Association of gut microbial dysbiosis with disease severity, response to therapy and disease outcomes in Indian patients with COVID-19.

BACKGROUND: Severe coronavirus disease 2019 (COVID-19) is associated with systemic hyper-inflammation. An adaptive interaction between gut microbiota and host immune systems is important for intestinal homeostasis and systemic immune regulation. The association of gut microbial composition and functions with COVID-19 disease severity is sparse, especially in India. We analysed faecal microbial diversity and abundances in a cohort of Indian COVID-19 patients to identify key signatures in the gut microbial ecology in patients with severe COVID-19 disease as well as in response to different therapies. The composition of the gut microbiome was characterized using 16Sr RNA gene sequences of genomic DNA extracted from faecal samples of 52 COVID-19 patients. Metabolic pathways across the groups were predicted using PICRUSt2. All statistical analyses were done using Vegan in the R environment. Plasma cytokine abundance at recruitment was measured in a multiplex assay. RESULTS: The gut microbiome composition of mild and severe patients was found to be significantly different. Immunomodulatory commensals, viz. Lachnospiraceae family members and Bifidobacteria producing butyrate and short-chain fatty acids (SCFAs), were under represented in patients with severe COVID-19, with an increased abundance of opportunistic pathogens like Eggerthella. The higher abundance of Lachnoclostridium in severe disease was reduced in response to convalescent plasma therapy. Specific microbial genera showed distinctive trends in enriched metabolic pathways, strong correlations with blood plasma cytokine levels, and associative link to disease outcomes. CONCLUSION: Our study indicates that, along with SARS-CoV-2, a dysbiotic gut microbial community may also play an important role in COVID-19 severity through modulation of host immune responses.

Deficient Phagocytosis in Circulating Monocytes from Patients with COVID-19-Associated Mucormycosis.

Cases of rhino-orbital mucormycosis in patients suffering from severe coronavirus disease 2019 (COVID-19) were reported in different parts of the world, especially in India. However, specific immune mechanisms that are linked to susceptibility to COVID-19-associated mucormycosis (CAM) remain largely unexplored. We aimed to explore whether the differential regulation of circulating cytokines in CAM patients had any potential pathogenic links with myeloid phagocyte function and susceptibility to mucormycosis. A small cohort of Indian patients suffering from CAM (N = 9) as well as COVID-19 patients with no evidence of mucormycosis (N = 5) were recruited in the study. Venous blood was collected from the patients as well as from healthy volunteers (N = 8). Peripheral blood mononuclear cells and plasma were isolated. Plasma samples were used to measure a panel of 48 cytokines. CD14+ monocytes were isolated and used for a flow cytometric phagocytosis assay as well as a global transcriptome analysis via RNA-sequencing. A multiplex cytokine analysis of the plasma samples revealed reduction in a subset of cytokines in CAM patients, which is known to potentiate the activation, migration, or phagocytic activity of myeloid cells, compared to the COVID-19 patients who did not contract mucormycosis. Compared to monocytes from healthy individuals, peripheral blood CD14+ monocytes from CAM patients were significantly deficient in phagocytic function. The monocyte transcriptome also revealed that pathways related to endocytic pathways, phagosome maturation, and the cytoskeletal regulation of phagocytosis were significantly downregulated in CAM patients. Thus, the study reports a significant deficiency in the phagocytic activity of monocytes, which is a critical effector mechanism for the antifungal host defense, in patients with CAM. This result is in concordance with results regarding the specific cytokine signature and monocyte transcriptome. IMPORTANCE A number of cases of mucormycosis, often fatal, were reported among severe COVID-19 patients from India as well as from some other parts of the world. However, specific immunocellular mechanisms that underlie susceptibility to this fungal infection in COVID-19 remain largely unexplored. Our study reports a deficiency in phagocytosis by monocytes in COVID-19 patients who are concomitantly afflicted with mucormycosis, with this deficiency being linked to a characteristic monocyte transcriptome as well as a circulating cytokine signature. The functional phenotype and cytokine signature of the monocytes may provide useful biomarkers for detecting potential susceptibility to mucormycosis in COVID-19 as well as in other viral infections.

Circulating Interleukin-8 Dynamics Parallels Disease Course and Is Linked to Clinical Outcomes in Severe COVID-19.

Severe COVID-19 frequently features a systemic deluge of cytokines. Circulating cytokines that can stratify risks are useful for more effective triage and management. Here, we ran a machine-learning algorithm on a dataset of 36 plasma cytokines in a cohort of severe COVID-19 to identify cytokine/s useful for describing the dynamic clinical state in multiple regression analysis. We performed RNA-sequencing of circulating blood cells collected at different time-points. From a Bayesian Information Criterion analysis, a combination of interleukin-8 (IL-8), Eotaxin, and Interferon-γ (IFNγ) was found to be significantly linked to blood oxygenation over seven days. Individually testing the cytokines in receiver operator characteristics analyses identified IL-8 as a strong stratifier for clinical outcomes. Circulating IL-8 dynamics paralleled disease course. We also revealed key transitions in immune transcriptome in patients stratified for circulating IL-8 at three time-points. The study identifies plasma IL-8 as a key pathogenic cytokine linking systemic hyper-inflammation to the clinical outcomes in COVID-19.

Clinical Trial Subgroup Analyses to Investigate Clinical and Immunological Outcomes of Convalescent Plasma Therapy in Severe COVID-19.

Objective: To assess the clinical and immunological benefits of passive immunization using convalescent plasma therapy (CPT). Materials and Methods: A series of subclass analyses were performed on the previously published outcome data and accompanying clinical metadata from a completed randomized controlled trial (RCT) (Clinical Trial Registry of India, number CTRI/2020/05/025209). The subclass analyses were performed on the outcome data and accompanying clinical metadata from a completed RCT (patient recruitment between May 15, 2020 and October 31, 2020). Data on the plasma abundance of a large panel of cytokines from the same cohort of patients were also used to characterize the heterogeneity of the putative anti-inflammatory function of convalescent plasma (CP) in addition to passively providing neutralizing antibodies. Results: Although the primary clinical outcomes were not significantly different in the RCT across all age groups, significant immediate mitigation of hypoxia, reduction in hospital stay, and significant survival benefit were registered in younger (<67 years in our cohort) patients with severe coronavirus disease 2019 and acute respiratory distress syndrome on receiving CPT. In addition to neutralizing the antibody content of CP, its anti-inflammatory proteome, by attenuation of the systemic cytokine deluge, significantly contributed to the clinical benefits of CPT. Conclusion: Subgroup analyses revealed that clinical benefits of CPT in severe coronavirus disease 2019 are linked to the anti-inflammatory protein content of CP apart from the anti-severe acute respiratory syndrome coronavirus 2 neutralizing antibody content.

Development and Validation of a Treatment Benefit Index to Identify Hospitalized Patients With COVID-19 Who May Benefit From Convalescent Plasma.

Importance: Identifying which patients with COVID-19 are likely to benefit from COVID-19 convalescent plasma (CCP) treatment may have a large public health impact. Objective: To develop an index for predicting the expected relative treatment benefit from CCP compared with treatment without CCP for patients hospitalized for COVID-19 using patients' baseline characteristics. Design, Setting, and Participants: This prognostic study used data from the COMPILE study, ie, a meta-analysis of pooled individual patient data from 8 randomized clinical trials (RCTs) evaluating CCP vs control in adults hospitalized for COVID-19 who were not receiving mechanical ventilation at randomization. A combination of baseline characteristics, termed the treatment benefit index (TBI), was developed based on 2287 patients in COMPILE using a proportional odds model, with baseline characteristics selected via cross-validation. The TBI was externally validated on 4 external data sets: the Expanded Access Program (1896 participants), a study conducted under Emergency Use Authorization (210 participants), and 2 RCTs (with 80 and 309 participants). Exposure: Receipt of CCP. Main Outcomes and Measures: World Health Organization (WHO) 11-point ordinal COVID-19 clinical status scale and 2 derivatives of it (ie, WHO score of 7-10, indicating mechanical ventilation to death, and WHO score of 10, indicating death) at day 14 and day 28 after randomization. Day 14 WHO 11-point ordinal scale was used as the primary outcome to develop the TBI. Results: A total of 2287 patients were included in the derivation cohort, with a mean (SD) age of 60.3 (15.2) years and 815 (35.6%) women. The TBI provided a continuous gradation of benefit, and, for clinical utility, it was operationalized into groups of expected large clinical benefit (B1; 629 participants in the derivation cohort [27.5%]), moderate benefit (B2; 953 [41.7%]), and potential harm or no benefit (B3; 705 [30.8%]). Patients with preexisting conditions (diabetes, cardiovascular and pulmonary diseases), with blood type A or AB, and at an early COVID-19 stage (low baseline WHO scores) were expected to benefit most, while those without preexisting conditions and at more advanced stages of COVID-19 could potentially be harmed. In the derivation cohort, odds ratios for worse outcome, where smaller odds ratios indicate larger benefit from CCP, were 0.69 (95% credible interval [CrI], 0.48-1.06) for B1, 0.82 (95% CrI, 0.61-1.11) for B2, and 1.58 (95% CrI, 1.14-2.17) for B3. Testing on 4 external datasets supported the validation of the derived TBIs. Conclusions and Relevance: The findings of this study suggest that the CCP TBI is a simple tool that can quantify the relative benefit from CCP treatment for an individual patient hospitalized with COVID-19 that can be used to guide treatment recommendations. The TBI precision medicine approach could be especially helpful in a pandemic.

A phase 2 single center open label randomised control trial for convalescent plasma therapy in patients with severe COVID-19.

A single center open label phase 2 randomised control trial (Clinical Trial Registry of India No. CTRI/2020/05/025209) was done to assess clinical and immunological benefits of passive immunization using convalescent plasma therapy. At the Infectious Diseases and Beleghata General Hospital in Kolkata, India, 80 patients hospitalized with severe COVID-19 disease and fulfilling the inclusion criteria (aged more than 18 years, with either mild ARDS having PaO2/FiO2 200-300 or moderate ARDS having PaO2/FiO2 100-200, not on mechanical ventilation) were recruited and randomized into either standard of care (SOC) arm (N = 40) or the convalescent plasma therapy (CPT) arm (N = 40). Primary outcomes were all-cause mortality by day 30 of enrolment and immunological correlates of response to therapy if any, for which plasma abundance of a large panel of cytokines was quantitated before and after intervention to assess the effect of CPT on the systemic hyper-inflammation encountered in these patients. The secondary outcomes were recovery from ARDS and time taken to negative viral RNA PCR as well as to report any adverse reaction to plasma therapy. Transfused convalescent plasma was characterized in terms of its neutralizing antibody content as well as proteome. The trial was completed and it was found that primary outcome of all-cause mortality was not significantly different among severe COVID-19 patients with ARDS randomized to two treatment arms (Mantel-Haenszel Hazard Ratio 0.6731, 95% confidence interval 0.3010-1.505, with a P value of 0.3424 on Mantel-Cox Log-rank test). No adverse effect was reported with CPT. In severe COVID-19 patients with mild or moderate ARDS no significant clinical benefit was registered in this clinical trial with convalescent plasma therapy in terms of prespecified outcomes.

Plasma Gradient of Soluble Urokinase-Type Plasminogen Activator Receptor Is Linked to Pathogenic Plasma Proteome and Immune Transcriptome and Stratifies Outcomes in Severe COVID-19.

Disease caused by SARS-CoV-2 coronavirus (COVID-19) led to significant morbidity and mortality worldwide. A systemic hyper-inflammation characterizes severe COVID-19 disease, often associated with acute respiratory distress syndrome (ARDS). Blood biomarkers capable of risk stratification are of great importance in effective triage and critical care of severe COVID-19 patients. Flow cytometry and next-generation sequencing were done on peripheral blood cells and urokinase-type plasminogen activator receptor (suPAR), and cytokines were measured from and mass spectrometry-based proteomics was done on plasma samples from an Indian cohort of COVID-19 patients. Publicly available single-cell RNA sequencing data were analyzed for validation of primary data. Statistical analyses were performed to validate risk stratification. We report here higher plasma abundance of suPAR, expressed by an abnormally expanded myeloid cell population, in severe COVID-19 patients with ARDS. The plasma suPAR level was found to be linked to a characteristic plasma proteome, associated with coagulation disorders and complement activation. Receiver operator characteristic curve analysis to predict mortality identified a cutoff value of suPAR at 1,996.809 pg/ml (odds ratio: 2.9286, 95% confidence interval 1.0427-8.2257). Lower-than-cutoff suPAR levels were associated with a differential expression of the immune transcriptome as well as favorable clinical outcomes, in terms of both survival benefit (hazard ratio: 0.3615, 95% confidence interval 0.1433-0.912) and faster disease remission in our patient cohort. Thus, we identified suPAR as a key pathogenic circulating molecule linking systemic hyperinflammation to the hypercoagulable state and stratifying clinical outcomes in severe COVID-19 patients with ARDS.

Nature and Dimensions of Systemic Hyperinflammation and its Attenuation by Convalescent Plasma in Severe COVID-19.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing coronavirus disease 2019 (COVID-19), has led to significant morbidity and mortality. While most suffer from mild symptoms, some patients progress to severe disease with acute respiratory distress syndrome (ARDS) and associated systemic hyperinflammation. METHODS: First, to characterize key cytokines and their dynamics in this hyperinflammatory condition, we assessed abundance and correlative expression of a panel of 48 cytokines in patients progressing to ARDS as compared to patients with mild disease. Then, in an ongoing randomized controlled trial of convalescent plasma therapy (CPT), we analyzed rapid effects of CPT on the systemic cytokine dynamics as a correlate for the level of hypoxia experienced by the patients. RESULTS: We identified an anti-inflammatory role of CPT independent of its neutralizing antibody content. CONCLUSIONS: Neutralizing antibodies, as well as reductions in circulating interleukin-6 and interferon-γ-inducible protein 10, contributed to marked rapid reductions in hypoxia in response to CPT. CLINICAL TRIAL REGISTRY OF INDIA: CTRI/2020/05/025209. http://www.ctri.nic.in/.

A prospective study on COVID-19 convalescent plasma donor (CCP) recruitment strategies in a resource constrained blood centre.

Background and Objectives: The COVID-19 pandemic has spread across 87 million people with more than 1·8 million deaths in the world. As there is no definite treatment modality, the use of convalescent plasma has become increasingly popular worldwide. This study aimed to identify an appropriate strategy of donor recruitment and to evaluate the appropriateness of pre-set plasma donation guidelines. Material and Methods: In this prospective study conducted from May to September 2020, the donors were recruited under the following two circumstances: Group I, patients in the post-COVID-19 follow-up in the clinic, and Group II, patients recovered from COVID-19 recruited through mass and electronic media. A pre-set donor selection criteria and laboratory investigation was designed according to national and international guidelines. Approximately 500 ml of COVID-19 convalescent plasma (CCP) was collected from recovered individuals in each group by two different cell separators. The overall donor's attendance rate, deferral rate, adverse events and donor compliance was analysed and compared between the two groups. Results: There was a significant difference in attendance in relation to registration between the groups (P < 0·0001). Donor deferral was significantly higher in group II compared with group I. The single most frequent cause of donor deferral was low antibody index (P = 0·0001). The total donor adverse event rate in CCP donation was significantly lower compared with routine plateletpheresis procedures. The donor's compliance to blood centre's protocol was satisfactory in both the groups. Conclusion: Recruitment of patients in the post-COVID-19 follow-up in the clinic was more effective than the general recruitment through mass and electronic media for convalescence plasma donation in a resource-constrained blood centre.

Overview of Immune Response During SARS-CoV-2 Infection: Lessons From the Past.

After the 1918 flu pandemic, the world is again facing a similar situation. However, the advancement in medical science has made it possible to identify that the novel infectious agent is from the coronavirus family. Rapid genome sequencing by various groups helped in identifying the structure and function of the virus, its immunogenicity in diverse populations, and potential preventive measures. Coronavirus attacks the respiratory system, causing pneumonia and lymphopenia in infected individuals. Viral components like spike and nucleocapsid proteins trigger an immune response in the host to eliminate the virus. These viral antigens can be either recognized by the B cells or presented by MHC complexes to the T cells, resulting in antibody production, increased cytokine secretion, and cytolytic activity in the acute phase of infection. Genetic polymorphism in MHC enables it to present some of the T cell epitopes very well over the other MHC alleles. The association of MHC alleles and its downregulated expression has been correlated with disease severity against influenza and coronaviruses. Studies have reported that infected individuals can, after recovery, induce strong protective responses by generating a memory T-cell pool against SARS-CoV and MERS-CoV. These memory T cells were not persistent in the long term and, upon reactivation, caused local damage due to cross-reactivity. So far, the reports suggest that SARS-CoV-2, which is highly contagious, shows related symptoms in three different stages and develops an exhaustive T-cell pool at higher loads of viral infection. As there are no specific treatments available for this novel coronavirus, numerous small molecular drugs that are being used for the treatment of diseases like SARS, MERS, HIV, ebola, malaria, and tuberculosis are being given to COVID-19 patients, and clinical trials for many such drugs have already begun. A classical immunotherapy of convalescent plasma transfusion from recovered patients has also been initiated for the neutralization of viremia in terminally ill COVID-19 patients. Due to the limitations of plasma transfusion, researchers are now focusing on developing neutralizing antibodies against virus particles along with immuno-modulation of cytokines like IL-6, Type I interferons (IFNs), and TNF-α that could help in combating the infection. This review highlights the similarities of the coronaviruses that caused SARS and MERS to the novel SARS-CoV-2 in relation to their pathogenicity and immunogenicity and also focuses on various treatment strategies that could be employed for curing COVID-19.