Spatiotemporal dynamics of Plasmodium falciparum histidine-rich protein 2 and 3 deletions in Peru

Abstract

Peru was the first country where pfhrp2 and pfhrp3 gene deletions were detected despite the fact that rapid diagnostics tests are not commonly used for confirmatory malaria diagnosis. This context provides a unique scenario to study the dynamics of pfhrp2 and pfhrp3 gene deletions without apparent RDTs selection pressure. In this study we characterized the presence of pfhrp2 and pfhrp3 genes on 325 P. falciparum samples collected in Iquitos and surrounding communities between 2011 and 2018 in order to understand the dynamics of gene deletion prevalence, potential associations with clinical symptomatology and parasite genetic background. P. falciparum presence was confirmed by microscopy and PCR of 18 s rRNA, pfmsp1 and pfmsp2. Gene deletions were assessed by amplification of exon1 and exon2 of pfhrp2 and pfhrp3 using gene specific PCRs. Confirmation of absence of HRP2 expression was assessed by ELISA of HRP2 and pLDH. Genotyping of 254 samples were performed using a panel of seven neutral microsatellite markers. Overall, pfhrp2 and pfhrp3 dual gene deletions were detected in 67% (217/324) parasite samples. Concordance between pfhrp2 deletion and negligible HRP2 protein levels was observed (Cohen's Kappa = 0.842). Prevalence of gene deletions was heterogeneous across study sites (adjusted p < 0.005) but there is an overall tendency towards increase through time in the prevalence of dual pfhrp2/3-deleted parasites between 2011 (14.3%) and 2016 (88.39%) stabilizing around 65% in 2018. Dual deletions increase was associated with dominance of a single new parasite haplotype (H8) which rapidly spread to all study sites during the 8 study years. Interestingly, participants infected with dual pfhrp2/3-deleted parasites had a significantly lower parasitemias than those without gene deletions in this cohort. Our study showed the increase of pfhrp2/3 deletions in the absence of RDTs pressure and a clonal replacement of circulating lines in the Peruvian Amazon basin. These results suggest that other factors linked to the pfhrp2/3 deletion provide a selective advantage over non-deleted strains and highlight the need for additional studies and continuing surveillance.

Figure 1

Collection sites. Study sites located in the city of Iquitos and surrounding communities. Orange circles showed the different health centers where samples were collected. The map was created using open data obtained from GADM database of Global Administrative Areas, version 3.6. URL: https://www.gadm.org.

Figure 2

Flow chart illustrating study procedures.

Figure 3

Prevalence of pfhrp2 and pfhrp3 deletions in Iquitos and surrounding areas. The figure shows the prevalence of single, dual and non-deleted parasites for pfhrp2 and pfhrp3. Significant differences were found in the prevalence of Amazon Hope compared to Bellavista, Apoyo Hospital, Moranococha, Padrecocha and Santa Clara and for Apoyo Hospital against Padreocha according to pairwise Fisher’s exact test. Tupac and Puerto America were excluded from statistical analysis due to small sample numbers.

Figure 4

Comparison of HRP2 and pLDH protein levels in parasites with different pfhrp2 and pfhrp3 status. Solid dots: HRP2; circles: pLDH.

Figure 5

Temporal distribution of pfhrp2 and pfhrp3 deletions between 2011 and 2018. The figure shows a continuous increase the prevalence of the double pfhrp2/3 deletion across time.

Figure 6

Dynamics of parasite haplotypes between 2011 and 2018.

Figure 7

Genetic relatedness of parasites with different phrp2/3 status. (A) PHYLOViZ showing that that most dual deleted parasites grouped in a single cluster and (B) DAPC result showing that samples from this study collected between 2011 and 2018 do not cluster with Peruvian samples collected in the early 2000s. Brown circles are samples from a published study from Eritrea which were used as controls for the analysis.