Genomic analysis of SARS-CoV-2 Circulating during Second and Third Wave of COVID-19 in Nepal.

BACKGROUND: In Nepal, since the first detection of COVID-19 case in January 2020, the total cases have rose to almost a million with more than 12,000 deaths. Till now, WHO has classified 5 variants of SARS-Cov2 as variant of concerns at different time points causing many waves in different countries and regions at different time points. Nepal had also faced three distinct waves of COVID-19 caused by different variant of COVID 19. The objective of this study was to perform whole-genome sequencing of SARS-CoV-2 circulating in different waves of COVID-19 in Nepal and investigate its variant or lineage. METHODS: In this study, samples from 49 SARS-CoV-2 infected subjects from May 2021 to January 2022, were investigated. The methodology followed RNA extraction, real-time PCR for confirmation and whole-genome sequencing. The consensus genomes were interpreted with appropriate bioinformatics tools and databases. RESULTS: Sequence analysis of 49 genomes revealed to be of Delta (n=27) and Omicron Variant (n=22). The mutations in the consensus genomes contained the defining mutations of the respective lineages/variants. There were 20 genomes of Omicron sub-lineage BA.2, 1 of BA.1.1 and 1 of B.1.1.529. CONCLUSIONS: This study provides concise genomic evidence of presence of Delta and Omicron variant of COVID-19 in Nepal. Delta and Omicron variants were driving the second wave and the third wave of COVID-19 respectively in Nepal. Therefore, the genomic surveillance must be increased to clearly map out the pandemic and strategize vaccination approaches in the country.

Plasmodium DDI1 is a potential therapeutic target and important chromatin-associated protein.

DNA damage inducible 1 protein (DDI1) is involved in a variety of cellular processes including proteasomal degradation of specific proteins. All DDI1 proteins contain a ubiquitin-like (UBL) domain and a retroviral protease (RVP) domain. Some DDI1 proteins also contain a ubiquitin-associated (UBA) domain. The three domains confer distinct activities to DDI1 proteins. The presence of a RVP domain makes DDI1 a potential target of HIV protease inhibitors, which also block the development of malaria parasites. Hence, we investigated the DDI1 of malaria parasites to identify its roles during parasite development and potential as a therapeutic target. DDI1 proteins of Plasmodium and other apicomplexan parasites share the UBL-RVP domain architecture, and some also contain the UBA domain. Plasmodium DDI1 is expressed across all the major life cycle stages and is important for parasite survival, as conditional depletion of DDI1 protein in the mouse malaria parasite Plasmodium berghei and the human malaria parasite Plasmodium falciparum compromised parasite development. Infection of mice with DDI1 knock-down P. berghei was self-limiting and protected the recovered mice from subsequent infection with homologous as well as heterologous parasites, indicating the potential of DDI1 knock-down parasites as a whole organism vaccine. Plasmodium falciparum DDI1 (PfDDI1) is associated with chromatin and DNA-protein crosslinks. PfDDI1-depleted parasites accumulated DNA-protein crosslinks and showed enhanced susceptibility to DNA-damaging chemicals, indicating a role of PfDDI1 in removal of DNA-protein crosslinks. Knock-down of PfDDI1 increased susceptibility to the retroviral protease inhibitor lopinavir and antimalarial artemisinin, which suggests that simultaneous inhibition of DDI1 could potentiate antimalarial activity of these drugs. As DDI1 knock-down parasites confer protective immunity and it could be a target of HIV protease inhibitors, Plasmodium DDI1 is a potential therapeutic target for malaria control.

Bazedoxifene, a Postmenopausal Drug, Acts as an Antimalarial and Inhibits Hemozoin Formation.

Despite a remarkable improvement in health care and continued drug discovery efforts, malaria control efforts are continuously challenged by the emergence of drug-resistant parasite strains. Given a long and risky development path of new drugs, repurposing existing drugs for the treatment of malaria is an attractive and shorter path. Tamoxifen, a selective estrogen receptor modulator (SERM) for the treatment and prevention of estrogen receptor-positive breast cancer, possesses antibacterial, antifungal, and antiparasitic activities. Hence, we assessed tamoxifen, raloxifene, and bazedoxifene, which represent the first-, second-, and third-generation SERMs, respectively, for antimalarial activity. Raloxifene and bazedoxifene inhibited the erythrocytic development of Plasmodium falciparum with submicromolar 50% inhibitory concentration (IC50) values. Among the three, bazedoxifene was the most potent and also decreased P. berghei infection in female mice but not in male mice. However, bazedoxifene similarly inhibited P. falciparum growth in erythrocytes of male and female origin, which highlights the importance of sex-specific host physiology in drug efficacy. Bazedoxifene was most potent on early ring-stage parasites, and about 35% of the treated parasites did not contain hemozoin in the food vacuole. Bazedoxifene-treated parasites had almost 34% less hemozoin content than the control parasites. However, both control and bazedoxifene-treated parasites had similar hemoglobin levels, suggesting that bazedoxifene inhibits hemozoin formation and that toxicity due to accumulation of free heme could be a mechanism of its antimalarial activity. Because bazedoxifene is in clinical use and bazedoxifene-chloroquine combination shows an additive antiparasitic effect, bazedoxifene could be an adjunctive partner of currently used antimalarial regimens. IMPORTANCE The emergence and spread of drug-resistant strains of the human malaria parasite Plasmodium falciparum has necessitated new drugs. Selective estrogen receptor modulators are in clinical use for the prevention and treatment of breast cancer and postmenopausal osteoporosis. We demonstrate that bazedoxifene, a third-generation selective estrogen receptor modulator, has potent inhibitory activity against both susceptible and drug-resistant strains of Plasmodium falciparum. It also blocked the development of Plasmodium berghei in mice. The inhibitory effect was strongest on the ring stage and resulted in the inhibition of hemozoin formation, which could be the major mechanism of bazedoxifene action. Hemozoin is a nontoxic polymer of heme, which is a by-product of hemoglobin degradation by the malaria parasite during its development within the erythrocyte. Because bazedoxifene is already in clinical use for the treatment of postmenopausal osteoporosis, our findings support repurposing of bazedoxifene as an antimalarial.

Antimicrobial Resistance in Typhoidal Salmonella in Nepal: Surveillance for Enteric Fever, 2016-2019.

BACKGROUND: Enteric fever (caused by Salmonella enterica) has been associated with poor hygiene and is endemic in the South-Asian countries. The increase in resistance to first line antimicrobials has been observed, while the emergence of multi/extremely drug resistance cases have been identified in several countries. The objective of this study is to analyze the current trend of antimicrobial resistance in Salmonella isolates in Nepal, and to identify the status of multi- and extremely- drug resistant isolates. METHODS: We recruited individuals at study hospitals with suspected enteric fever between September 2016 and August 2019 and performed blood cultures. The Salmonella isolates were tested for antimicrobial susceptibility and the antimicrobial resistance trend was evaluated. RESULTS: 1438 positive blood culture isolates were studied for antimicrobial resistance. 88% were culture positive for Salmonella Typhi and 12% for Salmonella Paratyphi. Multidrug resistant S. Typhi cases appeared mostly in December 2018 and January 2019, while there were no multidrug resistant S. Paratyphi cases. Also, extremely drug resistant S. Typhi cases were not observed during the study period. CONCLUSIONS: The Salmonella isolates were mostly susceptible to first-line antimicrobials, cephalosporins and others. Many fluoroquinolones non-susceptible Salmonella were obtained, nevertheless their overall trend seems to be declining. In addition, the S. Paratyphi total cases are reducing since September 2017. Among S. Typhi isolates, only few were multidrug resistant and there were no extremely drug resistant isolates.

Characterization of Plasmodium falciparum NEDD8 and identification of cullins as its substrates.

A variety of post-translational modifications of Plasmodium falciparum proteins, including phosphorylation and ubiquitination, are shown to have key regulatory roles during parasite development. NEDD8 is a ubiquitin-like modifier of cullin-RING E3 ubiquitin ligases, which regulates diverse cellular processes. Although neddylation is conserved in eukaryotes, it is yet to be characterized in Plasmodium and related apicomplexan parasites. We characterized P. falciparum NEDD8 (PfNEDD8) and identified cullins as its physiological substrates. PfNEDD8 is a 76 amino acid residue protein without the C-terminal tail, indicating that it can be readily conjugated. The wild type and mutant (Gly75Ala/Gly76Ala) PfNEDD8 were expressed in P. falciparum. Western blot of wild type PfNEDD8-expressing parasites indicated multiple high molecular weight conjugates, which were absent in the parasites expressing the mutant, indicating conjugation of NEDD8 through Gly76. Immunoprecipitation followed by mass spectrometry of wild type PfNEDD8-expressing parasites identified two putative cullins. Furthermore, we expressed PfNEDD8 in mutant S. cerevisiae strains that lacked endogenous NEDD8 (rub1Δ) or NEDD8 conjugating E2 enzyme (ubc12Δ). The PfNEDD8 immunoprecipitate also contained S. cerevisiae cullin cdc53, further substantiating cullins as physiological substrates of PfNEDD8. Our findings lay ground for investigation of specific roles and drug target potential of neddylation in malaria parasites.

Design, synthesis and biological evaluation of several aromatic substituted chalcones as antimalarial agents.

Malaria is a communicable disease which is caused by protozoan's mainly Plasmodium species (P. falciparum, P. ovale, P. vivax, P. malariae and P. knowlesi). The increasing resistance of Plasmodium to available malarial drugs poses a great responsibility for the researchers in the field of malaria. To overcome this problem of resistance, this study aimed to design and synthesize a new class of antimalarial agent with chalcone as the main moiety. Chalcones, a member of flavanoid family, consist of two aromatic rings of 1,3-diphenyl-2-propen-1-one linked by a three carbon α,ß-unsaturated carbonyl system. Five derivatives were designed and among them one was selected. The CC2 was then synthesized by esterification of Para amino acetophenone followed by treatment with hydrazide to form 2-(4 acetylphenoxy)acetohydrazide. This was then coupled with 2-Bromo substituted Diazotized esterified anilines, which was finally linked with substituted benzaldehyde to yield CC2. These were then structurally verified by Infra Red (IR) and Nuclear Magnetic Resonance (NMR) spectroscopy. The chalcone was then tested for in vitro growth inhibition assays using SYBR GREEN-1 Based assay and IC50 values were identified. The compound CC2 showed quite promising antimalarial activity by inhibiting cysteine protease enzyme. The acute toxicity studies of the compound were carried out as per OECD guideline 425 and the results showed no toxic signs and symptoms indicating CC2 as a safe and less toxic compound.

Synthesis and efficacy of pyrvinium-inspired analogs against tuberculosis and malaria pathogens.

Herein, we report the synthesis and evaluation of pyrvinium-based antimalarial and antitubercular compounds. Pyrvinium is an FDA approved drug for the treatment of pinworm infection, and it has been reported to have antiparasitic and antimicrobial activities. Pyrvinium contains quinoline core coupled with pyrrole. We replaced the pyrrole with various aryl or heteroaryl substituents to generate pyrvinium analogs. The profiling of these compounds against malaria parasite P. falciparum 3D7 revealed analogs with better antimalarial activity than pyrvinium pamoate. Compound 14 and 16 showed IC50 of 23 nM and 60 nM against P. falciparum 3D7, respectively. These compounds were also effective against drug-resistant malaria parasite P. falciparum Dd2 with IC50 of 53 nM and 97 nM, respectively. The cytotoxicity against CHO-K1, HEK and NRK-49F cells revealed better selectivity index for these new analogs compared to pyrvinium. Additionally, this series of compounds showed activity against M. tuberculosis H37Rv; particularly compounds 10, 13, 14 and 16 showed equipotent antitubercular activity to that of pyrvinium pamoate. The compounds 14 and 16 should be taken forward as leads for further optimization.