The online version of this article (doi:10.1186/1475-2875-11-35) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interests.
DAM designed the study, performed the experiments and statistical analysis, and wrote the manuscript. JV, JM, NCM, KGB, DMR performed the experiments. CV performed the statistical analysis. RFD, SKV, DEN, DJP, SFS, AKL, DVT, RCW, and PCS designed, developed, and validated the barcoding assay. KBS, SJG, SK, MEM, and TET provided clinical care and data collection for all patients. DFW designed the study and edited the manuscript. All authors read and approved the final manuscript.
Cerebral malaria, a severe form of Plasmodium falciparum infection, is an important cause of mortality in sub-Saharan African children. A Taqman 24 Single Nucleotide Polymorphisms (SNP) molecular barcode assay was developed for use in laboratory parasites which estimates genotype number and identifies the predominant genotype.
The 24 SNP assay was used to determine predominant genotypes in blood and tissues from autopsy and clinical patients with cerebral malaria.
Single genotypes were shared between the peripheral blood, the brain, and other tissues of cerebral malaria patients, while malaria-infected patients who died of non-malarial causes had mixed genetic signatures in tissues examined. Children with retinopathy-positive cerebral malaria had significantly less complex infections than those without retinopathy (OR = 3.7, 95% CI [1.51-9.10]).The complexity of infections significantly decreased over the malaria season in retinopathy-positive patients compared to retinopathy-negative patients.
Cerebral malaria patients harbour a single or small set of predominant parasites; patients with incidental parasitaemia sustain infections involving diverse genotypes. Limited diversity in the peripheral blood of cerebral malaria patients and correlation with tissues supports peripheral blood samples as appropriate for genome-wide association studies of parasite determinants of pathogenicity.
Additional file 1: Comparison of the number of msp-1 and - msp- 2 alleles found in peripheral blood and tissues of patients with different diagnosis with the number of heterozygous calls found using the molecular barcode demonstrates low complexity in cerebral malaria. Statistical comparison was made by ANOVA. Comparison of the average number of heterozygous calls found using the molecular barcode in the clinical peripheral blood samples shows a significant difference between retinopathy positive and retinopathy negative. The slightly higher average value of the retinopathy positives is due to the inclusion of patients who may be CM and SMA. Statistical comparison was made by t-test of means. (XLS 20 KB)12936_2011_2001_MOESM1_ESM.XLS
Additional file 2: Table S2. The barcodes for 112 consecutive patients with positive peripheral blood parasitaemia and sufficient DNA for performance of the molecular barcode are shown. The order of the patients displayed in the table is by study number and not by date (see Figure 4). (XLS 66 KB)
Additional file 3: The complete molecular barcodes (24 TaqMan SNP Assay) for 19 patients who had pathological evidence of cerebral malaria at autopsy. Peripheral blood DNA at the time of admission was available from five CM patients and three non-CM patients and generally showed the same genotype as tissues, with more variation in the CM + SMA patients and non-CM patients. Sample types include peripheral blood (PB), frontal lobe (FB), cerebellum (CB), brainstem (BS), heart (H), lung (L), and colon (C). The major alleles are presented in a white background and the minor alleles are presented with a grey background. The frequency of major and minor alleles for the population of Malawi parasites examined in this study was significantly different from those seen in the global population (Table 2). For a heterozygous call, the IUPAC code is used and BOTH bases denoted by the code are present (orange/light-grey background). Failed reactions are shown as "X". See Additional File 4 for IUPAC codes used in this context. (XLS 76 KB)
Additional file 4: Descriptions of the assays used in this study are shown including the derived major and minor allele frequencies for the Malawi data set as well as codes for heterozygous allele calls (both alleles present) in the standard IUPAC format. For a complete list of chromosome positions, primer sequences, and probe sequences, please see Additional File 3 in previous publication ([ https://www.biomedcentral.com/content/supplementary/1475-2875-7-223-S3.pdf]). (XLS 28 KB)
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- Human cerebral malaria and Plasmodium falciparum genotypes in Malawi
Danny A Milner Jr
Rachel F Daniels
Sarah K Volkman
Daniel E Neafsey
Daniel J Park
Stephen F Schaffner
Nira C Mahesh
Kayla G Barnes
David M Rosen
Amanda K Lukens
Daria Van Tyne
Roger C Wiegand
Pardis C Sabeti
Karl B Seydel
Simon J Glover
Malcolm E Molyneux
Terrie E Taylor
Dyann F Wirth
- BioMed Central
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