Plasmodium falciparum: underestimation of dihydrofolate reductase and dihydropteroate synthase polymorphism in field samples: a technical shortcoming of nested PCR assays with mutation-specific primers

Abstract

n a study parallel to the present one, we conducted a genotype characterization of Plasmodium falciparum based on isolates of patients infected with malaria, who come from a small location in Colombia. The analysis involved extraction of DNA from hematological smears, amplification of the dihydrofolate reductase gene and the dihydropteroate synthase (DHPS) gene for each sample by PCR, and detection through mutation-specific primers nested PCR of mutations associated with resistance to pyrimethamine and sulfadoxine. Given the difficulty in quantifying the DNA extracts due to the type of sample and its heterogeneity, different volumes of the product of the first PCR were tested as template for the nested PCR. Surprisingly, for some samples, we found contradictory results between determinations, which differed only in the amount of template used. This prompted a more general concern that in a natural isolate, where the parasite population is heterogeneous, the nested PCR with mutation-specific primers technique can produce erroneous results that underestimate the complexity of the sample. To test this hypothesis, we designed experiments in this study using position 581 of the DHPS gene as an indicator system and constructed samples simulating the heterogeneity of natural samples. In effect, our data show that the results obtained in the nested PCR can be altered by the amount of template used in the reaction and, therefore, some heterogeneous samples might be classified mistakenly as homogeneous, simple mutant or simple wild type. These observations may explain, at least in part, contradictory results found in the literature. Our data also suggest the need for a more cautious approach to interpretation of the results of nested PCR assays with mutation-specific primers and their implications in the definition of resistance to the pyrimethamine-sulfadoxine combination.