Research Article: Histidine-rich protein 2 (pfhrp2) and pfhrp3 gene deletions in Plasmodium falciparum isolates from select sites in Brazil and Bolivia

Date Published: March 16, 2017

Publisher: Public Library of Science

Author(s): Giselle Maria Rachid Viana, Sheila Akinyi Okoth, Luciana Silva-Flannery, Danielle Regina Lima Barbosa, Alexandre Macedo de Oliveira, Ira F. Goldman, Lindsay C. Morton, Curtis Huber, Arletta Anez, Ricardo Luiz Dantas Machado, Luís Marcelo Aranha Camargo, Suiane Costa Negreiros do Valle, Marinete Marins Póvoa, Venkatachalam Udhayakumar, John W. Barnwell, Michelle Louise Gatton.


More than 80% of available malaria rapid diagnostic tests (RDTs) are based on the detection of histidine-rich protein-2 (PfHRP2) for diagnosis of Plasmodium falciparum malaria. Recent studies have shown the genes that code for this protein and its paralog, histidine-rich protein-3 (PfHRP3), are absent in parasites from the Peruvian Amazon Basin. Lack of PfHRP2 protein through deletion of the pfhrp2 gene leads to false-negative RDT results for P. falciparum. We have evaluated the extent of pfhrp2 and pfhrp3 gene deletions in a convenience sample of 198 isolates from six sites in three states across the Brazilian Amazon Basin (Acre, Rondonia and Para) and 25 isolates from two sites in Bolivia collected at different times between 2010 and 2012. Pfhrp2 and pfhrp3 gene and their flanking genes on chromosomes 7 and 13, respectively, were amplified from 198 blood specimens collected in Brazil. In Brazil, the isolates collected in Acre state, located in the western part of the Brazilian Amazon, had the highest percentage of deletions for pfhrp2 25 (31.2%) of 79, while among those collected in Rondonia, the prevalence of pfhrp2 gene deletion was only 3.3% (2 out of 60 patients). In isolates from Para state, all parasites were pfhrp2-positive. In contrast, we detected high proportions of isolates from all 3 states that were pfhrp3-negative ranging from 18.3% (11 out of 60 samples) to 50.9% (30 out of 59 samples). In Bolivia, only one of 25 samples (4%) tested had deleted pfhrp2 gene, while 68% (17 out of 25 samples) were pfhrp3-negative. Among the isolates tested, P. falciparum pfhrp2 gene deletions were present mainly in those from Acre State in the Brazilian Amazon. These results indicate it is important to reconsider the use of PfHRP2-based RDTs in the western region of the Brazilian Amazon and to implement appropriate surveillance systems to monitor pfhrp2 gene deletions in this and other parts of the Amazon region.

Partial Text

The number of reported malaria cases in Brazil has steadily decreased from 637,000 in 1999 to approximately 178,546 in 2013 [1, 2]. Most of the malaria cases in Brazil are caused by Plasmodium vivax (82%), are transmitted by Anopheles darlingi and occur in the Amazon region [1, 3]. The percentage of malaria infections due to P. falciparum has also declined somewhat in the past few years, from 21.5% of reported cases in 2009 to 18% in 2013 [3]. Bolivia, which borders Brazil, is similar in that the majority of malaria cases are due to P. vivax and only 7% are due to P. falciparum infection. Only a total of 7,342 malaria cases were reported in Bolivia in 2013 [4].

A total of 212 DNA and plasma specimens were collected from P. falciparum-infected patients in Brazil between 2010 and 2012 from Acre, Para, and Rondonia states. These were sent to the IEC and CDC malaria laboratories for genetic analyses, while quantitative ELISA of plasma for PfHRP2 concentrations was conducted only at CDC. A total of 198 specimens met the inclusion criteria for this study by yielding positive results for the amplification of both 18S rRNA and msp2 genes and were, therefore, considered valid for further molecular analysis. Seventy-nine (39.9%) P. falciparum samples were collected in Acre, 59 (29.8%) in Para and 60 (30.3%) in Rondonia.

The findings from this study confirm that P. falciparum parasite populations with deletions of the pfhrp2 and pfhrp3 genes are present in Brazil and Bolivia. The highest proportion of pfhrp2-negative isolates (31.2%) was found in Cruzeiro do Sul municipality in Acre state, Brazil. This relatively high percentage of isolates with pfhrp2-deleted parasites is comparable to what has been observed previously in the neighboring Peruvian Amazon River basin, where the prevalence of pfhrp2-negative parasites has been reported to be between 30 and 40% [16, 17]. The specimens collected in Monte Negro, Rondonia State, had a much lower percentage of isolates with deletion of the pfhrp2 gene (3.3%). The proportion of P. falciparum isolates with deletion of pfhrp2 gene was also low (4%) in the Department of Beni in Bolivia, which borders Rondonia. In Para state, Brazil, which is situated at the eastern end of the Amazon River basin, no parasites (out of 59 samples) were detected with pfhrp2 deletions. Although these data may seem to suggest that pfhrp2-negative parasites are confined to the western border regions of Brazil, our study was not designed to provide representative information about the distribution or prevalence of pfhrp2 and pfhrp3 genetic variants. Given that the three states we surveyed accounted for approximately 56% of the reported cases of P. falciparum in the Brazilian Amazon Basin in 2012 [Brazilian National Reportable Disease Information System for Malaria (SIVEP)], a more comprehensive and representative survey should be conducted to evaluate pfhrp2 gene deletion in these 3 states as well as Amazonas, Roraima, and Amapa.