Assessment of copy number variation in genes related to drug resistance in Plasmodium vivax and Plasmodium falciparum isolates from the Brazilian Amazon and a systematic review of the literature

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Abstract

Background

Parasite resistance to anti-malarials represents a great obstacle for malaria elimination. The majority of studies have investigated the association between single-nucleotide polymorphisms (SNPs) and drug resistance; however, it is becoming clear that the copy number variation (CNV) is also associated with this parasite phenotype. To provide a baseline for molecular surveillance of anti-malarial drug resistance in the Brazilian Amazon, the present study characterized the genetic profile of both markers in the most common genes associated with drug resistance in Plasmodium falciparum and Plasmodium vivax isolates. Additionally, these data were compared to data published elsewhere applying a systematic review of the literature published over a 20-year time period.

Methods

The genomic DNA of 67 patients infected by P. falciparum and P. vivax from three Brazilian States was obtained between 2002 and 2012. CNV in P. falciparum multidrug resistance gene-1 ( pfmdr1), GTP cyclohydrolase 1 ( pfgch1) and P. vivax multidrug resistance gene-1 ( pvmdr1) were assessed by real-time PCR assays. SNPs in the pfmdr1 and pfcrt genes were assessed by PCR–RFLP. A literature search for studies that analysed CNP in the same genes of P. falciparum and P. vivax was conducted between May 2014 and March 2017 across four databases.

Results

All analysed samples of P. falciparum carried only one copy of pfmdr1 or pfgch1. Although the pfcrt K76T polymorphism, a determinant of CQ resistance, was present in all samples genotyped, the pfmdr1 N86Y was absent. For P. vivax isolates, an amplification rate of 20% was found for the pvmdr1 gene. The results of the study are in agreement with the low amplification rates for pfmdr1 gene evidenced in the Americas and Africa, while higher rates have been described in Southeast Asia. For P. vivax, very low rates of amplification for pvmdr1 have been described worldwide, with exceptions in French Guiana, Cambodia, Thailand and Brazil.

Conclusions

The present study was the first to evaluate gch1 CNV in P. falciparum isolates from Brazil, showing an absence of amplification of this gene more than 20 years after the withdrawal of the Brazilian antifolates therapeutic scheme. Furthermore, the rate of pvmdr1 amplification was significantly higher than that previously reported for isolates circulating in Northern Brazil.

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The online version of this article (doi:10.1186/s12936-017-1806-z) contains supplementary material, which is available to authorized users.

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Most cited references 87

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Emergence of artemisinin-resistant malaria on the western border of Thailand: a longitudinal study

Aung Pyae Phyo, Aung Phyo, Standwell Nkhoma … (2012)

Summary Background Artemisinin-resistant falciparum malaria has arisen in western Cambodia. A concerted international effort is underway to contain artemisinin-resistant Plasmodium falciparum, but containment strategies are dependent on whether resistance has emerged elsewhere. We aimed to establish whether artemisinin resistance has spread or emerged on the Thailand–Myanmar (Burma) border. Methods In malaria clinics located along the northwestern border of Thailand, we measured six hourly parasite counts in patients with uncomplicated hyperparasitaemic falciparum malaria (≥4% infected red blood cells) who had been given various oral artesunate-containing regimens since 2001. Parasite clearance half-lives were estimated and parasites were genotyped for 93 single nucleotide polymorphisms. Findings 3202 patients were studied between 2001 and 2010. Parasite clearance half-lives lengthened from a geometric mean of 2·6 h (95% CI 2·5–2·7) in 2001, to 3·7 h (3·6–3·8) in 2010, compared with a mean of 5·5 h (5·2–5·9) in 119 patients in western Cambodia measured between 2007 and 2010. The proportion of slow-clearing infections (half-life ≥6·2 h) increased from 0·6% in 2001, to 20% in 2010, compared with 42% in western Cambodia between 2007 and 2010. Of 1583 infections genotyped, 148 multilocus parasite genotypes were identified, each of which infected between two and 13 patients. The proportion of variation in parasite clearance attributable to parasite genetics increased from 30% between 2001 and 2004, to 66% between 2007 and 2010. Interpretation Genetically determined artemisinin resistance in P falciparum emerged along the Thailand–Myanmar border at least 8 years ago and has since increased substantially. At this rate of increase, resistance will reach rates reported in western Cambodia in 2–6 years. Funding The Wellcome Trust and National Institutes of Health.

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Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number.

Ric Price, Anne-Catrin Uhlemann, Alan Brockman … (2004)

The borders of Thailand harbour the world's most multidrug resistant Plasmodium falciparum parasites. In 1984 mefloquine was introduced as treatment for uncomplicated falciparum malaria, but substantial resistance developed within 6 years. A combination of artesunate with mefloquine now cures more than 95% of acute infections. For both treatment regimens, the underlying mechanisms of resistance are not known. The relation between polymorphisms in the P falciparum multidrug resistant gene 1 (pfmdr1) and the in-vitro and in-vivo responses to mefloquine were assessed in 618 samples from patients with falciparum malaria studied prospectively over 12 years. pfmdr1 copy number was assessed by a robust real-time PCR assay. Single nucleotide polymorphisms of pfmdr1, P falciparum chloroquine resistance transporter gene (pfcrt) and P falciparum Ca2+ ATPase gene (pfATP6) were assessed by PCR-restriction fragment length polymorphism. Increased copy number of pfmdr1 was the most important determinant of in-vitro and in-vivo resistance to mefloquine, and also to reduced artesunate sensitivity in vitro. In a Cox regression model with control for known confounders, increased pfmdr1 copy number was associated with an attributable hazard ratio (AHR) for treatment failure of 6.3 (95% CI 2.9-13.8, p<0.001) after mefloquine monotherapy and 5.4 (2.0-14.6, p=0.001) after artesunate-mefloquine therapy. Single nucleotide polymorphisms in pfmdr1 were associated with increased mefloquine susceptibility in vitro, but not in vivo. Amplification in pfmdr1 is the main cause of resistance to mefloquine in falciparum malaria. Multidrug resistant P falciparum malaria is common in southeast Asia, but difficult to identify and treat. Genes that encode parasite transport proteins maybe involved in export of drugs and so cause resistance. In this study we show that increase in copy number of pfmdr1, a gene encoding a parasite transport protein, is the best overall predictor of treatment failure with mefloquine. Increase in pfmdr1 copy number predicts failure even after chemotherapy with the highly effective combination of mefloquine and 3 days' artesunate. Monitoring of pfmdr1 copy number will be useful in epidemiological surveys of drug resistance in P falciparum, and potentially for predicting treatment failure in individual patients.

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Copy number variation: new insights in genome diversity.

Jennifer L R Freeman, George H Perry, Lars Feuk … (2006)

DNA copy number variation has long been associated with specific chromosomal rearrangements and genomic disorders, but its ubiquity in mammalian genomes was not fully realized until recently. Although our understanding of the extent of this variation is still developing, it seems likely that, at least in humans, copy number variants (CNVs) account for a substantial amount of genetic variation. Since many CNVs include genes that result in differential levels of gene expression, CNVs may account for a significant proportion of normal phenotypic variation. Current efforts are directed toward a more comprehensive cataloging and characterization of CNVs that will provide the basis for determining how genomic diversity impacts biological function, evolution, and common human diseases.

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Author and article information

Contributors

Gabriel Luíz Costa: gabriel.costa@cpqrr.fiocruz.br

Lara Cotta Amaral: laracotta@cpqrr.fiocruz.br

Cor Jesus Fernandes Fontes: corfontes@gmail.com

Luzia Helena Carvalho: lhcarvalho@cpqrr.fiocruz.br

Cristiana Ferreira Alves de Brito: cristiana@cpqrr.fiocruz.br

Taís Nóbrega de Sousa: tais@cpqrr.fiocruz.br

Journal

Journal ID (nlm-ta): Malar J

Journal ID (iso-abbrev): Malar. J

Title: Malaria Journal

Publisher: BioMed Central (London )

ISSN (Electronic): 1475-2875

Publication date (Electronic): 19 April 2017

Publication date PMC-release: 19 April 2017

Publication date Collection: 2017

Volume: 16

Electronic Location Identifier: 152

Affiliations

[1 ]ISNI 0000 0001 0723 0931, GRID grid.418068.3, Molecular Biology and Malaria Immunology Research Group, , Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz (FIOCRUZ), ; Belo Horizonte, Minas Gerais Brazil

[2 ]ISNI 0000 0001 2322 4953, GRID grid.411206.0, , Hospital Julio Muller, Universidade Federal de Mato Grosso, ; Cuiabá, Mato Grosso Brazil

Article

Publisher ID: 1806

DOI: 10.1186/s12936-017-1806-z

PMC ID: 5395969

PubMed ID: 28420389

SO-VID: bea8591a-6c80-42e2-a5c0-6a0647c63094

License:

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

History

Date received : 15 June 2016

Date accepted : 7 April 2017

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ScienceOpen disciplines: Infectious disease & Microbiology

Keywords: malaria,plasmodium falciparum,plasmodium vivax,drug resistance,copy number variation,single nucleotide polymorphism,systematic review

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ScienceOpen disciplines: Infectious disease & Microbiology

Keywords: malaria, plasmodium falciparum, plasmodium vivax, drug resistance, copy number variation, single nucleotide polymorphism, systematic review

Assessment of copy number variation in genes related to drug resistance in Plasmodium vivax and Plasmodium falciparum isolates from the Brazilian Amazon and a systematic review of the literature

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Malaria vaccine development

Most cited references 87

Emergence of artemisinin-resistant malaria on the western border of Thailand: a longitudinal study

Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number.

Copy number variation: new insights in genome diversity.

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