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Practical PCR genotyping protocols for Plasmodium vivax using Pvcs and Pvmsp1.

Imwong M, Pukrittayakamee S, Grüner AC, Rénia L, Letourneur F, Looareesuwan S, White NJ, Snounou G - Malar. J. (2005)

Bottom Line: Analysis revealed that P. vivax populations in Thailand are highly diverse genetically, with mixed genotype infections found in 26 % of the samples (average multiplicity of infection = 1.29).A large number of distinguishable alleles were found for the two markers, 23 for Pvcs and 36 for Pvmsp1.A total of 68 distinct genotypes could be enumerated in the 74 isolates with a multiplicity of infection of 1.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical Tropical Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. noi@tropmedres.ac

ABSTRACT

Background: Plasmodium vivax is the second most prevalent malaria parasite affecting more than 75 million people each year, mostly in South America and Asia. In addition to major morbidity this parasite is associated with relapses and a reduction in birthweight. The emergence and spread of drug resistance in Plasmodium falciparum is a major factor in the resurgence of this parasite. P. vivax resistance to drugs has more recently emerged and monitoring the situation would be helped, as for P. falciparum, by molecular methods that can be used to characterize parasites in field studies and drug efficacy trials.

Methods: Practical PCR genotyping protocols based on polymorphic loci present in two P. vivax genetic markers, Pvcs and Pvmsp1, were developed. The methodology was evaluated using 100 P. vivax isolates collected in Thailand.

Results and discussion: Analysis revealed that P. vivax populations in Thailand are highly diverse genetically, with mixed genotype infections found in 26 % of the samples (average multiplicity of infection = 1.29). A large number of distinguishable alleles were found for the two markers, 23 for Pvcs and 36 for Pvmsp1. These were generally randomly distributed amongst the isolates. A total of 68 distinct genotypes could be enumerated in the 74 isolates with a multiplicity of infection of 1.

Conclusion: These results indicate that the genotyping protocols presented can be useful in the assessment of in vivo drug efficacy clinical trials conducted in endemic areas and for epidemiological studies of P. vivax infections.

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Allele frequency of the distinct allelic variants of Pvcs observed in the 100 isolates from Thailand. Allelic types were defined according to repeat type (VK210 or VK247), fragment size and the presence or absence of defined pre- and post-repeat sequences.
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Figure 2: Allele frequency of the distinct allelic variants of Pvcs observed in the 100 isolates from Thailand. Allelic types were defined according to repeat type (VK210 or VK247), fragment size and the presence or absence of defined pre- and post-repeat sequences.

Mentions: Genotyping of the 100 P. vivax isolates collected from Thailand was achieved successfully using the protocols described above. In total, parasites with a Pvcs bearing the VK210 type repeats only were found in 90 isolates (representing seven size polymorphisms). Parasites with a Pvcs bearing the VK247-type repeats only were found in nine isolates (representing only three size polymorphisms), and in one isolate parasites of both types were found. Thus, 10 different allelic forms of Pvcs were detected by simple analyses of the fragment size and repeat type. When associated with RFLP analysis of the pre- and post-repeat types, these could be divided into 23 different allelic types (Table 2), 18 for the VK210 type and five for the VK247 type. Mixed genotype infections were found in 20 of the isolates, for each of which the multiplicity of infection (MOI), i.e. the total number of different allelic types observed, was two. Thus, for the samples analysed, a total of 120 bands were observed, with an mean MOI of 1.2. The allelic variants were generally randomly distributed between the 120 bands; the highest frequencies, 0.2 and 0.18, were found for VK210k and VK210n, respectively (Fig 2).


Practical PCR genotyping protocols for Plasmodium vivax using Pvcs and Pvmsp1.

Imwong M, Pukrittayakamee S, Grüner AC, Rénia L, Letourneur F, Looareesuwan S, White NJ, Snounou G - Malar. J. (2005)

Allele frequency of the distinct allelic variants of Pvcs observed in the 100 isolates from Thailand. Allelic types were defined according to repeat type (VK210 or VK247), fragment size and the presence or absence of defined pre- and post-repeat sequences.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1131918&req=5

Figure 2: Allele frequency of the distinct allelic variants of Pvcs observed in the 100 isolates from Thailand. Allelic types were defined according to repeat type (VK210 or VK247), fragment size and the presence or absence of defined pre- and post-repeat sequences.
Mentions: Genotyping of the 100 P. vivax isolates collected from Thailand was achieved successfully using the protocols described above. In total, parasites with a Pvcs bearing the VK210 type repeats only were found in 90 isolates (representing seven size polymorphisms). Parasites with a Pvcs bearing the VK247-type repeats only were found in nine isolates (representing only three size polymorphisms), and in one isolate parasites of both types were found. Thus, 10 different allelic forms of Pvcs were detected by simple analyses of the fragment size and repeat type. When associated with RFLP analysis of the pre- and post-repeat types, these could be divided into 23 different allelic types (Table 2), 18 for the VK210 type and five for the VK247 type. Mixed genotype infections were found in 20 of the isolates, for each of which the multiplicity of infection (MOI), i.e. the total number of different allelic types observed, was two. Thus, for the samples analysed, a total of 120 bands were observed, with an mean MOI of 1.2. The allelic variants were generally randomly distributed between the 120 bands; the highest frequencies, 0.2 and 0.18, were found for VK210k and VK210n, respectively (Fig 2).

Bottom Line: Analysis revealed that P. vivax populations in Thailand are highly diverse genetically, with mixed genotype infections found in 26 % of the samples (average multiplicity of infection = 1.29).A large number of distinguishable alleles were found for the two markers, 23 for Pvcs and 36 for Pvmsp1.A total of 68 distinct genotypes could be enumerated in the 74 isolates with a multiplicity of infection of 1.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical Tropical Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. noi@tropmedres.ac

ABSTRACT

Background: Plasmodium vivax is the second most prevalent malaria parasite affecting more than 75 million people each year, mostly in South America and Asia. In addition to major morbidity this parasite is associated with relapses and a reduction in birthweight. The emergence and spread of drug resistance in Plasmodium falciparum is a major factor in the resurgence of this parasite. P. vivax resistance to drugs has more recently emerged and monitoring the situation would be helped, as for P. falciparum, by molecular methods that can be used to characterize parasites in field studies and drug efficacy trials.

Methods: Practical PCR genotyping protocols based on polymorphic loci present in two P. vivax genetic markers, Pvcs and Pvmsp1, were developed. The methodology was evaluated using 100 P. vivax isolates collected in Thailand.

Results and discussion: Analysis revealed that P. vivax populations in Thailand are highly diverse genetically, with mixed genotype infections found in 26 % of the samples (average multiplicity of infection = 1.29). A large number of distinguishable alleles were found for the two markers, 23 for Pvcs and 36 for Pvmsp1. These were generally randomly distributed amongst the isolates. A total of 68 distinct genotypes could be enumerated in the 74 isolates with a multiplicity of infection of 1.

Conclusion: These results indicate that the genotyping protocols presented can be useful in the assessment of in vivo drug efficacy clinical trials conducted in endemic areas and for epidemiological studies of P. vivax infections.

Show MeSH
Related in: MedlinePlus