ABSTRACT
BACKGROUND: Current World Health Organization guidelines for conducting anti-malarial drug efficacy clinical trials recommend genotyping Plasmodium falciparum genes msp1 and msp2 to distinguish recrudescence from reinfection. A more recently developed potential alternative to this method is a molecular genotyping assay based on a panel of 24 single nucleotide polymorphism (SNP) markers. METHODS: Performance parameters of these two genotyping methods were compared using data from two recently completed drug efficacy trials. Blood samples from two anti-malarial therapeutic trials were analysed by both msp genotyping and the 24 SNP assay. Additionally, to conserve time and resources, the statistical program R was used to select the most informative SNPs for a set of unrelated Malawian samples to develop a truncated SNP-based assay for the region surrounding Blantyre, Malawi. The ability of this truncated assay to distinguish reinfection from recrudescence when compared to the full 24 SNP assay was then analysed using data from the therapeutic trials. RESULTS: A total of 360 samples were analysed; 66 for concordance of msp and SNP barcoding methodologies, and 294 for assessing the most informative of the 24 SNP markers. SNP genotyping performed comparably to msp genotyping, with only one case of disagreement among the 50 interpretable results, where the SNP assay identified the sample as reinfection and the msp typing as recrudescence. Furthermore, SNP typing was more robust; only 6% of samples were uninterpretable by SNP typing, compared to 19.7% when msp genotyping was used. For discriminating reinfection from recrudescence, a truncated 6 SNP assay was found to perform at 95.1% the accuracy of the full 24 SNP bar code. CONCLUSIONS: The use of SNP analysis has similar sensitivity to the standard msp genotyping in determining recrudescence from reinfection. Although more expensive, SNP typing is faster and less work intensive. Limiting the assay to those SNPs most informative in the geographical region of interest may further decrease the workload and the cost, making this technique a feasible and affordable alternative in drug efficacy trials.
Subject(s)
Antigens, Protozoan/genetics , Genotyping Techniques/methods , Malaria, Falciparum/diagnosis , Malaria, Falciparum/parasitology , Merozoite Surface Protein 1/genetics , Plasmodium falciparum/classification , Polymorphism, Single Nucleotide , Protozoan Proteins/genetics , Child, Preschool , Clinical Trials as Topic , Female , Genotype , Humans , Infant , Malawi , Male , Plasmodium falciparum/genetics , Plasmodium falciparum/isolation & purification , Recurrence , Sensitivity and Specificity , Time FactorsABSTRACT
Genotyping of allelic variants of Plasmodium falciparum merozoite surface proteins 1 and 2 (msp-1 and msp-2), and the glutamate-rich protein is the gold standard for distinguishing reinfections from recrudescences in antimalarial drug trials. We compared performance of the recently developed 24-single-nucleotide polymorphism (SNP) Barcoding Assay against msp-1 and msp-2 genotyping in a cluster-randomized effectiveness trial of artemether-lumefantrine and dihydroartemisinin-piperaquine in Malawi. Rates of recrudescence and reinfection estimated by the two methods did not differ significantly (Fisher's exact test; P = 0.887 and P = 0.768, respectively). There was a strong agreement between the two methods in predicting treatment outcomes and resolving the genetic complexity of malaria infections in this setting. These results support the use of this SNP assay as an alternative method for correcting antimalarial efficacy/effectiveness data.
Subject(s)
Antigens, Protozoan/genetics , Antimalarials/therapeutic use , Malaria, Falciparum/drug therapy , Merozoite Surface Protein 1/genetics , Merozoites/genetics , Plasmodium falciparum/drug effects , Protozoan Proteins/genetics , Artemether, Lumefantrine Drug Combination/therapeutic use , Artemisinins/therapeutic use , Child , Cluster Analysis , Drug Combinations , Female , Gene Expression , Genotype , Genotyping Techniques , Humans , Malaria, Falciparum/diagnosis , Malaria, Falciparum/parasitology , Malawi , Male , Merozoites/drug effects , Merozoites/growth & development , Plasmodium falciparum/genetics , Plasmodium falciparum/growth & development , Polymorphism, Single Nucleotide , Quinolines/therapeutic use , Recurrence , Treatment OutcomeABSTRACT
With support from the Global Fund, the United States President's Malaria Initiative (PMI) and other cooperating partners, Malawi is implementing a comprehensive malaria control programme involving indoor residual spraying in targeted districts, universal coverage with insecticide-treated bed nets, use of rapid diagnostic tests to confirm the clinical diagnosis of malaria and use of the highly effective artemisinin-based combination therapy, artemether-lumefantrine (AL), as the first-line treatment for malaria. We genotyped 24 genome-wide single nucleotide polymorphisms (SNPs) in Plasmodium falciparum infections (n=316) sampled from a single location in Malawi before (2006 and 2007) and after enhanced intervention (2008 and 2012). The SNP data generated were used to examine temporal changes in the proportion of multiple-genotype infections (MIs), mean number of heterozygous SNPs within MIs, parasite genetic diversity (expected heterozygosity and genotypic richness), multilocus linkage disequilibrium and effective population size (N(e)). While the proportion of MIs, expected heterozygosity, genotypic richness, multilocus linkage disequilibrium and Ne were unchanged over time, the mean number (±standard deviation) of heterozygous SNPs within MIs decreased significantly (p=0.01) from 9(±1) in 2006 to 7(±1) in 2012. These findings indicate that the genetic diversity of P. falciparum malaria parasites in this area remains high, suggesting that only subtle gains, if any, have been made in reducing malaria transmission. Continued surveillance is required to evaluate the impact of malaria control interventions in this area and the rest of Malawi, and to better target control interventions.