Molecular detection of drug resistant polymorphisms in Plasmodium falciparum isolates from Southwest, Nigeria

doi:10.21203/rs.3.rs-35555/v2

Download PDF

Research note

Molecular detection of drug resistant polymorphisms in Plasmodium falciparum isolates from Southwest, Nigeria

https://doi.org/10.21203/rs.3.rs-35555/v2

This work is licensed under a CC BY 4.0 License

Journal Publication

published 27 Oct, 2020

Read the published version in BMC Research Notes →

You are reading this older preprint version

Read the latest preprint version →

Objective: Nigeria bears 25% of global malaria burden despite concerted efforts towards its control and elimination. The emergence of drug resistance to first line drugs, artemisinin combination therapies (ACTs), indicates an urgent need for continuous molecular surveillance of drug resistance especially in high burden countries where drug interventions are heavily relied on. This study describes mutations in Plasmodium falciparum genes associated with drug resistance in malaria; Pfk13, Pfmdr1, PfATPase6 and Pfcrt in isolates obtained from 83 symptomatic malaria patients between 1-61 years old from Lagos, a Mega city in South-west Nigeria.

Results: Two Pfmdr1, N86 and Y184 variants were present at a prevalence of 56% and 13.25% of isolates respectively. There was one synonymous (S679S) and two non-synonymous (M699V, S769M) mutations in the Pfatp6 gene, while Pfcrt genotype (CVIET), had a prevalence of 45%. The Pfk13 C580Y mutant allele was suspected by allelic discrimination in two samples with mixed genotypes. Our findings calls for robust molecular surveillance of antimalarial drug resistance markers in west Africa especially with increased use of antimalarial drugs as prophylaxis for covid-19.

Infectious Diseases

Molecular Epidemiology

artemisinin

malaria

K13

drug resistance

Nigeria

Malaria infects over 220 million people with at least 405,000 deaths annually, 67% of those deaths occur in children under the age of five, and 94% of deaths according to 2018 World Health Organization data occurred in Africa (1). Drug resistance of malaria parasites to previously efficacious first line chemotherapies, chloroquine (CQ) and sulfadoxine-pyrimethamine (SP), in sub-Saharan Africa (SSA), led to replacement with artemisinin combination therapies (ACT), and complete removal of CQ (1,2). CQ resistance (Pfcrt) is as a result of a single point mutation K76T, in codons 72-76 and has been the main cause of resistance (3,4), while mutations in the dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) were responsible for SP resistance in parasites (5,6).

P. falciparum has also developed resistance to ACTs, which combines a fast-acting artemisinin (ART) derivative with a long-lasting partner such as Lumefantrine, Mefloquine or Amodiaquine. ACT resistance is widespread in the Greater Mekong subregion (GMS) in southeast Asia (1,7). Resistance to ART and its derivatives have been confirmed to be associated with mutations in PfK13. Mutations in PfK13 have been reported in GMS, Guyana, Rwanda, and Tanzania (1). In SSA, PfK13 mutations are yet to be associated with partial or complete ART-resistant parasite isolates, and ACT efficacy still remains very high. However, there have been increasing reports of ACT failures, or delayed parasite clearance within Africa suggesting the need to investigate the factors that might be responsible (8,9). Mutations in Pfmdr1 on codons 86, 184, 1246 and copy number amplifications have also been linked to susceptibility to drugs including ACTs. P. falciparum sarco-endoplasmic reticulum calcium-ATPase (SERCA) type protein encoded by a gene PfATPase6 has been described to modulate the susceptibility of parasites to ART. PfATPase6 protein of the parasite has been suggested to be the target of ARTs (10–12).

Following reports of PfK13 mutations in Rwanda, Tanzania, and delay in parasite clearance after ACT use, continuous genomic surveillance for drug resistant parasites is necessary to sustain malaria control and elimination efforts. Hence, it is vital to monitor for the emergence or introduction of mutations associated with ACT resistance particularly in Africa which bears >90% of the deaths globally. This study describes the molecular prevalence of mutations in the drug resistant genes Pfk13, Pfmdr1, PfATPase6 and Pfcrt from P. falciparum clinical samples in Southwest Nigeria, where Arthemeter Lumefantrine is the ACT used for treatment.

Study site

This study was carried out in two communities - Badagry (Lagos State) and Alajue (Ede, Osun State. Badagry (6^o25’N 2⁰53’E), is a coastal town with an area of 170m² and a human population of 241,093 (NPC, 2006) that borders the Republic of Benin. Alajue village (7⁰40’N 4⁰30’E), is an ancient Yoruba town with a total area of 130m² and a human population of 159,866 (NPC, 2006). Both towns are located in the south western part of Nigeria, with similar environmental conditions, occupation and lifestyle of the people in those communities.

Sampling

A total of 83 symptomatic malaria patients were recruited for this study. Following informed consent of the participants, parent or guardian, they were tested with malaria rapid diagnostic test (RDT) kit and 2 ml blood samples collected into RNAlater and dried blood spots (DBS) made on 3MM Whatman filter paper.

Nucleic acid extraction

DNA from DBS was extracted using the Qiagen Mini Kit (Qiagen) according to manufacturer’s instructions and stored at minus 20 ^oC until needed. Total RNA was isolated from whole blood stored in RNAlater was using PureLink^TM RNA Mini Kit (Invitrogen) following the manufacturer’s instructions.

Plasmodiumfalciparum molecular detection

DNA from DBS of each sample was used for molecular speciation of P. falciparum by nested PCR through amplification of the 18SrRNA following established protocols (13). Nest 1 amplified a large part of the 18S rRNA common to the Plasmodium genus, while the Nest 2 amplified a region in the genus specific for that species of Plasmodia. Differentiation of the species was based on the molecular weight with P. falciparum having a size of 205bp. PCR fragments. were detected and sized on the QIAXCEL automated electrophoresis system.

Quantitative PCR

To determine the gene expression levels of Pfk13 and PfATPase, total RNA was treated to remove genomic DNA by digesting with 2 µl of DNaseI (Fermentas) and 5 µl of reaction buffer, incubated at 37^oC for 30 minutes and inactivated with 5 µl, 25 mM EDTA, 65^oC for 10 minutes. RNA purity and concentration was determined using a NanoDrop 1000TM (Thermo Scientific). cDNA synthesis was carried out using the RNA Reverse Transcriptase kit (Invitrogen). Synthesized cDNA was quantified by qPCR performed on a CFX 96 (Bio-Rad) with the following cyclic conditions: 95^oC, 10 minutes, 49 cycles of 95 ^oC, 15 seconds and 60^oC for 90 seconds. Relative fold increase of specific mRNA transcripts in samples was compared to P. falciparum (3D7) wildtype control, normalised using the 18SrRNA housekeeping gene, and expression was calculated using 2^-^{ΔΔ Ct} method. Data was analysed using at least 3 independent experiments.

PfK13 and PfATPase genotyping

Alleles of Pfk13 propeller domain polymorphisms (Y493H, R539T, I543T, C580Y), and PfATPase6 (S679S, M699V, S769M) associated with delayed clearance were determined by Taqman allelic discrimination and sequencing and list of primers (Tables S1&S2), used provided in supplementary materials. For each sample a working Master Mix was prepared to include 1x of Taqman Universal PCR Master Mix (Life Technologies), 300nM of the forward and reverse primers, 200 nM of each allele specific probes (Table S3) and at least 5 ng of DNA from DBS in 25 µl reaction volume. Amplification was done on the Bio-Rad CFX96 real-time thermocycler set to detect fluorescent emissions for 6-carboxyfluorescein (6-FAM) (mutant) and hexachloro-6-carboxyfluorescein (HEX) (wild type). . Each SNP was amplified in a thermocycle of 50 ^oC for 2 min, 10 min of initial template denaturation and enzyme activation at 95^oC followed by 50 cycles of 92 ^oC for 15 seconds and 60 ^oC for 1 minute. Allelic discrimination analysis was performed with the Bio-Rad CFX manager with parameters set to subtract background and correct for fluorescent drift prior to clustering of wild or mutant amplicons. All PCRs and digests included as controls the DNA from P. falciparum 3D7 as wildtype control.

Data analysis

Descriptive statistics was carried out in Microsoft Excel 2010, sequence alignment (on CLC Main Workbench Version 6.7.1) and translation of nucleotide sequence to amino acid sequences and editing of sequences (MEGA 7.0.4). A P-value of ≤ 0.05 was considered statistically significant. Transcript level and allelic discrimination assays were analysed on the Bio-Rad CFX96 manager software.

Demographics

In this study, 83 patients, 36 (43.37%) male and 47 (56.63%) females presenting with symptoms of malaria were recruited in the study. The age distribution were as follows; 6 (0-5 yrs), 34 (6-19 yrs) 25 (20-35 yrs) and 18 were >35 yrs.

mRNA transcript levels and polymorphisms in Pfk13 and PfATPase6 genes

Following confirmation that all RDT positive samples were P. falciparum positive by PCR, we carried out gene expression and only Pfk13 wild type transcript was detected in samples. Relatively lower levels PfATPase transcript was also detected with both wild and mutant strains (Figure 1). By probe specific allelic discrimination, both wild type and mutant Pfk13 and PfATPase6 alleles were detected for known drug resistance SNPs (Figure 2). The PfK13 C580Y allele was suspected in 2 isolates, which were mixed infections. Sanger sequencing was carried out for amplicons from all isolates and these were analysed for SNPs against the reference strain P. falciparum 3D7 using DNASTAR LaserGene. Eight non-synonymous SNPs in Pfk13, all in single isolates. A deletion variant A557 was identified in 3.61% of isolates sequenced (Table S3).

Sequence analysis of PfATPase revealed 3 SNPs, one synonymous and two non-synonymous mutations (supplementary Table 2). The most reported ACT resistance related SNP (S769) was present in 3.6% of samples. Pfmdr1 wildtype N86 allele was present in 56% of isolates sequenced. Pfmdr1 Y184 was at much lower prevalence of 13.25% (supplementary Table 2). Pfcrt 72-76 variants were translated and haplotypes inferred. The CVIET haplotype associated with CQ resistance had a prevalence of 45%, while the wildtype CVMNK was found in 55% of isolates from the population (Figure 3).

Artemisinin based combination therapy was introduced into Nigeria in 2005 following the withdrawal of CQ. Chemotherapy is one of the main malaria control strategies implemented by the National Malaria Control program in Nigeria. Hence, there is need to continuously monitor drug resistant genes that are associated with reduced susceptibility. This study characterized four different genetic markers of resistance Pfcrt, Pfmdr1, PfATPase and Pfk13 that have been implicated in ACT resistance (2,3,9,14).

Analysis of mRNA transcripts demonstrated that most isolates expressed the wild type Pfk13 while both the wild and mutant PfATPase variants were expressed by different strains. The Pfk13 C580Y mutant suspected in the two mixed infections were in the minority and mRNA expression of Pfk13 mutants might have been masked by the predominant Pfk13 wildtype parasites. However, the possibility of the Pfk13 580Y resistance mutations is a call for concern as these have been shown to lead to reduction in parasite susceptibility to ACTs. Studies carried out in vitro using forward genetic approaches have implicated C580Y mutation in delayed parasite clearance (18,19). Few African countries have reported delayed ACT clearance, and the mechanisms of delayed clearance in African parasites is speculated to be different from that observed in south east Asia, raising questions about the roles of these mutations in African isolates. However, different Pfk13 haplotypes containing mutations at positions 578 and 581, close to the C580Y mutation are known to circulate in Africa. The A578S has been observed in Mali, Kenya, Gabon, Ghana and Uganda respectively (20). Similarly, prior to ACT introduction, K13-propeller mutations were found in parasites collected from Mali, suggesting that these haplotypes might have existed at low frequencies prior to the widespread use of ARTs (20).

These early molecular signs should be taken seriously to prevent the spread of Pfk13 and reduced efficacy of ACTs in Africa given increased fitness in a population which heavily relies on drugs against high levels of infection and morbidity.

We also detected polymorphisms of PfATPase6, the R₃₇K, A6₃₀S, I8₉8I found in Brazil, double mutation E431K, A623E in Senegal, and H₂₄₃Y in Central Africa. The PfATPase6 S769 SNP we detected has been widely reported as a potential molecular marker for P. falciparum resistance to artemether (15). This SNP is found within the cytoplasmic N (nucleotide binding) domain close to the conserved hinge, which in many species is important for structural transitions in the PfATPase cycle, calcium binding and release (14).

Other well-known characterized mutations in drug resistant genes such as Pfmdr1- N86Y and Y184F, and pfcrt- CVIET haplotype were also detected. Polymorphisms of Pfmdr1 (N86Y, Y184F, S1034C, N1042D, and D1246Y) and copy number amplifications have been described in field isolates and associated with reduced efficacy of artemether lumefantrine/mefloquine (21). Artemther-Lumefantrine is the most common ACT combination distributed in Nigeria and selects for wildtype N86 and mutant Y184, as shown for other populations in West Africa (22). Surprisingly the CVIET haplotype was recorded at high prevalence despite the withdrawal of CQ from the population over 10 years prior to this study. The high prevalence (45%) observed for Pfcrt-76 T resistance marker in southwest Nigeria is comparable to reports from the south-eastern part of the country, where prevalence was as high as 75% (23). Lumefantrine selects for wildtype Pfcrt K76, reversing CQ resistance that is strongly linked to the CVIET haplotype (3). This reversal to the CQ susceptible haplotypes CVMNK following CQ withdrawal has been reported across malaria endemic regions; in China (24), Tanzania (25), Kenya (26) and Malawi (27), or a decline as observed in Ghana (28). Exception are only seen in countries like Ethiopia (29) were CQ is still being administered to treat P. vivax, hence still exerting drug pressure on the parasite to retain the resistant haplotypes. In Nigeria, Chloroquine remains accessible through private drug supplies together with amodiaquine, both of which could be slowing the reversal of the resistant haplotype in the population.

This study provides a molecular profile of the drug resistance genes in malaria parasites from Nigeria, highlighting the need for continuous surveillance for antimalarial resistance using a panel of drug resistance molecular markers.

Our limited study locations and sample size is not sufficient to substantiate the spread of artemisinin related mutations in the population.

Availability of data and materials

All data generated or analysed during this study are included.

Ethics approval and consent to participate

Ethical approval for this study, IRB/13/218 was approved by the Institutional Review Board of the Nigerian Institute of Medical Research, Yaba, Lagos, Nigeria. Written informed consent was obtained from all participants in the study. All individuals positive for malaria were treated with Arthemeter-Lumefantrine by a nurse or clinician.

Consent for publication

All authors approved the final version of this manuscript for publishing.

Availability of data and materials

All data generated or analysed during this study are included in this published article [and its supplementary information files].

Competing interests

The authors declare that they have no competing interests

Funding

The work was supported by funds from an MRC Career Development fellowship (Award MR/K02440X/1) to Dr. Alfred Amambua Ngwa through the WT/MRC/DFID/EDCTP Global Health Scheme.

Authors' contributions

MT designed the study, performed experiments, analysed the data and wrote the first draft of the manuscript. OA wrote the manuscript, analysed the data and edited the final draft of the manuscript. ETI and STA, designed the study, supervised and approved the final draft. AAN designed the study, analysed the data, provided resources for the research and edited and approved the final draft of the manuscript.

Acknowledgements

We wish to acknowledge all the parents and participants of this study including the management and staff of the hospitals where samples were collected (especially the laboratory scientist) and the support of laboratory staff at the Public Health Division of the Nigerian Institute of Medical Research and MRCG at LSHTM The Gambia.

ART: Artemisinin

ACT: Artemisinin combination therapy

CQ: Chloroquine

SSA: Sub Saharan Africa

PCR: Polymerase chain reaction

NPC: National population commission

SNP: Single nucleotide polymorphisms

World Health Organisation. Malaria [Internet]. 2020. Available from: https://www.who.int/news-room/fact-sheets/detail/malaria
Ashley EA, Dhorda M, Fairhurst RM, Amaratunga C, Lim P, Suon S, et al. Spread of Artemisinin Resistance in Plasmodium falciparum Malaria. N Engl J Med. 2014 Jul 31;371(5):411–23.
Djimdé A, Doumbo OK, Cortese JF, Kayentao K, Doumbo S, Diourté Y, et al. A Molecular Marker for Chloroquine-Resistant Falciparum Malaria. N Engl J Med. 2001 Jan 25;344(4):257–63.
Fidock DA, Nomura T, Talley AK, Cooper RA, Dzekunov SM, Ferdig MT, et al. Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Mol Cell. 2000 Oct;6(4):861–71.
Kublin JG, Dzinjalamala FK, Kamwendo DD, Malkin EM, Cortese JF, Martino LM, et al. Molecular markers for failure of sulfadoxine-pyrimethamine and chlorproguanil-dapsone treatment of Plasmodium falciparum malaria. J Infect Dis. 2002 Feb 1;185(3):380–8.
van Eijk AM, Larsen DA, Kayentao K, Koshy G, Slaughter DEC, Roper C, et al. Effect of Plasmodium falciparum sulfadoxine-pyrimethamine resistance on the effectiveness of intermittent preventive therapy for malaria in pregnancy in Africa: a systematic review and meta-analysis. Lancet Infect Dis [Internet]. 2019 Mar [cited 2019 Apr 4]; Available from: https://linkinghub.elsevier.com/retrieve/pii/S1473309918307321
WWARN K13 Genotype-Phenotype Study Group. Association of mutations in the Plasmodium falciparum Kelch13 gene (Pf3D7_1343700) with parasite clearance rates after artemisinin-based treatments—a WWARN individual patient data meta-analysis. BMC Med. 2019 Dec;17(1):1.
Adams T, Ennuson NAA, Quashie NB, Futagbi G, Matrevi S, Hagan OCK, et al. Prevalence of Plasmodium falciparum delayed clearance associated polymorphisms in adaptor protein complex 2 mu subunit (pfap2mu) and ubiquitin specific protease 1 (pfubp1) genes in Ghanaian isolates. Parasit Vectors. 2018 Dec;11(1):175.
Borrmann S, Sasi P, Mwai L, Bashraheil M, Abdallah A, Muriithi S, et al. Declining Responsiveness of Plasmodium falciparum Infections to Artemisinin-Based Combination Treatments on the Kenyan Coast. Mueller I, editor. PLoS ONE. 2011 Nov 10;6(11):e26005.
Uhlemann A-C, Cameron A, Eckstein-Ludwig U, Fischbarg J, Iserovich P, Zuniga FA, et al. A single amino acid residue can determine the sensitivity of SERCAs to artemisinins. Nat Struct Mol Biol. 2005 Jul;12(7):628–9.
Eckstein-Ludwig U, Webb RJ, van Goethem IDA, East JM, Lee AG, Kimura M, et al. Artemisinins target the SERCA of Plasmodium falciparum. Nature. 2003 Aug;424(6951):957–61.
Duraisingh MT, Jones P, Sambou I, von Seidlein L, Pinder M, Warhurst DC. The tyrosine-86 allele of the pfmdr1 gene of Plasmodium falciparum is associated with increased sensitivity to the anti-malarials mefloquine and artemisinin. Mol Biochem Parasitol. 2000 Apr;108(1):13–23.
Snounou G, Viriyakosol S, Jarra W, Thaithong S, Brown KN. Identification of the four human malaria parasite species in field samples by the polymerase chain reaction and detection of a high prevalence of mixed infections. Mol Biochem Parasitol. 1993 Apr;58(2):283–92.
Jambou R, Legrand E, Niang M, Khim N, Lim P, Volney B, et al. Resistance of Plasmodium falciparum field isolates to in-vitro artemether and point mutations of the SERCA-type PfATPase6. The Lancet. 2005 Dec;366(9501):1960–3.
Pillai DR, Lau R, Khairnar K, Lepore R, Via A, Staines HM, et al. Artemether resistance in vitro is linked to mutations in PfATP6 that also interact with mutations in PfMDR1 in travellers returning with Plasmodium falciparum infections. Malar J. 2012;11(1):131.
Ariey F, Witkowski B, Amaratunga C, Beghain J, Langlois A-C, Khim N, et al. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature. 2014 Jan;505(7481):50–5.
World Health Organization. Artemisinin and artemisinin-based combination therapy resistance: status report. [Internet]. 2016 [cited 2019 Mar 17]. Available from: https://apps.who.int/iris/bitstream/handle/10665/208820/WHO_HTM_GMP_2016.5_eng.pdf?sequence=1&isAllowed=y
Straimer J, Gnadig NF, Witkowski B, Amaratunga C, Duru V, Ramadani AP, et al. K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates. Science. 2015 Jan 23;347(6220):428–31.
Ghorbal M, Gorman M, Macpherson CR, Martins RM, Scherf A, Lopez-Rubio J-J. Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system. Nat Biotechnol. 2014 Aug;32(8):819–21.
Ouattara A, Kone A, Adams M, Fofana B, Maiga AW, Hampton S, et al. Polymorphisms in the K13-propeller gene in artemisinin-susceptible Plasmodium falciparum parasites from Bougoula-Hameau and Bandiagara, Mali. Am J Trop Med Hyg. 2015 Jun;92(6):1202–6.
Foote SJ, Kyle DE, Martin RK, Oduola AMJ, Forsyth K, Kemp DJ, et al. Several alleles of the multidrug-resistance gene are closely linked to chloroquine resistance in Plasmodium falciparum. Nature. 1990 May;345(6272):255–8.
Amambua-Ngwa A, Okebe J, Mbye H, Ceesay S, El-Fatouri F, Joof F, et al. Sustained Ex Vivo Susceptibility of Plasmodium falciparum to Artemisinin Derivatives but Increasing Tolerance to Artemisinin Combination Therapy Partner Quinolines in The Gambia. Antimicrob Agents Chemother [Internet]. 2017 Dec [cited 2019 Mar 17];61(12). Available from: http://aac.asm.org/lookup/doi/10.1128/AAC.00759-17
Ikegbunam MN, Nkonganyi CN, Thomas BN, Esimone CO, Velavan TP, Ojurongbe O. Analysis of Plasmodium falciparum Pfcrt and Pfmdr1 genes in parasite isolates from asymptomatic individuals in Southeast Nigeria 11 years after withdrawal of chloroquine. Malar J. 2019 Dec;18(1):343.
Wang X, Mu J, Li G, Chen P, Guo X, Fu L, et al. Decreased prevalence of the Plasmodium falciparum chloroquine resistance transporter 76T marker associated with cessation of chloroquine use against P. falciparum malaria in Hainan, People’s Republic of China. Am J Trop Med Hyg. 2005 Apr;72(4):410–4.
Mohammed A, Ndaro A, Kalinga A, Manjurano A, Mosha JF, Mosha DF, et al. Trends in chloroquine resistance marker, Pfcrt-K76T mutation ten years after chloroquine withdrawal in Tanzania. Malar J. 2013;12(1):415.
Mwai L, Ochong E, Abdirahman A, Kiara SM, Ward S, Kokwaro G, et al. Chloroquine resistance before and after its withdrawal in Kenya. Malar J. 2009;8(1):106.
Kublin JG, Cortese JF, Njunju EM, G. Mukadam RA, Wirima JJ, Kazembe PN, et al. Reemergence of Chloroquine‐Sensitive Plasmodium falciparum Malaria after Cessation of Chloroquine Use in Malawi. J Infect Dis. 2003 Jun 15;187(12):1870–5.
Alam MT, de Souza DK, Vinayak S, Griffing SM, Poe AC, Duah NO, et al. Selective Sweeps and Genetic Lineages of Plasmodium falciparum Drug -Resistant Alleles in Ghana. J Infect Dis. 2011 Jan 15;203(2):220–7.
Golassa L, Enweji N, Erko B, Aseffa A, Swedberg G. High prevalence of pfcrt-CVIET haplotype in isolates from asymptomatic and symptomatic patients in south-central Oromia, Ethiopia. Malar J. 2014 Dec;13(1):120.

Download PDF

Journal Publication

published 27 Oct, 2020

Read the published version in BMC Research Notes →

Editorial decision: Minor revision
13 Sep, 2020
Review #2 received at journal
08 Sep, 2020
Reviewer #2 agreed at journal
21 Aug, 2020
Review #1 received at journal
21 Aug, 2020
Reviewers invited by journal
18 Aug, 2020
Reviewer #1 agreed at journal
18 Aug, 2020
Editor assigned by journal
17 Aug, 2020
Submission checks completed at journal
16 Aug, 2020
Editor invited by journal
16 Aug, 2020

You are reading this older preprint version

Read the latest preprint version →

Molecular detection of drug resistant polymorphisms in Plasmodium falciparum isolates from Southwest, Nigeria

Status:

Journal Publication

Version 2

Abstract

Figures

Introduction

Materials And Methods

Results

Discussion

Limitations

Declarations

Competing interests

The work was supported by funds from an MRC Career Development fellowship (Award MR/K02440X/1) to Dr. Alfred Amambua Ngwa through the WT/MRC/DFID/EDCTP Global Health Scheme.

Authors' contributions

Abbreviations

References

Supplementary Files

Status:

Journal Publication

Version 2