A new method for detection of pfmdr1 mutations in Plasmodium falciparum DNA using real-time PCR
© Purfield et al; licensee BioMed Central Ltd. 2004
Received: 06 February 2004
Accepted: 07 May 2004
Published: 07 May 2004
Surveillance for drug-resistant Plasmodium falciparum should be a component of malaria control programmes. Real-time PCR methods for the detection of parasite single-nucleotide polymorphisms (SNPs) and gene amplification could be useful survellance tools.
A real-time PCR assay has been developed that identifies single nucleotide polymorphisms (SNPs) at amino acids 86, 184, 1034 and 1042 in the P. falciparum multi-drug resistant (pfmdr 1) gene that may be associated with anti-malarial drug resistance.
This assay has a sensitivity and specificity of 94% and 100% when compared to traditional PCR methods for genotyping. Only 54 of 68 (79%) paired pre- and post-culture DNA samples were concordant at all four loci.
Real-time PCR is a sensitive and specific method to detect SNP's in pfmdr 1. Genotypes of parasites after in vitro culture may not reflect that seen in vivo.
The increasing prevalence of multi-drug resistant parasites threatens to impede efforts to control malaria world-wide . Current in vitro and in vivo methods to monitor the emergence of drug resistance are difficult, costly and labor-intensive. Molecular methods could prove to be useful alternatives .
For Plasmodium falciparum, the pfmdr 1 gene product, PGH1, is thought to play an integral role in the mechanism behind parasite resistance to multiple malarial drugs . Both increased gene copy number and single-nucleotide polymorphisms (SNP's) have been associated in epidemiological studies with changes in sensitivity to chloroquine and mefloquine (reviewed in [1, 4, 5]). In two studies from South-east Asia, both increased pfmdr 1 gene copy number and SNPs have been associated with in vitro resistance to mefloquine [6, 7].
In this manuscript, a real-time PCR assay has been developed for the detection of pfmdr 1 SNPs. This assay was developed to be used in conjunction with a previously developed assay for pfmdr 1 gene copy number  to assess molecular markers as predictors of mefloquine failure in a clinical study that took place in Sangkhlaburi, Thailand, in 2001–2. The results of the clinical study will be described in a subsequent publication.
Numerous studies have shown discrepancies between in vitro and in vivo tests for antimalarial drug resistance [8–11]. In order to understand why, pfmdr 1 genotypes were determined directly from patient blood and from cultures derived from those patients and compared.
Subject recruitment. This study received ethical approval from the University of North Carolina IRB and the Thai Ministry of Public Health. Patients presenting with slide-confirmed falciparum malaria to the free Ministry of Public Health malaria clinic or the Kwai River Christian Hospital clinic in Sangklaburi, Thailand from during July 2001 – August 2002 were enrolled. Patients with vivax infections, history of anti-malarial drug use within the past two weeks, bleeding tendency (by self-reported history or based on medical records), or severe/complicated malaria requiring prompt medical management for life support were excluded. In total, 74 patients consented. Blood samples were taken, aliquoted, stored in liquid nitrogen, and transported to the Armed Forces Research Institute for the Medical Sciences (AFRIMS) in Bangkok. At AFRIMS, aliquots were thawed for in vitro culture. 59 of the 74 patient blood samples were successfully cultured as previously described [6, 12]. Parasites were cultured for between 2 and 188 days (mean = 38.5; median = 23). DNA was extracted from patient blood ("pre-culture") and cultured parasites ("post-culture") using the QiaAmp DNA Blood Minikit Blood and Body Fluid Spin Protocol (Qiagen, Valencia, CA) and then shipped on dry ice to the University of North Carolina.
DNA was PCR-amplified using a MJ Thermocycler (MJ Research, Waltham, MA) as previously described . DNA sequencing was performed at the University of North Carolina Sequencing Core facility using a 3100 Genetic Analyser (Applied Biosystems, Foster City, CA). The sequencing reaction was done using the ABI PRISM™ BigDye™ Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq® DNA Polymerase, FS (Applied Biosystems).
Primers and probes used in real-time PCR assay
Oligonucleotide (5'--> 3')
Asn (wt*) probe
Tyr (mut**) probe
Tyr (wt) probe
Phe (mut) probe
Ser (wt) probe
Cys (mut) probe
Asn (wt) probe
Asp (mut) probe
Of the 74 patients enrolled, parasites were successfully cultured from 59. Following treatment, 20 of the 74 admission patients recrudesced. Parasites were successfully cultured from 13 of these 20 recrudescent patients.
Sensitivity and Ssecificity of Real-Time PCR
Prevalence in monoclonal culture samples (n = 20) of mutations detected by traditional PCR and sequencing (gold standard) and real-time PCR, and sensitivity and specificity of realtime PCR assay.
All mutations correct
Two of the 22 samples were found to be mixed infections when sequenced. One sample contained a mixture of Tyrl84 and Phel84. The other sample contained a mixture of Asn86 and His86. The real-time PCR method accurately identified the Tyrl84/Phel84 mixed population. However, because the probes were designed to detect only Asn86 or Tyr86, His86 was not detected by real-time PCR.
Comparison of SNPs pre- and post-culture
In order to determine how the process of culturing parasites in vitro alters genotype, a comparison was made between the pfmdr 1 genotypes of DNA extracted from pre-and post-culture parasites as measured by real-time PCR. Of the paired pre- and post-culture specimens, both members of 68 pairs were successfully amplified by real-time PCR. Fifty-eight of these pairs were obtained from admission patient blood while 10 pairs were derived from recrudescent patients.
Genotype results of DNA from pre-culture and post-culture DNA by real-time PCR
Pre-culture genotype results
Post-culture genotype results
Tyr & Phe
Asn & Asp
Tyr & Phe
Asn & Asp
Novel His86 mutation identified
In one DNA culture sample neither wild type nor mutant probes revealed amplification during real-time PCR. This post-culture sample was amplified by standard PCR methods and sequenced to genotype the position 86 region. Sequencing this sample revealed a His86.
In this paper, a real-time PCR method is described which accurately ascertains parasite pfmdr 1 genotype. This method has high sensitivity (94%) and specificity (100%) for detecting four pfmdr 1 SNPs associated with drug resistance. There was a 21% discordance in the real-time PCR genotype results between DNA samples obtained directly from patient blood and DNA samples obtained from subsequent in vitro cultures.
Real-time PCR offers several advantages over standard PCR methods for genotyping DNA. First, because of the small volume the material, costs for real-time PCR are much lower than for standard PCR and are as little as $0.40 per reaction. Second, using real-time PCR, a single technician can perform and analyse hundreds of reactions per day, thus reducing the labor cost as well. Third, this method reduces the opportunity for post-PCR contamination. Once the sample is prepared with the reagents, amplification and analysis are completed in a closed-tube system. Finally, real-time PCR analysis of genotype is easier and requires less scientific expertise for analysis. Thus, the initial cost of a real-time PCR instrument (~$40,000) would be offset by savings in labor, quality assurance, and materials in labs analysing large numbers of samples.
In this paper, a novel His86 mutation was identified in two samples. To our knowledge, this mutation has never been identified before and may or may not be associated with drug resistance. Further studies on this mutation are needed.
A previous study utilized a novel real-time PCR technology to identify pfmdr 1 SNPs in parasite DNA extracted from clinical blood samples . Although the chemistry of the assay differed from the technique described here, both studies yielded similar results and concluded that real-time PCR may accurately detect Asn86 → Tyr86, Tyr184 → Phe184, Ser1034 → Cys1034 and Asn1042 → Asp1042 in pfmdr 1. In addition, de Monbrison, et al., used this method to genotype Asp1246 → Tyr1246. SNPs at 1246 were not studied here because they have not been found in South-east Asia [6, 7].
Twenty-one percent of samples manifested differences in pfmdr 1 genotype pre- and post-culture. Since most patients are probably infected with a mixture of strains, the genotypes observed post-culture reflects the results of the selection of a subset of strains by in vitro culture conditions. This could manifest itself as loss of strains observed pre-culture or the appearance of strains not observed pre-culture because they were present at levels below detection limits. A much greater change in genotype (74.5%) was observed in a previous study looking at variations in the polymorphic regions of MSP1 and MSP2 (merozoite surface protein 1 and 2) as well as GLURP (glutamate-rich protein) , perhaps because these genes are more variable.
In addition to SNP detection, our group has previously described a real-time PCR method to measure pfmdr 1 gene amplification . Thus it is now possible to assess both pfmdr 1 gene copy number and SNPs using real-time PCR making it possible to carry out a complete assessment of pfmdr 1 genetics in large cohorts.
Real-time PCR is a sensitive and specific method to detect pfmdr 1 mutations and gene amplification. Because it is inexpensive and amenable to high-throughput, it could be a useful public health tool.
This work was supported by NIH grant R23 AI054590 and US Department of Defense-Global Emerging Infections Surveillance and Response Program (DoD-GEIS). We would like to acknowledge AFRIMS Malaria Field Team, Mark Fukuda, Paul Wilson, Charlotte Lanteri, Jesse Kwiek, Alisa Alker, the staff of Kwai River Christian Hospital and of the Vector Borne Diseases Control Unit #9 (Sangkhlaburi) for their assistance and support.
- Wongsrichanalai C, Pickard AL, Wernsdorfer WH, Meshnick SR: Epidemiology of drug-resistant malaria. Lancet Infect Dis. 2002, 2: 209-218. 10.1016/S1473-3099(02)00239-6.View ArticlePubMedGoogle Scholar
- Plowe CV: Monitoring antimalarial drug resistance: making the most of the tools at hand. J Exp Biol. 2003, 206: 3745-3752. 10.1242/jeb.00658.View ArticlePubMedGoogle Scholar
- Reed MB, Saliba KJ, Caruana SR, Kirk K, Cowman AF: Pgh1 modulates sensitivity and resistance to multiple antimalarials in Plasmodium falciparum. Nature. 2000, 403: 906-909. 10.1038/35002615.View ArticlePubMedGoogle Scholar
- Wernsdorfer WH, Noedl H: Molecular markers for drug resistance in malaria: use in treatment, diagnosis and epidemiology. Curr Opin Infect Dis. 2003, 16: 553-558. 10.1097/00001432-200312000-00007.View ArticlePubMedGoogle Scholar
- Talisuna AO, Bloland P, D'Alessandro U: History, dynamics, and public health importance of malaria parasite resistance. Clin Microbiol Rev. 2004, 17: 235-254. 10.1128/CMR.17.1.235-254.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Pickard AL, Wongsrichanalai C, Purfield A, Kamwendo D, Emery K, Zalewski C, Kawamoto F, Miller RS, Meshnick SR: Resistance to antimalarials in Southeast Asia and genetic polymorphisms in pfmdr1. Antimicrob Agents Chemother. 2003, 47: 2418-2423. 10.1128/AAC.47.8.2418-2423.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Price RN, Cassar C, Brockman A, Duraisingh M, van Vugt M, White NJ, Nosten F, Krishna S: The pfmdr1 gene is associated with a multidrug-resistant phenotype in Plasmodium falciparum from the western border of Thailand. Antimicrob Agents Chemother. 1999, 43: 2943-2949.PubMed CentralPubMedGoogle Scholar
- Takechi M, Matsuo M, Ziba C, MacHeso A, Butao D, Zungu IL, Chakanika I, Bustos MD: Therapeutic efficacy of sulphadoxine/pyrimethamine and susceptibility in vitro of Plasmodium falciparum isolates to sulphadoxine-pyremethamine and other antimalarial drugs in Malawian children. Trop Med Int Health. 2001, 6: 429-434. 10.1046/j.1365-3156.2001.00735.x.View ArticlePubMedGoogle Scholar
- Ringwald P, Basco LK: Comparison of in vivo and in vitro tests of resistance in patients treated with chloroquine in Yaounde, Cameroon. Bull World Health Organ. 1999, 77: 34-43.PubMed CentralPubMedGoogle Scholar
- Segurado AA, di Santi SM, Shiroma M: In vivo and in vitro Plasmodium falciparum resistance to chloroquine, amodiaquine and quinine in the Brazilian Amazon. Rev Inst Med Trop Sao Paulo. 1997, 39: 85-90.View ArticlePubMedGoogle Scholar
- Dua VK, Kar PK, Gupta NC, Kar I, Sharma VP: In vivo and in vitro sensitivity of Plasmodium falciparum to chloroquine in Chennai (Tamil Nadu), India. Indian J Malariol. 1997, 34: 1-7.PubMedGoogle Scholar
- Trager W, Jensen JB: Human malaria parasites in continuous culture. Science. 1976, 193: 673-675.View ArticlePubMedGoogle Scholar
- Livak KJ: Allelic discrimination using fluorogenic probes and the 5' nuclease assay. Genet Anal. 1999, 14: 143-149. 10.1016/S1050-3862(98)00019-9.View ArticlePubMedGoogle Scholar
- Afonina I, Zivarts M, Kutyavin I, Lukhtanov E, Gamper H, Meyer RB: Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder. Nucleic Acids Res. 1997, 25: 2657-2660. 10.1093/nar/25.13.2657.PubMed CentralView ArticlePubMedGoogle Scholar
- Holland PM, Abramson RD, Watson R, Gelfand DH: Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci U S A. 1991, 88: 7276-7280.PubMed CentralView ArticlePubMedGoogle Scholar
- de Monbrison F, Raynaud D, Latour-Fondanaiche C, Staal A, Favre S, Kaiser K, Peyron F, Picot S: Real-time PCR for chloroquine sensitivity assay and for pfmdr1-pfcrt single nucleotide polymorphisms in Plasmodium falciparum. J Microbiol Methods. 2003, 54: 391-401. 10.1016/S0167-7012(03)00086-1.View ArticlePubMedGoogle Scholar
- Viriyakosol S, Siripoon N, Zhu XP, Jarra W, Seugorn A, Brown KN, Snounou G: Plasmodium falciparum: selective growth of subpopulations from field samples following in vitro culture, as detected by the polymerase chain reaction. Exp Parasitol. 1994, 79: 517-525. 10.1006/expr.1994.1112.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.