Malaria molecular surveillance in the Peruvian Amazon with novel highly multiplexed Plasmodium falciparum Ampliseq assay

Background: Malaria molecular surveillance has great potential to support local national malaria control programs (NMCPs) to inform policy for malaria control and elimination. Molecular markers associated with drug resistance are good predictors of treatment responses. In addition, molecular detection of deletions in hrp2 and hrp3 genes are indicative of potential failure of HRP2-based rapid diagnostic tests. However, there is an urgent need for feasible, cost-effective and fast molecular surveillance tools that NMCPs can implement. Methods: Here we present a new 3-day workflow for targeted resequencing of markers in 13 resistance-associated genes, hrp2&3, a country-specific 28 SNP-barcode for population genetic analysis, and ama1. The assay was applied to control isolates and retrospective samples collected between 2003-2018 in the Loreto region (n = 254) in Peru. Pf AmpliSeq libraries were prepared using a multiplex PCR simultaneously amplifying a high number of targets from dried blood spots and sequenced at high coverage (median 1336, range 20-43795). Results: There was no evidence suggesting the emergence of artemisinin resistance in Peru. However, alleles in ubp1 and coronin contributed to recent genetic differentiation of the parasite population. After 2008, predominant parasite lineages in Peru are resistant to sulfadoxine-pyrimethamine (sextuple dhfr/dhps mutant) and chloroquine (SVMNT in crt and NDFCDY in mdr1) and can escape HRP2 based RDTs. Conclusions: These findings indicate a parasite population under drug pressure, and demonstrates the added value of molecular surveillance systems and offers a highly multiplexed surveillance tool. The targets in the assay can be easily adjusted to suit the needs of other settings. Funding: This work was funded by the Belgium Development Cooperation (DGD) under the Framework Agreement Program between DGD and ITM (FA4 Peru, 2017-2021) and the sample collections in 2018 were supported by VLIR-UOS (project PE2018TEA470A102; University of Antwerp). Funding for the sample collections lead by the U.S. Naval Medical Research Unit 6 (NAMRU-6) in 2011 and 2012 was provided by the Armed Forces Health Surveillance Division (AFHSD) and its Global Emerging Infections Surveillance and Response (GEIS) Section (P0144_20_N6_01, 2020-2021).

Reproducible Discovery of Cell-Binding Peptides "Lost" in Bulk Amplification via Emulsion Amplification in Phage Display Panning

Many pharmaceutically-relevant cell surface receptors are functional only in the context of intact cells. Phage display, while being a powerful method for the discovery of ligands for purified proteins often fails to identify a diverse set of ligands to receptors on a cell membrane mosaic. To understand this deficiency, we examined growth bias in naive phage display libraries and observed that it fundamentally changes selection outcomes: The presence of growth-biased (parasite) phage clones in a phage library is detrimental to selection and cell-based panning of such biased libraries is poised to yield ligands from within a small parasite population. Importantly, amplification of phage libraries in water-oil emulsions suppressed the amplification of parasites and steered the selection of biased phage libraries away from parasite population. Attenuation of the growth bias through the use of emulsion amplification reproducibly discovers the ligands for cell-surface receptors that cannot be identified in screen that use conventional "bulk" amplification.

Genetic diversity and population structure of Plasmodium falciparum in Nigeria: insights from microsatellite loci analysis

Background Malaria remains a public health burden especially in Nigeria. To develop new malaria control and elimination strategies or refine existing ones, understanding parasite population diversity and transmission patterns is crucial. Methods In this study, characterization of the parasite diversity and structure of Plasmodium falciparum isolates from 633 dried blood spot samples in Nigeria was carried out using 12 microsatellite loci of P. falciparum. These microsatellite loci were amplified via semi-nested polymerase chain reaction (PCR) and fragments were analysed using population genetic tools. Results Estimates of parasite genetic diversity, such as mean number of different alleles (13.52), effective alleles (7.13), allelic richness (11.15) and expected heterozygosity (0.804), were high. Overall linkage disequilibrium was weak (0.006, P < 0.001). Parasite population structure was low (Fst: 0.008–0.105, AMOVA: 0.039). Conclusion The high level of parasite genetic diversity and low population structuring in this study suggests that parasite populations circulating in Nigeria are homogenous. However, higher resolution methods, such as the 24 SNP barcode and whole genome sequencing, may capture more specific parasite genetic signatures circulating in the country. The results obtained can be used as a baseline for parasite genetic diversity and structure, aiding in the formulation of appropriate therapeutic and control strategies in Nigeria.

Dynamic Analysis of the Symbiotic Model of Commensalism and Parasitism with Harvesting in Commensal Populations

This article discussed about a dynamic analysis of the symbiotic model of commensalism and parasitism with harvesting in the commensal population. This model is obtained from a modification of the symbiosis commensalism model. This modification is by adding a new population, namely the parasite population. Furthermore, it will be investigated that the three populations can coexist. The analysis carried out includes the determination of all equilibrium points along with their existence and local stability along with their stability requirements. From this model, it is obtained eight equilibrium points, namely three population extinction points, two population extinction points, one population extinction point and three extinction points can coexist. Of the eight points, only two points are asymptotically stable if they meet certain conditions. Next, a numerical simulation will be performed to illustrate the model’s behavior. In this article, a numerical simulation was carried out using the RK-4 method. The simulation results obtained support the results of the dynamic analysis that has been done previously.This article discussed about a dynamic analysis of the symbiotic model of The dynamics of the symbiotic model of commensalism and parasitism with harvesting in the commensal population. is the main focus of this study. This model is obtained from a modification of the symbiosis commensalism model. This modification is by adding a new population, namely the parasite population. Furthermore, it will be investigated that the three populations can coexist. The analysis carried out includes the determination begins by identifying the conditions for the existence of all equilibrium points along with their existence and local stability along with their stability requirements. From this model, it is obtained eight equilibrium points, namely three population extinction points, two population extinction points, one population extinction point and three extinction points can coexist. Of the eight points, only two points are asymptotically stable if they meet certain conditions. Next, a numerical simulation will be performed to illustrate the model’s behavior. In this article, a numerical simulation was carried out using the RK-4 method. The simulation results obtained support the results of the dynamic analysis that has been done previously.[VM1] [VM1]To add a mathematical effect to the article. There can be added mathematical models produced in the study at the end of this section.

Diversity and within-host evolution of parasites from VL and VL/HIV patients in Northern Ethiopia

Visceral leishmaniasis (VL) is a fatal disease and a growing public health problem in East Africa, where Ethiopia has one of the highest VL burdens. The largest focus of VL in Ethiopia is driven by high prevalence in migrant agricultural workers and associated with a high rate of co-infection with HIV. This co-infection makes VL more difficult to treat successfully, and is associated with a high rate of relapse, with VL/HIV patients frequently experiencing many relapses of VL before succumbing to this infection. We present genome-wide data on Leishmania donovani isolates from a longitudinal study of cohorts of VL and VL/HIV patients reporting to a single clinic in Ethiopia. Extensive clinical data allows us to investigate the influence of co-infection and relapse on the populations of parasites infecting these patients. We find that the same parasite population is responsible for both VL and VL/HIV infections, and that in most cases, disease relapse is caused by recrudescence of the population of parasites that caused primary VL. Complex, multi-clonal infections are present in both primary and relapse cases, but the infrapopulation of parasites within a patient loses genetic diversity between primary disease presentation and subsequent relapses, presumably due to a population bottleneck induced by treatment. These data suggest that VL/HIV relapses are not caused by genetically distinct parasite infections, nor by re-infection. Treatment of VL does not lead to sterile cure, and in VL/HIV the infecting parasites are able to re-establish after clinically successful treatment, leading to repeated relapse of VL.ImportanceVisceral leishmaniasis (VL) is the second largest cause of deaths due to parasite infections, and a growing problem in East Africa. In Ethiopia, it is particularly associated with migrant workers moving from non-endemic regions for seasonal agricultural work, and frequently found as a co-infection with HIV, which leads to frequent VL relapse following treatment. Insight into the process of relapsing in these patients is thus key to controlling the VL epidemic in Ethiopia. We show that there is little genetic differentiation between the parasites infecting HIV positive and HIV negative VL patients. Moreover, we provide evidence that relapses are caused by the initially infecting parasite population, and that treatment induces a loss of genetic diversity in this population. We propose that restoring functioning immunity and improving anti-parasitic treatment may be key in breaking the cycle of relapsing VL in VL/HIV patients.

Parasite genetic diversity reflects continued residual malaria transmission in Vhembe District, a hotspot in the Limpopo Province of South Africa

Background South Africa aims to eliminate malaria transmission by 2023. However, despite sustained vector control efforts and case management interventions, the Vhembe District remains a malaria transmission hotspot. To better understand Plasmodium falciparum transmission dynamics in the area, this study characterized the genetic diversity of parasites circulating within the Vhembe District. Methods A total of 1153 falciparum-positive rapid diagnostic tests (RDTs) were randomly collected from seven clinics within the district, over three consecutive years (2016, 2017 and 2018) during the wet and dry malaria transmission seasons. Using 26 neutral microsatellite markers, differences in genetic diversity were described using a multiparameter scale of multiplicity of infection (MOI), inbreeding metric (Fws), number of unique alleles (A), expected heterozygosity (He), multilocus linkage disequilibrium (LD) and genetic differentiation, and were associated with temporal and geospatial variances. Results A total of 747 (65%) samples were successfully genotyped. Moderate to high genetic diversity (mean He = 0.74 ± 0.03) was observed in the parasite population. This was ascribed to high allelic richness (mean A = 12.2 ± 1.2). The majority of samples (99%) had unique multi-locus genotypes, indicating high genetic diversity in the sample set. Complex infections were observed in 66% of samples (mean MOI = 2.13 ± 0.04), with 33% of infections showing high within-host diversity as described by the Fws metric. Low, but significant LD (standardised index of association, ISA = 0.08, P < 0.001) was observed that indicates recombination of distinct clones. Limited impact of temporal (FST range − 0.00005 to 0.0003) and spatial (FST = − 0.028 to 0.023) variation on genetic diversity existed during the sampling timeframe and study sites respectively. Conclusions Consistent with the Vhembe District’s classification as a ‘high’ transmission setting within South Africa, P. falciparum diversity in the area was moderate to high and complex. This study showed that genetic diversity within the parasite population reflects the continued residual transmission observed in the Vhembe District. This data can be used as a reference point for the assessment of the effectiveness of on-going interventions over time, the identification of imported cases and/or outbreaks, as well as monitoring for the potential spread of anti-malarial drug resistance.

Parasite genetic diversity reflects continued residual malaria transmission in Vhembe District, a hotspot in the Limpopo Province of South Africa

Background South Africa aims to eliminate malaria transmission by 2023. However, despite sustained vector control efforts and case management interventions, the Vhembe District remains a malaria transmission hotspot. To better understand Plasmodium falciparum transmission dynamics in the area, this study characterized the genetic diversity of parasites circulating within the Vhembe District.Methods A total of 1153 falciparum-positive rapid diagnostic tests (RDTs) were randomly collected from seven clinics within the district, over three consecutive years (2016, 2017 and 2018) during the wet and dry malaria transmission seasons. Using 26 neutral microsatellite markers, differences in genetic diversity were described using a multiparameter scale of multiplicity of infection (MOI), inbreeding metric (Fws), number of unique alleles (A), expected heterozygosity (He), multilocus linkage disequilibrium (LD) and genetic differentiation, and were associated with temporal and geospatial variances. Results A total of 747 (65%) samples were successfully genotyped. Moderate to high genetic diversity (mean He = 0.74 ± 0.03) was observed in the parasite population. This was ascribed to high allelic richness (mean A = 12.2 ± 1.2). The majority of samples (99%) had unique multi-locus genotypes, indicating high genetic diversity in the sample set. Complex infections were observed in 66% of samples (mean MOI = 2.13 ± 0.04), with 33% of infections showing high within-host diversity as described by the Fws metric. Low, but significant LD (standardised index of association, ISA = 0.08, P < 0.001) was observed that indicates recombination of distinct clones. Limited impact of temporal (FST range -0.00005 to 0.0003) and spatial (FST = –0.028 to 0.023) variation on genetic diversity existed during the sampling timeframe and study sites respectively. Conclusion Consistent with the Vhembe District’s classification as a 'high' transmission setting within South Africa, P. falciparum diversity in the area was moderate to high and complex. This study showed that genetic diversity within the parasite population reflects the continued residual transmission observed in the Vhembe District. This data can be used as a reference point for the assessment of the effectiveness of on-going interventions over time, the identification of imported cases and/or outbreaks, as well as monitoring for the potential spread of anti-malarial drug resistance.

Parasite genetic diversity reflects continued residual malaria transmission in Vhembe District, a hotspot in the Limpopo Province of South Africa

Background: South Africa aims to eliminate malaria transmission by 2023. However, despite sustained vector control efforts and case management interventions, the Vhembe District remains a malaria transmission hotspot. To better understand Plasmodium falciparum transmission dynamics in the area, this study characterised the genetic diversity of parasites circulating within the Vhembe District.Methods: A total of 1153 falciparum-positive rapid diagnostic tests (RDTs) were randomly collected from seven clinics within the district, over three consecutive years (2016, 2017 and 2018) during the wet and dry malaria transmission seasons. Using 26 neutral microsatellite markers, differences in genetic diversity were described using a multiparameter scale of multiplicity of infection (MOI), inbreeding metric (Fws), number of unique alleles (A), expected heterozygosity (He), multilocus linkage disequilibrium (LD) and genetic differentiation, and were associated with temporal and geospatial variances. Results: A total of 747 (65%) samples were successfully genotyped. Moderate to high genetic diversity (mean He = 0.74 ± 0.03) was observed in the parasite population. This was ascribed to high allelic richness (mean A = 12.2 ± 1.2). The majority of samples (99%) had unique multi-locus genotypes, indicating high genetic diversity in the sample set. Complex infections were observed in 66% of samples (mean MOI = 2.13 ± 0.04), with 33% of infections showing high within-host diversity as described by the Fws metric. Low, but significant LD (standardised index of association, ISA = 0.08, P < 0.001) was observed that indicates recombination of distinct clones. Limited impact of temporal (FST range -0.00005 to 0.0003) and spatial (FST = –0.028 to 0.023) variation on genetic diversity existed during the sampling timeframe and study sites respectively. Conclusion: Consistent with the Vhembe District’s classification as a 'high' transmission setting within South Africa, P. falciparum diversity in the area was moderate to high and complex. This study showed that genetic diversity within the parasite population reflects the continued residual transmission observed in the Vhembe District. This data can be used as a reference point for the assessment of the effectiveness of on-going interventions over time, the identification of imported cases and/or outbreaks, as well as monitoring for the potential spread of antimalarial drug resistance.

Multi-locus genotyping reveals established endemicity of a geographically distinct Plasmodium vivax population in Mauritania, West Africa

Background Plasmodium vivax has been recently discovered as a significant cause of malaria in Mauritania, although very rare elsewhere in West Africa. It has not been known if this is a recently introduced or locally remnant parasite population, nor whether the genetic structure reflects epidemic or endemic transmission. Methodology/Principal findings To investigate the P. vivax population genetic structure in Mauritania and compare with populations previously analysed elsewhere, multi-locus genotyping was undertaken on 100 clinical isolates, using a genome-wide panel of 38 single nucleotide polymorphisms (SNPs), plus seven SNPs in drug resistance genes. The Mauritanian P. vivax population is shown to be genetically diverse and divergent from populations elsewhere, indicated consistently by genetic distance matrix analysis, principal components analyses, and fixation indices. Only one isolate had a genotype clearly indicating recent importation, from a southeast Asian source. There was no linkage disequilibrium in the local parasite population, and only a small number of infections appeared to be closely genetically related, indicating that there is ongoing genetic recombination consistent with endemic transmission. The P. vivax diversity in a remote mining town was similar to that in the capital Nouakchott, with no indication of local substructure or of epidemic population structure. Drug resistance alleles were virtually absent in Mauritania, in contrast with P. vivax in other areas of the world. Conclusions/Significance The molecular epidemiology indicates that there is long-standing endemic transmission that will be very challenging to eliminate. The virtual absence of drug resistance alleles suggests that most infections have been untreated, and that this endemic infection has been more neglected in comparison to P. vivax elsewhere.

Population genomics of Plasmodium vivax in Panama to assess the risk of case importation on malaria elimination

Malaria incidence in Panama has plateaued in recent years in spite of elimination efforts, with almost all cases caused by Plasmodium vivax. Notwithstanding, overall malaria prevalence remains low (fewer than 1 case per 1000 persons). We used selective whole genome amplification to sequence 59 P. vivax samples from Panama. The P. vivax samples were collected from two periods (2007–2009 and 2017–2019) to study the population structure and transmission dynamics of the parasite. Imported cases resulting from increased levels of human migration could threaten malaria elimination prospects, and four of the samples evaluated came from individuals with travel history. We explored patterns of recent common ancestry among the samples and observed that a highly genetically related lineage (termed CL1) was dominant among the samples (47 out of 59 samples with good sequencing coverage), spanning the entire period of the collection (2007–2019) and all regions of the country. We also found a second, smaller clonal lineage (termed CL2) of four parasites collected between 2017 and 2019. To explore the regional context of Panamanian P. vivax we conducted principal components analysis and constructed a neighbor-joining tree using these samples and samples collected worldwide from a previous study. Three of the four samples with travel history clustered with samples collected from their suspected country of origin (consistent with importation), while one appears to have been a result of local transmission. The small number of Panamanian P. vivax samples not belonging to either CL1 or CL2 clustered with samples collected from Colombia, suggesting they represent the genetically similar ancestral P. vivax population in Panama or were recently imported from Colombia. The low diversity we observe in Panama indicates that this parasite population has been previously subject to a severe bottleneck and may be eligible for elimination. Additionally, while we confirmed that P. vivax is imported to Panama from diverse geographic locations, the lack of impact from imported cases on the overall parasite population genomic profile suggests that onward transmission from such cases is limited and that imported cases may not presently pose a major barrier to elimination.