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

Abigail R. Tirrell,Lisa A. Checkley,Ashley M. Vaughan,Michael T. Ferdig,Sage Z. Davis,Marina McDew-White,Katelyn M. Vendrely,Timothy J. C. Anderson,Ian H. Cheeseman,François H. Nosten

doi:10.1186/s12936-019-2934-4

Abstract

BackgroundCompetitive 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.MethodsAn 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.ResultsTwenty-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.ConclusionsThis 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.

Highlights

Competitive outcomes between co-infecting malaria parasite lines can reveal fitness disparities in blood stage growth
To optimize a higher-throughput method to ascertain the relative fitness of many parasite lines, the previous protocol was adapted and optimized to a 96-well plate format and compared it to outcomes from standard 5 ml flask competition assays
This study describes and validates a plate-based, in vitro, competitive growth methodology and use it to determine fitness relationships among cloned parasite lines recently isolated from patients in Southeast Asia with a range of artemisinin resistance (Art-R)-associated kelch13 alleles

Summary

Introduction

Competitive outcomes between co-infecting malaria parasite lines can reveal fitness disparities in blood stage growth. For pathogens including HIV, fungi, and bacteria, the gold standard for measuring fitness in vitro and in vivo relies on labeling methods to assay the relative growth success of the competing lines [3] While both cooperative and competitive interactions can occur between co-infecting conspecific pathogens [4,5,6] head-to-head growth outcomes usually reflect the innate proliferation rates of each of the competing genotypes. Clinical artemisinin resistance (Art-R), manifests as a delayed clearance of parasites from a patient’s blood following drug treatment and is defined as a parasite clearance half-life ≥ than 5 h after drug treatment [13] Parasites isolated from these patients have been studied using competitive growth assays [14] and a few focused laboratory experiments have demonstrated a cost of Art-R mutations in P. falciparum lines grown in the absence of drug [15, 16]

Methods

Results

Discussion

Conclusion