Biology subjects

Cassady, Z.

Publications and source records attributed to Cassady, Z..

2 recordsLinked to original sources

bioRxiv2024

Measuring Growth, Resistance and Recovery after Artemisinin Treatment of Plasmodium falciparum in a semi-high-throughput Assay

BackgroundArtemisinin partial resistance (ART-R) has spread throughout Southeast Asia and mutations in pfKelch13, the molecular marker of resistance, are widely reported in East Africa. Effective in vitro assays and robust phenotypes are crucial for monitoring populations for the emergence and spread of resistance. The recently developed extended Recovery Ring-stage Survival Assay used a qPCR-based readout to reduce the labor intensiveness for in vitro phenotyping of ART-R and improved correlation with the clinical phenotype of ART-R. Here, we extend and refine this assay to include measurements of parasite growth and recovery after drug exposure. Clinical isolates and progeny from two genetic crosses were used to optimize and validate the reliability of a straight-from-blood, SYBR Green-based qPCR protocol in a 96-well plate format to accurately measure phenotypes for Growth, Resistance, and Recovery. ResultsThe assay determined growth between 6 h and 96 h, resistance at 120 h, and recovery from 120 h and 192 h. Growth can be accurately captured by qPCR and is shown by reproduction of previous growth phenotypes from HB3 x Dd2. Resistance measured at 120 h continually shows the most consistent phenotype for ring stage susceptibility. Recovery identifies an additional response to drug than parasites that are determined sensitive by Fold Change at 120 h. Comparison of progeny phenotypes for Growth vs Resistance showed a minor but significant correlation, whereas Growth vs Recovery and Resistance vs Recovery showed no significant correlation. Additionally, dried blood spot (DBS) samples matched Fold Change measured from liquid samples demonstrating Resistance can be easily quantified using either storage method. ConclusionsThe qPCR-based methodology provides the throughput needed to quickly measure large numbers of parasites for multiple relevant phenotypes. Growth can reveal fitness defects and illuminate relationships between proliferation rates and drug response. Recovery serves as a complementary phenotype to resistance that quantifies the ability of sensitive parasites to tolerate drug exposure. All three phenotypes offer a comprehensive assessment of parasite-drug interaction each with independent genetic determinants of main effect and overlapping secondary effects that should be further. By adapting our method to include DBS, readouts can be easily extended to ex vivo surveillance applications.

microbiology↗

bioRxiv2019

The extended recovery ring stage survival assay provides superior prediction of patient clearance half life and increases throughput

BackgroundTracking 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. MethodsP. falciparum cultures were synchronized with single layer Percoll during the schizont stage of the erythrocytic cycle. Cultures were left to reinvade to early ring-stage and parasitemia was quantified using flow cytometry. Cultures were diluted to 2% hematocrit and 0.5% parasitemia in a 96-well plate to start the assay, allowing for increased throughput and decreased variability between biological replicates. Parasites were treated with 700nM of dihydroartemisinin or an equivalent amount of 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. Results15 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. ConclusionseRRSA 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.

microbiology↗