key: cord-305856-xt3zxajf
authors: Shanmugam, Chandrakumar; Mohammed, Abdul Rafi; Ravuri, Swarupa; Luthra, Vishwas; Rajagopal, Narasimhamurthy; Karre, Saritha
title: COVID-2019 – A comprehensive pathology insight
date: 2020-09-18
journal: Pathol Res Pract
DOI: 10.1016/j.prp.2020.153222
sha: 
doc_id: 305856
cord_uid: xt3zxajf

Corona virus disease-2019 (COVID-19) caused by severe acute respiratory syndrome corona virus-2 (SARS CoV-2), a highly contagious single stranded RNA virus genetically related to SARS CoV. The lungs are the main organs affected leading to pneumonia and respiratory failure in severe cases that may need mechanical ventilation. Occasionally patient may present with gastro-intestinal, cardiac and neurologic symptoms with or without lung involvement. Pathologically, the lungs show either mild congestion and alveolar exudation or acute respiratory distress syndrome (ARDS) with hyaline membrane or histopathology of acute fibrinous organizing pneumonia (AFOP) that parallels disease severity. Other organs like liver and kidneys may be involved secondarily. Currently the treatment is principally symptomatic and prevention by proper use of personal protective equipment and other measures is crucial to limit the spread. In the midst of pandemic there is paucity of literature on pathological features including pathogenesis, hence in this review we provide the current pathology centered understanding of COVID-19. Furthermore, the pathogenetic pathway is pivotal in the development of therapeutic targets.

The current pandemic of corona virus disease-2019 (COVID-19) caused by severe acute respiratory syndrome corona virus-2 (SARS CoV-2) led to complete lockdown in many countries contributing to major socio-economic crisis and irreparable recession, globally. SARS CoV-2, a novel β CoV was first identified in adults presenting with acute lower respiratory tract infection of unexplained etiology in China. [1] Though no age group is spared, severe forms occur in patients older than 60 years specifically with co-morbidities. The majority of the infected individuals are asymptomatic or with mild form of disease and are potential transmitters. This disease is highly contagious and mainly spread through respiratory droplets, close contact with infected cases or materials (fomites) and nosocomially to other patients and health care workers in the hospitals. [2, 3] COVID-19 has a much lower case fatality ratio and significantly greater transmission rate than 2003 SARS pandemic. [4, 5] Currently RT-PCR of upper and lower respiratory swabs or samples is the gold standard diagnostic test. Serological tests based on antibody detection, though not helpful during the early phases of disease, can be used to confirm infection in later phase. A thorough literature search (PubMed, preprint servers and google scholar) using terms COVID-19 and pathology/pathogenesis, SARS CoV-2 and pathology/pathogenesis and 2019-nCoV and pathology/pathogenesis was done to maximize the yield of literature, which ended on 24 May 2020. In this review, we have comprehensively discussed all aspects of COVID-19 with special emphasis on the pathology including pathogenesis and therapeutic targets. It forms a ready resource for clinicians, pathologists, and researchers including epidemiologists aiding them in the diagnosis and treatment of these patients, and may also pave way to further research.

The earliest case of SARS CoV-2 infection currently known was reported on 31st

December 2019 in Wuhan, Hubei province of China. [1] After this it spread rapidly to other parts of China as well as internationally affecting over 185 countries as of 23 April 2020, leading to the current global pandemic. [2] The World Health Organization declared COVID-19 to be a Public Health Emergency of International Concern on 30 January 2020, and recognized it as a pandemic on 11 March 2020.[6,7] As of 24 May 2020, globally 5.48 million cases of COVID- 19 have been reported, resulting in 346,071 deaths and 2,290,776 people have recovered. [8] J o u r n a l P r e -p r o o f

The basic reproduction number (R0) of the SARS CoV-2 is estimated to be between 1.4 and 3.9, indicating its highly contagious nature. [9, 10] The R0 may be even higher in places of public gatherings like in cruise ships, religious/political/academic/business congregations as well as in hospitals non-compliant with personal protective measures. [9, 10, 11] The incubation period and serial interval is estimated at 5-6 days and 8 days, respectively, which is similar to that for SARS CoV and MERS CoV. [9, 12, 13, 14] Early in the pandemic, the case-fatality rate (CFR) was estimated to be between 0.9% and 3%, [15, 16] lower than other HCoVs (SARS CoV (6%-17%) and MERS CoV (20%-40%)). [17, 18, 19] However, by the 24 th of May 2020 many countries exhibited exponential rise in CFR.

[8] (Table:I) 

Unlike SARS CoV, the high percentage of SARS CoV-2 infected individuals manifest as asymptomatic or pauci-symptomatic infection who escape detection and become potential transmitters. [20, 21] It is important to note that, not all close contacts are infected suggesting a role for individual genetic susceptibility. [22, 23, 24] In humans, the virus usually gains entry through upper aero-digestive tract. More recently SARS CoV-2 was isolated from the feces of patients, indicating the possibility of fecal-oral spread. [24, 25] Furthermore, SARS CoV-2 infection in pregnant women raised a possibility of vertical transmission. [26] However, the vertical transmission was ruled out based on negative testing for the virus on the swabs collected from the amniotic fluid, cord blood, neonatal throat and breast milk of the six infected pregnant women. [26] The long range airborne transmission is also speculated which depends on flow dynamics of the virus from the infected person and also on ventilation status of the area. [27] Moreover, the expansion and spread of COVID-19 can be visualized by mapping techniques like cartograms. [28] The understanding of modes of transmission of SARS CoV-2 will enable application of appropriate containment measures.

Though there is generalized susceptibility to SARS CoV-2 infection for all age groups, body defense against infection as well as their underlying age related organ system compromise. [22, 31, 32, 33] Similar to SARS CoV, a recent study reported non-O blood group specifically group A had higher infection and death rates due to COVID-19 owing to absence of protective anti-A IgM antibodies. [34, 35] Many uncertainities still persist in the SARS CoV-2 epidemiology especially virus-host interaction including host susceptibility and the evolution of epidemic.

The corona viruses (CoVs) are classified into α and β (seen in mammals including humans); γ and δ (seen in avian species). [ to pangolin CoV with a difference of only one amino acid. [46] Recently another study suggested pangolin involvement in SARS CoV-2 origin due to evidence of re-assortment in CoVs. [47] SARS CoV-2 differs from other β CoVs by the presence of unique polybasic cleavage site that contributes to increased pathogenicity and transmissibility. [48] Each virion is a enveloped, non-segmented, positive sense single stranded RNA virus 

The SARS CoV-2 because of its similarity with SARS CoV is presumed to infect human cells through its densely glycosylated spike (S) proteins S1 fraction with receptor binding domain (RBD) which binds to the angiotensin-converting enzyme 2 receptor (ACE-2 R) with 10 to 20 fold higher affinity than SARS CoV. [ [56, 57] After the virus gets attached to this receptor, the SARS CoV-2 with its unique polybasic S1/S2 protease cleavage site with SPRR insertion on the spike protein which is recognized and cleaved by transmembrane protease serine (TMPRSSs) expressed on host cells to expose the fusion protein (S2 fraction) that enables the fusion of both viral and the host cell J o u r n a l P r e -p r o o f membrane. [56] It has been demonstrated that ACE-2 R and TMPRSSs are highly co-expressed in alveolar type 2 pneumocytes, epithelium of upper esophagus and absorptive enterocytes, forming the basis of speculation that the SARS CoV-2 can gain access into host through esophageal and intestinal epithelium apart from alveolar epithelium. Hence, the potential target tissues for SARS CoV-2 should co-express ACE-2 R and TMPRSSs. 

The current understanding of pathology stems from few case reports and autopsy case studies. The gross features include heavy and boggy lungs, patchy consolidation along with pleural fibrinous exudate and /or fibrosis, sometimes with purulent inflammation due to secondary bacterial infection with/without evidence of pericarditis. [62] The microscopic features depend on stage and severity of the disease. Early stages (asymptomatic/mildly symptomatic patients) show non-specific changes including pulmonary J o u r n a l P r e -p r o o f edema, focal pneumocyte hyperplasia, focal chronic inflammatory infiltrate and multinucleated giant cells with absence of prominent hyaline membrane formation. [63] As, the disease progress there is diffuse alveolar damage with transparent hyaline membrane formation and severe pulmonary edema. However, in SARS CoV-2, there is firbomyxoid exudates with visible fibrinous cords along with mucous plugging of bronchioles which has a bearing with respect to oxygen therapy. There is also widespread interstitial inflammatory infiltrates with severe epithelial damage,diffuse type II pneumocyte hyperplasia consistent with ARDS. [64, 65, 66, 67, 68] One study reported massive pulmonary interstitial fibrosis with variable degree of hemorrhagic necrosis, chronic inflammation with multinucleate giant cells and intracytoplasmic viral inclusion bodies in severe cases. [69] Interestingly, another study showed features of lymphocytic viral pneumonia in a patient who died early in the disease (5th day after development of symptoms), whereas five other patients who succumbed later (20th day after development of symptoms) exhibited acute fibrinous and organizing pneumonia (AFOP)

showing extensive fibrinous deposits forming balls/mounds but not hyaline membrane in their alveoli. These patients also showed prominent vascular injury evidenced by endothelial cell detachment and prominent intracytoplasmic vacuolization in small and medium-sized pulmonary blood vessels. [70] Also, severe COVID-19 infection has been associated with a novel pulmonary-specific vasculopathy known as pulmonary intravascular coagulopathy (PIC), that parallels disease severity. [71] These findings may be considered as important indicators of disease severity and prognosis.

The liver shows mild lobular lymphocytic infiltration and moderate micro-vesicular steatosis along with mild lobular activity, possibly related to the viral infection itself and ischemia. There were no obvious histological changes in heart tissue except for mild interstitial chronic mononuclear infiltrate. [57, 64, 65] Hence the changes in the liver and heart are more likely secondary or related to the underlying diseases. [64] The pathology in other organs have not been elucidated.

It is too early to determine the specificity and consistency of these histopathological findings with respect to the stage and severity of the COVID-19 owing to the paucity of information obtained from few biopsy/autopsy case reports. In addition, the histopathological features may be modified or altered by patients' immunity, presence of co-morbidities, secondary infections and therapy given to these patients especially steroids. Only few patients present with gastrointestinal symptoms like diarrhea (5.7%) and nausea/vomiting (6.1%). [72] In 26.7% of COVID-19 patients there was at least one underlying co-morbidity(hypertension, diabetes, chronic cardiovascular/ pulmonary/ renal disease and cancer). [72] The severe form is characterized by ARDS that necessitates mechanical ventilatory support in an intensive care unit (ICU), and also leads to multiorgan involvement resulting in shock, septicemia, and MODS with high mortality. [73, 74] A substantial proportion of patients developed diarrhea during hospitalization, potentially aggravated by various drugs including antibiotics. [75] These patients may also present with cardiac sounding chest pain due to myocarditis and myocardial infarction. 

Children are either asymptomatic or pauci-symptomatic (fever (50%), cough (38%), fatigue, rhinorrhoea or nasal congestion) and are less likely to have severe infections. [82, 83] Gastrointestinal symptoms like diarrhoea, abdominal cramps and vomiting, common in children, 

COVID-19 positive patients frequently exhibit hematologic abnormalities in the form of lymphopenia, leukopenia, and thrombocytopenia, along with elevated levels of liver enzymes, lactate dehydrogenase, prothrombin time and D-dimers. [72] Lymphopenia is associated with disease severity and mortality. [1] Acute phase reactants such as CRP, ferritin and procalcitonin and pro-inflammatory cytokine levels were higher in COVID-19 than healthy adults. [86] COVID-19 patients needing ICU management when compared to non-ICU patients had higher plasma levels of pro-inflammatory cytokines (IL2, IL7, IL10, GSCF, IP10, MCP1, MIP1A, and TNFα), increased total WBC and neutrophil counts, higher levels of D-dimer, creatine kinase, and creatinine. [31, 74] Similar laboratory findings were seen in Children with COVID-19. [29] 

Findings on chest imaging in SARS CoV-2 pneumonia seems to be similar to ordinary viral pneumonia, with some peculiarities. Chest X-ray and CT changes may be seen even before the detection of the virus from swab. In contrast, the chest X-ray may be normal in 31% of laboratory confirmed COVID-19 cases. [87] The commonest feature on chest X-ray is presence of bilaterally symmetrical ground glass opacities with or without associated consolidation in the posterior and peripheral lung fields. [32] However, the CT findings vary with the duration of symptoms. [88] In the initial Phase (Days 2-4) basal multifocal peripheral ground-glass opacities are noted. With disease progression (mid Phase (Days 4-7) there is linear opacities developing on a background of ground-glass opacities (crazy pavement pattern). In the late Phase (Days 8-14) the central ground-glass opacities become surrounded by denser crescentic shaped consolidation (forming more than three-fourths of a circle) or form complete ring of at least 2 mm in thicknesscalled as 'reversed halo sign' or 'atoll sign' [88] Children also exhibit similar radiologic features. [29] Chest CT suggesting COVID-19 had 97% sensitivity in concordance with positive These tests are complex, time consuming, expensive and need expertise to perform as well as to interpret. [91, 95, 96] The molecular test based on point of care testing using cartridges are rapid and needs less expertise. High throughput technologies including NGS can be used for simultaneous screening of large number of samples but its application is limited to research only due to high expenditure. [97] The serological tests detects either the viral antigens (spike protein and nucleo-capsid being target antigens), or the antibody response to the virus by immunochromatography and ELISA methods. Specifically, the antibody testing is not helpful in the early phases of infection. Though simple, cost effective, easy to perform and interpret, there are chances of false positives especially due to cross reactive antibodies against other HCoVs.

Additionally, a negative antibody test does not exclude SARS CoV-2 infection. [95, 98] The role of virus isolation and culture as well as detection of the virus by its cytopathic effects on cell lines is highly limited due to requirements of bio-safety level-3 facility. [99, 100] Hence it is not J o u r n a l P r e -p r o o f recommended by WHO for diagnostic purpose [101] Currently the diagnostic tests for detecting SARS CoV-2 infection is variable and non-uniform owing to the use of different probes, kits and reagents.

Though there are numerous reports claiming efficacy of various drugs and vaccine against COVID-19, none are effective and safe to receive approval by regulatory authorities. The management of COVID-19 mainly relies on effective implementation of infection preventive and control measures and delivery of timely supportive care including oxygen therapy and mechanical ventilation as and when indicated.

As the R0 value is >1 (range 1.4 to 3.9), [9, 10] Efficiency of intervention strategies such as screening of incoming people, wearing masks, quarantine for travellers has already been proved.

[102] Specifically, reducing travel volume to and from China has had a positive impact on transmission dynamics of covid-19. [103] .

Though preventive vaccines against SARS CoV-2 can be developed targeting the spike (S) glycoprotein or its receptor-binding domain (RBD), these are made ineffective due to generation of altered immunogens in the target proteins owing to rapid mutations and recombinations. [104, 105] In SARS CoV, live attenuated vaccine with the deleted structural E gene mutant was effective in producing neutralizing antibodies which lowered viral loads and reduced disease severity. [106] The development of inactivated vaccines against SARS CoV was hindered due to occurrence of harmful immune and/or inflammatory responses post challenge.

J o u r n a l P r e -p r o o f 13 [107, 108, 109] Sub-unit vaccines (purified proteins combined with adjuvants) and viral vector (adeno virus) vaccines against S glycoprotein or its RBD and N protein of SARS CoV and MERS CoV elicited higher humoral response as well as enhanced mucosal immunity with intranasal administration. [110, 111, 112] Furthermore, DNA based vaccine against S glycoprotein of MERS CoV also showed robust neutralizing antibody response and is currently under clinical trial. [113] Based on these reports vaccines for SARS CoV-2 is likely possible. However, its efficacy and safety has to be proved before approval.

In the absence of specific anti-viral therapy, treatment is mainly symptomatic and supportive that includes oxygen therapy, conservative fluid management, hemodynamic support and / or mechanical ventilation. Mechanical ventilatory support with low tidal volume and low inspiratory pressure is indicated when the respiratory distress is refractory to conventional oxygen therapy or NIV. [2] Extracorporeal membrane oxygenation (ECMO) is indicated in patients with refractory hypoxemia despite prone position mechanical ventilation. [2] A recent retrospective study identified older age, high sequential organ failure assessment score (SOFA) score, and D-dimer greater than 1 µg/mL as poor prognostic factors which aid the clinician early in instituting aggressive treatment and monitoring for such patients. [114] Steroids, and injudicious antibiotic use should be discouraged. Some studies report effective use of RNA polymerase inhibitors Remdesivir and Immucillin-A as prophylactic and therapeutic agents against HCoVs including SARS CoV-2. [115, 116] Anti-malarial drug chloroquine and its analogue may show protective effect against virus by decreasing intracellular pH but may cause cardiac arrythmias owing to prolonged QTc interval in some patients. [117] Monoclonal antibodies against interleukin-6 (IL-6) like Sarilumab, Siltuximab, Tocilizumab and interleukin-1 (IL-1) inhibitor like Anakinra may be useful in severe cases and may control the effects of SIRS which is the main culprit in the pathogenesis of severe cases. [118] Currently, there are 1,673 studies registered in clinical trials involving various investigational drugs and vaccine apart from those mentioned above and are still at phase-I level.

[119] Theoretically, molecules involved at each step of SARS CoV-2 pathogenesis may become potential therapeutic targets. (Figure: To conclude, SARS CoV-2 is highly infective and its control depends on strict implementation of preventive measures. Though RT-PCR is the gold standard for SARS CoV-2 diagnosis, the results are variable and there is scope of false negatives owing to either sampling errors or due to usage of different primers and reagents by different vendors. Serologic estimations of antibody titres though not helpful for diagnosis, may be useful for prognostication and follow-up. Though currently available pathologic data is limited, it is of prime importance to unveil the pathogenesis which will enable the development of therapeutic options. However, studies on larger cohorts are needed to validate the findings obtained for generalized application.

Importantly, due to limited availability of time and resources for research during the current emergency situation there is a huge lag and gap in understanding COVID-19; hence the current review removes the lag and bridges the gap and provides pathology centered understanding of the disease. Furthermore, the currently approved clinical trials that are in various stages of development on all aspects of COVID-19 including vaccines and potential therapeutic targets will shed light in future.

There are no conflicts of interest to disclose. 

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We thank Dr. Vaseemuddin Mohammad (Consultant nephrologist,

Tables: Table I