Repurposing FDA-approved drugs to target malaria through inhibition of dihydrofolate reductase in the folate biosynthesis pathway: A prospective approach.

Dihydrofolate reductase (DHFR) is a ubiquitous enzyme that regulates the biosynthesis of tetrahydrofolate among various species of Plasmodium parasite. It is a validated target of the antifolate drug pyrimethamine (Pyr) in Plasmodium falciparum (Pf), but its clinical efficacy has been hampered due to the emergence of drug resistance. This has made the attempt to screen Food & Drug Administration-approved drugs against wild- and mutant PfDHFR by employing an in-silico pipeline to identify potent candidates. The current study has followed a virtual screening approach for identifying potential DHFR inhibitors from DrugBank database, based on a structure similarity search of candidates, followed by absorption, distribution, metabolism, and excretion estimation. The screened drugs were subjected to various parameters like docking, molecular mechanics with generalized born and surface area solvation calculations, and molecular simulations. We have thus identified two potential drug candidates, duloxetine and guanethidine, which can be repurposed to be tested for their efficacy against wild type and drug resistant falciparum malaria.

Correction: India can consider integration of three eliminable disease control programmes on malaria, lymphatic filariasis, and visceral leishmaniasis.

[This corrects the article DOI: 10.1371/journal.ppat.1009492.].

The threat of increased transmission of non-knowlesi zoonotic malaria in humans: a systematic review.

Of the 5 human malarial parasites, Plasmodium falciparum and Plasmodium vivax are the most prevalent species globally, while Plasmodium malariae, Plasmodium ovale curtisi and Plasmodium ovale wallikeri are less prevalent and typically occur as mixed-infections. Plasmodium knowlesi, previously considered a non-human primate (NHP) infecting species, is now a cause of human malaria in Malaysia. The other NHP Plasmodium species, Plasmodium cynomolgi, Plasmodium brasilianum, Plasmodium inui, Plasmodium simium, Plasmodium coatneyi and Plasmodium fieldi cause malaria in primates, which are mainly reported in southeast Asia and South America. The non-knowlesi NHP Plasmodium species also emerged and were found to cross-transmit from their natural hosts (NHP) ­ to human hosts in natural settings. Here we have reviewed and collated data from the literature on the NHPs-to-human-transmitting non-knowlesi Plasmodium species. It was observed that the natural transmission of these NHP parasites to humans had been reported from 2010 onwards. This study shows that: (1) the majority of the non-knowlesi NHP Plasmodium mixed species infecting human cases were from Yala province of Thailand; (2) mono/mixed P. cynomolgi infections with other human-infecting Plasmodium species were prevalent in Malaysia and Thailand and (3) P. brasilianum and P. simium were found in Central and South America.

Structure of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase-dihydropteroate synthase from Plasmodium vivax sheds light on drug resistance.

The genomes of the malaria-causing Plasmodium parasites encode a protein fused of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) domains that catalyze sequential reactions in the folate biosynthetic pathway. Whereas higher organisms derive folate from their diet and lack the enzymes for its synthesis, most eubacteria and a number of lower eukaryotes including malaria parasites synthesize tetrahydrofolate via DHPS. Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) HPPK-DHPSs are currently targets of drugs like sulfadoxine (SDX). The SDX effectiveness as an antimalarial drug is increasingly diminished by the rise and spread of drug-resistant mutations. Here, we present the crystal structure of PvHPPK-DHPS in complex with four substrates/analogs, revealing the bifunctional PvHPPK-DHPS architecture in an unprecedented state of enzymatic activation. SDX's effect on HPPK-DHPS is due to 4-amino benzoic acid (pABA) mimicry, and the PvHPPK-DHPS structure sheds light on the SDX-binding cavity, as well as on mutations that effect SDX potency. We mapped five dominant drug resistance mutations in PvHPPK-DHPS: S382A, A383G, K512E/D, A553G, and V585A, most of which occur individually or in clusters proximal to the pABA-binding site. We found that these resistance mutations subtly alter the intricate enzyme/pABA/SDX interactions such that DHPS affinity for pABA is diminished only moderately, but its affinity for SDX is changed substantially. In conclusion, the PvHPPK-DHPS structure rationalizes and unravels the structural bases for SDX resistance mutations and highlights architectural features in HPPK-DHPSs from malaria parasites that can form the basis for developing next-generation anti-folate agents to combat malaria parasites.

Epidemiology of rare bacterial, parasitic, and fungal pathogens in India.

Rare human pathogens are infrequently observed clinically but can lead to undiagnosed infections, delays in treatment, severe complications, including death. Traditional diagnostic tools cannot routinely detect rare infections in public health settings. This study focuses on the incidence and outcomes of rare pathogenic microorganisms over 13 years (2010-2022) using PubMed database to obtain epidemiological data on rare bacterial, parasitic, and fungal infections in hospitals throughout India. A total of 974 articles were screened using case studies, datasets, comments, classical articles, letters, editorials, observational studies, and meta-analyses. Our analysis identified 28 rare bacteria, six parasites, and five fungal species infections in India. Fatal cases were associated with rare bacterial and fungal infections, including two from pan-drug-resistant bacteria (both from the Myroides genus). A total of 10 bacterial species displayed multi-drug resistance; one was extensively drug-resistant, and eight remained unclassified. Of the 83 patients with these rare infections, the mortality was ∼8.4% (seven of 83). Considering drug resistance and high mortality, prompt diagnosis of rare pathogens is crucial to controlling their spread. An increased awareness within the Indian health care system focusing on diagnostics, record keeping, and data sharing will be necessary to enhance surveillance.

A computational strategy for systematic virtual screening of plasmodium falciparum heme detoxification protein inhibitors from the Drugbank database.

Antimalarial drug resistance poses one of the greatest threats to malaria treatment, resulting in increased morbidity and mortality. Heme Detoxification Protein (HDP) is among the essential hemoglobinases of P. falciparum (Pf), a vital molecular target for the treatment of malaria. In this study, we utilized the virtual screening workflow tool of the Schrodinger suite to find the best hits for the PfHDP from the DrugBank library. A total of 14,942 compounds were identified against the PfHDP. The top compounds with the highest docking scores and least energy scores were subjected to molecular simulations for 500 nanosecond to check the stability of the protein-drug complexes. The top three DrugBank compounds were found to be stable over 500 ns, namely DB09298 (silibinin), DB07426 (1-Hydroxy-2-(1,1':3',1''-Terphenyl-3-Yloxy) Ethane-1,1-Diyl] Bis (Phosphonic Acid), and DB07410 [(2-(3-Dibenzofuran-4-yl-Phenyl)-1-Hydroxy-1-Phosphono-Ethyl]-Phosphonic Acid). Overall analysis suggests that the top three compounds, DB09298, DB07426, and DB07410, have good stability for 500 ns. Their scaffolds can be used to design and develop new analogs of the target HDP protein. Silibinin, the anti-cancer drug, was found to be highly stable for the entire simulation period as compared to the other compounds. DB07426 shows its therapeutic effect on bones, especially in the treatment of osteoporosis, and DB07410 has anti-tumor, antibacterial, anti-oxidative, and anti-viral activities. All three compounds can be considered for repurposing as antimalarial drugs to evaluate the binding capacity or inhibition potential of these compounds. Further in-vivo and in-vitro analysis against the PfHDP protein should be conducted.Communicated by Ramaswamy H. Sarma.

India Needs to Consider Planning a Change to Artemether-Lumefantrine to Treat Plasmodium falciparum Malaria.

As the malaria elimination target draws closer for India, it must be ensured that the country's policies, strategies, and tools remain effective. Artemisinin-based combination therapies are the mainstay of Plasmodium falciparum malaria management. India has a differential standard therapy for uncomplicated falciparum malaria in the form of artemether-lumefantrine in its northeastern states and artesunate + sulfadoxine-pyrimethamine in the rest of the country. The clinical failure of artesunate + sulfadoxine-pyrimethamine in the northeast regions were attributed primarily to parasite resistance resulting from mutations in the enzymes dihydropteroate synthase and dihydrofolate reductase. Artemether-lumefantrine was therefore substituted for artesunate + sulfadoxine-pyrimethamine in the region. The change has been a success, as evidenced by the therapeutic efficacy studies conducted at regular intervals in India. However, studies suggest that resistance may be emerging toward sulfadoxine-pyrimethamine in multiple parts of the nation. Hence, there is a possibility that the artesunate + sulfadoxine-pyrimethamine combination may be acting in part as a monotherapy, and this makes the longevity of the artesunate + sulfadoxine-pyrimethamine drug combination therapy uncertain. The increasing presence of drug-resistant mutants in P. falciparum dhps and dhfr genes suggests the need for a policy switch for uncomplicated P. falciparum malaria from artesunate + sulfadoxine-pyrimethamine to artemether-lumefantrine.

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.

Polymerase Chain Reaction-Based Malaria Diagnosis Can Be Increasingly Adopted during Current Phase of Malaria Elimination in India.

Despite commendable progress in the control of malaria in India and other countries, there are hidden reservoirs of parasites in human hosts that continually feed malaria transmission. Submicroscopic infections are known to be a significant proportion in low-endemic settings like India and these infections do possess transmission potential. Hence, these reservoirs of infection add to the existing roadblocks for malaria elimination. It is crucial that this submerged burden of malaria is detected and treated to curtail further transmission. The currently used diagnostic tools including the so-called "gold standard" of microscopy are incapable of detecting these submicroscopic infections and thus are suboptimal. It is an opportune time to usher in more sensitive molecular tools like polymerase chain reaction (PCR) for routine diagnosis at all levels of healthcare as an additional diagnostic tool in routine settings. Polymerase chain reaction assays have been developed into user-friendly formats for field diagnostics and are near point of care. In India, because of the COVID-19 pandemic, these are being used rampantly across the country. The facilities created for COVID-19 diagnosis can easily be co-opted and harnessed for malaria diagnosis to augment surveillance by the inclusion of molecular techniques like PCR in the routine national malaria control program.

Geographical spread and structural basis of sulfadoxine-pyrimethamine drug-resistant malaria parasites.

The global spread of sulfadoxine (Sdx, S) and pyrimethamine (Pyr, P) resistance is attributed to increasing number of mutations in DHPS and DHFR enzymes encoded by malaria parasites. The association between drug resistance mutations and SP efficacy is complex. Here we provide an overview of the geographical spread of SP resistance mutations in Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) encoded dhps and dhfr genes. In addition, we have collated the mutation data and mapped it on to the three-dimensional structures of DHPS and DHFR which have become available. Data from genomic databases and 286 studies were collated to provide a comprehensive landscape of mutational data from 2005 to 2019. Our analyses show that the Pyr-resistant double mutations are widespread in Pf/PvDHFR (P. falciparum ∼61% in Asia and the Middle East, and in the Indian sub-continent; in P. vivax ∼33% globally) with triple mutations prevailing in Africa (∼66%) and South America (∼33%). For PfDHPS, triple mutations dominate South America (∼44%), Asia and the Middle East (∼34%) and the Indian sub-continent (∼27%), while single mutations are widespread in Africa (∼45%). Contrary to the status for P. falciparum, Sdx-resistant single point mutations in PvDHPS dominate globally. Alarmingly, highly resistant quintuple and sextuple mutations are rising in Africa (PfDHFR-DHPS) and Asia (Pf/PvDHFR-DHPS). Structural analyses of DHFR and DHPS proteins in complexes with substrates/drugs have revealed that resistance mutations map proximal to Sdx and Pyr binding sites. Thus new studies can focus on discovery of novel inhibitors that target the non-substrate binding grooves in these two validated malaria parasite drug targets.

Trends of neglected Plasmodium species infection in humans over the past century in India.

BACKGROUND: Efforts for malaria elimination in India focus solely on the more prevalent human malaria parasites of Plasmodium falciparum (Pf) and Plasmodium vivax (Pv). The three non-Pf/Pv species - Plasmodium malariae (Pm), Plasmodium ovale (Po) and Plasmodium knowlesi (Pk) are seldom studied though they are often present as mixed infections with Pf/Pv and thus may be misdiagnosed. This study provides a comprehensive landscape of Pm, Po, and Pk infections from 1930 to 2020. METHODOLOGY: We systematically searched for published literature on Pm, Po, and Pk in India from PubMed database and collated data from 35 studies. The data, starting from 1930, were mapped decade-wise across India. The prevalence of the three neglected Plasmodium species and their proportional contribution to reported Plasmodium mixed-infection were also calculated and analysed. PRINCIPAL FINDINGS: Amongst the three non-Pf/Pv species, Pm infections have been reported in greater numbers across India and were mostly mono-infections till 1980. From 1983 onwards, reports of Pm mixed infections with Pf/Pv started to emerge. In contrast, reports on occurrence of Po are still rare barring few mixed infection studies. Further, Pk mono- and mixed cases were first reported in 2004 in India and Pk now has been found reported from four Indian states. CONCLUSION: This is the first account of country-wide assimilation of reported malaria parasite species data that covers Pm, Po, and Pk infection profiles from 1930 to 2020. This study illustrates the need to survey all 5 human malaria parasite species in India and to target them collectively during the malaria elimination phase.

Epidemiological profiles and associated risk factors of SARS-CoV-2 positive patients based on a high-throughput testing facility in India.

We describe the epidemiological characteristics and associated risk factors of those presenting at a large testing centre for SARS-CoV-2 infection. This is a retrospective record review of individuals who underwent SARS-CoV-2 testing by reverse transcription-polymerase chain reaction (RT-PCR) at a high-throughput national-level government facility located in the north of India. Samples collected from 6 April to 31 December 2020 are included in this work and represent four highly populous regions. Additionally, there was a prospective follow-up of 1729 cases through telephone interviews from 25 May 2020 to 20 June 2020. Descriptive analysis has been performed for profiling clinic-epidemiological aspects of suspect cases. Multivariable logistic regression analysis was undertaken to determine risk factors that are associated with SARS-CoV-2 test positivity and symptom status. A total of 125 600 participants' details have been included in this report. The mean (s.d.) age of the participants was 33.1 (±15.3) years and 66% were male. Among these tested, 9515 (7.6%) were positive for COVID-19. A large proportion of positive cases were asymptomatic. In symptomatic positive cases, the commonest symptoms were cough and fever. Increasing age (groups 20-59 and ≥60 years compared to age group less than 5 years), male sex, history of international travel, symptoms for SARS-CoV-2, and participants from Delhi and Madhya Pradesh were positively associated with SARS-CoV-2 test positivity. Having co-morbidity, risk behaviours and intra-familial positivity were associated with a positive odds ratio for exhibiting SARS-CoV-2 symptoms. Intensified testing and isolation of cases, identification of both asymptomatic and symptomatic individuals and additional care of those with co-morbidities and risk behaviours will all be collectively important for disease containment in India. Reasons for differentials in testing between men and women remain an important area for in-depth study. The increased deployment of vaccines is likely to impact the trajectory of COVID-19 in the coming time, and therefore our data will serve as a comparative resource as India experiences the second wave of infection in light of newer variants that are likely to accelerate disease spread.

Cytomorphological features as predictors of epidermal growth factor receptor mutation status in lung adenocarcinoma.

BACKGROUND: Epidermal growth factor receptor mutation-positive (EGFR-p) lung adenocarcinomas are sensitive to tyrosine kinase inhibitors. Although histopathological subtype is an independent predictor of mutation status, there is a paucity of data on the cytomorphological features correlating with the EGFR mutation status. Therefore, the aim of this study was to determine whether certain cytomorphological features correlate with EGFR mutation in lung adenocarcinoma. MATERIALS AND METHODS: A retrospective analysis of 48 lung adenocarcinoma cases diagnosed on fine needle aspiration cytology with known EGFR mutation status was conducted. All cytology smears with cellblock sections were reviewed. The cytomorphological features including tumor pattern, stromal features, nuclear and cytoplasmic features, and tumor grade were evaluated. Clinicoradiological features such as age, sex, smoking, tumor size, clinical stage, metastases, and presence of mass, nodule, lymphadenopathy, pleural effusion, and clinical outcome were also assessed. RESULTS: Of 48 cases, 19 were EGFR-p and 29 were negative. EGFR-p cases showed a positive and significant correlation with flat monolayered sheets and acini, mild nuclear atypia, fine chromatin and smooth nuclear margins and these tumors were well differentiated. EGFR-negative tumors were moderate to poorly differentiated with predominance of solid clusters, moderate to marked nuclear atypia, with irregular nuclear margins and coarse chromatin. Clinically, female sex, nonsmoking status, smaller tumor size, and good clinical outcome correlated with EGFR-p status. CONCLUSION: Certain cytomorphological features correlate with and may suggest EGFR mutation status in advanced lung adenocarcinoma in an appropriate clinical context.

Drug targeting of one or more aminoacyl-tRNA synthetase in the malaria parasite Plasmodium falciparum.

Malaria remains a major infectious disease and, despite incidence reduction, it threatens resurgence in drug-resistant forms. Antimalarial drugs remain the mainstay of therapeutic options and hence there is a constant need to identify and validate new druggable targets. Plasmodium falciparum aminoacyl-tRNA synthetases (Pf-aaRSs) drive protein translation and are potent targets for development of next-generation antimalarials. Here, we detail advances made in structural-biology-based investigations in Pf-aaRSs and discuss their distribution of druggable pockets. This review establishes a platform for systematic experimental dissection of malarial parasite aaRSs as a new focus for sustained drug development efforts against malaria.