Validation of microsatellite markers for use in genotyping polyclonal Plasmodium falciparum infections

Abstract

Genotyping methods for Plasmodium falciparum drug efficacy trials have not been standardized and may fail to accurately distinguish recrudescence from new infection, especially in high transmission areas where polyclonal infections are common. We developed a simple method for genotyping using previously identified microsatellites and capillary electrophoresis, validated this method using mixtures of laboratory clones, and applied the method to field samples. Two microsatellite markers produced accurate results for single-clone but not polyclonal samples. Four other microsatellite markers were as sensitive as, and more specific than, commonly used genotyping techniques based on merozoite surface proteins 1 and 2. When applied to samples from 15 patients in Burkina Faso with recurrent parasitemia after treatment with sulphadoxine-pyrimethamine, the addition of these four microsatellite markers to msp1 and msp2 genotyping resulted in a reclassification of outcomes that strengthened the association between dhfr 59R, an anti-folate resistance mutation, and recrudescence (P = 0.31 versus P = 0.03). Four microsatellite markers performed well on polyclonal samples and may provide a valuable addition to genotyping for clinical drug efficacy studies in high transmission areas.

Figure 1

Examples of missed alleles for msp1 and a microsatellite marker from a sample containing all six clones at the following parasite densities (parasites/μL): 3D7 (2,500), FCR3 (2,500), HB3 (2,500), K1 (10,000), W2 (2,500), V1/S (30,000). (A) Agarose gel images of K1- and MAD20-type family-specific PCR products for msp1. An arrow indicates the expected position of the missed 3D7 allele. (B) Electropherogram for microsatellite TA81. An arrow indicates the expected position of the missed HB3 allele. The stutter peaks were correctly filtered by the allele detection algorithm.

Figure 2

Examples of false positive alleles for msp2 and a microsatellite marker from a sample containing three clones at the following parasite densities (parasites/μL): HB3 (35,000), W2 (10,000), V1/S (5,000). (A) Agarose gel images of IC3D7- and FC27-type family-specific PCR products for msp2. (B) Electropherogram for microsatellite PfPK2. The stutter peaks were correctly filtered by the allele detection algorithm. Arrows in both A and B indicate PCR products identified as false-positive alleles that did not correspond to any clones present in the sample.