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An efficient PCR–SSCP-based method for detection of a chloroquine resistance marker in the PfCRT gene of Plasmodium falciparum
Transactions of the Royal Society of Tropical Medicine and Hygiene (2006) 100, 243—247
An efficient PCR—SSCP-based method for detection of a chloroquine resistance marker in the PfCRT gene of Plasmodium falciparum Sasmita Mishra, Dipak Kumar Raj, R.K. Hazra 1, A.P. Dash 2, Prakash C. Supakar ∗ Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar 751023, India Received 22 June 2004 ; received in revised form 21 April 2005; accepted 13 May 2005 Available online 8 November 2005 KEYWORDS Malaria; Plasmodium falciparum; PfCRT gene; PCR—SSCP; India
Summary The spread of chloroquine resistance throughout the world poses a major problem in combating malaria. In the present study, an efficient polymerase chain reaction—single strand conformational polymorphism (PCR—SSCP)-based assay detected the PfCRT K76T point mutation, which is a marker for chloroquine resistance. For the first time, we have used a PCR—SSCP-based technique to identify the mutation in a single-step labelling reaction during PCR and SSCP gel electrophoresis. This assay is 100% efficient, giving no false-positive or -negative results, and can be carried out within a short bench time. We have successfully analysed 120 natural isolates using the PCR—SSCP method for detection of the chloroquine resistance marker and found 91 of the 120 samples to show the PfCRT T76 mutation, and 71% (65 of the 91 samples) showed a positive correlation with chloroquine resistance from the clinical data of the patients. The PCR—SSCP technique can also be applied for the detection of new haplotypes of the PfCRT gene and surveillance of chloroquine-resistant malaria in malaria-endemic localities around the world. © 2005 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.
1. Introduction
∗
Corresponding author. Tel.: +91 674 2300137/2301476; fax: +91 674 2300728. E-mail address: [email protected] (P.C. Supakar). 1 Present address: Regional Medical Research Centre, Chandrasekharpur, Bhubaneswar 751023, India. 2 Present address: Malaria Research Center, 22 Shamnath Marg, New Delhi 110054, India.
Malaria, with an estimated 300—500 million cases and 1.5—2.7 million deaths per year, is a major health problem (WHO, 1996). Despite earlier successes in reducing human malaria, the disease is now rampant in many tropical and subtropical countries, taking a heavy toll on human life. Among the several drugs of choice, chloroquine is the most frequently chosen because it is less toxic,
0035-9203/$ — see front matter © 2005 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.trstmh.2005.05.020
244 inexpensive and highly effective against susceptible malaria parasites (Wellems and Plowe, 2001). Increasing rates of chloroquine resistance contribute to rising morbidity and mortality from malaria in most African and Asian countries. In vitro and molecular methods are available to test chloroquine resistance in Plasmodium falciparum (Babiker et al., 2001; Mehlotra et al., 2001; Thanh et al., 2001), of which the culture method is quite laborious and in many cases does not show any result (WHO, 1997). The PfCRT gene encodes a membrane transporter of P. falciparum, and a single point mutation at amino acid codon position 76 has been associated with chloroquine resistance in several field studies (Binder et al., 2002; Chen et al., 2001; Djimde et al., 2001; Sidhu et al., 2002). Sequencing and PCR—RFLP are used as the techniques of choice for detection of a point mutation at amino acid codon position 76 of the PfCRT gene (Fidock et al., 2000; Mehlotra et al., 2001). However, screening of the PfCRT gene by the sequencing method in a large number of individuals is quite cumbersome and expensive. Although PCR—RFLP can be used for mutation detection, it requires two rounds of PCR and a restriction endonuclease ApoI digestion of a 145 bp fragment. Therefore, we have used single PCR in combination with single strand conformational polymorphism (SSCP) for screening samples to detect mutations at codon positions 72—76 of the PfCRT gene. The 145 bp region of PfCRT examined in this study is semiconserved and mutations have only been reported at codon positions 72—76 (Mehlotra et al., 2001; Vathsala et al., 2004). In this study, PCR—SSCP was used for PfCRT T76 allelic typing and subsequently to screen P. falciparum isolates from the eastern Indian state of Orissa.
2. Materials and methods 2.1. Study area and P. falciparum isolates Orissa, a state of India, was selected as the study area. It has a longitude of 30.20.N and latitude of 85.54.E and the whole region comes under the endemic zone of malaria transmission. Orissa makes up 38% of India’s population and contributes 20% of P. falciparum cases and 40% of malarial deaths in India. The Institutional Ethical Committee, Institute of Life Sciences, Bhubaneswar, India, approved this study. Blood samples (N = 128) were collected from P. falciparum-infected individuals attending a malaria clinic at primary health centres of hyperendemic regions of Orissa. Blood was collected from
S. Mishra et al. consenting volunteers as 1 ml samples in 0.08 M EDTA. The presence of parasites was confirmed by microscopic examination and samples were stored at −70 ◦ C for further analysis. The patients’ blood samples taken for DNA isolation showed a wide range of parasitaemias (0.1—10%).
2.2. DNA isolation DNA was prepared from patient samples using a rapid DNA isolation method described earlier (Foley et al., 1992) with some modifications. Briefly, 500 ml of ice-cold 5 mM sodium phosphate (pH 8) was added to 20 l of venipuncture blood and vortexed. After centrifugation for 10 min in a microcentrifuge tube at 4 ◦ C, the supernatant was discarded. The pellet was suspended in 100 l PBS containing 0.01% saponin and washed twice with the same buffer by repeating the above steps of vortexing and centrifugation. After washing, the pellet was suspended in 50 l of sterile water, vortexed and then boiled for 20 min at 100 ◦ C. After centrifugation in a microcentrifuge tube for 10 min at 4 ◦ C, the supernatant was collected and 5 ml of supernatant was used for 20 l PCR reaction.
2.3. Adaptation of the PCR—SSCP method for typing of the PfCRT T76 allele The PCR—SSCP was performed as described by Raj et al. (2004) with modifications. Briefly, 5 l of genomic DNA was amplified for 40 cycles using primers TCRD1 and TCRD2 as described earlier (Djimde et al., 2001) in a 20 l reaction in the presence of [␣32 P] dATP. The reaction conditions were an initial denaturation for 5 min at 94 ◦ C followed by 40 cycles of 1 min denaturation at 94 ◦ C, 1 min annealing at 54 ◦ C and extension at 72 ◦ C for 1 min. Final extension was carried out at 72 ◦ C for 10 min. Then, 2 l of the [␣32 P]-labelled PCR product was mixed with 9 l of loading dye (98% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol). The samples were denatured at 95 ◦ C for 10 min and immediately chilled in ice for 5 min. Then, 1—2 l of the above samples were electrophoresed through a non-denaturing 5% polyacrylamide and 0.25% agarose composite gel containing 3% glycerol (Peng et al., 1995). Electrophoresis was performed at 250 V for 18—20 h at 25 ◦ C. The gel was then autoradiographed for 12—18 h at −70 ◦ C.
2.4. PCR—RFLP analysis Two rounds of PCR were performed to amplify a 145 bp region that contains a marker (point
Detection of chloroquine resistance marker by PCR—SSCP
245
Figure 1 A representative number of samples showing PCR—RFLP. Lanes 1 and 6: 100 bp ladder; lane 5: K76 allele; lanes 2—4 and 7—10: T76 alleles.
mutation) for chloroquine resistance. Approximately 5 l of genomic DNA was taken in a 20 l PCR reaction mixture containing 25 nmol of forward and reverse primers. The first set of primers (TCRP1 and TCRP2) corresponds to a 537 bp product. The second set of anchored primers (TCRD1 and TCRD2) was used to amplify the required 145 bp fragment as reported earlier (Djimde et al., 2001). The PfCRT gene mutations at codon position 76 (K76T) of P. falciparum were analysed using RFLP of the 145 bp PCR-amplified product as described earlier (Labbe et al., 2001).
2.5. Sequencing of the PCR product The PCR-amplified 145 bp double-stranded DNA was purified using a PCR product purification kit (Montage; Millipore Corp., Bedford, MA, USA). Approximately 5—7 ng of template DNA was sequenced in an automated DNA sequencer (CEQ-8000 Analyser; Beckman-Coulter, Fullerton, CA, USA).
3. Results and discussion In total, 128 samples were taken for analysis of drug resistance. Eight samples did not show any band pattern on the PCR—SSCP gel corresponding to the 145 bp fragment of the PfCRT gene, possibly owing to an incorrect microscopic diagnosis. Hence, 120 samples with a wide range of parasitaemias (0.1—10%) were analysed for detection of the drug resistance marker. All samples were analysed by PCR—SSCP, but it was not possible to analyse five samples by PCR—RFLP for the occurrence of the PfCRT K76T mutation. Failure to analyse the five samples by PCR—RFLP could be due to the relatively low sensitivity of PCR—RFLP and a low parasitaemia. From the 120 samples analysed by PCR—SSCP, 91 showed the PfCRT T76 allele. After RFLP of detectable amplified bands, it was possible
to detect the appropriate pattern for K76T mutation in the PfCRT gene (Figure 1) in 115 samples, and 86 samples were found to have the PfCRT T76 mutation. The PCR—RFLP method shows expected results and has some advantages such as accuracy, reproducibility and being less prone to variations; however, it is quite tedious and requires more bench time. Furthermore, PCR—RFLP requires two rounds of PCR and ApoI digestion for detection of the K76T mutation. PCR—SSCP analysis using a composite gel of acrylamide and agarose showed effective detection of PfCRT K76T mutation compared with a nondenaturing polyacrylamide gel (Peng et al., 1995). It was possible to analyse 100% (120/120) of the samples, compared with RFLP in which only 96% (115/120) could be analysed. The advantage of our method lies in the fact that a single-step PCR reaction is sufficient for detection of the K76T mutation. The SSCP method requires less consumables and the results are highly reproducible. The reported sequence of PfCRT genes from different geographical locations does not show any mutation other than the base change in positions 72—76 (Mehlotra et al., 2001). The PCR—SSCP analysis is a very sensitive technique in terms of detection of the K76T mutation in the PfCRT gene. Furthermore, all samples detected by PCR—SSCP were also identified by PCR—RFLP. In various regions of the world, a number of haplotypes such as CVIET, CVMNT and SVMNT have been reported (Mehlotra et al., 2001; Vathsala et al., 2004). However, in any geographical location, mixed haplotypes are rare (Mehlotra et al., 2001; Nagesha et al., 2003; Vathsala et al., 2004). In such circumstances, by taking different sets of haplotypes as a control, we can analyse the whole set of samples from a region by the PCR—SSCP method. PCR—SSCP has previously been used in the cg2 gene for analysis of drug-resistant parasites (Labbe et al., 2001). However, for the first time we have used the PCR—SSCP technique in the PfCRT gene for detection of the K76T mutation, which is an
246
Figure 2 A representative number of samples showing PCR—SSCP. Lane 1: undenatured DNA samples, showing only double-stranded (DS) DNA; lanes 2—7: DNA samples denatured with formamide, showing DS and singlestranded (SS) PCR product DNA; lane 2: control haplotype SVMNK (K76 allele); lane 3: control haplotype CVIET (T76 allele); lane 4: control haplotype SVMNT (T76 allele); lanes 5—7: unknown samples.
appropriate marker for chloroquine resistance. Our data showed the T76 allele in the samples (Figure 2, lanes 2, 3, 4, 5 and 7) that were also found to be resistant to ApoI digestion in PCR—RFLP (Figure 1). One sample taken from each of a set of samples representing a particular type of banding pattern was sequenced and then used as the controls for further studies. The band patterns observed in the PCR—SSCP gel for each of the 120 field isolates taken for the study were found to match with at least one of the controls, SVMNK, CVIET and SVMNT haplotypes (Figure 2, lanes 2, 3 and 4, respectively). Since the single strands of all the different haplotypes of the PfCRT gene show a different mobility in the PCR—SSCP gel (Figure 2), a mixed haplotype can also be detected. Previous studies showed that the majority of Indian isolates show SVMNT or SVMNK haplotypes and only a very few cases show CVIET (Vathsala et al., 2004). Our data show that the majority of the isolates (113 of 120) were either SVMNK or SVMNT, and 7 isolates showed band patterns that matched with the control CVIET (one shown in Figure 2, lane 3 as control). However, we were unable to find any altered banding pattern for CVIEK, which is the wild-type for haplotypes CVIET, because of low prevalence. In the area of the present study, we were unable to find a single isolate with CVMNK haplotype, so it did not affect our analysis by looking at the control pattern of PCR—SSCP. No abnormal banding pattern or
S. Mishra et al. mixed haplotype pattern was observed for the 120 isolates analysed in the PCR—SSCP gel. Correlation of drug resistance with the widespread K76T mutation around the world reveals that it can be used as a marker for surveillance of chloroquine-resistant malaria (Cox-Singh et al., 2003; Daily et al., 2003; Ochong et al., 2003). Our study showed that 65 of 91 patients (PfCRT T76 isolates) had taken chloroquine within the past 10 days (from their clinical data) but had healthy parasites in their fingerprick blood. The above finding indicates that the use of chloroquine as a first-line drug for the treatment of malaria should be withdrawn from the Indian state of Orissa, and alternatives such as artemisinin derivative drugs and sulfa drugs should be used. Again, our preliminary study on complicated malaria (cerebral malaria and multi-organ failure cases) shows a very high percentage of PfCRT T76 mutation (unpublished data). Taking the above facts into consideration, we strongly believe that the high death rate in Orissa (40% of all deaths due to malaria in India) may be due to the chloroquine-resistant parasites rather than the environmental factors. We have shown for the first time that the PCR—SSCP method can be effectively applied for surveillance of chloroquine resistance in a malaria-endemic area and, depending upon the data, appropriate drug administration can be undertaken to treat people in malaria-endemic areas. Conflicts of interest statement The authors have no conflicts of interest concerning the work reported in this paper.
Acknowledgements This work was supported by funds from the Department of Biotechnology, Government of India.
References Babiker, H.A., Pringle, S.J., Abdel-Muhsin, A., Mackinnon, M., Hunt, P., Walliker, D., 2001. High-level chloroquine resistance in Sudanese isolates of Plasmodium falciparum is associated with mutations in the chloroquine resistant transporter gene pfCRT and the multidrug resistant gene pfmdr1. J. Infect. Dis. 183, 1535—1538. Binder, R.K., Borrmann, S., Adegnika, A.A., Missinou, M.A., Kremsner, P.G., Kun, J.F., 2002. Polymorphisms in the parasite genes for pfCRT and pfmdr-1 as molecular markers for chloroquine resistance in Plasmodium falciparum in Lambarene, Gabon. Parasitol. Res. 88, 475—476. Chen, N., Russell, B., Staley, J., Kotecka, B., Nasveld, P., Cheng, Q., 2001. Sequence polymorphisms in pfCRT are strongly
Detection of chloroquine resistance marker by PCR—SSCP associated with chloroquine resistance in Plasmodium falciparum. J. Infect. Dis. 183, 1543—1545. Cox-Singh, J., Lu, H.Y., Davis, T.M., Ilett, K.F., Hackett, L.P., Matusop, A., Singh, B., 2003. Application of a multi-faceted approach for the assessment of treatment response in falciparum malaria: a study from Malaysian Borneo. Int. J. Parasitol. 33, 1545—1552. Daily, J.P., Roberts, C., Thomas, S.M., Ndir, O., Dieng, T., Mboup, S., Wirth, D.F., 2003. Prevalence of Plasmodium falciparum pfCRT polymorphisms and in vitro chloroquine sensitivity in Senegal. Parasitology 126, 401—405. 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. Fidock, D.A., Nomura, T., Talley, A.K., 2000. Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Mol. Cell 6, 861—871. Foley, M., Ranford-Cartwright, L.C., Babiker, H.A., 1992. Rapid and simple method for isolating DNA from finger prick samples of blood. Mol. Biochem. Parasitol. 53, 241— 244. Labbe, A.C., Bualombai, P., Pillai, D.R., Zhong, K.J.Y., Vanisaveth, V., Hongvathong, B., Looareesuwan, S., Kain, K.C., 2001. Molecular marker for chloroquine-resistant Plasmodium falciparum malaria in Thailand and Laos. Ann. Trop. Med. Parasitol. 95, 781—788. Mehlotra, R.K., Fujioka, H., Roepe, P.D., Janneh, O., Ursos, L.M., Jacobs-Lorena, V., McNamara, D.T., Bockarie, M.J., Kazura, J.W., Kyle, D.E., Fidock, D.A., Zimmerman, P.A., 2001. Evolution of a unique Plasmodium falciparum chloroquine-resistance phenotype in association with pfCRT polymorphism in Papua New Guinea and South America. Proc. Natl. Acad. Sci. USA 98, 12689—12694. Nagesha, H.S., Casey, G.J., Rieckmann, K.H., Fryauff, D.J., Laksana, B.S., Reeder, J.C., Maguire, J.D., Baird, J.K., 2003. New haplotypes of the Plasmodium falciparum chloroquine resistance transporter (pfCRT) gene among chloroquine-
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resistant parasite isolates. Am. J. Trop. Med. Hyg. 68, 398—402. Ochong, E.O., Van den Broek, I.V., Keus, K., Nzila, A., 2003. Short report: association between chloroquine and amodiaquine resistance and allelic variation in the Plasmodium falciparum multiple drug resistance 1 gene and the chloroquine resistance transporter gene in isolates from the upper Nile in southern Sudan. Am. J. Trop. Med. Hyg. 69, 184—187. Peng, H., Du, M., Ji, J., Isaacson, P.G., Pan, L., 1995. High resolution SSCP analysis using polyacrylamide agarose composite gel and a background-free silver staining method. Biotechnique 19, 411—414. Raj, D.K., Das, B.R., Dash, A.P., Supakar, P.C., 2004. Identification of a rare point mutation at C-terminus of merozoite surface antigen-1 gene of Plasmodium falciparum in eastern Indian isolates. Exp. Parasitol. 106, 45—49. Sidhu, A.B., Verdier-Pinard, D., Fidock, D.A., 2002. Chloroquine resistance in Plasmodium falciparum malaria parasites conferred by pfCRT mutations. Science 298, 210—213. Thanh, N.V., Cowman, A.F., Hipgrave, D., Kim, T.B., Phuc, B.Q., Cong, L.D., Biggs, B.A., 2001. Assessment of susceptibility of Plasmodium falciparum to chloroquine, quinine, sulfadoxine—pyrimethamine and artemisinin in southern Viet Nam. Trans. R. Soc. Trop. Med. Hyg. 95, 513—517. Vathsala, P.G., Pramanik, A., Dhanasekaran, S., Devi, C.U., Pillai, C.R., Subbarao, S.K., Ghosh, S.K., Tiwari, S.N., Sathyanarayan, T.S., Deshpande, P.R., Mishra, G.C., Ranjit, M.R., Dash, A.P., Rangarajan, P.N., Padmanaban, G., 2004. Widespread occurrence of the Plasmodium falciparum chloroquine resistance transporter (PfCRT) gene haplotype SVMNT in P. falciparum malaria in India. Am. J. Trop. Med. Hyg. 70, 256—259. Wellems, T.E., Plowe, C.V., 2001. Chloroquine resistant malaria. J. Infect. Dis. 184, 770—776. WHO, 1996. World malaria situation in 1993. Wkly. Epidemiol. Rec. 71, 17—22. WHO, 1997. In vitro microtest (Mark III) for the assessment of the response of Plasmodium falciparum to chloroquine, mefloquine, quinine, sulfadoxine/pyrimethamine and artemisinin. World Health Organization, Geneva, WHO/CTD/MAL/97.20.
An efficient PCR—SSCP-based method for detection of a chloroquine resistance marker in the PfCRT gene of Plasmodium falciparum Sasmita Mishra, Dipak Kumar Raj, R.K. Hazra 1, A.P. Dash 2, Prakash C. Supakar ∗ Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar 751023, India Received 22 June 2004 ; received in revised form 21 April 2005; accepted 13 May 2005 Available online 8 November 2005 KEYWORDS Malaria; Plasmodium falciparum; PfCRT gene; PCR—SSCP; India
Summary The spread of chloroquine resistance throughout the world poses a major problem in combating malaria. In the present study, an efficient polymerase chain reaction—single strand conformational polymorphism (PCR—SSCP)-based assay detected the PfCRT K76T point mutation, which is a marker for chloroquine resistance. For the first time, we have used a PCR—SSCP-based technique to identify the mutation in a single-step labelling reaction during PCR and SSCP gel electrophoresis. This assay is 100% efficient, giving no false-positive or -negative results, and can be carried out within a short bench time. We have successfully analysed 120 natural isolates using the PCR—SSCP method for detection of the chloroquine resistance marker and found 91 of the 120 samples to show the PfCRT T76 mutation, and 71% (65 of the 91 samples) showed a positive correlation with chloroquine resistance from the clinical data of the patients. The PCR—SSCP technique can also be applied for the detection of new haplotypes of the PfCRT gene and surveillance of chloroquine-resistant malaria in malaria-endemic localities around the world. © 2005 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.
1. Introduction
∗
Corresponding author. Tel.: +91 674 2300137/2301476; fax: +91 674 2300728. E-mail address: [email protected] (P.C. Supakar). 1 Present address: Regional Medical Research Centre, Chandrasekharpur, Bhubaneswar 751023, India. 2 Present address: Malaria Research Center, 22 Shamnath Marg, New Delhi 110054, India.
Malaria, with an estimated 300—500 million cases and 1.5—2.7 million deaths per year, is a major health problem (WHO, 1996). Despite earlier successes in reducing human malaria, the disease is now rampant in many tropical and subtropical countries, taking a heavy toll on human life. Among the several drugs of choice, chloroquine is the most frequently chosen because it is less toxic,
0035-9203/$ — see front matter © 2005 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.trstmh.2005.05.020
244 inexpensive and highly effective against susceptible malaria parasites (Wellems and Plowe, 2001). Increasing rates of chloroquine resistance contribute to rising morbidity and mortality from malaria in most African and Asian countries. In vitro and molecular methods are available to test chloroquine resistance in Plasmodium falciparum (Babiker et al., 2001; Mehlotra et al., 2001; Thanh et al., 2001), of which the culture method is quite laborious and in many cases does not show any result (WHO, 1997). The PfCRT gene encodes a membrane transporter of P. falciparum, and a single point mutation at amino acid codon position 76 has been associated with chloroquine resistance in several field studies (Binder et al., 2002; Chen et al., 2001; Djimde et al., 2001; Sidhu et al., 2002). Sequencing and PCR—RFLP are used as the techniques of choice for detection of a point mutation at amino acid codon position 76 of the PfCRT gene (Fidock et al., 2000; Mehlotra et al., 2001). However, screening of the PfCRT gene by the sequencing method in a large number of individuals is quite cumbersome and expensive. Although PCR—RFLP can be used for mutation detection, it requires two rounds of PCR and a restriction endonuclease ApoI digestion of a 145 bp fragment. Therefore, we have used single PCR in combination with single strand conformational polymorphism (SSCP) for screening samples to detect mutations at codon positions 72—76 of the PfCRT gene. The 145 bp region of PfCRT examined in this study is semiconserved and mutations have only been reported at codon positions 72—76 (Mehlotra et al., 2001; Vathsala et al., 2004). In this study, PCR—SSCP was used for PfCRT T76 allelic typing and subsequently to screen P. falciparum isolates from the eastern Indian state of Orissa.
2. Materials and methods 2.1. Study area and P. falciparum isolates Orissa, a state of India, was selected as the study area. It has a longitude of 30.20.N and latitude of 85.54.E and the whole region comes under the endemic zone of malaria transmission. Orissa makes up 38% of India’s population and contributes 20% of P. falciparum cases and 40% of malarial deaths in India. The Institutional Ethical Committee, Institute of Life Sciences, Bhubaneswar, India, approved this study. Blood samples (N = 128) were collected from P. falciparum-infected individuals attending a malaria clinic at primary health centres of hyperendemic regions of Orissa. Blood was collected from
S. Mishra et al. consenting volunteers as 1 ml samples in 0.08 M EDTA. The presence of parasites was confirmed by microscopic examination and samples were stored at −70 ◦ C for further analysis. The patients’ blood samples taken for DNA isolation showed a wide range of parasitaemias (0.1—10%).
2.2. DNA isolation DNA was prepared from patient samples using a rapid DNA isolation method described earlier (Foley et al., 1992) with some modifications. Briefly, 500 ml of ice-cold 5 mM sodium phosphate (pH 8) was added to 20 l of venipuncture blood and vortexed. After centrifugation for 10 min in a microcentrifuge tube at 4 ◦ C, the supernatant was discarded. The pellet was suspended in 100 l PBS containing 0.01% saponin and washed twice with the same buffer by repeating the above steps of vortexing and centrifugation. After washing, the pellet was suspended in 50 l of sterile water, vortexed and then boiled for 20 min at 100 ◦ C. After centrifugation in a microcentrifuge tube for 10 min at 4 ◦ C, the supernatant was collected and 5 ml of supernatant was used for 20 l PCR reaction.
2.3. Adaptation of the PCR—SSCP method for typing of the PfCRT T76 allele The PCR—SSCP was performed as described by Raj et al. (2004) with modifications. Briefly, 5 l of genomic DNA was amplified for 40 cycles using primers TCRD1 and TCRD2 as described earlier (Djimde et al., 2001) in a 20 l reaction in the presence of [␣32 P] dATP. The reaction conditions were an initial denaturation for 5 min at 94 ◦ C followed by 40 cycles of 1 min denaturation at 94 ◦ C, 1 min annealing at 54 ◦ C and extension at 72 ◦ C for 1 min. Final extension was carried out at 72 ◦ C for 10 min. Then, 2 l of the [␣32 P]-labelled PCR product was mixed with 9 l of loading dye (98% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol). The samples were denatured at 95 ◦ C for 10 min and immediately chilled in ice for 5 min. Then, 1—2 l of the above samples were electrophoresed through a non-denaturing 5% polyacrylamide and 0.25% agarose composite gel containing 3% glycerol (Peng et al., 1995). Electrophoresis was performed at 250 V for 18—20 h at 25 ◦ C. The gel was then autoradiographed for 12—18 h at −70 ◦ C.
2.4. PCR—RFLP analysis Two rounds of PCR were performed to amplify a 145 bp region that contains a marker (point
Detection of chloroquine resistance marker by PCR—SSCP
245
Figure 1 A representative number of samples showing PCR—RFLP. Lanes 1 and 6: 100 bp ladder; lane 5: K76 allele; lanes 2—4 and 7—10: T76 alleles.
mutation) for chloroquine resistance. Approximately 5 l of genomic DNA was taken in a 20 l PCR reaction mixture containing 25 nmol of forward and reverse primers. The first set of primers (TCRP1 and TCRP2) corresponds to a 537 bp product. The second set of anchored primers (TCRD1 and TCRD2) was used to amplify the required 145 bp fragment as reported earlier (Djimde et al., 2001). The PfCRT gene mutations at codon position 76 (K76T) of P. falciparum were analysed using RFLP of the 145 bp PCR-amplified product as described earlier (Labbe et al., 2001).
2.5. Sequencing of the PCR product The PCR-amplified 145 bp double-stranded DNA was purified using a PCR product purification kit (Montage; Millipore Corp., Bedford, MA, USA). Approximately 5—7 ng of template DNA was sequenced in an automated DNA sequencer (CEQ-8000 Analyser; Beckman-Coulter, Fullerton, CA, USA).
3. Results and discussion In total, 128 samples were taken for analysis of drug resistance. Eight samples did not show any band pattern on the PCR—SSCP gel corresponding to the 145 bp fragment of the PfCRT gene, possibly owing to an incorrect microscopic diagnosis. Hence, 120 samples with a wide range of parasitaemias (0.1—10%) were analysed for detection of the drug resistance marker. All samples were analysed by PCR—SSCP, but it was not possible to analyse five samples by PCR—RFLP for the occurrence of the PfCRT K76T mutation. Failure to analyse the five samples by PCR—RFLP could be due to the relatively low sensitivity of PCR—RFLP and a low parasitaemia. From the 120 samples analysed by PCR—SSCP, 91 showed the PfCRT T76 allele. After RFLP of detectable amplified bands, it was possible
to detect the appropriate pattern for K76T mutation in the PfCRT gene (Figure 1) in 115 samples, and 86 samples were found to have the PfCRT T76 mutation. The PCR—RFLP method shows expected results and has some advantages such as accuracy, reproducibility and being less prone to variations; however, it is quite tedious and requires more bench time. Furthermore, PCR—RFLP requires two rounds of PCR and ApoI digestion for detection of the K76T mutation. PCR—SSCP analysis using a composite gel of acrylamide and agarose showed effective detection of PfCRT K76T mutation compared with a nondenaturing polyacrylamide gel (Peng et al., 1995). It was possible to analyse 100% (120/120) of the samples, compared with RFLP in which only 96% (115/120) could be analysed. The advantage of our method lies in the fact that a single-step PCR reaction is sufficient for detection of the K76T mutation. The SSCP method requires less consumables and the results are highly reproducible. The reported sequence of PfCRT genes from different geographical locations does not show any mutation other than the base change in positions 72—76 (Mehlotra et al., 2001). The PCR—SSCP analysis is a very sensitive technique in terms of detection of the K76T mutation in the PfCRT gene. Furthermore, all samples detected by PCR—SSCP were also identified by PCR—RFLP. In various regions of the world, a number of haplotypes such as CVIET, CVMNT and SVMNT have been reported (Mehlotra et al., 2001; Vathsala et al., 2004). However, in any geographical location, mixed haplotypes are rare (Mehlotra et al., 2001; Nagesha et al., 2003; Vathsala et al., 2004). In such circumstances, by taking different sets of haplotypes as a control, we can analyse the whole set of samples from a region by the PCR—SSCP method. PCR—SSCP has previously been used in the cg2 gene for analysis of drug-resistant parasites (Labbe et al., 2001). However, for the first time we have used the PCR—SSCP technique in the PfCRT gene for detection of the K76T mutation, which is an
246
Figure 2 A representative number of samples showing PCR—SSCP. Lane 1: undenatured DNA samples, showing only double-stranded (DS) DNA; lanes 2—7: DNA samples denatured with formamide, showing DS and singlestranded (SS) PCR product DNA; lane 2: control haplotype SVMNK (K76 allele); lane 3: control haplotype CVIET (T76 allele); lane 4: control haplotype SVMNT (T76 allele); lanes 5—7: unknown samples.
appropriate marker for chloroquine resistance. Our data showed the T76 allele in the samples (Figure 2, lanes 2, 3, 4, 5 and 7) that were also found to be resistant to ApoI digestion in PCR—RFLP (Figure 1). One sample taken from each of a set of samples representing a particular type of banding pattern was sequenced and then used as the controls for further studies. The band patterns observed in the PCR—SSCP gel for each of the 120 field isolates taken for the study were found to match with at least one of the controls, SVMNK, CVIET and SVMNT haplotypes (Figure 2, lanes 2, 3 and 4, respectively). Since the single strands of all the different haplotypes of the PfCRT gene show a different mobility in the PCR—SSCP gel (Figure 2), a mixed haplotype can also be detected. Previous studies showed that the majority of Indian isolates show SVMNT or SVMNK haplotypes and only a very few cases show CVIET (Vathsala et al., 2004). Our data show that the majority of the isolates (113 of 120) were either SVMNK or SVMNT, and 7 isolates showed band patterns that matched with the control CVIET (one shown in Figure 2, lane 3 as control). However, we were unable to find any altered banding pattern for CVIEK, which is the wild-type for haplotypes CVIET, because of low prevalence. In the area of the present study, we were unable to find a single isolate with CVMNK haplotype, so it did not affect our analysis by looking at the control pattern of PCR—SSCP. No abnormal banding pattern or
S. Mishra et al. mixed haplotype pattern was observed for the 120 isolates analysed in the PCR—SSCP gel. Correlation of drug resistance with the widespread K76T mutation around the world reveals that it can be used as a marker for surveillance of chloroquine-resistant malaria (Cox-Singh et al., 2003; Daily et al., 2003; Ochong et al., 2003). Our study showed that 65 of 91 patients (PfCRT T76 isolates) had taken chloroquine within the past 10 days (from their clinical data) but had healthy parasites in their fingerprick blood. The above finding indicates that the use of chloroquine as a first-line drug for the treatment of malaria should be withdrawn from the Indian state of Orissa, and alternatives such as artemisinin derivative drugs and sulfa drugs should be used. Again, our preliminary study on complicated malaria (cerebral malaria and multi-organ failure cases) shows a very high percentage of PfCRT T76 mutation (unpublished data). Taking the above facts into consideration, we strongly believe that the high death rate in Orissa (40% of all deaths due to malaria in India) may be due to the chloroquine-resistant parasites rather than the environmental factors. We have shown for the first time that the PCR—SSCP method can be effectively applied for surveillance of chloroquine resistance in a malaria-endemic area and, depending upon the data, appropriate drug administration can be undertaken to treat people in malaria-endemic areas. Conflicts of interest statement The authors have no conflicts of interest concerning the work reported in this paper.
Acknowledgements This work was supported by funds from the Department of Biotechnology, Government of India.
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