Abstract
BackgroundCurrent 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.MethodsPerformance 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.ResultsA 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.ConclusionsThe 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.
Highlights
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
The Chikhwawa district in southern Malawi has an estimated inoculation rate of 183 infective bites per person per year [1]. In such high transmission settings, it is imperative to determine whether infections occurring during the course of therapeutic drug trials are due to a recurrence of the initial infection or a new infection
Study area and population To compare the barcode method versus msp genotyping, this study utilized 66 paired samples from cases of recurrent malaria infection during two therapeutic efficacy studies conducted among children age 6 to 59 months in Malawi between July 2011 and November 2012 at Machinga District Hospital and between March and July of 2014 at Machinga, Nkhotakota, and Karonga District Hospitals [7]
Summary
Current World Health Organization guidelines for conducting anti-malarial drug efficacy clinical trials recommend genotyping Plasmodium falciparum genes msp and msp to distinguish recrudescence from reinfection. The Chikhwawa district in southern Malawi has an estimated inoculation rate of 183 infective bites per person per year [1] In such high transmission settings, it is imperative to determine whether infections occurring during the course of therapeutic drug trials are due to a recurrence of the initial infection (recrudescence) or a new infection (reinfection). This is a critical distinction, as recrudescence signifies resistance of the infection to the anti-malarial regimen, while a reinfection merely reflects a high transmission setting To distinguish between these two possibilities, each infection is characterized or “fingerprinted” using molecular genotyping of highly polymorphic alleles. On the other hand, the molecular fingerprints at the two-time points differ
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