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Survey of Plasmodium falciparum multidrug resistance-1 and chloroquine resistance transporter alleles in Haiti
© ElBadry et al.; licensee BioMed Central Ltd. 2013
Received: 17 August 2013
Accepted: 25 October 2013
Published: 19 November 2013
In Haiti where chloroquine (CQ) is widely used for malaria treatment, reports of resistance are scarce. However, recent identification of CQ resistance genotypes in one site is suggestive of an emerging problem. Additional studies are needed to evaluate genetic mutations associated with CQ resistance, especially in the Plasmodium falciparum multi-drug resistance- 1 gene (pfmdr 1) while expanding the already available information on P. falciparum CQ transporter gene (pfcrt) in Haiti.
Blood samples were collected on Whatman filter cards (FTA) from eight clinics spread across Haiti. Following the confirmation of P. falciparum in the samples, PCR protocols were used to amplify regions of pfmdr 1and pfcrt codons of interest, (86, 184, 1034, 1042, and 1246) and (72-76), respectively. Sequencing and site-specific restriction enzyme digestions were used to analyse these DNA fragments for the presence of single nucleotide polymorphisms (SNPs) known to confer resistance to anti-malarial drugs.
P. falciparum infection was confirmed in160 samples by amplifying a segment of the P. falciparum 18S small subunit ribosomal RNA gene (pfssurrn a). The sequence of pfmdr1 in 54 of these samples was determined between codons 86,184 codons 1034, 1042 and 1246. No sequence differences from that of the NF54 clone 3D7 were found among the 54 samples except at codon 184, where a non-silent mutation was found in all samples predicted to alter the amino acid sequence replacing tyrosine with phenylalanine (Y184F). This altered sequence was also confirmed by restriction enzyme digestion. The sequence of pfmdr 1 at codons 86, 184, 1034 and 1042 encoded the NF SN haplotype. The sequence of pfcrt codons 72-76 from 79 samples was determined and found to encode CVMNK, consistent with a CQ sensitive genotype.
The presence of the Y184F mutation in pfmdr1 of P. falciparum parasites in Haiti may have implications for resistance to antimalarial drugs. The absence of mutation in pfcrt at codon 76 among 79 isolates tested suggests that sensitivity to CQ in Haiti remains common. Wide-spread screening of the pfmdr1 and pfcrt especially among patients experiencing treatment failure may be a useful tool in early detection of the emergence of antimalarial drug resistance in Haiti.
An estimated 30,000 cases of malaria infections are reported annually in Haiti [1, 2], where transmission is hypo-endemic with sporadic epidemics fueled by heavy rainfall. Malaria infections in Haiti are dominantly caused by Plasmodium falciparum[3, 4]. Chloroquine (CQ) has been in use as an anti-malarial since the 1950s , and is currently used extensively in the treatment of malaria in Haiti . Recently, a national malaria treatment policy revision added single dose primaquine to target gametocytes . Despite long-term use, there are scarce reports of CQ-resistant P. falciparum malaria in Haiti [6–8]. However, the recent reports of CQ-resistant P. falciparum parasites harbouring known resistant alleles of pfcrt[9, 10] are alarming and could present serious challenges to clinical management of malaria in Haiti and diminish prospects for its elimination from Hispaniola (the Caribbean island where both Haiti and the Dominican Republic are located).
Research studies conducted more than three decades ago found only CQ sensitive P. falciparum strains [6, 7], but very few follow-up studies have been conducted during the intervening years. In the recent past, a few studies identified the CQ-resistance-associated K76T mutation in the pfcrt at a very low prevalence in samples from the Artibonite valley, from Leogane, and from travellers returning from Haiti [8–10]. The potential role of resistant alleles of pfmdr1 in CQ resistance in Haiti has yet to be investigated. In addition, there is a need to screen for the CQ resistance-associated pfcrt and pfmdr1 alleles in other parts of Haiti.
Single nucleotide polymorphisms (SNPs) in the pfmdr1 have been associated with resistance to CQ and other anti-malarial drugs including amodiaquine (AQ) and artemether-lumefantrine (AL). The P. falciparum glycoprotein homologue-1 found in the digestive vacuole of the malaria parasites is encoded by pfmdr1 gene . CQ diffuses to parasite food vacuole (FV) membrane neutrally where it protonates by acids present, which prevents free diffusion out the vacuole and results in accumulation of CQ in sensitive parasites. In CQ resistant parasites, changes in FV membrane proteins encoded by pfcrt and pfmdr1 genes are thought to play a role in reducing accumulation of drug inside the vacuole [11–14]. The role played by pfmdr1 mutations (N86Y, Y184F, S1034C and D1246Y) in mediating in vivo and in vitro CQ resistance has received a lot of research interest [13, 15–23]. No previous reports have described alleles of pfmdr1 that may be associated with anti-malarial drug resistance in Haiti. Therefore, this research study evaluated in Haiti the mutations in pfmdr1 that in other regions of the world are associated with resistance to CQ and expanded the surveillance locations in Haiti for CQ resistance mutations in pfcrt. This study complements the anti-malarial drug resistance surveillance efforts of the Haitian government.
Plasmodium falciparum malaria infected blood samples were collected by finger prick and preserved as dried blood spots (DBS) on FTA cards (Whatman GE Healthcare, MA, USA) between May 2010 and June 2012. Samples were obtained from patients attending study clinics, meeting the inclusion criteria (any patient above the age of two year old with malaria-like symptoms) who tested positive with rapid diagnostic test (CareStat™First Response Malaria Ag pLDH/HRP2 Combo card, Premier Medical Corporation, India) and microscopy for P. falciparum and agreed to sign a consent form. All patients received treatment according to the Haitian approved standard of care available at the clinics. The Haiti Ethical Review Board, University of Florida Institutional Review Board and the Office of Research Protections, US Army Medical Research and Material Command approved this study.
Plasmodium falciparum DNA was extracted from the DBS with a methanol extraction wash protocol as previously described [26, 27]. A single disc 3 mm in diameter was punched from the DBS and placed in a 0.2 ml micro-centrifuge tube. A volume of 100 μl of absolute methanol was added and the tube incubated at room temperature for 15 min. Methanol was then decanted and the 3 mm disc left to air dry for 30 min. Sixty-five microlitres of sterile DNA-free grade water was then added to the dried disc, agitated slightly and then heated to 97°C for 30 min. The tube was then centrifuged at low speed and the supernatant stored at −20°C for use in the PCR amplification protocol.
Detection of Plasmodium falciparum and amplification of pfmdr1 by PCR
The PCR amplification primers list for pfmr1 and pfcrt used in the study
Restriction enzyme digestion for pfmdr1 mutations
The pfmdr1 amplification products were subjected to restriction enzyme digestion. The restriction enzymes used included Apo I/Afl III, Dra I, Dde I, AseI and EcoRV for analysis of sequences at codons 86, 184, 1034, 1042 and 1246, respectively. All enzymes were purchased from New England Biolabs (Ipswich, MA, USA). Technique was performed as previously described [28, 29]. Digestion was carried out according to manufacturer instructions. The product of digestion was resolved on a 2.5% agarose gel and visualized with BioRad Transilluminator (Universal Hood II).
Amplification of pfcrt
Samples positive for pfssurrna were used to amplify codons 72–76 of pfcrt gene as previously described [10, 30]. A one-step PCR was carried out using Phusion flash high fidelity master mix from (New England Biolabs, Ipswich, MA,USA) with a 25 μl final volume of PCR reaction composed of 1x PCR Master Mix containing 0.2 mM dNTPs, 0.5 μM each primer, 2.5 mM MgCl2, and 1 μl of DNA extract. The cycling temperature protocol used was: two cycles at 95°C for 5 min, amplification for 40 cycles (94°C for 30 sec, 60°C for 30 sec, and 72°C for 30 sec) and a final extension at 72°C for 10 min. Products of amplification were then electrophoresed on 2% agarose gel, stained with EtBr and visualized with BioRad Transilluminator (Universal Hood II).
Sequencing and alignment
Amplified segments of pfmdr1 and pfcrt were sequenced by the Sanger chain-termination method to determine polymorphic sites associated with CQ or other anti-malarial resistance. Sequencing was done on a Biosystem 3730 Genetic Analyzer at the University of Florida’s Interdisciplinary Center for Biotechnology Research DNA Sequencing Core Laboratory. All sequences results were aligned to P. falciparum 3D7 pfmdr1 and pfcrt sequences. [GenBank: NC_004326.1 and NC_004328.2] respectively.
Presents summary of PCR amplification results and geographic distribution of samples collected in the study, number of samples amplified for different genes pfssurrna , pfcrt , and differential data on no. of samples amplified for different codons of pfmdr1
No. of samples positive for microscopy
Positive for pfssurrna
Positive for pfcrt
No. of samples amplified for codons 86-1042
Although this study was limited by small sample size, it provides preliminary information about the pfmdr1 alleles in Haiti and adds additional data on pfcrt alleles in parasites from both previously sampled regions of Haiti and from regions not previously sampled. This study is the first to examine in Haiti the pfmdr1 genetic mutations linked to anti-malarial drug resistance in other regions of the world. This study found neither the K76T mutation in pfcrt nor the N86Y, S1034C, N1042D or D1246Y mutations in pfmdr1, that have been reported to confer resistance to CQ [22, 23, 31–33]. The absence of mutations at codons 1034 and 1042 in pfmdr1 suggests that the P. falciparum parasites analysed in this study likely remain sensitive to mefloquine  and AL . Interestingly, all the samples tested had a Y− > F mutation in codon 184 in pfmdr1. The widespread occurrence of this single mutation in pfmdr1 is not unique in the literature [36, 37]. Previous studies of clinical isolates of P. falciparum in Cambodia reported a similar widespread occurrence of single point mutations, and the mutation resulting in the Y184F mutation was predominant over other pfmdr1 mutations .
Although the Y184F mutation in pfmdr1 has been associated mainly with low level resistance to AL , there is no consensus in the literature for its role in CQ resistance [28, 34, 40]. In a previous study , the Y184F polymorphism was shown to play a role in CQ resistance in close association with the K76T mutation in pfcrt. The Y184F mutation is thought to alter the kinetics of anti-malarial drug transport [32, 41], and thus efficacy. The absence of the K76T mutations in the study sample indicates that the Y184F mutation in pfmdr1 is rarely found in Haiti in combination with K76T in pfcrt. Although this pfmdr1 allele may not alone increase CQ resistance, it’s high prevalence could have an impact if the frequency of the pfcrt K76T allele were to increase in Haiti . Other studies have shown a modification of resistance to AL in P. falciparum with the Y184F mutation under a N86Y and D1246Y background . The absence of N86Y, S1034C, N1042D or D1246Y in pfmdr1 alleles in this study sustains CQ or AL sensitivity in Haiti. Therefore, the findings would seem to indicate that the presence of the Y184F mutation alone would not contribute greatly to anti-malarial resistance in Haiti in the absence of wide spread K76T mutations in pfcrt or additional mutations in pfmdr1. Routine molecular surveillance of the anti-malarial drug resistance genes in Haiti combined with in vitro drug sensitivity studies are needed to ascertain the sensitivity of Haitian P. falciparum strains to the major anti-malarial medications in use in Haiti and other parts of world.
The recent reports of CQ resistance genotypes in Haiti are alarming for public health officials in Haiti and concerted efforts are needed to monitor the anti-malaria drug resistance genotypes in Haiti. The results of this study have provided background data on pfmdr1, which may be referenced in discussions of future P. falciparum anti-malarial resistance patterns in Haiti. This study also provides additional data on the prevalence of the pfcrt K76T allele in Haiti, but additional studies are needed to increase the sample size and provide an accurate estimate for K76T allele prevalence and distribution. Nonetheless, the findings in this study have increased the knowledge of two important anti-malaria drug resistance markers in Haiti, where widespread drug resistance has not yet been observed.
The small sample size is indeed the largest limitation of this study, however due to the difficulty of the study initiation; proper sample storage for the FTA cards was lacking which affected the quality of DNA. The study lack adequate sample distribution, were some sites failed to enroll sufficient number of patients, this can be attributed to change in site administration which affected the study enrollment.
The authors thank the Haiti Ministry of Sanitation and Public Practice (MSPP), Christianville Foundation Haiti, Family Health Ministries, Haiti, Hospital Sainte Croix and Blanchard Clinic in Haiti for their support in the execution of this work. The work in this paper was supported by a grant from the Department of Defense (DoD) Global Emerging Infections Surveillance & Response System (GEIS) Grant # P0103_13__UN awarded to BAO.
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