Sanmitra Ghosh, Author at Adamas University

#MicrobiologyNext: Epidemiology of Infectious diseases: An offshoot of Medical Microbiology

Medical microbiology is a subdivision of microbiology that concerns the study of pathogenesis and epidemiology of microorganisms that can infect and cause disease symptoms in human beings. This subject specialization evolved to complement medical science with its hands-on applications as diagnostic and therapeutic guidance in disease management. The clinical facet of the field emphasizes on detecting the occurrence of microbial infections and its spread in individuals, the prognosis of the disease, and protocols for treatment. This individualistic concept derived from clinical microbiology must be elaborated to public health as most of the time the infectious diseases do not remain confined in one or few subjects, rather can spread, persist and affect a large number of people over time. This is true for all infectious diseases irrespective of the ailments being sporadic, endemic, epidemic, or pandemic. A cornerstone to this public health is the second facet of medical microbiology known as epidemiology. The word “Epidemiology”, is derived from Greek ‘epi’, meaning ‘upon, among’, ‘demos’, meaning ‘people, district’, and ‘logos’, meaning ‘study. Therefore epidemiology is the study of where-about of a disease and is considered to be the basic science of preventive medicine. An epidemiologist moves ahead of the biology of understanding the disease processes, and also relies on social sciences to elucidate proximate and distal causes, engineering for assessment of exposure to the disease and statistics to utilize the data/observation to reach apt inferences. This is because, for the prevention of infectious disease, it is crucial to understand the causative agents, risk factors, and circumstances that lead to a specific disease, and even simple interventions may break the chain of transmission. Clinical microbiology and epidemiology operate in cooperative and interdependent ways to determine the dynamics of infection and also accurate tracking of etiological agents.

Ignaz Semmelweis was the first to demonstrate the significance of epidemiology who by careful analysis of statistical data alone inferred about the mode of transmission of streptococcal puerperal fever. He also formulated handwashing to be a way out to prevent transmission decades before the discovery of the causal agent itself (Streptococcus pyogenes). Since then, each organism can be assigned with its profile of vital statistics contributing significantly to morbidity and mortality of human beings globally. Some agents are transmitted by air or water, some by food, and others by insects or other animals; many are en route from one person to another. Some agents occur globally, and others are limited in specific geographic locations or ecologic niche. Knowledge regarding the mode of transmission of the causal organism in the target population is crucial to understanding the disease. Considering the advent of new diseases, it is also essential to determine whether the etiological agent is truly novel or derivatized from previously existing ones. Unraveling mysterious outbreaks or identification of new epidemiologic patterns have often led to the path of isolating new agents.

Epidemiologists concentrate their study on population groups rather than on individuals. It can cohort studies where subjects are selected based on their exposure status, i.e., the study participants should be at risk of the outcome under investigation as the study initiates and will be followed through time to assess their later outcome status. Infectious disease epidemiology also considers the interaction between individuals within the population group in two ways-

(i) case-series study involving the qualitative study of the outcome in a single patient/ small group of patients with comparable diagnosis/ a statistical factor to cause illness with periods when they are unexposed. Considering a single patient or patients with a similar diagnosis, no inference can be drawn about the general population of patients. However, the identification of a unique feature of disease/ patient history may lead to the proposition of a new hypothesis regarding the natural history of the disease or possible causal factors.

(ii) case-control study which is a retrospective study of subjects who are disease positive (case) w.r.t. group of disease negative people (control) within the same population.

The Changing Picture of Infectious Disease Epidemiology

More than forty new pathogens have been identified in the last three decades, some of which are of global importance. Causative agents for infectious diseases are specific, but they may change over time. The best illustration of an etiological agent to undergo modifications that enable them to reinfect subjects that have already been infected and immune is the Influenza virus owing to antigenic shift and antigenic drift in surface antigens (H and/or N).

In recent times all over the world, there has been an increase in the number of people who are at high risk for infectious disease. Improvement in health –care and increased life expectancy in developed countries have led to an increase in the number of people who are aged and /or immune-deficient (cancer survivors, transplant patients, or people on immunosuppressive drugs for long-term autoimmune diseases) predisposed to infectious diseases and developing life-threatening complications. In populations that were originally at low risk, adoption of altered lifestyles has increased opportunities for disease transmission. Intravascular drug injections, air travel, consumption of raw fish, ethnic foods expanded the area of distribution of a variety of infectious agents.

Evolutionary biology

Infectious disease epidemiology is aimed at curtailing the influence of pathogens on public health. As both pathogens and their hosts have evolved, knowledge of evolutionary biology is pertinent to decipher the nature of their interactions for fitness, as well as to understand the history of pathogen transmission. Mathematical models can be used to study the effects of different selective markers (measurable biological and behavioral risk factors), whereas molecular data can be used to define different strains and their genetic variations. Recent progressions, particularly in the rapid determination of sequence data, have increased the propensity of evolutionary biology towards clinical microbiology.

Molecular strain typing

The practical science of infectious disease epidemiology involves the authentication of infectious etiologies through repeated isolation of a specific microorganism from patients with a given disease or syndrome as well as from animals, vectors, and environmental sources to identify reservoirs and modes of transmission. To prescribe effective therapy often it is essential to identify species of infecting microorganisms through phenotypic and genotypic characterization. Phenotypic methods like biotyping of biochemical profiles, phage typing, and serological typing help to characterize the products of gene expression to differentiate strains. But they are inadequate to provide enough unrelated parameters for proper reflection of genotype. Also, phenotypic methods tend to give variable results as many gene expressions are affected by spontaneous mutations, environmental conditions, or reversible phenotypic changes; therefore, they have been replaced by genotypic methods that rely on the analysis of the genetic structure of an organism. Highly sensitive and specific molecular techniques have recently been adopted to facilitate epidemiologic studies through the detection and characterization of the genetic variability of infectious agents (bacteria, fungi, protozoa, viruses). It necessitates the selection of molecular method(s) that can discriminate genetic variants at different hierarchical levels namely, above the level of species, between species, between intraspecific variants, etc. coupled with the selection of a region of nucleic acid suitable for interrogation. For bacterial fingerprinting RFLP-PFGE, RFLP+ probe & ribotyping have been the most commonly used. RAPD and karyotyping have been used for fingerprinting fungi while MLEE, RAPD, and PCR-RFLP have been used for parasitic protozoa. Except for whole-genome sequencing, the other molecular methods analyze only a small portion of the organisms’ genetic complement. For this reason, only whole-genome sequencing would provide the unequivocal data required to distinguish isolates exhibiting similar patterns despite being unrelated epidemiologically.

Omics and epidemiology

Application of omics to infectious disease epidemiology breaks free the study beyond single organisms. Next-generation sequencing technologies like the 454 Pyrosequencing (Roche, Branford, CT) and massively parallel sequencing by synthesis (Illumina, San Diego, CA) allow the characterization of the structure and functionality of microbial communities based on genetic sequence based on the sequence of the ribosomal genes ( rDNA ). Rapid sequencing technologies provide adequate data to characterize the microbial communities living on and in the human body, how they vary over periods, respond to treatment, and interact with pathogens. The Human Microbiome Project is an on-going effort funded by the National Institutes of Health to provide these parameters. Taxonomy based on the ribosomal gene sequence gives rise to operational taxonomic units (OTUs) or phylotypes that enable identification and characterization of minute variations in candidate species in a microbial community in terms of species richness, evenness, and diversity.

In the pretext of COVID-19 pandemic, interrogating epidemiology of viral and other infectious diseases has gained the utmost priority through the study of the complex relationships among hosts and infectious agents to reduce a load of pathogenicity on the public health sector globally. Epidemiologists are concerned with virus spread or transmission, with or without the disease. Viral epidemiologists try to envisage the potential for the development of epidemics, with the ultimate aim to define the kinds of interventions that could contain a virus outbreak. To model virus transmission, epidemiologists must try to consider a variety of attributes involving both host and virus. Similar objectives and modeling are applicable for any other type of infectious disease transmission too. Factors that can impact microbial transmission and spread of infection include: a) prevalence of the causal agent within the population, b) mode of transmission of the agent, c) duration of the infection, d) proportion of susceptible and resistant individuals in the population, e) population density, f) modes of travel or associations, g) lifestyle and h) climate and/or season. Once all the parameters are taken into account a disease model can be envisioned that will complement the clinical findings to formulate appropriate disease management protocol for infectious diseases.

Globalization and spread of infectious disease

Globalization involves accelerated transcontinental streaming of goods, people, capital, information, and energy across borders, frequently empowered by technological developments. The dissemination of infectious diseases worldwide has followed a parallel trend. Since the prehistoric era, an enormous breeding ground was created as Europe, Asia, and North America became connected by world trade routes. During the Antonine Plague, smallpox emerged in Rome killing millions of Roman people from 165 AD to 180 AD. After 300 years Europe was afflicted by the bubonic plague as the Plague of Justinian (542-543 AD). This bubonic plague re-emerged in the 14^th century in more severe form as the Black Death with the initiation of a new trade route with China that prompted rapid transmission of flea-infested furs from plague-ridden Central Asia.

Before the expansion of world trade routes, the spread of human pathogens predominantly occurred in two ways.

With the livelihood as a hunter-gatherer, people lived a life in motion. So it was challenging for the microorganisms to come in contact with their human host for a long period. As people started colonizing in large numbers in the same space with farming as an occupation, they were in prolonged contact with each other and also with their fecal matter that increased the chance of disease transmission.
With the establishment of towns and cities even larger numbers of people came in closer proximity under even worse sanitary conditions.

After two millennia as the human civilization proudly embraced a new era of globalization in terms of faster travel over greater distances and worldwide trade, the human pathogens also took the golden opportunity to disseminate following the same route. At the beginning of19^thcentury, the emergence of plague epidemics in several port cities all over the world demonstrated the remarkable influence of increased trade and travel on infectious diseases. In Johannesburg, the incidence of the plague led to the relocation of black residents as white colonists believed them to be the source of the disease. Almost at the same time, human civilization witnessed the death of millions of people worldwide due to the influenza pandemic. Thus with the current era of globalization, population density is becoming higher, millions of people are moving around the world very fast (by choice or by force), food, animals, capitals and other daily commodities are freely getting transported across political boundaries. In the course of this process, pathogens are getting plenty of opportunities to hitch rides all around the world using humans, commodities, airplanes, and other conveyances as vehicles. Regardless of the successful endeavors of the developed world to control and even eradicate several infectious diseases, every year around 13 million people still die from such diseases.

In the last two centuries, the average distance of human travel with speed has amplified a thousand-fold together with the number of people traveling at a time, while incubation periods for infectious diseases continued to be the same as before. Previously what could have been a merely localized outbreak, now can progress into a large, worldwide epidemic in a matter of days. The global spread of AIDS is a singular, but the most devastating, instance of the impact of this tremendous human mobility on infectious disease.

Open borders and international travel opportunities complemented by advances in transportation technology are availed not only by the tourists; but also by millions of other people who leave their homeland in search of a job or an improved quality of life, and several more who are forcefully displaced by war. Such migrant populations are among the most vulnerable to emerging infectious diseases. The emergence and re-emergence of infectious diseases like multidrug-resistant tuberculosis in developed countries are often reported to be associated with the massive influx of immigrants from poor countries with a higher prevalence of such diseases. It is expected that the situation will deteriorate more in the future with the growth of the world population along with an increase in demographic and economic gaps between the developed and developing worlds that will instigate people to move out in search of a better quality of life. In the context of transportation, an airplane transporting an infected mosquito vector in the wheel wells was hypothesized to be the vehicle for the introduction of the West Nile virus into the United States in 1999 for the first time.

Previously, most food products have been manufactured and consumed locally. However, over the last few decades, with the rise in consumer demand and adaptation of westernized lifestyle together with food production and processing activities being geographically more fragmented, the epidemiology of the foodborne disease has changed significantly. Before the establishment of WTO in 1995, trade in animals and animal products were conducted following a policy of zero risks. But at present, imported products are treated similarly to domestic products, even as animal health restrictions are considered. The experience of bovine spongiform encephalopathy in the United Kingdom and Europe exemplifies the remarkable threat that accompanies the unlimited growth and opportunity offered by new international trade regulations enforced by WTO. Many countries do not have all-inclusive food safety programs incorporated into their public health strategies. Outbreaks of foodborne illness have revealed many shortcomings in food safety regulation practices not only in developing countries but also in developed countries like the United States. The free flow of food also elevates serious concerns about the global spread of antibiotic resistance associated with the consumption of antibiotic-fed food animals.

Similar aspects make the situation more complex as novel diseases emerge and spread out very fast causing unprecedented pandemics like SARS in 2002-2004 and COVID 19 in 2019-2020. Thus as we dream to progress using technological advancement as a vehicle towards a borderless world through globalization, often we are forced to halt and take several steps back as the pandemics are jeopardizing public health and socio-economic conditions all around the world. We hope, utilizing the seamless global communication and scientific development as devices of globalization in real-time, proper treatment measures will soon be invented and implemented to control, if not eradicate the life-threatening diseases from the world.

Biomedical implications of metagenomics: Advances in the field of Microbiology

Disease-causing microorganisms pose a constant threat to public health and global economics due to the incessant evolution of known pathogens and the emergence of novel ones. Between 1940 and 2010 almost 160 new infectious diseases caused by bacteria have been identified. Patho-mechanism of most of the disease-causing microorganisms can be demonstrated. However, the evolutionary origin of virulence is not well-deciphered. The virulence factors attributing to disease symptoms may originate from complex interaction of the pathogen with host, abiotic, and biotic components of the environment as well as other related or unrelated microorganisms. The presence of virulence factors in soil and freshwater environments has been reported in several studies employing PCR and traditional microbial culture-based techniques. However, a much elaborate investigation encompassing multiple environmental microbiomes is essential for an in-depth understanding of –

(i) the importance of virulence gene cognate for microbial persistence in non-human environments and

(ii) the contribution of these environments in the evolution of microbial pathogens.

So, microbial metagenomics is now being implemented to target the environmental gene pool of pathogenic traits. Microbial metagenomics is the culture-independent sequence and function-based analysis of the heterogeneous microbial genomes of a microbial community from their natural habitats, through the application of contemporary high-throughput genomic techniques. Metagenomics has radically transformed in the approach of the researchers to explore the community structure, composition, and distribution of populations of microorganisms from a diverse form of environments, from the mundane to the extreme and human to non-human together with the identification of genes that encode a function of interest. Landmarks in metagenomic studies have extended from outcomes with significant biotechnological impact to surprising revelations of high biomedical relevance, unraveling hidden molecular components, and connections between microbial communities and complex diseases. Metagenomic research was successfully initiated with Sanger sequencing technology. Now the advent of next-generation sequencing (NGS) technologies with several updated sequencing platforms capable of simultaneous sequencing of millions of DNA fragments and therefore the generation of abundant data have revolutionized our understanding of microbiomes in our surroundings in the following ways:

a) Comparison and correlation of sequencing data obtained help in the identification of a novel protein family with implications in virulence such as its overexpression in disease conditions, its association with virulence phenotypes, its prevalence in a pathogenicity island, or its overrepresentation within genomes of pathogenic or host-associated species.

b) Several virulence factors display homologies to host proteins, allowing them to interfere with host functions. Identification of genes encoding such “mimicry” proteins in bacterial genomes is another promising approach for virulence factor detection.

c) Comparison of DNA signatures of isolated microorganisms with those that probe the biology of that environment in which they inhabit may lead to the identification of environment-exclusive gene clusters encoding for new protein families that impeccably correlated with pathogenicity regimes.

Application of all these approaches revealed the presence of dynamic interactions and genetic exchange occurring between bacterial members of the same or other species and also between bacteria and their viruses. By the process of horizontal gene transfer mediated by transmission of phage DNA or plasmid such novel genes get transferred in bacterial population rendering them pathogenic. Transmission of these unique genes influences the population dynamics of bacteria making them ecologically fit. At the same time, they encode exotoxins or lytic enzymes that impart virulence property in recipient microorganisms.

Several metagenome-wide association studies (MGWAS) revealed that multiple complex diseases like Crohn’s disease, rheumatoid arthritis, obesity, type 1 and type 2 diabetes, and atherosclerosis are associated with the gut microbiome. These observations have led to the identification of microbiome-based signatures that are of biomedical relevance for use in diagnostics, prognostics, and treatment of patients. One such MGWASled to the identification of more than 20 microbial gene markers including those from Fusobacteriumnucleatum and Peptostreptococcus stomatis specific for colorectal cancer. Thereafter a panel of four such gene markers was shortlisted and validated as a non-invasive signature biomarker for early detection of colorectal cancer in patient cohorts from China, France, Austria, and Denmark. A similar MGWAS with 2045 individuals from four countries Spain, Belgium, UK, and Germany led to the identification of a microbial signature of eight groups of microorganisms that could be used to discriminate between two inflammatory bowel diseases with similar prognosis- Crohn’s disease and ulcerative colitis. Likewise, unique gut microbiome signatures can be used for the diagnosis of advanced fibrosis in individuals with non-alcoholic fatty liver and also distinguish between type 2 diabetic and non-diabetic individuals.

Environmental disruption is driven by global climate change, urbanization and other human interventions may wreak havoc with the biodiversity. Loss of biodiversity facilitates the microbial ecosystem in the evolution and transmission of infectious diseases that affect plants, animals, and humans. The use of metagenomics to assess the microbial landscape before and after human activities provides an approach to examine the influence of such changes. For instance, the decline of biodiversity associated with the reduction in natural forests and their inhabitants were reported to be a major cause of the spread of Lyme disease in the United States. Also, the increasing proximity of humans to wild and domesticated animals along with human population growth has been related to the emergence of the Nipah virus in Malaysia, as well as the worldwide increase in food-borne illnesses.

Diseases of viral origin have largely contributed to many epidemics and pandemics taking a toll on human life globally. The evolution of viral virulence can be better understood within a phylogenomic outline if relevant data are available and strong links are formed between genomics, phylogenetic, epidemiology, and experimental studies of virus virulence and fitness. Therefore, a concurrent combination of clinical and epidemiological metadata with that of the sequencing of virus genomes from clinical samples is essential. We should also emphasize the significance of gathering concurrent and historical data from probable reservoir species as these may provide a more complete insight into virulence evolution and determining the full range of microorganisms that infect a particular species, as well as their interactions which can successfully be achieved by metagenomic studies. Similar studies are now being effectively carried out worldwide to elucidate the role of the viral evolution of SARS-CoV2 in the severity and progression of the current pandemic of COVID19.

For analysis of genome and metagenome along with the characterization of epidemiology and clinical attributes of diseases caused by microorganisms require in-depth knowledge of Microbiology. Proficiency in this subject will enable the characterization of virulence factors of novel pathogens which will facilitate understanding of patho-mechanism of the disease and subsequent disease management.

Targeting the Spike glycoprotein of SARS-CoV2: From the standpoint of Immunology

Student contributors: Subhrajyoti Misra & Dhrubajyoti Chatterjee [Department. Of Microbiology(UG IV)]

Introduction:

With the significant increase in the number of positive cases of COVID-19 everywhere, overriding one hospital after another and pushing the global death toll past 2.2 lakhs, the sprint to identify the patho-mechanism of SARS-CoV-2 has hastened radically. SARS-CoV-2 is the seventh member of the family Coronaviridae that can cause human disease.While infection with HKU1, NL63, OC43, and 229E can show trivial symptoms, that of SARS-CoV, MERS-CoV, and SARS-CoV2 cause severe disease.The identification of virulence factors of SARS-CoV-2 will enable us to put an end to the atrocity of COVID19 through targeted drug therapy or vaccines.

How does the virus invade the host body?

SARS-CoV-2 entry into host cells is a complex process mediated by the homo-trimeric protrusion of trans-membrane spike (S) glycoprotein on the viral surface. The S protein is classified into 3 subparts: (a) large ectodomain(160Å long, 2.8 Å in diameter, classified into S1 & S2 subdomains) (b) a single-pass transmembrane domain & (c) an intracellular tail. The concerted action of the receptor-binding domain in the distal S1 subunit together with proteolytic processing of the S2 subunit containing the fusion machinery to stimulate virus-cell fusion through ACE2 receptor on lung and of the respiratory tract cells allows the entry of coronavirus in susceptible cells.Once internalized, the viral RNA gets incorporated in the host cell’s protein synthesis machinery, produces thousands of new viruses which when released causing cell death or damage and contribute to disease spread. So the disease can be stopped by preventing the virus from entering the cell. Vaccines, being a pure preparation of one/more viral components can train the human body to identify and attack the virus before it can infect healthy human cells by presenting a preview of the virus without causing disease.Therefore, spike protein is the primary antigenic target of neutralizing antibodies and vaccines.

SARS-CoV2 is different from the rest:

Although SARS-CoV2 shares significant structural and genetic similarity with SARS-CoV and MERS-CoV, epitope analysis has shown significant variability in the antigenicity of the spike protein of SARS-CoV2 from the other two coronaviruses. The variability is sequestered in the non-conserved domain.Some unique features of the receptor-binding domain of SARS-CoV2 are as follows:

The Receptor Binding Domain(RBD) of spike protein is the most variable part encoded by the COVID-19 genome, having binding domains like L455, F486, Q493, S494, N501, Y505.Five of these six residues are unique to SARS-CoV2 and these unique epitopes, are derived from either the non-conserved regions or the combinations of the conserved and non-conserved regions of the genome.
The sequence variability of these epitopes attributes to the stronger binding of SARS-CoV2in the interaction interface with ACE2.

Another feature exclusive of SARS-CoV2that determines viral infectivity is a polybasic cleavage site at the junction of S1 and S2, targeted by furin and other proteases. The presence of a proline residue that creates a turn in the cleavage site to enable the addition of O-linked glycans to S673, T678, and S686 also contributes to the novelty of this domain. (1)

Potential candidature for therapy:

Interestingly, a recent study revealed the lack of antibody cross-reactivity by monoclonal antibodies specific for SARS-CoV RBD against SARS-CoV2 RBD. SARS-CoV2 spike protein is reported to have ~ 24.5% amino acid sequence non-conserved to that of SARS-CoV. Moreover, despite five epitopes being shared between SARS-CoV and SARS-CoV2, apparent dominances of unique epitopes in SRAS-CoV (83.9%) and SRAS-CoV-2 (85.3%) have been demonstrated (2). So by exploiting the novelty of the interactive domains in SARS-CoV-2 RBD, the vaccine can be designed with monoclonal antibodies specific for these surface antigens of SARS-CoV2.

However, the production of protein vaccines demands large-scale production of purified viral proteins. Viral growth and protein purification at clinically acceptable pharmaceutical scales is a time-consuming process that may not be very ideal at the current period of a public health emergency. As an alternative approach, synthetic mRNA vaccines carrying molecular instructions for protein synthesis can be an attractive candidate that can be used by the host body to produce the viral proteins on their own. The mRNA vaccines would also be safer than the attenuated viral or protein-based vaccines as they are devoid of the risk of reactivation of the injected virus, or protein contamination. Inspired by this strategy, an experimental COVID-19 mRNA vaccine called mRNA-1273 has been produced and is being used for clinical trials in humans.

COVID19 and Immunology

To understand the patho-physiology of viral pathogen and significance of immunization as a probable tool for disease management, the concept of microbiology and immunology is essential. An elaborate knowledge of the subject not only makes a student proficient in addressing disease prognosis but also helps them in tailoring disease management. Thus the students can progress in his/her career in the field of academics, research, drug designing, pharmaceutical industry, etc. At Adamas University, the School of Life Science and Biotechnology offers B.Sc and M.Sc courses in Microbiology where Immunology is a major component in both under-graduate and postgraduate levels. We hope the immunologists will come up with an anti-COVID19 vaccine soon as ammunition to fight back the crisis created by the COVID19 pandemic.

References:

Kristian G. Andersen, Andrew Rambaut, W. Ian Lipkin, Edward C. Holmes & Robert F. Garry. The proximal origin of SARS-CoV-2.Nature Medicine volume 26, pages450–452(2020).
Ming Zheng&Lun Song. Novel antibody epitopes dominate the antigenicity of spike glycoprotein in SARS-CoV-2 compared to SARS-CoV. Cellular & Molecular Immunology volume 17, pages536–538(2020).

Targeting the cytokine storm: An Immunologic perspective to address the role of Hydroxychloroquine in COVID-19 treatment

Student contributors: Megha Dutta, Shatarupa Biswas , Sayandip Jana

When we hear the word “trending” it reminds us of some form of new entertainment, fashion, incidence, or habit. But in the current scenario, the prevailing trend is to divert all aspects of our life towards the only seemingly persistent difficulty of our life and that is the pandemic of COVID-19 caused by the novel coronavirus, officially known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The disease has been jeopardizing human civilization since December 2019 to date.

The immune system in COVID-19 patients

In all afflicted parts of the world, the apparent mild cases of COVID-19 rapidly transmute into severe cases with pulmonary involvements. Patients infected with COVID-19 are reported to have elevated amounts of proinflammatory cytokines in serum (e.g., IL1B, IFNγ, IP10, IL-1Ra, IL-2Ra, IL-6, IL-10, IL-18, HGF, MCP-3, MIG, M-CSF, G-CSF, MIG-1a and MCP1), that probably leads to activated T-helper-1 (T_H1) cell responses. As a result of this cytokine profile similar to SARS and MERS-CoV, critically ill patients with COVID-19 suffer from pulmonary inflammation and substantial lung damage. Interstitial mononuclear inflammatory infiltrates in the lungs, dominated by lymphocytes together with increased concentration of IL-1Ra, IL-2Ra, MCP-3, and IL-6 in serum are also reported to be associated with the COVID-19 disease severity and prognosis. Over-activation of T cells accounts for the severe immune injury characteristic of the disease. Moreover, patients requiring respiratory support are observed to have increased concentrations of GCSF, IP10, MCP1, MIP1A, and TNFα than less severe patients, suggesting that the disease severity could be associated with a cytokine storm.

What is a cytokine storm?

The cytokines are a group of low molecular weight secreted proteins/glycoproteins that mediate the complex cellular interactions involving cells of immune, inflammatory, and hematopoietic systems. A cytokine storm is an overproduction of immune cells and their activating compounds cytokines—namely, GCSF, IFNϒ-induced protein 10, MCP1, MIP1α, and TNFα, which is often associated with an outpouring of activated immune cells into the lungs. The subsequent lung inflammation and fluid accumulation can progress to respiratory distress and can be worsened by ancillary bacterial pneumonia. Also, the surge in immune molecules can contribute to fatal multi-organ failure. As a result, the mortality rate is increased.

Treatment – Is there any?

Despite an untiring attempt of the scientists and clinicians, till now this situation no proper treatment measures are available to confront this state of “public health emergency”. Several antiviral drugs like remdesivir, favipiravir, ivermectin, and antiparasitic drug like the chloroquine (CQ) and its derivative hydroxychloroquine(HCQ) with/without Azithromycin have been tested. Among them, CQ and HCQ in combination with azithromycin have shown some promising effects.

Mode of action of CQ and HCQ

Chloroquine is well-known for its use in the treatment of malaria and amoebiasis. Hydroxychloroquine (HCQ) sulfate, a derivative of CQ with an additional hydroxyl group, was demonstrated to have less toxicity than the parent compound. HCQ is still widely used for the management of autoimmune diseases like systemic lupus erythematosus and rheumatoid arthritis. Being a weak base and immune system modulator, CQ, and HCQ are potential candidates of therapeutic activity against COVID19with following mode of actions:

CQ increases endosomal pH and interferes with the glycosylation of the cellular receptor of SARS-CoV. CQ also inhibits the enzyme quinone reductase that is involved in sialic acid biosynthesis. These attributes confer this drug with a wide-spectrum antiviral activity.
CQ alters the pH of lysosome and inhibits cathepsin that results in the formation of autophagosome where SARS-CoV 2 spike protein is cleaved off.
CQ inhibits MAP-kinase and alters virion assembly, budding, and interfering with proteolytic action of the M protein(membrane protein).
CQ can also bind the angiotensin-converting enzyme 2(ACE2) receptor glycosylation thus prevents SARS-CoV-2 attachment to the target cell.

Proposed Immunological Aspects of HCQ treatment of COVID-19:

There is an uncontrolled cytokine release as COVID-19 symptoms transform into acute respiratory distress syndrome (ARDS). HCQ inhibits the cytokine storm of proinflammatory factors by suppressing T cell activation. Moreover, since acidification is essential for maturation and function of the endosome, and hence membrane fusion, it is assumed that HCQ therapy might suppress endosome maturation at intermediate stages of endocytosis, failing further transport of virions to the eventual releasing site. Therefore, chemoprophylaxis with CQ or HCQ can prevent COVID-19 associated pneumonia and thus block the transmission of disease by decreasing the number of the asymptomatic carrier by inhibiting viral transmission.

Cautions and contraindication with CQand HCQ:

Use of both the drugs need some precaution to be undertaken that include frequent monitoring of hematological parameter(RBC, WBC, platelet count), serum electrolyte, blood glucose, hepatic as well as renal function.
Before starting the therapy with this drug routine electrocardiography is required.
All doctors must know contraindication before they prescribed these drugs like hypersensitivity, epilepsy, retinopathy, Glucose-6-phosphate dehydrogenase (G6PD) deficiency, recent myocardial infarction.
HCQ is less toxic than CQ, but prolonged usage or overdose can cause poisoning.

Conclusions:

Evidence of effectivity of CQ and HCQ in COVID-19 treatment is limited based on in vitro experiments and only two small human trials. A recent report also claims no effect of HCQ in reducing the risk of mechanical ventilation in patients with COVID-19, although they could not infer about asymptomatic and mildly symptomatic patients. Although according to the theoretical concept of immunology, HCQ is a promising drug to delimit the disease spread from both asymptomatic and symptomatic patients; more experimental evidence in a larger cohort of the patient population is needed to confirm the effectivity of the drug for COVID-19 treatment in practicality.

An overview of dietary habit and food components to strengthen our immune system against COVID-19

COVID 19 scenario at present and us:

After spending twenty-one days of lockdown we are currently at the brink of extending a restricted lifestyle for another fifteen days. Every day we are witnessing the assault of global humanity in the form of death or severe ailment of an ever-increasing number of people. With no proper immunization currently available to overthrow the virus, our only option is social distancing to prevent the spread of the disease. As a consequence, our daily life has lost its normal rhythm, leaving us unstable and uncertain in every aspect of life: be it personal or professional. Being confined in isolation, either with family or without, we are witnessing a critical phase of social, economic and medical insecurity that has left us worried about the well-being of our family members and associates. The biology of COVID-19 is gradually unraveling and now we know that a weaker immune system predisposes an individual to this respiratory disorder. That is why people with older age and associated comorbidities such as diabetes, hypertension and cardiovascular disease are at higher risk.

But we must fight back.

However, we need to keep the stress at bay and win over this period of adversity with a positive attitude. It will not only help us to survive and cheer up with our near and dear ones, but also to optimize our performance, engagement, and focus in life. In the absence of well-defined medicaments, we should adopt some natural heal to improve our immunity so that we can fight against the crisis on our own. In isolation, an important way to do so is to avail a healthy nutritious diet that is easily available, nutritious as well as immunologically beneficial.

Secluded lifestyle and the temporary closing of businesses may affect normal food-related practices and availability. In midst of the limitation in access to purchase and variability of food products in these hot summer days, let us identify some easily available foods and food practices that will help in making our immunity strong and bring variation in our diet regime.

First and foremost: Food habits

Avoid over-purchasing: WHO observed that panic buying may have adverse consequences like an increase in food prices, overconsumption of food and unequal distribution of products. It is better to plan our meal, be considerate about others and use short shelf-life food to bring variation in food habits and also to avoid food waste.

Home-cooked family meal: As we are spending considerable periods at home we can try a variety of healthy and delicious recipes that can be found online with accessible ingredients. It will help to manage time also. Family meals are a golden opportunity to spend quality time with each other, have fun and strengthen the bonding.

Safe food-handling: In the well-sanitized kitchen with clean utensils, we should cook food thoroughly using safe water and ingredients that are properly washed. Frequent washing of hands with soap following WHO guidelines must be followed. Raw food should always be separated from cooked food. Food wastes should be disposed of in a sanitary way.

Smoking and alcohol consumption to be avoided: Smoking and alcohol are not a necessary part of our diet and not part of a healthy lifestyle. Rather this is a perfect time to give up the habits of smoking and alcohol consumption. As smoking enhances inflammation, impairing ciliary movement in lungs the propensity of the immune-deficient condition and autoimmune disorders increase. Alcohol consumption disrupts the gut barrier by damaging epithelial cells, and immune cells like T cells, macrophages and neutrophils and facilitating leakage of microbes into the circulation. Thus it enhances the chance of bacterial and viral infections and also an autoimmune response by inhibiting cytokine production. Therefore it is better not to make our immune system more susceptible at the time when it needs to gear up.

Avoid overeating and do the physical exercise: Being at home for extended periods, especially with limited activities can also lead to overeating and hence obesity. Physical exercise for at least 30 minutes will keep us healthy and refresh our minds.

Frequent hydration: At least 8-9 glasses of safe water are advised for consumption so that our mucous membranes remain hydrated lowering the chance of catching a cold and our innate immune system is strengthened. But frequent consumption of strong coffee, strong tea should be avoided as it may disturb our sleeping habits.

Say no to excess fat, sugar, and salt: WHO recommends a maximum salt intake of 5 g/day and also less than 5% of total energy intake for adults coming from free sugars. While a high-salt diet is one of the major risk factors in the development of hypertension, kidney injury, and cardiovascular diseases, excess of sugar can be linked to obesity and several metabolic diseases, including diabetes mellitus type II, NAFLD, and cardiovascular diseases. WHO also endorses limiting total fat intake to less than 30% of total energy intake, of which no more than 10% should come from saturated fat which otherwise will disrupt the intestinal barrier by attracting pathogenic bacteria.

What are the food choices?
Good health is largely affected by nutrients. Fresh products, especially fruits, vegetables, and reduced-fat dairy products should be prioritized over other types of fruits. A rainbow of nutrients, i.e., a colorful source of food is considered to be an ideal example of a balanced diet that we may narrow down to three colors similar to our National flag – saffron/orange, white and green.

Orange food: In the orange group we consider all red, yellow, orange-colored fruits and vegetables. In this season we can easily get papaya, oranges, pomegranates, red onions, sweet potatoes, tomatoes, carrots, mangoes, pumpkin, and watermelon that are rich in Beta-carotene, zeaxanthin, flavonoids, lycopene, potassium, glutathione and vitamin C, A, B6. These nutrients contribute to immunity build-up by lower blood pressure, reduce LDL cholesterol levels, scavenge harmful free-radicals, encourage alkaline balance and strengthen gut microbiota. Citrus fruits help in WBC production. A healthy gut microflora ensures proper functioning of gut-associated lymphoid tissues that contain a major fraction of our immunocytes.

White food: A wide variety of food ranging from fruits and vegetables like bananas, garlic, ginger, potato, etc. and whole-grain products like oats, barley to cereals like rice, wheat and dairy products like milk, home-made curd are included in this category. Poultry products also come under this category. White food of plant origin contain β-glucans, EGCG, SDG, and lignans that provide powerful immune-boosting activity by activating natural killer B and T cells. Garlic’s immune-boosting properties seem to come from a heavy concentration of sulfur-containing compounds, such as allicin. Vitamin D in home-made curd can enhance natural immunity and the probiotic microorganisms present help to maintain a healthy gut microflora. Proteins, essential amino acids and Vitamin B6 make chicken and egg an essential component in our dining platter to give us protection against flu and other diseases as well as to increase the erythrocyte count. Stock or broth made by boiling chicken bones contains gelatin, chondroitin, and other nutrients helpful for gut healing and immunity.

Green food: All green and leafy vegetables and fruits contain nutrients like Chlorophyll, fiber, lutein, zeaxanthin, calcium, folate, vitamin C, calcium, and β-carotene that lower blood pressure and LDL cholesterol levels, normalize digestion time, fight harmful free-radicals, and boost immune system activity.

With all these resources we can prepare different platters that will bring some variations in our present monotonous life and at the same time help build up a strong defense mechanism of our own. Good food practice also encourages positive thinking. So eat well, be optimistic and stay safe. If we stay healthy we can apprehend to overcome this temporary phase of gloominess and start afresh our schedule in the light of hopes and possibilities very soon.