Parasite Clones Research Articles

Antibodies to PfEMP1 and variant surface antigens: Protection after controlled human malaria infection in semi-immune Kenyan adults

Acquisition of antibodies to Plasmodium falciparum variant surface antigens (VSA) expressed on infected red blood cells (iRBCs) is associated with naturally acquired immunity to malaria. We have previously shown that antibodies to VSA on iRBCs are associated with protection against parasite growth in the context of controlled human malaria infection (CHMI). This study explored whether antibodies to recombinant antigens derived from PfEMP1 domains were independently associated with protection during CHMI in semi-immune Kenyan adults. We used a multiplex bead assay to measure levels of IgG antibody against a panel of 27 recombinant PfEMP1 antigens derived from the PfEMP1 repertoire of the 3D7 parasite clone. We measured IgG levels in plasma samples collected from the CHMI participants before inoculation with Sanaria® PfSPZ Challenge, on the day of diagnosis, and 35 days post-inoculation. Univariable and multivariable Cox regression analysis was used to evaluate the relationship between the levels of antibodies to the antigens and CHMI outcome. We also adjusted for previous data including antibodies to VSA on iRBCs, and we assessed the kinetics of antibody acquisition to the different PfEMP1 recombinant antigens over time. All study participants had detectable antibodies to multiple PfEMP1 proteins before inoculation. All PfEMP1 antigens were associated with protection against parasite growth to the threshold criteria for treatment in CHMI, albeit with substantial collinearity. However, individual PfEMP1 antigens were not independently associated with protection following adjustment for breadth of reactivity to VSA on iRBCs and schizont extract. In addition, antibodies to PfEMP1 antigens derived from group B PfEMP1 were induced and sustained in the participants who could not control parasite growth. This study shows that the breadth of antibody response to VSA on iRBCs, and not to specific PfEMP1 antigens, is predictive of protection against malaria in CHMI.

Genotyping Plasmodium falciparum gametocytes using amplicon deep sequencing

BackgroundUnderstanding the dynamics of gametocyte production in polyclonal Plasmodium falciparum infections requires a genotyping method that detects distinct gametocyte clones and estimates their relative frequencies. Here, a marker was identified and evaluated to genotype P. falciparum mature gametocytes using amplicon deep sequencing.MethodsA data set of polymorphic regions of the P. falciparum genome was mined to identify a gametocyte genotyping marker. To assess marker resolution, the number of unique haplotypes in the marker region was estimated from 95 Malawian P. falciparum whole genome sequences. Specificity of the marker for detection of mature gametocytes was evaluated using reverse transcription-polymerase chain reaction of RNA extracted from NF54 mature gametocytes and rings from a non-gametocyte-producing strain of P. falciparum. Amplicon deep sequencing was performed on experimental mixtures of mature gametocytes from two distinct parasite clones, as well as gametocyte-positive P. falciparum field isolates to evaluate the quantitative ability and determine the limit of detection of the genotyping approach.ResultsA 400 bp region of the pfs230 gene was identified as a gametocyte genotyping marker. A larger number of unique haplotypes was observed at the pfs230 marker (34) compared to the sera-2 (18) and ama-1 (14) markers in field isolates from Malawi. RNA and DNA genotyping accurately estimated gametocyte and total parasite clone frequencies when evaluating agreement between expected and observed haplotype frequencies in gametocyte mixtures, with concordance correlation coefficients of 0.97 [95% CI: 0.92–0.99] and 0.92 [95% CI: 0.83–0.97], respectively. The detection limit of the genotyping method for male gametocytes was 0.41 pfmget transcripts/µl [95% CI: 0.28–0.72] and for female gametocytes was 1.98 ccp4 transcripts/µl [95% CI: 1.35–3.68].ConclusionsA region of the pfs230 gene was identified as a marker to genotype P. falciparum gametocytes. Amplicon deep sequencing of this marker can be used to estimate the number and relative frequency of parasite clones among mature gametocytes within P. falciparum infections. This gametocyte genotyping marker will be an important tool for studies aimed at understanding dynamics of gametocyte production in polyclonal P. falciparum infections.

Quantification of sporozoite expelling by Anopheles mosquitoes infected with laboratory and naturally circulating P. falciparum gametocytes

It is currently unknown whether all Plasmodium falciparum-infected mosquitoes are equally infectious. We assessed sporogonic development using cultured gametocytes in the Netherlands and naturally circulating strains in Burkina Faso. We quantified the number of sporozoites expelled into artificial skin in relation to intact oocysts, ruptured oocysts, and residual salivary gland sporozoites. In laboratory conditions, higher total sporozoite burden was associated with shorter duration of sporogony (p<0.001). Overall, 53% (116/216) of infected Anopheles stephensi mosquitoes expelled sporozoites into artificial skin with a median of 136 expelled sporozoites (interquartile range [IQR], 34–501). There was a strong positive correlation between ruptured oocyst number and salivary gland sporozoite load (ρ = 0.8; p<0.0001) and a weaker positive correlation between salivary gland sporozoite load and number of sporozoites expelled (ρ = 0.35; p=0.0002). In Burkina Faso, Anopheles coluzzii mosquitoes were infected by natural gametocyte carriers. Among salivary gland sporozoite positive mosquitoes, 89% (33/37) expelled sporozoites with a median of 1035 expelled sporozoites (IQR, 171–2969). Again, we observed a strong correlation between ruptured oocyst number and salivary gland sporozoite load (ρ = 0.9; p<0.0001) and a positive correlation between salivary gland sporozoite load and the number of sporozoites expelled (ρ = 0.7; p<0.0001). Several mosquitoes expelled multiple parasite clones during probing. Whilst sporozoite expelling was regularly observed from mosquitoes with low infection burdens, our findings indicate that mosquito infection burden is positively associated with the number of expelled sporozoites. Future work is required to determine the direct implications of these findings for transmission potential.

Quantification of sporozoite expelling by Anopheles mosquitoes infected with laboratory and naturally circulating P. falciparum gametocytes.

It is currently unknown whether all Plasmodium falciparum-infected mosquitoes are equally infectious. We assessed sporogonic development using cultured gametocytes in the Netherlands and naturally circulating strains in Burkina Faso. We quantified the number of sporozoites expelled into artificial skin in relation to intact oocysts, ruptured oocysts, and residual salivary gland sporozoites. In laboratory conditions, higher total sporozoite burden was associated with shorter duration of sporogony (p<0.001). Overall, 53% (116/216) of infected Anopheles stephensi mosquitoes expelled sporozoites into artificial skin with a median of 136 expelled sporozoites (interquartile range [IQR], 34-501). There was a strong positive correlation between ruptured oocyst number and salivary gland sporozoite load (ρ = 0.8; p<0.0001) and a weaker positive correlation between salivary gland sporozoite load and number of sporozoites expelled (ρ = 0.35; p=0.0002). In Burkina Faso, Anopheles coluzzii mosquitoes were infected by natural gametocyte carriers. Among salivary gland sporozoite positive mosquitoes, 89% (33/37) expelled sporozoites with a median of 1035 expelled sporozoites (IQR, 171-2969). Again, we observed a strong correlation between ruptured oocyst number and salivary gland sporozoite load (ρ = 0.9; p<0.0001) and a positive correlation between salivary gland sporozoite load and the number of sporozoites expelled (ρ = 0.7; p<0.0001). Several mosquitoes expelled multiple parasite clones during probing. Whilst sporozoite expelling was regularly observed from mosquitoes with low infection burdens, our findings indicate that mosquito infection burden is positively associated with the number of expelled sporozoites. Future work is required to determine the direct implications of these findings for transmission potential.

Whole genome sequencing identifies novel mutations in malaria parasites resistant to artesunate (ATN) and to ATN + mefloquine combination.

The global evolution of resistance to Artemisinin-based Combination Therapies (ACTs) by malaria parasites, will severely undermine our ability to control this devastating disease. Here, we have used whole genome sequencing to characterize the genetic variation in the experimentally evolved Plasmodium chabaudi parasite clone AS-ATNMF1, which is resistant to artesunate + mefloquine. Five novel single nucleotide polymorphisms (SNPs) were identified, one of which was a previously undescribed E738K mutation in a 26S proteasome subunit that was selected for under artesunate pressure (in AS-ATN) and retained in AS-ATNMF1. The wild type and mutated three-dimensional (3D) structure models and molecular dynamics simulations of the P. falciparum 26S proteasome subunit Rpn2 suggested that the E738K mutation could change the toroidal proteasome/cyclosome domain organization and change the recognition of ubiquitinated proteins. The mutation in the 26S proteasome subunit may therefore contribute to altering oxidation-dependent ubiquitination of the MDR-1 and/or K13 proteins and/or other targets, resulting in changes in protein turnover. In light of the alarming increase in resistance to artemisin derivatives and ACT partner drugs in natural parasite populations, our results shed new light on the biology of resistance and provide information on novel molecular markers of resistance that may be tested (and potentially validated) in the field.

Plasmodium falciparum transmission in the highlands of Ethiopia is driven by closely related and clonal parasites.

Malaria cases are frequently recorded in the Ethiopian highlands even at altitudes above 2000 m. The epidemiology of malaria in the Ethiopian highlands, and, in particular, the role of importation by human migration from the highly endemic lowlands is not well understood. We sequenced 187 Plasmodium falciparum samples from two sites in the Ethiopian highlands, Gondar (n = 159) and Ziway (n = 28), using a multiplexed droplet digital PCR (ddPCR)-based amplicon sequencing method targeting 35 microhaplotypes and drug resistance loci. Here, we characterize the parasite population structure and genetic relatedness. We identify moderate parasite diversity (mean HE : 0.54) and low infection complexity (74.9% monoclonal). A significant percentage of infections share microhaplotypes, even across transmission seasons and sites, indicating persistent local transmission. We identify multiple clusters of clonal or near-clonal infections, highlighting high genetic relatedness. Only 6.3% of individuals diagnosed with P. falciparum reported recent travel. Yet, in clonal or near-clonal clusters, infections of travellers were frequently observed first in time, suggesting that parasites may have been imported and then transmitted locally. 31.1% of infections are pfhrp2-deleted and 84.4% pfhrp3-deleted, and 28.7% have pfhrp2/3 double deletions. Parasites with pfhrp2/3 deletions and wild-type parasites are genetically distinct. Mutations associated with resistance to sulphadoxine-pyrimethamine or suggested to reduce sensitivity to lumefantrine are observed at near-fixation. In conclusion, genomic data corroborate local transmission and the importance of intensified control in the Ethiopian highlands.

Abstract B029: Centuries of genome instability and evolution in soft-shell clam (Mya arenaria) transmissible cancer

Abstract Transmissible cancers are infectious parasitic clones that metastasize to new hosts, living past the death of the founder animal in which the cancer initiated. Using genomic and transcriptomic analyses, we investigated the evolutionary history of a recently identified transmissible cancer lineage that has spread though the soft-shell clam (Mya arenaria) population. We first assembled a chromosome-scale soft-shell clam reference genome and characterized somatic mutations in the cancer lineage, discovering a novel mutational signature that estimates the lineage to be &amp;gt;200 years old and observing a wide variety of mutation types indicative of an extremely unstable cancer genome. We next quantified gene expression, observing differential expression of stereotypical cancer hallmarks in addition to key pathways that may have facilitated the cancer’s ability to survive seawater transfer to new hosts and evade immune rejection. Finally, we developed genetic assays to track bivalve transmissible neoplasia in the soft-shell clam and identify a novel lineage in the Baltic clam (Macoma balthica). Taken together, this study reveals the long-term survival of an invertebrate cancer lineage and identifies adaptive mechanisms to evade physical and immune barriers to cancer transmission, which may have been facilitated in part by the adaptive potential of its unstable genome. Citation Format: Samuel F.M. Hart, Michael J. Metzger. Centuries of genome instability and evolution in soft-shell clam (Mya arenaria) transmissible cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr B029.

Nanoparticle tracking analysis of natural hemozoin from Plasmodium parasites

BackgroundHemozoin is a byproduct of hemoglobin digestion crucial for parasite survival. It forms crystals that can be of interest as drug targets or biomarkers of malaria infection. However, hemozoin has long been considered as an amorphous crystal of simple morphology. Studying the consequences of biomineralization of this crystal during the parasite growth may provide more comprehensive evidence of its role during malaria. ObjectivesThis study aimed to investigate the interest of nanoparticles tracker analysis for measuring the concentration and size of hemozoin particles produced from different parasite sources and conditions. MethodsHemozoin was extracted from several clones of Plasmodium falciparum both asexual and sexual parasites. Hemozoin was also extracted from blood samples of malaria patients and from saliva of asymptomatic malaria carriers. Nanoparticles tracking analysis (NTA) was performed to assess the size and concentration of hemozoin. ResultsNTA data showed variation in hemozoin concentration, size, and crystal clusters between parasite clones, species, and stages. Among parasite clones, hemozoin concentration ranged from 131 to 2663 particles/infected red blood cell (iRBC) and size ranged from 149.6 ± 6.3 nm to 234.8 ± 40.1 nm. The mean size was lower for Plasmodium vivax (176 ± 79.2 nm) than for Plasmodium falciparum (254.8 ± 74.0 nm). Sexual NF54 parasites showed a 7.5-fold higher concentration of hemozoin particles (28.7 particles/iRBC) compared to asexual parasites (3.8 particles/iRBC). In addition, the mean hemozoin size also increased by approximately 60 % for sexual parasites. Compared to in vitro cultures of parasites, blood samples showed low hemozoin concentrations. ConclusionsThis study highlights the potential of NTA as a useful method for analyzing hemozoin, demonstrating its ability to provide detailed information on hemozoin characterization. However, further research is needed to adapt the NTA for hemozoin analysis.

Centuries of genome instability and evolution in soft-shell clam, Mya arenaria, bivalve transmissible neoplasia

Transmissible cancers are infectious parasitic clones that metastasize to new hosts, living past the death of the founder animal in which the cancer initiated. We investigated the evolutionary history of a cancer lineage that has spread though the soft-shell clam (Mya arenaria) population by assembling a chromosome-scale soft-shell clam reference genome and characterizing somatic mutations in transmissible cancer. We observe high mutation density, widespread copy-number gain, structural rearrangement, loss of heterozygosity, variable telomere lengths, mitochondrial genome expansion and transposable element activity, all indicative of an unstable cancer genome. We also discover a previously unreported mutational signature associated with overexpression of an error-prone polymerase and use this to estimate the lineage to be >200 years old. Our study reveals the ability for an invertebrate cancer lineage to survive for centuries while its genome continues to structurally mutate, likely contributing to the evolution of this lineage as a parasitic cancer.

High Prevalence of Polyclonal Plasmodium falciparum Infections and Association with Poor IgG Antibody Responses in a Hyper-Endemic Area in Cameroon.

Malaria remains a major public health problem worldwide, with eradication efforts thwarted by drug and insecticide resistance and the lack of a broadly effective malaria vaccine. In continuously exposed communities, polyclonal infections are thought to reduce the risk of severe disease and promote the establishment of asymptomatic infections. We sought to investigate the relationship between the complexity of P. falciparum infection and underlying host adaptive immune responses in an area with a high prevalence of asymptomatic parasitaemia in Cameroon. A cross-sectional study of 353 individuals aged 2 to 86 years (median age = 16 years) was conducted in five villages in the Centre Region of Cameroon. Plasmodium falciparum infection was detected by multiplex nested PCR in 316 samples, of which 278 were successfully genotyped. Of these, 60.1% (167/278) were polyclonal infections, the majority (80.2%) of which were from asymptomatic carriers. Host-parasite factors associated with polyclonal infection in the study population included peripheral blood parasite density, participant age and village of residence. The number of parasite clones per infected sample increased significantly with parasite density (r = 0.3912, p < 0.0001) but decreased with participant age (r = -0.4860, p < 0.0001). Parasitaemia and the number of clones per sample correlated negatively with total plasma levels of IgG antibodies to three highly reactive P. falciparum antigens (MSP-1p19, MSP-3 and EBA175) and two soluble antigen extracts (merozoite and mixed stage antigens). Surprisingly, we observed no association between the frequency of polyclonal infection and susceptibility to clinical disease as assessed by the recent occurrence of malarial symptoms or duration since the previous fever episode. Overall, the data indicate that in areas with the high perennial transmission of P. falciparum, parasite polyclonality is dependent on underlying host antibody responses, with the majority of polyclonal infections occurring in persons with low levels of protective anti-plasmodial antibodies.

Genomic variation during culture adaptation of genetically complex Plasmodium falciparum clinical isolates.

Experimental studies on the biology of malaria parasites have mostly been based on laboratory-adapted lines, but there is limited understanding of how these may differ from parasites in natural infections. Loss-of-function mutants have previously been shown to emerge during culture of some Plasmodium falciparum clinical isolates in analyses focusing on single-genotype infections. The present study included a broader array of isolates, mostly representing multiple-genotype infections, which are more typical in areas where malaria is highly endemic. Genome sequence data from multiple time points over several months of culture adaptation of 28 West African isolates were analysed, including previously available sequences along with new genome sequences from additional isolates and time points. Some genetically complex isolates eventually became fixed over time to single surviving genotypes in culture, whereas others retained diversity, although proportions of genotypes varied over time. Drug resistance allele frequencies did not show overall directional changes, suggesting that resistance-associated costs are not the main causes of fitness differences among parasites in culture. Loss-of-function mutants emerged during culture in several of the multiple-genotype isolates, affecting genes (including AP2-HS, EPAC and SRPK1) for which loss-of-function mutants were previously seen to emerge in single-genotype isolates. Parasite clones were derived by limiting dilution from six of the isolates, and sequencing identified de novo variants not detected in the bulk isolate sequences. Interestingly, several of these were nonsense mutants and frameshifts disrupting the coding sequence of EPAC, the gene with the largest number of independent nonsense mutants previously identified in laboratory-adapted lines. Analysis of genomic identity by descent to explore relatedness among clones revealed co-occurring non-identical sibling parasites, illustrative of the natural genetic structure within endemic populations.

Genetic diversity of Toxoplasma gondii isolates from birds in the world: A systematic review.

Toxoplasma gondii (T. gondii) is one of the most important foodborne pathogens that infects a large number of vertebrate species and has a cosmopolitan distribution. Birds as intermediate hosts are very important in the life cycle of T. gondii and they can be a main source of infection for humans and felids, as well as other animals. Most species of birds feed from the ground and are the best indicator for soil contamination with T. gondii oocysts. Hence, T. gondii strains isolated from birds can represent different genotypes circulating in the environment and their main predators and consumers. The recent systematic review tries to represent the population structure of T. gondii in birds around the world. Six English language databases were searched from 1990 to 2020 to find the related studies and overall, 1275 isolates of T. gondii were separated from the analyzed samples in birds. The results of our study revealed that atypical genotypes were predominant (58.8%, 750 out of 1275). Types II, III, and I had less frequency with prevalence rates of 23.4%, 13.8%, and 2%, respectively. No isolates of Type I were reported from Africa. Summarizing ToxoDB genotypes circulating in birds around the world manifested that ToxoDB #2 was the most common (101/875), followed by ToxoDB #1 (80/875), and #3 (63/875). Totally, the results of our review represented the high genetic diversity of T. gondii with circulating non-clonal strains in birds from South and North America, while clonal parasites with low genetic diversity were predominant in Europe, Asia, and Africa.

Cytoplasmic isoleucyl tRNA synthetase as an attractive multistage antimalarial drug target.

Development of antimalarial compounds into clinical candidates remains costly and arduous without detailed knowledge of the target. As resistance increases and treatment options at various stages of disease are limited, it is critical to identify multistage drug targets that are readily interrogated in biochemical assays. Whole-genome sequencing of 18 parasite clones evolved using thienopyrimidine compounds with submicromolar, rapid-killing, pan-life cycle antiparasitic activity showed that all had acquired mutations in the P. falciparum cytoplasmic isoleucyl tRNA synthetase (cIRS). Engineering two of the mutations into drug-naïve parasites recapitulated the resistance phenotype, and parasites with conditional knockdowns of cIRS became hypersensitive to two thienopyrimidines. Purified recombinant P. vivax cIRS inhibition, cross-resistance, and biochemical assays indicated a noncompetitive, allosteric binding site that is distinct from that of known cIRS inhibitors mupirocin and reveromycin A.Our data show that Plasmodium cIRS is an important chemically and genetically validated target for next-generation medicines for malaria.

Genetic Characterization of Trypanosoma cruzi I Populations from an Oral Chagas Disease Outbreak in Venezuela: Natural Resistance to Nitroheterocyclic Drugs.

The oral transmission of Chagas disease (oCD) in Venezuela announced its appearance in 2007. Different from other populations affected by oCD and despite close supervision during treatment with nitroheterocyclic drugs, the result was treatment failure. We studied genetic features of natural bloodstream parasite populations and populations after treatment of nine patients of this outbreak. In total, we studied six hemoculture isolates, eight Pre-Tx blood samples, and 17 samples collected at two or three Post-Tx time-points between 2007 and 2015. Parasitic loads were determined by quantitative polymerase chain reaction (qPCR), and discrete typing units (DTU), minicircle signatures, and Tcntr-1 gene sequences were searched from blood samples and hemocultures. Half-maximal inhibitory concentration (IC50) values were measured from the hemocultures. All patients were infected by TcI. Significant decrease in parasitic loads was observed between Pre-Tx and Post-Tx samples, suggesting the evolution from acute to chronic phase of Chagas disease. 60% of intra-DTU-I variability was observed between Pre-Tx and Post-Tx minicircle signatures in the general population, and 43 single-nucleotide polymorphisms (SNPs) were detected in a total of 12 Tcntr-1 gene sequences, indicative of a polyclonal source of infection. SNPs in three post-Tx samples produced stop codons giving rise to putative truncated proteins or displaced open reading frames, which would render resistance genes. IC50 values varied from 5.301 ± 1.973 to 104.731 ± 4.556 μM, demonstrating a wide range of susceptibility. The poor drug response in the Pre-Tx parasite populations may be associated with the presence of naturally resistant parasite clones. Therefore, any information that can be obtained on drug susceptibility from in vitro assays, in vivo assays, or molecular characterization of natural populations of Trypanosoma cruzi becomes essential when therapeutic guidelines are designed in a given geographical area.

Atypical life cycle does not lead to inbreeding or selfing in parasites despite clonemate accumulation in intermediate hosts.

Many parasites utilize asexual and sexual reproduction and multiple hosts to complete their life cycles. How these taxa avoid inbreeding is an essential question for understanding parasite evolution and ecology. Aquatic trematodes that require multiple host species may benefit from diverse genetic parasite assemblages accumulating within second intermediate hosts prior to sexual reproduction in definitive hosts. However, Cotylurus species are able to utilize the same snail species as first and second intermediate hosts, potentially resulting in the accumulation of genetically identical clones (clonemates) prior to sexual reproduction. In this study, we developed and analysed novel microsatellite loci to determine if clones are accumulating within snail hosts prior to ingestion by bird hosts and the effects this could have on parasite inbreeding. Contrary to previous studies of aquatic trematodes, significantly large numbers of clonemates were present within snails, but full-sibs were not. Genetic structure was present over a relatively small geographical scale despite the use of vagile definitive hosts. Phylogenetic analysis identified the Cotylurus sp. clones as belonging to a single species. Despite the presence of clones within snails, mating between clones/selfing was not common and heterozygosity is maintained within individuals. Potential issues with clones mating may be mitigated by the presence of snails with numerous clones, the consumption of many snails by bird hosts and parasite clone recognition/avoidance. Use of the same host species for multiple life stages may have advantages when parasites are able to avoid inbreeding and the required hosts are common.

Identifying Antigenic Switching by Clonal Cell Barcoding and Nanopore Sequencing in Trypanosoma brucei.

Many organisms alternate the expression of genes from large gene sets or gene families to adapt to environmental cues or immune pressure. The single-celled protozoan pathogen Trypanosoma brucei spp. periodically changes its homogeneous surface coat of variant surface glycoproteins (VSGs) to evade host antibodies during infection. This pathogen expresses one out of ~2,500 VSG genes at a time from telomeric expression sites (ESs) and periodically changes their expression by transcriptional switching or recombination. Attempts to track VSG switching have previously relied on genetic modifications of ES sequences with drug-selectable markers or genes encoding fluorescent proteins. However, genetic modifications of the ESs can interfere with the binding of proteins that control VSG transcription and/or recombination, thus affecting VSG expression and switching. Other approaches include Illumina sequencing of the VSG repertoire, which shows VSGs expressed in the population rather than cell switching; the Illumina short reads often limit the distinction of the large set of VSG genes. Here, we describe a methodology to study antigenic switching without modifications of the ES sequences. Our protocol enables the detection of VSG switching at nucleotide resolution using multiplexed clonal cell barcoding to track cells and nanopore sequencing to identify cell-specific VSG expression. We also developed a computational pipeline that takes DNA sequences and outputs VSGs expressed by cell clones. This protocol can be adapted to study clonal cell expression of large gene families in prokaryotes or eukaryotes. Key features • This protocol enables the analysis of variant surface glycoproteins (VSG) switching in T. brucei without modifying the expression site sequences. • It uses a streamlined computational pipeline that takes fastq DNA sequences and outputs expressed VSG genes by each parasite clone. • The protocol leverages the long reads sequencing capacity of the Oxford nanopore sequencing technology, which enables accurate identification of the expressed VSGs. • The protocol requires approximately eight to nine days to complete.

Short-term cryopreservation and thawing have minimal effects on Plasmodium falciparum ex vivo invasion profile.

Ex vivo phenotyping of P. falciparum erythrocyte invasion diversity is important in the identification and down selection of potential malaria vaccine targets. However, due to the lack of appropriate laboratory facilities in remote areas of endemic countries, direct processing of P. falciparum clinical isolates is usually not feasible. Here, we investigated the combined effect of short-term cryopreservation and thawing processes on the ex vivo invasion phenotypes of P. falciparum isolates. Ex-vivo or in vitro invasion phenotyping assays were performed with P. falciparum clinical isolates prior to or following culture adaptation, respectively. All isolates were genotyped at Day 0 for parasite clonality. Subsequently, isolates that were successfully culture-adapted were genotyped again at Days 7, 15, 21, and 28-post adaptation. Invasion phenotyping assays were performed in isogenic isolates revived at different time points (3, 6, and 12 months) post-cryopreservation and the resulting data were compared to that from ex-vivo invasion data of matched isogenic parental isolates. We also show that short-term culture adaptation selects for parasite clonality and could be a driving force for variation in invasion phenotypes as compared to ex vivo data where almost all parasite clones of a given isolate are present. Interestingly, our data show little variation in the parasites’ invasion phenotype following short-term cryopreservation. Altogether, our data suggest that short-term cryopreservation of uncultured P. falciparum clinical isolates is a reliable mechanism for storing parasites for future use.

Fitness Costs of pfhrp2 and pfhrp3 Deletions Underlying Diagnostic Evasion in Malaria Parasites.

Rapid diagnostic tests based on detection of histidine-rich proteins (HRPs) are widely used for malaria diagnosis, but parasites carrying pfhrp deletions can evade detection and are increasing in frequency in some countries. Models aim to predict conditions under which pfhrp2 and/or pfhrp3 deletions will increase, but a key parameter-the fitness cost of deletions-is unknown. We removed pfhrp2 and/or pfhrp3 from a Malawian parasite clone using gene editing approaches) and measured fitness costs by conducting pairwise competition experiments. We observed significant fitness costs of 0.087 ± 0.008 (1 standard error) per asexual cycle for pfhrp2 deletion and 0.113 ± 0.008 for the pfhrp2/3 double deletion, relative to the unedited progenitor parasite. Selection against deletions is strong and comparable to that resulting from drug resistance mutations. Prior modeling suggested that diagnostic selection may drive increased frequency of pfhrp deletions only when fitness costs are mild. Our experiments show that costs of pfhrp deletions are higher than these thresholds, but modeling and empirical results can be reconciled if the duration of infection is short. These results may inform future modeling to understand why pfhrp2/3 deletions are increasing in some locations (Ethiopia and Eritrea) but not in others (Mekong region).

A single point mutation in the Plasmodium falciparum 3\u2032\u20135\u2032 exonuclease does not alter piperaquine susceptibility

BackgroundThe rise in Plasmodium falciparum resistance to dihydroartemisinin–piperaquine (DHA–PPQ) treatment has been documented in the Greater Mekong Subregion with associations with mutations in the P. falciparum chloroquine resistance transporter (pfcrt) and plasmepsin 2 (pfpm2) genes. However, it is unclear whether other genes also play a role with PPQ resistance, such as the E415G mutation in the exonuclease (pfexo) gene. The aim of this study was to investigate the role of this mutation in PPQ resistance by generating transgenic parasites expressing the pfexo-E415G mutant allele.MethodsTransgenic parasite clones carrying the E415G mutation in PfEXO of the B5 isolate were derived by CRISPR-Cas9 gene editing and verified using PCR and gene sequencing. Polymorphisms of pfkelch-13, pfcrt, and pfexo were examined by PCR while the copy number variations of pfpm2 were examined by both relative quantitative real-time PCR and the duplication breakpoint assay. Drug sensitivity against a panel of antimalarials, the ring-stage survival assay (RSA), the PPQ survival assay (PSA), and bimodal dose-response curves were used to evaluate antimalarial susceptibility.ResultsThe transgenic line, B5-rexo-E415G-B8, was successfully generated. The PPQ-IC90, %PPQ survival, and the bimodal dose-response clearly showed that E415G mutation in PfEXO of B5 isolate remained fully susceptible to PPQ. Furthermore, growth assays demonstrated that the engineered parasites grew slightly faster than the unmodified parental isolates whereas P. falciparum isolates harbouring pfkelch-13, pfcrt, and pfexo mutations with multiple copies of pfpm2 grew much more slowly.ConclusionsInsertion of the E415G mutation in PfEXO did not lead to increased PPQ-IC90 and %PPQ survival, suggesting that this mutation alone may not be associated with PPQ resistance, but could still be an important marker if used in conjunction with other markers for monitoring PPQ-resistant parasites. The results also highlight the importance of monitoring and evaluating suspected genetic mutations with regard to parasite fitness and resistance.

Plasmodium falciparum\xa0multiplicity of infection and pregnancy outcomes in Congolese women from southern Brazzaville, Republic of Congo

BackgroundInvestigating whether the multiplicity of Plasmodium falciparum infection (MOI) is related to pregnancy outcomes, is of interest in sub-Saharan area where malaria is highly endemic. The present study aimed to characterize the genetic diversity of P. falciparum in women at delivery from Southern Brazzaville, and investigate whether the MOI is associated with maternal anaemia, preterm delivery, or low birth weight.MethodsThis was a cross sectional study carried out with samples collected between March 2014 and April 2015 from 371 women recruited at delivery at a Health Centre in southern Brazzaville, Republic of Congo. Matched peripheral, placental, and cord blood collected from each of the women at delivery were used for the detection of P. falciparum microscopic and submicroscopic parasitaemia, and parasite DNA genotyping by nested PCR.ResultsFrom 371 recruited women, 27 were positive to microscopic malaria parasitaemia while 223 women harboured submicroscopic parasitaemia. All msp-1 block 2 family allelic types (K1, MAD20 and RO33) were observed in all the three compartments of blood, with K1 being most abundant. K1 (with 12, 10, and 08 alleles in the peripheral, placental, and cord blood respectively) and MAD20 (with 10, 09, and 06 alleles in the respective blood compartments) were more diverse compared to RO33 (with 06, 06, and 05 alleles in the respective blood compartments). From the 250 women with microscopic and/or submicroscopic parasitaemia, 38.5%, 30.5%, and 18.4% of peripheral, placental and cord blood sample, respectively, harboured more than one parasite clone, and polyclonal infection was more prevalent in the peripheral blood of women with microscopic parasitaemia (54.5%) compared to those with submicroscopic parasitaemia (36.7%) (p = 0.02). The mean multiplicity of genotypes per microscopic and submicroscopic infection in peripheral blood was higher in anemic women (2.00 ± 0.23 and 1.66 ± 0.11, respectively) than in non-anaemic women (1.36 ± 0.15 and 1.45 ± 0.06, respectively) (p = 0.03 and 0.06). In logistic regression, women infected with four or more clones of the parasite were 9.4 times more likely to be anaemic than women harbouring one clone. This association, however, was only observed with the peripheral blood infection. No significant association was found between the MOI and low birth weight or preterm delivery.ConclusionsThese results indicate that the genetic diversity of P. falciparum is high in pregnant women from southern Brazzaville in the Republic of Congo, and the multiplicity of the infection might represent a risk for maternal anaemia.