We designed a set of 23 HRM assays using the 3′-blocked LunaProbe technology to genotype SNPs across six genes previously associated with reduced drug sensitivity. These assays failed traditional TaqMan primer/probe design requirements, but HRM design was successful. The LunaProbe technology has been shown to increase assay sensitivity: the addition of 3′-blocked probes covering short (less than 50-bp) regions within the primer amplicon results in enhanced peak melting-temperature (Tm
) separation between alleles, especially class IV SNPS (A→T or T→A) that are a challenge to differentiate with traditional HRM methodologies (A and B). LunaProbes also allow separation of SNPs located very close or adjacent to each other in the genome (B and C). This improved design resulted in better peak separation than previously reported with HRM (1
). We confirmed the genotypes determined by HRM analysis through direct sequencing of the amplicons, finding 100% correspondence of the observed genotypes to the reported sequences.
Fig 1 Representative melting peaks of HRM assays. (A) Example of a single-SNP probe assay; Pfcrt H97 shown. The probe is designed to be a perfect match to the wild type (3D7). Any mutations in this probe region lower the melting peak. Shown here is TM90C6B (more ...)
Our genotyping assays proved to be robust and specific. They were able to detect and differentiate alleles with low concentrations of template material even in the presence of excess contaminating genomic material. Using cultured isolates without excess human material, as would be found in filter-extracted patient samples, the limit of detection was 10−5
ng, similar to that found by other groups (A) (1
) and also similar to other PCR-based methodologies and comfortably within the yield from extraction of patient samples collected on filter paper in the field. The assays remained specific for their target regions in the presence of an excess of human genomic material that we introduced in order to mimic the ratios of human and parasite DNAs obtained from patient samples; however, the absolute limit of detection with contaminating human DNA was reduced to approximately 10 pg (B).
Fig 2 Limit of detection and performance with human genomic material. (A) Limit of detection of assays. A representative assay (pfdhps 436/437) shows the limit of detection. The melting peaks are still distinguishable, with 10−5 ng of Plasmodium template (more ...)
The genotyping assays are also sensitive and were made more so by our technology modifications. We were able to detect alleles comprising 2% to 5% of the total sample; by introducing the novel amplification method MAAB on a LightScanner-32 instrument using glass capillary tubes, we were able to increase the sensitivity to detect mutant alleles present at less than 1% of the mixture (B). There is some vertical spread between the mixture peaks; however, the inflection for each mixture does not change compared to the negative or peak curve seen with the unmixed samples. This inflection difference is consistent across repeated trials and is reliable as long as unmixed controls are present.
Fig 3 Assay performance with mixtures of genomes and MAAB. (A) Mixture of three genomes showing clear differentiation between fractions: 3D7-7G8-Dd2. The pfcrtK72-76 assay is shown. (B) MAAB. Shown in the pfcrt K72-76 assay, MAAB in the LightScanner-32 increases (more ...)
Remarkably, we could discriminate individual alleles even in more complex mixtures containing three or more alleles using our modified HRM approach (A). These results demonstrate that the assays are sensitive and specific and are able to identify alleles present at less than 1% of a mixture when multiple alleles exist in a sample. Comparable amplification technologies, such as TaqMan, fail to consistently detect mutant alleles at less than 10% of the sample mixture (8
). Without the refinements to HRM using blocked probes, the typical sensitivity for detection is still an improvement at 2 to 5%.
Additional methods, such as nested-PCR amplification and RFLP analysis, rely on more subjective and complicated interpretation of gel bands. Here, we show that HRM analysis results in clearly separated and unambiguous melting peaks to determine sample genotypes.
To assess the accuracy and reliability of these assays, we applied them to genotype 44 independent, culture-adapted parasites from a number of different geographical regions and verified our results by sequencing (Genewiz, Inc., South Plainfield, NJ) (). From our assays, we found that 40% of the samples had the mutant genotype for pfcrt
K76 and 18% and 40% displayed variants at dhps
residues 436 and 437, respectively. For dhfr
, 65% were mutants at residues 51 and 59, and 70% were mutants at residue 108. While many other SNPs associated with drug resistance were primarily wild-type genotypes, there were variant polymorphisms in this international collection for each of the following loci: pfcrt
220 (40%) and 356 (12.5%), pfmdr1
86 (32.5%) and 184 (70%), and PfATPase6
(40%). All mutations detected by genotyping assays were confirmed by sequencing (Genewiz, Inc., South Plainfield, NJ), and our assays showed 100% correlation with the sequencing results, as well as those reported on PlasmoDB version 6.3 (http://www.plasmodb.org
). The genotyping assays are also sensitive and were made more so by technology improvements. These data validate the assays as able to reliably detect previously characterized mutations in the six drug resistance loci.
Sample and genotype information
We next tested the correspondence of our genotyping assays with drug phenotypes among the culture-adapted parasites (2
). We evaluated the K76 locus of pfcrt
associated with chloroquine resistance and the N51, C59, and S108 loci of dhfr
associated with pyrimethamine resistance. We observed 100% correspondence between the pfcrt
K76T mutation and chloroquine resistance among culture-adapted parasites. We also observed 100% correspondence between the N51I allele (100%) and the C59R allele (100%) in dhfr
for pyrimethamine resistance. For the S108N allele, four of the parasites deviated from direct correspondence with pyrimethamine resistance. Three of the parasites that had the S108N allele tested sensitive to pyrimethamine. These parasites were wild type for the N51 and C59 alleles and had pyrimethamine resistance levels near the cutoff (2,000 nM) for resistance (3
). One parasite that had the S108 allele tested resistant to pyrimethamine. This parasite, however, was mutant for the N51I and C59R alleles and also had a level of pyrimethamine resistance that was just over the resistance cutoff level of 2,000 nM. Thus, we observed almost complete correspondence between the genotyped alleles and the expected drug responses for chloroquine and pyrimethamine among the culture-adapted parasites in this analysis, confirming that these molecular markers are useful for interpreting drug responses in P. falciparum
We then demonstrated the usefulness of our HRM assays on materials derived from blood collected on filter paper from patients seeking treatment for malaria at the Thies, Senegal, clinic. shows the prevalences of mutations in the following loci: pfcrt
76 (15%), dhps
436 (26%) and 437 (48%), and dhfr
51 and 59 (85%) and 108 (93%). Other SNPs with variant alleles include pfcrt
220 (26%) and 356 (22%), pfmdr1
86 (7%), pfmdr1
184 (70%), and PfATPase6
623 and 431 (7%). Based upon comparison to data from previous years, there has been a decrease in the prevalence of mutations associated with chloroquine resistance and an increase in mutations associated with pyrimethamine resistance. The decrease in chloroquine resistance but increase in pyrimethamine resistance is consistent with the use of these drugs for malaria treatment in Senegal. While chloroquine use has largely been discontinued, use of pyrimethamine has continued (initially in a combination treatment with sulfadoxine and amodiaquine) and remains the mainstay treatment for intermittent preventative treatment for pregnant women (IPTp) in Senegal to date (30
While traditional probe-based technology approaches fail to amplify if novel variants appear within the amplicon region, our assays allowed us to detect both known and novel haplotypes and mutations in genetic loci associated with drug resistance. Techniques such as TaqMan require prior knowledge of all sequence variants for successful genotyping in order to design labeled probes for every polymorphism. We have demonstrated that our method, on the other hand, is far more robust and can identify novel variants. First, we detected previously unreported haplotypes in Senegal for pfdhps
436/437 in three of the culture-adapted parasite lines, including the 436A/437A haplotype for two parasites (P05.02 and Th105.07), as well as the 436Y/437G haplotype for another parasite (Th28.04) (A). Second, we detected a new mutation in the cytB
locus from a single patient sample (B). This novel mutation, confirmed by sequencing (Genewiz, Inc., South Plainfield, NJ), is the M270I variant, located near the amino acid associated with atovaquone resistance in the cytB
gene at Y268 (17
). Because this novel allele was detected in direct patient material, we were unable to test for any biological effects of the mutation on drug sensitivity. Because the probe-based analysis also allows study of the larger amplicon, we were able to discover SNPs 22 bp upstream of the probe region but still within the amplicon targeted by the ATPase6 431 assay in D10 (MRA-201) (data not shown). The additional utility of focusing on genomic regions known to be under selection is the increased likelihood that additional mutations associated with reduced drug sensitivity will be found in the regions targeted by the assay primers that may confer drug resistance. While samples with variant melting peaks must be sequenced to characterize the emerging mutation, HRM technologies offer the unique ability to quickly and economically scan samples for emerging variants.
Fig 4 Detection of emerging and new mutations. (A) New SNP haplotype detected in Senegal patient samples. The pfdhps 436A/437A and 436Y/437G haplotypes have not previously been reported in Senegal, though they have been reported in other regions. (B) Novel (more ...)
In addition, the assays differentiated between copies of a genetic locus. The pfmdr1
locus is known to be present in multiple copies in some drug-resistant parasites, particularly those resistant to mefloquine (13
). As part of this analysis, we observed different genotypes for the pfmdr1
locus in the Dd2 parasite and confirmed the presence of the newly reported pfmdr1
N86F mutation in Dd2 (4
). This demonstrates the ability of these assays to detect mutations among loci subject to copy number variation, which has been implicated in some drug-resistant parasites (11
). Copy number variation has also been identified as a source of reduced sensitivity to antimalarial drugs. Instruments with real-time capacity (Idaho Technology LightScanner-32 and Qiagen Rotorgene-Q) offer additional utility of these assays for direct detection of these variations.
Finally, we demonstrated that our assays distinguish between single and mixed alleles, in both culture-adapted and clinical samples. Multiple melting peaks appearing in single-genome infections determined by molecular barcode or other methods (8
) suggest that there are variant copies of the genetic locus in the parasite genome ().