Exposing malaria in-host diversity and estimating population diversity by capture-recapture using massively parallel pyrosequencing

Abstract

Malaria infections commonly contain multiple genetically distinct variants. Mathematical and animal models suggest that interactions among these variants have a profound impact on the emergence of drug resistance. However, methods currently used for quantifying parasite diversity in individual infections are insensitive to low-abundance variants and are not quantitative for variant population sizes. To more completely describe the in-host complexity and ecology of malaria infections, we used massively parallel pyrosequencing to characterize malaria parasite diversity in the infections of a group of patients. By individually sequencing single strands of DNA in a complex mixture, this technique can quantify uncommon variants in mixed infections. The in-host diversity revealed by this method far exceeded that described by currently recommended genotyping methods, with as many as sixfold more variants per infection. In addition, in paired pre- and posttreatment samples, we show a complex milieu of parasites, including variants likely up-selected and down-selected by drug therapy. As with all surveys of diversity, sampling limitations prevent full discovery and differences in sampling effort can confound comparisons among samples, hosts, and populations. Here, we used ecological approaches of species accumulation curves and capture-recapture to estimate the number of variants we failed to detect in the population, and show that these methods enable comparisons of diversity before and after treatment, as well as between malaria populations. The combination of ecological statistics and massively parallel pyrosequencing provides a powerful tool for studying the evolution of drug resistance and the in-host ecology of malaria infections.

Fig. 1.

msp2 genotype discovery curves. These figures show genotype discovery curves and rarefaction curves for several patient samples. The genotype discovery curves are generated by plotting the number of unique sequence variants in a sample (y axis) against the rank order of sequences reads generated (x axis). Therefore, the x axis illustrates the cumulative number of sequences determined for a sample at a given site (number of sequence reads or fold coverage of the site), which are ordered by the sequential well number of the 454 picotiter plate, which reflects increasing sampling effort. (A) Number of msp2 variants discovered with increasing sequencing effort in six samples. Patient codes and sample numbers cross-reference with those in Table 1. As expected, the number of variants discovered is a function of sequencing effort, but for a given effort, some samples contain more variants than others. For six samples, expected variant richness (rarefaction), curves (thick lines), and 95% CI (thin lines) for (B) patient 1/014 before and after drug treatment [light blue (Upper) and brown (Lower) lines, respectively; samples 1 and 3], (C) patient 1/009 before and after drug treatment (pink and dark blue lines, respectively; samples 1 and 2), and (D) msp2 diversity in two different patients [2/041 I and 2/044 I, black (Upper) and red (Lower) lines, respectively].

Fig. 2.

msp2 variant dynamics between longitudinal patient samples from Malawi. (A) Change in variant population in a pre- and posttreatment sample pair (1/009I and 1/009 II). The green and blue circles represent unique variants detected in the initial parasitemia and recurrent parasitemias, respectively. The brown, orange, yellow, and red circles represent population sizes of variants that are shared between the two samples. (B) Genotypes of three infections in patient 1/014. In each case, treatment appears to have cleared all of the genotypes as none of the samples contained a shared genotype within the same patient. However, all of the samples did contain a genotype identified in other patients in the study. Of note, there was a significant reduction in the complexity of infection between the first and second infection with sulfadoxine–pyrimethamine treatment.