Association of the C allele of rs479200 in the EGLN1 gene with COVID-19 severity in Indian population: a novel finding.

The present study investigated two single nucleotide polymorphisms (SNPs)-rs479200 and rs516651 in the host EGLN1/PHD2 gene for their association with COVID-19 severity. A retrospective cohort of 158 COVID-19 patients from the Indian population (March 2020 to June 2021) was enrolled. Notably, the frequency of C allele (0.664) was twofold higher than T allele (0.336) in severe COVID-19 patients. Here, we report a novel finding that the C allele of rs479200 in the EGLN1 gene imparts a high risk of severe COVID-19 (odds ratio-6.214 (1.84-20.99) p = 0.003; 9.421 (2.019-43.957) p = 0.004), in additive inheritance model (adjusted and unadjusted, respectively).

Single-Nucleotide Polymorphisms in Glucose-6-Phosphate Dehydrogenase and their Relevance for the Deployment of Primaquine as a Radical Cure for Malaria.

Malaria remains an important public health problem despite efforts to control it. Besides active transmission, relapsing malaria caused by dormant liver stages of Plasmodium vivax and Plasmodium ovale hypnozoites is a major hurdle in malaria control and elimination programs. Primaquine (PQ) is the most widely used drug for radical cure of malaria. Due to its anti-hypnozoite and gametocidal activity, PQ plays a key role in malaria relapse and transmission. The human enzyme glucose-6-phosphate dehydrogenase (G6PD) is crucial in determining the safety of PQ because G6PD-deficient individuals are prone to hemolysis if treated with PQ. Therefore, there is a need to study the prevalence of G6PD-deficient genetic variants in endemic populations to assess the risk of PQ treatment and the necessity to develop alternative treatments. In this work, we discuss the common G6PD variants, their varying enzymatic activity, and their distribution on the three-dimensional structure of G6PD. Our work highlights the important G6PD variants and the need for large-scale G6PD gene polymorphism studies to predict populations at risk of PQ-induced toxicity.

Age-specific malaria vulnerability and transmission reservoir among children.

Purpose: The pediatric population, especially under-five children, is highly susceptible to malaria and accounts for 76 % of global malaria deaths according to the World Malaria Report 2022. The purpose of this manuscript is to discuss the various factors involved in the susceptibility of the pediatric population to Malaria and the importance of this age group for malaria elimination. Methodology: Data on pediatric malaria epidemiology that includes prevalence, risk factors, immune factors, socioeconomic factors, control methods, etc. were extracted from published literature using PubMed and Google Scholar. This data was further correlated with malaria incidence data from the World Health Organization (WHO) and the National Center for Vector Borne Diseases Control (NCVBDC). Results: The younger age group is vulnerable to severe malaria due to an immature immune system. The risk of infection and clinical disease increases after the waning of maternal immunity. In the initial years of life, the developing brain is more susceptible to malaria infection and its after-effects. The pediatric population may act as a malaria transmission reservoir due to parasite density and asymptomatic infections. WHO recommended RTS,S/AS01 has limitations and may not be applicable in all settings to propel malaria elimination. Conclusion: The diagnosis of malaria is based on clinical suspicion and confirmed with microscopy and/or rapid diagnostic testing. The school-age pediatric population serves as a transmission reservoir in the form of asymptomatic malaria since they have acquired some immunity due to exposure in early childhood. Targeting the hidden reservoir in the pediatric population and protecting this vulnerable group will be essential for malaria elimination from the countries targeting elimination.

Profiles of host immune impairment in Plasmodium and SARS-CoV-2 infections.

Over the past two decades, many countries have reported a steady decline in reported cases of malaria, and a few countries, like China, have been declared malaria-free by the World Health Organization. In 2020 the number of deaths from malaria has declined since 2000. The COVID-19 pandemic has adversely affected overall public health efforts and thus it is feasible that there might be a resurgence of malaria. COVID-19 and malaria share some similarities in the immune responses of the patient and these two diseases also share overlapping early symptoms such as fever, headache, nausea, and muscle pain/fatigue. In the absence of early diagnostics, there can be a misdiagnosis of the infection(s) that can pose additional challenges due to delayed treatment. In both SARS-CoV-2 and Plasmodium infections, there is a rapid release of cytokines/chemokines that play a key role in disease pathophysiology. In this review, we have discussed the cytokine/chemokine storm observed during COVID-19 and malaria. We observed that: (1) the severity in malaria and COVID-19 is likely a consequence primarily of an uncontrolled 'cytokine storm'; (2) five pro-inflammatory cytokines (IL-6, IL-10, TNF-α, type I IFN, and IFN-γ) are significantly increased in severe/critically ill patients in both diseases; (3) Plasmodium and SARS-CoV-2 share some similar clinical manifestations and thus may result in fatal consequences if misdiagnosed during onset.

Effects of Host Genetic Polymorphisms on the Efficacy of the Radical Cure Malaria Drug Primaquine.

Malaria is a major cause of death in low-income countries. Malaria relapses are caused by Plasmodium vivax-induced latent liver stage hypnozoites, and relapses contribute significantly to the total disease burden. The goal of malaria elimination is threatened in countries where P. vivax is endemic and relapses remain a key aspect of concern. Targeting of the hypnozoites is crucial for radical cure and this is achieved by primaquine (PQ). In addition to its anti-hypnozoite effects, PQ also possesses gametocidal activity against all malaria causing Plasmodium species and is hence a useful tool to curtail malaria transmission. It is well known that host glucose-6-phosphate dehydrogenase (G6PD) deficiency is associated with hemolysis after treatment with PQ. Multiple other host polymorphisms impact on PQ metabolism, potentially affecting drug efficacy. Being a prodrug, PQ requires host factors cytochrome P450 2D6 (CYP2D6), cytochrome P450 NADPH: oxidoreductase (CPR) and monoamine oxidase (MAO) for its metabolism and conversion to active form. The efficacy of PQ in the host is therefore dependent on genetic polymorphisms of these three host genes. The efficacy of PQ is important for clearing reservoirs of P. vivax infection. Here, we have analyzed the known spectrum of genetic polymorphisms for host genes that enable PQ metabolism. It is vital to delineate the polymorphisms that determine the ultimate efficacy of PQ for formulating better malaria elimination strategies in countries with severe malaria burden. Thus population-based studies of these gene variants will provide new insights into the role of host genetics on PQ treatment outcomes.

Inhalable particles containing isoniazid and rifabutin as adjunct therapy for safe, efficacious and relapse-free cure of experimental animal tuberculosis in one month.

We investigated the preclinical efficacy and safety/tolerability of biodegradable polymeric particles containing isoniazid (INH) and rifabutin (RFB) dry powder for inhalation (DPI) as an adjunct to oral first-line therapy. Mice and guinea pigs infected with Mycobacterium tuberculosis H37Rv (Mtb) were treated with ∼80 and ∼300 µg of the DPI, respectively, for 3-4 weeks starting 3, 10, and 30 days post-infection. Adjunct combination therapy eliminated culturable Mtb from the lungs and spleens of all but one of 52 animals that received the DPI. Relapse-free cure was not achieved in one mouse that received DPI + oral, human-equivalent doses (HED) of four drugs used in the Directly Observed Treatment, Short Course (DOTS), starting 30 days post-infection. Oral doses (20 mg/Kg/day, each) of INH + RFB reduced Mtb burden from ∼106 to ∼103 colony-forming units. Combining half the oral dose with DPI prevented relapse of infection four weeks after stopping the treatment. The DPI was safe in rodents, guinea pigs, and monkeys at 1, 10, and 100 µg/day doses over 90 days. In conclusion, we show the efficacy and safety/tolerability of the DPI as an adjunct to oral chemotherapy in three different animal models of TB.

Host-directed Therapy: A New Arsenal to Come.

The emergence of drug-resistant strains among the variety of pathogens worsens the situation in today's scenario. In such a situation, a very heavy demand for developing the new antibiotics has arisen, but unfortunately, very limited success has been achieved in this arena till now. Infectious diseases usually make their impression in the form of severe pathology. Intracellular pathogens use the host's cell machinery for their survival. They alter the gene expression of several host's pathways and endorse to shut down the cell's innate defense pathway like apoptosis and autophagy. Intracellular pathogens are co-evolved with hosts and have a striking ability to manipulate the host's factors. They also mimic the host molecules and secrete them to prevent the host's proper immune response against them for their survival. Intracellular pathogens in chronic diseases create excessive inflammation. This excessive inflammation manifests in pathology. Host directed therapy could be alternative medicine in this situation; it targets the host factors, and abrogates the replication and persistence of pathogens inside the cell. It also provokes the anti-microbial immune response against the pathogen and reduces the exacerbation by enhancing the healing process to the site of pathology. HDT targets the host's factor involved in a certain pathway that ultimately targets the pathogen life cycle and helps in eradication of the pathogen. In such a scenario, HDT could also play a significant role in the treatment of drugsensitive as well with drug resistance strains because it targets the host's factors, which favors the pathogen survival inside the cell.

Exploring links between vitamin D deficiency and COVID-19.

Coronavirus Disease 2019 (COVID-19) pandemic remains a major public health threat in most countries. The causative severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus can lead to acute respiratory distress syndrome and result in mortality in COVID-19 patients. Vitamin D is an immunomodulator hormone with established effectiveness against various upper respiratory infections. Vitamin D can stall hyper-inflammatory responses and expedite healing process of the affected areas, primarily in the lung tissue. Thus, there are ecological and mechanistic reasons to promote exploration of vitamin D action in COVID-19 patients. As no curative drugs are available currently for COVID-19, we feel that the potential of vitamin D to alter the course of disease severity needs to be investigated. Clinical studies may be undertaken to address the value of vitamin D supplementation in deficient, high-risk COVID-19 patients.

Challenges with Mosquito-borne Viral Diseases: Outbreak of the Monsters.

BACKGROUND: The viruses responsible for mosquito-borne diseases are on an exploring mode, expanding their horizon, adapting to the situation and comfortably making their presence felt globally, from South Africa to Asia, Europe and United States. The current global scenario and recent documentations indicate towards the real monsters, outbreak of Zika, dengue and chikungunya viruses. Zika, dengue and chikungunya viruses are positive sense single-stranded RNA arbovirus and so their initial symptoms are almost 80% similar and all three are spread by mosquitos which bite during the day. Zika virus may damage brain by targeting the neuron cells in babies, and thereby it is very perilous to pregnant women. Dangerous Type: A less common but highly dangerous type of dengue is one which causes haemorrhagic fever and shock syndrome which are lethal. Chikungunya is not as lethal as Zika and dengue are, but it triggers joints pain which could last for months and even for years. CONCLUSION: The vaccines against Zika, dengue and chikungunya viruses are at different stages of development. The challenges associated with the epidemic wave of Zika, dengue and chikungunya viruses have been explained and the current status of drug/ vaccine development against these viruses has been reviewed.

Particulate pulmonary delivery systems containing anti-tuberculosis agents.

There is renewed interest in delivering anti-tuberculosis (TB) drugs to the lungs by inhalation. Several groups have investigated particulate pulmonary drug delivery formulations containing anti-TB agents, prepared using a variety of design approaches and processes. This review summarizes trends that indicate feasibility and translation of research efforts aimed at developing inhaled therapies for TB. Whereas formulations intended for reconstitution as solutions prior to nebulization can be produced with relative ease, particle engineering for powder formulations is more specialized. Spray drying and emulsion methods used to prepare particulate pulmonary delivery systems of anti-TB agents are compared. Pharmaceutical characterization is outlined. Administration of repeated inhalations to laboratory animals, especially under Animal Biosafety Level-3 (ABSL-3) containment as required for TB research, is another major challenge. Techniques employed by different groups are reviewed in the context of suitability for drug delivery and amenability towards use in ABSL-3 settings. It is concluded that spray drying is suitable for production of inhalable particles, rigorous physicochemical characterization is necessary for developing inhaled therapies as drug products, and pulmonary delivery of formulations containing anti-TB drugs to animals infected with Mycobacterium tuberculosis can best be carried out using handheld devices.

Inhalable microparticles of nitric oxide donors induce phagosome maturation and kill Mycobacterium tuberculosis.

Nitric oxide (NO) kills Mycobacterium tuberculosis (Mtb) in vitro, but gaseous NO is difficult to administer to patients. We evaluated the consequences of intracellular delivery of NO using inhalable microparticles (MP) containing NO donors. MP containing 10% w/w of NO donors alone, or in addition to 25% each of isoniazid (INH) and rifabutin (RFB) in a polylactide-co-glycolide (PLGA) matrix were prepared by spray drying. THP-1-derived macrophages infected with Mtb H37Rv were exposed to MP or soluble NO donors. Phagosome-lysosome fusion (PLF) and bacterial killing were monitored. Colony forming units (cfu) in lungs and spleen of mice infected with a low-dose aerosol and administered inhalations of MP were enumerated. Bacterial DNA in these tissues was estimated by real-time PCR. In vitro studies indicated a bacteriostatic effect of NO donors despite significant enhancement of PLF. Daily inhalation of MP containing 10% diethylenetriamine nitric oxide adduct (DETA/NO) alone reduced log10 cfu in the lungs from 6.1 to 4.4 at the highest dose in four weeks, but did not significantly affect cfu in the spleen. Inhalations of MP containing DETA/NO in combination with INH and RFB significantly (P < 10(-5), ANOVA) reduced cfu in lungs and spleens by 4 log. Gross morphology and histology of the lungs and spleen indicated that inhaled particles were well-tolerated. Inhalable MP containing NO donors need further investigation as an adjunct to standard anti-tuberculosis chemotherapy.

Inhaled microparticles containing clofazimine are efficacious in treatment of experimental tuberculosis in mice.

Inhalable clofazimine-containing dry powder microparticles (CFM-DPI) and native clofazimine (CFM) were evaluated for activity against Mycobacterium tuberculosis in human monocyte-derived macrophage cultures and in mice infected with a low-dose aerosol. Both formulations resulted in 99% killing at 2.5 µg/ml in vitro. In mice, 480 µg and 720 µg CFM-DPI inhaled twice per week over 4 weeks reduced numbers of CFU in the lung by as much as log(10) 2.6; 500 µg oral CFM achieved a log(10) 0.7 reduction.

Inhalable microparticles containing nitric oxide donors: saying NO to intracellular Mycobacterium tuberculosis.

Although nitric oxide (NO) is a bactericidal component of the macrophage's innate response to intracellular infections such as tuberculosis (TB), prolonged inhalation of NO gas has little benefit in chemotherapy of TB. The impact of controlled release of NO through intracellular delivery of NO donors to macrophages infected in vitro with Mycobacterium tuberculosis (Mtb) was investigated. Inhalable microparticles (MP) were prepared by spray-drying. Isosorbide mononitrate (ISMN), sodium nitroprusside (SNP), and diethylenetriamine nitric oxide adduct (DETA/NO) were incorporated in poly(lactic-co-glycolic acid) (PLGA) with encapsulation efficiencies of >90% to obtain MP yields of ∼70%. The mass median aerodynamic diameter (MMAD) of the MP was 2.2-2.4 µm within geometric standard deviations (GSD) of ≤0.1 µm. MP were phagocytosed by THP-1 derived macrophages in culture and significantly (P < 0.05) sustained NO secretion into culture supernatant from 6 to 72 h in comparison to equivalent amounts of drugs in solution. Significantly (P < 0.05) higher dose-dependent killing of intracellular Mtb by MP compared to equivalent amounts of drugs in solution was observed on estimation of colony forming units (CFU) surviving 24 h after exposure to drugs or MP. The cytotoxicity of MP toward macrophages was lower than that of dissolved drugs. It was concluded that inhalable MP can target NO donors to the macrophage, control NO release in the macrophage cytosol, and reduce Mtb CFU by up to 3-log in 24 h, at doses that are much lower than those required for cardiovascular effects.

The devil's advocacy: when and why inhaled therapies for tuberculosis may not work.

Factors that are inimical to the success of inhaled therapies for tuberculosis (TB) include: (i) lack of access of inhaled therapies to poorly-aerated areas of the tubercular lung; (ii) limited ability to penetrate biofilms formed by extracellular bacilli; (iii) selection for resistant bacilli on account of administration of low doses of anti-TB agents; (iv) induction of inflammation and/or immunopathology in the airways and lungs; and (v) anomalies in antigen processing and presentation of vaccines delivered to the lungs. Further, the claim that inhaled therapies rescue alternatively-activated macrophages may not be applicable to all individuals. Fortunately, there are ways and means to address each of the above factors.

Inhaled therapies for tuberculosis and the relevance of activation of lung macrophages by particulate drug-delivery systems.

Pathogenic strains of Mycobacterium tuberculosis (Mtb) induce 'alternative activation' of lung macrophages that they colonize, in order to create conditions that promote the establishment and progression of infection. There is some evidence to indicate that such macrophages may be rescued from alternative activation by inhalable microparticles containing a variety of drugs. This review summarizes the experience of various groups of researchers, relating to observations of induction of a number of classical macrophage activation pathways. Restoration of a 'respiratory burst' and upregulation of reactive oxygen species and nitrogen intermediates through the phagocyte oxidase and nitric oxide synthetase enzyme systems; induction of proinflammatory macrophage cytokines; and finally induction of apoptosis rather than necrosis of the infected macrophage are discussed. It is suggested that there is scope to co-opt host responses in the management of tuberculosis, through the route of pulmonary drug delivery.

Microparticles induce variable levels of activation in macrophages infected with Mycobacterium tuberculosis.

Activation of human macrophages infected with Mycobacterium tuberculosis was investigated following exposure to microparticles (MP) possessing high anti-tubercular efficacy in mice. A small set of innate responses (cytokine profiles, NO production, Annexin-V staining and caspase-8, caspase-9 and caspase-3 activities) of differentiated THP-1 cells or human monocyte-derived macrophages infected 1:10 in vitro were compared. Cytokines of THP-1 macrophages were comparable in trends, but not in magnitude, with five human genotypes studied. MP reversed suppression of tumor necrosis factor induced by infection, and transiently upregulated gamma-interferon. Drug-free MP surprisingly induced gamma-interferon, but not tumor necrosis factor. Primary cells responded to MP, regardless of drug content, by upregulation of NO; but THP-1 cells did not respond to drug-free MP. About 19% of infected cells exposed to MP underwent apoptosis compared to approximately 11% cells treated otherwise. Cell death induced by drug-free MP was caspase independent. Intracellular bacterial survival varied between individuals. Untreated infection resulted in survival of 900+/-141 cfu; exposure to soluble drugs, drug-containing and blank microparticles respectively, reduced CFU counts to <10, <10 and 102+/-139. These observations indicate that despite variations in magnitude between cells from different sources, innate responses conducive to killing intracellular bacteria were evoked by inhalable MP.

Inhalable microparticles containing isoniazid and rifabutin target macrophages and 'stimulate the phagocyte' to achieve high efficacy.

Macrophage responses to infection with Mycobacterium tuberculosis (MTB) and treatment with soluble isoniazid (INH) plus rifabutin (RFB) versus microparticles containing equivalent amounts of drugs were compared. It was investigated whether macrophages driven to alternative activation upon infection with MTB could be rescued to display the classical activation phenotype. It was established that microparticles sustain high levels of drugs in cytosol of macrophages for longer period as compared to soluble drugs. Microparticles co-localized with intracellular bacteria, and induced a variety of innate bactericidal responses, including induction of free radicals, alteration of mitochondrial membrane potential and apoptosis. The data strongly suggest that additional benefit may be derived from the nature of the drug delivery system, which fulfils Koch's dictum 'stimulate the phagocyte' for curing tuberculosis.