E intraclass correlation coefficient (ICC) for agreement. Only sigmoidal curves had been analysed, to avoid biases on account of lack of fitness of different IC50 curve-fitting models. For all drugs, mean variations in IC50 values amongst replicates have been around zero, as anticipated. Except to get a couple of outliers (fewer than five points for every drug), variations amongst replicates had been compact compared together with the IC50 selection of every drug. Statistical analyses were performed in GraphPad Prism (v5.0d, San Diego, CA) and R-2.11.1. IC50 values measured ex vivo have been when compared with in vitro IC50 values from culture-adapted parasites by calculating the intraclass correlation coefficient (ICC) for consistency (R package irr), and by linear regression. To monitor population drug sensitivity, IC50 variations over time had been measured by means of linear regression with log10-transformed IC50 values. Major analysis focused on non-linear trends (making use of indicator variables for many years) but benefits have been confirmed by assessing linear trends. Various regression models have been applied to measure regardless of whether IC50 values changed considerably over time right after accounting for the effect of potentialVan Tyne et al. Malaria Journal 2013, 12:441 http://www.malariajournal/content/12/1/Page four ofconfounders (clonality, haematocrit, parasitaemia, age, and temperature). Given that parasites with lowered drug sensitivity may possibly first arise in subpopulations that exhibit bigger IC50 values, the 90th percentile among all IC50 values isreported for each year. Adjustments inside the prevalence of drug resistance markers over time have been measured by FisherHamilton exact test, and 95 confidence intervals for marker prevalence are based around the logit (R packageFigure 1 Validation in the DAPI ex vivo drug assay.Flunarizine A.Halofuginone Fluorescence intensity of maximum growth wells versus initial parasitaemia for parasites tested within the DAPI ex vivo drug assay. Pearson = 0.47, linear slope P 0.0001. B. Box plots showing signal-to-noise ratio (SNR) and Z’-factor for all assays. C-F. Bland-Altman plots showing variations between IC50 values of every single technical replicate vs. average IC50 values for amodiaquine (C), artemisinin (D), chloroquine (E), and mefloquine (F). Horizontal lines indicate the imply distinction in IC50 values among replicates. Intra-class correlation coefficients (ICC, with corresponding 95 self-confidence intervals) are displayed on each graph. G-I. Comparison of ex vivo with in vitro IC50 values for artemisinin (G), chloroquine (H), and mefloquine (I), among culture-adapted monoclonal parasites collected in 2009.PMID:24516446 Mean in vitro IC50 values are plotted with error bars displaying the regular error of a minimum of two biological replicates. denotes the Pearson correlation coefficient.Van Tyne et al. Malaria Journal 2013, 12:441 http://www.malariajournal/content/12/1/Page five ofbinom). Lastly, associations involving IC50 values along with the occurrence of drug resistance-associated mutations in pfcrt and pfmdr1 have been assessed by means of the Wilcoxon rank-sum test.ResultsDAPI ex vivo assay validationThe usefulness on the DAPI-based ex vivo assay for monitoring drug sensitivity amongst the parasites circulating in Thi , Senegal was assessed by measuring the dynamic variety and reliability of the assay. This ex vivo assay may very well be made use of to accurately calculate anti-malarial 50-percent inhibitory concentrations (IC50 values) in direct patient samples, as evidenced by the dynamic range of the assay (Figure 1A and B), and by testing for reproducibility between te.
