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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

A. Schematic representation of the Pvmsp1 gene for the localization of interallele conserved blocks (blank boxes) and variable blocks (black boxes). The position of the three amplified segments (F1, F2 and F3) is indicated by the horizontal bold lines. B. Gel electrophoresis of PCR products of a selection of fragments, corresponding to the three segments, amplified from different isolates. The molecular size of the largest and smallest bands are indicated. C. Analysis of the diversity of the F2 segment by PCR-RFLP using Alu I or Mnl I. A selection of lanes is labelled to indicate the type of variant observed (corresponding to the nomenclature in Table 3). A 100 bp ladder was used a molecular weight marker (M) for all the gels.
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Figure 3: A. Schematic representation of the Pvmsp1 gene for the localization of interallele conserved blocks (blank boxes) and variable blocks (black boxes). The position of the three amplified segments (F1, F2 and F3) is indicated by the horizontal bold lines. B. Gel electrophoresis of PCR products of a selection of fragments, corresponding to the three segments, amplified from different isolates. The molecular size of the largest and smallest bands are indicated. C. Analysis of the diversity of the F2 segment by PCR-RFLP using Alu I or Mnl I. A selection of lanes is labelled to indicate the type of variant observed (corresponding to the nomenclature in Table 3). A 100 bp ladder was used a molecular weight marker (M) for all the gels.

Mentions: The Pvmsp1 gene encodes a polypeptide of about 1,720 amino acids [25,26], and sequence comparison revealed 13 regions of interallele conserved blocks and variable blocks [27]. Three main regions of sequence divergence were found through comparison of the full length Pvmsp1 sequences from two distinct P. vivax lines (Sal-1 and Belem). Three segments (labelled F1 to F3) corresponding to these regions were thus amplified for further analysis (Fig. 3A).


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)

A. Schematic representation of the Pvmsp1 gene for the localization of interallele conserved blocks (blank boxes) and variable blocks (black boxes). The position of the three amplified segments (F1, F2 and F3) is indicated by the horizontal bold lines. B. Gel electrophoresis of PCR products of a selection of fragments, corresponding to the three segments, amplified from different isolates. The molecular size of the largest and smallest bands are indicated. C. Analysis of the diversity of the F2 segment by PCR-RFLP using Alu I or Mnl I. A selection of lanes is labelled to indicate the type of variant observed (corresponding to the nomenclature in Table 3). A 100 bp ladder was used a molecular weight marker (M) for all the gels.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: A. Schematic representation of the Pvmsp1 gene for the localization of interallele conserved blocks (blank boxes) and variable blocks (black boxes). The position of the three amplified segments (F1, F2 and F3) is indicated by the horizontal bold lines. B. Gel electrophoresis of PCR products of a selection of fragments, corresponding to the three segments, amplified from different isolates. The molecular size of the largest and smallest bands are indicated. C. Analysis of the diversity of the F2 segment by PCR-RFLP using Alu I or Mnl I. A selection of lanes is labelled to indicate the type of variant observed (corresponding to the nomenclature in Table 3). A 100 bp ladder was used a molecular weight marker (M) for all the gels.
Mentions: The Pvmsp1 gene encodes a polypeptide of about 1,720 amino acids [25,26], and sequence comparison revealed 13 regions of interallele conserved blocks and variable blocks [27]. Three main regions of sequence divergence were found through comparison of the full length Pvmsp1 sequences from two distinct P. vivax lines (Sal-1 and Belem). Three segments (labelled F1 to F3) corresponding to these regions were thus amplified for further analysis (Fig. 3A).

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