COVID-19: laboratory diagnosis for clinicians. An updating article

ABSTRACT COVID-19 (coronavirus disease 2019) is an infectious disease caused by the new coronavirus associated with severe acute respiratory syndrome 2 (SARS-CoV-2). Coronaviridae comprises a large family, of which at least seven members are known to cause respiratory diseases in humans. Coronaviruses have the ability to infect virtually all major groups of animals and, eventually, can infect humans. SARS-CoV-2 is the third coronavirus to cross the species barrier and infect humans. This virus was identified in an outbreak of pneumonia cases in Wuhan city, Hubei province, China, in December 2019. Its entire genome is inscribed on a single strand of ribonucleic acid. Some proteins present on the surface of the virus act as facilitators for its entry into host cells, while others, apparently, are related to its pathogenesis. Coronaviruses are responsible for respiratory infections in humans and some animals. The infection is often mild to moderate in intensity, but some coronaviruses may cause serious illnesses, such as severe acute respiratory syndrome (SARS), which occurred in 2002, and the Middle East respiratory syndrome (MERS). Coronaviruses can activate an excessive and unregulated immune response, which may promote SARS development. Although the lungs are one of the target organs, the hypoxia mechanism is systemic and other organs begin to suffer both through lack of oxygen and through deregulation of inflammation control mechanisms.


COVID-19 (coronavirus disease 2019) is an infectious disease caused by the new coronavi-
rus associated with severe acute respiratory syndrome 2 (SARS-CoV-2). Coronaviridae comprises a large family, and at least seven coronaviruses are well known for causing respiratory diseases in humans. Coronaviruses have the ability to infect virtually all major groups of animals, among which some host other species that may infect humans. The current understanding is that SARS-CoV-2 is the third zoonotic coronavirus to have crossed the barrier between species and become capable of infecting humans over the past two decades.

THE CORONAVIRUS THAT CAUSES COVID-19
SARS-CoV-2 is a new betacoronavirus belonging to the large viral family of Coronaviridae, first identified in an outbreak of pneumonia cases in Wuhan city, Hubei province, China, in December 2019. The name COVID-19 was chosen as the name for this new infection as an acronym of coronavirus disease 2019, i.e. from corona "co", virus "vi", disease "d" and the number 19 indicating the year of its appearance. Efforts are being made by the World Health Organization (WHO) to ensure that the nomenclature of viruses and their infections no longer refers to geographical locations, as it did traditionally, in order to combat the stigma resulting from this practice.
The entire SARS-CoV-2 genome is inscribed on a single strand of RNA (ribonucleic acid). This type of virus undergoes genetic mutations more frequently than DNA (deoxyribonucleic acid) viruses, given that RNA viruses have less ability to correct any transcription errors. 1 SARS-CoV-2, in particular, is a single-stranded RNA virus that is capable of synthesizing about 29 different proteins. Some of these proteins are present on the surface of the virus and act as facilitators of its entry into host cells, while others, apparently, are related to its pathogenesis.  SARS-CoV-2 binds to the human host cell via the ACE2 receptor (angiotensin-converting enzyme 2), and its input mechanism depends on sequential action by the serine protease TMPRSS2 enzyme. These data suggest that several therapeutic targets are possible, including the interleukin (IL)-6-STAT3 axis, which is associated with cytokine release syndrome (CRS).
Another structural protein of the virus is thought to have the ability to displace the iron that is present in hemoglobin. This would reduce the oxygen transportation capacity and provide the low level of saturation that is observed in some of the patients who evolve poorly. Additionally, release of iron ions in high quantities would cause oxidative damage, thus triggering an intense inflammatory process that might result in the condition known as a cytokine storm. Although the lungs are one of the target organs, the hypoxia mechanism is systemic and other organs start to suffer, both through lack of oxygen and through deregulation of the inflammation control mechanisms. Two other organs strongly affected are the liver and kidneys.
The clinical manifestations of COVID-19 can range from none, i.e. a totally asymptomatic state (which may be the case in up to 89% of individuals who become infected), to a situation characterized by mild to critical and fatal symptoms. The symptoms can develop between 2 to 14 days after exposure to the virus, 2 with an average incubation period of 5.1 days. Hence, the recommended quarantine period is usually 14 days.
Although the proportions may vary in the different populations affected, data provided by the Chinese Center for Disease Control and Prevention (CCDC) have shown that 81% of the patients had mild clinical manifestations; 14% of them had severe manifestations, including hypoxia; 5% of the cases were critical, with respiratory failure, multiple organ dysfunction and shock; and 2.3% of the cases were fatal. 3 The course of the disease can last for around 16 days after a short incubation period, in mild to moderate cases; or can last for up to 10 weeks if there is a longer incubation period and a severe or fatal outcome. The following clinical scenarios have been estimated from various publications.
• Incubation period of 2 to 14 days (average 5 to 6 days) after infection • Mild cases: duration of two weeks • Severe and recovered cases: duration of three to six weeks A person can be a transmitter even before symptoms appear, and can continue to be a transmitter until they disappear. The peak period for transmission is around five days after the onset of symptoms. It should be noted that because the pandemic is still new, the information provided here may change. 4

LABORATORY DIAGNOSIS
When an infected person exhales droplets containing SARS-CoV-2 virions and they are inhaled by someone else, these droplets lodge on the nasal mucosa, which is formed by cells covered with ACE2 receptors. The virus binds with these receptors and thus is able to penetrate and hijack cell machinery in order to produce other virions and infect new cells. While viral multiplication is occurring, this person eliminates virions in large quantities, especially in the first week. This period can be asymptomatic or paucisymptomatic, with fever, dry cough, sore throat, anosmia, ageusia and severe headaches or body pain. 5 If the immune system is unable to stop the infection at this point, the virus progresses through the respiratory tract to the pulmonary alveoli, which are rich in ACE2 receptors. In the alveoli, leukocytes migrate due to the action of cytokines, which results in disruption of gas exchange, with pneumonia, characterized by productive cough, fever and dyspnea. 6 In outpatients with flu-like symptoms, the chemosensory disorder causes loss of the sense of the smell and taste that is strongly associated with SARS-CoV-2 infection. This sign should be con- The virus, probably with help from the immune response to it, can cause damage to the following other organs: • Liver: about half of hospitalized patients show signs of liver changes.
• Kidneys: kidney damage, including kidney failure and the need for dialysis, is common in severe cases.
• Intestines: there may be a clinical presentation characterized by gastrointestinal symptoms, especially vomiting, diarrhea and abdominal pain. A "stomach" pain that may be associated with inflammation of the base of the lungs and diaphragm has been described. The lower gastrointestinal tract is rich in ACE2 receptors and about 20% of patients report diarrhea.
• Central nervous system: there have been reports of stroke, possibly due to formation of microthrombi, occurrences of seizures and cognitive changes.
• Eyes: in severe cases, occurrence of conjunctivitis has been described.
• Heart: an increase in cardiovascular events, notably acute myocardial infarction and thromboembolic events, and even disseminated intravascular coagulation, has been described.

MOLECULAR TESTS FOR SARS-COV-2: REAL-TIME REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION (RRT-PCR)
The real-time reverse transcription-polymerase chain reac- Initially, laboratory confirmation depended on detection of two genetic markers but, considering the current high rate of virus circulation, confirmation can be given through detection of a single genetic marker.
The recommendation for laboratory confirmation of cases is that two different genetic markers should be detected (for example, the E gene followed by the RdRP gene, as previously described for the Charité protocol This laboratory test is very specific. However, its sensitivity can vary, mainly due to pre-analytical variables such as: • Stage of infection and viral load in secretions and excretions, mainly with regard to upper respiratory tract samples collected less than three days and more than ten days since the beginning of the contamination; • Place of collection: It is known that materials from the lower respiratory tract (sputum or bronchoalveolar lavage) tend to be more positive than those from the upper respiratory tract In order to ensure that the tests that are requested are performed, it is essential that the medical order is clear and objective.
It is recommended that the requesting physician should indicate the material that is to be collected (for example, secretions from the oropharynx or nasopharynx, sputum, tracheal aspirate or bronchoalveolar lavage) and explain which test is to be performed, i.e.

MOLECULAR PANELS FOR OTHER RESPIRATORY VIRUSES
According to guidance from the Ministry of Health, 8  A Chinese study reported that coinfection with other respiratory viruses would be rare. 9 Another study on the incidence of coinfection showed that molecular detection of a pathogen other than SARS-CoV-2 was not sufficient to ensure absence of infection with the new coronavirus. These results do not support routine use of virome panels, given their low effectiveness in this context. However, one possible exception could be the use of neuraminidase inhibitors in patients infected or coinfected by influenza. 10 Considering that viral coinfections are relatively frequent, according to the guide of the Influenza Surveillance Laboratory Network of Brazil, 11 all patients presenting suspected influenza-like illness (ILI) or SARS should be tested for the COVID-19 virus and other respiratory pathogens, using usual laboratory procedures.

IMMUNOLOGICAL LABORATORY TESTS
In the light of the WHO guidance on the need for mass testing of Some time is needed for production of these antibodies.
On average, the time required is 7 to 10 days after the onset of symptoms for the IgM class antibodies, and 10 days or more for IgG. These numbers clearly indicate that early detection of antibodies is possible , but only in a limited number of patients. As the days go by, the concentrations of both antibody classes increase, and the chance of false-negative results decreases. While the search for viral particles is carried out mainly using secretions and washings, the search for and quantification of antibodies can be carried out using capillary blood, whole blood, serum or plasma.
Thus, blood collection is required, either from a fingertip for the rapid test, or from a vein to obtain whole blood. In early April, the WHO recommended that rapid tests should be used for epidemiological purposes only and contraindicated their use for diagnosis. In cases in which RT-PCR tests are non-reactive but COVID-19 infection is suspected, serological tests performed on sequential samples can assist in clarifying the diagnosis. 12

SEROPOSITIVITY CERTIFICATE
At the present time, the best definition for an "immunity passport", would perhaps be that this is a "seropositivity certificate". There is strong pressure for social life to return to the old "normal" pattern, i.e. for there to be a loosening of social distancing. With the recent development of laboratory tests that detect the presence of antibodies, an expectation has been created that people in whom antibodies against SARS-CoV-2 are potentially detected could be released to resume their usual activities.
Studies in this direction are being carried out in some countries of the European Union, especially Germany, and in China and the United States. At the moment, the following elements need to be considered: • Antibodies start to be detected only one to two weeks after infection; • It is not yet known whether antibodies detectable through current tests are capable of conferring long-lasting immunity (i.e. whether they would be protective antibodies), or for how long; • The new tests have been released in a speeded-up manner and still need to be carefully validated.
In the case of SARS in 2002, antibodies were present for two to three years and in the case of MERS, for one year. In the case of COVID-19, this information would be important for making it possible to determine, for example, the retest interval that would be required in order to enable certification of immunity on an ongoing basis and to organize vaccination programs.
It is not certain that the appearance of antibodies indicates that the person is no longer a transmitter or that he/she becomes immune, and for how long. Some people have been shown to carry the virus for a long time (a few weeks). There are ethical issues. Even if the percentage of false-reactants is not large, non-immune people would be considered immune and exposed, and susceptible people could be discriminated against in the job market. And lastly, it is necessary to avoid enabling trade in this type of certificate, including through fraud.

LABORATORY EVALUATION OF THE INFLAMMATORY STORM
Cytokines are low molecular weight proteins that are released by various types of cells, especially those that make up the immune system, and have a role in intercellular signaling. Among various actions, the cytokines released by the cells of the immune system act to modulate the inflammatory response, from the time when the organism is attacked by infectious agents. The term "cytokine" is derived from two Greek words "cyto" for cell, and "kinos" for movement. The rationale for this nomenclature, which emphasizes the mobile nature of these cells, is based on the fact that, because they are small molecules, they have great mobility in body fluids and have the ability to recruit different cells of the immune system to act together.
The inflammatory response is an extremely complex event, involving numerous cellular and humoral agents. Among other purposes, it seeks to identify, isolate, neutralize and eliminate agents that are harmful to the body. It involves participation of cellular and humoral elements that act in a coordinated manner, with self-modulation of their intensity of response to an invading agent. However, dysregulation of the inflammatory response may sometimes occur. In these situations, excessive quantities of cytokines are released, and these activate and recruit cells of the immune system such that an oversized response is generated, thus resulting in hyperinflammation. This condition is called a "cytokine storm" and results in generation of a microenvironment that is harmful to the organism itself.
The cytokine storm concept apparently began to be studied in the early 1990s. It was correlated with several viral infections and was recognized during the SARS outbreak in 2002, as a factor presenting a high risk of mortality. The term was only coined in 2005, when avian influenza caused by the H5N1 virus occurred, and its occurrence was linked to a high mortality rate due to an exacerbated pro-inflammatory response. In some non-infectious diseases, such as multiple sclerosis, cytokine storms can also be observed. Since then, cytokine storms have been described in relation to several respiratory diseases caused by viruses of the coronavirus family, such as MERS in 2012 and, more recently, SARS-CoV-2. 13 The main cause of death in cases of COVID-19 is respiratory failure caused by SARS. 14 Another syndrome related to the immune response is secondary hemophagocytic lymphohistiocytosis (sHLH), a little-known condition that is characterized by fulminant hypercytokinemia and rapidly evolves to multiple organ failure. It has been described in about 3.7% to 4.3% of sepsis cases, 15 but it is more often described in viral infections. 16 Clinically, it is characterized by the presence of constant fever, cytopenia and high levels of ferritin, interleukins (IL), granulocyte-colony stimulating factor (GCSF), interferon-γ inducible protein 10, monocyte chemoattractant protein-1, macrophage inflammatory protein 1-α and tumor necrosis factor-α. 17 SARS occurs in about 50% of patients with sHLH. 18,19 Comparison of groups of patients with SARS in association with the new coronavirus via laboratory results has shown significant differences between survivors and non-survivors. The most striking differential parameters include leukocyte counts, absolute lymphocyte and platelet counts and the concentrations of albumin, total bilirubin, serum urea, creatinine, myoglobin, cardiac troponin, C-reactive protein (CRP) and interleukin-6 (IL-6).
In patients with COVID-19, ferritin and IL-6 levels have been shown to be good predictors of fatality, as shown in Table 1  inflammation markers such as CRP, ferritin, troponin and brain natriuretic peptide (BNP) will be at very high levels, thus characterizing an inflammatory storm.