Dataset Information

Limitations of haemozoin-based diagnosis of Plasmodium falciparum using dark-field microscopy.

ABSTRACT:

Background

The 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.

Methods

Thin 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.

Results

The 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.

Discussion

Although 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.

SUBMITTER: Delahunt C

PROVIDER: S-EPMC4021049 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Publications

Limitations of haemozoin-based diagnosis of Plasmodium falciparum using dark-field microscopy.

Delahunt Charles C Horning Matthew P MP Wilson Benjamin K BK Proctor Joshua L JL Hegg Michael C MC

Malaria journal 20140417

<h4>Background</h4>The 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 ord ...[more]

PMID: 24739286

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Project description:BACKGROUND:Plasmodium falciparum haemozoin, a detoxification product of digested haemoglobin from infected erythrocytes, is released into the bloodstream upon schizont rupture and accumulates in leukocytes. High levels of haemozoin correlate with disease severity. Some studies have shown that concentrations of the substrate of inducible nitric oxide synthase (iNOS), L-arginine, as well as nitric oxide are low in patients infected with P. falciparum malaria. The present study investigates, in vitro, the role of P. falciparum haemozoin on nitric oxide production, iNOS expression in macrophages, and the possible interaction between L-arginine and haemozoin. METHODS:Plasmodium falciparum haemozoin was obtained from in vitro cultures through magnetic isolation. Phagocytosis of haemozoin by immortalized bone marrow derived macrophages was detected by confocal reflection combined with fluorescence microscopy. Nitrite concentrations in the supernatants was evaluated by Griess assay as a standard indication of nitric oxide production, while iNOS expression was detected on cell extracts by western blotting. Detection of L-arginine in haemozoin-treated or untreated media was achieved by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS:Haemozoin synergizes in vitro with interferon-gamma to produce nitric oxide. However, when mouse macrophages were stimulated with haemozoin, a proportional increase of nitric oxide was observed up to 25 ?M of haemozoin, followed by a decrease with doses up to 100 ?M, when nitric oxide release was completely abrogated. This was not due to reactive oxygen species production, nor to an effect on iNOS activity. Interestingly, when at 24 h, haemozoin-treated macrophages were washed and incubated in fresh medium for further 24 h, the nitric oxide production was restored in a dose-response manner. Similar results were seen when L-arginine-enriched media was used in the stimulation. Moreover, muramyldipeptide, a strong nitric oxide inducer, was unable to activate macrophages to release nitric oxide in the presence of haemozoin-treated medium. By LC-MS/MS a complete depletion of L-arginine was observed in this haemozoin-treated, conditioned medium. CONCLUSIONS:It is proposed that haemozoin interacts with L-arginine reducing its availability for iNOS, and thus decreasing nitric oxide production. The clinical (or pathological) implications of these results are discussed.

Dataset Information

Limitations of haemozoin-based diagnosis of Plasmodium falciparum using dark-field microscopy.

Background

Methods

Results

Discussion

Publications

Limitations of haemozoin-based diagnosis of Plasmodium falciparum using dark-field microscopy.

Similar Datasets

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