Genetic diversity in merozoite surface protein (MSP)-1 and MSP-2 genes of Plasmodium falciparum in a major endemic region of Iran

Merozoite surface protein-1 (MSP-1) and merozoite surface protein-2 (MSP-2) were used to develop vaccines and to investigate the genetic diversity in Plasmodium falciparum malaria in Iran. Nested polymerase chain reaction amplification was used to determine polymorphisms of block 2 of the MSP-1 and the central domain of MSP-2 genes. A total of 67 microscopically positive P. falciparum infected individuals from a major endemic region, southeast Iran, were included in this trial. Nine alleles of MSP-1 and 11 alleles of MSP-2 were identified. The results showed that amplified product from these surface antigen genes varied in size and there was specific pattern for each isolate. Besides, regarding this pattern, 23 multiple infections with at least 2 alleles were observed. While the endemic regions of malaria in Iran is classified in low to moderate group, but extensive polymorphism was observed for each marker and the MSP-2 central repeat was the most diverse that could be considered in designing malaria vaccine.     

Imbalanced distribution of Plasmodium falciparum MSP-1 genotypes related to sickle-cell trait.

BACKGROUND: The sickle-cell trait protects against severe Plasmodium falciparum malaria and reduces susceptibility to mild malaria but does not prevent infection. The exact mechanism of this protection remains unclear. We have hypothesized that AS individuals are protected by virtue of being less susceptible to a subset of parasite strains; thus we compared some genetic characteristics of parasites infecting AS and AA subjects. MATERIALS AND METHODS: Blood was collected from asymptomatic individuals living in two different regions of Africa. The polymorphic MSP-1 and MSP-2 loci were genotyped using a PCR-based methodology. Individual alleles were identified by size polymorphism, amplification using family-specific primers, and hybridization using family-specific probes. Multivariate logistic regression was used to analyze allele distribution. RESULTS: In Senegalese carriers, age and hemoglobin type influenced differently the distribution of the three MSP-1 families and had an impact on distinct individual alleles, whereas the distribution of MSP-2 alleles was marginally affected. There was no influence of other genetic traits, including the HLA Bw53 genotype, or factors such as place of residence within the village. In a cohort of Gabonese schoolchildren in which the influence of age was abrogated, a similar imbalance in the MSP-1 allelic distribution but not of MSP-2 allelic distribution by hemoglobin type was observed. CONCLUSIONS: The influence of the host's hemoglobin type on P. falciparum genotypes suggests that parasite fitness for a specific host is strain-dependent, which is consistent with our hypothesis that innate resistance might result from reduced fitness of some parasite strains for individuals with sickle-cell traits.     

Differential antibody recognition of four allelic variants of the merozoite surface protein-2 (MSP-2) of Plasmodium falciparum

The merozoite surface protein-2 (MSP-2) is a major vaccine candidate for the asexual blood stage of Plasmodium falciparum. MSP-2 is essentially dimorphic, and allelic families are named after the representative isolates FC27 and IC1. The polymorphic central region contains immunodominant repeats, which vary in number, length, and sequence within and between allelic families. We have examined the antibody recognition of repeat regions from both MSP-2 allelic families expressed as recombinant fusion peptides. The results are summarized as follows. (1) Immunization of mice with the fusion peptides elicited IgG antibodies that cross-reacted with the native MSP-2 molecule in an allelic family-specific manner. (2) These mouse antibodies recognized the recombinant proteins in both a variant-specific and a family-specific manner, as shown in inhibition immunoassays. Antibodies raised against the peptide FC27 seemed to be essentially variant-specific, since the soluble form of the S20 antigen (a member of FC27 family) had relatively little inhibitory effect on them. (3) The overall pattern of human IgG antibody responses to MSP-2 in Karitiana Indians, a population continuously exposed to hypoendemic malaria in the Brazilian Amazon Region, differs from that described in hyperendemic areas in Africa and Papua New Guinea in two important features: there was no clear age-dependent increase in the prevalence and mean concentration of specific IgG antibodies, and there was no skewing towards the IgG3 subclass in antibody responses. (4) The relatively poor correlation between concentrations of IgG antibodies that are specific for members of the same allelic family suggests that recognition of MSP-2 peptides by naturally acquired antibodies was largely variant-specific in this population. The potential role of naturally acquired variant-specific antibodies in immune evasion, by selecting mutant parasites carrying insertions or deletions of repeat sequences, is briefly discussed.     

Merozoite surface protein 2 allelic variation influences the specific antibody response during acute malaria in individuals from a Brazilian endemic area

The antibody response to Plasmodium falciparum parasites of naturally infected population is critical to elucidate the role of polymorphic alleles in malaria. Thus, we evaluated the impact of antigenic diversity of repetitive and family dimorphic domains of the merozoite surface protein 2 (MSP-2) on immune response of 96 individuals living in Peixoto de Azevedo (MT-Brazil), by ELISA using recombinant MSP-2 proteins. The majority of these individuals were carrying FC27-type infections. IgG antibody responses were predominantly directed to FC27 parasites and were correlated to the extension of polymorphism presented by each MSP-2 region. This finding demonstrated the impact of the genetic polymorphism on antibody response and therefore, its importance on malaria vaccine efficacy.     

Genetic Diversity of Plasmodium falciparum Populations in Malaria Declining Areas of Sabah,East Malaysia

Malaysia has a national goal to eliminate malaria by 2020. Understanding the genetic diversity of malaria parasites in residual transmission foci can provide invaluable information which may inform the intervention strategies used to reach elimination targets. This study was conducted to determine the genetic diversity level of P. falciparum isolates in malaria residual foci areas of Sabah. Malaria active case detection was conducted in Kalabakan and Kota Marudu. All individuals in the study sites were screened for malaria infection by rapid diagnostic test. Blood from P. falciparum-infected individuals were collected on filter paper prior to DNA extraction. Genotyping was performed using merozoite surface protein-1 (MSP-1), merozoite surface protein-2 (MSP-2), glutamate rich protein (GLURP) and 10 neutral microsatellite loci markers. The size of alleles, multiplicity of infection (MOI), mean number of alleles (Na), expected heterozygosity (He), linkage disequilibrium (LD) and genetic differentiation (F_ST) were determined. In Kalabakan, the MSP-1 and MSP-2 alleles were predominantly K1 and FC27 family types, respectively. The GLURP genotype VI (751–800 bp) was predominant. The MOI for MSP-1 and MSP-2 were 1.65 and 1.20, respectively. The Na per microsatellite locus was 1.70. The He values for MSP-1, MSP-2, GLURP and neutral microsatellites were 0.17, 0.37, 0.70 and 0.33, respectively. In Kota Marudu, the MSP-1 and MSP-2 alleles were predominantly MAD20 and 3D7 family types, respectively. The GLURP genotype IV (651–700 bp) was predominant. The MOI for both MSP-1 and MSP-2 was 1.05. The Na per microsatellite locus was 3.60. The He values for MSP-1, MSP-2, GLURP and neutral microsatellites were 0.24, 0.25, 0.69 and 0.30, respectively. A significant LD was observed in Kalabakan (0.495, p<0.01) and Kota Marudu P. falciparum populations (0.601, p<0.01). High genetic differentiation between Kalabakan and Kota Marudu P. falciparum populations was observed (F_ST = 0.532). The genetic data from the present study highlighted the limited diversity and contrasting genetic pattern of P. falciparum populations in the malaria declining areas of Sabah.     

Plasmodium falciparum: worldwide sequence diversity and evolution of the malaria vaccine candidate merozoite surface protein-2 (MSP-2)

We examined patterns and putative mechanisms of sequence diversification in the merozoite surface protein-2 (MSP-2) of Plasmodium falciparum, a major dimorphic malaria vaccine candidate antigen, by analyzing 448 msp-2 alleles from all continents. We describe several nucleotide replacements, insertion and deletion events, frameshift mutations, and proliferations of repeat units that generate the extraordinary diversity found in msp-2 alleles. We discuss the role of positive selection exerted by naturally acquired type- and variant-specific immunity in maintaining the observed levels of polymorphism and suggest that this is the most likely explanation for the significant excess of nonsynonymous nucleotide replacements found in dimorphic msp-2 domains. Hybrid sequences created by meiotic recombination between alleles of different dimorphic types were observed in few (3.1%) isolates, mostly from Africa. We found no evidence for an extremely ancient origin of allelic dimorphism at the msp-2 locus, predating P. falciparum speciation, in contrast with recent findings for other surface malarial antigens.     

Plasmodium falciparum: diversity studies of isolates from two Colombian regions with different endemicity

The population structure of Plasmodium falciparum has been widely studied in diverse epidemiological contexts, but emphasis has been made in regions with high and stable transmission. In order to establish the genetic structure of P. falciparum in areas of Colombia with different degree of endemicity, we studied 100 samples from malaria patients of two different municipalities. The frequency of multiclonal infection in these areas and the correlation with the endemicity were carried out by comparison of the amplified products from polymorphic segments of MSP-1, MSP-2, and GLURP genes. We found low size polymorphism of the studied genes: 1 MSP-1 allele, 3 MSP-2 alleles, and 4 GLURP alleles. We conclude that the P. falciparum population in the regions studied is genetically homogeneous.     

Site-based study on polymorphism of Plasmodium falciparum MSP-1 and MSP-2 genes in isolates from two villages in Central Africa.

We investigated Plasmodium falciparum genetic diversity in isolates collected from school-going residents aged from 5 to 15 years in the village of Pouma (Cameroon, Central Africa). Seventy-six children were grouped according to the clinical status. Asymptomatic status was defined as parasite carriage in the absence of any clinical symptom and malaria symptomatic status with patent parasitemia over 5000 parasites/microliter of blood and an axillary temperature > 37.5 degrees C. Parasite DNA was analysed prior to malaria treatment. Genotyping of the P. falciparum merozoite surface proteins (MSP) 1 and 2 was performed by polymerase chain reaction using allele-specific primers. K1, MAD20, Ro33 and 3D7/CAMP, FC27 allelic families were attributed to MSP-1 and MSP-2 genes, respectively. No association was found between P. falciparum MSP-1 and MSP-2 genotypes and the clinical status of children. Mixed P. falciparum infections were detected in 78% of overall samples and all isolates from symptomatic children contained more than 1 clone. The results obtained in the village of Pouma were compared to those of the village of Dienga in Gabon where a similar study, using the same genotyping methods, had been carried out in the same age group of schoolchildren. Data are interpreted in the context of malaria epidemiology in both settings.     

Acquired immune responses to Plasmodium falciparum merozoite surface protein-1 in the human fetus.

Infants born in areas of stable malaria transmission are relatively protected against severe morbidity and high density Plasmodium falciparum blood-stage infection. This protection may involve prenatal sensitization and immunologic reactivity to malaria surface ligands that participate in invasion of red cells. We examined cord blood T and B cell immunity to P. falciparum merozoite surface protein-1 (MSP-1) in infants born in an area of stable malaria transmission in Kenya. T cell cytokine responses to the C-terminal 19-kDa fragment of MSP-1 (MSP-1(19)) were detected in 24 of 92 (26%) newborns (4-192 IFN-gamma and 3-88 IL-4-secreting cells per 10(6)/cord blood lymphocytes). Peptide epitopes in the N-terminal block 3 region of MSP-1 also drove IFN-gamma and/or IL-13 production. There was no evidence of prenatal T cell sensitization to liver-stage Ag-1. A total of 5 of 86 (6%) newborns had cord blood anti-MSP-1(19) IgM Abs, an Ig isotype that does not cross the placenta and is therefore of fetal origin. The frequency of neonatal B cell sensitization was higher than that indicated by serology alone, as 5 of 27 (18%) cord blood samples contained B cells that produced IgG when stimulated with MSP-1(19) in vitro. Neonatal B cell IgG responses were restricted to the Q-KNG allele of MSP-1(19), the major variant in this endemic area, whereas T cells responded to all four MSP-1(19) alleles evaluated. In utero sensitization to MSP-1 correlated with the presence of malaria parasites in cord blood (chi(2) = 20, p < 0.0001). These data indicate that prenatal sensitization to blood-stage Ags occurs in infants born in malaria endemic areas.     

Origins of sequence diversity in the malaria vaccine candidate merozoite surface protein-2 (MSP-2) in Amazonian isolates of Plasmodium falciparum

The recent evolution of Plasmodium falciparum is at odds with the extensive polymorphism found in most genes coding for antigens. Here, we examined the patterns and putative mechanisms of sequence diversification in the merozoite surface protein-2 (MSP-2), a major malarial repetitive surface antigen. We compared the msp-2 gene sequences from closely related clones derived from sympatric parasite isolates from Brazilian Amazonia and used microsatellite typing to examine, in these same clones, the haplotype background of chromosome 2, where msp-2 is located. We found examples of msp-2 sequence rearrangements putatively created by nonreciprocal recombinational events, such as replication slippage and gene conversion, while maintaining the chromosome haplotype. We conclude that these nonreciprocal recombination events may represent a major source of antigenic diversity in MSP-2 in P. falciparum populations with low rates of classical meiotic recombination.     

Plasmodium falciparum: polymorphism in the MSP-1 gene in Indian isolates and predominance of certain alleles in cerebral malaria

Polymorphism in the block-2 region of merozoite surface protein-1 gene in 69 North Indian Plasmodium falciparum isolates was studied by PCR and RFLP using Dra-1 endonuclease. On the basis of molecular weight of the PCR products, considerable size polymorphism in target gene was seen and 69 isolates were classified into five allelic types. On RFLP, the isolates in three allelic types were further divided into two sub-allelic types each and thus eight genetic types could be identified. Interestingly, all five allelic types were identified in 47 isolates from uncomplicated (non-cerebral) malaria patients while only two allelic types (Type 2 and 3) were seen amongst 22 isolates from cerebral malaria patients. Furthermore, on RFLP, one subtype (2A) was predominantly seen in cerebral malaria patients and one subtype (3A) was exclusively found in cerebral malaria patients. These observations suggest that a few, comparatively more virulent isolates prevalent in an area may cause severe disease (cerebral malaria) which can be identified by molecular techniques like PCR-RFLP.     

Full-genome sequences of hepatitis B virus subgenotype D3 isolates from the Brazilian Amazon Region

The Brazilian Amazon Region is a highly endemic area for hepatitis B virus (HBV). However, little is known regarding the genetic variability of the strains circulating in this geographical region. Here, we describe the first full-length genomes of HBV isolated in the Brazilian Amazon Region; these genomes are also the first complete HBV subgenotype D3 genomes reported for Brazil. The genomes of the five Brazilian isolates were all 3,182 base pairs in length and the isolates were classified as belonging to subgenotype D3, subtypes ayw2 (n = 3) and ayw3 (n = 2). Phylogenetic analysis suggested that the Brazilian sequences are not likely to be closely related to European D3 sequences. Such results will contribute to further epidemiological and evolutionary studies of HBV.     

Relationship between in vivo synchronicity of Plasmodium falciparum and allelic diversity

Plasmodium falciparum cells tend to grow in synchronicity during their cyclic intraerythrocytic development in vivo. Both host and parasite factors appear to be involved in this synchronization. We examined the link between mixed-allelic-family P. falciparum infection and synchronicity in parasitized red blood cells (PRBC) from symptomatic children.The distribution of rings and trophozoites in each PRBC sample was determined by standard microscopy. P. falciparum was genotyped by using a polymerase chain reaction (PCR) targeting three loci (merozoite surface proteins (MSP) 1 and 2, and 175-kD erythrocyte binding antigen (EBA), allowing us to distinguish parasite clones belonging to a single-allelic family (SAF) and those belonging to a mixed-allelic family (MAF). Parasite development was considered synchronous when peripheral blood contained at least 95% of rings or 95% of trophozoites.Parasite development was synchronous in 22 (21.2%) of the 104 children studied. Twenty (90.9%) of these infections were SAF and two (9.1%) were MAF. Rings and trophozoites predominated in respectively 12 (60%) and 8 (40%) SAF infections. Respectively 17.1% and 82.9% of the 82 asynchronous cases corresponded to SAF and MAF infection. Parasite synchronicity was therefore significantly related to single-allelic-family infection (p < 2 × 10^− 10).Twenty different MSP-1 alleles and thirteen different MSP-2 alleles were identified. Only three isolates from patients with SAF infection comprised a single allele or genotype, the other isolates harboring at least two alleles. The mean number of alleles or clones was respectively 3.0 and 10.0 in SAF and MAF infection. These results reflect the allelic diversity of the MSP loci and show that SAF infection can correspond to multiple parasite clones (or genotypes) but, in general, fewer than in MAF infection (p ≤ 0.0007).These results confirm the extensive polymorphism of P. falciparum vaccine candidates MSP-1 and -2 in southeastern Gabon and demonstrate that parasite synchronicity in vivo is strongly associated with single-allelic-family infection.     

A Plasmodium vivax vaccine candidate displays limited allele polymorphism, which does not restrict recognition by antibodies.

BACKGROUND: The 19 kDa C-terminal region of the merozoite surface protein 1 (MSP1(19)) has been suggested as candidate for part of a subunit vaccine against malaria. A major concern in vaccine development is the polymorphism observed in different plasmodial strains. The present study examined the extension and immunological relevance of the allelic polymorphism of the MSP1(19) from Plasmodium vivax, a major human malaria parasite. MATERIALS AND METHODS: We cloned and sequenced 88 gene fragments representing the MSP1(19) from 28 Brazilian isolates of P. vivax. Subsequently, we evaluated the reactivity of rabbit polyclonal antibodies, a monoclonal antibody, and a panel of 80 human sera to bacterial and yeast recombinant proteins representing the two allelic forms of P. vivax MSP1(19) described thus far. RESULTS: We observed that DNA sequences encoding MSP1(19) were not as variable as the equivalent region of other species of Plasmodium, being conserved among Brazilian isolates of P. vivax. Also, we found that antibodies are directed mainly to conserved epitopes present in both allelic forms of the protein. CONCLUSIONS: Our findings suggest that the use of MSP1(19) as part of a subunit vaccine against P. vivax might be greatly facilitated by the limited genetic polymorphism and predominant recognition of conserved epitopes by antibodies.     

设施用厚皮甜瓜品种SSR标记遗传多样性分析

使用分布于甜瓜12条染色体上的72对SSR引物,对我国中东部设施内栽培的30个厚皮甜瓜品种进行分析;56对SSR引物在30个品种间表现为多态性。共检测到138个等位变异,每对引物的等位变异数变幅为2～6个,平均为2.6个。有效等位变异为86.16个,平均为2.25。每个SSR位点的多态性信息量(PIC)变化范围为0.045～0.725,平均为0.390。30个品种间遗传相似系数变幅为0.274～0.974之间,平均值为0.665,且90.4%的供试品种其遗传相似系数在0.474～0.824之间,亲缘关系较近;以遗传相似系数为原始数据,按UPGMA方法将30个品种划分为3大类群,结合系谱分析结果表明,我国中东部设施适宜种植的甜瓜品种遗传多样性不够丰富,多数品种间的亲缘关系较近,欲进一步提高中东部地区设施甜瓜产量和品质还需要拓宽亲本选择范围,扩大遗传背景。     

Plasmodium falciparum: sequence diversity and antibody recognition of the Merozoite surface protein-2 (MSP-2) in Brazilian Amazonia

The merozoite surface protein-2 (MSP-2) of Plasmodium falciparum comprises repeats flanked by dimorphic domains defining the allelic families FC27 and IC1. Here, we examined sequence diversity at the msp-2 locus in Brazil and its impact on MSP-2 antibody recognition by local patients. Only 25 unique partial sequences of msp-2 were found in 61 isolates examined. The finding of identical msp-2 sequences in unrelated parasites, collected 6-13 years apart, suggests that no major directional selection is exerted by variant-specific immunity in this malaria-endemic area. To examine antibody cross-reactivity, recombinant polypeptides derived from locally prevalent and foreign MSP-2 variants were used in ELISA. Foreign IC1-type variants, such as 3D7 (currently tested for human vaccination), were less frequently recognized than FC27-type and local IC1-type variants. Antibodies discriminated between local and foreign IC1-type variants, but cross-recognized structurally different local IC1-type variants. The use of evolutionary models of MSP-2 is suggested to design vaccines that minimize differences between local parasites and vaccine antigens.     

Virus-immune dynamics determined by prey-predator interaction network and epistasis in viral fitness landscape

The emergence and persistence of polymorphism within populations generally requires specific regimes of natural or sexual selection. Here, we develop a unified theoretical framework to explore how polymorphism at targeted loci can be generated and maintained by either disassortative mating choice or balancing selection due to, for example, heterozygote advantage. To this aim, we model the dynamics of alleles at a single locus A in a population of haploid individuals, where reproductive success depends on the combination of alleles carried by the parents at locus A. Our theoretical study of the model confirms that the conditions for the persistence of a given level of allelic polymorphism depend on the relative reproductive advantages among pairs of individuals. Interestingly, equilibria with unbalanced allelic frequencies were shown to emerge from successive introduction of mutants. We then investigate the role of the function linking allelic divergence to reproductive advantage on the evolutionary fate of alleles within the population. Our results highlight the significance of the shape of this function for both the number of alleles maintained and their level of genetic divergence. Large number of alleles are maintained with substantial replacement of alleles, when disassortative advantage slowly increases with allelic differentiation . In contrast, few highly differentiated alleles are predicted to be maintained when genetic differentiation has a strong effect on disassortative advantage. These opposite effects predicted by our model explain how disassortative mate choice may lead to various levels of allelic differentiation and polymorphism, and shed light on the effect of mate preferences on the persistence of balanced and unbalanced polymorphism in natural population.

     

Entamoeba histolytica: genetic diversity of clinical isolates from Bangladesh as demonstrated by polymorphisms in the serine-rich gene.

The varied organ tropisms and clinical presentations of infection by Entamoeba histolytica have stimulated interest in the role of parasite genetic diversity in virulence. We investigated genetic diversity among 54 E. histolytica isolates from Bangladesh by analyzing polymorphism in the serine-rich gene by nested PCR on DNA extracted from stool and liver aspirate pus. We detected both size and restriction site polymorphisms among the isolates within this endemic area. A combination of the nested PCR results and the AluI digestion of the PCR products examined yielded 25 distinct DNA banding patterns among the 42 stool isolates and an additional 9 distinct patterns among the 12 liver abscess isolates. Approximately half of the isolates had unique polymorphisms. Interestingly, the majority of E. histolytica from the liver had polymorphisms which were not present in intestinal isolates from the same geographic area. These data are consistent with the existence of genetic differences between E. histolytica which cause intestinal and those which cause hepatic disease. We conclude that there is genetic diversity within E. histolytica isolates from an endemic population as reflected in serine-rich E. histolytica protein gene polymorphism. The correlation of genetic differences with the pathogenic potential of E. histolytica strains and the implications of genetic diversity for the immunoprophylaxis of amebiasis require further study.     

Diversity and evolution of the Mhc-DRB1 gene in the two endemic Iberian subspecies of Pyrenean chamois, Rupicapra pyrenaica

Major histocompatibility complex class II locus DRB variation was investigated by single-strand conformation polymorphism analysis and sequence analysis in the two subspecies of Pyrenean chamois (Rupicapra pyrenaica) endemic to the Iberian Peninsula. Low levels of genetic variation were detected in both subspecies, with seven different alleles in R. p. pyrenaica and only three in the R. p. parva. After applying the rarefaction method to cope with the differences in sample size, the low allele number of parva was highlighted. The low allelic repertoire of the R. p. parva subspecies is most likely the result of bottlenecks caused by hunting pressure and recent parasitic infections by sarcoptic mange. A phylogenetic analysis of both Pyrenean chamois and DRB alleles from 10 different caprinid species revealed that the chamois alleles form two monophyletic groups. In comparison with other Caprinae DRB sequences, the Rupicapra alleles displayed a species-specific clustering that reflects a large temporal divergence of the chamois from other caprinids, as well as a possible difference in the selective environment for these species.     

Analysis of the genetic variability of PvMSP-3α among Plasmodium vivax in Brazilian field isolates

Reliable molecular markers are essential for a better understanding of the molecular epidemiology of Plasmodium vivax, which is a neglected human malaria parasite. The aim of this study was to analyze the genetic diversity of P. vivax isolates from the Brazilian Amazon using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the highly polymorphic merozoite surface protein-3alpha (PvMSP-3α) gene. To accomplish this, 60 isolates of P. vivax from different endemic areas in the Brazilian Amazon were collected. The PvMSP-3α gene was amplified by nested-PCR. Three major types of the PvMSP-3α locus were detected at different frequencies: type A (68%), B (15%) and C (17%). A single sample showed two PCR fragments, which corresponded to infection with types A and C. PCR-RFLP analysis using the HhaI restriction enzyme for 52 isolates clearly identified 11 haplotypes, eight of which were from type A, two from type B and only one from type C. Seven other isolates did not show a clear pattern using PCR-RFLP. This result might be due to multiple clone infections. This study showed a high diversity of the PvMSP-3α gene among P. vivax isolates from the Brazilian Amazon, but also indicated that the detection performance of PCR-RFLP of the PvMSP-3α gene may not be sufficient to detect multiple clone infections.