Dados do Trabalho

Título

Different molecular methods produce distinct and conflicting results when analyzing deletions in the Plasmodium falciparum pfhrp2 and pfhrp3 genes

Introdução

Malaria, especially Plasmodium falciparum, is a diagnostic challenge in remote tropical areas. Rapid diagnostic tests (RDT) detecting HRP2 and HRP3 proteins are widely used, but their effectiveness is threatened by deletions in pfhrp2, which affects RDT’s efficiency. Molecular methods such as qPCR are more accurate and sensitive, detecting low parasite densities. Several qPCR protocols are published, targeting different genomic regions and thus exhibiting different efficiencies, perhaps due to deletions. The variability of these deletions might have a negative impact in the use of RDT for malaria diagnosis, severely impacting the efforts to control the disease.

Objetivo (s)

To analyze different molecular (qPCR) protocols for the evaluation of deletions of pfhrp2 and pfhrp3 in P. falciparum positive samples from Rondônia. 

Material e Métodos

This study, carried out in Rondônia, was approved by CEP CEPEM-RO (CAAE: 52853021.6.0000.0011 - Group 1; CAAE: 29217820.1.0000.0011 - Group 2). Samples from 63 patients (51 group 1 and 12 group 2) positive for Plasmodium falciparum (qPCR 18S rRNA) were analyzed. For detection of deletions of the pfhrp2 and pfhrp3, we evaluated three different qPCR protocols (P1 - Grignard et al, 2020; P2 - Kreidenweiss et al, 2019; P3 - Schindler et al, 2019). Kappa coefficient and Spearman correlation were used to analyze the results. 

Resultados e Conclusão

The qPCR protocols used to identify deletions in pfhrp2 and pfhrp3 showed excellent efficiency in detecting low-parasitemia samples. Reaction efficiencies were similar to original studies. Analysis of clinical samples revealed deletions in pfhrp2 in 9.5% of the samples, with important variation among protocols: 7.9% (P1), 0% (P2) and 9.5% (P3). Spearman correlation among P1 and P3 was ρ = 0.969 with a Kappa coefficient of 0.9, indicating strong agreement. For pfhrp3, deletions were found in 95.2% of the samples, with important variation between protocols: 71.4% (P3), 3.1% (P1) and 12.7% (P2 and P3). The correlation between P1 and P2 was ρ = 0.923, with a Kappa coefficient of 0.9, indicating strong agreement. These results indicate significant disagreement among qPCR protocols and highlight the need to harmonize methods for detecting pfhrp2/3 genes, considering the genetic diversity of P. falciparum, the presence of mixed infections, the prevalence of pfhrp3 gene deletions, and the correlation between transmission intensity and genetic diversity.

Palavras Chave

Plasmodium falciparum; pfhrp2; pfhrp3; qPCR; deletions; malária