Antimalarial drug resistance: linking Plasmodium falciparum parasite biology to the clinic

Abstract

The global adoption of artemisinin-based combination therapies (ACTs) in the early 2000s heralded a new era in effectively treating drug-resistant Plasmodium falciparum malaria. However, several Southeast Asian countries have now reported the emergence of parasites that have decreased susceptibility to artemisinin (ART) derivatives and ACT partner drugs, resulting in increasing rates of treatment failures. Here we review recent advances in understanding how antimalarials act and how resistance develops, and discuss new strategies for effectively combatting resistance, optimizing treatment and advancing the global campaign to eliminate malaria.

Figure 1

Plasmodium’s life cycle and its relationship to drug resistance. (a) Human infection begins when infective female Anopheles mosquitoes deliver fewer than 100 sporozoites into the dermis during blood feeding. These motile forms migrate rapidly into the bloodstream and to the liver, where they invade hepatocytes. A prodigious phase of replication over a week results in an estimated 10,000–30,000 merozoite progeny per intracellular parasite. (b) Liberated merozoites (~10⁵–10⁶ in total) then invade human mature red blood cells (RBCs), developing inside a parasitophorous vacuole and initiating ~48-h cycles of asexual blood stage (ABS) parasite growth, egress and reinvasion. (c) ABS parasites, typically 10⁸–10¹², are responsible for disease manifestations. (d) About 1–2% of intra-erythrocytic parasites enter an alternative program of sexual development, a process that over ~10–12 d produces mature male and female gametocytes that are transmissible to Anopheles mosquitoes. (e) An estimated 10³–10⁴ mature gametocytes are taken up during a blood meal. (f–i) These gametocytes then form male and female gametes (~10²–10³) that undergo sexual recombination (f), forming ookinetes (<100; g) and then oocysts (typically 1–2; h) before completing their life cycle by forming 10³ to 10⁴ sporozoites that migrate to salivary glands (i), ready for further human infection. In acute cases, ABS parasites can infect up to 10–20% of all erythrocytes (i.e., >10¹²). Primary causes of death include severe malaria anemia, or cerebral malaria that causes brain herniation and respiratory arrest. Immunity is acquired slowly and is nonsterilizing; its maintenance is dependent on continued infection. Selective forces that drive the emergence and spread of drug resistance differ throughout the life cycle. Important factors include the parasite numbers and drug pressure at different stages, stage specificity of drug action, the essentiality of the targeted pathways in the mosquito vector and vertebrate host, host immunity, multiplicity of infection, and local factors that affect therapeutics use and compliance. The pathogenic ABS reproduction cycle experiences the highest parasitemias and drug pressure, whereas the lower numbers of clinically silent liver-stage parasites provide much less fertile ground for the emergence of resistance. Human-to-mosquito transmission is possible only if sufficient densities of mutant gametocytes are produced, which can be triggered in some instances by drug treatment. Parasite number estimates were derived from refs. ,–. Stages targeted by current and former first-line drugs used to treat P. falciparum are shown.

Figure 2

Molecular targets of and mechanisms of resistance to major antimalarial drugs. Frequently targeted biological pathways include heme detoxification in the digestive vacuole, folate and pyrimidine biosynthesis in the cytosol, and electron transport in the mitochondrion. The 4- aminoquinolines, including CQ and AQ, as well as PPQ, the Mannich base pyronaridine (PND), and to some degree the aryl-amino alcohol quinine (QN), are all thought to concentrate in the digestive vacuole, where they bind reactive heme and interfere with its detoxification through incorporation into chemically inert hemozoin. Ferrous (Fe²⁺) iron-heme—liberated after parasite protease-mediated degradation of imported host hemoglobin (Hb)—can cleave and thereby activate the endoperoxide bridge of ART derivatives (star symbol). Point mutations (pink circles) in the transporters PfCRT and PfMDR1 are important determinants of resistance to 4-aminoquinolines. Resistance to PPQ is associated with increased expression of the hemoglobinases plasmepsin 2 and 3 (PM2/PM3, in the digestive vacuole), and might in some instances involve mutant PfCRT. Copy-number changes in pfmdr1, as well as PfCRT and PfMDR1 sequence variants, also affect the parasite’s susceptibility to the aryl-amino alcohols quinine (QN), lumefantrine (LMF) and mefloquine (MFQ) and can modulate ART potency. Variant forms of K13, thought to localize at the ER and in vesicular structures, are the primary mediator of ring-stage parasite resistance to ART. Mutations in two key enzymes of the folate biosynthesis pathway, dihydropteroate synthetase (DHPS) and dihydrofolate reductase (DHFR), can confer resistance respectively to the antifolates sulfadoxine (SDX) and both pyrimethamine (PYR) and cycloguanil (CYC). Atovaquone (ATQ) inhibits mitochondrial electron transport, and a single point mutation in the cytochrome b subunit (CYTb) of the bc1 complex can confer resistance to this drug. The ETC is important in ABS parasites because of its role in providing electrons for the ubiquinone-dependent dihydroorotate dehydrogenase (DHODH), an enzyme essential for de novo pyrimidine biosynthesis. Antibiotics such as clindamycin (CLD) and doxycycline (DOX) inhibit protein translation inside the apicoplast. CLD resistance is mediated by a point mutation in the apicoplast-encoded 23S rRNA.

Figure 3

History of the introduction of the principal antimalarials and of the first emergence of resistance in the field. Single bars refer to monotherapies; double- and triple-bar boxes denote combination therapies. Colors refer to the chemical classes to which the antimalarials belong. Quinine, first imported into Europe in the 1630s to treat malaria, encountered partial resistance in the early twentieth century, and later, was replaced by chloroquine (CQ), a former gold standard used massively until resistance appeared a decade later. Resistance to proguanil was detected within a year of clinical use. The replacement drug sulfadoxine–pyrimethamine (SP) quickly encountered resistance, and today is used primarily for intermittent preventive treatment during pregnancy and for seasonal malaria chemoprevention in combination with AQ (SPAQ, not shown) and as first-line treatment in combination with artesunate (ASSP) in some SP-sensitive areas. Artemisinins (ARTs) were first used in monotherapy (and injectable artesunate is still used for severe malaria), although their short half-life in plasma and issues of resistance led to the development of artemisinin-based combination therapies (ACTs) for uncomplicated malaria. Several ACT partner drugs (such as amodiaquine and mefloquine) had been used as monotherapies and remained in use as single agents long after resistance was first found. Piperaquine and pyronaridine were introduced in China as a replacement of CQ ~40 years ago^,,. Resistance to piperaquine monotherapy was reported there in the late 1980s. ART resistance (as manifested by delayed parasite clearance following treatment with an artesunate monotherapy or with an ACT) was first reported in 2008 (ref. 10) but was already present several years earlier in western Cambodia. Treatment failure owing to resistance to one or both components of an ACT has been documented first for ASMQ and, more recently, for DHA-PPQ^,,,,. The WHO has recently reported AL treatment failures in Laos. Atovaquone -proguanil (Malarone) is currently prescribed as a prophylactic agent for travelers to malaria-endemic areas. AL, artemether + lumefantrine; ASMQ, artesunate + mefloquine; ASAQ, artesunate + amodiaquine; DHA-PPQ, dihydroartemisinin + piperaquine; PA, artesunate + pyronaridine; ASSP, artesunate + SP.

Figure 4

Emergence and spread of P. falciparum resistance to CQ, pyrimethamine and ART derivatives. Resistance to CQ is thought to have arisen in multiple sites and spread globally (black arrows) owing to the selection pressure of CQ on mutant pfcrt alleles, resulting in a selective sweep at this locus. CQ-resistant parasites in Southeast Asia near the Thai–Cambodian border are thought to have seeded the introduction of CQ-resistant parasites into Africa, carried by individuals with the infection. Resistance to pyrimethamine also emerged in Southeast Asia as well as in South America, and with triple-mutant dhfr Asian alleles, spread to Africa (red arrows)^,. Pyrimethamine-resistantdhfr alleles also emerged independently in Africa^,. Arrows are overlaid onto a 2010 map of P. falciparum endemicity on the basis of P. falciparum parasite rate (PfPR) surveys, in 2–10 year olds, using model-based geostatistics. ART resistance was first detected in Cambodia (inset), driven by the emergence of mutant k13 alleles, and has since been detected in multiple countries in the region. Taken with permission from “Artemisinin and artemisinin-based combination therapy resistance,” October 2016 Status Report from the Global Malaria Programme of the World Health Organization, document WHO/HTM/GMP/2016.11; available at http://apps.who.int/iris/bitstream/10665/250294/1/WHO-HTM-GMP-2016.11-eng.pdf.