients for two to three years. schools, dormitories, or correction 2002 to interfere with SARS-CoV entry The presence of SARS-CoV-2 facilities; about returning people to into cells – but does not benefit patients antibodies does not guarantee immunity. work; or about changes to clinical with COVID-19. The FDA recently It is not currently known which antibody practice or the use of PPE. revoked the emergency authorization of responses – if any – are protective or hydroxychloroquine and chloroquine to sustained. Of COVID-19 patients How is COVID-19 treated? treat COVID-19 because neither drug who developed antibodies during The care of patients with COVID-19 demonstrated benefits that outweighed hospitalization, one in three lacked is similar to that of patients with the risks of dangerous cardiac arrhythmias.

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On March 24, 2020, the FDA approved prior to donation, or at least 14 days As many as 123 different SARS-CoV-2 the investigational use of convalescent prior to donation and a negative vaccine candidates are under development plasma, which contains antibodies to nasopharyngeal swab by PCR. worldwide, 10 of which are in human trials. SARS-CoV-2, for patients with serious • Compliance with all blood donor Many have not been tested in animals. In or life-threatening disease. COVID-19 eligibility requirements. July, the National Institutes of Health convalescent plasma (CCP) is a potentially (NIH) will begin randomized phase safe and effective, but unproven, therapeutic Donors are tested for blood type and III trials to determine if any of these 10 modality for COVID-19. The FDA infectious diseases. Women who have vaccines prevent SARS-CoV-2 infection. requires clinical application of CCP to been pregnant are also tested for HLA They plan to enroll 20,000 individuals who be conducted under one of three defined antibodies; if present, their plasma is will receive a vaccine and 10,000 who will pathways: i) an investigational new drug not used for transfusion. Each unit of receive a placebo. (IND) application to support research – the CCP contains approximately 200 mL Conventional vaccines rely on the traditional approach for clinical trials; ii) and should be ABO compatible with the production of either live attenuated virus or an emergency use IND for compassionate recipient. The dose is one to two units per inactivated virus. Live attenuated vaccines use in an individual patient with severe or patient. Transfusion rate is 100 to 250 mL use a weakened form of the virus to produce immediately life-threatening COVID-19; per hour or per hospital policy. CCP units an immune response without causing or iii) a government-led initiative providing can be frozen and stored for one year; after serious illness. Because they use live virus, expanded access program (EAP) IND to thawing, plasma may be stored for up to these vaccines need extensive safety testing. participating institutions under a master five days at 1–6°C. Possible adverse events Some live viruses can be transmitted to treatment protocol with modest data- are the same as other plasma components. other people, which is a concern for people reporting requirements. Mayo Clinic leads At the moment, CCP is for investigational who are immunocompromised. Inactivated the EAP IND for hospitalized patients use only. virus vaccines use a killed virus, which with severe or life-threatening COVID-19. may be safer, but often produces a weaker Several trials have been proposed to When will there be a vaccine? immune response. These vaccines require evaluate CCP for: On May 1, 2020, the US federal multiple doses and boosters to provide long- government launched “Operation Warp term immunity. Some vaccines also require • post-exposure prophylaxis among Speed” to deliver a COVID-19 vaccine adjuvants to enhance the immune response adults with close contact exposure by January 2021, years ahead of standard – and work is already underway on licensed to COVID-19 who have not yet vaccine timelines. Usually, a vaccine is first adjuvants for use with COVID-19 vaccines. manifested symptoms tested in animals. If it appears safe and A vaccine that targets the SARS- • treatment of patients with confirmed effective, then the three phases of human CoV-2 spike protein should theoretically mild disease clinical trials begin. Phase I evaluates the prevent the virus from binding to human • treatment of moderately ill, safety of the vaccine in humans; some cells and reproducing. The advent of hospitalized patients who have vaccines can make a viral infection more genetic engineering may allow scientists not been admitted to the intensive virulent by a mechanism called antibody- to produce novel vaccines that specifically care unit admission or required dependent enhancement. Phase II target this antigen. A gene for a single mechanical ventilation establishes the formulation and doses of SARS-CoV-2 protein can be introduced • rescue therapy for patients requiring the vaccine to optimize its effectiveness. into cell cultures, which synthesize large mechanical ventilation Phase III then tests the safety and efficacy quantities of relatively pure protein to • safety and pharmacokinetics in high- of the vaccine in a larger group of people. serve as a vaccine. Alternatively, the gene risk pediatric patients. Only after a vaccine passes all three phases can be inserted into an innocuous virus, • Potential CCP donors must meet the is it licensed, manufactured, distributed, such as adenovirus, which is then injected following criteria: and administered to humans. Because into people. The genetically engineered • Documented evidence of COVID-19 of the urgency of the pandemic, some adenovirus infects human cells, replicates, diagnosis by PCR at the time of scientists propose replacing this method and expresses the spike protein to prompt illness or positive SARS-CoV-2 with faster “challenge trials,” which an immune response. antibody test after recovery if PCR deliberately expose vaccinated volunteers Some companies are attempting to was not performed. to the virus and could determine a vaccine’s produce nucleic-acid vaccines, in which • Symptom-free for at least 28 days effectiveness in weeks instead of years. a gene for a SARS-CoV-2 antigen is Special Feature  13

introduced directly as a segment of either illustrates the beneficial effect of mitigation will continue to circulate in the human DNA or RNA. Such vaccines should carry – also known as “flattening the curve.” The population and will synchronize to less risk of contamination, because they do Center for Infectious Disease Research and a seasonal pattern with diminished not require cultured cells or viruses, but Policy (CIDRAP) has predicted that the severity over time, as with other less no RNA or DNA vaccine has ever been COVID-19 pandemic will last for 18 to 24 pathogenic coronaviruses. licensed for use in humans anywhere in the months and will not be halted until 60 to 70 world. DNA plasmid vaccines transfer the percent of the population becomes immune Fred Plapp is Clinical Professor and Medical genetic blueprint for RNA into cells, which (10). Depending on control measures, cases Director of Clinical Laboratories and then synthesize spike antigens; one such may come in waves of varying impact and Executive Vice Chair for Clinical Affairs in vaccine was developed for MERS, but never at different intervals as illustrated by the the Department of Pathology and Laboratory manufactured. RNA vaccines eliminate the following three scenarios. Medicine, University of Kansas School of need for DNA plasmids by embedding Scenario 1: The first wave of COVID-19 Medicine, Kansas City, USA. RNA into lipid globules that can merge (spring 2020) is followed by a series with cell membranes. Human cells then of repetitive smaller waves that occur References synthesize the corresponding antigen. through the summer and then consistently 1. J Guarner, “Three emerging coronaviruses in two RNA vaccines may produce more potent over a one- to two-year period, gradually decades”, Am J Clin Pathol, 153, 420 (2020). immunity than DNA plasmids, but they diminishing in 2021. These waves may vary PMID: 32053148. are less stable and must be stored frozen. geographically and may depend on what 2. C Del Rio, PN Malani, “COVID-19 – new mitigation measures are in place and how insights on a rapidly changing epidemic”, JAMA, Stopping COVID-19 – suppression they are eased. Depending on the height of [Epub ahead of print] (2020). PMID: or mitigation? the wave peaks, this scenario could require 32108857. Some countries attempted to reduce periodic reinstitution and subsequent 3. Center for Systems Science and Engineering at the infectivity of the pandemic to R0 by relaxation of mitigation measures over the Johns Hopkins University, “Coronavirus enforcing suppression. An R0 below 1 next one to two years. COVID-19 Global Cases” (2020). Available at: indicates that each infected person transmits Scenario 2: The first wave of COVID-19 https://bit.ly/3b6cQWl. SARS-CoV-2 to less than one other person. is followed by a larger wave in late 4. Z Xu et al., “Pathological findings of COVID-19 Successful suppression requires early and 2020 and one or more smaller waves associated with acute respiratory distress widespread testing – including of people in 2021. This pattern will require the syndrome”, Lancet Respir Med, [Epub ahead of without symptoms. Those who positive are reinstitution of mitigation measures in print] (2020). PMID: 32085846. isolated so that they cannot infect others. the autumn to decrease the spread of 5. IDSA, “Infectious Diseases Society of America A failure to implement early testing in infection and prevent healthcare systems Guidelines on the Diagnosis of COVID-19” other countries has forced them to rely from being overwhelmed. (2020). Available at: https://bit.ly/2NE3SpB. on mitigation, rather than suppression, to Scenario 3: The first wave of COVID-19 6. P Horby et al., “Effect of dexamethasone in slow the spread of disease. Mitigation is followed by a slow burn of ongoing hospitalized patients with COVID-19: efforts include handwashing, school transmission and case occurrence, but preliminary report” (2020). Available at: https:// and business closings, travel limitations, without a clear wave pattern. Again, bit.ly/2BTL6rB. mask wearing, and social distancing to this pattern may vary somewhat 7. JH Beigel et al., “Remdesivir for the treatment of decrease the likelihood of person-to- geographically and may be influenced COVID-19 – preliminary report”, N Engl J Med, person transmission. Mitigation focuses by the degree of mitigation measures [Epub ahead of print] (2020). PMID: 32445440. on protecting the most vulnerable from in place in various areas. This scenario 8. Pharmaceutical Technology, “Favilavir approved the effects of a disease that is already would likely not require the reinstitution as experimental coronavirus drug” (2020). widespread throughout the community. of mitigation measures, although cases Available at: https://bit.ly/2NAYvaK. By reducing the number of active cases at and deaths will continue to occur. 9. US Food and Drug Administration, “Coronavirus any given time, health care providers can Whichever scenario the pandemic (COVID-19) update: daily roundup, March 24, respond without becoming overwhelmed follows, a significant level of COVID-19 2020” (2020). Available at: https://bit. (see Figure 8). infection is likely to continue worldwide, ly/2Vt9IP3. The steep, dotted curve represents the with hotspots popping up periodically in 10. Center for Infectious Disease Research and Policy, occurrence of cases over time without diverse geographic areas. As the pandemic “COVID-19: the CIDRAP viewpoint” (2020). protective measures. The flatter, solid peak wanes, it is likely that SARS-CoV-2 Available at: https://bit.ly/2CSlP1H.