In this study, the prevalence of Enterocytozoon bieneusi in China was investigated. Twelve genotypes of E. bieneusi were identified, of which 10 were novel genotypes. Further, 41.6% of the genotypes were found in both humans and animals. This is the first report of E. bieneusi in China.Microsporidia are a diverse group of obligate intracellular pathogens consisting of approximately 1,300 formally described species in 160 genera that infect a wide range of invertebrate and vertebrate hosts, including humans (8,16). Enterocytozoon bieneusi, the most frequently diagnosed microsporidial species in humans, was first reported in an AIDS patient in 1985 (4). Over the last 2 decades, E. bieneusi has been detected in humans, other mammals, and birds in more than 30 countries (1,6,10,12). However, the prevalence of this parasite in China has been unclear.Currently, sequencing of the internal transcribed spacer (ITS) region of the rRNA gene is regarded as the standard method for species identification and genotyping of E. bieneusi isolates (17). More than 90 genotypes or variants of E. bieneusi that infect humans and animals have been identified (16). Recent molecular epidemiological studies indicated that some genotypes are host specific (17). Further, 8 genotypes (WL15, D, Peru6, EbpC, Peru10, Peru16, WL11, and Type IV) have been found in both humans and animals, indicating the potential for zoonotic transmission and the importance for surveillance of the epidemiology of E. bieneusi (17).We investigated the prevalence of E. bieneusi infection in humans and animals in China. A total of 220 fecal samples were collected. Among them, 40 fecal samples were from diarrheal children in the First Hospital of Jilin University in Changchun City, northeast China, 61 were from pigs in a livestock production facility, 26 were from dogs in a pet market, and 93 were from cows. Both human and animal samples were collected in the same area around Changchun City; however, the human samples were not from the same farm as the animal samples. The collection of human and animal stool samples was approved by the Ethics Committee of the Institute of Zoonosis, Jilin University. DNA was extracted from each fecal sample with a modified protocol as described previously (7).A nested PCR with primers based on the specific ITS sequences of E. bieneusi was applied for pathogen identification as previously reported (2, 5). E. bieneusi ITS sequences were determined, and a multiple alignment was performed using the ClustalX program. To assess the extent of genetic diversity and evolutionary relationships among the previously known genotypes of Enterocytozoon spp. and the novel genotypes, a neighbor-joining tree based on the evolutionary distances calculated by the Kimura two-parameter model (15) was constructed using the MEGA 4.0 program (19).E. bieneusi-specific sequences were amplified from 56 of the 220 fecal samples (9 from humans, 2 from dogs, 35 from cows, and 10 from pigs) ( Table 1). The 56 sequences were classified into 12 genotypes, which include...

cArtemisinin resistance in Plasmodium falciparum parasites in Southeast Asia is a major concern for malaria control. Its emergence at the China-Myanmar border, where there have been more than 3 decades of artemisinin use, has yet to be investigated. Here, we comprehensively evaluated the potential emergence of artemisinin resistance and antimalarial drug resistance status in P. falciparum using data and parasites from three previous efficacy studies in this region. These efficacy studies of dihydroartemisinin-piperaquine combination and artesunate monotherapy of uncomplicated falciparum malaria in 248 P. falciparum patients showed an overall 28-day adequate clinical and parasitological response of >95% and day 3 parasite-positive rates of 6.3 to 23.1%. Comparison of the 57 K13 sequences (24 and 33 from day 3 parasite-positive and -negative cases, respectively) identified nine point mutations in 38 (66.7%) samples, of which F446I (49.1%) and an N-terminal NN insertion (86.0%) were predominant. K13 propeller mutations collectively, the F446I mutation alone, and the NN insertion all were significantly associated with day 3 parasite positivity. Increased ring-stage survival determined using the ring-stage survival assay (RSA) was highly associated with the K13 mutant genotype. Day 3 parasite-positive isolates had ϳ10 times higher ring survival rates than day 3 parasitenegative isolates. Divergent K13 mutations suggested independent evolution of artemisinin resistance. Taken together, this study confirmed multidrug resistance and emergence of artemisinin resistance in P. falciparum at the China-Myanmar border. RSA and K13 mutations are useful phenotypic and molecular markers for monitoring artemisinin resistance.

BackgroundThe malaria parasites in the genus Plasmodium have a very complicated life cycle involving an invertebrate vector and a vertebrate host. RNA-binding proteins (RBPs) are critical factors involved in every aspect of the development of these parasites. However, very few RBPs have been functionally characterized to date in the human parasite Plasmodium falciparum.MethodsUsing different bioinformatic methods and tools we searched P. falciparum genome to list and annotate RBPs. A representative 3D models for each of the RBD domain identified in P. falciparum was created using I-TESSAR and SWISS-MODEL. Microarray and RNAseq data analysis pertaining PfRBPs was performed using MeV software. Finally, Cytoscape was used to create protein-protein interaction network for CITH-Dozi and Caf1-CCR4-Not complexes.ResultsWe report the identification of 189 putative RBP genes belonging to 13 different families in Plasmodium, which comprise 3.5 % of all annotated genes. Almost 90 % (169/189) of these genes belong to six prominent RBP classes, namely RNA recognition motifs, DEAD/H-box RNA helicases, K homology, Zinc finger, Puf and Alba gene families. Interestingly, almost all of the identified RNA-binding helicases and KH genes have cognate homologs in model species, suggesting their evolutionary conservation. Exploration of the existing P. falciparum blood-stage transcriptomes revealed that most RBPs have peak mRNA expression levels early during the intraerythrocytic development cycle, which taper off in later stages. Nearly 27 % of RBPs have elevated expression in gametocytes, while 47 and 24 % have elevated mRNA expression in ookinete and asexual stages. Comparative interactome analyses using human and Plasmodium protein-protein interaction datasets suggest extensive conservation of the PfCITH/PfDOZI and PfCaf1-CCR4-NOT complexes.ConclusionsThe Plasmodium parasites possess a large number of putative RBPs belonging to most of RBP families identified so far, suggesting the presence of extensive post-transcriptional regulation in these parasites. Taken together, in silico identification of these putative RBPs provides a foundation for future functional studies aimed at defining a unique network of post-transcriptional regulation in P. falciparum.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2092-1) contains supplementary material, which is available to authorized users.

Mutations in the Plasmodium falciparum Kelch 13 (PfK13) protein are associated with artemisinin resistance. PfK13 is essential for asexual erythrocytic development, but its function is not known. We tagged the PfK13 protein with green fluorescent protein in P. falciparum to study its expression and localization in asexual and sexual stages. We used a new antibody against PfK13 to show that the PfK13 protein is expressed ubiquitously in both asexual erythrocytic stages and gametocytes and is localized in punctate structures, partially overlapping an endoplasmic reticulum marker. We introduced into the 3D7 strain four PfK13 mutations (F446I, N458Y, C469Y, and F495L) identified in parasites from the China-Myanmar border area and characterized the in vitro artemisinin response phenotypes of the mutants. We found that all the parasites with the introduced PfK13 mutations showed higher survival rates in the ring-stage survival assay (RSA) than the wild-type (WT) control, but only parasites with N458Y displayed a significantly higher RSA value (26.3%) than the WT control. After these PfK13 mutations were reverted back to the WT in field parasite isolates, all revertant parasites except those with the C469Y mutation showed significantly lower RSA values than their respective parental isolates. Although the 3D7 parasites with introduced F446I, the predominant PfK13 mutation in northern Myanmar, did not show significantly higher RSA values than the WT, they had prolonged ring-stage development and showed very little fitness cost in in vitro culture competition assays. In comparison, parasites with the N458Y mutations also had a prolonged ring stage and showed upregulated resistance pathways in response to artemisinin, but this mutation produced a significant fitness cost, potentially leading to their lower prevalence in the Greater Mekong subregion. IMPORTANCE Artemisinin resistance has emerged in Southeast Asia, endangering the substantial progress in malaria elimination worldwide. It is associated with mutations in the PfK13 protein, but how PfK13 mediates artemisinin resistance is not completely understood. Here we used a new antibody against PfK13 to show that the PfK13 protein is expressed in all stages of the asexual intraerythrocytic cycle as well as in gametocytes and is partially localized in the endoplasmic reticulum. By introducing four PfK13 mutations into the 3D7 strain and reverting these mutations in field parasite isolates, we determined the impacts of these mutations identified in the parasite populations from northern Myanmar on the ring stage using the in vitro ring survival assay. The introduction of the N458Y mutation into the 3D7 background significantly increased the survival rates of the ring-stage parasites but at the cost of the reduced fitness of the parasites. Introduction of the F446I mutation, the most prevalent PfK13 mutation in northern Myanmar, did not result in a significant increase in ring-stage survival after exposure to dihydroartemisinin (DHA), but these parasites showed extended ring-stage development. Further, parasites with the F446I mutation showed only a marginal loss of fitness, partially explaining its high frequency in northern Myanmar. Conversely, reverting all these mutations, except for the C469Y mutation, back to their respective wild types reduced the ring-stage survival of these isolates in response to in vitro DHA treatment.

Drug resistance has emerged as one of the greatest challenges facing malaria control. The recent emergence of resistance to artemisinin (ART) and its partner drugs in ART-based combination therapies (ACT) is threatening the efficacy of this front-line regimen for treating Plasmodium falciparum parasites. Thus, an understanding of the molecular mechanisms that underlie the resistance to ART and the partner drugs has become a high priority for resistance containment and malaria management. Using genome-wide association studies, we investigated the associations of genome-wide single nucleotide polymorphisms with in vitro sensitivities to 10 commonly used antimalarial drugs in 94 P. falciparum isolates from the China-Myanmar border area, a region with the longest history of ART usage. We identified several loci associated with various drugs, including those containing pfcrt and pfdhfr. Of particular interest is a locus on chromosome 10 containing the autophagy-related protein 18 (ATG18) associated with decreased sensitivities to dihydroartemisinin, artemether and piperaquine – an ACT partner drug in this area. ATG18 is a phosphatidylinositol-3-phosphate binding protein essential for autophagy and recently identified as a potential ART target. Further investigations on the ATG18 and genes at the chromosome 10 locus may provide an important lead for a connection between ART resistance and autophagy.