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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 the F2 segment of Pvmsp1 observed in the 100 isolates from Thailand. Allelic variants were defined according to the RFLP patterns observed. A. Variants were divided according to the digestions patterns obtained individually with each of the two restriction enzymes Alu I (a1, a2, etc...) or Mnl I (m1, m2, etc...) for A or B, the two different sized fragments amplified, as in Table 3. B) Variants were classed according to size and the combined RFLP patterns obtained for both restriction enzymes (Alu I and Mnl I), as in Table 4.
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Figure 5: Allele frequency of the distinct allelic variants of the F2 segment of Pvmsp1 observed in the 100 isolates from Thailand. Allelic variants were defined according to the RFLP patterns observed. A. Variants were divided according to the digestions patterns obtained individually with each of the two restriction enzymes Alu I (a1, a2, etc...) or Mnl I (m1, m2, etc...) for A or B, the two different sized fragments amplified, as in Table 3. B) Variants were classed according to size and the combined RFLP patterns obtained for both restriction enzymes (Alu I and Mnl I), as in Table 4.

Mentions: The F2 segment, which was comparatively poorly polymorphic in size in the Thai isolates, encompassed regions established as polymorphic in previous studies [27]. An RFLP strategy was thus adopted to distinguish between the different allelic variants. The restriction endonucleases Alu I and Mnl I which recognize multiple sites in F2, were used to reveal extensive polymorphism at the nucleotide level (Fig. 3C). Thus, nine different Alu I and eight different Mnl I RFLP patterns were observed in the 100 Thai isolates (Table 3). The occurrence of patterns indicative of partial digestion, or the presence of mixed F2 genotypes in individual samples were excluded since the sum of the RFLP fragments' sizes was not found to be greater than that of the uncut product for any isolate and the patterns observed were not unique to isolates where mixed genotypes were detected through size polymorphism of the F1 or F3 segments. When the data from both analyses (size and RFLP) were combined, 36 Pvmsp1 F2 allelic variants could be differentiated (Table 4). When the frequency of the allelic variants was considered individually for the two RFLP patterns, 50 % and 38 % of the isolates were of a single Alu I- and Mnl I-classified allelic variant, respectively. The remaining isolates were randomly distributed between the different variants (Fig. 5A). However, by combining the results for both restriction enzymes, the most dominant allele was found in only 27 % of the isolates (Fig. 5B), while the remainder were found at lower frequency (5 % or less).


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 the F2 segment of Pvmsp1 observed in the 100 isolates from Thailand. Allelic variants were defined according to the RFLP patterns observed. A. Variants were divided according to the digestions patterns obtained individually with each of the two restriction enzymes Alu I (a1, a2, etc...) or Mnl I (m1, m2, etc...) for A or B, the two different sized fragments amplified, as in Table 3. B) Variants were classed according to size and the combined RFLP patterns obtained for both restriction enzymes (Alu I and Mnl I), as in Table 4.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Allele frequency of the distinct allelic variants of the F2 segment of Pvmsp1 observed in the 100 isolates from Thailand. Allelic variants were defined according to the RFLP patterns observed. A. Variants were divided according to the digestions patterns obtained individually with each of the two restriction enzymes Alu I (a1, a2, etc...) or Mnl I (m1, m2, etc...) for A or B, the two different sized fragments amplified, as in Table 3. B) Variants were classed according to size and the combined RFLP patterns obtained for both restriction enzymes (Alu I and Mnl I), as in Table 4.
Mentions: The F2 segment, which was comparatively poorly polymorphic in size in the Thai isolates, encompassed regions established as polymorphic in previous studies [27]. An RFLP strategy was thus adopted to distinguish between the different allelic variants. The restriction endonucleases Alu I and Mnl I which recognize multiple sites in F2, were used to reveal extensive polymorphism at the nucleotide level (Fig. 3C). Thus, nine different Alu I and eight different Mnl I RFLP patterns were observed in the 100 Thai isolates (Table 3). The occurrence of patterns indicative of partial digestion, or the presence of mixed F2 genotypes in individual samples were excluded since the sum of the RFLP fragments' sizes was not found to be greater than that of the uncut product for any isolate and the patterns observed were not unique to isolates where mixed genotypes were detected through size polymorphism of the F1 or F3 segments. When the data from both analyses (size and RFLP) were combined, 36 Pvmsp1 F2 allelic variants could be differentiated (Table 4). When the frequency of the allelic variants was considered individually for the two RFLP patterns, 50 % and 38 % of the isolates were of a single Alu I- and Mnl I-classified allelic variant, respectively. The remaining isolates were randomly distributed between the different variants (Fig. 5A). However, by combining the results for both restriction enzymes, the most dominant allele was found in only 27 % of the isolates (Fig. 5B), while the remainder were found at lower frequency (5 % or less).

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