Practical PCR genotyping protocols for Plasmodium vivax using Pvcs and Pvmsp1

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.

Figures

Figure 1

A. Schematic representation of the Pvcs gene, which consists of a conserved region (blank box) flanking a repeated region (grey box) which is itself flanked by pre- and post-repeat specific sequences (cross-hatched boxes). The bold horizontal line represents the PCR-amplified fragment used for further analysis. B. Example of the fragments (denoted A to G) of distinct size observed in different isolates, as single or as mixed infections (some lanes were not labelled because of lack of space). C. Fragments digested (D) or undigested (U) with Alu I or BstN I, two restriction enzymes with multiples sites in VK 210 type or VK247 type repeat sequences, respectively. Digestion cuts the fragment in small fragments of less than 150 bp in size. D. PCR-RFLP using specific restriction enzymes analysis for the presence/absence (yes/no) of specific pre- and post-repeat sequences in fragments carrying the VK210 or VK247 type repeats. Undigested fragments are denoted by (U) and Pr and Po denote digests to determine the presence of pre- and post-repeat sequence types, respectively. A 100 bp ladder, where the 600 bp band stains most intensely, was used a molecular weight marker (M) for all the gels.

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.

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.

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).

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.