Multiplex 5′ Nuclease Quantitative Real-Time PCR for Clinical Diagnosis of Malaria and Species-Level Identification and Epidemiologic Evaluation of Malaria-Causing Parasites, Including Plasmodium knowlesi

Abstract

Molecular diagnosis of malaria offers many potential advantages over microscopy, including identification of malaria to the species level in an era with few experienced microscopists. We developed high-throughput multiplex 5= nuclease quantitative PCR (qPCR) assays, with the potential to support large studies, to specifically identify Plasmodium falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. We compared qPCR to microscopy and confirmed discordant results with an alternative target PCR assay. The assays specifically detected 1 to 6 parasites/l of blood. The clinical sensitivities (95% confidence intervals [CIs]) of the 4-plex assay to detect microscopically confirmed malaria were 95.8% (88.3 to 99.1%) for P. falciparum, 89.5% (75.2 to 97.1%) for P. vivax, 94.1% (71.3 to 99.9%) for P. ovale, and 100% (66.4 to 100%) for P. malariae. The specificities (95% CIs) were 98.6% (92.4 to 100%) for P. falciparum, 99% (84.8 to 100%) for P. vivax, 98.4% (94.4 to 99.8%) for P. ovale, and 99.3% (95.9 to 100%) for P. malariae. The clinical specificity for samples without malaria was 100%. The clinical sensitivity of the 5-plex assay for confirmed P. knowlesi malaria was 100% (95% CI, 69.2 to 100%), and the clinical specificity was 100% (95% CI, 87.2 to 100%). Coded retesting and testing with an alternative target PCR assay showed improved sensitivity and specificity of multiplex qPCR versus microscopy. Additionally, 91.7% (11/12) of the samples with uncertain species by microscopy were identified to the species level identically by both our multiplex qPCR assay and the alternative target PCR assay, including 9 P. falciparum infections. Multiplex qPCR can rapidly and simultaneously identify all 5 Plasmodium species known to cause malaria in humans, and it offers an alternative or adjunct to microscopy for clinical diagnosis as well as a needed high-throughput tool for research.

A Sensitive, Colorimetric, High-Throughput Loop-Mediated Isothermal Amplification Assay for the Detection of Plasmodium knowlesi

“…Tests that have been reported include nested PCR assays targeting either the 18S ribosomal small subunit 8 or the cytochrome b mitochondrial gene. 9 Assays have been configured as single-step PCR, 10 real-time PCR, 11 multiplex PCR, 12 or semi-nested multiplex PCR. 13 However, due to its cost and requirements for infrastructure and technical expertise, PCR remains a reference laboratory confirmation tool and is generally inaccessible in regional health-care facilities, making it unsuitable as a point-of-care diagnostic tool.…”

Simple Real-Time PCR and Amplicon Sequencing Method for Identification of Plasmodium Species in Human Whole Blood

“…In fact, pairwise comparisons of representative 18S rRNA sequences for the five species revealed interspecies sequence identities of 77% to 87%, and the divergent regions of the gene were not optimal for primer design due to high A/T content (data not shown). Additionally, when we attempted to adopt several published 18S rRNA targeting primers, we were not able to verify their species specificity in a SYBR green assay (data not shown), similar to what has been reported by others (15). Targeting less conserved genomic regions may improve specificity; however, for most non-falciparum species, there is a paucity of publicly available sequences, which compromises the ability to adequately assess intraspecies sequence conservation.…”

“…Thus, for non-18S rRNA targets, there is a risk of inadvertently designing primers in genomic regions that may be genetically divergent to an unanticipated level, leading to reduced sensitivity. It is also important to recognize that whereas initial studies may report high species specificities when tested with relatively small numbers of clinical samples, those may not be verifiable as additional strains are tested, and this applies for 18S and non-18S primers (13)(14)(15).…”

“…These assays have increased specificity compared to microscopy and are superior for detecting mixed infections (11), which can account for Ͼ5% of malarial infections (12). However, most existing molecular assays employ PCR primers against the Plasmodium 18S rRNA gene, which have been reported to cross-react among species (13)(14)(15). For example, published primers targeting the P. knowlesi 18S rRNA gene have been shown to cross-react with some P. vivax isolates (13).…”

Comparison of Three Molecular Methods for the Detection and Speciation of Five Human Plasmodium Species

“…However, the detection limits for the multiplex single-round protocol were found to be 4 p/μL for P. ovale and 40 p/μL for P. malariae and P. vivax. In addition, a high-throughput multiplex 5 nuclease quantitative PCR (qPCR) assay was recently developed by Reller and others 17 for clinical diagnosis of human malaria species. This method was reported to be highly sensitive for all five Plasmodium species, showing a detection limit of 1-6 p/μL blood.…”