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Molecular quantification of Plasmodium parasite density from the blood retained in used RDTs.

ABSTRACT: Most malaria-endemic countries are heavily reliant upon rapid diagnostic tests (RDT) for malaria case identification and treatment. RDT previously used for malaria diagnosis can subsequently be used for molecular assays, including qualitative assessment of parasite species present or the carriage of resistance markers, because parasite DNA can be extracted from the blood inside the RDT which remains preserved on the internal components. However, the quantification of parasite density has not previously been possible from used RDT. In this study, blood samples were collected from school-age children in Western Kenya, in the form of both dried blood spots on Whatman filter paper, and the blood spot that is dropped into rapid diagnostic tests during use. Having first validated a robotic DNA extraction method, the parasite density was determined from both types of sample by duplex qPCR, and across a range of densities. The methods showed good agreement. The preservation of both parasite and human DNA on the nitrocellulose membrane inside the RDT was stable even after more than one year's storage. This presents a useful opportunity for researchers or clinicians wishing to gain greater information about the parasite populations that are being studied, without significant investment of resources.

SUBMITTER: Robinson A

PROVIDER: S-EPMC6434039 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Molecular quantification of Plasmodium parasite density from the blood retained in used RDTs.

Robinson Ailie A Busula Annette O AO Muwanguzi Julian K JK Powers Stephen J SJ Masiga Daniel K DK Bousema Teun T Takken Willem W de Boer Jetske G JG Logan James G JG Beshir Khalid B KB Sutherland Colin J CJ

Scientific reports 20190325 1

Most malaria-endemic countries are heavily reliant upon rapid diagnostic tests (RDT) for malaria case identification and treatment. RDT previously used for malaria diagnosis can subsequently be used for molecular assays, including qualitative assessment of parasite species present or the carriage of resistance markers, because parasite DNA can be extracted from the blood inside the RDT which remains preserved on the internal components. However, the quantification of parasite density has not pre ...[more]

PMID: 30911048

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Project description:Sequencing large numbers of individual samples is often needed for countrywide antimalarial drug resistance surveillance. Pooling DNA from several individual samples is an alternative cost and time saving approach for providing allele frequency (AF) estimates at a population level. Using 100 individual patient DNA samples of dried blood spots from a 2017 nationwide drug resistance surveillance study in Haiti, we compared codon coverage of drug resistance-conferring mutations in four Plasmodium falciparum genes (crt, dhps, dhfr, and mdr1), for the same deep sequenced samples run individually and pooled. Samples with similar real-time PCR cycle threshold (Ct) values (+/- 1.0 Ct value) were combined with ten samples per pool. The sequencing success for samples in pools were higher at a lower parasite density than the individual samples sequence method. The median codon coverage for drug resistance-associated mutations in all four genes were greater than 3-fold higher in the pooled samples than in individual samples. The overall codon coverage distribution for pooled samples was wider than the individual samples. The sample pools with < 40 parasites/μL blood showed more discordance in AF calls for dhfr and mdr1 between the individual and pooled samples. This discordance in AF estimation may be due to low amounts of parasite DNA, which could lead to variable PCR amplification efficiencies. Grouping samples with an estimated ≥ 40 parasites/μL blood prior to pooling and deep sequencing yielded the expected population level AF. Pooling DNA samples based on estimates of > 40 parasites/μL prior to deep sequencing can be used for rapid genotyping of a large number of samples for these four genes and possibly other drug resistant markers in population-based studies. As Haiti is a low malaria transmission country with very few mixed infections and continued chloroquine sensitivity, the pooled sequencing approach can be used for routine national molecular surveillance of resistant parasites.

Project description:BACKGROUND: The carbon metabolism of the blood stages of Plasmodium falciparum, comprising rapidly dividing asexual stages and non-dividing gametocytes, is thought to be highly streamlined, with glycolysis providing most of the cellular ATP. However, these parasitic stages express all the enzymes needed for a canonical mitochondrial tricarboxylic acid (TCA) cycle, and it was recently proposed that they may catabolize glutamine via an atypical branched TCA cycle. Whether these stages catabolize glucose in the TCA cycle and what is the functional significance of mitochondrial metabolism remains unresolved. RESULTS: We reassessed the central carbon metabolism of P. falciparum asexual and sexual blood stages, by metabolically labeling each stage with 13C-glucose and 13C-glutamine, and analyzing isotopic enrichment in key pathways using mass spectrometry. In contrast to previous findings, we found that carbon skeletons derived from both glucose and glutamine are catabolized in a canonical oxidative TCA cycle in both the asexual and sexual blood stages. Flux of glucose carbon skeletons into the TCA cycle is low in the asexual blood stages, with glutamine providing most of the carbon skeletons, but increases dramatically in the gametocyte stages. Increased glucose catabolism in the gametocyte TCA cycle was associated with increased glucose uptake, suggesting that the energy requirements of this stage are high. Significantly, whereas chemical inhibition of the TCA cycle had little effect on the growth or viability of asexual stages, inhibition of the gametocyte TCA cycle led to arrested development and death. CONCLUSIONS: Our metabolomics approach has allowed us to revise current models of P. falciparum carbon metabolism. In particular, we found that both asexual and sexual blood stages utilize a conventional TCA cycle to catabolize glucose and glutamine. Gametocyte differentiation is associated with a programmed remodeling of central carbon metabolism that may be required for parasite survival either before or after uptake by the mosquito vector. The increased sensitivity of gametocyte stages to TCA-cycle inhibitors provides a potential target for transmission-blocking drugs.

Dataset Information

Molecular quantification of Plasmodium parasite density from the blood retained in used RDTs.

Publications

Molecular quantification of Plasmodium parasite density from the blood retained in used RDTs.

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