In November 2013, a Plasmodium falciparum malaria outbreak of 11 cases occurred in Cusco, southern Peru, where falciparum malaria had not been reported since 1946. Although initial microscopic ...diagnosis reported only Plasmodium vivax infection in each of the specimens, subsequent examination by the national reference laboratory confirmed P. falciparum infection in all samples. Molecular typing of four available isolates revealed identity as the B-variant (B sub(V1)) strain that was responsible for a malaria outbreak in Tumbes, northern Peru, between 2010 and 2012. The P. falciparum B sub(V1) strain is multidrug resistant, can escape detection by PfHRP2-based rapid diagnostic tests, and has contributed to two malaria outbreaks in Peru. This investigation highlights the importance of accurate species diagnosis given the potential for P. falciparum to be reintroduced to regions where it may have been absent. Similar molecular epidemiological investigations can track the probable source(s) of outbreak parasite strains for malaria surveillance and control purposes.
Determining the source of malaria outbreaks in Ecuador and identifying remaining transmission foci will help in malaria elimination efforts. In this study, the genetic signatures of Plasmodium ...falciparum isolates, obtained from an outbreak that occurred in northwest Ecuador from 2012 to 2013, were characterized.
Molecular investigation of the outbreak was performed using neutral microsatellites, drug resistance markers and pfhrp2 and pfhrp3 genotyping.
A majority of parasite isolates (31/32) from this outbreak were of a single clonal type that matched a clonal lineage previously described on the northern coast of Peru and a historical isolate from Ecuador. All but one isolate carried a chloroquine-resistant pfcrt genotype and sulfadoxine- and pyrimethamine-sensitive pfdhps and pfdhfr genotypes. Pfmdr1 mutations were identified in codons 184 and 1042. In addition, most samples (97 %) showed presence of pfhrp2 gene.
This study indicates that parasites from a single clonal lineage largely contributed to this outbreak and this lineage was found to be genetically related to a lineage previously reported in the Peruvian coast and historical Ecuadorian parasites.
Plasmodium vivax is the causative infectious agent of 80–300 million annual cases of malaria. Many aspects of this parasite's biology remain unknown. To further elucidate the interaction of P. vivax ...with its Saimiri boliviensis host, we obtained detailed proteomes of infected red blood cells, representing the trophozoite-enriched stage of development. Data from two of three biological replicate proteomes, emphasized here, were analyzed using five search engines, which enhanced identifications and resulted in the most comprehensive P. vivax proteomes to date, with 1375 P. vivax and 3209 S. boliviensis identified proteins. Ribosome subunit proteins were noted for both P. vivax and S. boliviensis, consistent with P. vivax's known reticulocyte host–cell specificity. A majority of the host and pathogen proteins identified belong to specific functional categories, and several parasite gene families, while 33% of the P. vivax proteins have no reported function. Hemoglobin was significantly oxidized in both proteomes, and additional protein oxidation and nitration was detected in one of the two proteomes. Detailed analyses of these post-translational modifications are presented. The proteins identified here significantly expand the known P. vivax proteome and complexity of available host protein functionality underlying the host–parasite interactive biology, and reveal unsuspected oxidative modifications that may impact protein function.
Plasmodium vivax malaria is a serious neglected disease, causing an estimated 80 to 300 million cases annually in 95 countries. Infection can result in significant morbidity and possible death. P. vivax, unlike the much better-studied Plasmodium falciparum species, cannot be grown in long-term culture, has a dormant form in the liver called the hypnozoite stage, has a reticulocyte host–cell preference in the blood, and creates caveolae vesicle complexes at the surface of the infected reticulocyte membranes. Studies of stage-specific P. vivax expressed proteomes have been limited in scope and focused mainly on pathogen proteins, thus limiting understanding of the biology of this pathogen and its host interactions. Here three P. vivax proteomes are reported from biological replicates based on purified trophozoite-infected reticulocytes from different Saimiri boliviensis infections (the main non-human primate experimental model for P. vivax biology and pathogenesis). An in-depth analysis of two of the proteomes using 2D LC/MS/MS and multiple search engines identified 1375 pathogen proteins and 3209 host proteins. Numerous functional categories of both host and pathogen proteins were identified, including several known P. vivax protein family members (e.g., PHIST, eTRAMP and VIR), and 33% of protein identifications were classified as hypothetical. Ribosome subunit proteins were noted for both P. vivax and S. boliviensis, consistent with this parasite species' known reticulocyte host–cell specificity. In two biological replicates analyzed for post-translational modifications, hemoglobin was extensively oxidized, and various other proteins were also oxidized or nitrated in one of the two replicates. The cause of such protein modification remains to be determined but could include oxidized heme and oxygen radicals released from the infected red blood cell's parasite-induced acidic digestive vacuoles. In any case, the data suggests the presence of distinct infection-specific conditions whereby both the pathogen and host infected red blood cell proteins may be subject to significant oxidative stress.
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•We use 2D LC/MS/MS to obtain Plasmodium vivax trophozoite-enriched proteomes.•In two biological replicates, 5 search engines identify 1375 P. vivax proteins.•3209 host S. boliviensis proteins are also identified.•In both replicates extensive hemoglobin oxidation was observed.•Oxidation/nitration of other proteins in 1 replicate could alter protein function.
The recent emergence of artemisinin resistance in the Greater Mekong Subregion poses a major threat to the global effort to control malaria. Tracking the spread and evolution of artemisinin-resistant ...parasites is critical in aiding efforts to contain the spread of resistance. A total of 417 patient samples from the year 2007, collected during malaria surveillance studies across ten provinces in Thailand, were genotyped for the candidate Plasmodium falciparum molecular marker of artemisinin resistance K13. Parasite genotypes were examined for K13 propeller mutations associated with artemisinin resistance, signatures of positive selection, and for evidence of whether artemisinin-resistant alleles arose independently across Thailand. A total of seven K13 mutant alleles were found (N458Y, R539T, E556D, P574L, R575K, C580Y, S621F). Notably, the R575K and S621F mutations have previously not been reported in Thailand. The most prevalent artemisinin resistance-associated K13 mutation, C580Y, carried two distinct haplotype profiles that were separated based on geography, along the Thai-Cambodia and Thai-Myanmar borders. It appears these two haplotypes may have independent evolutionary origins. In summary, parasites with K13 propeller mutations associated with artemisinin resistance were widely present along the Thai-Cambodia and Thai-Myanmar borders prior to the implementation of the artemisinin resistance containment project in the region.
Plasmodium vivax and P.cynomolgi produce numerous caveola-vesicle complex (CVC) structures within the surface of the infected erythrocyte membrane. These contrast with the electron-dense knob ...protrusions expressed at the surface of Plasmodium falciparum-infected erythrocytes. Here we investigate the three-dimensional (3-D) structure of the CVCs and the identity of a predominantly expressed 95kDa CVC protein. Liquid chromatography - tandem mass spectrometry analysis of immunoprecipitates by monoclonal antibodies from P.cynomolgi extracts identified this protein as a member of the Plasmodium helical interspersed subtelomeric (PHIST) superfamily with a calculated mass of 81kDa. We named the orthologous proteins PvPHIST/CVC-8195 and PcyPHIST/CVC-8195, analysed their structural features, including a PEXEL motif, repeated sequences and a C-terminal PHIST domain, and show that PHIST/CVC-8195 is most highly expressed in trophozoites. We generated images of CVCs in 3-D using electron tomography (ET), and used immuno-ET to show PHIST/CVC-8195 localizes to the cytoplasmic side of the CVC tubular extensions. Targeted gene disruptions were attempted in vivo. The pcyphist/cvc-8195 gene was not disrupted, but parasites containing episomes with the tgdhfr selection cassette were retrieved by selection with pyrimethamine. This suggests that PHIST/CVC-8195 is essential for survival of these malaria parasites.
Plasmodium vivax
and
P. cynomolgi
produce numerous caveolae-vesicle complex (CVC) structures within the surface of the infected erythrocyte membrane. These contrast with the electron-dense knob ...protrusions expressed at the surface of
P. falciparum
infected erythrocytes. Here we investigate the 3-dimensional structure of the CVCs and the identity of a predominantly expressed 95 kDa CVC protein. Liquid chromatography - tandem mass spectrometry analysis of immunoprecipitates by monoclonal antibodies from
P. cynomolgi
extracts identified this protein as a member of the
Plasmodium
helical interspersed sub-telomeric (PHIST) superfamily with a calculated mass of 81 kDa. We named the orthologous proteins PvPHIST/CVC-81
95
and PcyPHIST/CVC-81
95
, analyzed their structural features, including a PEXEL motif, repeated sequences and a C-terminal PHIST domain, and show that PHIST/CVC-81
95
is most highly expressed in trophozoites. We generated images of CVCs in 3-D using electron tomography, and used immuno-Electron Tomography (ET) to show PHIST/CVC-81
95
localizes to the cytoplasmic side of the CVC tubular extensions. Targeted gene disruptions were attempted
in vivo
. The
pcyphist/cvc-81
95
gene was not disrupted, but parasites containing episomes with the
tgdhfr
selection cassette were retrieved by selection with pyrimethamine. This suggests that PHIST/CVC-81
95
is essential for survival of these malaria parasites.
Background.
Atovaquone-proguanil (AP) is the most commonly used treatment for uncomplicated Plasmodium falciparum malaria in the United States. Apparent AP treatment failures were reported 7 months ...apart in 2 American travelers who stayed in the same compound for foreign workers in Rivers State, Nigeria.
Methods.
We analyzed pretreatment (day 0) and day of failure samples from both travelers for mutations in the P falciparum cytochrome B (pfcytb) and dihydrofolate reductase (pfdhfr) genes associated with resistance to atovaquone and cycloguanil, the active metabolite of proguanil, respectively. We genotyped the parasites and sequenced their mitochondrial genomes.
Results.
On day 0, both travelers had proguanil-resistant genotypes but atovaquone-sensitive cytb sequences. Day of failure samples exhibited mutations in cytb for both travelers. One traveler had the common Y268S mutation, whereas the other traveler had a previously unreported mutation, I258M. The travelers had unrelated parasite genotypes and different mitochondrial genomes.
Conclusions.
Despite the infections likely having been contracted in the same site, there is no evidence that the cases were related. The mutations likely arose independently during the acute infection or treatment. Our results highlight the importance of genotyping parasites and sequencing the full cytb and dhfr genes in AP failures to rule out transmission of AP-resistant strains and identify novel mechanisms of AP resistance.
Plasmodium parasites modify their host red blood cell (RBC) antigenically and structurally, enabling parasite protein export, nutrient import, and RBC adherence. While knobs and Maurer’s clefts are ...observed in P. falciparum-infected erythrocytes, RBCs infected with other Plasmodium species display alterations such as caveolae and caveola-vesicle complexes (CVCs). Information on the antigenic makeup and function of these ultrastructures is scarce. This dissertation utilizes genomics, proteomics and biochemical tools as well as the simian malaria species P. knowlesi and P. cynomolgi to characterize and gain functional insights into these structures. This dissertation first focuses on investigations of glyceraldehyde-3-phosphate dehydrogenase, an enzyme involved in glycolysis for ATP production. We identified Plasmodium-specific amino acid substitutions within the functional domains of this protein that may prove to be of importance, thus providing insights for antimalarial drug-targeting studies. Secondly, we identified the gene encoding a protein known to be part of the CVCs of P. vivax, and believed to have an ortholog in the related parasite, P. cynomolgi. Immunoprecipitation of detergent extracts followed by trypsin peptide cleavage and mass spectrometric analysis identified this protein as an 81 kDa member of the Plasmodium helical interspersed sub-telomeric (PHIST) superfamily, termed PcyPHIST-81, with homologs in five other Plasmodium species. All PHIST-81 homologs have a Plasmodium export motif, conserved tryptophans, and four consecutive alpha helices in their amino acid sequences. Immunoelectron tomography studies on P. cynomolgi-infected RBCs using rabbit antisera to recombinant P. vivax PHIST-81 revealed that PcyPHIST-81 localizes to the tubular extensions of CVCs. Finally, we utilized a global approach to identify proteins that are localized to the membranes of P. cynomolgi and P. knowlesi-infected erythrocytes. Mass spectrometric analysis of parasite-infected RBC membrane ghosts identified 109 P. cynomolgi proteins and 129 P. knowlesi proteins, which included members of the PHIST and Pk-fam-c protein families as well as hypothetical proteins.
A number of studies have analyzed the performance of malaria rapid diagnostic tests (RDTs) in Colombia with discrepancies in performance being attributed to a combination of factors such as parasite ...levels, interpretation of RDT results and/or the handling and storage of RDT kits. However, some of the inconsistencies observed with results from Plasmodium falciparum histidine-rich protein 2 (PfHRP2)-based RDTs could also be explained by the deletion of the gene that encodes the protein, pfhrp2, and its structural homolog, pfhrp3, in some parasite isolates. Given that pfhrp2- and pfhrp3-negative P. falciparum isolates have been detected in the neighboring Peruvian and Brazilian Amazon regions, we hypothesized that parasites with deletions of pfhrp2 and pfhrp3 may also be present in Colombia. In this study we tested 100 historical samples collected between 1999 and 2009 from six Departments in Colombia for the presence of pfhrp2, pfhrp3 and their flanking genes. Seven neutral microsatellites were also used to determine the genetic background of these parasites. In total 18 of 100 parasite isolates were found to have deleted pfhrp2, a majority of which (14 of 18) were collected from Amazonas Department, which borders Peru and Brazil. pfhrp3 deletions were found in 52 of the100 samples collected from all regions of the country. pfhrp2 flanking genes PF3D7_0831900 and PF3D7_0831700 were deleted in 22 of 100 and in 1 of 100 samples, respectively. pfhrp3 flanking genes PF3D7_1372100 and PF3D7_1372400 were missing in 55 of 100 and in 57 of 100 samples. Structure analysis of microsatellite data indicated that Colombian samples tested in this study belonged to four clusters and they segregated mostly based on their geographic region. Most of the pfhrp2-deleted parasites were assigned to a single cluster and originated from Amazonas Department although a few pfhrp2-negative parasites originated from the other three clusters. The presence of a high proportion of pfhrp2-negative isolates in the Colombian Amazon may have implications for the use of PfHRP2-based RDTs in the region and may explain inconsistencies observed when PfHRP2-based tests and assays are performed.
Guyana and Suriname have made important progress in reducing the burden of malaria. While both countries use microscopy as the primary tool for clinical diagnosis, malaria rapid diagnostic tests ...(RDTs) are useful in remote areas of the interior where laboratory support may be limited or unavailable. Recent reports indicate that histidine-rich protein 2 (PfHRP2)-based diagnostic tests specific for detection of P. falciparum may provide false negative results in some parts of South America due to the emergence of P. falciparum parasites that lack the pfhrp2 gene, and thus produce no PfHRP2 antigen. Pfhrp2 and pfhrp3 genes were amplified in parasite isolates collected from Guyana and Suriname to determine if there were circulating isolates with deletions in these genes. Pfhrp3 deletions were monitored because some monoclonal antibodies utilized in PfHRP2-based RDTs cross-react with the PfHRP3 protein. We found that all 97 isolates from Guyana that met the inclusion criteria were both pfhrp2- and pfhrp3-positive. In Suriname (N = 78), 14% of the samples tested were pfhrp2-negative while 4% were pfhrp3-negative. Furthermore, analysis of the genomic region proximal to pfhrp2 and pfhrp3 revealed that genomic deletions extended to the flanking genes. We also investigated the population substructure of the isolates collected to determine if the parasites that had deletions of pfhrp2 and pfhrp3 belonged to any genetic subtypes. Cluster analysis revealed that there was no predominant P. falciparum population substructure among the isolates from either country, an indication of genetic admixture among the parasite populations. Furthermore, the pfhrp2-deleted parasites from Suriname did not appear to share a single, unique genetic background.
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