Plasmodium falciparum histidine rich protein 2 (pfhrp2): an additional genetic marker suitable for anti-malarial drug efficacy trials

Background Genotyping of the three Plasmodium falciparum polymorphic genes, msp1, msp2 and glurp, has been adopted as a standard strategy to distinguish recrudescence from new infection in drug efficacy clinical trials. However, the suitability of a particular gene is compromised in areas where its allelic variants distribution is significantly skewed, a phenomenon that might occur in isolated parasite populations or in areas of very low transmission. Moreover, observation of amplification bias has diminished the value of glurp as a marker. Methods The suitability of the polymorphic P. falciparum histidine-rich protein 2 (pfhrp2) gene was assessed to serve as an alternative marker using a PCR-sequencing or a PCR–RFLP protocol for genotyping of samples in drug efficacy clinical trials. The value of pfhrp2 was validated by side-by-side analyses of 5 admission-recrudescence sample pairs from Yemeni malaria patients. Results The outcome of the single pfhrp2 gene discrimination analysis has been found consistent with msp1, msp2 and glurp pool genotyping analysis for the differentiation of recrudescence from new infection. Conclusion The findings suggest that under the appropriate circumstances, pfhrp2 can serve as an additional molecular marker for monitoring anti-malarials efficacy. However, its use is restricted to endemic areas where only a minority of P. falciparum parasites lack the pfhrp2 gene.

Distinguishing gene flow between malaria parasite populations

Measuring gene flow between malaria parasite populations in different geographic locations can provide strategic information for malaria control interventions. Multiple important questions pertaining to the design of such studies remain unanswered, limiting efforts to operationalize genomic surveillance tools for routine public health use. This report examines the use of population-level summaries of genetic divergence (FST) and relatedness (identity-by-descent) to distinguish levels of gene flow between malaria populations, focused on field-relevant questions about data size, sampling, and interpretability of observations from genomic surveillance studies. To do this, we use P. falciparum whole genome sequence data and simulated sequence data approximating malaria populations evolving under different current and historical epidemiological conditions. We employ mobile-phone associated mobility data to estimate parasite migration rates over different spatial scales and use this to inform our analysis. This analysis underscores the complementary nature of divergence- and relatedness-based metrics for distinguishing gene flow over different temporal and spatial scales and characterizes the data requirements for using these metrics in different contexts. Our results have implications for the design and implementation of malaria genomic surveillance studies.

An Evolution From a Predominant K1 Allelic Variant to MAD20 of msp1 Gene Between 2015 to 2019 in Metehara, Southeast Ethiopia

Background: The complexity and quantity of parasite populations circulating in a specific location are reflected in the genetic diversity of malaria parasites (s). Between 2015 and 2019, this study in Metehara, South east, Ethiopia. set out to investigate the temporal dynamics of genetic diversity and multiplicity as a result of evolutionary change in the genes that contribute to Plasmodium falciparum infection elimination. Method: Between 2015 and 2019, a cross-sectional study was carried out. from eighty-three dry blood spots from malaria patients who were screened for P. falciparum mono-infection by QPCR. From this seventy confirmed P. falciparum were genotyping to merozoite surface protein 1,2 and glutamate-rich protein using nested PCR.Result: Between 2015 and 2019, seventy (84.3%) of the isolates were successfully genotyped for all three target genes in both years. In 2015 and 2019, the allelic distributions of the three genes differed significantly (P= 0.001). Overall, the most common allelic families for msp1 and msp2 were K1 and FC27 respectively. For glurp, eight distinct genotypes were identified. In 2015, the genotyping of msp1, msp2 and glurp was 25 (86.2%), 25 (86.2%) and 24 (82.2%) respectively. K1, MAD20 and RO33 all have 19(65.5%), 3(10.3%) and 3(10.3%) msp1 allelic families respectively. In 2019 the genes were 30 (73.2%), 39 (95.1%) and 30 (73.2%). K1, MAD20, and RO33 were genotyped for 6 (14.6 percent), 18 (43.9 percent) and 6 (14.6 percent) genotyping respectively. Over all the multiplicity of infection was 1.67 (95 percent CI 1.54-1.74) and the heterozygosity index for msp1, msp2, and glurp was 0.48, 0.70, and 0.55 respectively.Conclusion: This study provides current information on the genetic diversity of P. falciparum populations in Metehara over five-year intervals, The progression of the dominant K1 variant from 2015 to MAD20 variant in 2019 was observed in this study.

Genetic Diversity of Plasmodium vivax Reticulocyte Binding Protein 2b in Global Parasite Populations

BackgroundPlasmodium vivax reticulocyte binding protein 2b (PvRBP2b) plays a critical role in parasite invasion of reticulocytes by binding the transferring receptor 1. PvRBP2b is a vaccine candidate since the antibody titers against PvRBP2b recombinant proteins are negatively correlated with the parasitemia and risk of vivax malaria. This study aims to analyze the genetic diversity of the PvRBP2b gene in the global P. vivax populations. MethodsThe near full-length PvRBP2b nucleotide sequences (190-8349 bp) were obtained from 88 P. vivax isolates collected from the China–Myanmar border (n=44) and Thailand (n=44). Additional 224 sequences of PvRBP2b were retrieved from genome sequences from the global parasite populations. The genetic diversity, neutral selections, haplotypes distribution and genetic differentiation of PvRBP2b were examined. ResultsThe genetic diversity of PvRBP2b was distributed unevenly with the peak in the reticulocyte binding region in the N-terminus and subjected to the balancing selection. Several amino acid variants were found in all or nearly all endemic fields. However, the critical residues responsible for reticulocyte binding were highly conserved. There was substantial population differentiation according to the geographical separation. The distribution of haplotypes in the reticulocyte binding region varied among regions; even the two major haplotypes Hap_6 and Hap_8 were found in only five populations. ConclusionsOur data showed considerable genetic variations of PvRBPb in global parasite populations, and the geographic divergence may pose a challenge to PvRBP2b-based vaccine development.

Temporal Evolution of the Resistance Genotypes of Plasmodium Falciparum in Isolates From Equatorial Guinea During 20 Years (1999 to 2019).

Background: Malaria is one of the deadliest disease in the world, particularly in Africa. The resistance to antimalarial drugs is one of the most important problems to global malaria control. The study assess the evolution of the different resistance markers over time and the possible influence of the interventions and treatment changes that have been made in Equatorial Guinea.Methods: 1223 biological samples distributed from 1999 to 2019 were included in the study. The screening of the mutations in pfdhfr, pfdhps, pfmdr1 and pfcrt genes were carried out by nested PCR and RFLPs, and the study of pfk13 genes was carried out by nested PCR, followed by sequencing to determine the presence of mutations Results: the haplotypes partially and fully resistant (pfdhfr + pfdhps) increase over time. In 2019, the fully resistant is increasing although super resistant remains lower. There is also a continued decline over time in pfmdr1 and pfcrt gene mutations. Since 2008, when ACTs were introduced in the country, the number of mutations detected in pfk13 has been increasing over time, with more mutations being observed in 2019. In this year, were detected 2 synonymous and 5 non-synonymous mutations, although are not related to resistance to ACTs. In addition, the non-synonymous A578S mutation, the most frequent on the African continent, was detected in 2013, although it has not been detected in the following years.Conclusions: The withdrawal of the use of CQ as a treatment in the country has been effective over time, as wild-type parasite populations outnumber mutant populations. The upward trend observed in SP resistance markers evidence its misuse alone or in combination with AS or AQ in some areas of the country, this allows selective pressure from SP to continue. SNPs 540E and 581G do not exceed the limit of 50% and 10% respectively, which means that SP as an IPT is still effective in the country. As for the pfk13 gene, no mutations have been detected in relation to resistance to ACTs. However, in 2019 there is a greater accumulation of non-synonymous mutations compared to years prior to 2008.

Thinking outside the blood: Perspectives on tissue-resident Trypanosoma brucei

Trypanosoma brucei is a protozoan parasite that causes human and animal African trypanosomiases (HAT and AAT). In the mammalian host, the parasite lives entirely extracellularly, in both the blood and interstitial spaces in tissues. Although most T. brucei research has focused on the biology of blood- and central nervous system (CNS)-resident parasites, a number of recent studies have highlighted parasite reservoirs in the dermis and adipose tissue, leading to a renewed interest in tissue-resident parasite populations. In light of this renewed interest, work describing tissue-resident parasites can serve as a valuable resource to inform future investigations of tissue-resident T. brucei. Here, we review this body of literature, which describes infections in humans, natural hosts, and experimental animal models, providing a wealth of information on the distribution and biology of extravascular parasites, the corresponding immune response in each tissue, and resulting host pathology. We discuss the implications of these studies and future questions in the study of extravascular T. brucei.

Systemic host inflammation induces stage-specific transcriptomic modification and slower maturation in malaria parasites

Maturation rate of malaria parasites within red blood cells (RBC) can be influenced by host nutrient status or circadian rhythm. Here, we observed in mice that systemic host inflammation, induced by lipopolysaccharide (LPS) conditioning or ongoing acute malaria infection, slowed the progression of a single cohort of parasites from one generation of RBC to the next. LPS-conditioning and acute infection both triggered substantial changes to the metabolomic composition of plasma in which parasites circulated. This altered plasma directly slowed parasite maturation in a manner that could not be rescued by supplementation, consistent with the presence of inhibitory factors. Single-cell transcriptomic assessment of mixed parasite populations, exposed to a short period of systemic host inflammation in vivo, revealed specific impairment in the transcriptional activity and translational capacity of trophozoites compared to rings or schizonts. Thus, we provide in vivo evidence of transcriptomic and phenotypic plasticity of asexual blood-stage Plasmodium parasites when exposed to systemic host inflammation.

Single-nucleotide Polymorphisms of Artemisinin Resistance-related pfubp1 and pfap2mu Genes in Imported Plasmodium Falciparum to Wuhan, China

BackgroundMolecular markers for monitoring resistance could help improve malaria treatment policies. Delayed clearance of Plasmodium falciparum by Artemisinin-based Combination Therapies (ACTs) has been reported in several countries. In addition to the PfKelch13 (pfk13), new drug resistance genes, the ubiquitin-specific protease 1 (pfubp1) and the eadaptor protein complex 2 mu subunit (pfap2mu) have been identified as being linked to ACTs. This study investigated the prevalence of single-nucleotide polymorphisms (SNPs) in clinical P. falciparum isolates pfubp1 and pfap2mu imported from Africa and Southeast Asia (SEA) to Wuhan, China, to provide baseline data for antimalarial resistance monitoring in this region.MethodsPeripheral Blood samples were collected in Wuhan, China, from August 2011 to December 2019. The SNPs of Pfubp1 and pfap2mu of P. falciparum were determined by nested PCR and Sanger sequencing. ResultsIn total, 296 samples were collected. Subsequently, 92.23% (273/296) were successfully amplified and sequenced for the Pfubp1. There were 60.07% (164/273) wild strains and 39.93% (109/273) mutant strains. For the pfap2mu gene, it was divided into three fragments for amplification, 82.77% (245/296), 90.20% (267/296) and 94.59% (280/296) were sequenced successfully respectively. Genotypes reportedly associated with ACTs resistance detected in this study included pfubp1 D1525E as well as E1528D and pfap2mu S160N. The mutation prevalence rates were 10.99% (30/273), 13.19% (36/273) and 11.24% (30/267), respectively. These are all focused on Congo, Nigeria and Angola. And known delayed-clearance parasite mutation has also been found in SEA.ConclusionsThe existence of mutation sites of known clearance genes detected in the isolates in this study, including D1525E and E1528D in the pfubp1 gene, and S160N in the pfap2mu gene, further proved the risk of ACTs resistance. Constant vigilance is therefore needed to protect the effectiveness of ACTs, and to prevent the spread of drug-resistant P. falciparum. Further studies in malaria-endemic countries are needed to further validate potential genetic markers for monitoring parasite populations in Africa and SEA.

Single-Nucleotide Polymorphisms of Artemisinin Resistance-Related Pfubp1 and Pfap2mu Genes in Plasmodium Falciparum, Central China

BackgroundMolecular markers for monitoring resistance could help improve malaria treatment policies. Delayed clearance of Plasmodium falciparum by Artemisinin-based Combination Therapies (ACTs) has been reported in several countries. In addition to the PfKelch13 (pfk13), new drug resistance genes, the ubiquitin-specific protease 1 (pfubp1) and the eadaptor protein complex 2 mu subunit (pfap2mu) have been identified as being linked to ACTs. This study investigated the prevalence of single-nucleotide polymorphisms (SNPs) in clinical Plasmodium falciparum isolates pfubp1 and pfap2mu imported from Africa and Southeast Asia (SEA) to Wuhan, China, to provide baseline data for antimalarial resistance monitoring in this region.MethodsPeripheral Blood samples were collected in Wuhan, China, from August 2011 to December 2019. The SNPs of Pfubp1 and pfap2mu of P. falciparum were determined by nested PCR and Sanger sequencing. ResultsIn total, 296 samples were collected. Subsequently, 92.23% (273/296) were successfully amplified and sequenced for the Pfubp1. There were 60.07% (164/273) wild strains and 39.93% (109/273) mutant strains. For the pfap2mu gene, it was divided into three fragments for amplification, 82.77% (245/296), 90.20% (267/296) and 94.59% (280/296) were sequenced successfully respectively. Genotypes reportedly associated with ACTs resistance detected in this study included pfubp1 D1525E as well as E1528D and pfap2mu S160N. The mutation prevalence rates were 10.99% (30/273), 13.19% (36/273) and 11.24% (30/267), respectively. ConclusionsThe existence of mutation sites of known clearance genes detected in the isolates in this study, including D1525E and E1528D in the pfubp1 gene, and S160N in the pfap2mu gene, further proved the risk of ACTs resistance. Constant vigilance is therefore needed to protect the effectiveness of ACTs, and to prevent the spread of drug-resistant P. falciparum. Further studies in malaria-endemic countries are needed to further validate potential genetic markers for monitoring parasite populations in Africa and SEA.

Newly identified parasitic nematode beta-tubulin alleles confer resistance to benzimidazoles

Infections by parasitic nematodes cause large health and economic burdens worldwide. We use anthelmintic drugs to reduce these infections. However, resistance to anthelmintic drugs is extremely common and increasing worldwide. It is essential to understand the mechanisms of resistance to slow its spread. Recently, four new parasitic nematode beta-tubulin alleles have been identified in benzimidazole (BZ) resistant parasite populations: E198I, E198K, E198T, and E198stop. These alleles have not been tested for the ability to confer resistance or for any effects that they might have on organismal fitness. We introduced these four new alleles into the sensitive C. elegans laboratory-adapted N2 strain and exposed these genome-edited strains to both albendazole and fenbendazole. We found that all four alleles conferred resistance to both BZ drugs. Additionally, we tested for fitness consequences in both control and albendazole conditions over seven generations in competitive fitness assays. We found that none of the edited alleles had deleterious effects on fitness in control conditions and that all four alleles conferred strong and equivalent fitness benefits in BZ drug conditions. Because it is unknown if previously validated alleles confer a dominant or recessive BZ resistance phenotype, we tested the phenotypes caused by five of these alleles and found that none of them conferred a dominant BZ resistance phenotype. Accurate measurements of resistance, fitness effects, and dominance caused by the resistance alleles allow for the generation of better models of population dynamics and facilitate control practices that maximize the efficacy of this critical anthelmintic drug class.