Research paperAnalysis of genetic diversity in the chloroquine-resistant gene Pfcrt in field Plasmodium falciparum isolates from five regions of the southern Cameroon
Introduction
Emergence and spread of multidrug resistance in Plasmodium falciparum remain a foremost obstacle and a pending problem for a successful chemotherapeutic control of the disease. This malaria parasite (considered as the most dangerous one) has developed resistance to almost all the antimalarial drugs available with the common ones being resistance to chloroquine (CQ) and sulphadoxine-pyrimethamine (SP) widespread in sub-Saharan Africa, South Asia and South America (Ngassa et al., 2014, WHO, 2015). Of particular concern is the reduced responsiveness of P. falciparum to the quinoline derivatives (such as chloroquine, amodiaquine, mefloquine, halofantrine and quinine). These antimalarial drugs which have been used in malaria chemotherapy for more than a century are still indispensable, serving as partner drugs to artemisinin derivatives. Much more interest is given to sub-Saharan Africa where the majority of malaria cases are registered (about 90% of the worldwide record) (WHO, 2015). Hence, monitoring drug resistance appears to be indispensable for early detection/later prevention of the spread of drug resistance by timely changes of treatment policy. There are three possibilities to study drug resistance (WHO, 2003): in vitro tests, in vivo tests and study of molecular markers (major interest of the present study).
CQ has so far been the most successful/used antimalarial drug (Cui et al., 2015) and understanding the mechanism of chloroquine resistance (CQR) remains crucial for tracking the malaria resistance progression. This is true since it has been involved in the spread of malaria in new areas where the disease was previously eradicated (Awasthi and Das, 2013). Several studies have then been conducted and genetic basis of the CQR was discovered (Wellems et al., 1991, Fidock et al., 2000). P. falciparum chloroquine resistance transporter (Pfcrt) gene was pinpointed as the main target for CQR, specially a mutation occurring at the 76th amino acid position (T for threonine instead of K for lysine) in the second exon of the gene. Furthermore, three others mutations (at the amino acids position 72, 74 and 75), adjoining this 76th T mutation have also been suggested for playing a compensatory role to maintain the fitness of CQ-resistant strains (Awasthi et al., 2011, Awasthi et al., 2012, Cui et al., 2015). With this respect, studies have been done in majority of the malaria endemic parts of the world including Cameroon (a west-central African country), to genotype these four mutations. Since mutations in Pfcrt gene are also known to be mediators of altered sensitivity to multiple artemisinin partner drugs including amodiaquine (an artemisinin partner used in Cameroon) (Cui et al., 2015), additional parasite polymorphisms are likely important in drug responsiveness, and an improved understanding of the roles of these polymorphisms is needed.
Cameroon is highly endemic for P. falciparum-malaria and CQR prevails in the whole country (Ngassa Mbenda and Das, 2014). Population genetic studies of the Pfcrt gene in Cameroonian field P. falciparum isolates are mostly restricted to screening/genotyping of amino acid residues at the positions 72, 73, 74, 75 and 76 due to their substantial implication with chloroquine resistance (Cui et al., 2015, Pelleau et al., 2015). However, studies with direct sequencing of the entire second exon of the Pfcrt gene in view to detect new mutations in field isolates in and around these known mutations are so far almost inexistent in the literature of Cameroonian P. falciparum. Considering that malaria is a multifactorial disease with the epidemiological outcomes differing in different ecotypes of the globe (Das et al., 2012, Kaewwaen and Bhumiratana, 2015, Kar et al., 2014, Sharma et al., 2015a), researchers have highlighted the necessity to evaluate whether different local eco-climatic factors have influenced genetic changes at the genes conferring resistance to different antimalarials (Sorosjinda-Nunthawarasilp and Bhumiratana, 2014, Kar et al., 2016). In regard to this, Cameroon projected as “Africa in miniature” for malaria epidemiology (Ngassa et al., 2014) therefore represents a suitable place for such studies.
We have decided in the present study to scrutinize the genetic variability of the Cameroonian Pfcrt by sequencing fully its second exon in six populations of P. falciparum from five distinct regions of the southern country (the most malaria endemic part), differing from each other eco-climatically and having different malaria endemicity (Ngassa et al., 2014): East, Littoral, Centre, South, border of Cameroon-Gabon-Equatorial Guinea.
Section snippets
Samples collection and molecular methods
180 finger prick blood samples from malaria patients infected by P. falciparum have been screened in this study. The malaria parasites species were confirmed by Nested Polymerase Chain Reaction as described in previous studies (Johnston et al., 2006, Chauhan et al., 2013, Ngassa Mbenda and Das, 2014), after genomic DNA isolation following Qiagen protocol (Germany). The samples were collected in 2012 (8 years after the official adoption of ACTs in Cameroon) from six different district hospitals
PCR amplification and sequencing of the exon 2 of Cameroonian Pfcrt gene
The PCR amplification and DNA sequencing of the second exon of Pfcrt gene (264 bp) in a total of 180 field isolates from six different field populations of Cameroonian P. falciparum have been successfully performed. Multiple sequence alignment of these 180 DNA data revealed the presence of seven point mutations (six non-synonymous and one synonymous, Fig. 1), out of which two (one non-synonymous and one synonymous) were completely new (never reported in Cameroon so far). The six different
Discussion
Monitoring the level of P. falciparum resistance against anti-malarial drugs seems to be one of the keys to a successful malaria control in malaria endemic parts of the globe in general, and in Cameroon in particular. Even though controlled clinical trials are the best available tool for assessing the relevance of anti-malarial treatments, molecular monitoring offers some advantages (e.g. Studies on single-nucleotide polymorphisms (SNPs) can be carried out with more ease and are less
Funding information
H.G.M.N. thanks the Department of Biotechnology (DBT), New Delhi, India and the Third World Academy of Sciences (TWAS), Trieste, Italy for the award of Postgraduate Fellowship (2008 DBT-TWAS Postgraduate Fellowship FR number: 3240201438). AD thanks the ICMR for intramural funding support. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Competing interests
The authors declare no conflict of financial interest.
Acknowledgments
This study would not have been possible without the willingness of all the patients to participate in the study; for this we are indebted. We are highly grateful to the Ethical Committee of Cameroon for the Ethical Clearance for this study and the Director of National Institute of Malaria Research (NIMR) for facilities. The authors are thankful to Prof. Inocent Gouado of the University of Douala in Cameroon-Faculty of Sciences, to Dr. Mfopou Soule of New-Bell District Hospital, Douala, Cameroon
References (55)
- et al.
Population genetic analyses of Plasmodium falciparum chloroquine receptor transporter gene haplotypes reveal the evolutionary history of chloroquine-resistant malaria in India
Int. J. Parasitol.
(2011) - et al.
Analyses of genetic variations at microsatellite loci present in-and-around the Pfcrt gene in Indian Plasmodium falciparum
Infect. Genet. Evol.
(2013) The distinctive features of Indian malaria parasites
Trends Parasitol.
(2015)- et al.
Malaria in India: the center for the study ofcomplex malaria in India
Acta Trop.
(2012) - et al.
Variations in the sequence and expression of the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and their relationship to chloroquine resistance in vitro
Mol. Biochem. Parasitol.
(2004) - et al.
Assessing the effect of natural selection in malaria parasites
Trends Parasitol.
(2004) - et al.
Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance
Mol. Cell
(2000) - et al.
Comparative assessment on the prevalence of mutations in the Plasmodium falciparum drug-resistant genes in two different ecotypes of Odisha state, India
Infect. Genet. Evol.
(2016) On the number of segregating sites in genetic models without recombination
Theor. Popul. Biol.
(1975)- et al.
Amodiaquine-Artesunate versus artemether-lumefantrine against uncomplicated malaria in children less than 14 years in Ngaoundere, North Cameroon: efficacy, safety, and baseline drug resistant mutations in pfcrt, pfmdr1, and pfdhfr genes
Malar. Res. Treat.
(2013)
Microsatellite markers reveal a spectrum of population structures in the malaria parasite Plasmodium falciparum
Mol. Biol. Evol.
Effect of recombination on the accuracy of the likelihood method for detecting positive selection at amino acid sites
Genetics
Genetics of chloroquine-resistant malaria: a haplotypic view
Mem. Inst. Oswaldo Cruz
Pfcrt haplotypes and the evolutionary history of chloroquine-resistant Plasmodium falciparum
Mem. Inst. Oswaldo Cruz
Haploview: analysis and visualization of LD and haplotype maps
Bioinformatics
Origin and dissemination of chloroquine-resistant Plasmodium falciparum with mutant pfcrt alleles in the Philippines
Antimicrob. Agents Chemother.
Malaria variorum
Nature
Entomological monitoring and evaluation: diverse transmission settings of ICEMR projects will require local and regional malaria elimination strategies
Am.J.Trop. Med. Hyg.
Antimalarial drug resistance: literature review and activities and findings of the ICEMR network
Am.J.Trop. Med. Hyg.
Sequence analysis of coding DNA fragments of pfcrt and pfmdr-1 genes in Plasmodium falciparum isolates from Odisha, India
Mem. Inst. Oswaldo Cruz
Inferring the population structure and demography of Drosophila ananassae from multilocus data
Genetics
Statistical tests of neutrality of mutations against population growth, hitchhiking and background selection
Genetics
Statistical tests of neutrality of mutations
Genetics
Molecular evolution of PvMSP3α block II in Plasmodium vivax from diverse geographic origins
PLoS One
Detecting the genetic signature of natural selection in human populations: models, methods, and data
Annu. Rev. Anthropol.
PCR as a confirmatory technique for laboratory diagnosis of malaria
J. Clin. Microbiol.
Landscape ecology and epidemiology of malariaassociated with rubber plantations in Thailand: integrated approaches to malariaecotoping
Interdiscip. Perspect. Infect. dis.
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- 1
Currently Postdoctoral Fellow in Pain Laboratory, Brain Function Research Group, School of Physiology, Faculty of Health Sciences, University of Witwatersrand, Johannesburg, South Africa.
- 2
Currently Director of the Centre for Research in Medical Entomology, Plot No. 4, Sarojini Street, Chinna Chokkikulam, Madurai - 625002, Tamilnadu State, India.