A Comprehensive Analysis of the Genetic Diversity of Plasmodium falciparum Histidine-Rich Protein 2 (PfHRP2) in the Brazilian Amazon

Early diagnosis and treatment are fundamental to the control and elimination of malaria. In many endemic areas, routine diagnosis is primarily performed microscopically, although rapid diagnostic tests (RDTs) provide a useful point-of-care tool. Most of the commercially available RDTs detect histidine-rich protein 2 (HRP2) of Plasmodium falciparum in the blood of infected individuals. Nonetheless, parasite isolates lacking the pfhrp2 gene are relatively frequent in some endemic regions, thereby hampering the diagnosis of malaria using HRP2-based RDTs. To track the efficacy of RDTs in areas of the Brazilian Amazon, we assessed pfhrp2 deletions in 132 P. falciparum samples collected from four malaria-endemic states in Brazil. Our findings show low to moderate levels of pfhrp2 deletion in different regions of the Brazilian Amazon. Overall, during the period covered by this study (2002-2020), we found that 10% of the P. falciparum isolates were characterized by a pfhrp2 deletion. Notably, however, the presence of pfhrp2-negative isolates has not been translated into a reduction in RDT efficacy, which in part may be explained by the presence of polyclonal infections. A further important finding was the discrepancy in the proportion of pfhrp2 deletions detected using two assessed protocols (conventional PCR versus nested PCR), which reinforces the need to perform a carefully planned laboratory workflow to assess gene deletion. This is the first study to perform a comprehensive analysis of PfHRP2 sequence diversity in Brazilian isolates of P. falciparum. We identified 10 PfHRP2 sequence patterns, which were found to be exclusive of each of the assessed regions. Despite the small number of PfHRP2 sequences available from South America, we found that the PfHRP2 sequences identified in Brazil and neighboring French Guiana show similar sequence patterns. Our findings highlight the importance of continuously monitoring the occurrence and spread of parasites with pfrhp2 deletions, while also taking into account the limitations of PCR-based testing methods associated with accuracy and the complexity of infections.

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Evaluation of Histidine-Rich Proteins 2 and 3 Gene Deletions in Plasmodium falciparum in Endemic Areas of the Brazilian Amazon

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph18010123 ◽

2020 ◽

Vol 18 (1) ◽

pp. 123

Author(s):

Leandro Góes ◽

Nathália Chamma-Siqueira ◽

José Mário Peres ◽

José Maria Nascimento ◽

Suiane Valle ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Gene Deletion ◽

Brazilian Amazon ◽

Amazon Region ◽

The State ◽

Molecular Surveillance ◽

Gene Deletions ◽

Exon 2 ◽

Brazilian Amazon Region ◽

Endemic Areas

Histidine-rich proteins 2 and 3 gene (pfhrp2 and pfhrp3) deletions affect the efficacy of rapid diagnostic tests (RDTs) based on the histidine-rich protein 2 (HRP2), compromising the correct identification of the Plasmodium falciparum species. Therefore, molecular surveillance is necessary for the investigation of the actual prevalence of this phenomenon and the extent of the disappearance of these genes in these areas and other South American countries, thus guiding national malaria control programs on the appropriate use of RDTs. This study aimed to evaluate the pfhrp2 and pfhrp3 gene deletion in P. falciparum in endemic areas of the Brazilian Amazon. Aliquots of DNA from the biorepository of the Laboratory of Basic Research in Malaria, Evandro Chagas Institute, with a positive diagnosis for P. falciparum infection as determined by microscopy and molecular assays, were included. Monoinfection was confirmed by nested-polymerase chain reaction assay, and DNA quality was assessed by amplification of the merozoite surface protein-2 gene (msp2). The pfhrp2 and pfhrp3 genes were amplified using primers for the region between exons 1 and 2 and for all extension of exon 2. Aliquots of DNA from 192 P. falciparum isolates were included in the study, with 68.7% (132/192) from the municipality of Cruzeiro do Sul (Acre) and 31.3% (60/192) from Manaus (Amazonas). Of this total, 82.8% (159/192) of the samples were considered of good quality. In the state of Acre, 71.7% (71/99) showed pfhrp2 gene deletion and 94.9% (94/99) showed pfhrp3 gene deletion, while in the state of Amazonas, 100.0% (60/60) of the samples showed pfhrp2 gene deletion and 98.3% (59/60) showed pfhrp3 gene deletion. Moreover, 79.8% (127/159) of isolates displayed gene deletion. Our findings confirm the presence of a parasite population with high frequencies of pfhrp2 and pfhrp3 gene deletions in the Brazilian Amazon region. This suggests reconsidering the use of HRP2-based RDTs in the Acre and Amazonas states and calls attention to the importance of molecular surveillance and mapping of pfhrp2/pfhrp3 deletions in this area and in other locations in the Amazon region to guarantee appropriate patient care, control and ultimately contribute to achieving P. falciparum malaria elimination.

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Multiple genetic origins of histidine-rich protein 2 gene deletion in Plasmodium falciparum parasites from Peru

Scientific Reports ◽

10.1038/srep02797 ◽

2013 ◽

Vol 3 (1) ◽

Cited By ~ 45

Author(s):

Sheila Akinyi ◽

Tonya Hayden ◽

Dionicia Gamboa ◽

Katherine Torres ◽

Jorge Bendezu ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Gene Deletion ◽

Genetic Origins

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Evaluation of the genetic polymorphism of Plasmodium falciparum P126 protein (SERA or SERP) and its influence on naturally acquired specific antibody responses in malaria-infected individuals living in the Brazilian Amazon

Malaria Journal ◽

10.1186/1475-2875-7-144 ◽

2008 ◽

Vol 7 (1) ◽

pp. 144 ◽

Cited By ~ 9

Author(s):

Lilian Pratt-Riccio ◽

Selma Sallenave-Sales ◽

Joseli de Oliveira-Ferreira ◽

Bruno T da Silva ◽

Monick Guimarães ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Genetic Polymorphism ◽

Specific Antibody ◽

Brazilian Amazon ◽

Antibody Responses

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The tadpole of Amazophrynella teko (Anura: Bufonidae) from the eastern Amazon, Brazil

Zootaxa ◽

10.11646/zootaxa.4830.3.7 ◽

2020 ◽

Vol 4830 (3) ◽

pp. 592-596

Author(s):

MARCELO MENIN ◽

MARCOS ROBERTO DIAS-SOUZA ◽

CARLOS EDUARDO COSTA-CAMPOS

Keyword(s):

French Guiana ◽

Brazilian Amazon ◽

Wide Distribution ◽

Amazon Region ◽

The State ◽

Taxonomic Classification ◽

Nominal Species

The genus Amazophrynella Fouquet, Recoder, Teixeira, Cassimiro, Amaro, Camacho, Damasceno, Carnaval, Moritz, and Rodrigues, is represented by 12 nominal species and distributed in the Amazon region of Brazil, Bolivia, Colombia, Ecuador, Guyana, French Guiana, Peru, and Venezuela (Frost 2020). In the last eight years, ten species from this genus have been described. However, despite the wide distribution and diversity of these species, only the tadpole of Amazophrynella manaos Rojas, Carvalho, Ávila, Farias, and Hrbek from the Brazilian Amazon (Menin et al. 2014) and A. siona Rojas, Fouquet, Ron, Hernández-Ruz, Melo-Sampaio, Chaparro, Vogt, Carvalho, Pinheiro, Ávila, Farias, Gordo, and Hrbek from Ecuador have been formally described (Duellman & Lynch 1969; Rojas et al. 2018). Literature about tadpole morphology, reproduction, and bioacoustics of Amazophrynella is scarce and necessary to a comprehensive taxonomic classification (Kaefer et al. 2019). Herein, we describe the tadpole of the recently described species Amazophrynella teko Rojas, Fouquet, Ron, Hernández-Ruz, Melo-Sampaio, Chaparro, Vogt, Carvalho, Pinheiro, Ávila, Farias, Gordo, and Hrbek, found in the northeastern Amazon, in the State of Amapá, Brazil, and in French Guiana.

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Ultrasensitive and label-free biosensor for the detection of Plasmodium falciparum histidine-rich protein II in saliva

Scientific Reports ◽

10.1038/s41598-019-53852-5 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 5

Author(s):

Gita V. Soraya ◽

Chathurika D. Abeyrathne ◽

Christelle Buffet ◽

Duc H. Huynh ◽

Shah Mukim Uddin ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Malaria Elimination ◽

Point Of Care ◽

Turnaround Time ◽

Label Free ◽

Global Public Health ◽

Interdigitated Electrode ◽

Sensor Platform ◽

Electrode Sensor ◽

Phosphate Buffered Saline

AbstractMalaria elimination is a global public health priority. To fulfil the demands of elimination diagnostics, we have developed an interdigitated electrode sensor platform targeting the Plasmodium falciparum Histidine Rich Protein 2 (PfHRP2) protein in saliva samples. A protocol for frequency-specific PfHRP2 detection in phosphate buffered saline was developed, yielding a sensitivity of 2.5 pg/mL based on change in impedance magnitude of the sensor. This protocol was adapted and optimized for use in saliva with a sensitivity of 25 pg/mL based on change in resistance. Further validation demonstrated detection in saliva spiked with PfHRP2 from clinical isolates in 8 of 11 samples. With a turnaround time of ~2 hours, the label-free platform based on impedance sensors has the potential for miniaturization into a point-of-care diagnostic device for malaria elimination.

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A Novel Tool for the Generation of Conditional Knockouts To Study Gene Function across the Plasmodium falciparum Life Cycle

mBio ◽

10.1128/mbio.01170-19 ◽

2019 ◽

Vol 10 (5) ◽

Cited By ~ 10

Author(s):

Marta Tibúrcio ◽

Annie S. P. Yang ◽

Kazuhide Yahata ◽

Pablo Suárez-Cortés ◽

Hugo Belda ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Life Cycle ◽

Genetic Modification ◽

Gene Deletion ◽

Genetically Modify ◽

Complex Life Cycle ◽

Mosquito Vector ◽

Parasite Line ◽

Content Type ◽

First Time

ABSTRACT Plasmodium falciparum has a complex life cycle that involves interaction with multiple tissues inside the human and mosquito hosts. Identification of essential genes at all different stages of the P. falciparum life cycle is urgently required for clinical development of tools for malaria control and eradication. However, the study of P. falciparum is limited by the inability to genetically modify the parasite throughout its life cycle with the currently available genetic tools. Here, we describe the detailed characterization of a new marker-free P. falciparum parasite line that expresses rapamycin-inducible Cre recombinase across the full life cycle. Using this parasite line, we were able to conditionally delete the essential invasion ligand AMA1 in three different developmental stages for the first time. We further confirm efficient gene deletion by targeting the nonessential kinase FIKK7.1. IMPORTANCE One of the major limitations in studying P. falciparum is that so far only asexual stages are amenable to rapid conditional genetic modification. The most promising drug targets and vaccine candidates, however, have been refractory to genetic modification because they are essential during the blood stage or for transmission in the mosquito vector. This leaves a major gap in our understanding of parasite proteins in most life cycle stages and hinders genetic validation of drug and vaccine targets. Here, we describe a method that supports conditional gene deletion across the P. falciparum life cycle for the first time. We demonstrate its potential by deleting essential and nonessential genes at different parasite stages, which opens up completely new avenues for the study of malaria and drug development. It may also allow the realization of novel vaccination strategies using attenuated parasites.

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