A PCR-RFLP Technique to Assess the Geographic Origin of Plasmodium falciparum Strains in Central America

Abstract

The elimination of malaria requires strengthening diagnosis and offering adequate and timely treatment. Imported cases of falciparum malaria represent a major challenge for pre-elimination areas, such as Central America, where chloroquine and primaquine continue to be used as first-line treatment. The pfs47 gene has been previously described as a precise molecular marker to track the geographic origin of the parasite. The aim of this study was to design a simple and low-cost technique using the polymorphic region of pfs47 to assess the geographic origin of P. falciparum strains. A PCR-RFLP technique was developed and evaluated using the MseI enzyme that proved capable of discriminating, with reasonable precision, the geographical origin of the parasites. This method could be used by national surveillance laboratories and malaria elimination programs in countries such as Honduras and Nicaragua in cases of malaria where an origin outside the Central American isthmus is suspected.

Keywords: Central America; Honduras; Plasmodium falciparum; malaria; pfs47; pfs48/45; pvs47.

Figure 1

94 bp fragment of the pfs47 gene indicating the primer target sites and four MseI cutting points.

Figure 2

Restriction profiles of the pfs47 gene digested with MseI in relation to the geographic origin of the isolates.

Figure 3

(a) Structure of the Plasmodium falciparum cysteine-rich protein PfS47, showing three domains and a variable region within domain 2. The letter “C” within the polypeptide chain indicates the approximate position of the cysteine residues. (b) Multiple nucleotide sequence alignment of the polymorphic region of the pfs47 gene. (c) Multiple amino acid sequence alignment of the polymorphic region of the PfS47 protein. Red arrows indicate polymorphic sites.

Figure 4

Cladograms constructed with the UPGMA method using (a) 56 sequences of 1282 nucleotides of the pfs47 gene; (b) sequences of 94 nucleotides from domain 2 of the pfs47 gene; (c) sequences of 427 residues of the putative protein PfS47; and (d) 56 sequences of 31 predicted residues of domain 2 of the PfS47 protein. Colors indicate the geographic origin of Plasmodium falciparum isolates.

Figure 5

Polyacrylamide gel electrophoresis shows the restriction patterns of a fragment of the pfs47 gene digested with the enzyme MseI. Lanes 1, 5, and 15 show undigested amplification products, lanes 2 and 10 show strain 04/176, lane 18 shows a parasite of African origin isolated in Honduras, lane 3: strain K1, lanes 4, 6–9, 12–14, 16–17, 19–20: strains from Honduras and Nicaragua, lane 11: strain Dd2, lane 21: strain 7G8, and lane 22: strain HB3. MW: molecular weight marker of 25 bp.