Earlier this month, a paper by IV Lab’s Malaria Diagnostics team was published in the Malaria Journal. The paper – Limitations of haemozoin-based diagnosis of Plasmodium falciparum using dark-field microscopy – discusses IV Lab’s investigation into the hemozoin crystal as a biomarker for malaria diagnostics.
The good news: Hemozoin, which is very specific to malaria, can be readily detected using dark-field microscopy when it is present. A highly accurate, fully-automated detection algorithm is presented in the paper.
The bad news: Hemozoin is not present in detectable amounts in early ring stage parasites (0 – 6 hours old). This is a key finding because P. falciparum-infected blood samples, due to sequestration and synchronization effects, often contain only early ring stage parasites, and thus do not contain hemozoin.
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.
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.
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.
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.
Delahunt ,Charles B., Horning, Matthew P., Wilson, Benjamin K., Proctor, Joshua L., and Hegg, Michael C. “Limitations of haemozoin-based diagnosis of Plasmodium falciparum using dark-field microscopy.” Malaria Journal (2014) 13:147 doi:10.1186/1475-2875-13-147