A study on the morbid histopathological changes in COVID-19 patients with or without comorbidities using minimally invasive tissue sampling

Introduction: COVID-19 causes morbid pathological changes in different organs including lungs, kidney, liver,etc especially in those who succumb. Though clinical outcomes in those with comorbidities are known to be different from those without - not much is known about the differences at histopathological level. Aim(s): It was to compare the morbid histopathological changes in COVID-19 patients between those who were immunocompromised(Gr 1), malignancy(Gr 2) or had cardiometabolic conditions (hypertension, diabetes or coronary artery disease)(Gr 3). Method(s): Post-mortem tissue sampling (MITS) was done from the lungs, kidney, heart, and liver using biopsy gun within two hours of death. Routine (H & E stain) and special stains (AFB, SM, PAS) were done besides immunohistochemistry. Result(s): A total of 100 patients underwent MITS and data of 92 were included (immunocompromised: 27, maligancy:18, cardiometabolic conditions:71). Within lung histopathology, capillary congestion was more in those with malignancy while others like diffuse alveolar damage, microthrombi, pneumocyte hyperplasia etc was equally distributed. Within liver, architecture distortion was significantly different in immunocompromised while steatosis, portal inflammation, Kupffer cell hypertrophy, confluent necrosis were equally distributed. There was a trend towards higher acute tubular injury in those with cardiometabolic conditions as compared to the other groups. No significant histopathological differences in heart was discerned. Conclusion(s): Certain histopathological features are markedly different in different groups (Gr 1,2 and3)of COVID-19 patients with fatal outcome.

Immune dysregulation in COVID-19 and its therapeutic implications

Many countries in the world are affected by severe acute respiratory syndrome (SARS) coronavirus-2 (SARS-CoV-2) disease-2019 (COVID-19) pandemic. Approximately 80% of the cases are mild symptomatic, 15% are severe and approximately 5% are critically ill. The mortality among severe and critically ill patients ranges from 17% to 78%. Elderly and patients with comorbidities have higher chances of progression to severe disease and subsequent mortality. There are no proven antiviral agents available for the management of COVID-19. Besides the viral cytopathic effects, dysregulation in immunity also contributes substantially to the pathogenesis. Treatment with immunomodulatory agents such as interleukin-6 blockers, glucocorticoids and mesenchymal stem cell therapy has been observed to be potentially beneficial. In this review, the immune response in SARS-CoV-2, the mechanism of immune dysregulation as well as potential therapeutic targets for immunomodulatory therapies are discussed.

Utility of nasal swabs as samples for the diagnosis of Mucormycosis using molecular methods for detection

Objectives: Mucormycosis is an aggressive, life-threatening infection caused by fungi in the order Mucorales. There was an explosion of new cases of rhino-sino-orbital mucormycosis following the COVID pandemic in India, and the need for easy and rapid diagnostics was felt. The current diagnosis of mucormycosis relies on mycological cultures, radiology, and histopathology. These methods lack sensitivity and are most definitive later in the course of infection, resulting in the failure of timely intervention. A real-time multiplex PCR platform is commercially available for the detection of Rhizopus spp., Mucor spp.Rhizomucor spp., Lichtheimia spp., and Cunninghamella spp. (PN-700, MucorGenius , PathoNostics , Maastricht, The Netherlands) This real-time PCR has been validated to identify these fungal pathogens from bronchoalveolar lavage, tissue, and serum samples. This study aimed to validate this PCR-based system to detect Mucorales from nasal swab samples and evaluate its utility in the detection of Mucorales from nasal cavities of high-risk patients developing signs and symptoms of mucormycosis. Method(s): A single-center cross-sectional observational study was conducted on 50 hospitalized adult patients with signs and symptoms of mucormycosis. Nasal swabs were taken for PCR analysis once there was a clinical suspicion and were com-pared with the results of the gold standard.The gold standard for the diagnosis of mucormycosis was the conventional method (KOHmountedmicroscopy/HPE).Demographicdetails andrisk factorsfor thesepatients wererecorded, andthe RTPCR-based test was run on the nasal swab samples of all these 50 patients. The workflow is depicted graphically in Fig. 1 (Created with BioRender.com). Result(s): The study population mean (SD) age was 50 (16) years and consisted of 32 (64%) males. A total of 39 (78%) patients were known cases of diabetes mellitus, 48 (96%) patients had amphotericin B intake, and 20 (40%) had posaconazole intake. In all, 21 (42%) patients had a past history of COVID-19 infection;14 patients had received steroids and 10 patients received oxygen support. PCR for Mucorales was positive in 15 (30%) patients while the KOH mount was positive in 4 (8%) patients. Conclusion(s): These results are not encouraging for the use of nasal swabs as the sample for diagnosis of mucormyco-sis. Though the PCR performed better on the swab samples than KOH preparation and culture techniques, it highlights the importance of using standard sampling methods.

Diagnostic performance of MucorGenius® real-time polymerase chain reaction assay on tissue samples for the diagnosis of mucormycosis

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM:   Objectives: To assess the diagnostic utility of MucorGenius® real-time PCR in tissue samples for the diagnosis of mucormycosis in patients suspected of having invasive mucormycosis (IM) during the second wave of the COVID-19 pandemic. Methods: A total of 193 clinically suspected cases of IM presenting at our tertiary care center from May to July 2021 were included and defined as proven, probable, possible, or negative for invasive fungal disease (IFD) according to EORTC/MSGERC guidelines. One sample from each patient (nasal/sinus biopsy, nasal crust, or orbital tissue) was subjected to conventional methods for diagnosis of IM and MucorGenius® real-time PCR (hereafter called ‘the assay’). Results: A total of 5 (1.92%), 124 (47.6%), and 44 (16.9%) cases respectively were classified as having proven, probable, and possible IM. The remaining 20 (7.69%) were classified as not having invasive fungal infections and were used as controls to calculate the specificity of the test. The majority of cases were classified as ‘probable’ because specimens received included biopsy from the nasal or sinus cavity.According to radiological findings, sino-nasal involvement was seen in 26/173 (15.02%), sino-orbital involvement in 122/173 (70.5%), and additional intracranial extension in 25/173 (14.4%) of the 173 cases of IM.Among 129 proven and probable cases, direct microscopy of samples showed only aseptate hyphae in 70 cases, and both aseptate and septate hyphae in 36 cases;the assay was positive in 53 and 13 of these cases respectively. In the remaining 23 cases, direct microscopy of samples showed only septate hyphae and the assay was negative.Additionally, the assay was able to detect the presence of Mucorales among 44 possible cases of IM in which direct microscopy of samples showed no fungal elements, but the patients displayed clinical and radiological features of IM and improved with antifungal therapy.The overall sensitivity and specificity of the assay were 63.21% and 90.48% respectively.The sensitivity of the assay in proven and probable cases of IM was 60% and 66.7% respectively, while specificity was 90% for both, using the presence of aseptate hyphae in direct microscopy as a gold standard. Sensitivity and specificity in possible cases were 27.27% and 90% respectively, using the presence of clinical and radiological features of IM and response to antifungal treatment as a reference.When sensitivity and specificity were determined independently in cases of mucormycosis and mixed infection (mucormycosis + aspergillosis), they increased to 75.71% and 90.48% respectively in the former, and both decreased to 38% in the latter. Conclusion: The MucorGenius® real-time PCR performs well in detecting IM as a single infection, especially in cases of possible IM which are not detected by conventional methods. However, it is inefficient in detecting co-infections of invasive mucormycosis and aspergillosis, possibly because Aspergillus can suppress the growth of Mucorales. With further studies using the results to guide clinical intervention and measuring the impact on the outcome, it can be a useful tool to make an early diagnosis of mucormycosis in patients with a high index of clinical suspicion.

COVID-19-associated mucormycosis presenting to the Emergency Department-an observational study of 70 patients.

BACKGROUND: Mucormycosis (MM) is a deadly opportunistic fungal infection and a large surge in COVID-19-associated mucormycosis (CAM) is occurring in India. AIM: Our aim was to delineate the clinico-epidemiological profile and identify risk factors of CAM patients presenting to the Emergency Department (ED). DESIGN: This was a retrospective, single-centre, observational study. METHODS: We included patients who presented with clinical features or diagnosed MM and who were previously treated for COVID-19 in last 3 months of presentation (recent COVID-19) or currently being treated for COVID-19 (active COVID-19). Information regarding clinical features of CAM, possible risk factors, examination findings, diagnostic workup including imaging and treatment details were collected. RESULTS: Seventy CAM patients (median age: 44.5 years, 60% males) with active (75.7%) or recent COVID-19 (24.3%) who presented to the ED in between 6 May 2021 and 1 June 2021, were included. A median duration of 20 days (interquartile range: 13.5-25) was present between the onset of COVID-19 symptoms and the onset of CAM symptoms. Ninety-three percent patients had at least one risk factor. Most common risk factors were diabetes mellitus (70%) and steroid use for COVID-19 disease (70%). After clinical, microbiological and radiological workup, final diagnosis of rhino-orbital CAM was made in most patients (68.6%). Systemic antifungals were started in the ED and urgent surgical debridement was planned. CONCLUSION: COVID-19 infection along with its medical management have increased patient susceptibility to MM.

Clinicopathological correlation in fatal COVID-19 infection using postmortem minimally invasive tissue sampling: The first case series from India

Introduction: The COVID-19 pandemic began in China in December 2019. India is the second most affected country, as of November 2020 with more than 8.5 million cases. COVID-19 infection primarily involves the lung with the severity of illness varying from influenza-like illness to acute respiratory distress syndrome. Other organs have also been found to be variably affected. Studies evaluating the histopathological changes of COVID-19 are critical in providing a better understanding of the disease pathophysiology and guiding treatment. Minimally invasive biopsy techniques (MITS/B) provide an easy and suitable alternative to complete autopsies. In this prospective single-center study, we present the histopathological examination of 37 patients who died with complications of COVID-19. Materials and methods: This was an observational study conducted in the Intensive Care Unit of JPN Trauma Centre AIIMS. A total of 37 patients who died of COVID-19 were enrolled in the study. Postmortem percutaneous biopsies were taken with the help of surface landmarking/ultrasonography guidance from lung, heart, liver, and kidneys;after obtaining ethical consent. The biopsy samples were then stained with hematoxylin and eosin stain. Immunohistochemistry (IHC) was performed using CD61 and CD163 in all lung cores. The SARS-CoV-2 virus was detected using IHC with primary antibodies in selected samples. Details regarding demographics, clinical parameters, hospital course, treatment details, and laboratory investigations were also collected for clinical correlation. Results: A total of 37 patients underwent post-mortem minimally invasive tissue sampling. Mean age of the patients was 48.7years and 59.5% of them were males. Respiratory failure was the most common complication seen in 97.3%. Lung histopathology showed acute lung injury and diffuse alveolar damage in 78% patients. Associated bronchopneumonia was seen in 37.5% patients and scattered microthrombi were visualised in 21% patients. Immunostaining with CD61 and CD163 highlighted megakaryocytes, and increased macrophages in all samples. Immunopositivity for SARS-CoV-2 was observed in Type II pneumocytes. Acute tubular injury with epithelial vacuolization was seen in 46% of the renal biopsies but none of them showed evidence of microvascular thrombosis. 71% of the liver tissue cores showed evidence of Kupfer cell hyperplasia. 27.5% had evidence of submassive hepatic necrosis and 14% had features of acute on chronic liver failure. All the heart biopsies showed nonspecific features such as hypertrophy with nucleomegaly with no evidence of myocardial necrosis in any of the samples. Discussions: Minimally invasive autopsies (MIA) or MITS/B are a simplified method of conducting postmortem sampling, originally devised to investigate the causes of death in low-resource settings. Due to the potential risks associated with conducting traditional postmortem examinations in COVID-19 patients, this method has been adopted to study morbid pathological changes in COVID-19 patients. The typical findings described in pulmonary histopathology of COVID-19 patients include epithelial, vascular, fibrotic, and other changes. The epithelial changes described include diffuse alveolar damage with or without hyaline membranes, metaplasia of alveolar epithelium, desquamation/reactive hyperplasia of pneumocytes, viral cytopathic changes, and multinucleated giant cells. Common vascular changes include capillary congestion, thrombosis in microvasculatures, alveolar hemorrhage, capillary changes (proliferation, thickening, fibrin deposition, endothelial detachment), peri- or intravascular inflammatory infiltrates. In our series, the most common histopathological pattern seen on lung samples was DAD (diffuse alveolar damage), with nearly equal numbers of patients showing DAD in the acute exudative phase and organizing phase. The incidence of vascular changes were only 21%, which was less than that reported. Several factors could have contributed to this observation including regular use of anticoagulants in the coh rt as a part of national policies, lower thrombotic complications in the population studied due to genetic or climatic factors. Histopathological examination of the liver in COVID-19 patients had typically revealed mild steatosis, focal hepatic necrosis, Kupffer cell hyperplasia, and sinusoidal dilatation as reported in the literature. In our series, the most common features identified include Kupffer cell hypertrophy in 21 (72.41%) patients, acute submassive hepatic necrosis (27.5%) followed by acute on chronic liver failure (13.7%) in a background of chronic liver diseases, features of NCPF (10.3%) and cholestasis (24%). Though in our cohort of liver biopsies, hepatic lobular inflammatory cell infiltrates were not prominent, changes of hepatocyte degeneration as ballooning, acidophil bodies, MDBs (Mallory-Denk bodies), and microvesicular steatosis were prominent. Lack of inflammatory cell infiltrates in the liver has been described in earlier reports on fatal COVID-19 infections. Our findings disagree with the observation of Sonzogni et al. that the liver is not a primary target of COVID- 19 infection and only vascular changes in the liver are observed. In this cohort, we identified substantial histological changes of hepatocyte necrosis, degeneration, Kupffer cell hypertrophy, micro, and macrovesicular steatosis, more than the vascular changes. Renal histopathology in COVID-19 is reported to show changes including prominently acute tubular injury (more prominent in the proximal tubules), arteriosclerosis, or glomerulosclerosis (both as features of underlying comorbid conditions like hypertension), focal segmental glomerulosclerosis with a collapsing phenotype, and tubulointerstitial inflammation. Renal vascular changes reported are relatively less common and include fibrin thrombi, thrombotic angiopathy, and lymphocytic endothelitis. In our cohort of patients, the most common finding on renal histopathology was acute tubular injury and preexisting renal conditions were only evident in 18%. None of the cases showed significant vascular changes. Conclusion: The most common finding in this cohort is the diffuse alveolar damage with a demonstration of SARS-CoV-2 protein in the acute phase of DAD. Microvascular thrombi were rarely identified in the lung, liver, and kidney. Substantial hepatocyte necrosis, hepatocyte degeneration, Kupffer cell hypertrophy, micro, and macrovesicular steatosis unrelated to microvascular thrombi suggests that the liver might be a primary target of COVID-19. This study highlights the importance of MITS/B in better understanding the pathological changes associated with COVID-19.