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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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Related in: MedlinePlus

Alignment of amino acid sequences of amplified F1 and F3 fragments of Pvmsp1. Sequences were aligned against the largest fragment obtained; dots represent identical residues and dashes represent gaps. Sequences obtained during this study are labelled by their GenBank accession numbers, while the Belem and Sal-1 sequences had been previously published (AF435594 and AF435593, respectively).
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Figure 4: Alignment of amino acid sequences of amplified F1 and F3 fragments of Pvmsp1. Sequences were aligned against the largest fragment obtained; dots represent identical residues and dashes represent gaps. Sequences obtained during this study are labelled by their GenBank accession numbers, while the Belem and Sal-1 sequences had been previously published (AF435594 and AF435593, respectively).

Mentions: A selection of amplified product from F1 (n = 18) and F3 (n = 8) representative of all the size variants were sequenced and compared to previously published sequences (Fig. 4). The number of distinct F1 variants was found to be higher than that revealed by electrophoretic separation, since 11 different allelic variants were observed for the 18 fragments derived from the Thai isolates. Sequence differences were observed within the variants assigned to the B, D or E size classes. Some of the bands within each class might be distinguished by the use of higher resolution agarose gels, though others had the same size and only exhibited subtle sequence differences. A similar pattern was observed for the F3 fragments, where sequencing distinguished six different variants.


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)

Alignment of amino acid sequences of amplified F1 and F3 fragments of Pvmsp1. Sequences were aligned against the largest fragment obtained; dots represent identical residues and dashes represent gaps. Sequences obtained during this study are labelled by their GenBank accession numbers, while the Belem and Sal-1 sequences had been previously published (AF435594 and AF435593, respectively).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Alignment of amino acid sequences of amplified F1 and F3 fragments of Pvmsp1. Sequences were aligned against the largest fragment obtained; dots represent identical residues and dashes represent gaps. Sequences obtained during this study are labelled by their GenBank accession numbers, while the Belem and Sal-1 sequences had been previously published (AF435594 and AF435593, respectively).
Mentions: A selection of amplified product from F1 (n = 18) and F3 (n = 8) representative of all the size variants were sequenced and compared to previously published sequences (Fig. 4). The number of distinct F1 variants was found to be higher than that revealed by electrophoretic separation, since 11 different allelic variants were observed for the 18 fragments derived from the Thai isolates. Sequence differences were observed within the variants assigned to the B, D or E size classes. Some of the bands within each class might be distinguished by the use of higher resolution agarose gels, though others had the same size and only exhibited subtle sequence differences. A similar pattern was observed for the F3 fragments, where sequencing distinguished six different variants.

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