Genetic composition of Plasmodium falciparum in relation to drug resistance and disease transmission in Sri Lanka

Abstract

Malaria has been an important communicable disease in Sri Lanka. Even though P. vivax is the predominant species, there has been an increase in the proportion of chloroquine resistant P. falciparum infections in the country since the early 1990s. According to national data, the highest number of P. falciparum cases has been reported from the Northern Province for several years. This study examined the species distribution, antimalarial resistance, transmission potential and genetic structure of P. falciparum populations among security forces personnel in an operational area of the Mannar District of the Northern Province in 2002 and 2004 to understand their inter-related effects on clinical disease and transmission of malaria in the country. Some of these aspects were compared with those from other areas of the country to determine how they differed in the Northern Province. An attempt was also made for the first time to identify appropriate markers for molecular surveillance of sulfadoxine-pyrimethamine (S-P) resistance of P. falciparum in Sri Lanka. Screening of 1271 patients by light microscopy revealed that P. falciparum was the predominant species in the Northern Province study area. Clinical characteristics of P. falciparum infections (n = 151) suggested that parasite isolates, especially chloroquine resistant ones, in the Northern Province could be more virulent than isolates in other areas. Extended in vivo (n = 76) and in vitro micro test assays (n = 29) for chloroquine showed a treatment failure rate of more than 50 percent among P. falciparum infections in the Northern Province with a 3-fold rise in IC50 values for chloroquine within the two year period. Recrudescent infections caused significantly less severe disease and showed greater potential for transmission than sensitive infections suggesting that antimalarial resistance confers a survival advantage to P. falciparum isolates in the study area. None of the patients (n = 30) who completed in vivo assay for S-P showed evidence of treatment failure. Polymerase Chain Reaction (PCR) based genotyping of P. falciparum populations using size polymorphism in merozoite surface protein-1 (MSP-1), merozoite surface protein-2 (MSP-2) and glutamate rich protein (GLURP) showed a more complex parasite population in the Northern Province. Block 2 of MSP-1, MSP-2 and GLURP were identified as potential markers for genotyping of P. falciparum isolates in Sri Lanka. The proportion of mixed genotype infections and genotype composition of 'pure' P. falciparum infections appears to reflect the transmission intensity in the Northern Province study area. Longitudinal variation in genotype composition of 'pure' infections showed selection of genotypes 2 and 9, which were associated with resistance to chloroquine, providing indirect evidence for the presence of intrahost dynamics in this parasite population. PCR-restriction fragment length polymorphism based mutation analysis of P. falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes that confer resistance to pyrimethamine and sulfadoxine respectively showed that the majority of field isolates in the Northern Province (88 percent) as well as in other areas of the country (75 percent) were double mutants (C59R + S108N) in Pfdhfr. Overall, 18 percent were also mutants in Pfdhps, especially at codon 581 (A581G). Despite the absence of clinical failures with S-P, in vitro yeast expression assay showed a 860-fold higher mean IC50 value for pyrimethamine in the double mutants than in wild type isolates in the Northern Province. In vitro response of those double mutants closely resembled that of yeast cells dependent upon a triple mutant (N51I + C59R + S108N) reference isolate, indicating impending clinical failure to S-P in the country. These results suggest that the mutant codon at residue 51 of Pfdhfr will be the potential marker of imminent S-P clinical failure in Sri Lanka. In conclusion, recrudescent infections appear to drive the phase of tolerance during the evolutionary process of antimalarial resistance as suggested in the "STR model", while primary infections appear to drive the phase of complete resistance in this area. By causing more clinical episodes, chloroquine resistant primary infections probably induce immunity against malaria among individuals, optimizing the evolutionary process of virulence at an intermediate level. A close inter-relationship was evident in P. falciparum genetic structure, antimalarial resistance, clinical disease and disease transmission in the Northern Province. Continued use of chloroquine probably drives the vicious cycle of increasing antimalarial resistance and virulence in the Northern Province. Thus, these results highlight the need to change the current antimalarial therapy in Sri Lanka, particularly in the Northern Province in order to contain the further spread of antimalarial resistance in the country.