Open Access Highly Accessed Research article

Artemisinin resistance in Plasmodium falciparum is associated with an altered temporal pattern of transcription

Sachel Mok1, Mallika Imwong23, Margaret J Mackinnon4, Joan Sim1, Ramya Ramadoss1, Poravuth Yi9, Mayfong Mayxay56, Kesinee Chotivanich2, Kek-Yee Liong1, Bruce Russell7, Duong Socheat9, Paul N Newton58, Nicholas PJ Day38, Nicholas J White38, Peter R Preiser1, François Nosten108, Arjen M Dondorp38* and Zbynek Bozdech1*

Author Affiliations

1 School of Biological Sciences, Nanyang Technological University, Singapore

2 Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, Thailand

3 Mahidol-Oxford Research Unit, Faculty of Tropical Medicine, Mahidol University, Thailand

4 KEMRI-Wellcome Trust Research Programme, Kilifi, Kenya

5 Wellcome Trust-Mahosot Hospital-Oxford University Tropical Medicine Research Collaboration, Mahosot Hospital, Vientiane, Lao People's Democratic Republic

6 Faculty of Postgraduate Studies and Research, University of Health Sciences, Vientiane, Lao People's Democratic Republic

7 Singapore Immunology Network, Biopolis, Agency for Science Technology and Research (ASTAR), Singapore

8 Centre for Clinical Vaccinology and Tropical Medicine, Churchill Hospital, Oxford, UK

9 The National Center for Parasitology, Entomology, and Malaria Control, Phnom Penh, Cambodia

10 Shoklo Malaria Research Unit, Mae Sot, Thailand

For all author emails, please log on.

BMC Genomics 2011, 12:391  doi:10.1186/1471-2164-12-391

Published: 3 August 2011

Additional files

Additional file 1:

Parasite density over course of treatment and treatment outcomes. Plot of the parasite densities (relative to starting parasite numbers on admission) from the time of patient's 1st admission date up to 100 hours after admission. Of the 4 isolates from Pailin, Cambodia, only 3 (CP025 (red), CP037 (green), CP040 (blue)) display significant delayed parasite clearance time (pct) of 78 and 96 hours from the patients after treatment while CP022 (purple) parasites was cleared earlier at 54 hours after treatment. The initial parasite numbers upon admission (para0) and parasite reduction ratio at 24 hours (prr24) and 48 hours (prr48) are included in the table. Artesunate and mefloquine treatment regimes administered to the patients are listed (box).

Format: PDF Size: 839KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 2:

Information about the sampling times, parasitemia of the ring, trophozoite and schizont stages of the 11 isolates. na: information not available.

Format: XLS Size: 29KB Download file

This file can be viewed with: Microsoft Excel Viewer

Open Data

Additional file 3:

Ex-vivo transcriptomes generated of the 11 P. falciparum field isolates from South East Asia. Transcriptomes of the ex-vivo IDC of all 11 field isolates from the 3 geographical locations measured over 48 hour sampling time. Only genes with at least 80% of time points with a positive signal were included for each of these transcriptomes. The phaseograms were constructed by ordering the mean-centered log2 microarray expression ratios to the genes ordered by phase calculated from the Fast Fourier Transformation of the Dd2 reference in vitro lifecycle.

Format: PDF Size: 773KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 4:

Distribution of Pearson correlations between all isolates for the 3 stages. Including isolate time points that correspond to the 3 stages - 14 hpi, 26 hpi and 34 hpi - graphs shown are average Pearson Correlations calculated from multiple pair wise comparisons between all isolates (graphs on left panel) and the average of the PCC for the resistant and sensitive parasites (graphs on right panel) at 14 hpi (a), at 26 hpi (b) and at 34 hpi (c). Error bars represent the standard deviation of all the pair-wise comparisons.

Format: PDF Size: 444KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 5:

Sheets 1-3 lists log2 expression data of all genes for the isolates grouped by ring (14 hpi), trophozoite (26 hpi) and schizont (34 hpi) stage and their corresponding z-scores (signal to noise) and p-values using student t-distribution. Genes with significant difference in expression between the resistant and sensitive isolates (p-value < 0.01) are highlighted in orange. Sheet "rank product score" lists all genes including putative transcriptional regulators (including those with zinc finger, bromodomain, etc., domains predicted by PFAM, Histone/HDAC/HAT-related, AP2-domain containing and potential cell cycle regulators) used in the analysis and their corresponding z-scores and rank product scores for ring (14 hpi), trophozoite (26 hpi) and schizont (34 hpi) stages.

Format: XLS Size: 4MB Download file

This file can be viewed with: Microsoft Excel Viewer

Open Data

Additional file 6:

Functional analyses of differential expression in all stages of the artemisinin resistant parasites. For each of the 3 stages, genes were ranked according to the z-score by correlating the expression profiles to the phenotypic class. The mean-centered log2 ratios for each gene of the resistant (CP025, CP037, and CP040) and sensitive (CP022, BMT061, BMT076, BMT077, XPN003, NHP2094, NHP4459, NHP4460) isolates are represented in these clusters. Gene Set Enrichment Analysis [24] of the ranked clusters gave rise to gene sets down-regulated in rings and trophozoites and up-regulated in schizonts in the resistant parasites as shown ordered by the nominal p-value, false discovery rate (FDR) q-value and Normalized Enrichment Score (NES). Significant gene sets were based on cut-off p-value of 0.05 and FDR q-value of 0.25.

Format: PDF Size: 1.4MB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 7:

Functional pathways with significant differential expression in rings, trophozoites and schizonts of artemisinin resistant and sensitive parasites. Gene sets (functional groups) which are denoted in the graphs are obtained from various data sets of previous studies [63,66,70] and plotted are all pathways that are significantly differentially expressed in artemisinin resistant parasites in at least one IDC stage. For each pathway, each data point at ring (14 hpi), trophozoite (26 hpi) and schizont (34 hpi) of the resistant (green triangle) and sensitive (blue diamond) series was calculated from taking the average of the expression log2 ratio of a gene across all isolates in that phenotypic group (resistant or sensitive) and then averaged for all the genes associated with that pathway. Best fit polynomial curves were plotted (lines). Error bars indicate the standard deviation among the isolates for that pathway. Included separately are the average expression ratios of all genes belonging to that pathway for CP022 isolate (purple circle) from Pailin, Cambodia and reference in vitro strain (red square).

Format: PDF Size: 8.8MB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 8:

Relative expression of individual genes associated with four functional pathways that have significant differential expression in artemisinin resistant parasite. Top: Example of four functional gene sets with significant differential expression between resistant and sensitive parasites and the relative gene expression of all members of the gene sets at the three stages. Each set of data points are the average log2 expression ratios of the isolates in a group and averaged for all the genes in that pathway at 14, 26 and 34 hpi. The curves are the best fit polynomial curves to the data points. Bottom: Each of the 3 graphs plotted depict the average log2 gene expression ratios of the isolates in the resistant (green triangle) or sensitive (blue diamond) group with the standard deviation represented by error bars in each particular pathway at14, 26 or 34 hpi.

Format: PDF Size: 2.5MB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 9:

Transcriptional profiles of the 3 genes in the artemisinin resistant parasites compared with previous data published [18]. The mean expression log2 ratio (data point) and SD (error bars) among the resistant (green triangle) and sensitive (blue diamond) isolates for each stage are plotted for the genes: PF10_0121 - hypoxanthine phosphoribosyltransferase (rank 3207/4029; p-value = 0.1), PF08_0054 - Heat Shock Protein 70 kDa (rank 14/4041; p-value = 0.0004) and PFE1415w- cell cycle regulator (rank 3837/4041; p-value = 0.01). The polynomial represents the best fit curve through the data points. The arrow indicates the approximate stage in which significant increased or lowered expression was observed in artesunate-tolerant parasites and the fold change [18] (Witkowski et al., 2010).

Format: PDF Size: 783KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 10:

Functional analysis and clustering based on general differences in gene expression among field isolates without phenotypic classification. (a) Clusters are represented by the log2 expression ratios for all genes ordered according to the standard deviation (SD) for each gene in a descending manner. GSEA [24] performed on this pre-ranked list of genes identified these functional gene sets as differentially expressed among field isolates without any phenotypic classification for the 3 stages. (b) Hierarchically clustered isolates for the genes showing greatest variation in expression ratios (taking genes with SD value at the 95th percentile cut off). Each color denotes the location: Laos, Mae Sot or Pailin that the isolates originate from. The raw data reported in this paper has been deposited in the NCBI's Gene Expression Omnibus database [71] and are accessible through GEO Series accession number GSE25883. http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE25883 webcite

Format: PDF Size: 1.3MB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data