Villena et al. Malar J (2020) 19:450 https://doi.org/10.1186/s12936-020-03519-8 Malaria Journal RESEARCH Open Access Molecular surveillance of the Plasmodium vivax multidrug resistance 1 gene in Peru between 2006 and 2015 Fredy E. Villena1* , Jorge L. Maguiña2, Meddly L. Santolalla2,3, Edwar Pozo4, Carola J. Salas1, Julia S. Ampuero1, Andres G. Lescano2, Danett K. Bishop1 and Hugo O. Valdivia1 Abstract Background: The high incidence of Plasmodium vivax infections associated with clinical severity and the emergence of chloroquine (CQ) resistance has posed a challenge to control efforts aimed at eliminating this disease. Despite con- flicting evidence regarding the role of mutations of P. vivax multidrug resistance 1 gene (pvmdr1) in drug resistance, this gene can be a tool for molecular surveillance due to its variability and spatial patterns. Methods: Blood samples were collected from studies conducted between 2006 and 2015 in the Northern and Southern Amazon Basin and the North Coast of Peru. Thick and thin blood smears were prepared for malaria diagnosis by microscopy and PCR was performed for detection of P. vivax monoinfections. The pvmdr1 gene was subsequently sequenced and the genetic data was used for haplotype and diversity analysis. Results: A total of 550 positive P. vivax samples were sequenced; 445 from the Northern Amazon Basin, 48 from the Southern Amazon Basin and 57 from the North Coast. Eight non-synonymous mutations and three synonymous mutations were analysed in 4,395 bp of pvmdr1. Amino acid changes at positions 976F and 1076L were detected in the Northern Amazon Basin (12.8%) and the Southern Amazon Basin (4.2%) with fluctuations in the prevalence of both mutations in the Northern Amazon Basin during the course of the study that seemed to correspond with a malaria control programme implemented in the region. A total of 13 pvmdr1 haplotypes with non-synonymous mutations were estimated in Peru and an overall nucleotide diversity of π = 0.00054. The Northern Amazon Basin was the most diverse region (π = 0.00055) followed by the Southern Amazon and the North Coast (π = 0.00035 and π = 0.00014, respectively). Conclusion: This study showed a high variability in the frequencies of the 976F and 1076L polymorphisms in the Northern Amazon Basin between 2006 and 2015. The low and heterogeneous diversity of pvmdr1 found in this study underscores the need for additional research that can elucidate the role of this gene on P. vivax drug resistance as well as in vitro and clinical data that can clarify the extend of CQ resistance in Peru. Keywords: Malaria, Plasmodium vivax, Single nucleotide polymorphisms, Drug resistance Background In the Americas, Plasmodium falciparum and Plasmo- dium vivax are responsible for 25.9% and 74.1% of all malaria cases reported in the region, respectively [1]. *Correspondence: fredy.e.villena.ctr@mail.mil Although P. falciparum is associated with higher mor- 1 Department of Parasitology, U.S. Naval Medical Research Unit No, 6 (NAMRU-6), Lima, Peru tality rates, P. vivax is attributed with the highest mor- Full list of author information is available at the end of the article bidity in the continent and particularly in the Amazon © The Author(s) 2020. 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The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Villena et al. Malar J (2020) 19:450 Page 2 of 10 Basin with a ratio of at least 4:1 cases in relation to P. In addition, other studies have shown that changes in falciparum [2]. gene copy number and expression profiles of pvmdr1 and According to the Peruvian CDC, the Northern Ama- pvcrt could be associated with CQ resistance [23, 24]. The zon Basin accounted for most of all malaria cases association in the case of pvcrt is further supported reported in 2018 (Loreto region; 95%) followed by the by recent evidence that 5′UTR and intronic changes North Coast (Piura and Tumbes Region; 0.02%) and in pvcrt are linked with increased pvcrt expression in the Southern Amazon Basin (Madre de Dios; 0.01%). parasite CQ resistant cross lines tested in non-human Since 2006, the Northern Amazon Basin experienced primate models [25]. In the specific case of pvmdr1, its a reduction in the incidence of P. vivax malaria from role in P. vivax resistance is also under discussion in the 56,171 cases in that year to 26,846 cases in 2010 due to scientific community with some studies suggesting con- the implementation of the Global fund’s Malaria Pro- vergent evolution without an implication in resistance ject (PAMAFRO), a community programme focused on [26–28] whereas others propose a direct association with active case detection and treatment [3]. After the end P. vivax resistance [22]. In this regard, studies showed an of the programme in 2010, malaria cases increased each association between the prevalence of the pvmdr1 976F year from 11,779 in 2011 up to 60,268 in 2015. mutation with increased tolerance to CQ and decreased At the time when samples were collected, there were in vitro susceptibility to MQ and ART in areas with high two treatment schemes for P. falciparum that were levels of CQ resistance and the opposite in areas where administered according to the geographical area. In CQ continues to be used as primary treatment [22, 29, the North coast, standard treatment consisted of sul- 30]. This evidence suggests the presence of competitive fadoxine–pyrimethamine (SP) in combination with evolutionary pressures on pvmdr1 [29] caused by CQ, artesunate (ART), while in the rest of the country it MQ and ART and that pvmdr1 mutations might have sig- was changed to ART and mefloquine (MQ) as first-line nificant fitness costs [31]. treatment after a brief period of SP [4, 5]. Many of the initial associations of pvmdr1 with resist- Cases caused by P. vivax are primarily treated with ance to CQ were primarily based on molecular tests and CQ and primaquine (PQ) in order to eliminate sexual, epidemiological or clinical information. However, these asexual and dormant P. vivax stages. However, there assessments suffer from important confounding factors is accumulating evidence suggesting emergence of P. [32]. Stronger evidence have been obtained from studies vivax anti-malarial resistance in Peru. For instance, that combined molecular data from pvmdr1 (SNPs and four cases of P. vivax recurrence on days 21 and 28 after changes in gene copy number) with in vitro drug suscep- oral anti-malarial treatment with CQ (10  mg/kg on tibility tests against anti-malarial drugs used in the study days 0 and 1 and 5 mg/kg on day 2) and PQ (0.5 mg/kg/ area such as CQ and MQ [22, 29] or with clinical data day for seven days) were described during a study con- [18, 33]. ducted in 177 patients in the Peruvian Amazon region This study aimed to extend the genetic characteriza- between 1998 and 2001 [6]. Additionally, Graf et  al., tion of pvmdr1 in South America by assessing changes reported in 2012 a possible case of chloroquine-resist- over time in the prevalence of pvmdr1 polymorphisms ant P. vivax which presented recrudescence on day 28th in samples collected across the Amazon basin and other after treatment with a combination of CQ (25 mg base/ endemic regions from Peru between 2006 and 2015. kg divided into single daily doses over 3 days) and PQ The information collected will provide a picture of the (0.25 mg/kg daily for 14 days) [7]. dynamics of genetic variability of pvmdr1 in Peru, which Cases of CQ drug resistance in P. vivax were first can be used as a baseline for surveillance in response reported in 1989 in Papua New Guinea [8] that subse- to new regional initiatives of malaria control and quently extended to other endemic regions including elimination. South America [9–11]. However, the exact mechanisms that modulate drug resistance are still poorly under- Methods stood. Single nucleotide polymorphisms (SNPs) in Study design pfmdr1, pfdhps, pfdhfr and pfcrt have been shown to Samples were collected from four IRB-approved studies be involved in P. falciparum anti-malarial resistance conducted in Peru between 2006 and 2015 in different in  vivo and in  vitro [12–14]. Likewise, mutations on communities of the Northern Amazon Basin (region of the P. vivax orthologous genes pvmdr1, pvdhps, pvdhfr Loreto; 2006–2015), Southern Amazon Basin (region of and pvcrt have also been associated with anti-malarial Madre de Dios; 2012–2013) and North Coast (regions resistance although a clear role in treatment failure of Piura and Tumbes; 2008–2010) (Fig. 1). Although the remains to be fully elucidated [15–22]. Peruvian Amazon and the North Coast of Peru are dif- ferent environments, they share common characteristics Villena et al. Malar J (2020) 19:450 Page 3 of 10 Fig. 1 Geographic distribution of the pvmdr1 haplotypes. The graphic was made using 550 samples collected in the Northern Amazon Basin (n = 445), the Southern Amazon Basin (n = 48) and the North Coast (n = 57). Pie charts indicate the pvmdr1 haplotype present in a region and their prevalence. The M908L/T958M haplotype was distributed in all sites while the T958M/F1076L haplotype was specific for the North Coast such as proximity to massive water sources like rivers and Identification of single nucleotide polymorphisms (SNP) lakes and having a warm humid climate. in the pvmdr1 gene The whole pvmdr1 gene was amplified by PCR in three Sample collection and preparation overlapping fragments (Additional file 1: Fig. S1, Addi- Blood samples were collected by finger prick or veni- tional file  2: Table  S1). PCR reaction was carried out puncture and thick and thin blood smears were prepared in a 50  µl reaction volume containing 5  µl of gDNA for malaria microscopy and read at the Naval Medi- (~ 25  ng), 1X buffer, 2  mM MgCL2, 125  µM dNTP’s, cal Research Unit 6 (NAMRU-6) facilities. DNA was 250  nM of each primer and 1 unit of Taq Polymer- extracted from either 200  µl venous blood or ~ 100  µl ase (Invitrogen). PCR products were purified using dried blood spots using the QIAamp® DNA Blood Mini QIAquick™ silica‐based spin columns (GmbH, Hilden, Kit (GmbH, Hilden, Germany) following the manufac- Germany) and 30  ng of purified PCR product was turer’s instructions and stored at − 20  °C. Plasmodium used to amplify in a standard reaction (20  µl) of Big- vivax monoinfection was confirmed by polymerase chain Dye™ Terminator v3.1. The resulting products were reaction of the 18S small subunit ribosomal RNA gene sequenced on an ABI3130xl (Applied Biosystems) and (18S SSU rRNA) as previously described [34]. analysed in the program Sequencher 4.1 (Gen Codes Villena et al. Malar J (2020) 19:450 Page 4 of 10 Corporation) using the reference Sal I strain of P. vivax Results (Salvador I, GenBank accession number AY571984). Polymorphisms on the pvmdr1 gene The pvmdr1 gene was sequenced on 550 Plasmodium vivax samples with no mixed genotypes from the three Data analysis study areas. The majority of sequenced samples were col- Data was entered in Microsoft Excel and analysed in lected from the Northern Amazon Basin (n = 445, 80.9%) STATA 13.0 for Windows. Absolute and relative fre- followed by the Northern Coast (n = 57, 10.4%) and the quency of mutant and wild type alleles and haplotypes Southern Amazon basin (n = 48, 8.7%). of the pvmdr1 gene were calculated for each region. A total of eleven mutations were found in pvmdr1 Chi square and 2-tailed Fisher’s exact tests were used (eight non-synonymous and three synonymous muta- to assess statistical significant differences in propor- tions) (Additional file 3: Table S3). The non-synonymous tions according to geographic regions. Nucleotide mutations were L186W (2.5%), V221L (12.4%), D500N diversity, Tajima´s D test and FST values were calcu- (5.5%), M908L (99.1%), T958M (94.0%), Y976F (7.5%), lated in dnaSP v5.1 [28, 35]. In addition, Mega v7.0 was F1070L (4.0%) and F1076L (10.5%). From all these muta- used to assess for natural selection using the modi- tions, statistical significant differences on the prevalence fied Nei-Gojobori method [36]. Finally, a haplotype of SNPs across regions were found on five polymor- network was constructed in PopArt 1.7 [37] to assess phisms (Fig. 2 and Table 1). the relatedness of all isolates based on their pvmdr1 The Y976F and F1076L mutations that are associ- sequences. ated with CQ resistance were present in the North- ern Amazon Basin in the 908L/958  M/976F/1076L and 908L/958  M/1076L haplotypes (Y976F + F1076L = 9.2%, Fig. 2 Haplotype network for P. vivax mdr1 using 8 non-synonymous mutations for the study areas of Peru (n = 550). Each circle represent an independent haplotype, the lines connect nearby haplotypes and the cross line represent one non-synonymous mutation V illena et al. Malar J (2020) 19:450 Page 5 of 10 Table 1 Prevalence of nonsynonymous mutations by regions 2006–2015 SNPs Regions p-valuea Total (n = 550) Northern Amazon Southern Amazon North Coast (n = 57) Basin (n = 445) Basin (n = 48) n % n % n % n % L186W (TTG–> TGG) 14 3.1 0 0.0 0 0.0 0.269 14 2.5 V221L (GTG–> CTG)b 58 13.0 0 0.0 10 17.5 0.001 68 12.4 D500N (GAT–> AAT) 29 6.5 0 0.0 1 1.8 0.062 30 5.5 M908L (ATG–> CTG) 441 99.1 47 97.9 57 100.0 0.437 545 99.1 T958M (ACG–> ATG)b 413 92.8 48 100.0 56 98.2 0.039 517 94.0 Y976F (TAC–> TTC)b 41 9.2 0 0.0 0 0.0 0.001 41 7.5 F1070L (TTC–> CTC)b 12 2.7 0 0.0 10 17.5 0.001 22 4.0 F1076L (TTT–> CTT)b 56 12.6 2 4.2 0 0.0 0.001 58 10.5 a Chi square test to compare SNPs frequency across regions b Mutations with statistical significant differences across regions F1076L = 3.4%) and in the 908L/958  M/1076L and The frequencies of the Y976F and F1076L mutations 958  M/1076L haplotypes from the Southern Amazon fluctuated over time during the course of the study Basin (F1076L = 2.1%) (Additional file 3: Table S3). Syn- (Fig. 3). In this regard, there was a continuing decrease onymous mutations on pvmdr1 were found at positions in the distribution of both mutations from 17.8% in T529 (ACA > ACG), L1022 (CTA > TTA) and K1355 2006 to less than 2% in 2009. The highest prevalence for (AAA > AAG), which were present in 57.8%, 18.0% and both mutations was recorded in 2011 (Y976F = 16.7% 3.4% of the samples, respectively. Fig. 3 Fluctuation of non-synonymous mutations in the Northern Amazon Basin between 2006 and 2015. The graphic shows the dynamics of the 976F and 1076L polymorphisms and the number of reported cases in the Northern Amazon Basin. The “y” axis on the left shows the number of reported cases for the barplot. The “y” axis on the right depicts the percentage of the 976F and 1076L mutations for the lineplot whereas the “x” axis indicate the years. (1) Global fund’s Malaria project “PAMAFRO” (2005–2010). Plasmodium vivax data from 2010 was not included in the graphic because of the low sample size for that year (n = 3) Villena et al. Malar J (2020) 19:450 Page 6 of 10 and F1076L = 20.8%) followed by a downward trend haplotypes were closely related with most of them being until 2013 (Y976F = 4% and F1076L = 6.7%). only one mutational step from each other (Fig. 2). Genetic diversity of the pvmdr1 locus Geographic distribution of pvmdr1 haplotypes The overall nucleotide diversity of pvmdr1 in Peru was Twenty-seven haplotypes consisting of either synony- π = 0.00054 with differences according to geographical mous and non-synonymous mutations were identified location. In this regard, the Northern Amazon Basin was in pvmdr1 (Additional file 3: Table S3). Out of those, 13 the most diverse (π = 0.00055) followed by the South- haplotypes consisting of non-synonymous mutation were ern Amazon basin (π = 0.00035) and the North coast identified on pvmdr1 with all haplotypes but one present- (π = 0.00014). ing at least one non-synonymous mutation (Table 2). FST values were high across all regions: 0.399 between Significant differences were found in the distribu- the North coast and the Southern Amazon basin, 0.227 tion of the 908L/958M haplotype among the three sites between North Coast and the Northern Amazon Basin, (p < 0.05). The Northern Amazon Basin presented the and 0.316 between the Southern Amazon basin and the highest number of different haplotypes (92.3%) followed Northern Amazon Basin. Moreover, Tajima´s D test was by the Southern Amazon basin (23%) and the North positive (1.321) although not statistically significant when Coast (23%) (Fig. 1, Table 2). A common haplotype that samples were analyzed globally. At the regional level, was differentially (p < 0.05) distributed in all sites was Tajima’s D was also not significant across all regions, M908L/T958M, which accounted for 61.1% of cases the Northern Amazon Basin (2.072), Southern Ama- in the Northern Amazon Basin, 95.8% in the Southern zon basin (0.925) and North coast (− 0.612). This is Amazon and 80.7% in the North coast. also supported by a non-significant dN-dS ratio for Peru Single mutation was observed in 11 isolates (2%), dou- (− 0.709; P = 1.000) and at the regional level: the North- ble mutation in 389 (70.7%), triple mutation in 90 (16.2%) ern Amazon Basin (− 0.720; P = 1.000), the North Coast and quadruple mutation in 59 samples (10.7%). The (− 1.106; P = 1.000) and Southern Amazon Basin (1.587; F1076L mutation was present in three haplotypes (10.6% P = 0.058). of the samples), while the combination Y976F/F1076L was present in one haplotype (908L/958M/976F/1076L) Discussion exclusive from the Northern Amazon Basin (7.5% of the Different studies indicate that P. vivax has recently suf- samples). fered an important evolutionary pressure driven by the The haplotype network of pvmdr1 that comprised use of antifolate drugs [38, 39]. Furthermore, in areas 8 non-synonymous mutations showed that all the with P. vivax CQ susceptibility and P. falciparum CQ Table 2 Prevalence of haplotypes by regions 2006–2015 Haplotypes Regions Total (n = 550) Northern Amazon Basin Southern Amazon Basin North Coast (n = 57) (n = 445) (n = 48) n % n % n % n % Sal 1 like 1 0.2 0 0.0 0 0.0 1 0.2 908L 8 1.8 0 0.0 0 0.0 8 1.5 958 M 3 0.7 0 0.0 0 0.0 3 0.6 958 M/1076L 0 0.0 1 2.1 0 0.0 1 0.2 908L/958 Ma 272 61.1 46 95.8 46 80.7 364 66.2 500 N/908L 23 5.2 0 0.0 1 1.8 24 4.4 221L/908L/958 M 50 11.2 0 0.0 0 0.0 50 9.1 908L/958 M/1070L 4 0.9 0 0.0 0 0.0 4 0.7 908L/958 M/1076L 15 3.4 1 2.1 0 0.0 16 2.9 186 W/908L/958 M 14 3.1 0 0.0 0 0.0 14 2.6 500 N/908L/958 M 6 1.3 0 0.0 0 0.0 6 1.1 221L/908L/958 M/1070L 8 1.8 0 0.0 10 17.5 18 1.5 908L/958 M/976F/1076L 41 9.2 0 0.0 0 0.0 41 7.5 a Haplotypes with statistical significant differences across regions V illena et al. Malar J (2020) 19:450 Page 7 of 10 resistance, P. vivax is subjected to indirect selection pres- the Northern Amazon Basin from 48% in 2006 to 71% in sures during the treatment of P. falciparum or mixed 2015. No major changes in the frequencies of the other infections [40]. haplotypes were found. In this regard, studies carried Although most mutations in pvmdr1 play no role in out in P. falciparum suggest that CQ-wildtype alleles in parasite resistance, the Y976F and F1076L mutations pfmdr1 and pfcrt have a selective advantage over resist- are still controversial in the scientific community due to ant genotypes when CQ pressure is not exerted [45–47]. conflicting evidence [17, 18, 21, 22, 24, 41]. For instance, In this regard, regions where CQ was discontinued expe- Thailand, whose anti-malarial treatment regime for P. rienced an increase of wildtype pfcrt and pfmdr1 P. fal- vivax is based in CQ and PQ as in Peru [42], presented ciparum strains [45, 46, 48]. However, this is unlikely a frequency of 25% of the Y976F mutation [22], 21% to happen with the P. vivax population in the Peruvian of pvmdr1 amplifications [29], CQ susceptibility and Amazon due to the continued use of CQ as first-line reduced susceptibility to MQ and ART in P. vivax [43] treatment. Therefore, it is possible that the changes in the In contrast, countries such as Indonesia or New Guinea prevalence of pvmdr1 genotypes correspond to expan- showed up to 95% of the 976F mutation in pvmdr1, no sion or contraction of the wildtype parasite population evidence of pvmdr1 amplification, CQ resistance and sus- in response to local and temporal variations of malaria ceptibility to MQ and ART [29]. Furthermore, the Y976F control. mutation was associated with a fourfold higher chloro- The M908L and T958M mutations that are two of the quine IC50 and 5 to 8 folds lower IC50 for ART and MQ, most frequent polymorphisms in our study (99.1% and respectively [22]. A similar situation has been reported 94.0%, respectively) were reported with 100% prevalence in French Guyana that presented a decrease in pvmdr1 in Thailand and Madagascar and with 28% and 100% amplification from 71.3% when MQ was used against P. prevalence in Brazil, respectively [9, 15, 24, 49]. Also, falciparum (1995–2002) to 12.8% after a change in the these studies suggested that these polymorphisms do not anti-malarial regime. This change was accompanied by have any association with resistance to chloroquine and an increase in 976F haplotypes after the use of MQ [40]. are likely to be related to the evolutionary history of P. Unfortunately, the present study was not able to assess vivax [9, 15, 24, 26, 27, 49]. changes in pvmdr1 copy number and evaluate if there In terms of diversity, the Northern Amazon Basin was a relation between 976F haplotypes and pvmdr1 presented 12 of the 13 haplotypes found in Peru amplification. being the region with the highest diversity for pvmdr1 In the case of Peru, it appears that drug pressure and (π = 0.00055) compared to the global value of pvmdr1 changes in the incidence of malaria have also affected the in Peru (π = 0.00054) [50]. This level of diversity is lower prevalence of the Y976F and F1076L genotypes in the to the ones from other endemic areas such as India Northern Amazon Basin, which decreased from 17.8% (π = 0.0012), Brazil (π = 0.0016) and Ecuador (π = 0.0009) in 2006 to 1.2% in 2008. This decrease coincides with the [28]. In this regard, the low diversity in Peru and Ecua- implementation of a radical P. vivax treatment scheme in dor could be explained by frequent inbreeding and low 2005 in the Northern Peruvian Amazon, which changed recombination rates which are characteristic of low from 3 days of CQ and 14 days of PQ (0.25 mg/kg/day) transmission regions in contrast to the high recombina- to 3  days of CQ and 7  days of PQ (0.5  mg/kg/day) [2, tion rates between P. vivax genotypes in India and Brazil 44]. Therefore, it is highly likely that the new treatment [39, 51, 52]. scheme coupled with increased access to diagnosis and The North Coast (π = 0.00014) and Southern Ama- treatment accessibility provided by PAMAFRO could zon Basin (π = 0.00035) had a lower diversity which is have exerted strong selection pressures in the P. vivax consistent with previous studies in the North Coast that population. This is further supported by the fact that Peru showed a low parasite diversity and a homogeneous pop- reached a malaria incidence rate lower than 1 case/1,000 ulation structure [52]. This is likely due to the presence inhabitants towards the end of PAMAFRO in 2010 [2]. of the Andes mountain range that acts as a geographical However, after the end of PAMAFRO, the frequency of barrier that blocks migration of infected Amazonian vec- the Y976F and F1076L genotypes radically increased tors. Another factor that could influence the low parasite until reaching 20.83% in 2011 to initiate a gradual reduc- diversity in the North Coast is the drastic fluctuation of tion down to 5% in 2015. This rapid fluctuation in the malaria prevalence over time due to the El Niño South- frequencies of these genotypes could be due to local vari- ern Oscillation, the presence of a different malaria vec- ability in malaria control activities rather than reversion tor than the one in the Peruvian Amazon basin and to the of the mutated genotype. low human migration between both sites [2]. The 908L/958M haplotype that was the most fre- In the case of the Southern Amazon basin, distribu- quent in our study presented a continuous increase in tion and transmission of vector-borne diseases including Villena et al. Malar J (2020) 19:450 Page 8 of 10 malaria has suffered rapid fluctuations during the last Acknowledgments decade due to illegal mining, logging and agriculture, We would like to express our gratitude to “Dirección Regional de Salud Piura” for the logistic support and in executing the blood samples collection for the which have drastically changed the environment [2, 53]. “NMRCD.2010.0010” protocol. In addition, it is also possible that differences in diversity AGL is sponsored by the training grant D43 TW007393 awarded by the could be explained by the genetic background of circulat- Fogarty International Center of the US National Institutes of Health awarded to Emerge, the Emerging Diseases and Climate Change Research Unit of the ing populations in the distinct regions of Peru. School and Public Health Administration at Universidad Peruana Cayetano However, it is important to consider that a single Heredia. Meddly Santolalla received a scholarship from CAPES – Coordination marker and a potential target under selective pressure for the Improvement of Higher Education Personnel. Jorge L. Maguiña is a doctoral student studying Epidemiological Research at Universidad Peruana might have low resolution to accurately estimate genetic Cayetano Heredia under FONDECYT/CIENCIACTIVA scholarship EF033-235- diversity. This is particularly important in low transmis- 2015 and supported by training grant D43 TW007393 awarded by the Fogarty sion regions such as Peru where there is already strong International Center of the US National Institutes of Health. We thank to Dionicia Gamboa, PhD and Joseph Vinetz, MD and the evidence of low genetic diversity. Therefore, further stud- investigators, field team members and study participants from the Amazonia ies targeting multiple markers or using next generation Center of Excellence in Malaria Research, funded by cooperative agreement sequencing are needed to confirm our results for pvmdr1. U19AI089681 from the United States Public Health Service, NIH/NIAID. Finally, although there is a strong association between Authors’ contributions molecular markers and resistance to anti-malarials in P. FV, JM, MS, JA, AL, participated in all components of the study; design, inter- falciparum, this relationship is not clear for P. vivax and pretation and data analysis. EP, blood sample collection. DB, HV performed the genetic analysis. All authors contributed to the writing of the manuscript. All its orthologs [22, 28, 47]. This demands further research authors read and approved the final manuscript. to assess their effect at the clinical level in order to con- firm variants associated with resistance and therapeutic Funding This study was supported by funding from the US Department of Defense failure. Furthermore, continuous monitoring of pvmdr1 Health Agency-Armed Forces Health Surveillance Division (AFHSD), Global together with in  vitro susceptibility tests would help to Emerging Infection Surveillance (GEIS) under PROMIS ID P0106_18_N6_02. assess changes in the transmission of malaria overtime as Availability of data and materials a result of human and environmental variables to support All data generated and/or analyzed during this study are included in this initiatives for malaria control and elimination. published article and its additional files. Ethics approval and consent to participate The study protocols were approved by the Naval Medical Research Center Conclusion Institutional Review Board in compliance with all applicable Federal This research showed a regional diversification of pvmdr1 regulations governing the protection of human subjects (protocols NMRCD.2010.0010, NMRCD.2007.0004, NAMRU6.2008.0004, NMRCD.2005.0005 across endemic malaria regions in Peru and changes over and NMRCD.2012.0006). time in the frequency of pvmdr1 genotypes in the North- ern Amazon Basin between 2006 and 2015. This informa- Consent for publication Not relevant. tion is relevant for future epidemiological surveillance to measure the emergence of resistance and changes on Competing interests the parasite population in this and other endemic areas. The authors declare that no competing interests exist. However, additional research is needed to elucidate the Disclaimer role of pvmdr1 in P. vivax resistance complemented with The views expressed in this article are those of the authors and do not neces- ex vivo and phenotypic data. sarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the U.S. Government. Supplementary information Author details 1 Department of Parasitology, U.S. Naval Medical Research Unit No, 6 Supplementary information accompanies this paper at https: //doi. (NAMRU-6), Lima, Peru. 2 Emerge, Emerging Diseases and Climate Change org/10.1186/s1293 6-020-03519 -8. Research Unit, School of Public Health and Administration, Universidad Peru- ana Cayetano Heredia, Lima, Peru. 3 Departamento de Parasitología, Instituto Additional file 1: Fig. S1. Amplified and sequenced regions of the de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, pvmdr1 gene. MG, Brazil. 4 Piura Sanitary Intelligence Unit, Piura Health Region, Piura, Peru. Additional file 2: Table S1. Primers for conventional PCR, Nested PCR and Received: 4 February 2020 Accepted: 25 November 2020 sequencing ofpvmdr1 gene. Additional file 3: Table S3. Prevalence of haplotypes consisting of syn- onymous and nonsynonymousmutation. References Abbreviations 1. WHO Global malaria programme. World malaria report 2017. Geneva: IRB: Institutional review board; PAMAFRO: Global Fund Malaria Project; PCR: World Health Organization; 2018. Polymerase chain reaction; CQ: Chloroquine; PQ: Primaquine; ART : Artesunate; 2. Rosas-Aguirre A, Gamboa D, Manrique P, Conn JE, Moreno M, Lescano AG, SP: Sulfadoxine-pyrimethamine; MQ: Mefloquine. et al. Epidemiology of Plasmodium vivax malaria in Peru. Am J Trop Med Hyg. 2016;95:133–44. V illena et al. Malar J (2020) 19:450 Page 9 of 10 3. Isturiz O, Arias K, Restrepo B, Rosas-Aguirre A, Vargas D, Alvariño G. Com- 23. 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