- Email: [email protected]
Association between chloroquine resistance phenotypes and point mutations in pfcrt and pfmdr1 in Plasmodium falciparum isolates from Thailand
Acta Tropica 109 (2009) 37–40
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Association between chloroquine resistance phenotypes and point mutations in pfcrt and pfmdr1 in Plasmodium falciparum isolates from Thailand Kanchana Rungsihirunrat a,∗ , Wanna Chaijareonkul b , Aree Seugorn a , Kesara Na-Bangchang b , Sodsri Thaithong a a b
The College of Public Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand Graduate Program in Biomedical Sciences, Faculty of Allied Health sciences, Thammasat University, Patumthanee 12121, Thailand
a r t i c l e
i n f o
Article history: Received 25 December 2007 Received in revised form 11 September 2008 Accepted 17 September 2008 Available online 30 September 2008 Keywords: Plasmodium falciparum pfcrt pfmdr1 Quinoline antimalarials Drug resistance
a b s t r a c t The relationship between the in vitro susceptibility of Plasmodium falciparum isolates to the quinoline antimalarials chloroquine (CQ), mefloquine (MQ), and quinine (QN), and pfcrt and pfmdr1 gene polymorphisms were investigated. Field isolates (110 samples) were collected from various endemic areas of Thailand throughout 2002–2004. The pfcrt 76T allele was identified in 109 isolates (99.1%) while pfcrt 76K was found in a single (0.9%) isolate. The pfmdr 86N, 86Y, and the combination (86N + 86Y) alleles were identified in 83 (75.5%), 22 (20%), and 5 (4.5%) isolates, respectively. The pfmdr1 1042N, 1042D alleles and a mixture (1042N + 1042D) of the alleles were found in 94 (85.5%), 12 (10.9%) and 4 (3.6%) isolates, respectively. The pfmdr1 1246Y allele was detected in a single (0.9%) isolate. The pfmdr1 gene polymorphisms (86-1042-1246) was grouped into seven haplotypes as follows: N-N-D (68 isolates; 61.2%), Y-N-D (22 isolates; 19.8%), N-D-D (11 isolates; 9.9%), N-D-Y (1 isolate; 0.9%), N/Y-N-D (4 isolates; 3.6%), N-N/D-D (3 isolates; 2.7%), and N/Y-N/D-D (1 isolate; 0.9%). Eight different combinations of pfcrt–pfmdr1 genotypes were observed. Only one CQ-, MQ- and QN-sensitive isolate was found at the Thai–Laos border and no cases of QN resistance were found in this study. Crown Copyright © 2008 Published by Elsevier B.V. All rights reserved.
1. Introduction Malaria is a major health problem in several Southeast Asian countries and is exacerbated by the development and spread of antimalarial drug resistance. While a recent annual report on malaria in Thailand showed a downward trend in cases of the disease over the past 10 years (Na-Bangchang and Congpuong, 2007), the control of disease remains problematic, particularly along the country’s international borders where multidrug resistance is prevalent (Sornmani et al., 1983; Wongsrichanalai et al., 2001). Transporter genes have become attractive candidates for molecular markers of antimalarial drug resistance. The well-studied genes pfcrt, which encodes the transmembrane protein PfCRT, and the pfmdr1 gene, which encodes a P-glycoprotein homologue 1 (Pgh1), have been linked to chloroquine (CQ)-resistant parasites (Fidock et al., 2000; Duraisingh and Cowman, 2005; Sharma, 2005). The CQ resistance phenotype has been shown to be strongly associ-
∗ Corresponding author at: Malaria Research Unit, The College of Public Health Sciences, Chulalongkorn University, Phayathai Road, Bangkok 10330, Thailand. Tel.: +66 2218 5261; fax: +66 2255 7734. E-mail address: [email protected] (K. Rungsihirunrat).
ated with specific point mutations at codon 76 within the pfcrt gene. However, the relationship between pfmdr1 and CQ resistance remains unclear and is a matter of controversy (Wellems, 1991). Selection for mefloquine (MQ) resistance has been associated with a decreased resistance to CQ and an amplification of the pfmdr1 gene (Reed et al., 2000). This amplification of pfmdr1 gene copy numbers may also be associated with the observed higher in vitro IC50 (50% inhibitory concentration) for MQ in resistant Plasmodium falciparum isolates (Price et al., 2004). This is supported by observations from prior studies in Thailand and other Southeast Asian countries which demonstrate an association between increased pfmdr1 gene copy number and in vitro MQ resistance (Price et al., 1999; Wilson et al., 1993), although this relationship could not be confirmed by other researchers (Chaiyaroj et al., 1999; Basco et al., 1995). Despite these controversial findings, there are ongoing attempts to identify molecular markers for quinoline antimalarials and mutations in pfcrt and pfmdr1 which could prove useful in epidemiological studies monitoring the spread of CQ resistance. Since quinoline antimalarials are the mainstay for antimalarial chemotherapy, a more thorough understanding of the molecular basis of quinoline antimalarial resistance could facilitate effective malaria control programs and the development of new promising antimalarial drugs for the treatment of multidrug-resistant malaria.
0001-706X/$ – see front matter. Crown Copyright © 2008 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2008.09.011
38
K. Rungsihirunrat et al. / Acta Tropica 109 (2009) 37–40
The aim of the present study was to determine the relationship, if any, between pfcrt and pfmdr1 genetic polymorphisms P. falciparum resistance in Thailand toward the quinoline antimalarials CQ, MQ, and quinine (QN). Allelic variations in pfcrt and pfmdr1 and drug susceptibilities were investigated in P. falciparum isolates collected form various malaria-endemic regions in Thailand.
2.5. Statistical analysis The Pearson Chi-square test was used to determine significant associations between the in vitro drug susceptibilities of P. falciparum isolates to QN, MQ and QN and the pfcrt and pfmdr1 gene polymorphisms. Statistical significance was set at ˛ = 0.05. 3. Results
2. Material and methods
3.1. In vitro susceptibility of P. falciparum to antimalarials
2.1. Collection of P. falciparum isolates
A total of 110 P. falciparum field isolates were evaluated for in vitro susceptibilities. The MIC values [median (interquartile range)] for CQ, MQ, and QN were 500 (0), 200 (107.5), and 300 nM (200), respectively. Isolates exhibiting MIC values greater than 80, 320 and 2560 nM were classified as being resistant to CQ, MQ and QN, respectively. Thresholds for the in vitro susceptibilities were implemented according to WHO cut-off values (WHO, 2001). Based on these values, 1 (0.9%), 89 (80.9%) and 110 (100%) isolates, respectively, were considered to be sensitive to CQ, MQ, and QN.
P. falciparum-infected blood samples were collected from malaria patients residing in different geographical areas along the international Thai borders (Thai–Myanmar, Thai–Cambodia, Thai–Malaysia and Thai–Laos) during 2002–2004. Written informed consent for study participation was obtained from all patients. The study protocol was reviewed and approved by the Ethics Committee of Ministry of Public Health, Thailand. Following microscopic confirmation of blood films, 100–200 l of infected blood samples, irrespective of parasitaemia, were collected by finger-pricks and immediately mixed with 1 ml of transporting medium (RPMI 1640, 5%NaHCO3 and 10 units/ml heparin) (Thaithong et al., 1994). Blood-medium mixture was then used directly for in vitro drug susceptibility tests, as described previously (Thaithong and Beale, 1981).
2.2. In vitro drug susceptibility tests P. falciparum in vitro drug susceptibilities to CQ, MQ, and QN were determined using growth inhibition tests and using the minimum inhibitory concentration (MIC: minimum concentration of drug that inhibits parasite growth by 100%) as the endpoint (Thaithong and Beale, 1981).
2.3. Detection of pfcrt and pfmdr1 polymorphisms
3.2. Detection of pfcrt and pfmdr 1 polymorphisms Of the total of 110 P. falciparum isolates analyzed, the pfcrt 76T allele was detected in 109 isolates (99.1%) while the pfcrt 76K allele was detected in only one isolate (0.9%). The pfmdr1 86N, 86Y, and the mixture (86N + 86Y) of alleles were observed in 85 (75.5%), 22 (20%), and 5 (4.7%) isolates, respectively. The pfmdr1 1042N, 1042D and the mixture (1042N + 1042D) of alleles were found in 94 (85.5%), 12 (10.9%) and 4 (3.6%) isolates, respectively. The pfmdr1 1246Y allele was detected in a single (0.9%) isolate. The pfmdr1 gene polymorphism (86-1042-1246) was grouped into seven haplotypes as follows: N-N-D (68 isolates: 61.2%), Y-N-D (22 isolates: 19.8%), N-D-D (11 isolates: 9.9%), N-D-Y (1 isolate: 0.9%), N/Y-N-D (4 isolates: 3.6%), N-N/D-D (3 isolates: 2.7%), and N/Y-N/D-D (1 isolate: 0.9%). Eight different combinations of pfcrt–pfmdr1 genotypes were found and the frequency of each genotype is shown in Fig. 1.
Genomic DNA was extracted by a standard phenol/chloroform method (Sambrook et al., 1989). Polymorphisms in pfcrt at codon 76 and pfmdr1 at codons 86, 1042 and 1246 were detected using PCR–RFLP. PCR was performed in a total volume of 20 l with the following reaction mixture: 0.1 M of each primer, 2.5 mM MgCl2 , 100 M deoxynucleotides (dNTPs), 1× PCR buffer (100 mM KCl, 20 mM Tris–HCl pH 8.0), 2 l of DNA (used as a template), and 0.5 unit of Taq DNA polymerase (PromegaTM ). The primers and the reaction conditions used are according to Lopes et al. (2002).
2.4. PCR–RFLP genotyping Identification of the pfcrt 76K and pfmdr1 86N gene variants were determined by ApoI (New England Bio-Labs) digestion of the relevant PCR fragments. The pfmdr1 1042 and the pfmdr1 1246 variants were detected in this way but by using VspI (PromegaTM ), and EcoRV (New England Bio-Labs) enzymes, respectively. 3D7, HB3 and K1 P. falciparum clones were included as positive controls for the PCR–RFLP genotyping. Restriction digests were carried out according to the suppliers’ instructions. Digestion products for the pfmdr1 86 and 1246 variants were resolved on 2% agarose gels while the pfmdr1 1042 and pfcrt 76 digests were electrophoresed on 8% acrylamide gels.
Fig. 1. Prevalence of pfcrt–pfmdr1 genotypes in 110 P. falciparum isolates.
K. Rungsihirunrat et al. / Acta Tropica 109 (2009) 37–40
39
Fig. 2. Distribution of drug susceptibility profiles of P. falciparum isolates to CQ, MQ, and QN in different endemic areas of Thailand.
3.3. Association between pfcrt and pfmdr 1 haplotypes and in vitro susceptibility of P. falciparum isolates to quinoline antimalarials A strong (positive association, r2 = 1.00) and significant (p < 0.001) association was observed between the pfcrt 76 mutation and the in vitro susceptibility of P. falciparum isolates to CQ, and also between the pfmdr1 86 mutation and susceptibility of parasites to MQ (negative association, r2 = −0.332; p < 0.001). One isolate carrying the K76 genotype was classified as CQ-sensitive, whereas the remaining 109 isolates carrying the T76 mutation were classified as CQ-resistant. No gene variant was found to be associated with QN susceptibility. 3.4. Relationship between the susceptibility of P. falciparum isolates to quinoline antimalarials and the origin of isolates The distribution of susceptibility profiles of P. falciparum isolates collected from the four border areas of Thailand, i.e., Thai–Myanmar (66 isolates), Thai–Cambodian (26 isolates), Thai–Malaysian (10 isolates), and Thai–Laos (eight isolates) borders is shown in Fig. 2. A single CQ-sensitive isolate was found at the Thai–Laos border. MQ resistance was found only at the Thai–Myanmar and Thai–Cambodian borders, areas renowned for high levels of multidrug P. falciparum resistance. None of the isolates were resistant to QN. 4. Discussion CQ resistance in P. falciparum was first reported about 50 years ago, but the impact of this drug on malaria parasite genomes has been difficult to evaluate to date. Extended parasite exposure to CQ has placed a selective force upon the characteristics of P. falciparum population genetics. Many field and laboratory studies have attempted to link CQ resistance to pfcrt and pfmdr1 gene mutations (Duraisingh et al., 2000; Fidock et al., 2000; Djimde et al., 2001). Although the involvement of pfmdr1 mutation is currently under debate, the N86Y mutation (in conjunction with pfcrt mutations) is thought to modulate levels of CQ resistance to a higher degree (Foote et al., 1990; Reed et al., 2000; Djimde et al., 2001). In attempting to evaluate the emergence of CQ resistance patterns in field isolates, we analyzed mutations associated with CQ resistance for genes which encode the P. falciparum digestive food vacuole membrane proteins, pfcrt and pfmdr1, in 110 field isolates from various endemic areas of Thailand. Mutations in both pfcrt and pfmdr1 genes are believed to be very useful molecular markers for epidemiological studies when monitoring the spread of CQ
resistance (Duraisingh et al., 2000; Djimde et al., 2001). A single pfcrt mutation, K76T, is sufficient to cause CQ resistance. Replacement of the positively charged lysine by an uncharged threonine removes the electrostatic charge which then prohibits the protonated and positively charged CQ drug molecule from pore entry (Sanchez et al., 2007). The high prevalence of the pfcrt 76T variant in our study is in agreement with reports from other areas where clinical CQ failure frequently occurs (Dorsey et al., 2001; Congpuong et al., 2005). Eight different genotypes in pfcrt76–pfmdr86-1042-1246 were observed with a high prevalence of the TNND genotype. We also investigated the association between the in vitro susceptibility of P. falciparum isolates and pfcrt–pfmdr1 genotypes. The pfcrt K76 allele, with or without the N86Y mutation, was found in CQ-sensitive parasites. Conversely, the K76T mutation, with or without the pfmdr1 N86Y mutation, was resistant to CQ. It has been proposed that mutations in pfmdr1 may not be sufficient in producing the CQ resistance phenotype, however the N86Y mutation can moderate drug responses (Foote et al., 1990; Reed et al., 2000; Djimde et al., 2001; Duraisingh et al., 2000). For the pfcrt gene, a strong and significant association between the single point mutation in codon 76 of the pfcrt gene and in vitro susceptibility to CQ was found. On the other hand, the pfmdr1 mutation in codon 86 was significantly associated with MQ susceptibility. Our findings indicate that N86Y mutation may enhance the parasite susceptibility to MQ. Therefore, in areas subjected to MQ pressure, wild-type 86N was found more frequently. No association was observed between pfcrt and pfmdr1 mutations and susceptibility to QN. When considering the relationship between the susceptibility of P. falciparum isolates to quinoline antimalarials and the origin of parasite isolates, we note high levels of CQ-resistant malaria emerging in Thailand. The prevalence of MQ-sensitive isolates was generally higher than MQ-resistant isolates and was found in all areas, whereas MQ-resistant isolates were found only at the Thai–Myanmar and Thai–Cambodian borders. No QN-resistant isolates were found in our study. Although CQ-resistance is highly prevalent in Africa and Southeast Asia, QN and MQ remain effective against clinical isolates (Wernsdorfer and Payne, 1991; Ringwald et al., 1999). In Thailand, MQ resistance continues to progress in Mae Sot (Thai–Myanmar border) and Bo Rai (Thai–Cambodian border) (Wongsrichanalai et al., 2001). The introduction of an artesunate and mefloquine combination treatment is believed to delay the emergence of multidrug resistance in these areas. Thus, the detection of pfmdr1 gene polymorphisms may not be a useful surveillance tool for the identification of antimalarial drugresistant malaria in Thailand. Despite the fact that CQ has been withdrawn from clinical use as a first-line drug for acute uncomplicated falciparum malaria for more than 50 years in Thailand,
40
K. Rungsihirunrat et al. / Acta Tropica 109 (2009) 37–40
the malaria parasite has been exposed to CQ (as first-line drug for Plasmodium vivax malaria) or other quinoline-containing drugs. Resistance to CQ, MQ and QN would be expected to compromise the clinical effectiveness of the current first-line combination treatment with MQ and artesunate for falciparum malaria in Thailand. Further study is required to clarify this issue. Acknowledgements We are grateful to all the participants and the staff of Malaria Clinics for their kind assistance. This study was founded by Malaria Research fund, The College of Public Health Sciences, Chulalongkorn University and partial supported by Thailand Research fund. References Basco, L.K., Le Bras, J., Rhoades, Z., Wilson, C.M., 1995. Analysis of pfmdr1 and drug susceptibility in fresh isolates of Plasmodium falciparum from Subsaharan Africa. Mol. Biochem. Parasitol. 74, 157–166. Chaiyaroj, S.C., Buranakiti, A., Angkasekwinai, P., Looareesuwan, S., Cowman, A.F., 1999. Analysis of mefloquine resistance and amplification of pfmdr1 in multidrug-resistant Plasmodium falciparum isolates from Thailand. Am. J. Trop. Med. Hyg. 61, 780–783. Congpuong, K., Na-Bangchang, K., Mungthin, M., Bualombai, P., Wernsdorfer, W.H., 2005. Molecular epidemiology of drug resistance markers of Plasmodium falciparum malaria in Thailand. Trop. Med. Int. Health 10, 717–722. Djimde, A., Doumbo, O.K., Cortese, J.F., Kayentao, K., Doumbo, S., Diourte, Y., Dicko, A., Su, X.Z., Nomura, T., Fidock, D.A., Wellems, T.E., Plowe, C.V., Coulibaly, D., 2001. A molecular marker for chloroquine-resistant falciparum malaria. N. Engl. J. Med. 344, 257–263. Dorsey, G., Kamya, M.R., Singh, A., Rosenthal, P.J., 2001. Polymorphisms in the Plasmodium falciparum pfcrt and pfmdr1 genes and clinical response to chloroquine in Kampala. Uganda. J. Infect. Dis. 183, 1417–1420. Duraisingh, M.T., Roper, C., Walliker, D., Warhurst, D.C., 2000. Increased sensitivity to the antimalarials mefloquine and artemisinin is conferred by mutations in the pfmdr1 gene of Plasmodium falciparum. Mol. Microbiol. 36, 955–961. Duraisingh, M.T., Cowman, A.F., 2005. Contribution of the pfmdr1 gene to antimalarial drug-resistance. Acta Trop. 94, 181–190. Fidock, D.A., Nomura, T., Talley, A.K., Cooper, R.A., Dzekunov, S.M., Ferdig, M.T., Ursos, L.M., Sidhu, A.B., Naude, B., Deitsch, K.W., Su, X.Z., Wootton, J.C., Roepe, P.D., Wellems, T.E., 2000. Mutations in the Plasmodium falciparum digestive vacuole transmembrane protein Pfcrt and evidence for their role in chloroquine resistance. Mol. Cell 6, 861–871. Foote, S.J., le, D.E., Martin, R.K., Oduola, A.M., Forsyth, K., Kemp, D.J., Cowman, A.F., 1990. Several alleles of the multidrug-resistance gene are closely linked to chloroquine resistance in Plasmodium falciparum. Nature 345, 255–258.
Lopes, D., Rungsihirunrat, K., Nogueira, F., Seugorn, A., Gil, J., Do Rosario, V.E., Cravo, P., 2002. Molecular characterization of drug resistant Plasmodium falciparum from Thailand. Malar. J. 1, 1–12. Na-Bangchang, K., Congpuong, K., 2007. Current malaria status and distribution of drug resistance in East and Southeast Asia with special focus to Thailand. Tohoku J. Exp. Med. 211, 99–113. Price, R.N., Cassar, C., Brockman, A., Duraisingh, M., Van Vugt, M., White, N.J., Nosten, F., Krishna, S., 1999. The pfmdr1 gene is associated with a multidrug-resistant phenotype in Plasmodium falciparum from the western border of Thailand. Antimicrob. Agents Chemother. 43, 2943–2949. Price, R.N., Uhlemann, A.C., Brockman, A., McGready, R., Ashley, E., Phaipun, L., Patel, R., Laing, K., Looareesuwan, S., White, N.J., Nosten, F., Krishna, S., 2004. Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number. Lancet 364, 438–447. Reed, M.B., Saliba, K.J., Caruana, S.R., Kirk, K., Cowman, A.F., 2000. Pgh1 modulates sensitivity and resistance to multiple antimalarials in Plasmodium falciparum. Nature 403, 906–909. Ringwald, P., Bickii, J., Basco, L.K., 1999. In vitro activity of dihydroartemisinin against clinical isolates of Plasmodium falciparum in Yaounde, Cameroon. Am. J. Trop. Med. Hyg. 61, 187–192. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, second Edition. Cold Spring Harbour Laboratory, Cold Spring Harbour, New York. Sanchez, C.P., Stein, W.D., Lanzer, M., 2007. Is PfCRT a channel or a carrier? Two competing models explaining chloroquine resistance in Plasmodium falciparum. Trends Parasitol. 23, 332–339. Sharma, Y.D., 2005. Genetic alteration in drug resistance markers of Plasmodium falciparum. Indian J. Med. Res. 121, 13–22. Sornmani, S., Butraporn, P., Fungladda, W., Okanurak, K., Dissapongsa, S., 1983. Migration and disease problem: a study of pattern of migration in an endemic area of malaria in Thailand. Southeast Asian J. Trop. Med. Public Health 14, 64–68. Thaithong, S., Beale, G., 1981. Resistance of ten Thai isolates of Plasmodium falciparum to chloroquine and pyrimethamine by in vitro tests. Trans. R. Soc. Trop. Med. Hyg. 75, 271–273. Thaithong, S., Seugorn, A., Beale, G.H., 1994. Culturing Plasmodium falciparum from finger-prick samples of infected blood. Trans. R. Soc. Trop. Med. Hyg. 88, 490. Wellems, T.E., 1991. Molecular genetics of drug resistance in Plasmodium falciparum malaria. Parasitol. Today 7, 110–112. Wernsdorfer, W.H., Payne, D., 1991. The dynamics of drug resistance in Plasmodium falciparum. Pharmacol. Ther. 50, 95–121. World Health Organization, 2001. In vitro micro-test (Mark III) for the assessment of the response of Plasmodium falciparum to chloroquine, mefloquine, quinine, amodiaquine, sulfadoxine/pyrimethamine and artemisinin WHO document CTD/MAL/97.2, Rev. 2, WHO, Geneva. Wilson, C., Volkman, S., Thaithong, S., Martin, R., Kyle, D., Milhous, W., Wirth, D., 1993. Amplification of Pfmdr1 associated with mefloquine and halofantrine resistance in Plasmodium falciparum from Thailand. Mol. Biochem. Parasitol. 57, 151– 160. Wongsrichanalai, C., Sirichaisinthop, J., Karwacki, J.J., Congpuong, K., Miller, R.S., Pang, L., Thimasarn, K., 2001. Drug resistance malaria on the Thai–Myanmar and Thai–Cambodian borders. Southeast Asian J. Trop. Med. Public Health 32, 41–49.
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Association between chloroquine resistance phenotypes and point mutations in pfcrt and pfmdr1 in Plasmodium falciparum isolates from Thailand Kanchana Rungsihirunrat a,∗ , Wanna Chaijareonkul b , Aree Seugorn a , Kesara Na-Bangchang b , Sodsri Thaithong a a b
The College of Public Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand Graduate Program in Biomedical Sciences, Faculty of Allied Health sciences, Thammasat University, Patumthanee 12121, Thailand
a r t i c l e
i n f o
Article history: Received 25 December 2007 Received in revised form 11 September 2008 Accepted 17 September 2008 Available online 30 September 2008 Keywords: Plasmodium falciparum pfcrt pfmdr1 Quinoline antimalarials Drug resistance
a b s t r a c t The relationship between the in vitro susceptibility of Plasmodium falciparum isolates to the quinoline antimalarials chloroquine (CQ), mefloquine (MQ), and quinine (QN), and pfcrt and pfmdr1 gene polymorphisms were investigated. Field isolates (110 samples) were collected from various endemic areas of Thailand throughout 2002–2004. The pfcrt 76T allele was identified in 109 isolates (99.1%) while pfcrt 76K was found in a single (0.9%) isolate. The pfmdr 86N, 86Y, and the combination (86N + 86Y) alleles were identified in 83 (75.5%), 22 (20%), and 5 (4.5%) isolates, respectively. The pfmdr1 1042N, 1042D alleles and a mixture (1042N + 1042D) of the alleles were found in 94 (85.5%), 12 (10.9%) and 4 (3.6%) isolates, respectively. The pfmdr1 1246Y allele was detected in a single (0.9%) isolate. The pfmdr1 gene polymorphisms (86-1042-1246) was grouped into seven haplotypes as follows: N-N-D (68 isolates; 61.2%), Y-N-D (22 isolates; 19.8%), N-D-D (11 isolates; 9.9%), N-D-Y (1 isolate; 0.9%), N/Y-N-D (4 isolates; 3.6%), N-N/D-D (3 isolates; 2.7%), and N/Y-N/D-D (1 isolate; 0.9%). Eight different combinations of pfcrt–pfmdr1 genotypes were observed. Only one CQ-, MQ- and QN-sensitive isolate was found at the Thai–Laos border and no cases of QN resistance were found in this study. Crown Copyright © 2008 Published by Elsevier B.V. All rights reserved.
1. Introduction Malaria is a major health problem in several Southeast Asian countries and is exacerbated by the development and spread of antimalarial drug resistance. While a recent annual report on malaria in Thailand showed a downward trend in cases of the disease over the past 10 years (Na-Bangchang and Congpuong, 2007), the control of disease remains problematic, particularly along the country’s international borders where multidrug resistance is prevalent (Sornmani et al., 1983; Wongsrichanalai et al., 2001). Transporter genes have become attractive candidates for molecular markers of antimalarial drug resistance. The well-studied genes pfcrt, which encodes the transmembrane protein PfCRT, and the pfmdr1 gene, which encodes a P-glycoprotein homologue 1 (Pgh1), have been linked to chloroquine (CQ)-resistant parasites (Fidock et al., 2000; Duraisingh and Cowman, 2005; Sharma, 2005). The CQ resistance phenotype has been shown to be strongly associ-
∗ Corresponding author at: Malaria Research Unit, The College of Public Health Sciences, Chulalongkorn University, Phayathai Road, Bangkok 10330, Thailand. Tel.: +66 2218 5261; fax: +66 2255 7734. E-mail address: [email protected] (K. Rungsihirunrat).
ated with specific point mutations at codon 76 within the pfcrt gene. However, the relationship between pfmdr1 and CQ resistance remains unclear and is a matter of controversy (Wellems, 1991). Selection for mefloquine (MQ) resistance has been associated with a decreased resistance to CQ and an amplification of the pfmdr1 gene (Reed et al., 2000). This amplification of pfmdr1 gene copy numbers may also be associated with the observed higher in vitro IC50 (50% inhibitory concentration) for MQ in resistant Plasmodium falciparum isolates (Price et al., 2004). This is supported by observations from prior studies in Thailand and other Southeast Asian countries which demonstrate an association between increased pfmdr1 gene copy number and in vitro MQ resistance (Price et al., 1999; Wilson et al., 1993), although this relationship could not be confirmed by other researchers (Chaiyaroj et al., 1999; Basco et al., 1995). Despite these controversial findings, there are ongoing attempts to identify molecular markers for quinoline antimalarials and mutations in pfcrt and pfmdr1 which could prove useful in epidemiological studies monitoring the spread of CQ resistance. Since quinoline antimalarials are the mainstay for antimalarial chemotherapy, a more thorough understanding of the molecular basis of quinoline antimalarial resistance could facilitate effective malaria control programs and the development of new promising antimalarial drugs for the treatment of multidrug-resistant malaria.
0001-706X/$ – see front matter. Crown Copyright © 2008 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2008.09.011
38
K. Rungsihirunrat et al. / Acta Tropica 109 (2009) 37–40
The aim of the present study was to determine the relationship, if any, between pfcrt and pfmdr1 genetic polymorphisms P. falciparum resistance in Thailand toward the quinoline antimalarials CQ, MQ, and quinine (QN). Allelic variations in pfcrt and pfmdr1 and drug susceptibilities were investigated in P. falciparum isolates collected form various malaria-endemic regions in Thailand.
2.5. Statistical analysis The Pearson Chi-square test was used to determine significant associations between the in vitro drug susceptibilities of P. falciparum isolates to QN, MQ and QN and the pfcrt and pfmdr1 gene polymorphisms. Statistical significance was set at ˛ = 0.05. 3. Results
2. Material and methods
3.1. In vitro susceptibility of P. falciparum to antimalarials
2.1. Collection of P. falciparum isolates
A total of 110 P. falciparum field isolates were evaluated for in vitro susceptibilities. The MIC values [median (interquartile range)] for CQ, MQ, and QN were 500 (0), 200 (107.5), and 300 nM (200), respectively. Isolates exhibiting MIC values greater than 80, 320 and 2560 nM were classified as being resistant to CQ, MQ and QN, respectively. Thresholds for the in vitro susceptibilities were implemented according to WHO cut-off values (WHO, 2001). Based on these values, 1 (0.9%), 89 (80.9%) and 110 (100%) isolates, respectively, were considered to be sensitive to CQ, MQ, and QN.
P. falciparum-infected blood samples were collected from malaria patients residing in different geographical areas along the international Thai borders (Thai–Myanmar, Thai–Cambodia, Thai–Malaysia and Thai–Laos) during 2002–2004. Written informed consent for study participation was obtained from all patients. The study protocol was reviewed and approved by the Ethics Committee of Ministry of Public Health, Thailand. Following microscopic confirmation of blood films, 100–200 l of infected blood samples, irrespective of parasitaemia, were collected by finger-pricks and immediately mixed with 1 ml of transporting medium (RPMI 1640, 5%NaHCO3 and 10 units/ml heparin) (Thaithong et al., 1994). Blood-medium mixture was then used directly for in vitro drug susceptibility tests, as described previously (Thaithong and Beale, 1981).
2.2. In vitro drug susceptibility tests P. falciparum in vitro drug susceptibilities to CQ, MQ, and QN were determined using growth inhibition tests and using the minimum inhibitory concentration (MIC: minimum concentration of drug that inhibits parasite growth by 100%) as the endpoint (Thaithong and Beale, 1981).
2.3. Detection of pfcrt and pfmdr1 polymorphisms
3.2. Detection of pfcrt and pfmdr 1 polymorphisms Of the total of 110 P. falciparum isolates analyzed, the pfcrt 76T allele was detected in 109 isolates (99.1%) while the pfcrt 76K allele was detected in only one isolate (0.9%). The pfmdr1 86N, 86Y, and the mixture (86N + 86Y) of alleles were observed in 85 (75.5%), 22 (20%), and 5 (4.7%) isolates, respectively. The pfmdr1 1042N, 1042D and the mixture (1042N + 1042D) of alleles were found in 94 (85.5%), 12 (10.9%) and 4 (3.6%) isolates, respectively. The pfmdr1 1246Y allele was detected in a single (0.9%) isolate. The pfmdr1 gene polymorphism (86-1042-1246) was grouped into seven haplotypes as follows: N-N-D (68 isolates: 61.2%), Y-N-D (22 isolates: 19.8%), N-D-D (11 isolates: 9.9%), N-D-Y (1 isolate: 0.9%), N/Y-N-D (4 isolates: 3.6%), N-N/D-D (3 isolates: 2.7%), and N/Y-N/D-D (1 isolate: 0.9%). Eight different combinations of pfcrt–pfmdr1 genotypes were found and the frequency of each genotype is shown in Fig. 1.
Genomic DNA was extracted by a standard phenol/chloroform method (Sambrook et al., 1989). Polymorphisms in pfcrt at codon 76 and pfmdr1 at codons 86, 1042 and 1246 were detected using PCR–RFLP. PCR was performed in a total volume of 20 l with the following reaction mixture: 0.1 M of each primer, 2.5 mM MgCl2 , 100 M deoxynucleotides (dNTPs), 1× PCR buffer (100 mM KCl, 20 mM Tris–HCl pH 8.0), 2 l of DNA (used as a template), and 0.5 unit of Taq DNA polymerase (PromegaTM ). The primers and the reaction conditions used are according to Lopes et al. (2002).
2.4. PCR–RFLP genotyping Identification of the pfcrt 76K and pfmdr1 86N gene variants were determined by ApoI (New England Bio-Labs) digestion of the relevant PCR fragments. The pfmdr1 1042 and the pfmdr1 1246 variants were detected in this way but by using VspI (PromegaTM ), and EcoRV (New England Bio-Labs) enzymes, respectively. 3D7, HB3 and K1 P. falciparum clones were included as positive controls for the PCR–RFLP genotyping. Restriction digests were carried out according to the suppliers’ instructions. Digestion products for the pfmdr1 86 and 1246 variants were resolved on 2% agarose gels while the pfmdr1 1042 and pfcrt 76 digests were electrophoresed on 8% acrylamide gels.
Fig. 1. Prevalence of pfcrt–pfmdr1 genotypes in 110 P. falciparum isolates.
K. Rungsihirunrat et al. / Acta Tropica 109 (2009) 37–40
39
Fig. 2. Distribution of drug susceptibility profiles of P. falciparum isolates to CQ, MQ, and QN in different endemic areas of Thailand.
3.3. Association between pfcrt and pfmdr 1 haplotypes and in vitro susceptibility of P. falciparum isolates to quinoline antimalarials A strong (positive association, r2 = 1.00) and significant (p < 0.001) association was observed between the pfcrt 76 mutation and the in vitro susceptibility of P. falciparum isolates to CQ, and also between the pfmdr1 86 mutation and susceptibility of parasites to MQ (negative association, r2 = −0.332; p < 0.001). One isolate carrying the K76 genotype was classified as CQ-sensitive, whereas the remaining 109 isolates carrying the T76 mutation were classified as CQ-resistant. No gene variant was found to be associated with QN susceptibility. 3.4. Relationship between the susceptibility of P. falciparum isolates to quinoline antimalarials and the origin of isolates The distribution of susceptibility profiles of P. falciparum isolates collected from the four border areas of Thailand, i.e., Thai–Myanmar (66 isolates), Thai–Cambodian (26 isolates), Thai–Malaysian (10 isolates), and Thai–Laos (eight isolates) borders is shown in Fig. 2. A single CQ-sensitive isolate was found at the Thai–Laos border. MQ resistance was found only at the Thai–Myanmar and Thai–Cambodian borders, areas renowned for high levels of multidrug P. falciparum resistance. None of the isolates were resistant to QN. 4. Discussion CQ resistance in P. falciparum was first reported about 50 years ago, but the impact of this drug on malaria parasite genomes has been difficult to evaluate to date. Extended parasite exposure to CQ has placed a selective force upon the characteristics of P. falciparum population genetics. Many field and laboratory studies have attempted to link CQ resistance to pfcrt and pfmdr1 gene mutations (Duraisingh et al., 2000; Fidock et al., 2000; Djimde et al., 2001). Although the involvement of pfmdr1 mutation is currently under debate, the N86Y mutation (in conjunction with pfcrt mutations) is thought to modulate levels of CQ resistance to a higher degree (Foote et al., 1990; Reed et al., 2000; Djimde et al., 2001). In attempting to evaluate the emergence of CQ resistance patterns in field isolates, we analyzed mutations associated with CQ resistance for genes which encode the P. falciparum digestive food vacuole membrane proteins, pfcrt and pfmdr1, in 110 field isolates from various endemic areas of Thailand. Mutations in both pfcrt and pfmdr1 genes are believed to be very useful molecular markers for epidemiological studies when monitoring the spread of CQ
resistance (Duraisingh et al., 2000; Djimde et al., 2001). A single pfcrt mutation, K76T, is sufficient to cause CQ resistance. Replacement of the positively charged lysine by an uncharged threonine removes the electrostatic charge which then prohibits the protonated and positively charged CQ drug molecule from pore entry (Sanchez et al., 2007). The high prevalence of the pfcrt 76T variant in our study is in agreement with reports from other areas where clinical CQ failure frequently occurs (Dorsey et al., 2001; Congpuong et al., 2005). Eight different genotypes in pfcrt76–pfmdr86-1042-1246 were observed with a high prevalence of the TNND genotype. We also investigated the association between the in vitro susceptibility of P. falciparum isolates and pfcrt–pfmdr1 genotypes. The pfcrt K76 allele, with or without the N86Y mutation, was found in CQ-sensitive parasites. Conversely, the K76T mutation, with or without the pfmdr1 N86Y mutation, was resistant to CQ. It has been proposed that mutations in pfmdr1 may not be sufficient in producing the CQ resistance phenotype, however the N86Y mutation can moderate drug responses (Foote et al., 1990; Reed et al., 2000; Djimde et al., 2001; Duraisingh et al., 2000). For the pfcrt gene, a strong and significant association between the single point mutation in codon 76 of the pfcrt gene and in vitro susceptibility to CQ was found. On the other hand, the pfmdr1 mutation in codon 86 was significantly associated with MQ susceptibility. Our findings indicate that N86Y mutation may enhance the parasite susceptibility to MQ. Therefore, in areas subjected to MQ pressure, wild-type 86N was found more frequently. No association was observed between pfcrt and pfmdr1 mutations and susceptibility to QN. When considering the relationship between the susceptibility of P. falciparum isolates to quinoline antimalarials and the origin of parasite isolates, we note high levels of CQ-resistant malaria emerging in Thailand. The prevalence of MQ-sensitive isolates was generally higher than MQ-resistant isolates and was found in all areas, whereas MQ-resistant isolates were found only at the Thai–Myanmar and Thai–Cambodian borders. No QN-resistant isolates were found in our study. Although CQ-resistance is highly prevalent in Africa and Southeast Asia, QN and MQ remain effective against clinical isolates (Wernsdorfer and Payne, 1991; Ringwald et al., 1999). In Thailand, MQ resistance continues to progress in Mae Sot (Thai–Myanmar border) and Bo Rai (Thai–Cambodian border) (Wongsrichanalai et al., 2001). The introduction of an artesunate and mefloquine combination treatment is believed to delay the emergence of multidrug resistance in these areas. Thus, the detection of pfmdr1 gene polymorphisms may not be a useful surveillance tool for the identification of antimalarial drugresistant malaria in Thailand. Despite the fact that CQ has been withdrawn from clinical use as a first-line drug for acute uncomplicated falciparum malaria for more than 50 years in Thailand,
40
K. Rungsihirunrat et al. / Acta Tropica 109 (2009) 37–40
the malaria parasite has been exposed to CQ (as first-line drug for Plasmodium vivax malaria) or other quinoline-containing drugs. Resistance to CQ, MQ and QN would be expected to compromise the clinical effectiveness of the current first-line combination treatment with MQ and artesunate for falciparum malaria in Thailand. Further study is required to clarify this issue. Acknowledgements We are grateful to all the participants and the staff of Malaria Clinics for their kind assistance. This study was founded by Malaria Research fund, The College of Public Health Sciences, Chulalongkorn University and partial supported by Thailand Research fund. References Basco, L.K., Le Bras, J., Rhoades, Z., Wilson, C.M., 1995. Analysis of pfmdr1 and drug susceptibility in fresh isolates of Plasmodium falciparum from Subsaharan Africa. Mol. Biochem. Parasitol. 74, 157–166. Chaiyaroj, S.C., Buranakiti, A., Angkasekwinai, P., Looareesuwan, S., Cowman, A.F., 1999. Analysis of mefloquine resistance and amplification of pfmdr1 in multidrug-resistant Plasmodium falciparum isolates from Thailand. Am. J. Trop. Med. Hyg. 61, 780–783. Congpuong, K., Na-Bangchang, K., Mungthin, M., Bualombai, P., Wernsdorfer, W.H., 2005. Molecular epidemiology of drug resistance markers of Plasmodium falciparum malaria in Thailand. Trop. Med. Int. Health 10, 717–722. Djimde, A., Doumbo, O.K., Cortese, J.F., Kayentao, K., Doumbo, S., Diourte, Y., Dicko, A., Su, X.Z., Nomura, T., Fidock, D.A., Wellems, T.E., Plowe, C.V., Coulibaly, D., 2001. A molecular marker for chloroquine-resistant falciparum malaria. N. Engl. J. Med. 344, 257–263. Dorsey, G., Kamya, M.R., Singh, A., Rosenthal, P.J., 2001. Polymorphisms in the Plasmodium falciparum pfcrt and pfmdr1 genes and clinical response to chloroquine in Kampala. Uganda. J. Infect. Dis. 183, 1417–1420. Duraisingh, M.T., Roper, C., Walliker, D., Warhurst, D.C., 2000. Increased sensitivity to the antimalarials mefloquine and artemisinin is conferred by mutations in the pfmdr1 gene of Plasmodium falciparum. Mol. Microbiol. 36, 955–961. Duraisingh, M.T., Cowman, A.F., 2005. Contribution of the pfmdr1 gene to antimalarial drug-resistance. Acta Trop. 94, 181–190. Fidock, D.A., Nomura, T., Talley, A.K., Cooper, R.A., Dzekunov, S.M., Ferdig, M.T., Ursos, L.M., Sidhu, A.B., Naude, B., Deitsch, K.W., Su, X.Z., Wootton, J.C., Roepe, P.D., Wellems, T.E., 2000. Mutations in the Plasmodium falciparum digestive vacuole transmembrane protein Pfcrt and evidence for their role in chloroquine resistance. Mol. Cell 6, 861–871. Foote, S.J., le, D.E., Martin, R.K., Oduola, A.M., Forsyth, K., Kemp, D.J., Cowman, A.F., 1990. Several alleles of the multidrug-resistance gene are closely linked to chloroquine resistance in Plasmodium falciparum. Nature 345, 255–258.
Lopes, D., Rungsihirunrat, K., Nogueira, F., Seugorn, A., Gil, J., Do Rosario, V.E., Cravo, P., 2002. Molecular characterization of drug resistant Plasmodium falciparum from Thailand. Malar. J. 1, 1–12. Na-Bangchang, K., Congpuong, K., 2007. Current malaria status and distribution of drug resistance in East and Southeast Asia with special focus to Thailand. Tohoku J. Exp. Med. 211, 99–113. Price, R.N., Cassar, C., Brockman, A., Duraisingh, M., Van Vugt, M., White, N.J., Nosten, F., Krishna, S., 1999. The pfmdr1 gene is associated with a multidrug-resistant phenotype in Plasmodium falciparum from the western border of Thailand. Antimicrob. Agents Chemother. 43, 2943–2949. Price, R.N., Uhlemann, A.C., Brockman, A., McGready, R., Ashley, E., Phaipun, L., Patel, R., Laing, K., Looareesuwan, S., White, N.J., Nosten, F., Krishna, S., 2004. Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number. Lancet 364, 438–447. Reed, M.B., Saliba, K.J., Caruana, S.R., Kirk, K., Cowman, A.F., 2000. Pgh1 modulates sensitivity and resistance to multiple antimalarials in Plasmodium falciparum. Nature 403, 906–909. Ringwald, P., Bickii, J., Basco, L.K., 1999. In vitro activity of dihydroartemisinin against clinical isolates of Plasmodium falciparum in Yaounde, Cameroon. Am. J. Trop. Med. Hyg. 61, 187–192. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, second Edition. Cold Spring Harbour Laboratory, Cold Spring Harbour, New York. Sanchez, C.P., Stein, W.D., Lanzer, M., 2007. Is PfCRT a channel or a carrier? Two competing models explaining chloroquine resistance in Plasmodium falciparum. Trends Parasitol. 23, 332–339. Sharma, Y.D., 2005. Genetic alteration in drug resistance markers of Plasmodium falciparum. Indian J. Med. Res. 121, 13–22. Sornmani, S., Butraporn, P., Fungladda, W., Okanurak, K., Dissapongsa, S., 1983. Migration and disease problem: a study of pattern of migration in an endemic area of malaria in Thailand. Southeast Asian J. Trop. Med. Public Health 14, 64–68. Thaithong, S., Beale, G., 1981. Resistance of ten Thai isolates of Plasmodium falciparum to chloroquine and pyrimethamine by in vitro tests. Trans. R. Soc. Trop. Med. Hyg. 75, 271–273. Thaithong, S., Seugorn, A., Beale, G.H., 1994. Culturing Plasmodium falciparum from finger-prick samples of infected blood. Trans. R. Soc. Trop. Med. Hyg. 88, 490. Wellems, T.E., 1991. Molecular genetics of drug resistance in Plasmodium falciparum malaria. Parasitol. Today 7, 110–112. Wernsdorfer, W.H., Payne, D., 1991. The dynamics of drug resistance in Plasmodium falciparum. Pharmacol. Ther. 50, 95–121. World Health Organization, 2001. In vitro micro-test (Mark III) for the assessment of the response of Plasmodium falciparum to chloroquine, mefloquine, quinine, amodiaquine, sulfadoxine/pyrimethamine and artemisinin WHO document CTD/MAL/97.2, Rev. 2, WHO, Geneva. Wilson, C., Volkman, S., Thaithong, S., Martin, R., Kyle, D., Milhous, W., Wirth, D., 1993. Amplification of Pfmdr1 associated with mefloquine and halofantrine resistance in Plasmodium falciparum from Thailand. Mol. Biochem. Parasitol. 57, 151– 160. Wongsrichanalai, C., Sirichaisinthop, J., Karwacki, J.J., Congpuong, K., Miller, R.S., Pang, L., Thimasarn, K., 2001. Drug resistance malaria on the Thai–Myanmar and Thai–Cambodian borders. Southeast Asian J. Trop. Med. Public Health 32, 41–49.