Phenotypic and genotypic characterization of Plasmodium falciparum field isolates lacking pfhrp2 gene

The Plasmodium falciparum phenotypic characteristics (cytoadherence and rosetting), and the concentration of Histidine Rich Protein 2 (HRP2) in serum are associated with severe malaria in regions like Africa and Asia. Also, HRP2 has recently been associated as a P. falciparum virulence factor. Parasites lacking pfhrp2 gene were first reported in the Peruvian Amazon, however, there are not yet reports of the study of their phenotypes. For this research we decided to characterize the phenotype and genotype of P. falciparum field isolates from the Peruvian Amazon with different pfhrp2 patterns (positive or negative) cultivated in vitro.

P. falciparum field isolates (n = 5) from the Peruvian Amazon region were isolated and cultured in vitro. Three isolates had a positive result or pfhrp2 positive pattern and two of them were negatives (pfhrp2 negative pattern) by HRP2 Rapid diagnostic test. Then, these results were confirmed by conventional PCR. The following phenotypic characterization assays were tested: (i) Cytoadherence capacity (evaluated against Chondroitin Sulfate A (CSA) protein and Human umbilical vein endothelial cells (HUVEC cells)), and (ii) rosetting formation. Genotypic characterization assays were carried out by PCR (gene detection) and qPCR assays for pfhrp2 and pfhrp3 gene expression. Additionally, we evaluate the pfhrp2/3 genes and flanking genes stability using long-term cultures of these field isolates during a year.

Two of the three isolates with pfhrp2 positive pattern showed rosetting formation (R + , CS-, Hu-) and cytoadherence to CSA protein (R-, CS + , Hu-), on the other hand the two field isolates with pfhrp2 negative pattern showed adhesion to HUVEC cells (R-, CS-, Hu +). 3D7 strain was used for normalization during qPCR assays. We identified that pfhrp2 gene expression levels were higher in the field isolates with pfhrp2 positive pattern, while pfhrp3 gene expression levels were similar or lower. All cultured field isolates showed genomic stability of pfhrp2 gene and flanking genes during the long-term in vitro culture. However, one field isolate showed two pfhrp3 patterns during the culture due to the presence of two parasite genotypes populations, as corroborated by microsatellite markers.

P. falciparum Peruvian field isolates with pfhrp2 positive pattern showed cytoadherence to Chondroitin sulfate A protein (R-, CS + , Hu-) or positive characteristics for rosetting (R + , CS-, Hu-); pfhrp2 negative parasites showed cytoadherence to HUVEC cells (R-, CS-, Hu +). In this small set of Peruvian field isolates, a clear cytoadherence and resolution profile was observed for the different pfhrp2 patterns. These findings should be interpreted as preliminary and require further verification in larger isolated panels.