Multiple genetic origins of histidine-rich protein 2 gene deletion in Plasmodium falciparum parasites from Peru

Abstract

The majority of malaria rapid diagnostic tests (RDTs) detect Plasmodium falciparum histidine-rich protein 2 (PfHRP2), encoded by the pfhrp2 gene. Recently, P. falciparum isolates from Peru were found to lack pfhrp2 leading to false-negative RDT results. We hypothesized that pfhrp2-deleted parasites in Peru derived from a single genetic event. We evaluated the parasite population structure and pfhrp2 haplotype of samples collected between 1998 and 2005 using seven neutral and seven chromosome 8 microsatellite markers, respectively. Five distinct pfhrp2 haplotypes, corresponding to five neutral microsatellite-based clonal lineages, were detected in 1998-2001; pfhrp2 deletions occurred within four haplotypes. In 2003-2005, outcrossing among the parasite lineages resulted in eight population clusters that inherited the five pfhrp2 haplotypes seen previously and a new haplotype; pfhrp2 deletions occurred within four of these haplotypes. These findings indicate that the genetic origin of pfhrp2 deletion in Peru was not a single event, but likely occurred multiple times.

Figure 1. Location of pfhrp2 and its flanking genes.

Pfhrp2 is located subtelomerically (position 1,374,236 to 1,375,299) on chromosome 8. The seven microsatellites flanking pfhrp2 are shown in boxes; each value indicates the distance (in kilobases) of a microsatellite locus either upstream (negative values) or downstream (positive values) of the start and stop codons of pfhrp2, respectively. The two genes flanking pfhrp2 are shown and their old gene IDs (Mal7p1.xxx) are included below the current PlasmoDB designations. Gene location and information were obtained from PlasmoDB v.9.1 (http://plasmodb.org/plasmo/).

Figure 2. Bayesian cluster analysis of Iquitos samples.

The predicted number of likely clusters (K) for (a) samples collected in 1998–2001 (N = 92) was K = 5 while for (b) samples collected in 2003–2005 (N = 96) was K = 8. Each color corresponds to a population classified by Structure v 2.3.3 and each individual isolate is represented by a vertical bar. The Y axis represents the estimated proportion of membership of an individual to each predicted population cluster.

Figure 3. Median joining network analysis of P. falciparum samples collected in Iquitos in (a) 1998–2001 and (b) 2003–2005.

The genetic relationships among parasites were constructed using the seven neutral microsatellite loci shown on Table 2. The distinct lineages are rendered in different colors and circle sizes are proportional to the number of samples assigned to a particular lineage. The number of samples assigned to each lineage by Structure analysis are shown in parentheses.

Figure 4. Prevalence of pfhrp2 among the clonal lineages identified in Iquitos.

Clinical samples were collected in 1998–2001 (N = 92) and 2003–2005 (N = 96). Dark grey boxes represent the proportion of pfhrp2-positive samples while light grey boxes represent pfhrp2-negative isolates. The clonal lineage assignments are indicated along the x-axis.

Figure 5. Prevalence of pfhrp2 deletion among the P. falciparum haplotypes found in Peru.

The genetic relationships among parasite isolates collected in (a) 1998–2001 and (b) 2003–2005 were constructed using the seven neutral microsatellite loci shown on Table 2. Blue color represents the proportion of pfhrp2-positive samples while yellow indicates the proportion of pfhrp2-negative samples. Additionally, the pfhrp2 microsatellite-based haplotypes are labeled and highlighted. (b) Circle circumferences are proportional to the number of samples that belonged to a particular haplotype.