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  Table of Contents    
REVIEW ARTICLE  
Year : 2020  |  Volume : 63  |  Issue : 3  |  Page : 358-366
COVID-19: An up-to-date review – from morphology to pathogenesis


1 Department of Pathology, PGIMER, Chandigarh, India
2 Department of Pathology, Rohilkhand Medical College and Hospital, Bareilly, Uttar Pradesh, India
3 Department of Pathology, Division of Cardiovascular and Thoracic, Seth GS Medical College, Mumbai, Maharashtra, India
4 Department of Pathology, All India Institute of Medical Sciences, New Delhi, India
5 Department of Microbiology, King George Medical University, Lucknow, Uttar Pradesh, India

Click here for correspondence address and email

Date of Submission25-Jun-2020
Date of Decision07-Jul-2020
Date of Acceptance20-Jul-2020
Date of Web Publication7-Aug-2020
 

   Abstract 


The entire world is under a devastating pandemic caused by COVID-19 with a high mortality rate. Knowledge of the viral structure, factors that help in its progression and spread, pathological findings, diagnostic methods and, treatment modalities helps in understanding the viral disease and also in treating the patients in a better way besides preventing the community spread of this deadly infection. The causative agent is a single- stranded RNA virus. The clinical spectrum varies in symptomatic and asymptomatic patients, who later become potential silent carriers, thus unknowingly spreading the virus. The virus constantly undergoes recombination, with reports of cross-species infections. Studies have indicated a strong immunological basis of COVID-19 infection. Not only does it weaken the immune system causing multi-organ involvement but also helps in its progression and spread to others.Multiple organs especially lungs, heart, kidney, gastrointestinal and hepatic system, brain and skin are affected varying in their severity. Similarly, persons with associated co-morbidities are likely to be affected more in terms of the number as well as in the severity. Real- time reverse transcription polymerase chain reaction confirms the presence of COVID-19 infection. Serological diagnosis helps in diagnosing an ongoing outbreak or retrospective infection. Furthermore, it also identifies individuals who have been infected or have recovered from the disease especially the asymptomatic. This helps in the development of an effective vaccine indicating the status of herd immunity in the community. Different treatment modalities are being tried and under trial. This review article thus highlights the global epidemiological status, characteristic of the virus, symptomatology of the patients, role of diagnostic tests available, organs affected including their morphological changes and the latest line of treatment of COVID-19.

Keywords: COVID-19, pandemic, pathology, RNA virus

How to cite this article:
Bal A, Agrawal R, Vaideeswar P, Arava S, Jain A. COVID-19: An up-to-date review – from morphology to pathogenesis. Indian J Pathol Microbiol 2020;63:358-66

How to cite this URL:
Bal A, Agrawal R, Vaideeswar P, Arava S, Jain A. COVID-19: An up-to-date review – from morphology to pathogenesis. Indian J Pathol Microbiol [serial online] 2020 [cited 2020 Oct 27];63:358-66. Available from: https://www.ijpmonline.org/text.asp?2020/63/3/358/291689





   Introduction Top


Many viruses through aerosols, droplets, and droplet nuclei utilize the respiratory passages to establish not only localized respiratory tract infections but also systemic disease. The coronaviruses (CoV) are no exception. They are a group of zoonotic viruses originally found in avian and mammalian species and are responsible for 20% of the “common cold.” However, in their more ominous avatar, they can occasionally cause severe and serious diseases in humans. The two most common illnesses that occurred in the recent past were severe acute respiratory syndrome (SARS, 2003) and Middle East respiratory syndrome (MERS, 2012).[1] The current pandemic, which broke out in the late December 2019, has been a major threat to the global public health due to significant morbidity and mortality, akin to snapping of Thanos' fingers. The novel coronavirus was initially named as the 2019-novel CoV (2019-nCoV), but because of nearly 80% genetic homology to SARS-CoV, the Coronavirus Study Group of International Committee rechristened this virus as SARS-CoV-2.[1] The disease was named as coronavirus disease 2019 (COVID-19) on January 12, 2020 by the World Health Organization (WHO).[2] According to the Advisory Committee on dangerous pathogens UK, COVID-19 is assigned as hazardous group-3 organism, meaning that it can cause severe human disease.[3] The present review highlights the latest concepts published in the literature for better understanding of COVID-19.


   Epidemiology Top


The first case of human transmission of COVID-19 was identified in the city of Wuhan, Hubei province in China. Epidemiologists suggest that the initial outbreak was associated with a seafood market, where other wild animals were also sold for human consumption.[4] Subsequent viral genomic and structural studies from samples of infected individuals confirmed that the infective virus is a new seventh member of CoV. Bats were considered a source of infection with high diversity of CoVs.[4] Pangolins act as the intermediate hosts, which is supported by a genetic study done by Xu et al.[5] The cause for a trans-species transmission is still not clearly known. Epidemiologists confirmed that there was a person-to-person transmission through close contact, by droplets and aerosols in the initial outbreak. The timing of the second and third phases of the spread outside Wuhan city to other parts of China and other countries (more than 200) coincided with the mass movement during the celebrations of the Chinese New Year.[3],[6] With an explosive increase in the number of cases, the WHO declared this outbreak a public health emergency of international concern on January 30, 2020, and as a pandemic on March 11, 2020.[7] According to the WHO, the global confirmed cases, as on July 18, 2020, are 13,824,739 with a mortality of 591,666. The top most affected regions in the descending order are America, Europe, Eastern Mediterranean, and Western Pacific. According to the WHO update, as on July 18, 2020, there were 1,038,716 total cases all over India with a mortality of 26,273.[8] The worst-hit states include Maharashtra, Tamil Nadu, Delhi, Gujarat, Uttar Pradesh, Rajasthan, and West Bengal. However, the mortality rate in India is one of the lowest (around 2.53%) as compared to the overall global mortality rate of around 4.28%.[8] The effective reproduction number (R) is a measure of the expected number of cases that is generated from one case. For COVID-19, it is between 2.8 and 3.3, indicating that it has higher rate of transmissibility and pandemic risk than that of SARS-CoV (R of 1.77).[9]


   About the Virus Top


CoVs are large viruses (approximately 80–220 nm in diameter) belonging to the order Nidovirales, sub-order Cornidovirineae, family Coronaviridae, sub-family Orthocoronavirinae, and genus Betacoronavirus; the latter also includes the SARS-CoV and MERS-CoV. They are enveloped, icosahedral, symmetrical particles with spike-like projections on their membranes that give them the shape of crown (“Corona” in Latin) and hence the name. They have large single-stranded, positive-sense, nonsegmented RNA genome, of about 26–32 kb size.[10] Till date, the detailed ultrastructure of this virus remains incompletely understood. From India, the first report on the ultrastructure of the virus was based on negative staining on a throat swab sample after fixation in glutaraldehyde, demonstrating that the virus is round with an average size of 70–80 nm and a cobbled surface structure having envelope projections.[11] SARS-CoV-2 mutates rapidly, undergoes frequent recombinations, and easily crosses the species barrier, causing frequent novel cross-species infections.[12] It is transmitted between humans through both direct (droplet and human-to-human transmission) and indirect (contaminated objects and airborne contagion) contacts from both symptomatic and asymptomatic patients.[13]


   Pathogenesis Top


The COVID infection has brought to the forefront the intricate interplay and balance between the inflammatory, immunological, and hemostatic responses. Furthermore, the basis for the discrepant symptomology and their severity may lie within the genetic and acquired differences in host immune system. Pathological features such as infiltration in the infected tissue by macrophages, and the presence of lymphopenia and neutrophilia on hematological examination, serve as an indirect evidence of a strong immunological component to this infection.[14] Hence, our understanding of the pathogenesis can form the basis of guiding treatment strategies and design vaccines.

The crucial step in the life cycle of any virus is an attachment and subsequent penetration into the host cell. It has been proven beyond doubt that the attachment of SARS-CoV-2 is achieved by an interaction between the spike surface glycoprotein S and angiotensin-converting enzyme 2 (ACE-2), a membrane carboxypeptidase that is ubiquitously distributed in a variety of human tissues.[15] It is to be noted that the affinity of the S protein to ACE-2 is said to be exceedingly higher (at least 10–20 times) as compared to that of a similar protein on SARS-CoV-1, and the ACE-2 expression depends on the genetic factors, age, and sex. This explains a very low case fatality rate (CFR) in pediatric patients compared to patients beyond 80 years of age (CFR of 0% for individuals under 8 years of age vs. 21.9% for patients above 80 years).[15] Unfortunately, the ACE-2 expression also increases in the presence of comorbid conditions such as obesity, preexisting chronic cardiopulmonary disease, cancer, and use of immunosuppressive drugs, and such patients are prone to develop severe disease.[16] The other possible receptors are CD-147 and glucose-regulated protein-78. Other than ACE-2, the protease furin and serine protease TMPRSS2 are essential for the viral entry.[15],[16]

The morbidity and mortality produced by this novel virus is mediated by the functional loss of ACE-2 and certain direct and indirect effects of the virus.[14],[16],[17] Although ACE-2 is a homolog of the ACE, it exerts effects (anti-inflammatory, antioxidative, and vasodilatatory) through its cleavage product angiotensin [1],[2],[3],[4],[5],[6],[7] derived from angiotensin II. On the other hand, ACE converts angiotensin to angiotensin II, which leads to pro-inflammatory, pro-oxidative, and vasoconstrictive effects, a sharp contrast to ACE-2. Furthermore, ACE-2 also plays a role in bradykinin metabolism and the dopamine–serotonin synthetic pathway. With internalization of the ACE-2 after viral interaction, there would be downregulation of the membrane protein and hence derangement of the renin–angiotensin–aldosterone axis with consequent deleterious effects. It is not surprising that the brunt of the viral onslaught falls on the distal airways and alveolated lung parenchyma, which have the highest expression of ACE-2, particularly the type 2 pneumocytes and, to some extent, the alveolar macrophages and the lung dendritic cells. Other viral targets are the vascular endothelium; apical membranes of nasal, oral, nasopharyngeal, and oropharyngeal epithelium; gut epithelia; cardiac pericytes; renal proximal tubular cells; skin; reticulo-endothelial system; and even the central nervous system.

The cytopathic effects initially induced by the viral replication in the pneumocytes and the associated capillary endothelial cells stimulate the innate and adaptive arms of the immune system, which can either restrict or lead to progression of the disease.[14],[16],[17] The innate immune cells, through their “pattern recognition receptors,” recognize the viral molecular markers and elaborate interferons to restrict viral replication and stimulate the lymphocytes. The T-lymphocytes jump into the fray, jostled into action by the antigen-presenting macrophages and dendritic cells with subsequent destruction of the virally infected cells and stimulation of the B-lymphocytes into specific neutralizing antibody production (immunoglobulin [Ig] M isotype within the 1st week followed by IgG isotype). The antibody-coated virus particles are recognized by macrophages and dissipated by phagocytosis.[14] During the early phase of the infection in healthy individuals or in those with a low viral load, phagocytosis is sufficient to eliminate the infected cells and clear the virus with minimal tissue damage. In addition, there is synchronous endothelial cytokine or viral-induced damage with increased capillary permeability. Both these factors lead to platelet activation, hypercoagulability, hypofibrinolysis, and complement overactivation, paving the way for immuno-thrombosis within the lung parenchyma–pulmonary phase. SARS-CoV-2 expresses proteins that are capable of inhibiting the production of interferon. Besides, in patients with a high viral load, older individuals, people with comorbidities, or those with altered immunity, there is T-cell lymphopenia, which serves as a biomarker of severity of infection.[16],[17]

Flow cytometric studies have shown decrease in total lymphocyte, CD4+ T cell, CD8+ T cell, B cell, and natural killer cell counts.[18] This absolute and functional lymphopenia occurs through direct cytopathic effects or apoptosis. A subsequent dysregulated interferon production along with antibody-dependent enhancement (ADE) elicits an exaggerated macrophage and neutrophilic response and their cytokine/chemokine release, particularly interleukin (IL)-6. The ADE is triggered by neutralizing and nonneutralizing antibodies. CD32a expressed on the surfaces of monocytes and macrophages plays the central role. Once aggregated by several IgG molecules, CD32a transduces its signal through the associated immunoreceptor tyrosine-based activation motif. A pro-inflammatory feedback loop is established and unchecked inflammatory reaction reigns, producing proteases and reactive oxygen species. This may finally result in hyperinflammation and the so-called cytokine storm, i.e., macrophage activation syndrome or secondary hemophagocytic lymphohistiocytosis in some patients, leading to multiorgan failure, the third phase of the infection.[18] The flowchart [Figure 1] briefly summarizes the events that take place when the novel SARS-CoV-2 virus infects cells and alters host immune response.
Figure 1: Flow diagram showing the mechanisms of immune-mediated damage in COVID-19 (DAMPS: Damage-associated molecular patterns; ADE: Antibody-dependent enhancement)

Click here to view



   Pathological Findings Top


Although lungs are the seat of significant morbidity and mortality, the evolution of COVID-19 has depicted multiorgan dysfunction and the best answer to this is a complete autopsy.[19] High rate of transmission, shortage of health-care personnel, biosafety concerns, and an elaborate autopsy suite setup are some of the major deterrents,[19],[20] and hence only limited clinical autopsy data is available in the literature. [Table 1] outlines the organ findings at autopsy (≥5 patients' study taken into consideration) in the COVID infection.[21],[22],[23],[24],[25],[26],[27],[28],[29],[30],[31],[32]
Table 1: Coronavirus disease 2019 autopsy findings

Click here to view


Pulmonary pathology

On gross examination, the lungs are heavy, firm, and severely congested with patchy to diffuse firm areas. In majority of the patients, the prominent histologic finding is diffuse alveolar damage (DAD),[21],[22],[23],[24],[25],[26],[28],[29],[30],[31],[32] showing severe capillary congestion accompanied by eosinophilic hyaline membranes along alveolar ducts and marked reactive Type II pneumocyte hyperplasia with some syncytial/multinucleate cells, representing the exudative phase.[26] On immunohistochemistry, these syncytial cells have been shown to be positive for Thyroid transcription factor -1, thus confirming these to be pneumocytes. Desquamated Type 2 pneumocytes within the alveolar spaces showing viral cytopathic effects consisting of cytomegaly, and enlarged nuclei with bright eosinophilic nucleoli, have been demonstrated.[23] However, as in other studies, no obvious intranuclear or intracytoplasmic viral inclusions were identified in their cases. Some cases with prolonged respiratory symptoms have shown organizing phase of DAD. Very often, the DAD is accompanied by thrombotic microangiopathy,[21],[25],[26],[29] which is consistent with complement-mediated microvascular injury. In addition, endothelialitis and a novel finding of intussusceptive angiogenesis have also been identified by Ackermann et al.[21] Some studies have reported the interstitial lymphocytic pneumonitis [21],[26] and on immunophenotyping, this lymphocytic infiltrate is composed of a mixture of CD4+ and CD8+ lymphocytes. Other lung pathologies that have been described include pulmonary edema and alveolar hemorrhage. Furthermore, central or peripheral pulmonary thrombi or thrombo-emboli observed in many COVID-19 patients can represent a histological correlate of coagulopathies occurring in this infection.[25],[31] Focal and diffuse bronchopneumonia has been observed as a result of bacterial superinfection, which is common in viral pneumonias.

Cardiac pathology

In view of the elevations of the cardiac injury biomarker, involvement of the heart appears to be highly prevalent and serves as a prognostic factor in COVID-19. There are varied mechanisms to explain this phenomenon. The first would be direct infection of the cardiomyocytes, leading to myocarditis.[24],[33] This effect is further augmented by damage to the pericytes and vascular endothelium with impairment of the microvascular circulation. The projected incidence of myocarditis in these patients is up to 7%.[33] Other mechanisms of cardiac injury may be related to hypoxemia-induced stress and effects of the cytokine storm. The clinical presentation is variable ranging from mild symptoms such as dyspnea or chest pain to more severe manifestations such as arrhythmias, right-sided heart failure, or cardiogenic shock.[34],[35] It is also to be noted that heightened thrombogenicity can also predispose some susceptible patients to acute coronary syndromes.[34],[35]

Renal pathology

A renal tropism was very elegantly demonstrated by Puelles et al.[32] Su et al.[27] analyzed kidney abnormalities in 26 autopsied cases of COVID-19. Acute tubular injury was the main histological finding that included dilatation of tubular lumina, loss of brush border, flattened tubular epithelium, and interstitial edema. On electron microscopy (EM), they could demonstrate clusters of CoVs in the tubular epithelium and podocytes. Furthermore, they demonstrated SARS-CoV nucleoprotein in the renal tubules by an indirect fluorescence method and demonstrated upregulation of ACE2 receptor in tubular epithelial cells. Menter et al.[26] in addition also demonstrated morphological features of disseminated intravascular coagulation with small fibrin thrombi in glomerular capillaries of a few patients.

Gastrointestinal pathology

Although there are many clinical studies on gastrointestinal manifestations of COVID-19 highlighting the presence of virus in feces, the pathology data are scarce. There has been a report of endotheliitis of the submucosal vessels with accumulation of inflammatory cells and evidence of endothelial and inflammatory cell death.[36] The presence of viral particles was also demonstrated within the endothelial cells.

Hepatic pathology

The liver specimens have shown nonspecific changes including microvesicular steatosis, sinusoidal dilatation, and mild lobular/portal tract inflammation.[25],[26],[27],[37]

Central nervous system pathology

Virus-like particles ranging from 80 to 110 nm with characteristic club-shaped projections on EM in neural and capillary endothelial cells in frontal lobe tissue obtained at postmortem examination have been demonstrated.[38] Microvascular thrombi with infarctions are known to occur,[29] with occasional cases of acute encephalomyelitis-like picture.[39]

Pathology in other organs

Skin biopsies examined in COVID-19 patients have shown superficial perivascular dermatitis with slight lymphocytic exocytosis and thrombi in small dermal vessels.[40] One case showed features of transient acantholytic dermatosis with ballooning multinucleated cells. These findings can be attributed to viral exanthema as a result of cytokine storm. Placentae of pregnant COVID-19-positive mothers revealed changes related to fetal vascular malperfusion such as intramural fibrin deposition, foci of villous stromal-vascular karyorrhexis, and intramural nonocclusive thrombi.[41] In this series, all infants tested negative for COVID-19 by RT-PCR, thus indicating that there is no vertical transmission of virus from mother to child. The bone marrow showed reactive erythropoiesis, shift-to-left myelopoiesis, hyperplasia of cytotoxic CD8-positive T-cells, and hemophagocytosis. Such changes may be reflected in the peripheral smear, which may show leukoerythroblastic reaction, monocytosis, nucleated red blood cells (NRBCs), lymphopenia, plasmacytoid lymphocytes, and thrombocytopenia with presence of giant platelets. Even the presence of circulating plasma cells and megakaryocytes has been demonstrated. The monocytes and lymphocytes may show cytoplasmic vacuolation, whereas NRBCs display features of dyserythropoiesis and basophilic stippling. The lymph nodes showed marked congestion, dilatation of medullary sinuses, lymphocyte depletion, as well as increased reactive plasmablasts, indicating activated immune response.[25],[29]

Detection of virus in organs

Up to 500,000 viral copies/1 × 106 RPPH 1 copies in lung tissue by SARS-CoV2-specific reverse transcription-quantitative polymerase chain reaction (RT-PCR) have been demonstrated.[26] However, in other organs (brain, heart, testicle, and kidney), predominantly low levels of RNA copy numbers were detected. Tian et al.[42] demonstrated virus by RT-PCR in heart and lung tissues of one case, however, liver and heart tissues from another case were negative. Presence of SARS-CoV-2 in the brain by RT-PCR of frozen tissue confirmed its presence.[38]

To the best of our knowledge till date, no clinical autopsy on COVID-19 has been performed in India. However, at the Postgraduate Institute of Medical Education and Research, Chandigarh, postmortem biopsies of lungs, liver, and kidneys were evaluated from two COVID-19-positive patients (unpublished data) and showed similar changes of exudative phase of DAD with microthrombi in the lungs and changes of shock in the liver (centrizonal hepatocyte necrosis) and kidneys (acute tubular necrosis). No viral RNA was detected in these postmortem samples on RT-PCR. These findings when compared with H1N1 influenza 2009–2010 hypothesized that COVID-19 viral antigens are localized in proximal airways (throat, nasopharynx, and trachea) and exert their effects on distal lung parenchyma via release of cytokines.[43] The pathological findings in extrapulmonary organs can be attributed to multiorgan dysfunction syndrome rather than a direct viral cytopathic effect.


   Clinical Presentation Top


COVID-19 shows a wide spectrum of clinical presentations with significant number of asymptomatic carriers. The exact rate of asymptomatic infection, however, is yet to be ascertained as most of them in due course of time become symptomatic. The median incubation period of the disease is 5.1 days, ranging from 2 to 14 days.[6] The symptoms commonly vary from mild-to-moderate upper respiratory tract infection in the form of fever with associated fatigue, cough, and sore throat, while in about 15% of patients, nonrespiratory symptoms such as palpitation, headache, watery diarrhea, abdominal pain, nausea, and vomiting precede the respiratory symptoms.[44],[45],[46] Severe disease, seen in approximately 15% of the cases, occurs with one of the following features: dyspnea, respiratory rate (RR) >30/min and oxygen saturation (SpO2) <93%, PaO2/FiO2 <300 mmHg, and/or lung infiltrates developing within 24–48 in >50% parenchyma. Patients with critical disease often have certain preexisting risk factors such as hypertension; diabetes mellitus; chronic heart, lung, liver, or renal diseases, cancer, and cellular immune deficiency; or may be smokers, obese, or elderly.[44],[45],[46] A study conducted in India reported similar clinical features and underlying risk factors in their cohort of 21 COVID-19-positive patients.[47] An infrequently reported risk factor for severity of disease is health-care workers' infections attributable to the high viral load during the first infection followed by repeated exposures. COVID-19 affecting the ethnic/racial minority groups can be explained by socioeconomic and environmental rather than biological explanations.

Patients with critical disease meet one of the following three criteria of respiratory failure, septic shock, and multiple organ failure, and approximately 5%–6% of these patients require ventilatory support and intensive care.[44],[46] The cause of death in COVID-19 patients is primarily due to acute respiratory distress syndrome (ARDS), septic shock, disseminated intravascular coagulation, and/or multiorgan failure. The onset of symptoms to death ranges from 6 to 41 days, with a median of 14 days. The Chinese Centers for Disease Control and Prevention have reported the CFR for critical patients to the tune of 49 %.[48] The fatality rate of COVID-19 in India as on June 20, 2020, is 5.50% (13,285 deaths out of 241,592 closed cases).[8]


   Laboratory Diagnosis Top


Certain biochemical and hematological parameters can be used to signal critical disease and high risk of fatality [Table 2]. The PaO2/FiO2 may be ≤300, 200, or 100 mmHg in mild, moderate, or severe ARDS, respectively. During the initial stage of the disease, the total white cell count may be normal or reduced along with lymphopenia, which in itself indicates a grim prognosis. Destruction of the T lymphocyte leads to a bad clinical outcome. The white cell parameters are altered as a result of cytokine release syndrome. As the severity increases, more lymphopenia occurs with a continuous reduction in its absolute values. Enzymes of liver and skeletal muscle, lactate dehydrogenase, and C-reactive protein levels are elevated. Procalcitonin levels help in differentiating COVID-19 from sepsis in which the levels are increased, whereas in COVID-19, the levels remain normal.
Table 2: Alterations of different laboratory parameters in coronavirus disease 2019

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Features of multiorgan dysfunction including raised amylase level and deranged coagulation markers, especially raised D-dimer at admission, are associated with increased mortality. Manifestation of coagulopathy in COVID-19 is indicated by increased levels of fibrinogen and D-dimer, while there is mild alteration in the prothrombin/activated partial thromboplastin times and platelet counts, especially during the initial stages of the infection. In a study on 191 patients, it was found that increased mortality has been associated with D-dimer values >1.0 μg/mL at admission, increased prothrombin time, elevated IL-6 and other markers of inflammation, and increased troponin level besides any other associated comorbidity.[49] Increasing IL-6 levels and fibrinogen levels correlate well with each other. Increased D-dimer levels are a marker of disease severity and also used to monitor the prognosis of the treatment outcome. MuLBSTA score system is a marker of mortality rate in viral pneumonia and comprises six indices – multilobular infiltration, lymphopenia, bacterial co-infection, smoking, hypertension, and age.[50]

Combined nasopharyngeal and oropharyngeal swabs are the most recommended samples. Swabs collected from both the nostrils and oropharynx are placed and transported in the same tube containing viral transport medium.[51] Nasopharyngeal wash in ambulatory patients and lower respiratory tract specimens such as sputum, endotracheal aspirate, broncho-alveolar lavage in patients with respiratory distress can be collected. Some of the test methods available for laboratory diagnosis of COVID-19 include:

Molecular testing

The laboratory confirmation of COVID-19 is based on detection of unique sequences of SARS-CoV-2 RNA by nucleic acid amplification test (NAAT) using real-time RT-PCR. RNA can be extracted using any standard extraction protocols. A variety of RNA gene targets are used by different manufacturers targeting one or more of the envelope, nucleocapsid, spike, RNA-dependent RNA polymerase (RdRp), and ORF1 genes. The recommended protocols are usually based on the detection of at least two targets in the virus genome: detection of E gene as a screening tool followed by confirmation of E gene-positive samples by detection of at least one more gene such as RdRp/Orf/N gene/S gene. The E assay is specific for all SARS-CoV-related coronaviruses (i.e., SARS-CoV, COVID-19 virus, and related bat viruses). The screening E gene PCR has higher sensitivity.[51] Molecular detection using well-designed protocols is usually very specific; thus, a positive result confirms the detection of viral RNA but may not necessarily indicate the presence of viable virus.[52] Similarly, a negative result might not always mean the absence of infection. If a negative result is obtained from a patient with a high index of suspicion, especially when only the upper respiratory tract specimens were collected, additional specimens from the lower respiratory tract should be collected and tested. With discordant results, the patient should be resampled and, if appropriate, sequencing of the virus from the original specimen or of an amplicon generated from an appropriate NAAT assay should be obtained for reliable test result. Few cases have also been reported positive after two consecutive negative PCR tests performed 24 h apart. It is not clear if it is a testing error, a reinfection, or a reactivation.[51] False-positive results may occur due to technical errors, reagent contamination, inappropriate timing of sample collection, inappropriate sampling technique, etc. Thermal inactivation showed no significant influence on qualitative results for specimens carrying high viral loads, but low viremic specimens may give false-negative results.[53]

Infectivity has been reported to be the highest in bronchoalveolar lavage specimens (93%), followed by sputum (72%), nasal swab (63%), and pharyngeal swab (32%).[54] Throat swabs may show positivity in the 1st week of the disease. Later, the virus can disappear in the throat but continue to multiply in the lungs. In the 2nd week, it is preferred to collect sample from the deep airways by a suction catheter, coughed-up sputum or even saliva.[54],[55] Isolation of SARS-CoV2-specific RNA in different organs has not been uniformly supported and requires further evaluation.

Viral load estimation is not needed for patient management. Hence, currently, no test is available to monitor the viral load. It is usually required for some research analysis. One indicator of viral load is cycle threshold (Ct) value. Ct value is the number of cycles required for the fluorescent signaling to cross the base level of the machine. A Ct value of 35–40 is clinically reported as PCR positive, depending on the manufacturer's recommendations. Low Ct value indicates a high viral RNA load and vice versa. Only 7% of COVID-19 patients have been reported to have a high viral load. The vast majority (84%) have a low infectivity, transmitting to <1 person on an average. The rest 9% has a moderate viral load.[53]

RT-PCR tests are accurate, but it takes too much time, energy, and trained personnel to run the tests. The viral dynamics in other samples such as stools and blood has not been fully characterized. Poor or failed sample extraction, improper handling, transportation, and/or storage are important factors to be considered. Presence of PCR inhibitors in the extracted RNA or sample collection at a time where the patient was not shedding sufficient amounts of virus could be other factors. Viral mutation status in that region might affect the sensitivity of the test.[55]

Serological tests

Both enzyme-linked immunosorbent assay and rapid diagnostic tests are flooding the market for detection of SARS-CoV-2-specific IgM/IgG antibodies. Serological methods have limited or no use in patient diagnosis due to cross-reactivity with other community-prevalent CoVs, resulting in interpretation errors.[56] Moreover, antibody production and response during the different stages of infection have not been fully verified, further limiting their use. During the first 6–7 days of symptom onset, <40% of patients have detectable antibodies. Similarly, the detection of antibodies after day 7 only indicates previous contact with the virus but does not confirm the presence or shedding of the virus.[57] IgG antibodies to SARS-CoV-2 generally become detectable 10–14 days after the onset of infection with a peak around 28 days. Antibodies require time to develop, so they are not the best marker of acute infection. Because they persist in blood for long, they are ideal for detecting past infections. Assays based on detecting IgM/IgG antibodies can help investigating an ongoing outbreak and a retrospective assessment of the attack and can identify people who were infected and have already recovered from COVID-19, including those who were asymptomatic including the level of herd immunity. In cases where NAAT assays are negative but there is a strong epidemiological suspicion of COVID-19 infection, paired serum samples (in the acute and convalescent phases) could support diagnosis.[57] The recommended dictum to be followed when testing by rapid antigen test is to report positive results as true positive. However, if the results are negative and the patient is symptomatic, then retesting by RT-PCR should be done. Also, if the patient is asymptomatic, then the result should be considered negative. The performance of these antibody test kits may be subject to variation under field conditions. Accordingly, the rapid antibody tests can be done using whole blood, serum, or plasma samples with the results available in <1/2 h. The test may remain positive following several weeks after the infection. A positive test indicates exposure to SARS-CoV-2, whereas a negative test does not rule out COVID-19 infection.


   Treatment Top


No specific therapeutic intervention has shown unequivocal success, and the treatment strategies are evolving. The primary treatment strategy remains supportive care until the patient improves. For asymptomatic individuals or those with mild upper respiratory symptoms, isolation is required with mainly supportive treatment. For individuals with lower respiratory tract symptoms, supplemental oxygen by facemask, high-flow nasal cannula, or noninvasive ventilation (NIV) may be attempted; some may require endotracheal intubation and mechanical ventilation. Most therapeutic interventions are mainly based on a weak level of evidence due to a lack of high-quality randomized controlled trials. Corticosteroids can be used for moderate-to-severe disease as anti-inflammatory agents. Drugs such as hydroxychloroquine and chloroquine are recommended as prophylaxis for close contacts and health-care professionals. Antivirals, in the form of lopinavir-ritonavir, as well as remdesvir, are being used in clinical trials. Tocilizumab, an IL-6 inhibitor, is being tried targeting the cytokine storm that is the hallmark of this disease. Convalescent plasma has also demonstrated some efficacy, although larger trials are underway. Some of the other candidate drugs under trials till date include ivermectin, favipiravir, interferon, and doxycycline.[58],[59] Vaccines may hold some promise. In India, Covaxin derived from a strain of SARS-CoV-2 has been isolated by the National Institute of Virology, Pune, and developed by Bharat Biotech, Hyderabad, and is now under human trial.

Acknowledgments

The authors thank Dr. DN Lanjewar, Professor and Head, Department of Pathology, Gujarat Adani Institute of Medical Sciences, Bhuj, and Dr. Saurabh Mittal, Assistant Professor, Department of Pulmonary Medicine, Critical Care and Sleep Medicine, All India Institute of Medical Sciences, for their help in preparing this manuscript.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Correspondence Address:
Pradeep Vaideeswar
Department of Pathology, Division of Cardiovascular and Thoracic, Seth GS Medical College, Mumbai, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/IJPM.IJPM_779_20

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