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Abstract Background The genetic diversity of malaria parasites contributes to their ability to adapt to environmental changes, develop drug resistance and circumvent the host immune system. This study aimed to analyse the genetic diversity of the Pfmsp1 and Pfmsp2 genes in Plasmodium falciparum and the Pvmsp-3α gene in Plasmodium vivax isolates from District Nowshera in Pakistan. Methods Blood samples from 124 consenting patients with uncomplicated malaria presenting to different hospitals from the Nowshera district were collected between March and August 2019, representing 28 P. falciparum and 96 P. vivax isolates. The genomic DNA extracted from the isolates were subjected to nested PCR and allele-specific analysis. Pvmsp-3α amplified fragments were further treated with restriction fragment length polymorphism (RFLP)-based Hha1 restriction enzyme. Results Of the analyzed P. falciparum, 21 distinct alleles were detected, including 14 alleles for Pfmsp-1 and 7 alleles for Pfmsp-2. The sub-allelic families MAD20 (50%) of Pfmsp-1and FC27 (75%) of Pfmsp-2 were predominant. The multiplicity of infection (MOI) was calculated as 1.4 and 1.2 for Pfmsp-1 and Pfmsp-2, respectively, with an overall mean MOI of 1.34. In P. vivax, 4 allelic variants, Pvmsp-3α types A, B, C and D, were detected, while RFLP digestion of amplicons, detected 9 sub-allelic variants (A1-A4, B1, B2, C1, C2 and D1) at the Pvmsp-3α locus. Conclusion This first ever report of molecular characterization of P. falciparum and P. vivax genotypes from District Nowshera, Pakistan reveals moderate to high allelic diversity in parasite population from District Nowshera, Pakistan.

Abstract Background Plasmodium falciparum is an obligate intracellular parasite of humans that causes malaria. Falciparum malaria is a major public health threat to human life responsible for high mortality. Currently, the risk of multi-drug resistance of P. falciparum is rapidly increasing. There is a need to address new anti-malarial therapeutics strategies to combat the drug-resistance threat. Methods The P. falciparum essential proteins were retrieved from the recently published studies. These proteins were initially scanned against human host and its gut microbiome proteome sets by comparative proteomics analyses. The human host non-homologs essential proteins of P. falciparum were additionally analysed for druggability potential via in silico methods to possibly identify novel therapeutic targets. Finally, the PfAp4AH target was prioritized for pharmacophore modelling based virtual screening and molecular docking analyses to identify potent inhibitors from drug-like compounds databases. Results The analyses identified six P. falciparum essential and human host non-homolog proteins that follow the key druggability features. These druggable targets have not been catalogued so far in the Drugbank repository. These prioritized proteins seem novel and promising drug targets against P. falciparum due to their key protein–protein interactions features in pathogen-specific biological pathways and to hold appropriate drug-like molecule binding pockets. The pharmacophore features based virtual screening of Pharmit resource predicted a lead compound i.e. MolPort-045–917-542 as a promising inhibitor of PfAp4AH among prioritized targets. Conclusion The prioritized protein targets may worthy to test in malarial drug discovery programme to overcome the anti-malarial resistance issues. The in-vitro and in-vivo studies might be promising for additional validation of these prioritized lists of drug targets against malaria.

Abstract Background The Plasmodium falciparum apical membrane antigen-1 (PfAMA1) is considered as an ideal vaccine candidate for malaria control due to its high level of immunogenicity and essential role in parasite survival. Among the three domains of PfAMA1 protein, hyper-variable region (HVR) of domain I is the most immunogenic. The present study was conducted to evaluate the extent of genetic diversity across HVR domain I of the pfama1 gene in P. falciparum isolates from Hazara division of Pakistan. Methods The HVR domain I of the pfama1 was amplified and sequenced from 20 P. falciparum positive cases from Hazara division of Pakistan. The sequences were analysed in context of global population data of P. falciparum from nine malaria endemic countries. The DNA sequence reads quality assessment, reads assembling, sequences alignment/phylogenetic and population genetic analyses were performed using Staden, Lasergene v. 7.1, MEGA7 and DnaSP v.5 software packages respectively. Results Total 14 mutations were found in Pakistani isolates with 12 parsimony informative sites. During comparison with global isolates, a novel non-synonymous mutation (Y240F) was found specifically in a single Pakistani sample with 5% frequency. The less number of mutations, haplotypes, recombination and low pairwise nucleotide differences revealed tightly linked uniform genetic structure with low genetic diversity at HVR domain I of pfama1 among P. falciparum isolates from Hazara region of Pakistan. This uniform genetic structure may be shaped across Pakistani P. falciparum isolates by bottleneck or natural selection events. Conclusion The Pakistani P. falciparum isolates were found to maintain a distinct genetic pattern at HVR pfama1 with some extent of genetic relationship with geographically close Myanmar and Indian samples. However, the exact pattern of gene flow and demographic events may infer from whole genome sequence data with large sample size of P. falciparum collected from broad area of Pakistan.

Background: The genetic diversity of malaria parasites contributes to their ability to adapt to environmental changes, develop drug resistance and circumvent the host immune system. This study aimed to analyse the genetic diversity of the Pfmsp1 and Pfmsp2 genes in Plasmodium falciparum and the Pvmsp-3α gene in Plasmodium vivax isolates from District Nowshera in Pakistan. Methods: Blood samples from 124 consenting patients with uncomplicated malaria presenting to different hospitals from the Nowshera district were collected between March and August 2019, representing 28 P. falciparum and 96 P. vivax isolates. The genomic DNA extracted from the isolates were subjected to nested PCR and allele-specific analysis. Pvmsp-3α amplified fragments were further treated with restriction fragment length polymorphism (RFLP)-based Hha1 restriction enzyme. Results: Of the analyzed P. falciparum, 21 distinct alleles were detected, including 14 alleles for Pfmsp-1 and 7 alleles for Pfmsp-2. The sub-allelic families MAD20 (50%) of Pfmsp-1and FC27 (75%) of Pfmsp-2 were predominant. The multiplicity of infection (MOI) was calculated as 1.4 and 1.2 for Pfmsp-1 and Pfmsp-2, respectively, with an overall mean MOI of 1.34. In P. vivax, 4 allelic variants, Pvmsp-3α types A, B, C and D, were detected, while RFLP digestion of amplicons, detected 9 sub-allelic variants (A1-A4, B1, B2, C1, C2 and D1) at the Pvmsp-3α locus. Conclusion: This first ever report of molecular characterization of P. falciparum and P. vivax genotypes from District Nowshera, Pakistan reveals moderate to high allelic diversity in parasite population from District Nowshera, Pakistan. [ABSTRACT FROM AUTHOR]