Parasite Populations Research Articles

Declining genetic polymorphism of the C-terminus Merozoite Surface Protein-1 amidst increased Plasmodium knowlesi transmission in Thailand

Abstract Background Recent reports from Thailand reveal a substantial surge in Plasmodium knowlesi cases over the past decades, with a more than eightfold increase in incidence by 2023 compared to 2018. This study investigates temporal changes in genetic polymorphism associated with the escalating transmission of P. knowlesi malaria in Thailand over time using the prominent vaccine candidate, pkmsp1 as a marker. Methods Twenty-five P. knowlesi samples collected in 2018–2023 were sequenced for the 42-kDa region of pkmsp1 and compared with 24 retrieved sequences in 2000–2009, focusing on nucleotide diversity, natural selection, recombination rate, and population differentiation. Results Seven unique haplotypes were identified in recent samples, compared to 15 in earlier samples. Nucleotide and haplotype diversity were lower in recent samples (π = 0.016, Hd = 0.817) than in earlier samples (π = 0.018, Hd = 0.942). Significantly higher synonymous substitution rates were observed in both sample sets (dS–dN = 2.77 and 2.43, p < 0.05), indicating purifying selection and reduced genetic diversity over time. Additionally, 8 out of 17 mutation points were located on predicted B-cell epitopes, suggesting an adaptive response by the parasites to evade immune recognition. Population differentiation analysis using the fixation index (Fst) revealed high genetic differentiation between parasite populations in central and southern Thailand or Malaysia. Conversely, the relatively lower Fst value between southern Thailand and Malaysia suggests a closer genetic relationship, possibly reflecting historical gene flow. Conclusion This study highlights a decline in genetic diversity and evidence of purifying selection associated with the recently increased incidence of P. knowlesi malaria in Thailand. The minor genetic differentiation between P. knowlesi populations from southern Thailand and Malaysia suggests a shared recent ancestry of these parasites and underscores the need for coordinated efforts between the two countries for the elimination of P. knowlesi.

Genetic and biological insights into Hydatigera taeniaeformis in invasive black rats from southern Chile

IntroductionThis study investigates the genetic variability of Hydatigera taeniaeformis in black rats (Rattus rattus), a common tapeworm that infects cats and rodents worldwide. Despite its widespread presence and zoonotic potential, little is known about the genetic diversity of this parasite in the Americas.MethodsWe conducted DNA barcoding analysis using mitochondrial cox1 gene sequences using samples collected from 171 invasive wild black rats, captured in the temperate rainforest of Southern Chile. We also included two adult parasites isolated from road killed Kodkods (Leopardus guigna), a small felid species native to the Americas.ResultsOur findings revealed only two haplotypes, suggesting low genetic variability in a parasite that arrived in the Americas with the Spanish colonization.DiscussionThese haplotypes are more closely related to parasite populations from Peru, Africa, Australia, and Europe, suggesting an origin linked to the Spanish colonization, possibly from North Africa via the Canary Islands. The study also analyzed infection rates, parasite size, and their correlation with host body size, age, and weight, revealing significant patterns. These results provide new insights into the biogeography and genetic diversity of H. taeniaeformis in a new geographical area, enhancing our understanding of its evolutionary history.

Effects of fasting on the interplay between temperature and Trypanosoma cruzi infection on the life cycle of the Chagas disease vector Rhodnius prolixus

Rhodnius prolixus, a triatomine insect, is one of the most important vectors of Chagas disease in South America. Its interaction with the parasite Trypanosoma cruzi, the causative agent of this disease, is known to be deeply affected by ambient temperature and the nutritional status of the insect vector. In this study, we investigated how starvation affects the life cycle of R. prolixus and the population dynamics of the parasite inside the intestine of the vector at four temperatures ranging from 24°C to 30°C. The weights and molting times of chronically infected and uninfected insects were monitored through repeated 30-day fasting periods from first instar to adult stage, assessing their capacity to retain blood meal weight between developmental stages and tracking parasite concentrations in their urine. Our results demonstrate that ambient temperature is a crucial factor affecting the resistance of R. prolixus to starvation, as survival, body weight, and weight retention greatly decreased in high temperature treatments. Furthermore, we showed that temperature significantly influenced whether T. cruzi established an infection in early instars, with few insects developing infections at the lowest and highest temperature treatments. Additionally, we discovered that a fasting period of 30 days induces a steady decrease in parasite populations in the vector over its lifetime. Infection by T. cruzi had no effect on the survival, molting time, and nutritional factors monitored in our protocol. Our results highlight the importance of nutrition as a determining factor for the development of the vector and the parasite, providing valuable insights for elucidating the complex interplay between temperature and nutrition in shaping the epidemiology of Chagas disease in a changing climate.

Parasites in a Changing World: Troublesome or in Trouble?

There are plenty of reasons to believe that parasite populations will respond to biodiversity loss, warming, pollution, and other forms of global change. But will global change enhance transmission, increasing the incidence of troublesome parasites that put people, livestock, and wildlife at risk? Or will parasite populations decline in abundance—or even become extinct—suggesting trouble on the horizon for parasite biodiversity? Here, I explain why answers have thus far eluded us and suggest new lines of research that would advance the field. Data collected to date suggest that parasites can respond to global change with increases or decreases in abundance, depending on the driver and the parasite. The future will certainly bring outbreaks of some parasites, and these should be addressed to protect human and ecosystem health. But troublesome parasites should not consume all of our research effort, because this changing world contains many parasite species that are in trouble.

Low nucleotide diversity of the Plasmodium falciparum AP2-EXP2 gene among clinical samples from Ghana.

PfAP2-EXP2 is located within chromosome 6 of Plasmodium falciparum recently identified to be undergoing an extensive selective sweep in West African isolates. The gene encoding this transcription factor, PfAP2-EXP2, is essential and thus likely subject to purifying selection that limits variants in the parasite population despite its genomic location. 72 Plasmodium falciparum field samples and 801 clinical sequences from the Pf6 MalariaGEN dataset of Ghanaian origin, were integrated and analysed. A total of 14 single nucleotide variants of which 5 were missense variants, were identified after quality checks and filtering. Except for one, all identified variants were rare among the clinical samples obtained in this study (Minor allelic frequency < 0.01). Further results revealed a considerably low dN/dS value (0.208) suggesting the presence of purifying selection. Further, all the mutant amino acids were wildtype residues in AP2-EXP2 orthologous proteins-tentatively suggesting a genus-level conservation of amino acid residues. Computational analysis and predictions corroborated these findings. Despite the recent extensive selective sweep within chromosome 6 of West African isolates, PfAP2-EXP2 of Ghanaian origin exhibits low nucleotide diversity and very low dN/dS consistent with purifying selection acting to maintain the function of an essential gene. The conservation of AP2-EXP2 is an important factor that makes it a potential drug target.

Emerging threat of artemisinin partial resistance markers (pfk13 mutations) in Plasmodium falciparum parasite populations in multiple geographical locations in high transmission regions of Uganda

BackgroundArtemisinin-based combination therapy (ACT) is currently recommended for treatment of uncomplicated malaria. However, the emergence and spread of partial artemisinin resistance threatens their effectiveness for malaria treatment in sub-Saharan Africa where the burden of malaria is highest. Early detection and reporting of validated molecular markers (pfk13 mutations) in Plasmodium falciparum is useful for tracking the emergence and spread of partial artemisinin resistance to inform containment efforts.MethodsGenomic surveillance was conducted at 50 surveillance sites across four regions of Uganda in Karamoja, Lango, Acholi and West Nile from June 2021 to August 2023. Symptomatic malaria suspected patients were recruited and screened for presence of parasites. In addition, dried blood spots (DBS) were collected for parasite genomic analysis with PCR and sequencing. Out of 563 available dried blood spots (DBS), a random subset of 240 P. falciparum mono-infections, confirmed by a multiplex PCR were selected and used for detecting the pfk13 mutations by Sanger sequencing using Big Dye Terminator method. Regional variations in the proportions of pfk13 mutations were assessed using the chi square or Fisher’s exact tests while Kruskal–Wallis test was used to compare absolute parasite DNA levels between wild type and mutant parasites.ResultsOverall, 238/240 samples (99.2%) contained sufficient DNA and were successfully sequenced. Three mutations were identified within the sequenced samples; pfk13 C469Y in 32/238 (13.5%) samples, pfk13 A675V in 14/238 (5.9%) and pfk13 S522C in (1/238 (0.42%) samples across the four surveyed regions. The prevalence of pfk13 C469Y mutation was significantly higher in Karamoja region (23.3%) compared to other regions, P = 0.007. The majority of parasite isolates circulating in West Nile are of wild type (98.3), P = 0.002. Relative parasite DNA quantity did not differ in samples carrying the wild type, C469Y and A675V alleles (Kruskal–Wallis test, P = 0.6373).ConclusionDetection of validated molecular markers of artemisinin partial resistance in multiple geographical locations in this setting provides additional evidence of emerging threat of artemisinin partial resistance in Uganda. In view of these findings, periodic genomic surveillance is recommended to detect and monitor levels of pfk13 mutations in other regions in parallel with TES to assess potential implication on delayed parasite clearance and associated treatment failure in this setting. Future studies should consider identification of potential drivers of artemisinin partial resistance in the different malaria transmission settings in Uganda.

Genetic diversity of Plasmodium vivax population in northeast Myanmar assessed by amplicon sequencing of PvMSP1 and PvMSP3α

This study aimed to assess the baseline genetic diversity of the Plasmodium vivax population in an endemic area of northeast Myanmar at the onset of the malaria elimination campaign in the Greater Mekong Subregion. We genotyped 125 P. vivax clinical samples at two merozoite surface protein loci, PvMSP1 and PvMSP3α, by amplicon deep sequencing. Our study revealed that the parasite population in this region was highly diverse, identifying 60 PvMSP1 and 98 PvMSP3α haplotypes, with haplotype diversity of 0.929 and 0.944, respectively. Remarkably, 97.6% (122/125) of the patients harbored multiclonal infections with a mean multiplicity of infection of 4.18, indicating a relatively high transmission intensity. Neutrality tests and network analysis suggested a recent parasite population expansion, consistent with the concurrent malaria outbreak in the region. These findings underscore the existence of a highly diverse P. vivax population at the China-Myanmar border, highlighting the need for effective malaria control strategies to achieve the goal of regional malaria elimination.

Use of the Micro-Agar Larval Development Test to Differentiate Resistant and Susceptible Cooperia spp. Isolates in Cattle Within the Context of Parasite Population Replacement

Gastrointestinal nematode infections are a global concern in grazing cattle production systems, even more so due to the widespread problem of anthelmintic resistance. In response, early anthelmintic resistance detection methods, such as the micro-agar larval development test (MALDT), and parasite management strategies, such as the replacement of resistant parasite populations with susceptible ones, have been developed. This study aimed to characterize ivermectin-susceptible and -resistant isolates of Cooperia spp. using MALDT in the context of a parasite population replacement strategy. Three Cooperia spp. field isolates were evaluated: a susceptible one (Coop-S), a resistant one (Coop-R), and a post-replacement one (Coop-PR). The MALDT was performed in 96-well plates with 12 known concentrations of eprinomectin (EPR) on an agar base. Each test was performed in quadruplicate. Data analysis included nonlinear regression to determine EC50, EC90, and EC99 values, resistance ratios (RRs), and R2. The results showed clear differentiation between the isolates, with RR values of 5.78 and 1.28 for Coop-R and Coop-PR, respectively, compared to Coop-S. The MALDT proved to be a reliable tool for differentiating ivermectin-susceptible from ivermectin-resistant isolates of Cooperia spp., and future evaluations of this test in mixed nematode populations are recommended for routine diagnosis of anthelmintic resistance.

Tissue spaces are reservoirs of antigenic diversity for Trypanosoma brucei.

The protozoan parasite Trypanosoma brucei evades clearance by the host immune system through antigenic variation of its dense variant surface glycoprotein (VSG) coat, periodically 'switching' expression of the VSG using a large genomic repertoire of VSG-encoding genes1-6. Recent studies of antigenic variation in vivo have focused near exclusively on parasites in the bloodstream6-8, but research has shown that many, if not most, parasites reside in the interstitial spaces of tissues9-13. We sought to explore the dynamics of antigenic variation in extravascular parasite populations using VSG-seq7, a high-throughput sequencing approach for profiling VSGs expressed in populations of T. brucei. Here we show that tissues, not the blood, are the primary reservoir of antigenic diversity during both needle- and tsetse bite-initiated T. brucei infections, with more than 75% of VSGs found exclusively within extravascular spaces. We found that this increased diversity is correlated with slower parasite clearance in tissue spaces. Together, these data support a model in which the slower immune response in extravascular spaces provides more time to generate the antigenic diversity needed to maintain a chronic infection. Our findings reveal the important role that extravascular spaces can have in pathogen diversification.

Genetic polymorphism of merozoite surface protein 1 and merozoite surface protein 2 in the Vietnam Plasmodium falciparum population

BackgroundPlasmodium falciparum merozoite surface proteins 1 (PfMSP1) and 2 (PfMSP2) are potential candidates for malaria vaccine development. However, the genetic diversity of these genes in the global P. falciparum population presents a significant challenge in developing an effective vaccine. Hence, understanding the genetic diversity and evolutionary trends in the global P. falciparum population is crucial.MethodsThis study analyzed the genetic variations and evolutionary changes of pfmsp1 and pfmsp2 in P. falciparum isolates from the Central Highland and South-Central regions of Vietnam. DNASTAR and MEGA7 programs were utilized for analyses. The polymorphic nature of global pfmsp1 and pfmsp2 was also investigated.ResultsA total of 337 sequences of pfmsp1 and 289 sequences of pfmsp2 were obtained. The pfmsp1 and pfmsp2 from Vietnam revealed a higher degree of genetic homogeneity compared to those from other malaria-endemic countries. Remarkably, the allele diversity patterns of Vietnam pfmsp1 and pfmsp2 differed significantly from those of neighboring countries in the Greater Mekong Subregion. Declines in allele diversity and polymorphic patterns of Vietnam pfmsp1 and pfmsp2 were observed.ConclusionsThe Vietnam P. falciparum population might be genetically isolated from the parasite populations in other neighboring GMS countries, likely due to geographical barriers and distinct evolutionary pressures. Furthermore, bottleneck effects or selective sweeps may have contributed to the genetic homogeneity of Vietnam pfmsp1 and pfmsp2.

Genetic diversity and molecular evolution of the internal transcribed spacer regions (ITS1–5.8S–ITS2) of Babesia vogeli

Canine babesiosis, a severe haemoparasitic disease caused by Babesia species, has a significant global presence and can be fatal if left untreated. The current study was aimed to perform the population genetic characterization of B. vogeli on the basis of the internal transcribed spacer regions (ITS1–5.8S–ITS2). A maximum likelihood tree constructed with the Hasegawa-Kishino-Yano model grouped all sequences into a single major clade (BvG1), with the exception of a Taiwanese isolate (EF186914), which branched separately. This Taiwanese isolate represented a novel genotype (BvG2) identified in the present study. Nucleotide sequences (n = 62) exhibited 92.5–100 % nucleotide identity among themselves. However, the BvG1 and BvG2 genotypes shared a lower identity of 92.5–93.8 % between them. Notably, the newly generated Indian sequences (n = 21) demonstrated a high degree of homology, with 98.3–100 % identity. Alignment of the nucleotide sequences revealed 58 variations across the dataset. Additionally, 32 sites exhibited variation within the BvG1 genotype, while 56 sites differed between BvG1 and BvG2 genotypes. Within different B. vogeli populations, the nucleotide diversity (π) was low, but the haplotype diversity (Hd) was high. The haplotype diversity of the Indian population, BvG1 genotype, and the combined dataset was ∼0.8 suggesting a high haplotype diversity. The median-joining haplotype network displayed a total of 21 haplotypes, out of which six haplotypes consisted of more than one sequence (2–25 sequences). Haplotype distribution showed significant geographical structuring, with most haplotypes confined to a single country. Only two haplotypes (9.52 %; Hap_1 and Hap_4) were shared between countries, whereas 19 haplotypes (90.48 %) were country-specific. Hap_1, Hap_6, and Hap_4 were the most representative haplotypes, comprising 25, 10, and four sequences, respectively. India exhibited the highest number of haplotypes (h = 13) followed by China (h = 4), the United States of America (h = 3), Taiwan and Tunisia (h = 2), and Thailand (h = 1). Both location-wise and genotype-wise median joining haplotype networks clustered the haplotypes in two groups, representing two distinct genotypes (BvG1 and BvG2). The B. vogeli populations between Thailand and Tunisia exhibited the highest genetic differentiation (FST = 0.80) with a low gene flow (Nm = 0.125) between them. Results of AMOVA revealed a higher genetic variation within populations (69.43 %) as compared to the variation between them (30.57 %). Neutrality indices and the mismatch distributions of the Indian population and the overall dataset of B. vogeli indicated a constant population size to population expansion and population expansion, respectively, with the presence of two distinct genotypes. These data provide information about parasite population genetics and highlight the importance of starting a long-term molecular surveillance program. In conclusion, a high genetic diversity along with the presence of two distinct genotypes of B. vogeli were observed on the basis of internal transcribed spacer regions (ITS1–5.8S–ITS2).

Abundant genetic variation is retained in many laboratory schistosome populations.

Schistosomes are obligately sexual blood flukes that can be maintained in the laboratory using freshwater snails as intermediate and rodents as definitive hosts. The genetic composition of laboratory schistosome populations is poorly understood: whether genetic variation has been purged due to serial inbreeding or retained is unclear. We sequenced 19 - 24 parasites from each of five laboratory Schistosoma mansoni populations and compared their genomes with published exome data from four S. mansoni field populations. We found abundant genomic variation (0.897 - 1.22 million variants) within laboratory populations: these retained on average 49% (π = 3.27e-04 - 8.94e-04) of the nucleotide diversity observed in the four field parasite populations (π = 1.08e-03 - 2.2e-03). However, the pattern of variation was very different in laboratory and field populations. Tajima's D was positive in all laboratory populations except SmBRE, indicative of recent population bottlenecks, but negative in all field populations. Current effective population size estimates of laboratory populations were lower (2 - 258) compared to field populations (3,174 - infinity). The distance between markers at which linkage disequilibrium (LD) decayed to 0.5 was longer in laboratory populations (59 bp - 180 kb) compared to field populations (9 bp - 9.5 kb). SmBRE was the least variable; this parasite also shows low fitness across the lifecycle, consistent with inbreeding depression. The abundant genetic variation present in most laboratory schistosome populations has several important implications: (i) measurement of parasite phenotypes, such as drug resistance, using laboratory parasite populations will determine average values and underestimate trait variation; (ii) genome-wide association studies (GWAS) can be conducted in laboratory schistosome populations by measuring phenotypes and genotypes of individual worms; (iii) genetic drift may lead to divergence in schistosome populations maintained in different laboratories. We conclude that the abundant genetic variation retained within many laboratory schistosome populations can provide valuable, untapped opportunities for schistosome research.

Presence of Plasmodium falciparum strains with artemisinin-resistant K13 mutation C469Y in Busia County, Western Kenya

Malaria remains a key health and economic problem, particularly in sub-Saharan Africa. The emergence of artemisinin drug resistance (ART-R) parasite strains poses a serious threat to the control and elimination of this scourge. This is because artemisinin-based combination therapies (ACTs) remain the first-line treatment in the majority of malaria-endemic regions in Sub-Saharan Africa. Certain single-nucleotide polymorphisms in the propeller domains of Plasmodium falciparum Kelch 13 protein (K13) have been associated with delayed parasite clearance in vivo and in vitro. These mutations serve as vital molecular markers for tracking the emergence and dispersion of resistance. Recently, there have been increasing reports of the emergence and spread of P. falciparum ART-R parasites in the Eastern Africa region. This necessitates continued surveillance to best inform mitigation efforts. This study investigated the presence of all reported mutations of K13 propeller domains in the parasite population in Busia County, Kenya, a known malaria-endemic region. Two hundred twenty-six participants with microscopically confirmed uncomplicated malaria were recruited for this study. They were treated with artemether–lumefantrine under observation for the first dose, and microscopic examination was repeated 1 day later after ensuring the participants had taken the second and third doses. P. falciparum DNA from all samples underwent targeted amplification of the K13 gene using a semi-nested PCR approach, followed by Sanger sequencing. The recently validated ART-R K13 mutation C469Y was identified in three samples. These three samples were among 63 samples with a low reduction in parasitemia on day 1, suggesting day 1 parasitemia reduction rate is a useful parameter to enrich the ART-R parasites for further analysis. Our findings highlight the need for continuous surveillance of ART-R in western Kenya and the region to determine the spread of ART-R and inform containment.

Genetic diversity in the Plasmodium falciparum next-generation blood stage vaccine candidate antigen PfCyRPA in Senegal.

The Plasmodium falciparum cysteine-rich protective antigen (PfCyRPA) is a promising target as a next-generation blood-stage malaria vaccine and together with PCRCR complex members, the reticulocyte binding-like homologous protein 5 (PfRh5) and the Rh5-interacting protein (PfRipr), are currently being evaluated in clinical trials. PfCyRPA is essential for merozoite invasion and appears to be highly conserved within the P. falciparum parasite populations. Here, we used a targeted deep amplicon next-generation sequencing approach to assess the breadth of PfCyRPA genetic diversity in 95 P. falciparum clinical isolates from Kédougou, an area with a high seasonal malaria transmission in Senegal. Our data show the dominant prevalence of PfCyRPA wild type reference allele, while we also identify a total of 15 single nucleotide polymorphisms (SNPs). Of these, only five have previously been reported, while the majority of the SNPs were present as singletons within our sampled population. Moreover, the variant read frequency of the identified SNPs varied from 2.6 to 100%, while the majority of the SNPs were present at frequencies greater than 25% in polygenomic samples. We also applied a structure-based modelling approach to thread these SNPs onto PfCyRPA crystal structures and showed that these polymorphisms have different predicted functional impacts on the interactions with binding partner PfRH5 or neutralizing antibodies. Our prediction revealed that the majority of these SNPs have minor effects on PfCyRPA antibodies, while others alter its structure, stability, or interaction with PfRH5. Altogether, our present findings reveal conserved PfCyRPA epitopes which will inform downstream investigations on next-generation structure-guided malaria vaccine design.

Application of a new highly multiplexed amplicon sequencing tool to evaluate Plasmodium falciparum antimalarial resistance and relatedness in individual and pooled samples from Dschang, Cameroon.

Resistance to antimalarial drugs remains a major obstacle to malaria elimination. Multiplexed, targeted amplicon sequencing is being adopted for surveilling resistance and dissecting the genetics of complex malaria infections. Moreover, genotyping of parasites and detection of molecular markers drug resistance in resource-limited regions requires open-source protocols for processing samples, using accessible reagents, and rapid methods for processing numerous samples including pooled sequencing. P lasmodium falciparum S treamlined M ultiplex A ntimalarial R esistance and R elatedness T esting ( Pf -SMARRT) is a PCR-based amplicon panel consisting of 15 amplicons targeting antimalarial resistance mutations and 9 amplicons targeting hypervariable regions. This assay uses oligonucleotide primers in two pools and a non-proprietary library and barcoding approach. We evaluated Pf -SMARRT using control mocked dried blood spots (DBS) at varying levels of parasitemia and a mixture of 3D7 and Dd2 strains at known frequencies, showing the ability to genotype at low parasite density and recall within-sample allele frequencies. We then piloted Pf -SMARRT to genotype 100 parasite isolates collected from uncomplicated malaria cases at three health facilities in Dschang, Western Cameroon. Antimalarial resistance genotyping showed high levels of sulfadoxine-pyrimethamine resistance mutations, including 31% prevalence of the DHPS A613S mutation. No K13 candidate or validated artemisinin partial resistance mutations were detected, but one low-level non-synonymous change was observed. Pf -SMARRT's hypervariable targets, used to assess complexity of infections and parasite diversity and relatedness, showed similar levels and patterns compared to molecular inversion probe (MIP) sequencing. While there was strong concordance of antimalarial resistance mutations between individual samples and pools, low-frequency variants in the pooled samples were often missed. Overall, Pf -SMARRT is a robust tool for assessing parasite relatedness and antimalarial drug resistance markers from both individual and pooled samples. Control samples support that accurate genotyping as low as 1 parasite per microliter is routinely possible. Malaria remains a critical global public health problem. Antimalarial drug resistance has repeatedly undermined control and the emergence of artemisinin partial resistance in Africa is the latest major challenge. Malaria molecular surveillance (MMS) has emerged as a powerful tool to monitor molecular markers of resistance and changes in the parasite population. Streamlined methods are needed that can be readily adopted in endemic countries. We developed P lasmodium falciparum S treamlined M ultiplex A ntimalarial R esistance and R elatedness T esting ( Pf -SMARRT), a multiplex amplicon deep sequencing approach that uses easily accessible products without proprietary steps and can be sequenced on any Illumina sequencer. We validated this tool using controls, including mocked dried blood spots, and then implemented it to evaluate resistance and parasite relatedness among 100 samples from Cameroon. The assay was able to reliably assess the within-sample allele frequency of antimalarial resistance markers and discriminate strains within and between individuals. We also evaluated a more cost-effective surveillance approach for antimalarial resistance polymorphisms using pooled samples. While within-pool frequencies of mutations were accurate in pools with higher numbers of samples, this resulted in the loss of the ability to detect variants uncommon in the pool. Overall Pf- SMARRT provides a new protocol for conducting MMS that is easily implementable in Africa.

Astrocytes Can Be Key Players Against Cerebral Leishmaniasis: InVitro Co-Culture Model for the Assessment of Infection.

Leishmaniasis is a neglected tropical disease, caused by protozoan parasites of Leishmania (L.), and is transmitted by bite of phlebotomine sandflies. There are several studies on central nervous system infection to indicate that Leishmania can cross the blood-brain barrier, resulting in neurological manifestations, known as "cerebral leishmaniasis." This study highlighted the notions: (i) polarisation of bone marrow-derived macrophages (BMDM) incubated following stimulation with lipopolysaccharide (LPS) or soluble Leishmania antigen (SLA), (ii) quantification of parasites within co-culture of Leishmania-infected macrophages, and astrocytes, and (iii) effect of interferon-gamma (IFN-γ) on the infection rate of co-culture populations. Accordingly, 83% of overall macrophage population was identified on day 7 for CD11b and F4/80 macrophage markers. Flow cytometry analysis revealed significant increases in CD11b and F4/80 surface markers in LPS and SLA-stimulated BMDMs at 24 h, compared to untreated cells. TNF-α levels increased significantly in both LPS and SLA-treated BMDMs after 48 h. Additionally, SLA treatment induced a more elongated, spindle-like shape in the cells, indicative of M2 macrophage polarisation over the M1 phenotype. When non-infected astrocytes with/without stimulation with IFN-γ before co-culture, gp63 FITC-labelled parasite populations (%) in co-culture decreased to 25% at 72 h, thus indicating a lower infection rate in a time-dependent manner. IFN-γ and IL-6 levels significantly increased to 71.66 ± 3.51 and 184 ± 14.42 pg/mL, resulting in the inflammatory response in the co-culture system at 48 h (p ≤ 0.0001), when compared to the control (30 ± 2.52 pg/mL for IFN-γ and 8.66 ± 2.37 pg/mL for IL-6) at 0 h of the incubation. It is the first study to emphasize the communication between Leishmania-infected macrophages and astrocytes regarding Leishmania parasite load. The results suggest that astrocytes can lead to the reduction in Leishmania parasites, thereby controlling the incidence of cerebral leishmaniasis.

Gastrointestinal parasites of wild Bornean orang-utans (Pongo pygmaeus) in a habitat affected by wildfire smoke

Wildfires are becoming increasingly frequent and severe in the tropical peatlands of Southern Borneo, with major consequences for both wildlife and people that inhabit them. Burning peat releases vast amounts of toxic smoke that, when inhaled, can cause a plethora of health-related issues. With some of the largest remaining populations of Bornean orang-utans (Pongo pygmaeus) being found in peatland forests, wildfires are becoming one of the greatest threats to this Critically Endangered ape, yet the effects of the toxic smoke on their health are unknown. Wildfire has long been known to influence wildlife disease by reducing populations of free-living parasites in the environment (thus altering host exposure), and there is a growing body of evidence to suggest that smoke may affect host susceptibility to such parasites by diminishing physiological condition and immune function. In this study, we investigated parasitic nematode infections in wild Bornean orang-utans inhabiting a drained peatland forest prone to fire and smoke. We identified four gastrointestinal nematode taxa that varied significantly in their prevalence and intensity. Overall prevalence of nematode infection was high, but intensity was relatively low. We present some evidence for an increase the prevalence and intensity of certain nematode taxa after the wildfire smoke event. Namely, an increase in the prevalence of Enterobius spp. and Trichuris spp., and an increase in the intensity of hookworm infections, after the smoke period. We identify the need for multi-year, multi-fire event research to increase sample sizes, along with measuring endocrinological markers of physiological condition, in tandem with parasitological monitoring, to gain a more comprehensive understanding of the impacts of environmental changes on orang-utan health.

Form-assortative mating behaviors of individuals from parasitic and non-parasitic populations of Arctic lamprey (Lethenteron camtschaticum)

Abstract Evolutionary theory predicts that assortative mating is crucial for sympatric speciation by generating reproductive isolation between diverging populations. Here, we investigate the potential of form-assortative mating, an assumed mating pattern in lampreys, for sympatric speciation. By continuously recording mating activity between anadromous and freshwater-resident forms of L. camtschaticum that greatly differ in body size, we show that lampreys tend to mate with individuals of similar size in experimental conditions. However, we highlight that this pattern does not result from a choice of same-form partner but is the result of the simultaneous action of a preference of males – whatever their size – for large anadromous females, a higher competitive ability of aggressive males and physical constraints on heteroform pairs. Moreover, we do not advocate that sympatric speciation, as the sole consequence of form-assortative mating through sexual selection, is a plausible mechanism for the diversification of lampreys as a significant number of sneaking behaviors were observed in freshwater-resident males toward large anadromous females. Broader attention should be given to mechanisms other than sexual selection that may lead to form-assortative mating, such as variations in spatial or temporal distribution of alternative forms during reproductive season.

Emerging public health strategies in malaria control: innovations and implications

Malaria remains a significant global health challenge, particularly in regions with limited resources and tropical climates. Despite extensive efforts, the disease continues to cause significant morbidity and mortality, with approximately 229 million cases and 409,000 deaths reported in 2020. However, recent years have seen promising advancements in public health strategies aimed at malaria control and elimination. Technological advancements have played a crucial role in improving malaria control efforts. Genomic surveillance techniques enable the monitoring of malaria parasite populations, aiding in the detection of drug resistance and informing targeted interventions. Additionally, innovative diagnostic technologies, such as rapid diagnostic tests (RDTs) and molecular assays, have enhanced the speed and accuracy of malaria diagnosis, facilitated prompt treatment and reduced transmission. These tools are instrumental in achieving the World Health Organization’s (WHO) goals of reducing malaria cases and deaths by at least 90% by 2030. Novel vector control methods offer innovative approaches to reduce malaria transmission. Insecticide-treated nets (ITNs) and indoor residual spraying (IRS) remain foundational strategies, with advancements including the development of next-generation insecticides and long-lasting insecticidal nets (LLINs). Furthermore, genetic modification of mosquitoes, such as gene drive technology, holds promise for reducing mosquito populations and interrupting malaria transmission. These vector control innovations complement other strategies, contributing to comprehensive malaria control efforts aimed at achieving sustainable disease reduction and eventual elimination.

Rapid phenotypic and genotypic change in a laboratory schistosome population.

Genomic analysis has revealed extensive contamination among laboratory-maintained microbes including malaria parasites, Mycobacterium tuberculosis and Salmonella spp. Here, we provide direct evidence for recent contamination of a laboratory schistosome parasite population, and we investigate its genomic consequences. The Brazilian Schistosoma mansoni population SmBRE has several distinctive phenotypes, showing poor infectivity, reduced sporocysts number, low levels of cercarial shedding and low virulence in the intermediate snail host, and low worm burden and low fecundity in the vertebrate rodent host. In 2021 we observed a rapid change in SmBRE parasite phenotypes, with a~10x increase in cercarial production and ~ 4x increase in worm burden. To determine the underlying genomic cause of these changes, we sequenced pools of SmBRE adults collected during parasite maintenance between 2015 and 2023. We also sequenced another parasite population (SmLE) maintained alongside SmBRE without phenotypic changes. While SmLE allele frequencies remained stable over the eight-year period, we observed sudden changes in allele frequency across the genome in SmBRE between July 2021 and February 2023, consistent with expectations of laboratory contamination. (i) SmLE-specific alleles rose in the SmBRE population from 0 to 41-46% across the genome between September and October 2021, documenting the timing and magnitude of the contamination event. (ii) After contamination, strong selection (s=~ 0.23) drove replacement of low fitness SmBRE with high fitness SmLE alleles. (iii) Allele frequency changed rapidly across the whole genome, except for a region on chromosome 4 where SmBRE alleles remained at high frequency. We were able to detect contamination in this case because SmBRE shows distinctive phenotypes. However, this would likely have been missed with phenotypically similar parasites. These results provide a cautionary tale about the importance of tracking the identity of parasite populations, but also showcase a simple approach to monitor changes within populations using molecular profiling of pooled population samples to characterize fixed single nucleotide polymorphisms. We also show that genetic drift results in continuous change even in the absence of contamination, causing parasites maintained in different labs (or sampled from the same lab at different times) to diverge.