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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.

SUBMITTER: Reller ME

PROVIDER: S-EPMC3754650 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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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.

Reller Megan E ME Chen Wan Hsin WH Dalton Justin J Lichay Marguerite A MA Dumler J Stephen JS

Journal of clinical microbiology 20130626 9

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 ...[more]

PMID: 23804387

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Project description:IntroductionThe imminent risk of zoonoses of non-human malaria parasites is not far from reality in India, as has been observed in the case of Plasmodium knowlesi (Pk), and so is possible with P. cynomolgi (Pc), already reported from South East Asian countries. Therefore, a novel multiplex qPCR assay was developed and evaluated for detection of non-human malaria parasites- Pk and Pc in populations at risk.MethodsThe qPCR primers were designed in-house with fluorescence labeled probes (HEX for Pk and FAM for Pc). DNA samples of Pk and Pc were used as templates and further the qPCR assay was evaluated in 250 symptomatic and asymptomatic suspected human blood samples from malaria endemic areas of North Eastern states of India.ResultsThe qPCR assay successfully amplified the target 18S rRNA gene segment from Pk and Pc and was highly specific for Pk and Pc parasites only, as no cross reactivity was observed with P. falciparum (Pf), P. vivax (Pv), P. malariae (Pm), and P. ovale (Po). Standard curves were generated to estimate the limit of detection (LOD) of Pk and Pc parasites DNA (0.00275 & 0.075 ng/μl, respectively). Due to COVID-19 pandemic situation during 2020-21, the sample accessibility was difficult, however, we managed to collect 250 samples. The samples were tested for Pf and Pv using conventional PCR- 14 Pf and 11 Pv infections were observed, but no Pk and Pc infections were detected. For Pk infections, previously reported conventional PCR was also performed, but no Pk infection was detected.DiscussionThe multiplex qPCR assay was observed to be robust, quick, cost-effective and highly sensitive as compared to the currently available conventional PCR methods. Further validation of the multiplex qPCR assay in field setting is desirable, especially from the high-risk populations. We anticipate that the multiplex qPCR assay would prove to be a useful tool in mass screening and surveillance programs for detection of non-human malaria parasites toward the control and elimination of malaria from India by 2030.

Project description:BackgroundWhile transmission of the human Plasmodium species has declined, a significant increase in Plasmodium knowlesi/Plasmodium malariae cases was reported in Hulu Selangor, Selangor, Malaysia. Thus, a study was undertaken to determine the epidemiology and the vectors involved in the transmission of knowlesi malaria.MethodsCases of knowlesi/malariae malaria in the Hulu Selangor district were retrospectively reviewed and analyzed from 2009 to 2013. Mosquitoes were collected from areas where cases occurred in order to determine the vectors. Leucosphyrus group of mosquitoes were genetically characterized targeting the nuclear internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome c oxidase subunit I (CO1). In addition, temporal and spatial analyses were carried out for human cases and vectors.ResultsOf the 100 microscopy diagnosed P. knowlesi/P. malariae cases over the 5 year period in the Hulu Selangor district, there was predominance of P. knowlesi/P. malariae cases among the young adults (ages 20-39 years; 67 cases; 67%). The majority of the infected people were involved in occupations related to agriculture and forestry (51; 51%). No death was recorded in all these cases.Five hundred and thirty five mosquitoes belonging to 14 species were obtained during the study. Anopheles maculatus was the predominant species (49.5%) followed by Anopheles letifer (13.1%) and Anopheles introlatus (11.6%). Molecular and phylogenetic analysis confirmed the species of the Leucosphyrus group to be An. introlatus. In the present study, only An. introlatus was positive for oocysts. Kernel Density analysis showed that P. knowlesi hotspot areas overlapped with areas where the infected An. introlatus was discovered. This further strengthens the hypothesis that An. introlatusis is the vector for P. knowlesi in the Hulu Selangor district.Unless more information is obtained on the vectors as well as macaque involved in the transmission, it will be difficult to plan effective control strategies. The utilization of modern analytical tools such as GIS (Geographic Information System) is crucial in estimating hotspot areas for targeted control strategies.ConclusionsAnopheles introlatus has been incriminated as vector of P. knowlesi in Hulu Selangor. The cases of P. knowlesi are on the increase and further research using molecular techniques is needed.

Dataset Information

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.

Publications

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.

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