A TaqMan real-time PCR assay for the detection and quantitation of Plasmodium knowlesi

<p>Abstract</p> <p>Background</p> <p>The misdiagnosis of <it>Plasmodium knowlesi </it>by microscopy has prompted a re-evaluation of the geographic distribution, prevalence and pathogenesis of this species using molecular diagnostic tools. In this report, a specific probe for <it>P. knowlesi</it>, that can be used in a previously described TaqMan real-time PCR assay for detection of <it>Plasmodium </it>spp., and <it>Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae </it>and <it>Plasmodium ovale</it>, was designed and validated against clinical samples.</p> <p>Methods</p> <p>A hydrolysis probe for a real-time PCR assay was designed to recognize a specific DNA sequence within the <it>P. knowlesi </it>small subunit ribosomal RNA gene. The sensitivity, linearity and specificity of the assay were determined using plasmids containing <it>P. knowlesi </it>DNA and genomic DNA of <it>P. falciparum, P. knowlesi, P. malariae, P. ovale </it>and <it>P. vivax </it>isolated from clinical samples. DNA samples of the simian malaria parasites <it>Plasmodium cynomolgi </it>and <it>Plasmodium inui </it>that can infect humans under experimental conditions were also examined together with human DNA samples.</p> <p>Results</p> <p>Analytical sensitivity of the <it>P. knowlesi</it>-specific assay was 10 copies/μL and quantitation was linear over a range of 10-10⁶copies. The sensitivity of the assay is equivalent to nested PCR and <it>P. knowlesi </it>DNA was detected from all 40 clinical <it>P. knowlesi </it>specimens, including one from a patient with a parasitaemia of three parasites/μL of blood. No cross-reactivity was observed with 67 <it>Plasmodium </it>DNA samples (31 <it>P. falciparum</it>, 23 <it>P. vivax</it>, six <it>P. ovale</it>, three <it>P. malariae</it>, one <it>P. malariae/P. ovale</it>, one <it>P. falciparum/P. malariae, one P. inui and one P. cynomolgi) </it>and four samples of human DNA.</p> <p>Conclusions</p> <p>This test demonstrated excellent sensitivity and specificity, and adds <it>P. knowlesi </it>to the repertoire of <it>Plasmodium </it>targets for the clinical diagnosis of malaria by real-time PCR assays. Furthermore, quantitation of DNA copy number provides a useful advantage over other molecular assays to investigate the correlation between levels of infection and the spectrum of disease.</p

High-Throughput Genotyping of Single Nucleotide Polymorphisms in the Plasmodium falciparum dhfr Gene by Asymmetric PCR and Melt-Curve Analysis▿

Mutations within the Plasmodium falciparum dihydrofolate reductase gene (Pfdhfr) contribute to resistance to antimalarials such as sulfadoxine-pyrimethamine (SP). Of particular importance are the single nucleotide polymorphisms (SNPs) within codons 51, 59, 108, and 164 in the Pfdhfr gene that are associated with SP treatment failure. Given that traditional genotyping methods are time-consuming and laborious, we developed an assay that provides the rapid, high-throughput analysis of parasite DNA isolated from clinical samples. This assay is based on asymmetric real-time PCR and melt-curve analysis (MCA) performed on the LightCycler platform. Unlabeled probes specific to each SNP are included in the reaction mixture and hybridize differentially to the mutant and wild-type sequences within the amplicon, generating distinct melting curves. Since the probe is present throughout PCR and MCA, the assay proceeds seamlessly with no further addition of reagents. This assay was validated for analytical sensitivity and specificity using plasmids, purified genomic DNA from reference strains, and parasite cultures. For all four SNPs, correct genotypes were identified with 100 copies of the template. The performance of the assay was evaluated with a blind panel of clinical isolates from travelers with low-level parasitemia. The concordance between our assay and DNA sequencing ranged from 84 to 100% depending on the SNP. We also directly compared our MCA assay to a published TaqMan real-time PCR assay and identified major issues with the specificity of the TaqMan probes. Our assay provides a number of technical improvements that facilitate the high-throughput screening of patient samples to identify SP-resistant malaria

Prevalence of Plasmodium falciparum Resistance Markers to Sulfadoxine-Pyrimethamine among Pregnant Women Receiving Intermittent Preventive Treatment for Malaria in Uganda

This study was to assess the prevalence of mutations in Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes among pregnant women using sulfadoxine-pyrimethamine (SP) as an intermittent preventive treatment (IPTp).The aim of this study was to assess the prevalence of mutations in Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes among pregnant women using sulfadoxine-pyrimethamine (SP) as an intermittent preventive treatment (IPTp). A molecular epidemiological study of P. falciparum parasite resistance markers to SP was conducted from August 2010 to February 2012 in Mukono district in central Uganda. DNA was extracted from 413 P. falciparum-positive samples. Real-time PCR, followed by melting curve analysis, was used to characterize point mutations in the Pfdhfr and Pfdhps genes that are associated with SP resistance. The prevalence of the single-nucleotide mutations in Pfdhfr at codons 51I, 59R, and 108N and in Pfdhps at codons 437G and 540E was high (>98%), reaching 100% fixation after one dose of SP, while the prevalence of 581G was 3.3% at baseline, reaching 12.5% after one dose of SP. At baseline, the prevalence of Pfdhfr and Pfdhps quintuple mutations was 89%, whereas the sextuple mutations (including 581G) were not prevalent (3.9%), reaching 16.7% after one dose of SP. However, the numbers of infections at follow-up visits were small, and hence there was insufficient statistical power to test whether there was a true rise in the prevalence of this allele. The overall high frequency of Pfdhfr and Pfdhps quintuple mutations throughout pregnancy excluded further analyses of possible associations between certain haplotypes and the risk of lower birth weight and anemia. However, women infected with P. falciparum had 1.3-g/dl-lower hemoglobin levels (P_0.001) and delivered babies with a 400-g-lower birth weight (P_0.001) compared to nonparasitemic women. Despite this, 44 women who were P. falciparum positive at baseline became negative after one or two doses of SP (i.e., 50.5%), implying that SP-IPTp still has some efficacy. P. falciparum resistance markers to SP are high in this population, whereas P. falciparum infection was associated with poor birth outcomes

Post-Arrival Screening for Malaria in Asymptomatic Refugees Using Real-Time PCR

Malaria is a significant health risk to refugee populations originating from endemic areas, but there is little consensus on screening and/or treatment approaches for malaria in this population. Furthermore, detection of malaria in semi-immune asymptomatic refugees is limited by the sensitivity of diagnostic tests used for screening. We determined the prevalence of malaria by microscopy and real-time polymerase chain reaction (PCR) in a consecutive population of 324 asymptomatic refugees examined in Edmonton, Canada, during 2009–2010. Although all thick and thin blood smear results were negative, 10 subjects (3.1%) tested PCR positive for Plasmodium DNA. Interestingly, 6 of 10 PCR positive subjects are at risk of malaria relapse by P. vivax or P. ovale infections. These results suggest that appropriate guidelines for malaria screening should consider the risk of relapsing infections, and they highlight the potential usefulness of real-time PCR in the diagnosis of asymptomatic malaria