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Comparison between Flow Cytometry, Microscopy, and Lactate Dehydrogenase-Based Enzyme-Linked Immunosorbent Assay for Plasmodium falciparum Drug Susceptibility Testing under Field Conditions.

ABSTRACT: Flow cytometry is an objective method for conducting in vitro antimalarial sensitivity assays with increasing potential for application in field sites. We examined in vitro susceptibility to seven anti-malarial drugs for 40 fresh P. falciparum field isolates via a flow cytometry method (FCM), a colorimetric LDH-based ELISA : DELI), and standard microscopic slide analysis of growth. For FCM, 184/280 (66%) assays met analytical acceptance criteria, compared to 166/280 (59%) for DELI. There was good agreement between FCM and microscopy, while DELI tended to produce higher half-maximal inhibition constants (IC50s) than FCM, with an overall bias of 2.2-fold (Bland-Altman comparison). Values for artesunate and dihydroartemisinin were most affected. Paradoxical increases in signal at very high concentrations of mefloquine and related compounds were more marked with the DELI assay, suggesting that off-target effects on LDH production may be responsible. Loss of FCM signal due to reinvasion or slow growth was observed in a small number of samples. These results extend previous work on use of flow cytometry to determine antimalarial susceptibility in terms of the number of samples, range of drugs, and comparison with other methods.

SUBMITTER: Woodrow CJ

PROVIDER: S-EPMC4572553 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Comparison between Flow Cytometry, Microscopy, and Lactate Dehydrogenase-Based Enzyme-Linked Immunosorbent Assay for Plasmodium falciparum Drug Susceptibility Testing under Field Conditions.

Woodrow Charles J CJ Wangsing Chirapat C Sriprawat Kanlaya K Christensen Peter R PR Nosten Francois F Rénia Laurent L Russell Bruce B Malleret Benoît B

Journal of clinical microbiology 20150812 10

Flow cytometry is an objective method for conducting in vitro antimalarial sensitivity assays with increasing potential for application in field sites. We examined in vitro susceptibility to seven anti-malarial drugs for 40 fresh P. falciparum field isolates via a flow cytometry method (FCM), a colorimetric LDH-based ELISA : DELI), and standard microscopic slide analysis of growth. For FCM, 184/280 (66%) assays met analytical acceptance criteria, compared to 166/280 (59%) for DELI. There was goo ...[more]

PMID: 26269616

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Project description:BackgroundAntigen-detecting rapid diagnostic tests (RDTs) have been recommended by the World Health Organization for use in remote areas to improve malaria case management. Lactate dehydrogenase (LDH) of Plasmodium falciparum is one of the main parasite antigens employed by various commercial RDTs. It has been hypothesized that the poor detection of LDH-based RDTs is attributed in part to the sequence diversity of the gene. To test this, the present study aimed to investigate the genetic diversity of the P. falciparum ldh gene in Thailand and to construct the map of LDH sequence diversity in P. falciparum populations worldwide.MethodsThe ldh gene was sequenced for 50 P. falciparum isolates in Thailand and compared with hundreds of sequences from P. falciparum populations worldwide. Several indices of molecular variation were calculated, including the proportion of polymorphic sites, the average nucleotide diversity index (π), and the haplotype diversity index (H). Tests of positive selection and neutrality tests were performed to determine signatures of natural selection on the gene. Mean genetic distance within and between species of Plasmodium ldh was analysed to infer evolutionary relationships.ResultsNucleotide sequences of P. falciparum ldh could be classified into 9 alleles, encoding 5 isoforms of LDH. L1a was the most common allelic type and was distributed in P. falciparum populations worldwide. Plasmodium falciparum ldh sequences were highly conserved, with haplotype and nucleotide diversity values of 0.203 and 0.0004, respectively. The extremely low genetic diversity was maintained by purifying selection, likely due to functional constraints. Phylogenetic analysis inferred the close genetic relationship of P. falciparum to malaria parasites of great apes, rather than to other human malaria parasites.ConclusionsThis study revealed the global genetic variation of the ldh gene in P. falciparum, providing knowledge for improving detection of LDH-based RDTs and supporting the candidacy of LDH as a therapeutic drug target.

Project description:BackgroundThe haemozoin crystal continues to be investigated extensively for its potential as a biomarker for malaria diagnostics. In order for haemozoin to be a valuable biomarker, it must be present in detectable quantities in the peripheral blood and distinguishable from false positives. Here, dark-field microscopy coupled with sophisticated image processing algorithms is used to characterize the abundance of detectable haemozoin within infected erythrocytes from field samples in order to determine the window of detection in peripheral blood.MethodsThin smears from Plasmodium falciparum-infected and uninfected patients were imaged in both dark field (DF) unstained and bright field (BF) Giemsa-stained modes. The images were co-registered such that each parasite had thumbnails in both BF and DF modes, providing an accurate map between parasites and DF objects. This map was used to find the abundance of haemozoin as a function of parasite stage through careful parasite staging and correlation with DF objects. An automated image-processing and classification algorithm classified the bright spots in the DF images as either haemozoin or non-haemozoin objects.ResultsThe algorithm distinguishes haemozoin from non-haemozoin objects in DF images with an object-level sensitivity of 95% and specificity of 97%. Ring stages older than about 6 hours begin to show detectable haemozoin, and rings between 10-16 hours reliably contain detectable haemozoin. However, DF microscopy coupled with the image-processing algorithm detect no haemozoin in rings younger than six hours.DiscussionAlthough this method demonstrates the most sensitive detection of haemozoin in field samples reported to date, it does not detect haemozoin in ring-stage parasites younger than six hours. Thus, haemozoin is a poor biomarker for field samples primarily composed of young ring-stage parasites because the crystal is not present in detectable quantities by the methods described here. Based on these results, the implications for patient-level diagnosis and recommendations for future work are discussed.

Dataset Information

Comparison between Flow Cytometry, Microscopy, and Lactate Dehydrogenase-Based Enzyme-Linked Immunosorbent Assay for Plasmodium falciparum Drug Susceptibility Testing under Field Conditions.

Publications

Comparison between Flow Cytometry, Microscopy, and Lactate Dehydrogenase-Based Enzyme-Linked Immunosorbent Assay for Plasmodium falciparum Drug Susceptibility Testing under Field Conditions.

Similar Datasets

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