A novel real-time PCR assay for the detection of Plasmodium falciparum and Plasmodium vivax malaria in low parasitized individuals

Hwang, Seung-Young; Kim, So-Hee; Lee, Ga-Young; Hằng, Vũ Thị Thu; Moon, Chisook; Shin, Jeong Hwan; Koo, Wan-Lim; Kim, Seong-Youl; Park, Hae-Joon; Park, Han-Oh; Kho, Weon-Gyu

doi:10.1016/j.actatropica.2011.05.006

Cited by 17 publications

(10 citation statements)

References 16 publications

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“…As shown in Figure 1, the standard curve generated from P. vivax genomic DNA showed a significant correlation between mean Ct value and parasite density, similar to that of another standard curve drawn with a synthetic amplicon [24]. Except for the multiplex PCR method (1.56 parasites/µL), the remaining 3 PCR-based assays—real-time PCR, nested PCR, and LAMP—had the same LOD (0.78 parasites/µL).…”

Section: Resultssupporting

confidence: 53%

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Development and Efficacy of Real-Time PCR in the Diagnosis of Vivax Malaria Using Field Samples in the Republic of Korea

Kim

Goo

et al. 2014

PLoS ONE

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The development of sensitive, rapid, and accurate diagnostic methods for vivax malaria is essential for the effective control of malaria in the Republic of Korea, where vivax malaria patients usually show low parasitemia. In this study, a TaqMan-based real-time polymerase chain reaction (PCR) method was established and compared with other PCR-based assays, including nested PCR, loop-mediated isothermal amplification, and multiplex PCR, using samples from febrile patients with suspected vivax malaria. The established real-time PCR had a high sensitivity (99.6%) and specificity (100%). Therefore, this sensitive, specific, rapid, and quantitative real-time PCR method could be used for the routine diagnosis of vivax malaria in the laboratory of the Korea National Institute of Health.

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Section: Resultssupporting

confidence: 53%

“…To date, nested PCR, loop-mediated isothermal amplification (LAMP), and real-time PCR methods have been developed to detect vivax malaria [14], [23], [24]. Compared with nested PCR, real-time PCR uses fluorescent labels that allow the continuous monitoring of amplicon (PCR product) formation throughout the reaction.…”

Section: Introductionmentioning

confidence: 99%

Development and Efficacy of Real-Time PCR in the Diagnosis of Vivax Malaria Using Field Samples in the Republic of Korea

Kim

Goo

et al. 2014

PLoS ONE

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“…intestinal protozoa (ten Hove et al 2007(ten Hove et al , 2009Taniuchi et al 2011;van Lieshout and Verweij 2010), Plasmodium spp. (Khairnar et al 2009;Veron et al 2009;Dormond et al 2011;Hwang et al 2011), Wuchereria bancrofti and Brugia malayi (Intapan et al 2009b), S. mansoni andS. haematobium (ten Hove et al 2008), Ancylostoma duodenale, Necator americanus and Oesophagostomum bifurcum , and O. viverrini and S. stercoralis (Janwan et al 2011).…”

Section: Discussionmentioning

confidence: 95%

Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini eggs in human fecal samples using a duplex real-time fluorescence resonance energy transfer PCR and melting curve analysis

et al. 2012

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We developed a single step duplex real-time fluorescence resonance energy transfer (FRET) PCR merged with melting curve analysis for the fast detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini eggs in human fecal samples. Two species of mitochondrial NADH dehydrogenase subunit 2 (nad2) DNA elements, the 165-bp nad2 product of C. sinensis and the 209-bp nad2 product of O. viverrini, were amplified by species-specific primers, and the fluorescence melting curve analyses were generated from hybrid of amplicons and two pairs of species-specific fluorophore-labeled probes. By their different fluorescence channels and melting temperatures, both C. sinensis and O. viverrini eggs in infected human fecal samples were detected and differentiated with high (100%) sensitivity and specificity. Detection limit was as little as a single C. sinensis egg and two O. viverrini eggs in 100 mg of fecal sample. The assay could distinguish the DNA of both parasites from the DNA of negative fecal samples and fecal samples with other parasitosis, as well as from the well-defined genomic DNA of human leukocytes and other parasites. It can reduce labor time of microscopic examination and is not prone to carry over contamination of agarose electrophoresis. Our duplex real-time FRET PCR method would be useful to determine the accurate range of endemic areas and/or to discover the co-endemic areas of two liver flukes, C. sinensis and O. viverrini, in Asia. This method also would be helpful for the differential diagnosis of the suspected cases of liver fluke infections among travelers who had visited the endemic countries of those parasites.

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“…For each sample, DNA from three 5-mm punches (equivalent to approximately 25 μL whole blood) was extracted and concentrated four-fold by glycogen-acetate precipitation. Plasmodium falciparum DNA was identified using a validated TaqMan multiplex real-time PCR assay for P. falciparum 18 s ribosomal DNA [33], a molecular method for P. falciparum detection that has sensitivity and specificity comparable to nested PCR protocols [34,35]. Isolates were considered P. falciparum- positive if 18 s PCR end-cycle fluorescence exceeded 300 RFU.…”

Section: Methodsmentioning

confidence: 99%

Molecular surveillance for drug-resistant Plasmodium falciparum in clinical and subclinical populations from three border regions of Burma/Myanmar: cross-sectional data and a systematic review of resistance studies

Brown

Smith

Oo³

et al. 2012

Malar J

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BackgroundConfirmation of artemisinin-delayed parasite clearance in Plasmodium falciparum along the Thai-Myanmar border has inspired a global response to contain and monitor drug resistance to avert the disastrous consequences of a potential spread to Africa. However, resistance data from Myanmar are sparse, particularly from high-risk areas where limited health services and decades of displacement create conditions for resistance to spread. Subclinical infections may represent an important reservoir for resistance genes that confer a fitness disadvantage relative to wild-type alleles. This study estimates the prevalence of resistance genotypes in three previously unstudied remote populations in Myanmar and tests the a priori hypothesis that resistance gene prevalence would be higher among isolates collected from subclinical infections than isolates collected from febrile clinical patients. A systematic review of resistance studies is provided for context.MethodsCommunity health workers in Karen and Kachin States and an area spanning the Indo-Myanmar border collected dried blood spots from 988 febrile clinical patients and 4,591 villagers with subclinical infection participating in routine prevalence surveys. Samples positive for P. falciparum 18 s ribosomal RNA by real-time PCR were genotyped for P. falciparum multidrug resistance protein (pfmdr1) copy number and the pfcrt K76T polymorphism using multiplex real-time PCR.ResultsPfmdr1 copy number increase and the pfcrt K76 polymorphism were determined for 173 and 269 isolates, respectively. Mean pfmdr1 copy number was 1.2 (range: 0.7 to 3.7). Pfmdr1 copy number increase was present in 17.5%, 9.6% and 11.1% of isolates from Karen and Kachin States and the Indo-Myanmar border, respectively. Pfmdr1 amplification was more prevalent in subclinical isolates (20.3%) than clinical isolates (6.4%, odds ratio 3.7, 95% confidence interval 1.1 - 12.5). Pfcrt K76T prevalence ranged from 90-100%.ConclusionsCommunity health workers can contribute to molecular surveillance of drug resistance in remote areas of Myanmar. Marginal and displaced populations under-represented among previous resistance investigations can and should be included in resistance surveillance efforts, particularly once genetic markers of artemisinin-delayed parasite clearance are identified. Subclinical infections may contribute to the epidemiology of drug resistance, but determination of gene amplification from desiccated filter samples requires further validation when DNA concentration is low.

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A novel real-time PCR assay for the detection of Plasmodium falciparum and Plasmodium vivax malaria in low parasitized individuals

Cited by 17 publications

References 16 publications

Development and Efficacy of Real-Time PCR in the Diagnosis of Vivax Malaria Using Field Samples in the Republic of Korea

Development and Efficacy of Real-Time PCR in the Diagnosis of Vivax Malaria Using Field Samples in the Republic of Korea

Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini eggs in human fecal samples using a duplex real-time fluorescence resonance energy transfer PCR and melting curve analysis

Molecular surveillance for drug-resistant Plasmodium falciparum in clinical and subclinical populations from three border regions of Burma/Myanmar: cross-sectional data and a systematic review of resistance studies

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