Aspects of molecular markers in drug resistant malaria

Abstract

BACKGROUND: There were an estimated 207 million cases of malaria in 2012 of which 91% were due to Plasmodium falciparum. Antimalarial drug resistance constitutes a major problem in the efforts to control malaria. Drug resistance typically arises by the gradual accumulation of genetic changes that enable parasites to tolerate gradually increasing drug concentrations until fully resistant parasites develop. The aim of this thesis was to determine temporal and geographic frequencies of genetic polymorphisms linked to P. falciparum and P. vivax resistance after exposure to various antimalarial drugs.

METHODS: Polymorphisms in P. falciparum and P. vivax multidrug resistance 1 (pfmdr1 and pvmdr1), dihydrofolate reductase (pfdhfr and pvdhfr) genes, P. falciparum chloroquine resistance transporter (pfcrt), dihydropteroate synthase (pfdhps), V-type H+ pyrophosphatase (pfvp2), Na+/H+ exchanger-1 (pfnhe1) and multidrug-resistant protein 1 (pfmrp1) genes were determined in field samples. P. falciparum and P. vivax samples from Honduras, where drug resistant malaria has not been detected, were analysed for paper I. Samples from Honduras, Colombia, Liberia, Guinea-Bissau, Tanzania, Iran, Thailand and Vanuatu that represented varying origins and frequency of chloroquine (CQ) resistant P. falciparum malaria were analysed for paper II. Samples from 4 villages in Liberia in 1978 after children were administered village specific treatments of CQ, pyrimethamine (PYR), chlorproguanil or placebo monthly for 2 years were used for paper III. Samples (n=1806) from virtually all children aged <15 years with uncomplicated malaria between 2003 and 2012 in Guinea- Bissau were analysed for paper IV. Artemether-lumefantrine replaced an effective high dose CQ treatment in Guinea-Bissau in 2008 but quinine was prescribed to 43% of the children between 2010 and 2012.

RESULTS and CONCLUSIONS: Paper I. No genetic polymorphisms associated with CQ or sulphadoxine-pyrimethamine (SP) resistance were found indicating that P. falciparum remains CQ sensitive in Honduras. CQ resistance associated pvmdr1 976F (7/37) and SP resistance associated pvdhfr 57L+58R (2/57) were detected suggesting a degree of tolerance to CQ and SP in P. vivax. Paper II. Pfvp2 SNPs were found in only 20/367 patient samples. Nevertheless, the V405 + K582 + P711 haplotype was associated with pfcrt 76T (P=0.007) in line with previous in vitro data. However, the limited variation suggests that the results should be interpreted with caution. Paper III. Pfcrt 72-76 CVIET without other downstream resistance associated SNPs occurred in 1/178 samples from the village using CQ in Liberia. CVIET might have developed in Liberia in 1978 but the haplotype was not detected in 1981 suggesting that it probably did not provide a selective advantage in this area. Pfdhfr 108N was significantly lower (1/46, P=0.001) in the Liberian village using PYR compared with other villages (64/123). Pfdhfr 108N probably occurred as a wild type allele prior to the introduction of antifolates in Liberia. Decreased pfdhfr 108N frequency concurrently with the development of in vivo PYR resistance suggests a resistant mechanism other than pfdhfr point mutations. Paper IV. Between 2003 and 2007 pfcrt K76T and pfmdr1 N86Y frequencies were stable in Guinea-Bissau. After 2007, mean annual pfcrt 76T (24->57%) and pfmdr1 86N (72->83%) frequencies increased (p<0.001). Increased pfcrt 76T was probably driven by quinine that was as commonly used artemether-lumefantrine. Pfmdr1 86+184 NF frequency increased (39->66%) between 2003 and 2011 (p=0.004), possibly driven by artemether-lumefantrine.