Study Samples

Informed consent, as approved by the ethics committees of the University of North Carolina and the Malawi College of Medicine/Ministry of Health, was obtained from all participants in this research study. The Malaria and HIV in Pregnancy Study (MHP) patient samples originated from a study of pregnant women attending Queen Elizabeth Central Hospital, an urban hospital in Blantrye. The complete characteristics of the cohort and study design have been described elsewhere (10). The Mepemba and Madziabango (MM) patient samples were also collected from pregnant women as part of a pilot randomized, open-label, efficacy study of intermittent preventive treatment in pregnancy at rural health clinics. The diversity of these infections, as well as a description of the cohort and study design, has been described elsewhere (3). The characteristics of the patient samples used in this study are outlined in Table 1. All samples analyzed in this study were from filter paper blood spots of peripheral blood.

Malaria DNA Stocks

All malaria DNA used in the experiments, other than clinical samples, was from MR-4. Wild type (pfcrt K76) was from P. falciparum strain 3d7 (MR-4, MRA-102G). Two strains of mutant DNA (pfcrt 76T) were used in these experiments: P. falciparum strain K1 (MR4, MRA-159G) was used for the CVIET-resistant haplotype, based on the amino acid sequence from codon 72 to codon 76, and P. falciparum strain 7g8 (MR4, MRA-152G) was used for the SVMNT-resistant haplotype.

Generation of Heteroduplex Tracking Assay (HTA) Probe

Wild-type P. falciparum DNA was amplified with a Peltier thermal cycler (MJ Research, Waltham, MA, USA) in a volume of 50 μL. The reaction conditions consisted of 5 μL DNA, 2.5 U HotStar Taq DNA polymerase (QIAGEN, Valencia, CA, USA), 5 μL 10× PCR buffer, 1 μL deoxynucleoside triphosphate mix (catalog no. U1511, Promega, Madison, WI, USA), and 2,000 nmol/L forward and reverse primers CRT HTA F: 5′-GGAAATGGCTCACGTTTAGG-3′, and CRT HTA R: 5′-TGTGAGTTTCGGATGTTACAAAA-3′. This reaction was amplified by preheating to 95°C for 15 min, followed by 35 cycles of 95°C for 30 s, 50°C for 30 s, and 72°C for 1 min. The reaction was completed with a 10-min hold at 72°C. The 250-bp PCR product was cloned into a PCR2.1 TOPO plasmid as described in the protocol of the Topo TA cloning kit (Invitrogen, Carlsbad, CA, USA), and the sequence was confirmed at the University of North Carolina-Chapel Hill Automated DNA Sequencing Facility. Mutations were randomly introduced into the construct at the –3, –1, +1, and +3 nt relative to the single nucleotide polymorphism involved with the pfcrt K76T mutation using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) (11). Potential probes were then amplified by colony PCR under the conditions noted above.

Potential probes were screened against wild-type and resistant PCR amplicons by using a heteroduplex mobility assay on an 8% polyacrylamide gel in 1% Tris-borate-EDTA buffer. After an annealing reaction, probes were evaluated for differential migration between the lanes containing the different amplicons. The annealing reaction containing 4 μL PCR product from a colony of a potential probe mixed with 4 μL PCR product of control DNA, 1 μL 100-pmol CRT HTA F primer, 1 μL 100-pmol CRT HTA R primer, and 2 μL 6× loading dye (Promega) was heated to 95°C for 2 min and then allowed to cool at 25°C for 5 min. The annealing reaction was then loaded into the wells of a nondenaturing acrylamide gel and run at a constant current of 17 mA for 5 h per gel with an SE 600 Gel Electrophoresis Unit (Amersham Biosciences, Piscataway, NJ, USA). DNA was visualized by UV after staining in an ethidium bromide solution for 20 min and destaining in double-distilled H₂O for 15 min. In all, 77 potential probes were screened. The successful probe was sequenced and showed a –1 A to C mutation (GenBank accession no. submission in process).

The plasmid containing the probe was harvested by using Promega Wizard Minipreps (Promega) and then amplified according to the conditions noted above. The PCR product was the blunt-end cloned into the pT7Blue vector with the Perfectly Blunt Cloning kit (Novagen, Inc., Madison, WI, USA). The probe was radiolabeled according to the methods of Ngrenngarmlert et al. (12).

HTA

The MSS-HTA was performed under the conditions noted by Ngrenngarmlert et al. with some modifications (12). An annealing reaction consisting of 4 μL PCR product (either a control or sample DNA) was mixed with 1 μL 10× annealing buffer (1 mol/L NaCl, 100 mmol/L Tris-HCl, pH 7.5, 20 mmol/L EDTA), 2 μL 6× loading dye, 500 nmol/L CRT HTA F primer, 500 nmol/L CRT HTA R primer, and 1 μL radiolabeled probe in a total volume of 12 μL. The annealing reaction and electrophoresis were carried out under the same conditions as the heteroduplex mobility assay noted above. All MSS-HTA gels included the following controls: water, a nontemplate control PCR, and PCRs from the 3 genomic DNA stocks. The gels were dried onto filter paper (Whatman, Florham Park, NJ, USA) and exposed to BioMax MR X-ray film (Eastman Kodak, Rochester, NY, USA) for ≈48 h at 25°C. In addition, the gels were exposed to a phosphorimager screen for 48 h, and band intensities were quantified by using a GE Storm 860 Phosphorimager (Amersham Biosciences) and ImageQuant version 5.2 software (Molecular Dynamics, Sunnyvale, CA, USA).

ARPCR

ARPCR detection of pfcrt 76T was performed according to the methods described by Djimde et al. (13). The primers for the assay were modified according to Wilson et al. (8).

Minority Variant Detection

MSS-HTA and ARPCR were both run against mixtures of control DNA in quadruplicate. Differing proportions of wild-type genomic DNA and CVIET-resistant haplotype genomic DNA were mixed to a final sample concentration of 0.1 ng/μL. If a band was not visible to the eye, or only visible in 1 replicate, it was not counted.

Detection of Minor Variants in Clinical Samples

MSS-HTA was used to screen clinical samples in duplicate. All MHP samples were assayed by MSS-HTA and ARPCR. MM samples were all initially assayed by MSS-HTA. ARPCR was performed on all samples positive by HTA and on a random selection of 20 MSS-HTA–negative samples.

Ten samples that were positive by MSS-HTA were selected and Topo TA–cloned (Invitrogen). Twenty-five colonies from each of these 10 samples were screened by colony real-time PCR to determine if the plasmid construct contained a wild-type or resistant pfcrt insert (8,12). A selection of 2 mutant and 4 wild-type plasmids isolated from these colonies was then sequenced.