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
© The Author(s) 2017
Received: 15 June 2016
Accepted: 7 April 2017
Published: 19 April 2017
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.
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.
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.
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.
KeywordsMalaria Plasmodium falciparum Plasmodium vivax Drug resistance Copy number variation Single nucleotide polymorphism Systematic review
Malaria is an endemic disease and 3.2 billion people are exposed to its transmission in 95 countries and territories around the world. This disease is caused by five species of Plasmodium, with Plasmodium vivax having a wider geographic distribution and Plasmodium falciparum responsible for the most severe malaria cases associated with high mortality rates . According to the latest estimates, in 2015, approximately 143,000 cases were registered in the Brazilian Amazon region, with 87% of cases due to P. vivax .
One of the greatest challenges of malaria control is the parasite’s resistance to anti-malarials, defined as the ability of a parasite to survive and/or multiply in the presence of a drug . In Brazil, the first case of drug resistance in P. falciparum malaria was reported in the early nineteenth century, to quinine (QN), the first drug used for malaria treatment . It was only in the 1960s that an increased number of cases of P. falciparum chloroquine (CQ) resistance began to be reported in the country, concomitant with other countries from South America and Asia [5, 6]. In the 1970s, the Brazilian government adopted sulfadoxine-pyrimethamine (SP) as the first-line treatment; however, in the late 1980s, the rate of SP resistance was close to 90% . From the late 1980s to 2000, Brazil applied various therapeutic schemes, and in 2007 the artemisinin-based combination therapy (ACT) was adopted according to the WHO recommendations to treat uncomplicated P. falciparum malaria . Despite the reports of low susceptibility in Asia [8–11] and South America , ACT remains effective in Brazil . Concerning P. vivax malaria, even though this species has been the most prevalent in Brazil for decades, it is only in the last few years that this species has developed low levels of drug resistance [14–17] and cases of severe malaria [18, 19] have been reported in the Amazon region. Thus, CQ plus primaquine (PQ) is still effective in this area .
Anti-malarial resistance studies and surveillance can be conducted using in vitro parasite culture, animal models, in vivo methods and molecular genotyping . The latter aims to identify and monitor genetic polymorphisms related to parasite resistance. Such markers are mainly SNPs, which are characterized by the substitution of a single nucleotide, and CNVs. CNV refers to an increase or decrease in the copy number of a gene in the genome; CNVs are extensively found in humans, mice, Drosophila and other eukaryotes [22, 23].
The importance of copy number variation in the Plasmodium genome was noticed when an elevated copy number of the mdr1 (multidrug resistance gene-1) gene was reported and associated with multidrug resistance in P. falciparum isolates in Asia in the late 1980s . The pfmdr1 gene encodes a protein localized in the parasite digestive vacuole named P-glycoprotein homolog 1 (P-gh1), and many studies have shown a strong association between pfmdr1 and the multidrug resistance phenotype, such as to mefloquine (MQ), QN and halofantrine (HF) [25–27]. Another gene related to SP resistance in P. falciparum and characterized by copy number variation is gch1 (GTP cyclohydrolase 1), a gene encoding the enzyme GTP cyclohydrolase 1 that catalyzes the first step in the folate biosynthesis pathway [28–30]. Regarding CNV in P. vivax, the amplification of pvmdr1, orthologous to pfmdr1, seems to also be related to drug resistance [31, 32]. Due to the challenges of conducting in vitro studies in P. vivax, the understanding concerning the resistance to anti-malarials for this species is less comprehensive.
Despite the increased importance of CNV over the last decades, SNPs are the first polymorphisms associated with drug resistance in Plasmodium. In P. falciparum some SNPs have a major role in the drug resistance phenotype, e.g., K76T polymorphism in the pfcrt (P. falciparum chloroquine-resistant transporter) gene and N86Y, Y184F, S1034C, N1042D, D1246Y in the pfmdr1 gene [33, 34], while only the mutation Y976F in the pvmdr1 gene was related to clinical resistance to CQ in P. vivax in Southeast Asia and Papua New Guinea [35–37].
Many questions remain about the genetic basis of resistance in P. falciparum and P. vivax. A majority of studies has investigated the association between SNPs in the genome of Plasmodium and resistance to anti-malarials. However, it has become clear that the copy number variation is also associated with this parasite phenotype. The present study characterized the genetic profile of CNV in genes associated with drug resistance in P. falciparum and P. vivax isolates to provide a baseline for molecular surveillance of anti-malarial drug resistance in the Brazilian Amazon region. Moreover, SNPs in the same P. falciparum genes were assayed and correlated to the genetic profile observed for CNV. Finally, the genetic variability described in Brazilian isolates was compared to data published elsewhere applying a systematic review of the literature.
Subjects and sample collection
Description of samples included in this study by period of collection, gender, age and parasitemia
Gender (male, %)
Median age, years (IQRc)
Parasitemia, geometric mean (range)d
Patients infected by P. falciparum a
Mato Grosso (18)
Patients infected by P. vivax a
Mato Grosso (13)
Copy number estimation of Plasmodium spp. genes
The copy number variation of Plasmodium genes was determined by quantitative real-time PCR (qPCR) using specific hydrolysis probes and oligonucleotide primers for each gene. Probes and primers previously described were used for amplification of pfmdr1 , pfgch1  and pvmdr1 . Amplification reactions were performed in a total volume of 10 µL in the presence of 5 µL of Taqman ® Universal PCR Master Mix 2× (Applied Biosystems, AB, Foster City, CA, USA), 1 µL DNA (≈100 ng/µL), 900 nM (pfmdr1, pfgch1 and pvmdr1) or 300 nM (pftubulin and pvtubulin) of forward primer, 900 nM of reverse primer (for all genes), and 200 nM (pfmdr1, pfgch1 and pvmdr1) or 250 nM (pftubulin and pvtubulin) of the probe. The cycling parameters for PCR were as follows: initial denaturation at 95 °C for 10 min, 40 cycles of 15 s at 95 °C and 1 min at 60 °C. The PCR was performed in triplicate in the Applied Biosystems Viia7 real-time PCR system (AB) in 384 plates. The single-copy β-tubulin gene was used as a reference gene (normalizer) [27, 31], and a field sample with a single copy of the target gene was used as a calibrator. The ΔΔCt method was used to estimate the copy number of pfmdr1 and pvmdr1 genes relative to a standard calibrator sample. For the pfgch1 gene, a calibration curve was used with plasmids containing the pfgch1 insert. The samples were considered to have a copy number equal to 1 when the value of the relative quantification was between 0.5 and 1.5, and values with a minimum relative quantification >1.5 were defined as amplified. Only samples with a cycle threshold <32 and a Ct standard deviation <0.3 were considered in the analysis. Each experiment was performed in triplicate, and gene amplification was determined by at least two independent experiments. The accuracy of the qPCR assay was determined from at least three independent experiments, each performed in triplicate, of 19% of the total samples assayed and for the reference laboratory isolate W2mef (2 copies for pfmdr1 and 1 copy for pfgch1) (Additional file 1).
Detection of single nucleotide polymorphisms in the pfcrt and pfmdr1 genes
PCR followed by restriction fragment length polymorphism analysis (RFLP) was performed to identify SNPs at codons 76 of pfcrt and 86 of pfmdr1, according to Durand et al.  and Lopes et al. , respectively. The amplicons were submitted to enzyme digestion with ApoI (New England BioLabs, Ipswich, MA, USA) for 2 h at 50 °C. Then, the enzyme was inactivated by exposure to a temperature of 80 °C for 20 min at and the fragments were visualized on a silver-stained 12% polyacrylamide gel.
A literature search was conducted between May 2014 and March 2017. An advanced search was made using key words (Plasmodium, copy number, resistance, gene, copy number resistance and gene copy). These key words were identified in the title, abstract and text, according the search algorithm of each database. Manual exclusion criteria included: studies with other Plasmodium species than P. falciparum and P. vivax; studies with laboratory strains grown in vitro; other systematic reviews; and studies with genes related to anti-malarial resistance other than pfmdr1, pfgch1 and pvmdr1. All duplicate articles and review articles were excluded; the clinical studies were included in this systematic review. The title, abstract and methods section of all 309 articles (Additional file 2) were scanned to identify the studies according to the inclusion criteria. From the eligible studies the following data were extracted: author, year of sample collection (when available), country where the study was performed, sample number and the frequency of CNV in parasite population by gene assayed.
Results and discussion
Copy number estimation of mdr1 and gch1 of Plasmodium falciparum
Polymorphisms in pfmdr1 are related to resistance to several anti-malarials, including CQ, MQ, QN and HF . An increase in the copy number of this gene is the most important determinant of resistance to MQ and to reduced artesunate sensitivity in vitro [26, 27, 42]. In Brazil, P. falciparum resistance to anti-malarials has been reported since 1910 (QN—1910; CQ—1960s; SP—1980s; amodiaquine and MQ—1990s) . Another study also assessed the presence of CNV in the pfmdr1 gene from samples collected over four decades in Brazil (from 1984 to 2011), in which patients were treated with QN plus tetracycline, MQ or ACT . During that period, the rate of pfmdr1 amplification reached 42%. Particularly, the usage of drugs such as MQ, which is related to selection of resistant parasites with pfmdr1 amplification, could be an explanation for the observed high rates of amplification. A noticeable difference between the present study and that by Inoue et al. is the region and the period of sample collection (2000s herein), which could lead to the observed differences in genetic diversity. Accordingly, geographical foci of P. falciparum with distinct population structures have been described in the Brazilian Amazon region . However, as data from the previous study have not been shown by region, differences in CNV distribution due to the location of the infection could not be assessed here. Alternatively, cases of a decline in resistant isolates by the withdrawal of drug pressure could have occurred as previously reported in Africa [45, 46]. The genome of P. falciparum is known to be plastic , which means that the parasite can experience rapid and extensive variation in response to changes in its environment; this is also known as phenotypic plasticity . In a short timescale, this phenotypic plasticity can modify or produce new phenotypes. Thus, this phenomenon enables parasites to maximize fitness by mechanisms such as differential gene expression . An example was shown by Preechapornkul et al. where strains containing a single copy of the gene preferentially survived in the absence of MQ compared to those who had multiple copies . Parasites that had fewer copies had a greater survival rate due to this energy demand generated by P-gh1. This protein acts as an efflux pump that transports substrates against a concentration gradient with ATP hydrolysis . In Brazil, MQ was officially introduced in 1987 for the treatment of CQ- and SP-resistant P. falciparum, and few years later, in 2001, MQ was substituted with the combination of QN plus doxycycline, followed by primaquine (PQ) as a first line therapy . At this time, MQ plus PQ was instituted as a second line drug combination, and only after 2006 did the ACT become adopted in Brazil . Thus, the lower pressure exerted by MQ may have led to selection of parasites bearing no genetic amplification after the first decade of the 2000s.
Plasmodium falciparum resistance to SP is associated with SNPs in two genes in the folate pathway, dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) [51, 52]. However, polymorphisms in these genes may alter their efficiencies. The pfgch1 gene encodes the first enzyme of this pathway and an increased copy number of this gene may act as a compensatory mechanism since it is associated with a greater number of point mutations in dhfr and dhps [28, 53, 54]. Few studies have evaluated the role of CNV in resistance to SP, particularly in the Americas. The present study was the first to evaluate gch1 CNV in P. falciparum isolates from Brazil. Samples analysed here date from the 2000s, more than twenty years after the withdrawal of SP in the Brazilian therapeutic scheme in the late 1980s. Thus, the absence of amplification of the pfgch1 gene in the Brazilian isolates may be the result of antifolates withdrawal, which could have favored the spread of parasites bearing a single copy of the gene. In fact, the presence of CNV in this gene seems to not be advantageous in the absence of the drug pressure, having impact on parasite growth . Still, since the evolution of antifolate-resistant parasites is multifaceted and complex and the activity of the gch1 gene is linked with other enzymes (e.g., dhps and dhfr), further analysis is required to confirm the significance of gch1 CNV on the gain in SP resistance.
Analysis of single nucleotide polymorphisms in the crt and mdr1 genes of Plasmodium falciparum
The samples of P. falciparum were assayed for two polymorphisms in pfcrt (K76T) and pfmdr1 genes (N86Y). Twenty-seven samples (84% [27/32]) were successfully genotyped (10 from Amapá, 16 from Mato Grosso and 1 from Rondônia) for both genes. For pfcrt gene, all samples carried the polymorphism at codon 76, while the substitution N86Y in the pfmdr1 gene was absent (Fig. 1). The fixation of the 76T allele is in agreement with other studies in Brazil [43, 55, 56], despite the withdrawal of CQ from national treatment guidelines in the mid-1980s . The presence of the 76Y allele is in accordance with the description of isolates resistant to CQ in the same regions analysed here [43, 55]. The K76T mutation has been proven essential to CQ resistance, as demonstrated by transfection experiments and it was suggested that this mutation in the pfcrt gene confers resistance to CQ by reducing the amount of drug in the digestive vacuole of the parasite [33, 34, 58, 59].
Field studies have observed a significant association between the pfcrt (76T) and pfmdr1 (86T) alleles, suggesting a joint contribution of these two genes to CQ resistance [60, 61]. Reinforcing these findings, the study by Veiga et al. using zinc-finger nucleases to genetically modify the pfmdr1 gene confirmed the contribution of the N86Y substitution to CQ and amodiaquine resistance. In contrast, this substitution increased parasite susceptibility to MQ and lumefantrine . Additionally, an association between the 86Y mutant and reduced susceptibility to artemether was found using in vitro tests , although other studies have reported contradictory results with the N86Y substitution associated with increased artemether sensitivity [45, 64, 65]. In samples analysed here, the N86Y substitution was absent, a similar result to that observed by other studies that have also reported low frequency or absence of this substitution in pfmdr1 in Brazil [43, 55, 56]. On the other hand, Inoue et al. reported the emergence of the 86Y mutant in 25 and 43% of isolates from Brazil and Guyana, respectively, over the last few decades. These findings led the authors to hypothesize that the increase in prevalence of the 86Y mutant could be related to the indiscriminate use of ACT in Guyana and the high flow of gold mine workers between Brazil and this neighboring country. Although not analysed here, other polymorphisms (e.g. Y184F, S1034C, N1042D and D1246Y) that influence parasite response to different drugs such as CQ, MQ, QN, HF and artemisinin [41, 66, 67] are found with high frequency in South America [43, 55, 68–70]. In this way, it seems that the markers of CQ resistance may differ between South America and other endemic areas. Thus, it is necessary to define better and more reliable polymorphisms to characterize South American samples.
Copy number estimation of mdr1 of Plasmodium vivax
To characterize the pattern of copy number variation of P. vivax isolates from the Brazilian Amazon region, thirty-five samples collected in various time periods and from two different areas (the states of Mato Grosso and Rondônia) were analysed. Overall, an amplification rate of 20% (7/35) was found for the pvmdr1 gene (Fig. 1). In Mato Grosso, six of 13 (46%) isolates had multiple copies of the pvmdr1 gene (Additional file 3C). Despite being collected in the state of Mato Grosso, the probable locality of infection for these individuals carrying isolates with multiple copies of pvmdr1 included others areas of the Amazon region, including the states of Pará and Rondônia, as well as the neighboring country French Guiana. In addition, the majority of these isolates were collected in the 2010s decade. For 22 samples collected in Rondônia only one (4%) had multiple copies of the pvmdr1 gene (Additional file 3C). Overall, pvmdr1 copy number variation was not related to parasitaemia (median: 3150 [IQR 2935-12,000 parasites/μ] for individuals carrying isolates with multiple copies vs. 2483 [992-6175 parasites/μ] for samples with one copy of pvmdr1; Mann–Whitney U test, P = 0.201) or age (mean: 32 [SD 14.8 years] for samples with multiple copies vs. 37 [15.1 years] for samples with one copy of pvmdr1; Student’s t test, P = 0.469). Among samples with multiple copies of pvmdr1, the recurrence of the disease was reported in two individuals from Mato Grosso in a period of 45 days to 3 months after the initial infection.
In Brazil and in several other malaria endemic areas CQ and PQ are still the main drugs used to treat P. vivax . However, resistance to CQ has been reported in the Brazilian Amazon [14–17]. Similar to P. falciparum, the copy number variation on pvmdr1 is supposed to be related to drug resistance . In Brazil, only one study described pvmdr1 copy number variation, showing an amplification rate of 0.9% in isolates from the state of Acre . Furthermore, the authors have not observed any relation between SNPs described in pvmdr1 and CQ resistance. In this study, pvmdr1 amplification was observed in 20% of patients infected in different areas of Northern Brazil. One individual was declared to have been infected in French Guiana, where a high number of isolates with multiple copies of the pvmdr1 gene has previously been reported [73, 74]. The clinical features of age and parasitaemia, which are important predictors of patient’s response to the treatment [27, 75, 76] did not differ between individuals carrying parasites with one or multiple copies of pvmdr1 in the present study. Thus, for the two individuals bearing isolates with multiple copies of the gene who also had episodes of recurrence, there is the possibility of treatment failure due to parasite resistance to anti-malarials. However, as the individuals were not followed up after drug therapy with plasma drug level and parasitaemia measurements, the authors could not exclude the possibility of inadequate drug absorption. Additionally, the authors could not exclude the possibility of recurrence due to CQ or PQ-impaired metabolism by variants of cytochrome P450 isoenzymes , as the genes that encode the enzymes were not assayed here. On the other hand, a new infection seems to be unlikely since these individuals reside in an area without active transmission of malaria and did not travel to other endemic areas in the Brazilian Amazon after their initial infections.
As previously suggested for other geographical locations, pvmdr1 amplification could be associated with MQ pressure [31, 78] in a mixed infection context, when this drug was used alone or in combination to treat P. falciparum malaria. Accordingly, P. vivax resistance to MQ has been reported in the Brazilian Amazon region [16, 55, 79]. In P. falciparum, resistance to MQ has been associated with increased copy number of pfmdr1 . The mechanisms of MQ resistance seem to be similar between P. vivax and P. falciparum. In areas of Southeast Asia with intense and sustained MQ pressure, gene amplifications of pvmdr1 were significantly more common than in those patients from other localities where there had been less parasite exposure to MQ . Additionally, Suwanarusk et al. reported amplification of 21% in the pvmdr1 gene in P. vivax isolates from Thailand, which was associated with a twofold increase in MQ IC50 . Mefloquine and CQ seem to exert selection pressure in opposite direction on pvmdr1, with gene amplification associated with an increase of susceptibility to CQ . Therefore, the observed pattern of pvmdr1 copy number variation in the Brazilian Amazon region and elsewhere would be the result of pressure exerted by the two drugs. For Brazilian samples, an increase in the prevalence of pvmdr1 amplification was observed over the 7-years period analysed. However, this finding requires further investigation as it might indicate that the P. vivax population is undergoing an increased susceptibility to CQ.
Copy number variation in Plasmodium spp.: a systematic review of the literature
A total of 309 articles were selected from four databases (PubMed, Malaria Journal, Science Direct and CAPES), and 82 of these were included in the systematic review according to the established criteria. The data of articles included in this study are available in supplementary material (Additional file 4). Studies from four continents constituted the review, where Asia and Africa were the regions with the majority of selected articles (88%). The majority of the studies were about P. falciparum, related to copy number variation in pfmdr1. Only two studies analysed the pfgch1 gene. For the pvmdr1 gene, eight studies were eligible for inclusion.
Regarding pfgch1, only two studies evaluated the profile of CNV in field isolates. In Africa, Kiwuwa et al. have not detected any amplification in the 21 isolates analysed . However, in Southeast Asia a significant difference in the rates of pfgch1 amplification was reported for isolates from Thailand (72%) and Laos (2%), countries that have contrasting selection histories with antifolate drugs . Whereas SP was not extensively used in Laos until 2006, despite their use as official second line treatment for malaria , Thailand has had a longstanding history of antifolate use . This geographical differentiation on pfgch1 CNV suggests local adaptations to drug pressure, which has been experimentally corroborated .
For P. falciparum isolates, no amplification was found for pfmdr1 or pfgch1, but the SNP K76T associated with chloroquine resistance was present. Notably, the rate of pvmdr1 amplification observed in this study was significantly higher than previously reported for isolates circulating in Northern Brazil. Furthermore, a wide variation in the amplification rate of pvmdr1 was observed between the two study sites in Brazil. In a global view, in the Americas and Africa the amplification rates of the mdr1 gene of P. falciparum were generally very low, with a few exceptions. In Asia, particularly in Thailand and Cambodia, the highest rates of pfmdr1 amplification were reported. In general, P. falciparum showed the highest rates of gene amplification; however, it is important to highlight that especially for P. vivax, the available information is restricted and may not reflect the actual picture of genetic variability for this species.
single nucleotide polymorphism
copy number variation
multidrug resistance gene-1
P-glycoprotein homolog 1
GTP cyclohydrolase 1
polymerase chain reaction followed by restriction fragment length polymorphism
quantitative polymerase chain reaction
artemisin-based combination therapy
- IC50 :
half maximal inhibitory concentration
TNS and CFAB designed the study. TNS, GLC and LCA performed the experiments and analysed the data. CFAB, LHC and CJFF contributed reagents/materials/analysis tools. TNS, CFAB, LHC and GLC wrote the manuscript. All authors read and approved the final manuscript.
We are grateful to all the patients who made this study possible. We also thank Dr. Rubens Lima do Monte Neto to help build maps. We thank the Programme for Technological Development in Tools for Health-PDTIS platform (FIOCRUZ) for Real-time PCR facilities. CJF, LHC and CAB thank CNPq for the research scholarship support. GLC thanks CAPES (Coordination of Superior Level Staff Improvement) for scholarship support and the Graduate Programme in Health Sciences of CPqRR.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Ethical approval for the study was obtained from the Ethics and Research Committee of René Rachou Research Center (Protocol Number 377.205). All participants signed a written informed consent, including the next of kin, caretakers, or guardians on behalf of the minors/children enrolled in the study.
This study has financial support from FAPEMIG (Fundação de Amparo à Pesquisa do estado de Minas Gerais) and CNPq (National Council for Scientific and Technological Development).
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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