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1.
BMC Genomics ; 23(1): 237, 2022 Mar 26.
Article in English | MEDLINE | ID: covidwho-1793988

ABSTRACT

BACKGROUND: Novel antimalarials should be effective across all species of malaria parasites that infect humans, especially the two species that bear the most impact, Plasmodium falciparum and Plasmodium vivax. Protein kinases encoded by pathogens, as well as host kinases required for survival of intracellular pathogens, carry considerable potential as targets for antimalarial intervention (Adderley et al. Trends Parasitol 37:508-524, 2021;  Wei et al. Cell Rep Med 2:100423, 2021). To date, no comprehensive P. vivax kinome assembly has been conducted; and the P. falciparum kinome, first assembled in 2004, requires an update. The present study, aimed to fill these gaps, utilises a recently published structurally-validated multiple sequence alignment (MSA) of the human kinome (Modi et al. Sci Rep 9:19790, 2019). This MSA is used as a scaffold to assist the alignment of all protein kinase sequences from P. falciparum and P. vivax, and (where possible) their assignment to specific kinase groups/families. RESULTS: We were able to assign six P. falciparum previously classified as OPK or 'orphans' (i.e. with no clear phylogenetic relation to any of the established ePK groups) to one of the aforementioned ePK groups. Direct phylogenetic comparison established that despite an overall high level of similarity between the P. falciparum and P. vivax kinomes, which will help in selecting targets for intervention, there are differences that may underlie the biological specificities of these species. Furthermore, we highlight a number of Plasmodium kinases that have a surprisingly high level of similarity with their human counterparts and therefore not well suited as targets for drug discovery. CONCLUSIONS: Direct comparison of the kinomes of Homo sapiens, P. falciparum and P. vivax sheds additional light on the previously documented divergence of many P. falciparum and P. vivax kinases from those of their human host. We provide the first direct kinome comparison between the phylogenetically distinct species of P. falciparum and P. vivax, illustrating the key similarities and differences which must be considered in the context of kinase-directed antimalarial drug discovery, and discuss the divergences and similarities between the human and Plasmodium kinomes to inform future searches for selective antimalarial intervention.


Subject(s)
Malaria, Falciparum , Malaria, Vivax , Humans , Malaria, Falciparum/parasitology , Malaria, Vivax/parasitology , Phylogeny , Plasmodium falciparum/genetics , Plasmodium vivax/genetics
2.
Anal Chem ; 94(6): 2926-2933, 2022 02 15.
Article in English | MEDLINE | ID: covidwho-1721378

ABSTRACT

Recombinase polymerase amplification (RPA) is a useful pathogen identification method. Several label-free detection methods for RPA amplicons have been developed in recent years. However, these methods still lack sensitivity, specificity, efficiency, or simplicity. In this study, we propose a rapid, highly sensitive, and label-free pathogen assay system based on a solid-phase self-interference RPA chip (SiSA-chip) and hyperspectral interferometry. The SiSA-chips amplify and capture RPA amplicons on the chips, rather than irrelevant amplicons such as primer dimers, and the SiSA-chips are then analysed by hyperspectral interferometry. Optical length increases of SiSA-chips are used to demonstrate RPA detection results, with a limit of detection of 1.90 nm. This assay system can detect as few as six copies of the target 18S rRNA gene of Plasmodium falciparum within 20 min, with a good linear relationship between the detection results and the concentration of target genes (R2 = 0.9903). Single nucleotide polymorphism (SNP) genotyping of the dhfr gene of Plasmodium falciparum is also possible using the SiSA-chip, with as little as 1% of mutant gene distinguished from wild-type loci (m/wt). This system offers a high-efficiency (20 min), high-sensitivity (6 copies/reaction), high-specificity (1% m/wt), and low-cost (∼1/50 of fluorescence assays for RPA) diagnosis method for pathogen DNA identification. Therefore, this system is promising for fast identification of pathogens to help diagnose infectious diseases, including SNP genotyping.


Subject(s)
Nucleic Acid Amplification Techniques , Recombinases , Interferometry , Nucleic Acid Amplification Techniques/methods , Nucleotidyltransferases , Plasmodium falciparum/genetics , Sensitivity and Specificity
3.
J Antimicrob Chemother ; 76(11): 2854-2862, 2021 10 11.
Article in English | MEDLINE | ID: covidwho-1537567

ABSTRACT

BACKGROUND: The first potential focus for artemisinin resistance in South America was recently confirmed with the presence of the C580Y mutation in the Plasmodium falciparum kelch 13 gene (pfk13) in Guyana. OBJECTIVES: This study aimed to strengthen pfk13 monitoring in the Amazon basin countries, to compile the available data and to evaluate the risk of spreading of mutations. METHODS: Sanger sequencing was done on 862 samples collected between 1998 and 2019, and a global map of pfk13 genotypes available for this region was constructed. Then, the risk of spreading of mutations based on P. falciparum case importation between 2015 and 2018 within countries of the Amazon basin was evaluated. RESULTS: No additional pfk13 C580Y foci were identified. Few mutations (0.5%, 95% CI = 0.3%-0.8%) in the propeller domain were observed in the general parasite population of this region despite a high proportion of K189T mutations (49.1%, 95% CI = 46.2%-52.0%) in the non-propeller domain. Case information revealed two patterns of intense human migration: Venezuela, Guyana and the Roraima State in Brazil; and French Guiana, Suriname and the Amapá State in Brazil. CONCLUSIONS: There are few pfk13 mutant foci, but a high risk of dispersion in the Amazon basin, mainly from the Guiana Shield, proportionate to mining activities. Therefore, access to prompt diagnosis and treatment, and continuous molecular monitoring is essential in these geographical areas.


Subject(s)
Malaria, Falciparum , Mutation , Plasmodium falciparum , Antimalarials/pharmacology , Antimalarials/therapeutic use , Brazil , Drug Resistance , Humans , Kelch Repeat , Plasmodium falciparum/genetics , Protozoan Proteins/genetics
4.
Elife ; 102021 07 19.
Article in English | MEDLINE | ID: covidwho-1513059

ABSTRACT

The emergence of mutant K13-mediated artemisinin (ART) resistance in Plasmodium falciparum malaria parasites has led to widespread treatment failures across Southeast Asia. In Africa, K13-propeller genotyping confirms the emergence of the R561H mutation in Rwanda and highlights the continuing dominance of wild-type K13 elsewhere. Using gene editing, we show that R561H, along with C580Y and M579I, confer elevated in vitro ART resistance in some African strains, contrasting with minimal changes in ART susceptibility in others. C580Y and M579I cause substantial fitness costs, which may slow their dissemination in high-transmission settings, in contrast with R561H that in African 3D7 parasites is fitness neutral. In Cambodia, K13 genotyping highlights the increasing spatio-temporal dominance of C580Y. Editing multiple K13 mutations into a panel of Southeast Asian strains reveals that only the R561H variant yields ART resistance comparable to C580Y. In Asian Dd2 parasites C580Y shows no fitness cost, in contrast with most other K13 mutations tested, including R561H. Editing of point mutations in ferredoxin or mdr2, earlier associated with resistance, has no impact on ART susceptibility or parasite fitness. These data underline the complex interplay between K13 mutations, parasite survival, growth and genetic background in contributing to the spread of ART resistance.


Subject(s)
Artemisinins/pharmacology , Drug Resistance/drug effects , Drug Resistance/genetics , Mutation , Plasmodium falciparum/drug effects , Plasmodium falciparum/genetics , Protozoan Proteins/genetics , Africa , Antimalarials/pharmacology , Asia , Cambodia , Humans , Malaria, Falciparum/epidemiology , Malaria, Falciparum/parasitology , Molecular Epidemiology
5.
Molecules ; 26(8)2021 Apr 15.
Article in English | MEDLINE | ID: covidwho-1302425

ABSTRACT

Malaria is one of the most life-threatening infectious diseases and constitutes a major health problem, especially in Africa. Although artemisinin combination therapies remain efficacious to treat malaria, the emergence of resistant parasites emphasizes the urgent need of new alternative chemotherapies. One strategy is the repurposing of existing drugs. Herein, we reviewed the antimalarial effects of marketed antibiotics, and described in detail the fast-acting antibiotics that showed activity in nanomolar concentrations. Antibiotics have been used for prophylaxis and treatment of malaria for many years and are of particular interest because they might exert a different mode of action than current antimalarials, and can be used simultaneously to treat concomitant bacterial infections.


Subject(s)
Antimalarials/therapeutic use , Drug Repositioning/methods , Animals , Anti-Bacterial Agents/therapeutic use , Drug Resistance/genetics , Humans , Malaria/physiopathology , Plasmodium falciparum/genetics , Plasmodium falciparum/pathogenicity
6.
mBio ; 12(4): e0121421, 2021 08 31.
Article in English | MEDLINE | ID: covidwho-1297963

ABSTRACT

As access to high-throughput sequencing technology has increased, the bottleneck in biomedical research has shifted from data generation to data analysis. Here, we describe a modular and extensible framework for didactic instruction in bioinformatics using publicly available RNA sequencing data sets from infectious disease studies, with a focus on host-parasite interactions. We highlight lessons learned from adapting this course for virtual learners during the coronavirus disease 2019 (COVID-19) pandemic.


Subject(s)
Computational Biology/education , Computational Biology/methods , Host-Parasite Interactions/physiology , Animals , COVID-19/pathology , Data Analysis , Genomics , High-Throughput Nucleotide Sequencing , Humans , Plasmodium falciparum/drug effects , Plasmodium falciparum/genetics , Plasmodium falciparum/physiology , Schistosoma mansoni/drug effects , Schistosoma mansoni/genetics , Schistosoma mansoni/physiology , Toxoplasma/drug effects , Toxoplasma/genetics , Toxoplasma/physiology
7.
Int J Infect Dis ; 108: 137-144, 2021 Jul.
Article in English | MEDLINE | ID: covidwho-1272471

ABSTRACT

OBJECTIVES: Our objective was to systematically investigate false-negative histidine-rich protein 2 rapid diagnostic tests (HRP2-RDT) in imported Plasmodium falciparum malaria cases from travelers to the UK and the Republic of Ireland (RoI). METHODS: Five imported malaria cases in travellers returning to the UK and RoI from East Africa were reported to the PHE Malaria Reference Laboratory as negative according to histidine-rich protein (HRP2)-RDT. The cases were systematically investigated using microscopic, RDT, molecular, genomic, and in in vitro approaches. RESULTS: In each case, HRP2-RDT was negative, whereas microscopy confirmed the presence of P. falciparum. Further analysis revealed that the genes encoding HRP2 and HRP3 were deleted in three of the five cases. Whole-genome sequencing in one of these isolates confirmed deletions in P. falciparum chromosomes 8 and 13. Our study produced evidence that the fourth case, which had high parasitemia at clinical presentation, was a rare example of antigen saturation ('prozone-like effect'), leading to a false negative in the HRP2-RDT, while the fifth case was due to low parasitemia. CONCLUSIONS: False-negative HRP2-RDT results with P. falciparum are concerning. Our findings emphasise the necessity of supporting the interpretation of RDT results with microscopy, in conjunction with clinical observations, and sets out a systematic approach to identifying parasites carrying pfhrp2 and pfhrp3 deletions.


Subject(s)
Malaria, Falciparum , Parasites , Animals , Antigens, Protozoan/genetics , Diagnostic Tests, Routine , Gene Deletion , Humans , Ireland/epidemiology , Malaria, Falciparum/diagnosis , Malaria, Falciparum/epidemiology , Plasmodium falciparum/genetics , Protozoan Proteins/genetics , United Kingdom/epidemiology
8.
BMC Res Notes ; 13(1): 497, 2020 Oct 27.
Article in English | MEDLINE | ID: covidwho-895028

ABSTRACT

OBJECTIVE: Nigeria bears 25% of global malaria burden despite concerted efforts towards its control and elimination. The emergence of drug resistance to first line drugs, artemisinin combination therapies (ACTs), indicates an urgent need for continuous molecular surveillance of drug resistance especially in high burden countries where drug interventions are heavily relied on. This study describes mutations in Plasmodium falciparum genes associated with drug resistance in malaria; Pfk13, Pfmdr1, PfATPase6 and Pfcrt in isolates obtained from 83 symptomatic malaria patients collected in August 2014, aged 1-61 years old from South-west Nigeria. RESULTS: Two Pfmdr1, N86 and Y184 variants were present at a prevalence of 56% and 13.25% of isolates respectively. There was one synonymous (S679S) and two non-synonymous (M699V, S769M) mutations in the PATPase6 gene, while Pfcrt genotype (CVIET), had a prevalence of 45%. The Pfk13 C580Y mutant allele was suspected by allelic discrimination in two samples with mixed genotypes although this could not be validated with independent isolation or additional methods. Our findings call for robust molecular surveillance of antimalarial drug resistance markers in west Africa especially with increased use of antimalarial drugs as prophylaxis for Covid-19.


Subject(s)
Artemether, Lumefantrine Drug Combination/therapeutic use , Calcium-Transporting ATPases/genetics , Malaria, Falciparum/drug therapy , Membrane Transport Proteins/genetics , Multidrug Resistance-Associated Proteins/genetics , Mutation , Plasmodium falciparum/drug effects , Protozoan Proteins/genetics , Adolescent , Adult , Antimalarials/therapeutic use , Artemisinins/therapeutic use , COVID-19 , Child , Child, Preschool , Coronavirus Infections/epidemiology , Coronavirus Infections/prevention & control , Drug Resistance/genetics , Female , Gene Expression , Genotype , Humans , Infant , Malaria, Falciparum/epidemiology , Malaria, Falciparum/parasitology , Middle Aged , Molecular Epidemiology , Nigeria/epidemiology , Pandemics/prevention & control , Plasmodium falciparum/genetics , Plasmodium falciparum/growth & development , Pneumonia, Viral/epidemiology , Pneumonia, Viral/prevention & control
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