Malaria parasites with pfgch1 mutations enhance resistance to Sulfadoxine-pyrimethamine


In the ongoing arms race between humans and the parasite that causes malaria, Taane Clark and colleagues at the London School of Hygiene and Tropical Medicine (LSHTM) report that new mutations that enhance resistance to a drug used to prevent malaria in pregnant women and children are already common in countries fighting the disease. The new results are published December 31 in PLOS Genetics.

Malaria causes about 435,000 deaths each year, primarily in young children in sub-Saharan Africa. Despite a long-term global response, efforts to control the disease are hampered by the rise of drug-resistant strains of the parasite species that cause malaria.

Sulfadoxine-pyrimethamine (SP), for example, was once a first-line anti-malaria treatment, but now primarily is used to prevent infection in pregnant women and children. Mutations in two genes in the parasite Plasmodium falciparum offer resistance to SP, but recently, mutations related to resistance were discovered in a third gene, pfgch1.

To understand the extent and spread of these new mutations, Clark and colleagues analyzed genome sequences from 4,134 blood samples collected from 29 countries where malaria is endemic.

They discovered at least ten different versions of pfgch1, which occur in about one quarter of the samples from Southeast Asia and in one third of the samples from Africa, where strains carrying the mutations may be on the rise.

The growth in the number of malaria parasites with pfgch1 mutations is concerning, because the mutations enhance resistance to SP and may encourage the evolution of new resistant strains.

As a result, their growth may threaten efforts to use SP to prevent malaria in vulnerable groups. With the identification of these pfgch1 mutations through the new study, however, scientists can monitor their presence in parasite populations, to understand where SP can be used effectively, and where rates of drug-resistance are already too high.

“We need to understand how these mutations work and monitor them as part of malaria surveillance programs,” says Clark.

Colin Sutherland, an author and co-Director of the LSHTM Malaria Centre, says, “SP is an established drug for malaria prevention and treatment in vulnerable groups such as pregnant women and children.

We may have underestimated its vulnerability to parasite resistance, as these new data show.”

Mutations in pfdhfr/pfdhsp and pfgch1 copy number fluctuated overtime through the study period. Altogether, 14 unique pfdhfr–pdfhps haplotypes collectively containing quadruple to octuple mutations were identified.

High variation in pfdhfr–pfdhps haplotypes and a high proportion of pfgch1 multiple copy number (51% (73/146)) were observed on the Thailand–Myanmar border compared to other parts of Thailand. Overall, the prevalence of septuple mutations was observed for pfdhfr–pfdhps haplotypes.

In particular, the prevalence of pfdhfr–pfdhps, septuple mutation was observed in the Thailand–Myanmar (50%, 73/146) and Thailand–Cambodia (65%, 26/40) border. In Thailand–Malaysia border, majority of the pfdhfr–pfdhps haplotypes transaction from quadruple (90%, 9/10) to quintuple (65%, 24/37) during 2008–2016.

Within the pfdhfr–pfdhps haplotypes, during 2008–2013 the pfdhps A/S436F mutation was observed only in Thailand–Myanmar border (9%, 10/107), while it was not identified later. In general, significant correlation was observed between the prevalence of pfdhfr I164L (ϕ = 0.213, p-value = 0.001) or pfdhps K540E/N (ϕ = 0.399, p-value ≤ 0.001) mutations and pfgch1 gene amplification.

Despite withdrawal of SP as anti-malarial treatment for 17 years, the border regions of Thailand continue to display high prevalence of antifolate and anti-sulfonamide resistance markers in falciparum malaria. Significant association between pfgch1 amplification and pfdhfr (I164L) or pfdhps (K540E) resistance markers were observed, suggesting a compensatory mutation.

Both falciparum and vivax malaria remains an important public health problem in border regions of Thailand. Resistance to anti-malarials presents a major hurdle for eradication of the disease [1]. Drug resistance in both Plasmodium falciparum and Plasmodium vivax has been reported as early as the 1950s [2].

Sulfadoxine–pyrimethamine (SP), a folate pathway inhibitor was deployed in Thailand for the treatment of uncomplicated falciparum malaria from 1973 until 1991 [3]. By 1991, substantial loss of the SP drug efficacy prompted a change in first-line treatment in Thailand [3].

Molecular investigations revealed that mutations in P. falciparum dihydrofolate reductase (pfdhfr) and P. falciparum dihydropteroate synthase (pfdhps) were associated with SP treatment failures and could be used as molecular markers for SP resistance [4, 5]. The mutations in pfdhfr and pfdhps often occurred in a step-wise progressive manner resulting in increased levels of drug resistance [4, 6].

Resistance to antifolates has also been linked to gene amplification of P. falciparum guanosine triphosphate cyclohydrolase 1 (pfgch1)-an enzyme responsible for coding a crucial enzyme in the folate pathway [7]. Parasites with pfgch1 amplification were reportedly less susceptible to antifolates as elevated expression of enzymes assisted antifolate resistance by competing with the drugs [7], and compensating the loss of fitness caused by mutations in pfdhfr and pfdhps by increasing the flux of metabolic products in the folate pathway [8].

An earlier study from Thailand reported a high proportion of parasites carrying multiple copies of pfgch1 and suggested an association between pfgch1 copy number variation (CNV) and the pfdhfr (I164L) mutations [8]. Several studies conducted between 1995 and 2008 have identified varying levels of triple or quadruple mutations in pfdhfr and pfdhps [5, 8, 9].

A more recent survey conducted in Ubonratchathani province close the Thailand–Cambodia borders, which had a lot of reports in many anti-malarial drug resistances [2, 3], showed high levels of pfdhfr (N51I, C59R, and S108N, ≥ 76%) and pfdhps (A437G, K540E, A581G or A437G, K540N, A581G or S436A, A437G, K540E, ≥ 90%) triple mutations [10].

These border areas are malaria endemic regions. Each site is geographically distant from other and often experiences high migration of diverse human population. However, data on the current status of antifolate and anti-sulfonamide resistance markers in P. falciparum in other major border regions of Thailand is scarce.

Presumably, the persistence of highly mutations on SP-resistant markers related to the using of other drugs that may also induced pressure on pfdhfr and pfdhps of malaria parasite. The trimethoprim–sulfamethoxazole, which is used to treat acute respiratory infections, presented cross-resistance with pyrimethamine and sulfadoxine [11, 12].

Reemergence of chloroquine-sensitive P. falciparum in Malawi after a decade-long cessation of drug use shows that for some anti-malarials restoration of drug sensitivity is possible after removal of the drug pressure [13]. However, several factors including drug target, nature of genes and host/parasite genetic background may differently affect the persistence of SP resistance after removal of SP use.

The present study is a retrospective molecular surveillance of three antifolate and anti-sulfonamide resistance markers in samples obtained from the malaria endemic border provinces of Thailand between 2008 and 2016. It aimed at describing the current status of resistance markers after long-term cessation of SP as an anti-malarial treatment in Thailand.

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More information: Turkiewicz A, Manko E, Sutherland CJ, Diez Benavente E, Campino S, Clark TG (2020) Genetic diversity of the Plasmodium falciparum GTP-cyclohydrolase 1, dihydrofolate reductase and dihydropteroate synthetase genes reveals new insights into sulfadoxine-pyrimethamine antimalarial drug resistance. PLoS Genet 16(12): e1009268. DOI: 10.1371/journal.pgen.1009268


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