Sequence-Based Optimization of a Quantitative Real-Time PCR Assay for Detection of Plasmodium ovale and Plasmodium malariae

Abstract

Although microscopic examination of Giemsa-stained blood smears remains the gold standard for the diagnosis of malaria, molecular detection using PCR is becoming increasingly popular. Due to discrepant PCR and microscopy results, we aimed to optimize our detection assays for Plasmodium malariae and Plasmodium ovale by sequencing the 18S rRNA region and developing a new primer and probe set for real-time quantitative PCR (qPCR). Clinical specimens positive for P. malariae (n ‫؍‬ 15) or P. ovale (n ‫؍‬ 33) underwent amplification and sequencing of the 18S rRNA region. Based on sequence discrepancies between our current primer/probe and clinical isolates, degenerate P. ovale primer and probe were developed to determine if their performance characteristics improved. The reference (gold) standard was microscopy. No 18S sequence heterogeneity was observed among the P. malariae isolates, and the sensitivity and specificity of our current P. malariae qPCR assay were both 100%. Compared to microscopy, the sensitivity and specificity of our current P. ovale qPCR assay were 72.7% and 100%, respectively. Five single nucleotide polymorphisms (SNPs) were identified in P. ovale. The sensitivity of the new P. ovale assay increased to 100% with 100% specificity. We therefore improved the performance characteristics of our P. ovale molecular detection assay through the development of a degenerate primer and probe set which accommodates 18S SNPs among the 2 subspecies of P. ovale. Given the suboptimal sensitivity of rapid diagnostic tests for non-falciparum malaria and the typically low parasitemia of P. malariae and P. ovale, a well-performing confirmatory molecular assay is imperative for clinical laboratories.

Spectrum of Viral Pathogens in Blood of Malaria-Free Ill Travelers Returning to Canada

“…We confirmed all malaria results at study initiation using a multiplex real-time PCR assay ( 19 ). A malaria-negative specimen was defined as one for which Giemsa-stained expert microscopy and rapid diagnostic test results were negative and the patient did not have a documented prior episode of malaria in the past year.…”

Failure of atovaquone-proguanil malaria chemoprophylaxis in a traveler to Ghana

“…DNA extraction and quantitative real time PCR (qPCR) were conducted to confirm species as previously described [1,2]. Single-nucleotide polymorphism (SNP) analysis for cytochrome b (cytb) codon 268 and dihydrofolate reductase (dhfr) codons 16, 50, 51, 59, 108 and 164 were performed by Sanger sequencing (ABI 3130xl) and Pyrosequencing (Qiagen PyroMark Q24) [3].…”

Evaluation of Ebola Virus Inactivation Procedures for Plasmodium falciparum Malaria Diagnostics

“…We used 12.5 l of TaqMan Universal PCR master mix (Life Technologies), 5 l of DNA, and primers and probes, with concentrations as previously reported (12), for a final volume of 25 l per reaction mixture. All qPCR amplification curves were analyzed using a manual threshold cycle (C T ) of 0.02 and an automatic baseline.…”

“…Plasmodium-specific real-time PCR targeting the 18S rRNA gene (quantitative PCR [qPCR]) was performed, as previously described (12,13), using the ABI 7900HT real-time PCR system and under the following conditions: 50°C for 2 min, 95°C for 10 min, followed by 45 cycles of 95°C for 15 s and then 60°C for 1 min. We used 12.5 l of TaqMan Universal PCR master mix (Life Technologies), 5 l of DNA, and primers and probes, with concentrations as previously reported (12), for a final volume of 25 l per reaction mixture.…”