Sensitive and accurate quantification of human malaria parasites using droplet digital PCR (ddPCR)

Koepfli, Cristian; Nguitragool, Wang; Hofmann, Natalie; Robinson, Leanne J.; Ome-Kaius, Maria; Sattabongkot, Jetsumon; Felger, Ingrid; Müeller, Ivo

doi:10.1038/srep39183

Cited by 96 publications

(103 citation statements)

References 23 publications

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“…Our microscopy positive results had signi cantly high level of parasitaemia ranging 7000-12000 parasites/µl which resembles 0.15-0.25% quanti ed by RBC count method. Similar study was conducted by Koep i C, Nguitragool W, Hofmann NE et al had revealed light microscopy were able to quantify only upto 0.25-0.0625 µl of parasitaemia based on skilled microscopist whereas real-time PCR had quanti ed less than 0.03 µl of parasites [25].…”

Section: Discussionsupporting

confidence: 70%

Challenges in laboratory diagnosis of Malaria in a low resource country among cases of acute febrile illness at tertiary care hospital in eastern Nepal: Comparative study on Conventional Vs Molecular approach

Dahal

Khanal

Rai

et al. 2020

Preprint

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Background For ongoing malaria elimination programme, available methods like microscopy and rapid diagnostic test (RDT) are not able to detect all the cases of malaria in acute febrile illness. These methods are entirely dependent on the course of infection, parasite load and skilled technical resources thus the study was carried out to compare performance of microscopy and RDT which are commonly used in a low resource country along with reference to real-time PCR. Methods Altogether 52 blood samples collected from patients with acute febrile illness were tested by microscopy, RDT and real-time PCR. The results were compared in terms of sensitivity and specificity. Results The test results were as follows: 5.8% positivity by Microscopy, 13.5% positivity by RDT and 27% by real time PCR. Taking into consideration of PCR as a gold standard method microscopy showed 21.4% sensitivity and 100% specificity. Likewise, RDT results revealed 28.6% sensitivity and 92.1% specificity. Conclusion Despite of various diagnostic tools available, microscopy stills remains the gold standard for the diagnosis and RDT is the user friendly to execute the test under the tree, but our preliminary results emphasized the need to implement the test with the higher sensitivity and specificity in context of malaria elimination programme which could be another important opportunity to understand the parasite circulation in case of low endemic region. However, these results should be further verified with the large study cohort in order to document the submicroscopic infection.

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Section: Discussionsupporting

confidence: 70%

Challenges in laboratory diagnosis of Malaria in a low resource country among cases of acute febrile illness at tertiary care hospital in eastern Nepal: Comparative study on Conventional Vs Molecular approach

Dahal

Khanal

Rai

et al. 2020

Preprint

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“…falciparum , and equal sensitivity for P . vivax compared to the previous report [37]. In the current study, the ddPCR assay was developed using duplex reactions for detection and quantification of P .…”

Section: Discussionmentioning

confidence: 99%

Four human Plasmodium species quantification using droplet digital PCR

et al. 2017

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Droplet digital polymerase chain reaction (ddPCR) is a partial PCR based on water-oil emulsion droplet technology. It is a highly sensitive method for detecting and delineating minor alleles from complex backgrounds and provides absolute quantification of DNA targets. The ddPCR technology has been applied for detection of many pathogens. Here the sensitive assay utilizing ddPCR for detection and quantification of Plasmodium species was investigated. The assay was developed for two levels of detection, genus specific for all Plasmodium species and for specific Plasmodium species detection. The ddPCR assay was developed based on primers and probes specific to the Plasmodium genus 18S rRNA gene. Using ddPCR for ultra-sensitive P. falciparum assessment, the lower level of detection from concentrated DNA obtained from a high volume (1 mL) blood sample was 11 parasites/mL. For species identification, in particular for samples with mixed infections, a duplex reaction was developed for detection and quantification P. falciparum/ P. vivax and P. malariae/ P. ovale. Amplification of each Plasmodium species in the duplex reaction showed equal sensitivity to singleplex single species detection. The duplex ddPCR assay had higher sensitivity to identify minor species in 32 subpatent parasitaemia samples from Cambodia, and performed better than real-time PCR. The ddPCR assay shows high sensitivity to assess very low parasitaemia of all human Plasmodium species. This provides a useful research tool for studying the role of the asymptomatic parasite reservoir for transmission in regions aiming for malaria elimination.

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“…For example, the Bio-Rad ddPCR system (drop generator, PCR cycler, and reader) costs $90K-$100K, and the reagent cost per sample is ~$5. 60 By comparison, qPCR instruments are in the $25K-$50K range, and the reagent cost is $2. However, the per sample cost in qPCR may be offset by the added costs of calibration standards, whereas dPCR is calibration-free.…”

Section: Detection Of Rare Targets a Hallmark Of Dpcr Is Itsmentioning

confidence: 99%

Digital Assays Part I: Partitioning Statistics and Digital PCR

2017

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A digital assay is one in which the sample is partitioned into many small containers such that each partition contains a discrete number of biological entities (0, 1, 2, 3, …). A powerful technique in the biologist's toolkit, digital assays bring a new level of precision in quantifying nucleic acids, measuring proteins and their enzymatic activity, and probing single-cell genotypes and phenotypes. Part I of this review begins with the benefits and Poisson statistics of partitioning, including sources of error. The remainder focuses on digital PCR (dPCR) for quantification of nucleic acids. We discuss five commercial instruments that partition samples into physically isolated chambers (cdPCR) or droplet emulsions (ddPCR). We compare the strengths of dPCR (absolute quantitation, precision, and ability to detect rare or mutant targets) with those of its predecessor, quantitative real-time PCR (dynamic range, larger sample volumes, and throughput). Lastly, we describe several promising applications of dPCR, including copy number variation, quantitation of circulating tumor DNA and viral load, RNA/miRNA quantitation with reverse transcription dPCR, and library preparation for next-generation sequencing. This review is intended to give a broad perspective to scientists interested in adopting digital assays into their workflows. Part II focuses on digital protein and cell assays.

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Sensitive and accurate quantification of human malaria parasites using droplet digital PCR (ddPCR)

Cited by 96 publications

References 23 publications

Challenges in laboratory diagnosis of Malaria in a low resource country among cases of acute febrile illness at tertiary care hospital in eastern Nepal: Comparative study on Conventional Vs Molecular approach

Challenges in laboratory diagnosis of Malaria in a low resource country among cases of acute febrile illness at tertiary care hospital in eastern Nepal: Comparative study on Conventional Vs Molecular approach

Four human Plasmodium species quantification using droplet digital PCR

Digital Assays Part I: Partitioning Statistics and Digital PCR

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