A new method for detection of pfmdr1 mutations in Plasmodium falciparum DNA using real-time PCR

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 [6]. 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).

For real-time PCR, pre-culture DNA and post-culture DNA were genotyped using an Applied Biosystems Prism 7000 Sequence Detection System (Applied Biosystems). This assay uses sequence-specific fluorescently-labelled "MGB™ " probes to distinguish SNPs [13‐15]. Primers and probes (Table 1) were designed using Primer Express Software, Version 2.0 (Applied Biosystems). Primers were obtained from Qiagen. Fluorescent MGB™ probes were produced by Applied Biosystems and labelled with reporter dyes, 6FAM or VIC, at their 5' ends and non-fluorescent quencher molecules at their 3' ends. Primer and probe concentrations were optimized according to the TaqMan Universal PCR Master Mix technical bulletin (Applied Biosystems) using DNA obtained from P. falciparum strain 3D7 or from patient specimens. 300 nM of both forward and reverse primers were found to be optimal for all SNPs. Optimal probe concentration was determined to be 250 nM for all MGB probes used in this study. Using 10-fold serial dilutions of 3D7 genomic P. falciparum DNA, as few as 8 copies (0.001 ng) of genomic P. falciparum DNA could be detected.

Each real-time PCR amplification reaction contained both the wild type probe labelled with FAM, and the mutant probe labelled with VIC. Presence of either SNP could be imputed from the relative increase of fluorescence of the two fluorophores (Fig. 1). All reactions were done in duplicate.

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.

The sensitivity and specificity of SNP detection using real-time PCR were measured in 22 post-culture DNA samples using DNA sequencing as the gold standard (Table 2). Nineteen cultures were derived from blood of patients upon admission to the study. The other three cultures were obtained from who failed treatment. Two samples contained mixtures of SNPs at single positions based on DNA sequencing. Of the 20 monoclonal samples, 19 were concordant for all pfmdr 1 SNPs when analysed using both methods. The discordant sample was not an artefact since both the real-time PCR and sequencing reactions gave identical results when repeated. The discordant sample contained a single discrepancy at position 184 (phenylalanine by sequencing and tyrosine by real-time PCR). Thus, the sensitivity of real-time PCR method to detect Tyr86 or Asp1042 SNPs was 100% and the sensitivity to detect Phel84 was 92%; there were no Cysl034 mutations detected. Real-time PCR was 100% specific for each mutation. The sensitivity and specificity of the assay to detect all four SNPs were 94% and 100%, respectively.

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.

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.

Fifty-eight of the pre-culture genotypes were monoclonal, nine pre-culture samples had mixtures of alleles at single positions, and one had mixed SNPs at two positions (184 and 1042). Table 3 shows the number of isolates with specific combinations of pfmdr 1 genotypes pre- and post-culture at each specific locus. Isolates with identical genotype at each locus before and after culture are enumerated in the shaded diagonal. Overall, fifty-four of 68 (79%) pre-culture DNA samples were completely concordant with post-culture DNA at all four polymorphic sites examined. While there was a wide variation in the length of time parasites were cultured before DNA was extracted, there was no correlation between genotype discordances and length of time in culture (data not shown). Five of the 14 discordant isolates exhibited single differences at position 184, and two were different at position 1042. Seven discrepant samples contained mixtures of alleles at one or more sites before and/or after culture. Six samples showed a mixture of alleles pre-culture and single alleles post-culture. This type of selection occurred at amino acid 184 for four isolates, and 1042 for two isolates. Two of these six also had changes at a second position, amino acid 86. Finally, one of the seven samples was monoclonal at aminoacid 184 pre-culture but polyclonal post-culture (and also exhibited a change from Asn to Asp at 1042).

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.