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

Abstract

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.

Towards ultrasensitive malaria diagnosis using surface enhanced Raman spectroscopy

“…Moreover, spontaneous Raman spectroscopy combined with other approaches such as confocal microscopy 24 or optical tweezers 25 have shown great promise for malaria diagnosis 26 . However, the amount of hemozoin produced in the early ring stages normally seen in peripheral blood is very small and parasites younger than 6 hours post invasion do not contain hemozoin detectable by the dark-field microscopy method 27 , even dark-field microscopy has improved sensitivity as compared to traditional light microscopy 28 . Therefore, the enhancement of Raman signals is crucial for the detection of hemozoin in early ring stages so as to provide the sensitivity needed for diagnosis 17 .…”

A Simple low-cost device enables four epi-illumination techniques on standard light microscopes

“…Due to its birefringence and excellent light scattering hemozoin has been suggested as a promising target for non-invasive fast detection of malaria in unstained blood smears by cross-polarizing (xP) microscopy 16 and FSDF 17 18 . FSDF gives relatively low contrast, with a signal-to-noise ratio (SNR=maximal hemozoin pixel value/maximal empty erythrocyte pixel value) around 1.5–3, which does not allow clear identification of infected cells in crowded blood smears 4 19 . xP microscopy is more sensitive than FSDF 20 , though polarizing filters are relatively expensive.…”

“…xP microscopy is more sensitive than FSDF 20 , though polarizing filters are relatively expensive. An alternative advanced BSDF method was reported to increase SNR up to 50 4 19 , which opens up the possibility of automatic detection of the infection. This method combines xP imaging with a sophisticated DFSR system with sample illumination angle varying between 15–45°.…”

Light depolarization measurements in malaria: A new job for an old friend

“…For example, Hz is higher in gametocytes than in most asexual forms of P. falciparum, providing aid in identification (6). Although Hz levels in early P. falciparum ring-forms are below the detection thresholds of both dark-field microscopy (49) and flow cytometry (31) when analyzing native samples, it may be detected in bulk in blood after rigorous lysis of the sample (50). Even though we have shown that free synthetic Hz can be easily detected by flow cytometry (Fig.…”