Dataset Information

Dihydrofolate-Reductase Mutations in Plasmodium knowlesi Appear Unrelated to Selective Drug Pressure from Putative Human-To-Human Transmission in Sabah, Malaysia.

ABSTRACT: BACKGROUND:Malaria caused by zoonotic Plasmodium knowlesi is an emerging threat in Eastern Malaysia. Despite demonstrated vector competency, it is unknown whether human-to-human (H-H) transmission is occurring naturally. We sought evidence of drug selection pressure from the antimalarial sulfadoxine-pyrimethamine (SP) as a potential marker of H-H transmission. METHODS:The P. knowlesi dihdyrofolate-reductase (pkdhfr) gene was sequenced from 449 P. knowlesi malaria cases from Sabah (Malaysian Borneo) and genotypes evaluated for association with clinical and epidemiological factors. Homology modelling using the pvdhfr template was used to assess the effect of pkdhfr mutations on the pyrimethamine binding pocket. RESULTS:Fourteen non-synonymous mutations were detected, with the most common being at codon T91P (10.2%) and R34L (10.0%), resulting in 21 different genotypes, including the wild-type, 14 single mutants, and six double mutants. One third of the P. knowlesi infections were with pkdhfr mutants; 145 (32%) patients had single mutants and 14 (3%) had double-mutants. In contrast, among the 47 P. falciparum isolates sequenced, three pfdhfr genotypes were found, with the double mutant 108N+59R being fixed and the triple mutants 108N+59R+51I and 108N+59R+164L occurring with frequencies of 4% and 8%, respectively. Two non-random spatio-temporal clusters were identified with pkdhfr genotypes. There was no association between pkdhfr mutations and hyperparasitaemia or malaria severity, both hypothesized to be indicators of H-H transmission. The orthologous loci associated with resistance in P. falciparum were not mutated in pkdhfr. Subsequent homology modelling of pkdhfr revealed gene loci 13, 53, 120, and 173 as being critical for pyrimethamine binding, however, there were no mutations at these sites among the 449 P. knowlesi isolates. CONCLUSION:Although moderate diversity was observed in pkdhfr in Sabah, there was no evidence this reflected selective antifolate drug pressure in humans.

SUBMITTER: Grigg MJ

PROVIDER: S-EPMC4773021 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:BACKGROUND: While Malaysia has had great success in controlling Plasmodium falciparum and Plasmodium vivax, notifications of Plasmodium malariae and the microscopically near-identical Plasmodium knowlesi increased substantially over the past decade. However, whether this represents microscopic misdiagnosis or increased recognition of P. knowlesi has remained uncertain. METHODS: To describe the changing epidemiology of malaria in Sabah, in particular the increasing incidence of P. knowlesi, a retrospective descriptive study was undertaken involving a review of Department of Health malaria notification data from 2012-2013, extending a previous review of these data from 1992-2011. In addition, malaria PCR and microscopy data from the State Public Health Laboratory were reviewed to estimate the accuracy of the microscopy-based notification data. RESULTS: Notifications of P. malariae/P. knowlesi increased from 703 in 2011 to 815 in 2012 and 996 in 2013. Notifications of P. vivax and P. falciparum decreased from 605 and 628, respectively, in 2011, to 297 and 263 in 2013. In 2013, P. malariae/P. knowlesi accounted for 62% of all malaria notifications compared to 35% in 2011. Among 1,082 P. malariae/P. knowlesi blood slides referred for PCR testing during 2011-2013, there were 924 (85%) P. knowlesi mono-infections, 30 (2.8%) P. falciparum, 43 (4.0%) P. vivax, seven (0.6%) P. malariae, six (0.6%) mixed infections, 31 (2.9%) positive only for Plasmodium genus, and 41 (3.8%) Plasmodium-negative. Plasmodium knowlesi mono-infection accounted for 32/156 (21%) and 33/87 (38%) blood slides diagnosed by microscopy as P. falciparum and P. vivax, respectively. Twenty-six malaria deaths were reported during 2010-2013, including 12 with 'P. malariae/P. knowlesi' (all adults), 12 with P. falciparum (seven adults), and two adults with P. vivax. CONCLUSIONS: Notifications of P. malariae/P. knowlesi in Sabah are increasing, with this trend likely reflecting a true increase in incidence of P. knowlesi and presenting a major threat to malaria control and elimination in Malaysia. With the decline of P. falciparum and P. vivax, control programmes need to incorporate measures to protect against P. knowlesi, with further research required to determine effective interventions.

Project description:BackgroundPrimarily impacting poor, rural populations, the zoonotic malaria Plasmodium knowlesi is now the main cause of human malaria within Malaysian Borneo. While data is increasingly available on symptomatic cases, little is known about community-level patterns of exposure and infection. Understanding the true burden of disease and associated risk factors within endemic communities is critical for informing evidence-based control measures.Methodology/principal findingsWe conducted comprehensive surveys in three areas where P. knowlesi transmission is reported: Limbuak, Pulau Banggi and Matunggung, Kudat, Sabah, Malaysia and Bacungan, Palawan, the Philippines. Infection prevalence was low with parasites detected by PCR in only 0.2% (4/2503) of the population. P. knowlesi PkSERA3 ag1 antibody responses were detected in 7.1% (95% CI: 6.2-8.2%) of the population, compared with 16.1% (14.6-17.7%) and 12.6% (11.2-14.1%) for P. falciparum and P. vivax. Sero-prevalence was low in individuals <10 years old for P. falciparum and P. vivax consistent with decreased transmission of non-zoonotic malaria species. Results indicated marked heterogeneity in transmission intensity between sites and P. knowlesi exposure was associated with agricultural work (OR 1.63; 95% CI 1.07-2.48) and higher levels of forest cover (OR 2.40; 95% CI 1.29-4.46) and clearing (OR 2.14; 95% CI 1.35-3.40) around houses. Spatial patterns of P. knowlesi exposure differed from exposure to non-zoonotic malaria and P. knowlesi exposed individuals were younger on average than individuals exposed to non-zoonotic malaria.Conclusions/significanceThis is the first study to describe serological exposure to P. knowlesi and associated risk factors within endemic communities. Results indicate community-level patterns of infection and exposure differ markedly from demographics of reported cases, with higher levels of exposure among women and children. Further work is needed to understand these variations in risk across a wider population and spatial scale.

Project description:INTRODUCTION:Plasmodium knowlesi has long been present in Malaysia, and is now an emerging cause of zoonotic human malaria. Cases have been confirmed throughout South-East Asia where the ranges of its natural macaque hosts and Anopheles leucosphyrus group vectors overlap. The majority of cases are from Eastern Malaysia, with increasing total public health notifications despite a concurrent reduction in Plasmodium falciparum and P. vivax malaria. The public health implications are concerning given P. knowlesi has the highest risk of severe and fatal disease of all Plasmodium spp in Malaysia. Current patterns of risk and disease vary based on vector type and competence, with individual exposure risks related to forest and forest-edge activities still poorly defined. Clustering of cases has not yet been systematically evaluated despite reports of peri-domestic transmission and known vector competence for human-to-human transmission. METHODS AND ANALYSIS:A population-based case-control study will be conducted over a 2-year period at two adjacent districts in north-west Sabah, Malaysia. Confirmed malaria cases presenting to the district hospital sites meeting relevant inclusion criteria will be requested to enrol. Three community controls matched to the same village as the case will be selected randomly. Study procedures will include blood sampling and administration of household and individual questionnaires to evaluate potential exposure risks associated with acquisition of P. knowlesi malaria. Secondary outcomes will include differences in exposure variables between P. knowlesi and other Plasmodium spp, risk of severe P. knowlesi malaria, and evaluation of P. knowlesi case clustering. Primary analysis will be per protocol, with adjusted ORs for exposure risks between cases and controls calculated using conditional multiple logistic regression models. ETHICS:This study has been approved by the human research ethics committees of Malaysia, the Menzies School of Health Research, Australia, and the London School of Hygiene and Tropical Medicine, UK.

Project description:BackgroundThe 2022 malaria WHO reported around 4000 P. knowlesi infections in the South-East Asia region. In the same period, 72 positive cases were reported by the Department of Disease Control in Thailand, suggesting a persistent infection. Little is known about dihydrofolate reductase (pkdhfr) and dihydropteroate synthase (pkdhps), putative antimalarial resistance markers for P. knowlesi. The relevant amplification and sequencing protocol are presently unavailable. In this study, we developed a protocol for amplifying and evaluating pkdhps mutations. The haplotype pattern of pkdhfr-pkdhps in Thai isolates was analyzed, and the effects of these pkdhps mutations were predicted by using a computer program.MethodsPkdhps were amplified and sequenced from 28 P. knowlesi samples collected in 2008 and 2020 from nine provinces across Thailand. Combining pkdhfr sequencing data from previous work with pkdhps data to analyze polymorphisms of pkdhfr and pkdhps haplotype. Protein modeling and molecular docking were constructed using two inhibitors, sulfadoxine and sulfamethoxazole, and further details were obtained through analyses of protein-ligand interactions by using the Genetic Optimisation for Ligand Docking program. A phylogenetic tree cluster analysis was reconstructed to compare the P. knowlesi Malaysia isolates.ResultsFive nonsynonymous mutations in the pkdhps were detected outside the equivalence of the binding pocket sites to sulfadoxine and sulfamethoxazole, which are at N391S, E421G, I425R, A449S, and N517S. Based on the modeling and molecular docking analyses, the N391S and N517S mutations located close to the enzyme-binding pocket demonstrated a different docking score and protein-ligand interaction in loop 2 of the enzyme. These findings indicated that it was less likely to induce drug resistance. Of the four haplotypes of pkdhfr-pkdhps, the most common one is the R34L pkdhfr mutation and the pkdhps quadruple mutation (GRSS) at E421G, I425R, A449S, and N517S, which were observed in P. knowlesi in southern Thailand (53.57%). Based on the results of neighbor-joining analysis for pkdhfr and pkdhps, the samples isolated from eastern Thailand displayed a close relationship with Cambodia isolates, while southern Thailand isolates showed a long branch separated from the Malaysian isolates.ConclusionsA new PCR protocol amplification and evaluation of dihydropteroate synthase mutations in Knowlesi (pkdhps) has been developed. The most prevalent pkdhfr-pkdhps haplotypes (53.57%) in southern Thailand are R34L pkdhfr mutation and pkdhps quadruple mutation. Further investigation requires additional phenotypic data from clinical isolates, transgenic lines expressing mutant alleles, or recombinant proteins.

Project description:BackgroundLand use changes disrupt ecosystems, altering the transmission of vector-borne diseases. These changes have been associated with increasing incidence of zoonotic malaria caused by Plasmodium knowlesi; however, the population-level distributions of infection and exposure remain unknown. We aimed to measure prevalence of serological exposure to P knowlesi and assess associated risk factors.MethodsWe did an environmentally stratified, population-based, cross-sectional survey across households in the Kudat, Kota Marudu, Pitas, and Ranau districts in northern Sabah, Malaysia, encompassing a range of ecologies. Using blood samples, the transmission intensity of P knowlesi and other malaria species was measured by specific antibody prevalence and infection detected using molecular methods. Proportions and configurations of land types were extracted from maps derived from satellite images; a data-mining approach was used to select variables. A Bayesian hierarchical model for P knowlesi seropositivity was developed, incorporating questionnaire data about individual and household-level risk factors with selected landscape factors.FindingsBetween Sept 17, 2015, and Dec 12, 2015, 10 100 individuals with a median age of 25 years (range 3 months to 105 years) were sampled from 2849 households in 180 villages. 5·1% (95% CI 4·8-5·4) were seropositive for P knowlesi, and marked historical decreases were observed in the transmission of Plasmodium falciparum and Plasmodium vivax. Nine Plasmodium spp infections were detected. Age, male sex, contact with macaques, forest use, and raised house construction were positively associated with P knowlesi exposure, whereas residing at higher geographical elevations and use of insecticide were protective. Agricultural and forest variables, such as proportions and fragmentation of land cover types, predicted exposure at different spatial scales from households.InterpretationAlthough few infections were detected, P knowlesi exposure was observed in all demographic groups and was associated with occupational factors. Results suggest that agricultural expansion and forest fragmentation affect P knowlesi exposure, supporting linkages between land use change and P knowlesi transmission.FundingUK Medical Research Council, Natural Environment Research Council, Economic and Social Research Council, and Biotechnology and Biosciences Research Council.

Dataset Information

Dihydrofolate-Reductase Mutations in Plasmodium knowlesi Appear Unrelated to Selective Drug Pressure from Putative Human-To-Human Transmission in Sabah, Malaysia.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets