Limitations of haemozoin-based diagnosis of Plasmodium falciparum using dark-field microscopy - PubMed (original) (raw)

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

Charles Delahunt et al. Malar J. 2014.

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.

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Figures

Figure 1

Figure 1

Typical DF haemozoin and non-haemozoin objects. Typical objects of interest. A: Haemozoin objects, 100× BF thumbnails (top row) and the corresponding 50× DF thumbnails (second row) showing the haemozoin within the parasites. (1–4) are rings, (5) is a gametocyte. Note in (4) that one ring/troph has visible haemozoin, while the other ring does not. B: Typical non-haemozoin objects, DF thumbnails. The larger rings are cell wall information due to haemoglobin scattering.

Figure 2

Figure 2

Template for staging rings. The template for staging rings into 0–6 hours, 6–16 hours, and 16–26 hours. From [21].

Figure 3

Figure 3

Age distributions of rings by sample. Age distribution of ring stages by sample. For each sample, the x-axis is the age of rings as follows: 1 = 0–6 hours, 2 = 6–16 hours, 3 = 16–26 hours; the y-axis shows the number of rings. Samples 1–5 and sample 10 show high synchronization. Samples 1–5 contained no detectable haemozoin objects.

Figure 4

Figure 4

Examples of ring stage parasites, BF and DF images. Typical examples of ring stages, BF and corresponding DF. Row 1 = 0–6 hours old, row 2 = 6–16 hours old, row 3 = 16–26 hours old. Haemozoin is visible in none of row 1, most of row 2, and all of row 3.

Figure 5

Figure 5

Percentages of parasites with haemozoin vs age. The percentage of various ages of ring parasites containing sufficient haemozoin signal to be detectable by the adaptive threshold test. The figure in the base of each column is the number of rings of that age in the sample set. On the age (x-) axis, 1 = 0–6 hrs, 2 = 6–16 hrs, 3 = 16–26 hrs. Values between these three integers correspond to split decisions among the observers and reflect edge cases. For example, 1.5 corresponds to the earlier end of 6–16 hrs, while 2.5 corresponds to the later end of 6–16 hrs.

Figure 6

Figure 6

Haemozoin peripheral blood dynamics. The probability densities for haemozoin (green), sequestered parasites (blue), and parasite age distribution (red) as a function of time post-invasion. The inset shows the probability of detecting an individual parasite (black) vs. time during the cyclical infection when blood is drawn.

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