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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.

SUBMITTER: Phuong M

PROVIDER: S-EPMC3993485 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Sequence-based optimization of a quantitative real-time PCR assay for detection of Plasmodium ovale and Plasmodium malariae.

Phuong Melissa M Lau Rachel R Ralevski Filip F Boggild Andrea K AK

Journal of clinical microbiology 20140115 4

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 ...[more]

PMID: 24430459

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Project description:BackgroundPlasmodium ovale is comprised of two genetically distinct subspecies, P. ovale curtisi and P. ovale wallikeri. Although P. ovale subspecies are similar based on morphology and geographical distribution, allelic differences indicate that P. ovale curtisi and P. ovale wallikeri are genetically divergent. Additionally, potential clinical and latency duration differences between P. ovale curtisi and P. ovale wallikeri demonstrate the need for investigation into the contribution of this neglected malaria parasite to the global malaria burden.MethodsIn order to detect all P. ovale subspecies simultaneously, we developed an inclusive P. ovale-specific real-time PCR assay based on conserved regions between P. ovale curtisi and P. ovale wallikeri in the reticulocyte binding protein 2 (rbp2) gene. Additionally, we characterized the P. ovale subspecies prevalence from 22 asymptomatic malaria infections using multilocus genotyping to discriminate P. ovale curtisi and P. ovale wallikeri.ResultsOur P. ovale rbp2 qPCR assay validation experiments demonstrated a linear dynamic range from 6.25 rbp2 plasmid copies/microliter to 100,000 rbp2 plasmid copies/microliter and a limit of detection of 1.5 rbp2 plasmid copies/microliter. Specificity experiments showed the ability of the rbp2 qPCR assay to detect low-levels of P. ovale in the presence of additional malaria parasite species, including P. falciparum, P. vivax, and P. malariae. We identified P. ovale curtisi and P. ovale wallikeri in Western Kenya by DNA sequencing of the tryptophan-rich antigen gene, the small subunit ribosomal RNA gene, and the rbp2 gene.ConclusionsOur novel P. ovale rbp2 qPCR assay detects P. ovale curtisi and P. ovale wallikeri simultaneously and can be utilized to characterize the prevalence, distribution, and burden of P. ovale in malaria endemic regions. Using multilocus genotyping, we also provided the first description of the prevalence of P. ovale curtisi and P. ovale wallikeri in Western Kenya, a region holoendemic for malaria transmission.

Project description:The Greater Mekong Subregion is aiming to achieve regional malaria elimination by 2030. Though a shift in malaria parasite species predominance by Plasmodium vivax has been recently documented, the transmission of the two minor Plasmodium species, Plasmodium malariae and Plasmodium ovale spp., is poorly characterized in the region. This study aims to determine the prevalence of these minor species in the China-Myanmar border area and their genetic diversity.Epidemiology study was conducted during passive case detection in hospitals and clinics in Myanmar and four counties in China along the China-Myanmar border. Cross-sectional surveys were conducted in villages and camps for internally displaced persons to determine the prevalence of malaria infections. Malaria infections were diagnosed initially by microscopy and later in the laboratory using nested PCR for the SSU rRNA genes. Plasmodium malariae and P. ovale infections were confirmed by sequencing the PCR products. The P. ovale subtypes were determined by sequencing the Pocytb, Pocox1 and Pog3p genes. Parasite populations were evaluated by PCR amplification and sequencing of the MSP-1 genes. Antifolate sensitivity was assessed by sequencing the dhfr-ts and dhps genes from the P. malariae and P. ovale isolates.Analysis of 2701 blood samples collected from the China-Myanmar border by nested PCR targeting the parasite SSU rRNA genes identified 561 malaria cases, including 161 Plasmodium falciparum, 327 P. vivax, 66 P. falciparum/P. vivax mixed infections, 4 P. malariae and 3 P. ovale spp. P. vivax and P. falciparum accounted for >60 and ~30% of all malaria cases, respectively. In comparison, the prevalence of P. malariae and P. ovale spp. was very low and only made up ~1% of all PCR-positive cases. Nevertheless, these two species were often misidentified as P. vivax infections or completely missed by microscopy even among symptomatic patients. Phylogenetic analysis of the SSU rRNA, Pocytb, Pocox1 and Pog3p genes confirmed that the three P. ovale spp. isolates belonged to the subtype P. ovale curtisi. Low-level genetic diversity was detected in the MSP-1, dhfr and dhps genes of these minor parasite species, potentially stemming from the low prevalence of these parasites preventing their mixing. Whereas most of the dhfr and dhps positions equivalent to those conferring antifolate resistance in P. falciparum and P. vivax were wild type, a new mutation S113C corresponding to the S108 position in pfdhfr was identified in two P. ovale curtisi isolates.The four human malaria parasite species all occurred sympatrically at the China-Myanmar border. While P. vivax has become the predominant species, the two minor parasite species also occurred at very low prevalence but were often misidentified or missed by conventional microscopy. These minor parasite species displayed low levels of polymorphisms in the msp-1, dhfr and dhps genes.

Dataset Information

Sequence-based optimization of a quantitative real-time PCR assay for detection of Plasmodium ovale and Plasmodium malariae.

Publications

Sequence-based optimization of a quantitative real-time PCR assay for detection of Plasmodium ovale and Plasmodium malariae.

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