Vivax Populations Research Articles

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.

Simple in vitro assay for determining the sensitivity of Plasmodium vivax isolates from fresh human blood to antimalarials in areas where P. vivax is endemic.

The aim of this study was to develop a simple, field-practical, and effective in vitro method for determining the sensitivity of fresh erythrocytic Plasmodium vivax isolates to a range of antimalarials. The method used is a modification of the standard World Health Organization (WHO) microtest for determination of P. falciparum drug sensitivity. The WHO method was modified by removing leukocytes and using a growth medium supplemented with AB(+) serum. We successfully carried out 34 in vitro drug assays on 39 P. vivax isolates collected from the Mae Sod malaria clinic, Tak Province, Thailand. The mean percentage of parasites maturing to schizonts (six or more merozoites) in control wells was 66.5% +/- 5.9% (standard deviation). This level of growth in the control wells enabled rapid microscopic determination (5 min per isolate per drug) of the MICs of chloroquine, dihydroartemisinin, WR238605 (tafenoquine), and sulfadoxine. P. vivax was relatively sensitive to chloroquine (MIC = 160 ng/ml, 50% inhibitory concentration [IC(50)] = 49.8 ng/ml) and dihydroartemisinin (MIC = 0.5 ng/ml, IC(50) = 0.47 ng/ml). The poor response of P. vivax to both tafenoquine (MIC = 14,000 ng/ml, IC(50) = 9,739 ng/ml) and sulfadoxine (MIC = 500,000 ng/ml, IC(50) = 249,000 ng/ml) was due to the slow action of these drugs and the innate resistance of P. vivax to sulfadoxine. The in vitro assay developed in our study should be useful both for assessing the antimalarial sensitivity of P. vivax populations and for screening new antimalarials in the absence of long-term P. vivax cultures.

Plasmodium vivax synonymous substitution frequencies, evolution and population structure deduced from diversity in AMA 1 and MSP 1 genes

Polymorphic regions of the genes encoding Plasmodium vivax apical membrane antigen 1 ( PvAMA1) and P. vivax merozoite surface protein 1 ( PvMSP1) were sequenced to examine population diversity both within and between geographical areas. Sequences were obtained for 219 isolates for PvAMA1 and for 175 isolates for PvMSP1 from Africa, China, India, Indonesia, Philippines, Papua New Guinea, Solomon Islands and Thailand. Over half of the isolates were obtained from different regions within the Philippines, and this was used to look at the diversity within a country. Sixty nine haplotypes and 22 polymorphic sites in a 414-bp region of PvAMA1 and 41 haplotypes and 34 polymorphic sites in a 249-bp fragment of PvMSP1 were detected. For both PvAMA1 and PvMSP1, four previously unreported polymorphic nucleotide positions were identified. Population analysis indicated that there were significant differences in allele frequencies between different regions but these differences were small compared to the diversity within populations (Fixation index, F ST, of 0.126 and 0.078 for PvAMA1 and PvMSP1, respectively). PvAMA1 and PvMSP1 had similar nonsynonymous substitution frequencies but surprisingly, the synonymous substitution frequency for PvMSP1 was eight times the frequency for PvAMA1 suggesting that synonymous substitutions in at least PvAMA1 are not neutral.

Polymorphism at the merozoite surface protein-3alpha locus of Plasmodium vivax: global and local diversity.

Allelic diversity at the Plasmodium vivax merozoite surface protein-3alpha (PvMsp-3alpha) locus was investigated using a combined polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) protocol. Symptomatic patient isolates from global geographic origins showed a high level of polymorphism at the nucleotide level. These samples were used to validate the sensitivity, specificity, and reproducibility of the PCR/RFLP method. It was then used to investigate PvMsp3alpha diversity in field samples from children living in a single village in a malaria-endemic region of Papua New Guinea, with the aim of assessing the usefulness of this locus as an epidemiologic marker of P. vivax infections. Eleven PvMsp-3alpha alleles were distinguishable in 16 samples with single infections, revealing extensive parasite polymorphism within this restricted area. Multiple infections were easily detected and accounted for 5 (23%) of 22 positive samples. Pairs of samples from individual children provided preliminary evidence for high turnover of P. vivax populations.

Two new genotypes of Plasmodium vivax circumsporozoite protein found in the Republic of Korea

The gene encoding Plasmodium vivax circumsporozoite protein (PvCSP) exhibits polymorphism in many geographical isolates. The present study was designed to investigate polymorphism in PvCSP gene of P. vivax isolates in Korea. Thirty isolates, obtained from indigenous cases in Yonchon-gun, Kyonggi-do in 1997, were subjected for sequencing and RFLP analysis of the repeat and post-repeat regions of PvCSP gene and two genotypes (SK-A and SK-B) were identified. The genotype of 19 isolates was SK-A and that of 11 isolates was SK-B. Although the number of 12-base repeats present in SK-A was three while two were found in a Chinese strain CH-5, the repeat sequence of SK-A was identical to that of CH-5 except for one base substitution. Compared with known data there was no identical isolates with SK-B, but the sequence of SK-B was similar to that of a North Korean (NK) isolate. These results indicate that two genotypes of PvCSP coexist in the present epidemic area of Korea and the present parasite may originate from East Asia. RFLP would be useful to classify genotypes of P. vivax population instead of gene sequencing.

Interallelic recombination in the merozoite surface protein 1( MSP-1) gene of Plasmodium vivax from Thai isolates

The merozoite of Plasmodium vivax possesses a high molecular mass surface protein called Pv-merozoite surface protein 1, PvMSP-1, which exhibits antigenic diversity among isolates. In this study, the extent of sequence variation in the polymorphic region and the flanking interspecies conserved blocks (ICBs) 5 and 6 of the PvMSP-1 gene was analyzed using the polymerase chain reaction to amplify the DNA fragment encompassing these regions, followed by sequencing. Twenty different alleles were obtained from 15 Thai isolates. Results revealed five distinct sequence types of the polymorphic region, two of which were newly identified in this study: one probably generated by intragenic recombination at a site different from that previously reported and the other by duplication of a 30 nucleotide (nt) sequence at the 3′ end of the region. On the other hand, almost all nucleotide substitutions in the flanking regions, ICB5 and ICB6, were dimorphic, creating microheterogeneity in the region. Furthermore, stretches of nucleotide substitutions were found to be linked in ICB6, suggesting the potential recombination sites between these stretches. It is also noted that extensive sequence variation in the PvMSP-1 gene and eoinfection with different PvMSP-1 alleles occurred among the P. vivax population in the endemic areas of Thailand.

Differences in sensitivity of KenyanTrypanosoma vivax populations to the prophylactic and therapeutic actions of isometamidium chloride in Boran cattle

Isometamidium chloride was administered as a single prophylactic dose of 0.5 mg kg-1 body weight to each of 10 Boran (Bos indicus) steers. At monthly intervals following drug administration, groups of five cattle each were challenged with one of two different Trypanosoma vivax populations transmitted by infected Glossina morsitans centralis; one with a stock (IL 2982) from Galana, Kenya and the other with a stock (IL 2986) from Likoni, Kenya. Prophylaxis was afforded for less than one month against the Galana T. vivax and for one month against the Likoni T. vivax. In a therapeutic study a further 10 Boran steers were similarly infected with either of the T. vivax populations; five steers per population. Eleven days after infection all animals were treated with 0.5 mg kg-1 isometamidium chloride and all were cured. These findings demonstrate that, as defined in the field, the two Kenyan T. vivax populations express a high level of resistance to the prophylactic action of isometamidium yet a low level of resistance to the therapeutic action of the drug. The results also indicate that differences in drug resistance between different isolates play a major role in determining the apparent period of prophylaxis afforded by isometamidium chloride.

Therapeutic and prophylactic activity of isometamidium chloride in Boran cattle against Trypanosoma vivax transmitted by Glossina morsitans centralis

Ten Boran steers were infected with Trypanosoma vivax, transmitted by Glossina morsitans centralis; five steers with a T vivax clone from Nigeria and five with a T vivax clone from Kenya. Eleven days after infection all 10 animals were treated with 0.5 mg kg-1 isometamidium chloride. Four steers infected with the Nigerian T vivax and all five infected with the Kenyan T vivax were completely cured. When different steers received a single prophylactic dose of 0.5 mg kg-1 isometamidium chloride and subjected to monthly tsetse-transmitted challenge with the same T vivax clones, complete protection was afforded for at least two months against challenge with the Nigerian T vivax, but for less than one month against the Kenyan T vivax. The findings indicate that the level of sensitivity of a T vivax population to the prophylactic activity of isometamidium chloride cannot be concluded from sensitivity studies based on the therapeutic action of the drug.

Trypanosoma vivax: Courses of infection with three stabilates in inbred mouse strains

The courses of infection in inbred mouse strains were compared following infection with three Stabilates of high, intermediate, and low virulence of Trypanosoma vivax stock Zaria Y486. Mouse strains could only be shown to differ in their resistance to T. vivax infections as judged by the height of the initial parasitemia and survival times when a trypanosome population of low or intermediate virulence was used. A T. vivax population of high virulence was uniformly lethal. Comparison of lytic antibody titers between groups of resistant ( C57 B1 6 ) and susceptible ( Balb c ) mice did not show any significant differences in titers of the surviving mice but the mice in either group which did not control the initial parasitemia had lower lytic antibody titers than those which did. A significantly larger number of Balb c mice failed to control the initial infection as compared to the C57 B1 6 . Treatment with cyclophosphamide did not ablate differences in susceptibility between the two strains. The use of congenic mice showed that these differences in susceptibility were not related to differences in the major histocompatibility complex between these strains.

Trypanosoma vivax: Sequence of antigenic variants in mice and goats

Three populations composed of different variable antigen types were derived from a clone of the recently isolated mouse-infective Trypanosoma vivax stock Zaria Y486. The antigenic composition of the trypanosome populations which appeared following infection of mice and goats with these populations was compared using the immune lysis test. Significant differences were observed in the antigenic composition of both the initial and relapse populations obtained from goats and mice. These observations suggest that there may be selective growth of different variable antigen types in different hosts with the result that the antigenic composition of a goat-derived population may be greatly altered following subinoculation into normal mice. This can be somewhat reduced however if goat derived populations are subinoculated into lethally irradiated mice. Two antigenically different T. vivax populations were also cyclically transmitted to individual goats in a preliminary study of the effect of tsetse transmission on the antigenic composition of the ingested populations. It was observed that the composition of the first detectable populations in the infected goats was similar but different from that ingested by the tsetse flies.