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.

FIG 1

The 18S rRNA gene was sequenced and aligned with 4 classic and 5 variant type sequences from GenBank for 33 specimens positive for P. ovale. P. ovale forward primer and probe (nucleotide positions 1112 to 1139 and 1141 to 1159, respectively, of GenBank sequence L48987.1) sequences of the current assay were shown. The new primer and probe designed in this study with degenerate nucleotides bind to the same location. Single nucleotide polymorphisms (denoted by * and labeled as SNPs 1 to 5) were identified on 3 loci to which the primer and 2 loci to which the probe hybridized. SNP 5, along with the double-G deletions three nucleotides downstream, was used to differentiate between classic and variant types. In specimen number 30, in the SNP 5 position, although a dominant C nt was observed in the chromatogram, a minor A was also evident (denoted by an M). The dominant nt of the SNPs were indicated here; however, analysis of the peak heights of chromatograms revealed that the other polymorphic nt was also evident in SNPs 1 to 3, suggesting coinfection of more than one strain of P. ovale.