The genetic polymorphism of Plasmodium vivax genes in endemic regions of Thailand

AbstractObjectiveTo investigate the genetic polymorphism of Plasmodium vivax (P. vivax) PvCSP and PvMSP1 genes from field isolates at four endemic regions (North, East, West and South) of Thailand.MethodsThe 152 P. vivax infected cases from dried blood spots were DNA extracted and confirmed by species-specific primer sets using multiplex PCR method. PvMSP1 fragments F2 and F3; PvCSP were genotyped using RFLP-PCR method.ResultsTotally amplified DNA which was multiple genotypes for PvMSP1 F2 and PvMSP1 F3 were 12.50% and 8.55%, respectively while PvCSP was 3.95%. The overall frequency of multiple genotypes was 25%. There were 12 allele types of PvMSP1 F2 using AluI enzyme digestion and 8 size variations were found in PvMSP1 F3. The isolates from western region was highly genetic diverse when compare among all isolates. The predominant variant type of PvCSP gene was VK210 type.ConclusionsThe multiple genotypes are common found in Thailand and it might hide the real genotype. PvCSP does not have extensive genetic diversity in this study. However, PvMSP1 marker due to multiple genotypes is difficult to be analyzed. The multiple genotypes findings might stem from population migration and vector species findings

High density of ‘spiky’ excrescences covering the surface of an erythrocyte infected with Plasmodium malariae

10.1111/j.1365-2141.2010.08261.xBritish Journal of Haematology15111-BJHE

Comparison of three molecular methods for the detection and speciation of Plasmodium vivax and Plasmodium falciparum

<p>Abstract</p> <p>Background</p> <p>Accurate diagnosis of <it>Plasmodium </it>spp. is essential for the rational treatment of malaria. Despite its many disadvantages, microscopic examination of blood smears remains the current "gold standard" for malaria detection and speciation. PCR assays offer an alternative to microscopy which has been shown to have superior sensitivity and specificity. Unfortunately few comparative studies have been done on the various molecular based speciation methods.</p> <p>Methods</p> <p>The sensitivity, specificity and cost effectiveness of three molecular techniques were compared for the detection and speciation of <it>Plasmodium falciparum </it>and <it>Plasmodium vivax </it>from dried blood spots collected from 136 patients in western Thailand. The results from the three molecular speciation techniques (nested PCR, multiplex PCR, and real-time PCR) were used to develop a molecular consensus (two or more identical PCR results) as an alternative gold standard.</p> <p>Results</p> <p>According to the molecular consensus, 9.6% (13/136) of microscopic diagnoses yielded false negative results. Multiplex PCR failed to detect <it>P. vivax </it>in three mixed isolates, and the nested PCR gave a false positive <it>P. falciparum </it>result in one case. Although the real-time PCR melting curve analysis was the most expensive method, it was 100% sensitive and specific and least time consuming of the three molecular techniques investigated.</p> <p>Conclusion</p> <p>Although microscopy remains the most appropriate method for clinical diagnosis in a field setting, its use as a gold standard may result in apparent false positive results by superior techniques. Future studies should consider using more than one established molecular methods as a new gold standard to assess novel malaria diagnostic kits and PCR assays.</p

Effective and cheap removal of leukocytes and platelets from Plasmodium vivax infected blood

<p>Abstract</p> <p>Background</p> <p>Investigations of <it>Plasmodium vivax </it>are restricted to samples collected from infected persons or primates, because this parasite cannot be maintained in <it>in vitro </it>cultures. Contamination of <it>P. vivax </it>isolates with host leukocytes and platelets is detrimental to a range of <it>ex vivo </it>and molecular investigations. Easy-to-produce CF11 cellulose filters have recently provided us with an inexpensive method for the removal of leukocytes and platelets. This contrasted with previous reports of unacceptably high levels of infected red blood cell (IRBC) retention by CF11. The aims of this study were to compare the ability of CF11 cellulose filters and the commercial filter Plasmodipur at removing leukocyte and platelet, and to investigate the retention of <it>P. vivax </it>IRBCs by CF11 cellulose filtration.</p> <p>Methods and Results</p> <p>Side-by-side comparison of six leukocyte removal methods using blood samples from five healthy donor showed that CF11 filtration reduced the mean initial leukocyte counts from 9.4 × 10³per μl [95%CI 5.2–13.5] to 0.01 × 10³[95%CI 0.01–0.03]. The CF11 was particularly effective at removing neutrophils. CF11 treatment also reduced initial platelet counts from 211.6 × 10³per μl [95%CI 107.5–315.7] to 0.8 × 10³per μl [95%CI -0.7–2.2]. Analysis of 30 <it>P. vivax </it>blood samples before and after CF11 filtration showed only a minor loss in parasitaemia (≤ 7.1% of initial counts). Stage specific retention of <it>P. vivax </it>IRBCs was not observed.</p> <p>Conclusion</p> <p>CF11 filtration is the most cost and time efficient method for the production of leukocyte- and platelet-free <it>P. vivax</it>-infected erythrocytes from field isolates.</p

Plasmodium vivax trophozoites insensitive to chloroquine

© 2008 Sharrock et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

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

The geographical distribution of allele polymorphisms of Plasmodium vivax in different regions of Thailand.

OBJECTIVE To investigate the genetic structure of Plasmodium vivax circumsporozoite surface protein (PvCSP) and P. vivax merozoite surface protein 1 (PvMSP1) genes from field isolates of malaria parasites from four different regions of Thailand. MATERIAL AND METHOD The data was collected by cross-sectional survey, consisting of 273 P. vivax infected blood samples from malaria clinics in 4 different border regions of Thailand from February 2008 to February 2009. The dried blood spots were extracted for DNA and Plasmodium species confirmed by species-specific primer sets. PvCSP and PvMSP1 genes were amplified and their population genetics were analyzed by using the Heterozygosity (H) formula, F-STAT and LIAN programs. RESULT There was considerable variation in the PvMSP1 gene within 2 fragments for which HE was 0.8303, whereas PvCSP showed low HE at 0.1418. Significant differences in allele frequencies between sites were quantified by Fst, Linkage disequilibrium (LD). The results showed PvMSP1 F2; Fst = 0.063, p = 0.07; PvMSP1 F2 RFLP pattern; Fst = 0.154, p = 0.005; PvMSP1 F3; Fst = 0.23, p = 0.005 and the overall loci showed Fst = 0.151, p = 0.005 (Fisher's exact test). All values of Index association (I(S)A) were non-significant. There was no evidence of LD within the P. vivax populations. CONCLUSION H(E) at each locus of the PvMSP1 gene showed significant differences in allele frequencies between sites

Salivary Glands Proteins Expression of Anopheles dirus A Fed on Plasmodium vivax- and Plasmodium falciparum-Infected Human Blood

Mosquitoes are able to adapt to feed on blood by the salivary glands which created a protein that works against the haemostasis process. This study aims to investigate the salivary glands proteins expression of 50 adult female An. dirus A mosquitoes, a main vector of malaria in Thailand, each group with an age of 5 days which were artificial membrane fed on sugar, normal blood, blood infected with P. vivax, and blood infected with P. falciparum. Then mosquito salivary gland proteins were analyzed by SDS-PAGE on days 0, 1, 2, 3, and 4 after feeding. The findings revealed that the major salivary glands proteins had molecular weights of 62, 58, 43, 36, 33, 30, and 18 kDa. One protein band of approximately 13 kDa was found in normal blood and blood infected with P. vivax fed on day 0. A stronger protein band, 65 kDa, was expressed from the salivary glands of mosquitoes fed with P. vivax- or P. falciparum-infected blood on only day 0, but none on days 1 to 4. The study shows that salivary glands proteins expression of An. dirus may affect the malaria parasite life cycle and the ability of mosquitoes to transmit malaria parasites in post-24-hour disappearance observation

The genetic polymorphism of Plasmodium vivax genes in endemic regions of Thailand.

The multiple genotypes are common found in Thailand and it might hide the real genotype. PvCSP does not have extensive genetic diversity in this study. However, PvMSP1 marker due to multiple genotypes is difficult to be analyzed. The multiple genotypes findings might stem from population migration and vector species findings

The prevalence of molecular markers of drug resistance in Plasmodium vivax from the border regions of Thailand in 2008 and 2014

The prevalence of Plasmodium vivax is increasing in the border regions of Thailand; one potential problem confounding the control of malaria in these regions is the emergence and spread of drug resistance. The aim of this study was to determine the genetic diversity in genes potentially linked to drug resistance in P. vivax parasites isolated from four different border regions of Thailand; Thai-Myanmar (Tak, Mae Hong Son and Prachuap Khiri Khan Provinces), and Thai-Cambodian borders (Chanthaburi Province). Isolates were collected from 345 P. vivax patients in 2008 and 2014, and parasite DNA extracted and subjected to nucleotide sequencing at five putative drug-resistance loci (Pvdhfr, Pvdhps, Pvmdr1, Pvcrt-o and Pvk12). The prevalence of mutations in Pvdhfr, Pvdhps and Pvmdr1 were markedly different between the Thai-Myanmar and Thai-Cambodian border areas and also varied between sampling times. All isolates carried the Pvdhfr (58R and 117N/T) mutation, however, whereas the quadruple mutant allele (I57R58M61T117) was the most prevalent (69.6%) in the Thai-Myanmar border region, the double mutant allele (F57R58T61N117) was at fixation on the Thai-Cambodian border (100%). The most prevalent genotypes of Pvdhps and Pvmdr1 were the double mutant (S382G383K512G553) (65.1%) and single mutant (M958Y976F1076) (46.5%) alleles, respectively on the Thai-Myanmar border while the single Pvdhps mutant (S382G383K512A553) (52.7%) and the triple Pvmdr1 mutant (M958F976L1076) (81%) alleles were dominant on the Thai-Cambodian border. No mutations were observed in the Pvcrt-o gene in either region. Novel mutations in the Pvk12 gene, the P. vivax orthologue of PfK13, linked to artemisinin resistance in Plasmodium falciparum, were observed with three nonsynonymous and three synonymous mutations in six isolates (3.3%). Keywords: Plasmodium vivax, Antimalarial drugs, Drug-resistant mutations, Genetic diversit