Circumsporozoite protein gene diversity among temperate and tropical Plasmodium vivax isolates from Iran

To date, there is no information on the genetic diversity of the circumsporozoite protein (CSP), a leading vaccine candidate, in Plasmodium vivax populations circulating in Iran. The gene for this protein, Pvcsp, was amplified from 374 P. vivax isolates collected in the temperate northern, and in the tropical southern endemic areas. PCR-RFLP analysis of the repeated central region revealed that the parasites collected in the northern area were almost exclusively of the VK210 type. Parasites collected in the south-eastern areas were of both VK210 and VK247 types. We detected VK210 parasite in 70.5% of the samples, VK247 parasites in 17.5% and mixed type infections in 12% of the isolates. Sequence analysis of 137 isolates obtained from both areas identified a total of 25 distinct genotypes. The degree of genetic diversity was generally higher for the tropical (21 genotypes) than the temperate (7 genotypes) P. vivax populations, a difference possibly reflecting the high cross-border exchanges between Afghanistan and Pakistan and southern Iran. Interestingly, all but two VK210 type isolates sequenced harboured a 36-bp post-repeat insert previously only observed in North Korea and China. This large-scale survey of parasite diversity in the Eastern Mediterranean Region provides a set of baseline data suitable for future molecular epidemiological studies of P. vivax.     

我国分离株间日疟原虫与红细胞黏附关键区DBPⅡ区基因多态性分析

目的　了解我国季节流行区间日疟原虫红内期候选抗原DBP基因多态性特点。方法　疟原虫基因组DNA提取。PCR扩增DBPRⅡ区基因片段,序列直接测序分析。结果　成功扩增18个DBPRⅡ区基因片段,发现9个核苷酸突变,产生4种基因型,导致9个氨基酸置换。其中G115 1-A116 9-A12 70为主导基因型,G384 -H390 -I4 2 4是主导氨基酸型。结论　我国季节流行区分离株DBP基因突变位点完全含盖在以往报道的位点中,同时表明我国分离株DBP基因突变位点有限,提示我国分离株红内期候选抗原DBP蛋白功能相对保守。     

Genetic diversity of Plasmodium vivax and Plasmodium falciparum in Kohat District, Pakistan

Malaria is one of the serious diseases threatening human health in Pakistan and contributes to a large proportion of the total malaria deaths in South Asia. However, little is known about the nature and extent of genetic diversity of the malarial parasites circulating in Pakistan. This study was designed to assess the infection status of Plasmodium and the genetic diversity of Plasmodium vivax and Plasmodium falciparum by analyzing msp-3α, msp-3β and msp-1, msp-2 genes respectively using allele specific nested PCR and RFLP assays. For this purpose, 130 field isolates were collected from the individuals who exhibited clinical symptoms associated with malaria in the Kohat region of Khyber Pakhtoonkhwa (KPK), Pakistan. Among 130 blood samples collected, P. vivax was detected in 105/130 (80.8%) and P. falciparum in 21/130 (16.2%). Mixed infections with both parasites were detected in 4/130 (3%) of the isolates. A large number of distinguishable alleles were found for msp genetic markers: 10 alleles for msp-3α and seven for msp-3β with one mixed infection in case of msp-3β. The genotyping of P. falciparum showed that K1+MAD20 mixed genotype was dominant in msp-1 and FC27 in msp-2. The results collectively suggest that P. vivax and P. falciparum populations in this region are highly polymorphic and mixed infections are prevalent.     

Genetic variability in Plasmodium vivax aldolase gene in Korean isolates and the sensitivity of the Binax Now malaria test

Introduction of rapid malaria diagnostic tests (RDT) initiated numerous field evaluations in various epidemiologic settings. But the efficiency of some RTD kits based on aldolase raised reservations for direct implementation of RDT into clinical settings. We performed Binax Now malaria test in 84 Korean Plasmodium vivax isolates and compared it with the traditional Giemsa stain microscopy test as the reference standard. The sensitivity of Binax Now was 62.0% for P. vivax cases (52/84, 95% CI 51.2-71.6%) with 100.0% specificity (50/50, 95% confidence interval 92.9-100%). After the aldolase gene sequence analysis of 84 isolates, two synonymous mutations in aldolase gene were identified in both Binax Now positive and negative samples. No significant association between the mutations and Binax Now malaria tests was found. Thus, the genetic variability would not explain the poor performance of P. vivax RDTs by detecting aldolase in ROK isolates.     

The analysis of circumsporozoite-protein gene sequences from South Korean isolates of Plasmodium vivax

The amino-acid sequences corresponding to the circumsporozoite protein (CSP) of Plasmodium vivax fall into two main types, VK210 and VK247, each of which has a characteristic tandem repeat. When the repetitive domains of the CSP gene from six isolates of P. vivax from South Korea were sequenced they were found to show a total of 20 tandem amino-acid repeats, and repeat patterns that are regular and distinct from those of other P. vivax isolates. The amino-acid sequences of the South Korean parasites were found to be most similar to those of three isolates from China (CH-3, CH-4, and CH-5) and, particularly, to one from North Korea. A sequence (AGGNAANKKAEDAGGNA) and two repeats of the sequence GGNA found in the North Korean parasites were found in all six isolates from South Korea. The South Korean parasites investigated appear phylogenetically identical and unique to the Korean peninsula.     

Dynamics of asymptomatic Plasmodium vivax infections and Duffy binding protein polymorphisms in relation to parasitemia levels in Papua New Guinean children

The interaction between Plasmodium vivax Duffy binding protein II (PvDBPII) and human erythrocyte Duffy antigen is necessary for blood stage infections. However, PvDBPII is highly polymorphic. We recently observed that certain recombinant DBPII variants bind better to erythrocytes in vitro. To examine the hypothesis that haplotypes with enhanced binding have increased parasitemia levels, we followed 206 Papua New Guinean children biweekly for six months with a total of 713 P. vivax samples genotyped. Twenty-seven PvDBPII haplotypes were identified, and 3 haplotypes accounted for 57% of the infections. The relative frequencies of dominant haplotypes remained stable throughout the study. There was no significant association with PvDBPII alleles or haplotypes with P. vivax parasitemia. The dominant haplotype (26% of samples), however, corresponded to a high-binding haplotype. Thus, common haplotypes are not likely to have arisen from increased fitness as measured by greater parasitemia levels. The restricted number of common haplotypes increases the feasibility of a PvDBPII-based vaccine.     

Genetic diversity and multiple infections of Plasmodium vivax malaria in Western Thailand

Using two polymorphic genetic markers, the merozoite surface protein-3alpha (MSP-3alpha) and the circumsporozoite protein (CSP), we investigated the population diversity of Plasmodium vivax in Mae Sod, Thailand from April 2000 through June 2001. Genotyping the parasites isolated from 90 malaria patients attending two local clinics for the dimorphic CSP gene revealed that the majority of the parasites (77%) were the VK210 type. Genotyping the MSP3-alpha gene indicated that P. vivax populations exhibited an equally high level of polymorphism as those from Papua New Guinea, a hyperendemic region. Based on the length of polymerase chain reaction products, three major types of the MSP-3alpha locus were distinguished, with frequencies of 74.8%, 18.7%, and 6.5%, respectively. The 13 alleles distinguished by restriction fragment length polymorphism analysis did not show a significant seasonal variation in frequency. Genotyping the MSP-3alpha and CSP genes showed that 19.3% and 25.6% of the patients had multiple infections, respectively, and the combined rate was 35.6%. Comparisons of MSP-3alpha sequences from nine clones further confirmed the high level of genetic diversity of the parasite and also suggested that geographic isolation may exist. These results strongly indicate that P. vivax populations are highly diverse and multiple clonal infections are common in this malaria-hypoendemic region of Thailand.     

Analysis of Plasmodium vivax merozoite surface protein-1 gene sequences from resurgent Korean isolates

The merozoite surface protein-1 (MSP-1) of Plasmodium vivax exhibits great antigenic diversity among different isolates of this parasite. This antigen is a useful genetic marker for studying the polymorphism of natural P. vivax parasite populations. One or more of these populations has been responsible for resurgent malaria now occurring in Korea. This paper reports the analysis of a highly polymorphic region between interspecies conserved blocks 5 and 6 of the MSP-1 gene, using the polymerase chain reaction to amplify the DNA fragment encompassing these regions from 25 Korean isolates, followed by sequencing. Almost all amino acid sequences of Korean isolates were nearly identical to that of Thai isolates TD525A (96.6-99.7%) and TD424 (96.3-99.5%), and very similar to that of the France-Belem strain when compared with other isolates (Sal-1, Sri Lanka, and Colombia). Interallelic recombination was found in the poly-Q repeat and a Sal-1 type amino acid structure was observed in all isolates. This study shows that the MSP gene nucleotide sequence of resurgent P. vivax in Korea is most similar to that of Thai isolates; however, the Korean strains are phylogenetically unique.     

Plasmodium vivax Duffy binding protein: baseline antibody responses and parasite polymorphisms in a well‐consolidated settlement of the Amazon Region

Objective  To investigate risk factors associated with the acquisition of antibodies against Plasmodium vivax Duffy binding protein (PvDBP) – a leading malaria vaccine candidate – in a well‐consolidated agricultural settlement of the Brazilian Amazon Region and to determine the sequence diversity of the PvDBP ligand domain (DBP_II) within the local malaria parasite population. Methods  Demographic, epidemiological and clinical data were collected from 541 volunteers using a structured questionnaire. Malaria parasites were detected by conventional microscopy and PCR, and blood collection was used for antibody assays and molecular characterisation of DBP_II. Results  The frequency of malaria infection was 7% (6% for P. vivax and 1% for P. falciparum), with malaria cases clustered near mosquito breeding sites. Nearly 50% of settlers had anti‐PvDBP IgG antibodies, as detected by enzyme‐linked immunosorbent assay (ELISA) with subject’s age being the only strong predictor of seropositivity to PvDBP. Unexpectedly, low levels of DBP_II diversity were found within the local malaria parasites, suggesting the existence of low gene flow between P. vivax populations, probably due to the relative isolation of the studied settlement. Conclusion  The recognition of PvDBP by a significant proportion of the community, associated with low levels of DBP_II diversity among local P. vivax, reinforces the variety of malaria transmission patterns in communities from frontier settlements. Such studies should provide baseline information for antimalarial vaccines now in development.     

Allelic dimorphism in the merozoite surface protein-3alpha in Korean isolates of Plasmodium vivax

To study the genetic diversity of re-emerging Plasmodium vivax in the Republic of Korea, nucleotide sequence variations at the merozoite surface protein-3alpha (PvMSP-3alpha) locus were analyzed using 24 re-emerging isolates and 4 isolates from imported cases. Compared with the well known Belem strain (Brazil), a large number of amino acid substitutions, deletions, and insertions were found at the locus of the isolates examined. The Korean isolates were divided into two allelic types; type I (15 isolates), similar to the Belem strain, and type II (9), similar to the Chess strain (New Guinea). Isolates from imported cases were classified into three types; type III (1 from Malaysia), similar to type B from western Thailand, type IV (1 each from Indonesia and India), and type V (1 from Pakistan), both being new types. Our results have shown that the MSP-3alpha locus of re-emerging Korean P. vivax is dimorphic with two allelic types coexisting in the endemic area.     

The Duffy blood group and resistance to Plasmodium vivax in Honduras.

To test the hypothesis that the Duffy blood group negative genotype is a factor in resistance to Plasmodium vivax, we determined the Duffy blood group, the malaria antibodies, and the slide-demonstrated infection rates with P. vivax and P. falciparum of 420 persons living in Nueva Armenia, Honduras. In all, 247 persons were Duffy negative. Demonstrated infections with P. falciparum were almost equally distributed between Duffy-positive (5,8%) and Duffy-negative (4.9%) persons. Similarly, Duffy-positive (25.6%) and Duffy-negative (28.2%) persons had equal proportions of indirect fluorescent antibody test titers suggestive of past or present P. falciparum infection. In contrast, all 14 P. vivax infections were found in Duffy-negative persons. There was no evidence suggesting that Duffy-positive and Duffy-negative persons had different exposures to malaria. The Duffy negative genotype FyFy appears to be a factor in resistance to P. vivax.     

Genetic diversity of Plasmodium vivax Pvcsp and Pvmsp1 in Guyana, South America

Approximately 55% of malaria infections in the Guyana Amazon region are attributed to Plasmodium falciparum while the other 45% are attributed to non-falciparum, mostly Plasmodium vivax. However, little is known about the P. vivax strain types circulating in the region. Using PCR for Plasmodium detection and two genetic markers specific to P. vivax to detect the polymorphic circumsporozoite protein (CSP) and the conserved 19-kDa region of the merozoite surface protein-1 (MSP-1), we investigated the overall Plasmodium strain distribution and population diversity within P. vivax in isolates collected from the blood of infected individuals in the interior Amazon region of Guyana, South America. Out of a total of 250 samples positive for Plasmodium, P. vivax was detected in 30% (76/250) and P. falciparum was detected in 76% (189/250). Mixed infections containing both P. falciparum and P. vivax constituted 6% (15/250) of the total positive samples. Further analysis of P. vivax strains showed that 92% (56/61) of the P. vivax samples hybridized with a probe specific to type VK210, 39% (24/61) hybridized with a probe specific for type VK247, and 25% (15/61) hybridized with a probe specific for the P. vivax-like CS genotype. DNA sequencing of the 19-kDa C-terminal domain in block 13 of MSP-1 amplified from 61 samples from patients infected with P. vivax demonstrated that this region is highly conserved, and all samples were identical at the nucleotide level to the Belem and Salvador-1 types. No synonymous or nonsynonymous mutations were observed in this region of the gene, indicating that current vaccine-development efforts based on the MSP-1(19) fragment would be applicable in Guyana.     

Genetic diversity of merozoite surface protein-1 gene of Plasmodium vivax isolates in mining villages of Venezuela (Bolivar State)

The merozoite surface protein-1 gene of Plasmodium vivax is highly polymorphic and so, currently used in epidemiological studies of P. vivax malaria. We sequenced the variable block 5 of the gene from 39 Venezuelan isolates, 18 of which were co-infected with Plasmodium falciparum. We observed a limited variability with 34 isolates belonging to the type Salvador I, none Belem type and only five recombinants. Among the recombinants, only two types of sequences were observed with, respectively, 18 and 21 poly-Q residues. Nucleotide substitutions explained the major differences of the 11 patterns observed. We could evidence neither specific MSP-1 genotype associated with co-infected samples, nor peculiar MSP-1 genotype distribution inside the investigated areas. In comparison with other low endemic regions in the world, our sampling has a lower genetic diversity, which could be mainly explained by the lack of Belem type. In fact, the variable repeats of poly-Q residues involved in the polymorphism of Belem type and recombinant isolates are responsible for a great part of variability observed in MSP-1 block 5.     

Characteristics of natural antibody responses to the circumsporozoite protein of Plasmodium vivax

The antibody response to the prototype circumsporozoite (CS) protein of Plasmodium vivax (CSPV) was studied in Thai soldiers experiencing occupational malaria. Seventy-four (65%) of 114 men followed during assignment to a malaria transmission area developed blood-stage infection with P. vivax. IgG antibodies against the central repeat region of the CSPV protein were quantitated by ELISA using the recombinant protein, NS181V20, as the capture antigen. One quarter of the subjects had detectable anti-CSPV antibodies at the beginning of the study. CSPV antibody seroconversion was documented in 16 of 26 subjects assessed during their first observed episodes of vivax malaria. This antibody response was of moderate magnitude, fell off after the first week post-diagnosis and appeared, at the low levels observed, to be unassociated with protection. Continued assessment of anti-CSPV antibody after subjects left the transmission area found no increase associated with release of P. vivax. These findings indicate that CS antibody responses to P. vivax during occupational malaria share many characteristics with responses to P. falciparum.     

Single-nucleotide polymorphisms and genome diversity in Plasmodium vivax

The study of genetic variation in malaria parasites has practical significance for developing strategies to control the disease. Vaccines based on highly polymorphic antigens may be confounded by allelic restriction of the host immune response. In response to drug pressure, a highly plastic genome may generate resistant mutants more easily than a monomorphic one. Additionally, the study of the distribution of genomic polymorphisms may provide information leading to the identification of genes associated with traits such as parasite development and drug resistance. Indeed, the age and diversity of the human malaria parasite Plasmodium falciparum has been the subject of recent debate, because an ancient parasite with a complex genome is expected to present greater challenges for drug and vaccine development. The genome diversity of the important human pathogen Plasmodium vivax, however, remains essentially unknown. Here we analyze an approximately 100-kb contiguous chromosome segment from five isolates, revealing 191 single-nucleotide polymorphisms (SNPs) and 44 size polymorphisms. The SNPs are not evenly distributed across the segment with blocks of high and low diversity. Whereas the majority (approximately 63%) of the SNPs are in intergenic regions, introns contain significantly less SNPs than intergenic sequences. Polymorphic tandem repeats are abundant and are more uniformly distributed at a frequency of about one polymorphic tandem repeat per 3 kb. These data show that P. vivax has a highly diverse genome, and provide useful information for further understanding the genome diversity of the parasite.     

Genetic polymorphism of Plasmodium vivax msp1p, a paralog of merozoite surface protein 1, from worldwide isolates

Plasmodium vivax msp1p, a paralog of the candidate vaccine antigen P. vivax merozoite surface protein 1, possesses a signal peptide at its N-terminus and two epidermal growth factor-like domains at its C-terminus with a glycosylphosphatidylinositol attachment site. The msp1p gene locus may have originated by a duplication of the msp1 gene locus in a common ancestor of the analyzed Plasmodium species and lost from P. yoelii, P. berghei, and P. falciparum during their evolutionary history. Full-length sequences of the msp1p gene were generally highly conserved; they had a few amino acid substitutions, one highly polymorphic E/Q-rich region, and a single-to-triple hepta-peptide repeat motif. Twenty-one distinguishable allelic types (A1-A21) of the E/Q-rich region were identified from worldwide isolates. Among them, four types were detected in isolates from South Korea. The length polymorphism of the E/Q-rich region might be useful as a genetic marker for population structure studies in malaria-endemic areas.     

Conserved sequences of thrombospondin-related adhesive protein gene of Plasmodium vivax in clinical isolates from Korea

Thrombospondin-related adhesive protein (TRAP) from Plasmodium vivax (P. vivax) became one of the important vaccine candidates for malaria, because P. vivax TRAP (PvTRAP) is responsible for the sporozoite-host interactions. PvTRAP polymorphisms in the isolates from Republic of Korea (ROK) were analysed, setting the valuable baseline data for the future vaccine developments and clinical trials with PvTRAP, as a strong vaccine candidate. A total of 54 isolates were collected in 2010. PvTRAP genes from above isolates were amplified and sequenced, and the results were analysed and compared against Sal-1 strain. Sequencing analysis of 1424-bp-size PvTRAP PCR products revealed one major allelic type with six non-synonymous substitutions, where S81T, E95D, I121V and T127R substitutions were found in region II, and K371N and A425E substitutions from region IV. The ROK isolates revealed the limited sequence polymorphisms in PvTRAP in comparison with the reported isolates from other nations.     

Response to chloroquine of Plasmodium vivax among South Korean soldiers

The response to standard chloroquine treatment was evaluated, by microscopical examination of blood-smears, among 81 soldiers diagnosed with Plasmodium vivax malaria in South Korea in 1996. The smears were prepared pre-treatment and 3, 14 and 28 days after starting chemotherapy. Parasitaemias were determined after staining the smears with Giemsa's stain. Blood samples from the patients who were not smear-negative by day 3 were carefully checked for parasites, by staining smears with Acridine Orange and by a PCR-based assay. Only two of the patients appeared to be parasitaemic on day 14 and were therefore considered treatment failures. Although both were apparently cured after additional therapy with the same regimen, one had a recurrence 8 months later. Most cases of recent, resurgent malaria in South Korea therefore appear to sensitive to chloroquine.     

Dramatic difference in diversity between Plasmodium falciparum and Plasmodium vivax reticulocyte binding-like genes

Malaria parasite proteins involved in erythrocyte invasion are considered important vaccine targets. Members of the reticulocyte binding-like (RBL) family of Plasmodium merozoite proteins are found in human, simian, and rodent malaria parasites and function in the initial steps of erythrocyte selection and invasion. The RBL genes are large, ranging in size from 7.7 to 10 kb, and the extent of any sequence diversity in parasite populations is unknown. We present the first assessment of sequence diversity within RBL genes from the two major human malaria parasites: Plasmodium falciparum and P. vivax. Polymorphism within the RBL genes is generally limited, except for P. vivax reticulocyte binding protein 2 (PvRBP2), which has nucleotide diversity levels 25-fold higher than the other RBL genes. The PvRBP2 haplotypes appear to fall into two distinct classes of alleles, suggesting large-scale dimorphism in this gene. Polymorphisms were frequently clustered, suggesting that different RBL domains may be evolving under different selection and functional pressures.     

Age-acquired immunity to a Plasmodium vivax invasion ligand,the duffy binding protein

To investigate the interactions of glycoconjugates with the innate immune system, peripheral blood mononuclear cells were stimulated with glycolipids derived from Schistosoma mansoni eggs and worms and with biochemically synthesized neoglycoconjugates. Egg glycolipids stimulated the production of interleukin (IL)--10, IL-6, and tumor necrosis factor--alpha in monocytes, whereas worm glycolipids failed to do so. When monoclonal antibodies that specifically recognize defined carbohydrate epitopes were used, the binding of a GalNAc beta 1-4(Fuc alpha 1-2Fuc alpha 1-3)GlcNAc (LDN-DF) reactive antibody was pronounced on egg glycolipids but was absent on worm glycolipids. The binding of antibodies that recognize Gal beta 1-4(Fuc alpha 1-3)GlcNAc (LewisX), GalNAc beta 1-4GlcNAc (LDN), and GalNAc beta 1-4(Fuc alpha 1-3)GlcNAc (LDN-F) was comparable for both preparations. Cytokine production in response to neoglycoconjugates containing enzymatically synthesized glycans also was measured. The LDN-DF neoglycoconjugate was the most potent cytokine inducer, which indicates that this difucosylated glycan can act at the host-parasite interface and can trigger innate immune responses.