Genetic diversity of the Plasmodium falciparum GTP-cyclohydrolase 1, dihydrofolate reductase and dihydropteroate synthetase genes reveals new insights into sulfadoxine-pyrimethamine antimalarial drug resistance

Author summary Malaria causes approximately 435,000 deaths per year, concentrated in sub-Saharan Africa and among children under the age of five years. Global efforts to control and eliminate malaria are hampered by the emergence of Plasmodium falciparum malaria parasites resistant to currently available antimalarial drugs. Sulfadoxine-pyrimethamine (SP) was used globally as a first-line treatment for malaria and, although replaced by artemisinin combinations, is still used for the prevention of malarial disease in vulnerable groups (e.g. pregnant women). SP resistance is caused by mutations in the P. falciparum parasite genes pfdhfr and pfdhps, but recently novel structural variants in and around the pfgch1 gene have been described. By analysing genome sequence data of 4,134 P. falciparum across 29 malaria endemic countries, we establish there are at least ten different pfgch1 structural variants, existing in the presence of pfdhfr and pfdhps mutations, and occur increasingly and at high frequency in some Southeast Asian and African populations. These pfgch1 structural variants may enhance the survival of those parasites with pfdhfr and pfdhps substitutions, potentially threatening the efficacy of SP for prevention of malaria in vulnerable groups. Therefore it is important that they are monitored in molecular surveillance studies. Our work will assist epidemiological studies, laboratory and surveillance activities looking at the diversity and role of pfgch1 structural variants and their prevalence across malaria endemic regions, especially in countries using SP regimens. Plasmodium falciparum parasites resistant to antimalarial treatments have hindered malaria disease control. Sulfadoxine-pyrimethamine (SP) was used globally as a first-line treatment for malaria after wide-spread resistance to chloroquine emerged and, although replaced by artemisinin combinations, is currently used as intermittent preventive treatment of malaria in pregnancy and in young children as part of seasonal malaria chemoprophylaxis in sub-Saharan Africa. The emergence of SP-resistant parasites has been predominantly driven by cumulative build-up of mutations in the dihydrofolate reductase (pfdhfr) and dihydropteroate synthetase (pfdhps) genes, but additional amplifications in the folate pathway rate-limiting pfgch1 gene and promoter, have recently been described. However, the genetic make-up and prevalence of those amplifications is not fully understood. We analyse the whole genome sequence data of 4,134 P. falciparum isolates across 29 malaria endemic countries, and reveal that the pfgch1 gene and promoter amplifications have at least ten different forms, occurring collectively in 23% and 34% in Southeast Asian and African isolates, respectively. Amplifications are more likely to be present in isolates with a greater accumulation of pfdhfr and pfdhps substitutions (median of 1 additional mutations; P<0.00001), and there was evidence that the frequency of pfgch1 variants may be increasing in some African populations, presumably under the pressure of SP for chemoprophylaxis and anti-folate containing antibiotics used for the treatment of bacterial infections. The selection of P. falciparum with pfgch1 amplifications may enhance the fitness of parasites with pfdhfr and pfdhps substitutions, potentially threatening the efficacy of this regimen for prevention of malaria in vulnerable groups. Our work describes new pfgch1 amplifications that can be used to inform the surveillance of SP drug resistance, its prophylactic use, and future experimental work to understand functional mechanisms.