Pairwise growth competitions identify relative fitness relationships among artemisinin resistant Plasmodium falciparum field isolates

Abstract

Background: Competitive outcomes between co-infecting malaria parasite lines can reveal fitness disparities in blood stage growth. Blood stage fitness costs often accompany the evolution of drug resistance, with the expectation that relatively fitter parasites will be more likely to spread in populations. With the recent emergence of artemisinin resistance, it is important to understand the relative competitive fitness of the metabolically active asexual blood stage parasites. Genetically distinct drug resistant parasite clones with independently evolved sets of mutations are likely to vary in asexual proliferation rate, contributing to their chance of transmission to the mosquito vector.

Methods: An optimized in vitro 96-well plate-based protocol was used to quantitatively measure-head-to-head competitive fitness during blood stage development between seven genetically distinct field isolates from a hotspot of emerging artemisinin resistance and the laboratory strain, NF54. These field isolates were isolated from patients in Southeast Asia carrying different alleles of kelch13 and included both artemisinin-sensitive and artemisinin-resistant isolates. Fluorescent labeled microsatellite markers were used to track the relative densities of each parasite throughout the co-growth period of 14-60 days. All-on-all competitions were conducted for the panel of eight parasite lines (28 pairwise competitions) to determine their quantitative competitive fitness relationships.

Results: Twenty-eight pairwise competitive growth outcomes allowed for an unambiguous ranking among a set of seven genetically distinct parasite lines isolated from patients in Southeast Asia displaying a range of both kelch13 alleles and clinical clearance times and a laboratory strain, NF54. This comprehensive series of assays established the growth relationships among the eight parasite lines. Interestingly, a clinically artemisinin resistant parasite line that carries the wild-type form of kelch13 outcompeted all other parasites in this study. Furthermore, a kelch13 mutant line (E252Q) was competitively more fit without drug than lines with other resistance-associated kelch13 alleles, including the C580Y allele that has expanded to high frequencies under drug pressure in Southeast Asian resistant populations.

Conclusions: This optimized competitive growth assay can be employed for assessment of relative growth as an index of fitness during the asexual blood stage growth between natural lines carrying different genetic variants associated with artemisinin resistance. Improved understanding of the fitness costs of different parasites proliferating in human blood and the role different resistance mutations play in the context of specific genetic backgrounds will contribute to an understanding of the potential for specific mutations to spread in populations, with the potential to inform targeted strategies for malaria therapy.

Keywords: Artemisinin resistance; Competitive growth; Fitness; Population genetics; kelch13.

Fig. 1

Consistent outcomes between 5 ml flask and 96-well plate methods in parasite competitive growth assays. Competition assays began with a parasite ratio of approximately 0.5:0.5. DNA microsatellite markers were used to track the competitive growth of parasites over time. Parasite competition P3 versus NHP4026 (n = 3) revealed the ability of NHP4026 to outcompete P3 by day 22 in both 5 ml flasks (a) and 96-well plates (b). The competition between P3 and P1 (n = 3) demonstrated the ability of P1 to outcompete P3 in 5 ml flasks by day 34 (c) and in 96-well plates by day 50 (d). Overall outcome results were consistent between 5 ml flask and 96-well plate, confirming the reliability of 96-well plate competitive growth assay methodology

Fig. 2

Win/loss records between parasite lines generate a fitness hierarchy. Win/loss records were determined for each parasite line and used to order lines from highest competitive fitness to lowest fitness. Arrows point from the winning (more fit) parasite to the losing (less fit) parasite. The win/loss record hierarchy ranked the kelch13-wt, slow clearance NHP4026 as having the highest fitness (7 wins, 0 losses: 7-0 record). Both NHP3032 and NHP4373 were unable to outcompete any other parasite, and the NHP3032 versus NHP4373 competition was unresolved, giving both isolates a record of 0 wins, 0 losses, 1 tie (0-0-1), suggesting potential fitness disadvantages in these isolates. NHP4302 had a record of 2-5. The hierarchy also shows that the kelch13 mutant isolates, NHP1337 (3-4 record), NHP4333 (4-3 record), and NHP4076 (5-2 record) have middle-range fitness and have a fitness cost relative to the uncharacterized resistance in NHP4026 and to the sensitive lab line, NF54 (6-1 record)

Fig. 3

Parasite line competitive growth assays suggest a fitness advantage in the NHP4026 line. NHP4026, a slow clearance kelch13-wt isolate, was able to fully outcompete all seven isolates it was matched with in pairwise competitions, suggesting a potential fitness advantage. In NHP4026 versus NF54 (kelch13-wt, fast clearance), NF54 initially held a greater ratio of parasitaemia, but NHP4026 overcame NF54 around day 30 and fully outcompeted NF54 by day 60 (n = 3; a). NHP4026 outcompeted NHP4076 (kelch13 E252Q, slow clearance) by day 24 (n = 3; b), and outcompeted NHP4333 (kelch13 G538V, slow-clearance) by day 40 (n = 3; c)

Fig. 4

The Art-R associated kelch13 E252Q allele shows a greater competitive fitness than other kelch13 mutations. NHP4076 (E252Q) was competed against two other kelch13 mutants, NHP1337 (C580Y) and NHP4333 (G538V). NHP4076 outcompeted NHP1337 by day 40 (n = 3; a) and NHP4333 by day 60 (n = 3; b). NHP4076 versus NHP4333 was set up unintentionally with NHP4076 only composing 20% of the total parasitaemia, yet was still able to outcompete NHP4333, giving reason to include this competition in the dataset. NHP4333 was also able to outcompete NHP1337 by day 40 (n = 3; c). The data suggests a competitive growth advantage of E252Q associated resistance over G538V and C580Y resistances, and a potential fitness disadvantage of C580Y resistance