Plasmodium vivax mdr1 genotypes in isolates from successfully cured patients living in endemic and non-endemic Brazilian areas
Malaria Journal volume 15, Article number: 96 (2016)
Plasmodium vivax is the most widely distributed species causing the highest number of malaria cases in the world. In Brazil, P. vivax is responsible for approximately 84 % of reported cases. In the absence of a vaccine, control strategies are based on the management of cases through rapid diagnosis and adequate treatment, in addition to vector control measures. The approaches used to investigate P. vivax resistance to chloroquine (CQ) were exclusively in vivo studies because of the difficulty in keeping parasites in continuous in vitro culture. In view of the limitations related to follow-up of patients and to assessing the plasma dosage of CQ and its metabolites, an alternative approach to monitor chemo-resistance (QR) is to use molecular markers. Single nucleotide polymorphisms (SNPs) in the multidrug resistance gene pvmdr1 are putative determinants of CQ resistance (CQR), but such SNPs in P. vivax isolates from patients with good response to treatment should be further explored. The aim of this study is to investigate the mutations in the gene, supposedly associated to QR, in P. vivax isolates from successfully cured patients, living in Brazilian endemic and non-endemic areas.
Blood samples were collected from 49 vivax malaria patients from endemic (Amazon Basin: 45) and non-endemic (Atlantic Forest: four) Brazilian regions and analysed for SNPs in the CQR-related P. vivax gene (pvmdr1), using PCR-based methods.
Among the 49 isolates genetically characterized for the gene pvmdr1, 34 (70 %) presented at least one mutation. T958M mutant alleles were the most frequent (73 %) followed Y976F (15 %) and F1076L (12 %). Single mutation was detected in 24 (70.5 %) isolates and double mutations in ten (29.5 %). The most common single mutant genotype was the 958M/Y976/F1076 (79 %), followed by 976F/F1076 (21 %) whereas 958M/Y976/1076L (60 %) and 976F/1076L (40 %) double mutant genotypes were detected. Single mutant profile was observed only in isolates from Amazon Basin, although double mutants were found both in the Amazon and Atlantic Forest regions. Interestingly, the genotype 958M/Y976/1076L was present in all isolates from the Atlantic Forest in the Rio de Janeiro State.
Considering that primaquine (PQ) efficacy is highly dependent on concurrent administration of a blood schizontocidal agent and that PQ could not circumvent CQR, together with the fact that no pvmdr1 mutation should be expected in successfully cured patients, these findings seem to indicate that the pvmdr1 gene is not a reliable marker of CQR. Further investigations are needed to define a reliable molecular marker for monitoring P. vivax CQR in P. vivax populations.
Almost 40 % (approximately three billion) of the world’s population is presently at risk of contracting malaria. The disease causes almost 200 million clinical cases and around 600,000 deaths each year [1, 2]. Plasmodium vivax is the most geographically widespread of the human malaria parasites, and a serious public health concern in South and Central America, Asia and Southwest Pacific .
In Brazil, endemic regions are restricted to the Legal Amazon (comprising Acre, Amapá, Amazonas, part of Maranhão, Mato Grosso, Pará, Rondonia, Roraima and Tocantis States), a region that presently accounts for 99.6 % of the countrywide malaria burden . Plasmodium vivax is the predominant species, responsible for 84 % of the reported cases , PNCM, SVS, MS, unpublished data 2015. Extra-Amazonian autochthonous cases account for only 0.04 % of all Brazilian total registered and correspond to the autochthonous malaria existing in the Atlantic Forest, located along the southeastern Atlantic Coast .
Chloroquine resistance (CQR) is the main challenge for national malaria control programmes to control vivax malaria. The first cases of P. vivax resistant to chloroquine (CQ) were described in Papua New Guinea  and thereafter observed in Indonesia , Oceania, Asian [9, 10] and South American countries, including Brazil [11, 12]. In Brazil, CQ treatment failures, presumably related to CQR, have been reported . The latest 28 day in vivo test conducted to assess the efficacy of standard supervised CQ therapy in 109 volunteers showed a proportion of 10.1 % of treatment failure (n = 11), despite an adequate absorption of CQ in these individuals on day 2 .
Molecular markers can represent a valuable tool for monitoring introduction and spread of drug resistance. Contrarily to Plasmodium falciparum, mutations at codons in the pfcrt orthologue (pvcg10) gene do not seem to mediate CQR in P. vivax . On the other hand, the polymorphisms at codons Y976F and F1076L in the multidrug-resistant gene 1 (pvmdr1) has been described as molecular marker associated to CQR . Indeed, in Thailand, Indonesia  and Myanmar , as well as in Mauritania  and Cambodia , it has been shown that 976F mutants were associated with clinical resistance to CQ. In Nepal and India, where P. vivax CQR has not been recorded, prevalence of the 976F mutation is very low (5 %)  or not detected , while in India the presence of the F1076L mutation was not associated to CQR. In addition, in Madagascar, despite 5 % of clinical failures more than 90 % of Y976F mutant parasites were detected .
These polymorphisms also seem to be relatively uncommon in Latin America, where P. vivax CQR remains relatively infrequent . In Brazil, different conclusions were drawn: either mutations in pvmdr1 were reported in CQ-sensitive P. vivax parasites [24–26] or not detected in resistant P. vivax isolates [25, 26], as well as P. vivax CQR being associated with pvmdr1 mutants only in patients with severe malaria .
In view of these different epidemiological data, the nucleotide polymorphisms (SNPs) of pvmdr1 gene in successfully cured vivax malaria patients living in endemic (Amazonian) and non-endemic (Extra-Amazonian) Brazilian areas, were investigated in the present study.
Study site, blood samples and DNA extraction
Blood samples were collected between 2010 and 2014 in patients presenting vivax malaria (n = 49) at the Laboratório de Doenças Febris Agudas, INI-IPEC, Fiocruz, the Reference Laboratory for Malaria in the Extra-Amazon to the Brazilian Ministry of Health. The inclusion criterion was patients with uncomplicated vivax malaria. After obtaining informed consent, venous blood collection was performed according to protocols previously approved by the Ethical Research Committees of Fiocruz (32839013.6.00005248). Genomic DNA was extracted from 1 mL whole blood using QIAamp midi columns, as described by the manufacturer (Qiagen). Plasmodium vivax samples were diagnosed by microscopic examination and by polymerase chain reaction (PCR) . All patients were treated with CQ plus primaquine (PQ) and followed up for 42 days and no treatment failure was detected during this period.
PCR and electrophoresis
The pvmdr1 gene was amplified by PCR using gene-specific primers. The PCR was performed as described elsewhere [16, 29] to amplify a partial DNA sequence containing three SNPs for pvmdr1 gene including: T958M, Y976F and F1076L.
DNA sequencing and SNP polymorphisms detection
After purification using the Wizard SV Gel and PCR Clean-Up System (Promega), the amplified fragments were sequenced using Big Dye® Terminator Cycle Sequencing Ready Reaction version 3.1 (Applied Biosystems) and ABI PRISM DNA Analyzer 3730 (Applied Biosystems)  at the Genomic Platform/PDTIS/Fiocruz. The direct DNA sequencing from PCR products was compared with the reference wild type Sal I GenBank accession nº AY571984 [24, 25]. Forward and reverse sequences were analysed using the free software Bioedit Sequence Alignment Editor version 7.2.5. Statistical significance of differences of pvmdr1 genotypes frequencies among Brazilian localities was assessed using Fisher’s tests.
The pvmdr1 gene was successfully amplified and DNA sequenced in 49 isolates from the Amazon Region (Acre, Amazonas, Pará, and Rondonia) and the Extra-Amazonian State of Rio de Janeiro.
Globally, 34 (69 %) showed non-synonymous (958M, 976F and 1076L) mutations. 958M mutant alleles were the more frequent (25/34; 73 %) while 976F (5/34; 15 %) and 1076L (10/34; 12 %) were detected at lower frequencies (Table 1). Single mutation was observed in 24 isolates (70.5 %, 24/34), while double mutations were recorded in ten (29.5 %, 10/34) P. vivax samples. In the isolates presenting single mutant genotype, the MYF profile was predominant (19/55 %) contrasting with the FF, which was found in only five isolates (Table 2).
The pvmdr1 wild-type allele was prevalent in Pará (54 %) followed by Acre (40 %), Amazonas (33 %), and Rondonia (24 %) states without statistically significant difference in proportion (p > 0.05). Single mutants were observed only in isolates from the Brazilian Amazon: the MFY allele was prevalent in Rondonia (77 %), followed by Amazonas (62 %) and Acre (67 %) (p > 0.05), although in Pará the FF single mutant was more frequent (66 %) than the MYF (p > 0.05). However, when double mutants were investigated, samples presenting FL (12 %) and MYL (20 %) in both Amazonian and Extra Amazonian States, were identified. Irrespective to the Brazilian State FL double mutant was the less frequent (Table 2) (p > 0.05). Interestingly, all isolates from the Extra Amazon (Rio de Janeiro State) showed double mutant genotype (MYL) contrasting with those from the Amazon Basin (6 %) (p = 0.01), where most of the isolates came from.
CQ and PQ remain the drugs of choice to treat vivax malaria, but recent studies have reported P. vivax cases of resistance to CQ in different regions of the world [9, 31], including Brazil [11–13]. Therefore, monitoring the efficacy of CQ in the treatment of vivax malaria is essential for early warning systems to promote drug policies.
To circumvent the limitations of in vivo and in vitro studies and to assess chemo-resistance, identification of mutations in target genes has been proposed, such as those in the pvmdr1 gene at codons 976 and 1076, as well as the increased expression of pvcrto transcripts [12, 32]. Similar to previous studies performed with samples from western Brazilian Amazon [24, 25], in this work the T958M mutant was found to be the more frequent in the Brazilian Amazon and even in isolates from the Extra Amazonian regions. Additionally, in Madagascar , Nepal  and Thailand , most of the samples presented mutations at 958 position, although all isolates have been obtained from individuals with successful response to CQ therapy. No later than November 2015, Schousbe and colleagues  reported a high prevalence of 958M (97.6 %) among P. vivax samples from six different geographical sites, suggesting that this allelic variant is most likely not associated with CQR and could be an allele characteristic of Asia and Africa isolates. The present data reinforce the lack of association of 958M with CQR, but are not in agreement with Asian and African geographical characteristic of this allele, since this allele was present in 51 % of the Brazilian (South American) samples.
Previous studies seemed to indicate that both SNP and amplification of pvmdr1 are associated with variation in in vitro CQ susceptibility of P. vivax [17, 32]. It has been shown that the geometric mean of the CQ inhibitory concentration 50 % (IC50) was significantly higher in isolates carrying the Y976F mutation when compared to wild-type isolates in samples from Indonesia and Thailand . However, the clear association between the clinical outcome following a three-day CQ treatment and non-synonymous mutations in this gene has never been demonstrated elsewhere. In fact, the single 976F mutant was not very common worldwide (7.4 %) , and the FF double mutant genotype was detected only in endemic regions of three countries: Brazil [36, 37], Honduras  and Papua New Guinea . In the samples herein analysed, no significant difference was observed between the presence of double 976F/1076L mutant (12 %) and single 976F mutant (15 %) and no single 1076L mutant was noted. These findings suggest that polymorphisms at codons 976 and 1076 may not be strong indicators of CQ resistance since all P. vivax isolates were obtained from patients with good response to CQ therapy. In addition, 976F and 1076L mutants were also detected in P. vivax isolates in several countries in Africa and in South America from patients with no history of CQ recrudescence . Probably, these mutations might have been introduced in these countries from Asia where these mutations are prevalent . Interestingly, the 976F mutation in P. vivax isolates from Extra-Amazonian were not detected in areas where autochthonous malaria cases from Brazilian Atlantic Forest can occur. Thus, it seems that 976F mutations are more associated to geographical characteristics than to CQR.
Concerning codon 958, only samples from the Amazon Basin showed the MYF single mutant genotype, and double MYL mutants were observed in Amazonas and Acre State isolates. On the other hand, all isolates from the Extra Amazon State of Rio de Janeiro had the double mutant 958+1076 (MYL) genotype and these samples were wild type for codon 976. Once again, the heterogeneity in these P. vivax populations could reflect the genetic diversity rather than an association with CQR in endemic areas with different endemic profiles .
Considering that pvmdr1 mutations should not be expected in CQ-sensitive parasites and that PQ efficacy is highly dependent on concurrent administration of a blood schizontocidal agent  and thus PQ could not circumvent CQR, the present findings seem to indicate that the pvmdr1 gene is not a reliable marker of CQR.
This study provides new data concerning the molecular characterization of P. vivax isolates from Brazilian Atlantic Forest. The SNP diversity observed in samples from New World is similar to those from Asia and Africa, and probably reflects a capacity for great functional variation, as already suggested . Very little is known about the molecular mechanisms underlying drug resistance in P. vivax and most loci that have been suggested to be responsible for P. vivax CQR derived from orthologue P. falciparum drug CQR genes. Further investigations are needed to define a reliable molecular marker for monitoring CQR in the Brazilian P. vivax population.
Mendis K, Sina BJ, Marchesini P, Carter R. The neglected burden of Plasmodium vivax malaria. Am J Trop Med Hyg. 2001;64:97–106.
Battle KE, Gething PW, Elyazar IR, Moyes CL, Sinka ME, Howes RE, et al. The global public health significance of Plasmodium vivax. Adv Parasitol. 2012;80:1–111.
Price RN, Tjitra E, Guerra CA, Yeung S, White NJ, Anstey NM. Vivax malaria: neglected and not benign. Am J Trop Med Hyg. 2007;77:79–87.
da Silva-Nunes M, Moreno M, Conn JE, Gamboa D, Abeles S, Vinetz JM, et al. Amazonian malaria: asymptomatic human reservoirs, diagnostic challenges, environmentally driven changes in mosquito vector populations, and the mandate for sustainable control strategies. Acta Trop. 2012;121:281–91.
Oliveira-Ferreira J, Lacerda MV, Brasil P, Ladislau JL, Tauil PL, Daniel-Ribeiro CT. Malaria in Brazil: an overview. Malar J. 2010;9:115.
de Pina-Costa A, Brasil P, Di Santi SM, de Araujo MP, Suárez-Mutis MC, Santelli AC, et al. Malaria in Brazil: what happens outside the Amazonian endemic region. Mem Inst Oswaldo Cruz. 2014;109:618–33.
Whitby M, Wood G, Veenendaal JR, Rieckmann K. Chloroquine-resistant Plasmodium vivax. Lancet. 1989;2:1395.
Baird JK, Basri H, Purnomo Bangs MJ, Subianto B, Patchen LC, et al. Resistance to chloroquine by Plasmodium vivax in Irian Jaya. Indones. Am J Trop Med Hyg. 1991;44:547–52.
Baird JK. Chloroquine resistance in Plasmodium vivax. Antimicrob Agents Chemother. 2004;48:4075–83.
Lee KS, Kim TH, Kim ES, Lim HS, Yeom JS, Jun G, Park JW. Chloroquine-resistant Plasmodium vivax in the Republic of Korea. Am J Trop Med Hyg. 2009;80:215–7.
Gonçalves LA, Cravo P, Ferreira MU. Emerging Plasmodium vivax resistance to chloroquine in South America: an overview. Mem Inst Oswaldo Cruz. 2014;109:534–9.
Melo GC, Monteiro WM, Siqueira AM, Silva SR, Magalhães BML, Alencar ACC, et al. Expression levels of pvcrt-o and pvmdr1 are associated with chloroquine resistance and severe Plasmodium vivax malaria in patients of the Brazilian Amazon. PLoS ONE. 2014;9:e105922.
Alecrim MG, Alecrim W, Macêdo V. Plasmodium vivax resistance to chloroquine (R2) and mefloquine (R3) in Brazilian Amazon region. Rev Soc Bras Med Trop. 1999;32:67–8.
de Santana Filho FS, Arcanjo AR, Chehuan YM, Costa MR, Martinez-Espinosa FE, Vieira JL, et al. Chloroquine-resistant Plasmodium vivax. Brazilian Amazon. Emerg Infect Dis. 2007;13:1125–6.
Nomura T, Carlton JM, Baird JK, del Portillo HA, Fryauff DJ, Rathore D, et al. Evidence for different mechanisms of chloroquine resistance in 2 Plasmodium species that cause human malaria. J Infect Dis. 2001;183:1653–61.
Brega S, Meslin B, de Monbrison F, Severini C, Gradoni L, Udomsangpetch R, et al. Identification of the Plasmodium vivax mdr-like gene (pvmdr1) and analysis of single-nucleotide polymorphisms among isolates from different areas of endemicity. J Infect Dis. 2005;15:272–7.
Suwanarusk R, Chavchich M, Russell B, Jaidee A, Chalfein F, Barends M, et al. Amplification of pvmdr1 associated with multidrug-resistant Plasmodium vivax. J Infect Dis. 2008;10:1558–64.
Lu F, Lim CS, Nam DH, Kim K, Lin K, Kim TS, Lee HW, et al. Genetic polymorphism in pvmdr1 and pvcrt-o genes in relation to in vitro drug susceptibility of Plasmodium vivax isolates from malaria-endemic countries. Acta Trop. 2011;117:69–75.
Mint Lekweiry K, Ould Mohamed Salem Boukhary A, Gaillard T, Wurtz N, Bogreau H, Hafid JE, et al. Molecular surveillance of drug-resistant Plasmodium vivax using pvdhfr, pvdhps and pvmdr1 markers in Nouakchott, Mauritania. J Antimicrob Chemother. 2012;67:367–74.
Lin JT, Patel JC, Kharabora O, Sattabongkot J, Muth S, Ubalee R, et al. Plasmodium vivax isolates from Cambodia and Thailand show high genetic complexity and distinct patterns of P. vivax multidrug resistance gene 1 (pvmdr1) polymorphisms. Am J Trop Med Hyg. 2013;88:1116–23.
Ranjitkar S, Schousboe ML, Thomsen TT, Adhikari M, Kapel CMO, Bygbjerg IC, et al. Prevalence of molecular markers of anti-malarial drug resistance in Plasmodium vivax and Plasmodium falciparum in two districts of Nepal. Malar J. 2011;10:75.
Shalini S, Chaudhuri S, Sutton PL, Mishra N, Srivastava N, David JK, et al. Chloroquine efficacy studies confirm drug susceptibility of Plasmodium vivax in Chennai. India Malar J. 2014;13:129.
Vargas-Rodríguez R del C, Silva Bastos M da, Menezes MJ, Orjuela-Sánchez P, Ferreira MU. Single-nucleotide polymorphism and copy number variation of the multidrug resistance-1 locus of Plasmodium vivax: local and global patterns. Am J Trop Med Hyg. 2012;87:813–21.
Chehuan YF, Costa MRF, Costa JS, Alecrim MGC, Nogueira F, Silveira H, et al. In vitro chloroquine resistance for Plasmodium vivax isolates from the Western Brazilian Amazon. Malar J. 2013;3:226.
Sá JM, Nomura T, Neves JDA, Baird JK, Wellems TE, Del Portillo HA. Plasmodium vivax: allele variants of the mdr1 gene do not associate with chloroquine resistance among isolates from Brazil, Papua, and monkey-adapted strains. Exp Parasitol. 2005;109:256–9.
Marques MM, Santana Costa MRF, Filho FS, Vieira JLF, Nascimento MTS, Brasil LW, et al. Plasmodium vivax chloroquine resistance and anemia in the Western Brazilian Amazon. Antimicrobial Agents Chemother. 2014;58:342–7.
Aguiar AC, Pereira DB, Amaral NS, De Marco L, Krettli AU. Plasmodium vivax and Plasmodium falciparum ex vivo susceptibility to anti-malarials and gene characterization in Rondônia, West Amazon. Brazil. Malar J. 2014;13:73.
Torres KL, Figueiredo DV, Zalis MG, Daniel-Ribeiro CT, Alecrim W, de Ferreira-da-Cruz M. F. Standardization of a very specific and sensitive single PCR for detection of Plasmodium vivax in low parasitized individuals and its usefulness for screening blood donors. Parasitol Res. 2006;98:519–24.
Brega S, de Monbrison F, Severini C, Udomsangpetch R, Sutanto I, Ruckert P, et al. Real-time PCR for dihydrofolate reductase gene single-nucleotide polymorphisms in Plasmodium vivax isolates. Antimicrob Agents Chemother. 2004;7:2581–7.
Otto TD, Vasconcellos EA, Gomes LH, Moreira AS, Degrave WM, Mendonça-Lima L, et al. ChromaPipe: a pipeline for analysis, quality control and management for a DNA sequencing facility. Genet Mol Res. 2008;23:861–71.
Añez A, Moscoso M, Laguna Á, Garnica C, Melgar V, Cuba M, et al. Resistance of infection by Plasmodium vivax to chloroquine in Bolivia. Malar J. 2015;14:261.
Fernández-Becerra C, Pinazo MJ, González A, Alonso PL, del Portillo HA, Gascón J. Increased expression levels of the pvcrt-o and pvmdr1 genes in a patient with severe Plasmodium vivax malaria. Malar J. 2009;8:55.
Barnadas C, Ratsimbasoa A, Tichit M, Bouchier C, Jahevitra M, Picot S, et al. Plasmodium vivax resistance to chloroquine in Madagascar: clinical efficacy and polymorphisms in pvmdr1 and pvcrt-o genes. Antimicrob Agents Chemother. 2008;52:4233–40.
Rungsihirunrat K, Muhamad P, Chaijaroenkul W, Kuesap J, Na-Bangchang K. Plasmodium vivax drug resistance genes; Pvmdr1 and Pvcrt-o polymorphisms in relation to chloroquine sensitivity from a malaria endemic area of Thailand. Korean J Parasitol. 2015;53:43–9.
Schousboe ML, Ranjitkar S, Rajakaruna RS, Amerasinghe PH, Morales F, Pearce R, et al. Multiple origins of mutations in the mdr1 gene: a putative marker of chloroquine resistance in P. vivax. PLoS Negl Trop Dis. 2015;9:e0004196.
Orjuela-Sánchez P, de Santana Filho FS, Machado-Lima A, Chehuan YF, Costa MR, Alecrim MD, et al. Analysis of single-nucleotide polymorphisms in the crt-o and mdr1 genes of Plasmodium vivax among chloroquine-resistant isolates from the Brazilian Amazon region. Antimicrob Agents Chemother. 2009;53:3561–4.
Gama BE, Oliveira NK, Souza JM, Daniel-Ribeiro CT, de Ferreira-da-Cruz M. F. Characterisation of pvmdr1 and pvdhfr genes associated with chemoresistance in Brazilian Plasmodium vivax isolates. Mem Inst Oswaldo Cruz. 2009;104:1009–11.
Jovel IT, Mejía RE, Banegas E, Piedade R, Alger J, Fontecha G, et al. Drug resistance associated genetic polymorphisms in Plasmodium falciparum and Plasmodium vivax collected in Honduras. Cent Am. Malar J. 2011;10:376.
Marfurt J, de Monbrison F, Brega S, Barbollat L, Müller I, Sie A, Goroti M, et al. Molecular markers of in vivo Plasmodium vivax resistance to amodiaquine plus sulfadoxine-pyrimethamine: mutations in pvdhfr and pvmdr1. J Infect Dis. 2008;198:409–17.
Fernando D, Rodrigo C, Rajapakse S. Primaquine in vivax malaria: an update and review on management issues. Malar J. 2011;10:351.
Neafsey DE, Galinsky K, Jiang RH, Young L, Sykes SM, Saif S, Gujja S, et al. The malaria parasite Plamodium vivax exhibits greater genetic diversity than Plasmodium falciparum. Nat Genet. 2012;44:1046–50.
MFFC carried out the study and the manuscript. LRG performed and analysis DNA sequencing and statistical analysis and drafted the manuscript. DM and CTDR participated in the discussions and reviewed the final manuscript. NKAO, SRFL and AL performed DNA extraction and PCRs. APC and PB recruited the patients. All authors read and approved the final manuscript.
CTDR and MFFC are recipients of a Research Productivity Fellowship from the National Brazilian Council for Scientific and Technological Development (CNPq) and from the Foundation for Research Support in the State of Rio de Janeiro (Faperj) as Cientistas do Nosso Estado. LRG receives a doctoral fellowship from CAPES, Brazilian Ministry of Education. The work was supported by the POM (Fiocruz), the PNCM, Secretary for Health Surveillance, Brazilian Ministry of Health and Fiocruz-Pasteur network. Sponsors had no role in the collection, analysis and interpretation of data, in the writing of the manuscript and in the decision to submit the manuscript for publication.
The authors declare that they have no competing interests.
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Gomes, L.R., Almeida-de-Oliveira, N.K., de Lavigne, A.R. et al. Plasmodium vivax mdr1 genotypes in isolates from successfully cured patients living in endemic and non-endemic Brazilian areas. Malar J 15, 96 (2016). https://doi.org/10.1186/s12936-016-1141-9