Cyclical regression covariates remove the major confounding effect of cyclical developmental gene expression with strain-specific drug response in the malaria parasite Plasmodium falciparum.

BACKGROUND: The cyclical nature of gene expression in the intraerythrocytic development cycle (IDC) of the malaria parasite, Plasmodium falciparum, confounds the accurate detection of specific transcriptional differences, e.g. as provoked by the development of drug resistance. In lab-based studies, P. falciparum cultures are synchronized to remove this confounding factor, but the rapid detection of emerging resistance to artemisinin therapies requires rapid analysis of transcriptomes extracted directly from clinical samples. Here we propose the use of cyclical regression covariates (CRC) to eliminate the major confounding effect of developmentally driven transcriptional changes in clinical samples. We show that elimination of this confounding factor reduces both Type I and Type II errors and demonstrate the effectiveness of this approach using a published dataset of 1043 transcriptomes extracted directly from patient blood samples with different patient clearance times after treatment with artemisinin. RESULTS: We apply this method to two publicly available datasets and demonstrate its ability to reduce the confounding of differences in transcript levels due to misaligned intraerythrocytic development time. Adjusting the clinical 1043 transcriptomes dataset with CRC results in detection of fewer functional categories than previously reported from the same data set adjusted using other methods. We also detect mostly the same functional categories, but observe fewer genes within these categories. Finally, the CRC method identifies genes in a functional category that was absent from the results when the dataset was adjusted using other methods. Analysis of differential gene expression in the clinical data samples that vary broadly for developmental stage resulted in the detection of far fewer transcripts in fewer functional categories while, at the same time, identifying genes in two functional categories not present in the unadjusted data analysis. These differences are consistent with the expectation that CRC reduces both false positives and false negatives with the largest effect on datasets from samples with greater variance in developmental stage. CONCLUSIONS: Cyclical regression covariates have immediate application to parasite transcriptome sequencing directly from clinical blood samples and to cost-constrained in vitro experiments.

Humanized Mice and the Rebirth of Malaria Genetic Crosses.

The first experimental crosses carried out with the human malaria parasite Plasmodium falciparum played a key role in determining the genetic loci responsible for drug resistance, virulence, invasion, growth rate, and transmission. These crosses relied on splenectomized chimpanzees to complete the liver stage of the parasite's life cycle and the subsequent transition to asexual blood stage culture followed by cloning of recombinant progeny in vitro. Crosses can now be routinely carried out using human-liver-chimeric mice infused with human erythrocytes to generate hundreds of unique recombinant progeny for genetic linkage mapping, bulk segregant analysis, and high-throughput 'omics readouts. The high number of recombinant progeny should allow for unprecedented power and efficiency in the execution of a systems genetics approach to study P. falciparum biology.

The extended recovery ring-stage survival assay provides a superior association with patient clearance half-life and increases throughput.

BACKGROUND: Tracking and understanding artemisinin resistance is key for preventing global setbacks in malaria eradication efforts. The ring-stage survival assay (RSA) is the current gold standard for in vitro artemisinin resistance phenotyping. However, the RSA has several drawbacks: it is relatively low throughput, has high variance due to microscopy readout, and correlates poorly with the current benchmark for in vivo resistance, patient clearance half-life post-artemisinin treatment. Here a modified RSA is presented, the extended Recovery Ring-stage Survival Assay (eRRSA), using 15 cloned patient isolates from Southeast Asia with a range of patient clearance half-lives, including parasite isolates with and without kelch13 mutations. METHODS: Plasmodium falciparum cultures were synchronized with single layer Percoll during the schizont stage of the intraerythrocytic development cycle. Cultures were left to reinvade to early ring-stage and parasitaemia was quantified using flow cytometry. Cultures were diluted to 2% haematocrit and 0.5% parasitaemia in a 96-well plate to start the assay, allowing for increased throughput and decreased variability between biological replicates. Parasites were treated with 700 nM of dihydroartemisinin or 0.02% dimethyl sulfoxide (DMSO) for 6 h, washed three times in drug-free media, and incubated for 66 or 114 h, when samples were collected and frozen for PCR amplification. A SYBR Green-based quantitative PCR method was used to quantify the fold-change between treated and untreated samples. RESULTS: 15 cloned patient isolates from Southeast Asia with a range of patient clearance half-lives were assayed using the eRRSA. Due to the large number of pyknotic and dying parasites at 66 h post-exposure (72 h sample), parasites were grown for an additional cell cycle (114 h post-exposure, 120 h sample), which drastically improved correlation with patient clearance half-life compared to the 66 h post-exposure sample. A Spearman correlation of - 0.8393 between fold change and patient clearance half-life was identified in these 15 isolates from Southeast Asia, which is the strongest correlation reported to date. CONCLUSIONS: eRRSA drastically increases the efficiency and accuracy of in vitro artemisinin resistance phenotyping compared to the traditional RSA, which paves the way for extensive in vitro phenotyping of hundreds of artemisinin resistant parasites.

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

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.