Prevalence of pfhrp2 and pfhrp3 gene deletions in Puerto Lempira, Honduras

Abstract

Background: Recent studies have demonstrated the deletion of the histidine-rich protein 2 (PfHRP2) gene (pfhrp2) in field isolates of Plasmodium falciparum, which could result in false negative test results when PfHRP2-based rapid diagnostic tests (RDTs) are used for malaria diagnosis. Although primary diagnosis of malaria in Honduras is determined based on microscopy, RDTs may be useful in remote areas. In this study, it was investigated whether there are deletions of the pfhrp2, pfhrp3 and their respective flanking genes in 68 P. falciparum parasite isolates collected from the city of Puerto Lempira, Honduras. In addition, further investigation considered the possible correlation between parasite population structure and the distribution of these gene deletions by genotyping seven neutral microsatellites.

Methods: Sixty-eight samples used in this study, which were obtained from a previous chloroquine efficacy study, were utilized in the analysis. All samples were genotyped for pfhrp2, pfhrp3 and flanking genes by PCR. The samples were then genotyped for seven neutral microsatellites in order to determine the parasite population structure in Puerto Lempira at the time of sample collection.

Results: It was found that all samples were positive for pfhrp2 and its flanking genes on chromosome 8. However, only 50% of the samples were positive for pfhrp3 and its neighboring genes while the rest were either pfhrp3-negative only or had deleted a combination of pfhrp3 and its neighbouring genes on chromosome 13. Population structure analysis predicted that there are at least two distinct parasite population clusters in this sample population. It was also determined that a greater proportion of parasites with pfhrp3-(and flanking gene) deletions belonged to one cluster compared to the other.

Conclusion: The findings indicate that the P. falciparum parasite population in the municipality of Puerto Lempira maintains the pfhrp2 gene and that PfHRP2-based RDTs could be considered for use in this region; however continued monitoring of parasite population will be useful to detect any parasites with deletions of pfhrp2.

Figure 1

Schematic of (A) Pfhrp2 and (B) Pfhrp3 and their respective flanking genes. In PlasmoDB version 8.0, from which gene information was obtained, pfhrp2 was located on chromosome 7 although in a recent update of PlasmoDB this gene and its flanking genes were reassigned to Chromosome 8. Pfhrp3 was reported to be immediately flanked by the indicated genes, all of which are on Chromosome 13. Arrows indicate the gene fragments that were amplified.

Figure 2

Prevalence of deletions in pfhrp3 and neighboring genes in P. falciparum isolates from Puerto Lempira, Nicaragua. The map shows the location of Honduras in relation to neighboring countries in Central America. All parasite isolates analysed were found to be positive for pfhrp2 and its flanking genes (not shown). The three pie charts shown illustrate the proportion of parasite isolates with deletions in pfhrp3 and its neighboring genes. The percentages shown represent proportions of samples out of the total samples that were 18S rRNA- and msp2-positive.

Figure 3

Bayesian cluster analysis of P. falciparum singly-infected samples collected from Puerto Lempira, Honduras (N = 65). The predicted number of likely clusters (K) for the samples was K = 2. Each color corresponds to a population cluster as classified by Structure v2.3.3, and each individual isolate is represented by a vertical bar. The Y axis represents the estimated proportion of membership of an individual to each population.