Geographical distribution and genetic diversity of Plasmodium vivax reticulocyte binding protein 1a correlates with patient antigenicity

Plasmodium vivax is the most widespread cause of human malaria. Recent reports of drug resistant vivax malaria and the challenge of eradicating the dormant liver forms increase the importance of vaccine development against this relapsing disease. P. vivax reticulocyte binding protein 1a (PvRBP1a) is a potential vaccine candidate, which is involved in red cell tropism, a crucial step in the merozoite invasion of host reticulocytes. As part of the initial evaluation of the PvRBP1a vaccine candidate, we investigated its genetic diversity and antigenicity using geographically diverse clinical isolates. We analysed pvrbp1a genetic polymorphisms using 202 vivax clinical isolates from six countries. Pvrbp1a was separated into six regions based on specific domain features, sequence conserved/polymorphic regions, and the reticulocyte binding like (RBL) domains. In the fragmented gene sequence analysis, PvRBP1a region II (RII) and RIII (head and tail structure homolog, 152–625 aa.) showed extensive polymorphism caused by random point mutations. The haplotype network of these polymorphic regions was classified into three clusters that converged to independent populations. Antigenicity screening was performed using recombinant proteins PvRBP1a-N (157–560 aa.) and PvRBP1a-C (606–962 aa.), which contained head and tail structure region and sequence conserved region, respectively. Sensitivity against PvRBP1a-N (46.7%) was higher than PvRBP1a-C (17.8%). PvRBP1a-N was reported as a reticulocyte binding domain and this study identified a linear epitope with moderate antigenicity, thus an attractive domain for merozoite invasion-blocking vaccine development. However, our study highlights that a global PvRBP1a-based vaccine design needs to overcome several difficulties due to three distinct genotypes and low antigenicity levels.     

Plasmodium vivax Reticulocyte Binding Proteins for invasion into reticulocytes

Plasmodium vivax is responsible for most of the malaria infections outside Africa and is currently the predominant malaria parasite in countries under elimination programs. P. vivax preferentially enters young red cells called reticulocytes. Advances in understanding the molecular and cellular mechanisms of entry are hampered by the inability to grow large numbers of P. vivax parasites in a long‐term in vitro culture. Recent progress in understanding the biology of the P. vivax Reticulocyte Binding Protein (PvRBPs) family of invasion ligands has led to the identification of a new invasion pathway into reticulocytes, an understanding of their structural architecture and PvRBPs as targets of the protective immune response to P. vivax infection. This review summarises current knowledge on the role of reticulocytes in P. vivax infection, the function of the PvRBP family of proteins in generating an immune response in human populations, and the characterization of anti‐PvRBP antibodies in blocking parasite invasion.     

Characterization of Plasmodium vivax Early Transcribed Membrane Protein 11.2 and Exported Protein 1

In Plasmodium, the membrane of intracellular parasites is initially formed during invasion as an invagination of the red blood cell surface, which forms a barrier between the parasite and infected red blood cells in asexual blood stage parasites. The membrane proteins of intracellular parasites of Plasmodium species have been identified such as early-transcribed membrane proteins (ETRAMPs) and exported proteins (EXPs). However, there is little or no information regarding the intracellular parasite membrane in Plasmodium vivax. In the present study, recombinant PvETRAMP11.2 (PVX_003565) and PvEXP1 (PVX_091700) were expressed and evaluated antigenicity tests using sera from P. vivax-infected patients. A large proportion of infected individuals presented with IgG antibody responses against PvETRAMP11.2 (76.8%) and PvEXP1 (69.6%). Both of the recombinant proteins elicited high antibody titers capable of recognizing parasites of vivax malaria patients. PvETRAMP11.2 partially co-localized with PvEXP1 on the intracellular membranes of immature schizont. Moreover, they were also detected at the apical organelles of newly formed merozoites of mature schizont. We first proposed that these proteins might be synthesized in the preceding schizont stage, localized on the parasite membranes and apical organelles of infected erythrocytes, and induced high IgG antibody responses in patients.     

Evaluation of the antigenicity of universal epitopes from PvDBPII in individuals exposed to Plasmodium vivax malaria

The Duffy-binding protein (PvDBP) mediates invasion of reticulocytes by the malaria parasite Plasmodium vivax. PvDBP has been recognized as a good vaccine candidate due to its ability to induce antibody responses capable of inhibiting target cell invasion after natural infections. For the development of subunit-based vaccines, it is important to identify universal epitopes that could be presented by different HLA-DR alleles to induce effective cellular and humoral immune responses. In this study, the antigenicity of universal epitopes from PvDBPII was evaluated by stimulating peripheral blood mononuclear cells (PBMCs) isolated from individuals with different degrees of P. vivax malaria exposure and distinct HLA-DR alleles. Peptides 1635 and 1638 induced lymphoproliferation and stimulated the production of IL-6 and IFN-γ. The results suggest that conserved peptides binding with high activity to red blood cells and with known affinity to HLA-DR proteins could be good components for a P. vivax vaccine.     

Identifying and characterizing a member of the RhopH1/Clag family in Plasmodium vivax

Plasmodium vivax malaria caused is a public health problem that produces very high morbidity worldwide. During invasion of red blood cells the parasite requires the intervention of high molecular weight complex rhoptry proteins that are also essential for cytoadherence. PfClag9, a member of the RhopH multigene family, has been identified as being critical during Plasmodium falciparum infection. This study describes identifying and characterizing the pfclag9 ortholog in P. vivax (hereinafter named pvclag7). The pvclag7 gene is transcribed at the end of the intraerythrocytic cycle and is recognized by sera from humans who have been infected by P. vivax. PvClag7 subcellular localization has been also determined and, similar to what occurs with PfClag9, it co-localize with other proteins from the Rhoptry high molecular weight complex.     

Red blood cells derived from peripheral blood and bone marrow CD34+ human haematopoietic stem cells are permissive to Plasmodium parasites infection

The production of fully functional human red cells in vitro from haematopoietic stem cells (hHSCs) has been successfully achieved. Recently, the use of hHSCs from cord blood represented a major improvement to develop the continuous culture system for Plasmodium vivax. Here, we demonstrated that CD34+hHSCs from peripheral blood and bone marrow can be expanded and differentiated to reticulocytes using a novel stromal cell. Moreover, these reticulocytes and mature red blood cells express surface markers for entrance of malaria parasites contain adult haemoglobin and are also permissive to invasion by P. vivax and Plasmodium falciparum parasites.     

Increased reticulocyte count from cord blood samples using hypotonic lysis

Human reticulocytes are one of the fundamental components needed to study the in vitro invasion processes of the human malaria parasite Plasmodium vivax. Additionally examinations of reticulocytes and their binding proteins are difficult in areas of the world that do not have access to advanced equipment or stem cell lines. These issues are particularly relevant to malaria vaccine candidate studies that are directed against surface proteins that the parasites use to gain entry into erythrocytes. Described here is a simple and inexpensive method to increase the reticulocyte count of cord blood samples. Exposure of cord blood to hypotonic saline (0.2%) for 5 min selectively lyses the non-reticulocytes resulting in an average 3.6-fold increase in reticulocyte count. Our studies show that this enrichment process does not damage the hemoglobin of the remaining erythrocytes which are still capable of supporting Plasmodium falciparum invasion and growth. This economical and rapid method of enrichment could facilitate studies of in vitro laboratory culturing of other malaria parasite species which preferentially invade reticulocytes such as P. vivax.     

Hematopoietic Stem/Progenitor Cell Sources to Generate Reticulocytes for Plasmodium vivax Culture

The predilection of Plasmodium vivax (P. vivax) for reticulocytes is a major obstacle for its establishment in a long-term culture system, as this requires a continuous supply of large quantities of reticulocytes, representing only 1–2% of circulating red blood cells. We here compared the production of reticulocytes using an established in vitro culture system from three different sources of hematopoietic stem/progenitor cells (HSPC), i.e. umbilical cord blood (UCB), bone marrow (BM) and adult peripheral blood (PB). Compared to CD34⁺-enriched populations of PB and BM, CD34⁺-enriched populations of UCB produced the highest amount of reticulocytes that could be invaded by P. vivax. In addition, when CD34⁺-enriched cells were first expanded, a further extensive increase in reticulocytes was seen for UCB, to a lesser degree BM but not PB. As invasion by P. vivax was significantly better in reticulocytes generated in vitro, we also suggest that P. vivax may have a preference for invading immature reticulocytes, which should be confirmed in future studies.     

Coexistence of Malaria and Thalassemia in Malaria Endemic Areas of Thailand

Hemoglobinopathy and malaria are commonly found worldwide particularly in malaria endemic areas. Thalassemia, the alteration of globin chain synthesis, has been reported to confer resistance against malaria. The prevalence of thalassemia was investigated in 101 malaria patients with Plasmodium falciparum and Plasmodium vivax along the Thai-Myanmar border to examine protective effect of thalassemia against severe malaria. Hemoglobin typing was performed using low pressure liquid chromatography (LPLC) and α-thalassemia was confirmed by multiplex PCR. Five types of thalassemia were observed in malaria patients. The 2 major types of thalassemia were Hb E (18.8%) and α-thalassemia-2 (11.9%). There was no association between thalassemia hemoglobinopathy and malaria parasitemia, an indicator of malaria disease severity. Thalassemia had no significant association with P. vivax infection, but the parasitemia in patients with coexistence of P. vivax and thalassemia was about 2-3 times lower than those with coexistence of P. falciparum and thalassemia and malaria without thalassemia. Furthermore, the parasitemia of P. vivax in patients with coexistence of Hb E showed lower value than coexistence with other types of thalassemia and malaria without coexistence. Parasitemia, hemoglobin, and hematocrit values in patients with coexistence of thalassemia other than Hb E were significantly lower than those without coexistence of thalassemia. Furthermore, parasitemia with coexistence of Hb E were 2 times lower than those with coexistence of thalassemia other than Hb E. In conclusion, the results may, at least in part, support the protective effect of thalassemia on the development of hyperparasitemia and severe anemia in malaria patients.     

Prevalence and Clinical Manifestations of Malaria in Aligarh,India

Malaria is one of the most widespread infectious diseases of tropical countries with an estimated 207 million cases globally. In India, there are endemic pockets of this disease, including Aligarh. Hundreds of Plasmodium falciparum and P. vivax cases with severe pathological conditions are recorded every year in this district. The aim of this study is to find out changes in liver enzymes and kidney markers. Specific diagnosis for P. falciparum and P. vivax was made by microscopic examination of Giemsa stained slides. Clinical symptoms were observed in both of these infections. Liver enzymes, such as AST, ALT, and ALP, and kidney function markers, such as creatinine and urea, were estimated by standard biochemical techniques. In Aligarh district, P. vivax, P. falciparum, and mixed infections were 64%, 34%, and 2%, respectively. In case of P. falciparum infection, the incidences of anemia, splenomegaly, renal failure, jaundice, and neurological sequelae were higher compared to those in P. vivax infection. Recrudescence and relapse rates were 18% and 20% in P. falciparum and P. vivax infections, respectively. Liver dysfunctions and renal failures were more common in P. falciparum patients, particularly in elderly patients. Artesunate derivatives must, therefore, be introduced for the treatment of P. falciparum as they resist to chloroquine as well as sulfadoxine-pyrimethamine combinations.     

Plasmodium vivax Drug Resistance Genes; Pvmdr1 and Pvcrt-o Polymorphisms in Relation to Chloroquine Sensitivity from a Malaria Endemic Area of Thailand

The aim of the study was to explore the possible molecular markers of chloroquine resistance in Plasmodium vivax isolates in Thailand. A total of 30 P. vivax isolates were collected from a malaria endemic area along the Thai-Myanmar border in Mae Sot district of Thailand. Dried blood spot samples were collected for analysis of Pvmdr1 and Pvcrt-o polymorphisms. Blood samples (100 μl) were collected by finger-prick for in vitro chloroquine susceptibility testing by schizont maturation inhibition assay. Based on the cut-off IC₅₀ of 100 nM, 19 (63.3%) isolates were classified as chloroquine resistant P. vivax isolates. Seven non-synonymous mutations and 2 synonymous were identified in Pvmdr1 gene. Y976F and F1076L mutations were detected in 7 (23.3%) and 16 isolates (53.3%), respectively. Analysis of Pvcrt-o gene revealed that all isolates were wild-type. Our results suggest that chloroquine resistance gene is now spreading in this area. Monitoring of chloroquine resistant molecular markers provide a useful tool for future control of P. vivax malaria.     

Host Erythrocyte Environment Influences the Localization of Exported Protein 2, an Essential Component of the Plasmodium Translocon

Malaria parasites replicating inside red blood cells (RBCs) export a large subset of proteins into the erythrocyte cytoplasm to facilitate parasite growth and survival. PTEX, the parasite-encoded translocon, mediates protein transport across the parasitophorous vacuolar membrane (PVM) in Plasmodium falciparum-infected erythrocytes. Proteins exported into the erythrocyte cytoplasm have been localized to membranous structures, such as Maurer''s clefts, small vesicles, and a tubovesicular network. Comparable studies of protein trafficking in Plasmodium vivax-infected reticulocytes are limited. With Plasmodium yoelii-infected reticulocytes, we identified exported protein 2 (Exp2) in a proteomic screen of proteins putatively transported across the PVM. Immunofluorescence studies showed that P. yoelii Exp2 (PyExp2) was primarily localized to the PVM. Unexpectedly, PyExp2 was also associated with distinct, membrane-bound vesicles in the reticulocyte cytoplasm. This is in contrast to P. falciparum in mature RBCs, where P. falciparum Exp2 (PfExp2) is exclusively localized to the PVM. Two P. yoelii-exported proteins, PY04481 (encoded by a pyst-a gene) and PY06203 (PypAg-1), partially colocalized with these PyExp2-positive vesicles. Further analysis revealed that with P. yoelii, Plasmodium berghei, and P. falciparum, cytoplasmic Exp2-positive vesicles were primarily observed in CD71⁺ reticulocytes versus mature RBCs. In transgenic P. yoelii 17X parasites, the association of hemagglutinin-tagged PyExp2 with the PVM and cytoplasmic vesicles was retained, but the pyexp2 gene was refractory to deletion. These data suggest that the localization of Exp2 in mouse and human RBCs can be influenced by the host cell environment. Exp2 may function at multiple points in the pathway by which parasites traffic proteins into and through the reticulocyte cytoplasm.     

Probability of Antibody Formation against Circumsporozoite Protein of Plasmodium vivax among Korean Malaria Patients

To evaluate the seroprevalence against circumsporozoite protein (CSP) of Plasmodium vivax in sera of Korean patients, the central repeating domain (CRD) of CSP was cloned and analyzed. From the genomic DNA of patient''s blood, 2 kinds of CSPs were identified to belong to a VK210 type, which is the dominant repeating of GDRA(D/A)GQPA, and named as PvCSPA and PvCSPB. Recombinantly expressed his-tagged PvCSPA or PvCSPB in Escherichia coli reacted well against sera of patients in western blot, with the detecting rate of 47.9% (58/121), which included 15 cases positive for PvCSPA, 6 cases positive for PvCSPB, and 37 cases for both. The mixture of PvCSPA and PvCSPB was loaded to a rapid diagnostic test kit (RDT) and applied with the same set of patient sera, which resulted in detection rates of 57.0% (69/121). When the protein sequences of PvCSPA were compared with those of P. vivax in endemic regions of India and Uganda, they were compatibly homologous to PvCSPA with minor mutations. These results suggested that the recombinant PvCSPA and PvCSPB loaded RDT may be a milestone in latent diagnosis which has been a hot issue of domestic malaria and important for radical therapy in overlapped infections with P. falciparum in tropical and subtropical areas. During the biological process of malarial infection, exposure of CSP to antigen-antibody reaction up to 57.0% is the first report in Korea.     

Genetic diversity in new members of the reticulocyte binding protein family in Thai Plasmodium vivax isolates

Background

Plasmodium vivax merozoites specifically invade reticulocytes. Until recently, two reticulocyte-binding proteins (Pvrbp1 and Pvrbp2) expressed at the apical pole of the P. vivax merozoite were considered to be involved in reticulocyte recognition. The genome sequence recently obtained for the Salvador I (Sal-I) strain of P. vivax revealed additional genes in this family, and in particular Pvrbp2a, Pvrbp2b (Pvrbp2 has been renamed as Pvrbp2c) and two pseudogenes Pvrbp2d and Pvrbp3. It had been previously found that Pvrbp2c is substantially more polymorphic than Pvrbp1. The primary goal of this study was to ascertain the level of polymorphism of these new genes.

Methodology/Principal Findings

The sequence of the Pvrbp2a, Pvrbp2b, Pvrbp2d and Pvrbp3 genes were obtained by amplification/cloning using DNA purified from four isolates collected from patients that acquired the infection in the four cardinal regions of Thailand (west, north, south and east). An additional seven isolates from western Thailand were analyzed for gene copy number variation. There were significant polymorphisms exhibited by these genes (compared to the reference Sal-I strain) with the ratio of mutations leading to a non-synonymous or synonymous amino acid change close to 3∶1 for Pvrbp2a and Pvrbp2b. Although the degree of polymorphism exhibited by these two genes was higher than that of Pvrbp1, it did not reach the exceptional diversity noted for Pvrbp2c. It was interesting to note that variations in the copy number of Pvrbp2a and Pvrbp2b occurred in some isolates.

Conclusions/Significance

The evolution of different members of the Pvrbp2 family and their relatively high degree of polymorphism suggests that the proteins encoded by these genes are important for parasite survival and are under immune selection. Our data also shows that there are highly conserved regions in rbp2a and rbp2b, which might provide suitable targets for future vaccine development against the blood stage of P. vivax.     

A Library of Plasmodium vivax Recombinant Merozoite Proteins Reveals New Vaccine Candidates and Protein-Protein Interactions

Background

A vaccine targeting Plasmodium vivax will be an essential component of any comprehensive malaria elimination program, but major gaps in our understanding of P. vivax biology, including the protein-protein interactions that mediate merozoite invasion of reticulocytes, hinder the search for candidate antigens. Only one ligand-receptor interaction has been identified, that between P. vivax Duffy Binding Protein (PvDBP) and the erythrocyte Duffy Antigen Receptor for Chemokines (DARC), and strain-specific immune responses to PvDBP make it a complex vaccine target. To broaden the repertoire of potential P. vivax merozoite-stage vaccine targets, we exploited a recent breakthrough in expressing full-length ectodomains of Plasmodium proteins in a functionally-active form in mammalian cells and initiated a large-scale study of P. vivax merozoite proteins that are potentially involved in reticulocyte binding and invasion.

Methodology/Principal Findings

We selected 39 P. vivax proteins that are predicted to localize to the merozoite surface or invasive secretory organelles, some of which show homology to P. falciparum vaccine candidates. Of these, we were able to express 37 full-length protein ectodomains in a mammalian expression system, which has been previously used to express P. falciparum invasion ligands such as PfRH5. To establish whether the expressed proteins were correctly folded, we assessed whether they were recognized by antibodies from Cambodian patients with acute vivax malaria. IgG from these samples showed at least a two-fold change in reactivity over naïve controls in 27 of 34 antigens tested, and the majority showed heat-labile IgG immunoreactivity, suggesting the presence of conformation-sensitive epitopes and native tertiary protein structures. Using a method specifically designed to detect low-affinity, extracellular protein-protein interactions, we confirmed a predicted interaction between P. vivax 6-cysteine proteins P12 and P41, further suggesting that the proteins are natively folded and functional. This screen also identified two novel protein-protein interactions, between P12 and PVX_110945, and between MSP3.10 and MSP7.1, the latter of which was confirmed by surface plasmon resonance.

Conclusions/Significance

We produced a new library of recombinant full-length P. vivax ectodomains, established that the majority of them contain tertiary structure, and used them to identify predicted and novel protein-protein interactions. As well as identifying new interactions for further biological studies, this library will be useful in identifying P. vivax proteins with vaccine potential, and studying P. vivax malaria pathogenesis and immunity.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00663546","term_id":"NCT00663546"}}NCT00663546     

Receptor–ligand and parasite protein–protein interactions in Plasmodium vivax: Analysing rhoptry neck proteins 2 and 4

Elucidating receptor–ligand and protein–protein interactions represents an attractive alternative for designing effective Plasmodium vivax control methods. This article describes the ability of P. vivax rhoptry neck proteins 2 and 4 (RON2 and RON4) to bind to human reticulocytes. Biochemical and cellular studies have shown that two PvRON2‐ and PvRON4‐derived conserved regions specifically interact with protein receptors on reticulocytes marked by the CD71 surface transferrin receptor. Mapping each protein fragment's binding region led to defining the specific participation of two 20 amino acid‐long regions selectively competing for PvRON2 and PvRON4 binding to reticulocytes. Binary interactions between PvRON2 (ligand) and other parasite proteins, such as PvRON4, PvRON5, and apical membrane antigen 1 (AMA1), were evaluated and characterised by surface plasmon resonance. The results revealed that both PvRON2 cysteine‐rich regions strongly interact with PvAMA1 Domains II and III (equilibrium constants in the nanomolar range) and at a lower extent with the complete PvAMA1 ectodomain and Domains I and II. These results strongly support that these proteins participate in P. vivax's complex invasion process, thus providing new pertinent targets for blocking P. vivax merozoites' specific entry to their target cells.     

Evaluation of splenic accumulation and colocalization of immature reticulocytes and Plasmodium vivax in asymptomatic malaria: A prospective human splenectomy study

BackgroundA very large biomass of intact asexual-stage malaria parasites accumulates in the spleen of asymptomatic human individuals infected with Plasmodium vivax. The mechanisms underlying this intense tropism are not clear. We hypothesised that immature reticulocytes, in which P. vivax develops, may display high densities in the spleen, thereby providing a niche for parasite survival.Methods and findingsWe examined spleen tissue in 22 mostly untreated individuals naturally exposed to P. vivax and Plasmodium falciparum undergoing splenectomy for any clinical indication in malaria-endemic Papua, Indonesia (2015 to 2017). Infection, parasite and immature reticulocyte density, and splenic distribution were analysed by optical microscopy, flow cytometry, and molecular assays. Nine non-endemic control spleens from individuals undergoing spleno-pancreatectomy in France (2017 to 2020) were also examined for reticulocyte densities. There were no exclusion criteria or sample size considerations in both patient cohorts for this demanding approach.In Indonesia, 95.5% (21/22) of splenectomy patients had asymptomatic splenic Plasmodium infection (7 P. vivax, 13 P. falciparum, and 1 mixed infection). Significant splenic accumulation of immature CD71 intermediate- and high-expressing reticulocytes was seen, with concentrations 11 times greater than in peripheral blood. Accordingly, in France, reticulocyte concentrations in the splenic effluent were higher than in peripheral blood. Greater rigidity of reticulocytes in splenic than in peripheral blood, and their higher densities in splenic cords both suggest a mechanical retention process. Asexual-stage P. vivax-infected erythrocytes of all developmental stages accumulated in the spleen, with non-phagocytosed parasite densities 3,590 times (IQR: 2,600 to 4,130) higher than in circulating blood, and median total splenic parasite loads 81 (IQR: 14 to 205) times greater, accounting for 98.7% (IQR: 95.1% to 98.9%) of the estimated total-body P. vivax biomass. More reticulocytes were in contact with sinus lumen endothelial cells in P. vivax- than in P. falciparum-infected spleens. Histological analyses revealed 96% of P. vivax rings/trophozoites and 46% of schizonts colocalised with 92% of immature reticulocytes in the cords and sinus lumens of the red pulp. Larger splenic cohort studies and similar investigations in untreated symptomatic malaria are warranted.ConclusionsImmature CD71⁺ reticulocytes and splenic P. vivax-infected erythrocytes of all asexual stages accumulate in the same splenic compartments, suggesting the existence of a cryptic endosplenic lifecycle in chronic P. vivax infection. Findings provide insight into P. vivax-specific adaptions that have evolved to maximise survival and replication in the spleen.

Dr. Anstey and co-authors found that P. vivax-infected immature reticulocytes and erythrocytes accumulate in the same splenic compartments, suggesting existence of a cryptic endosplenic lifecycle in chronic P. vivax infection that maximizes survival and replication in the spleen.     

Genetic Polymorphisms in VIR Genes among Indian Plasmodium vivax Populations

The vir genes are antigenic genes and are considered to be possible vaccine targets. Since India is highly endemic to Plasmodium vivax, we sequenced 5 different vir genes and investigated DNA sequence variations in 93 single-clonal P. vivax isolates. High variability was observed in all the 5 vir genes; the vir 1/9 gene was highly diverged across Indian populations. The patterns of genetic diversity do not follow geographical locations, as geographically distant populations were found to be genetically similar. The results in general present complex genetic diversity patterns in India, requiring further in-depth population genetic and functional studies.     

A Case of Plasmodium ovale wallikeri Infection in a Chinese Worker Returning from West Africa

In contrast to the gradual reduction in the number of locally transmitted malaria cases in China, the number of imported malaria cases has been increasing since 2008. Here, we report a case of a 39-year-old Chinese man who acquired Plasmodium ovale wallikeri infection while staying in Ghana, West Africa for 6 months in 2012. Microscopic examinations of Giemsa-stained thin and thick blood smears indicated Plasmodium vivax infection. However, the results of rapid diagnostic tests, which were conducted 3 times, were not in agreement with P. vivax. To further check the diagnosis, standard PCR analysis of the small-subunit rRNA gene was conducted, based on which a phylogeny tree was constructed. The results of gene sequencing indicated that this malaria is a variant of P. ovale (P. ovale wallikeri). The infection in this patient was not a new infection, but a relapse of the infection from the one that he had contracted in West Africa.