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Background  Optical detection of circulating haemozoin has been suggested as a needle free method to diagnose malaria using in vivo microscopy. Haemozoin is generated within infected red blood cells by the malaria parasite, serving as a highly specific, endogenous biomarker of malaria. However, phagocytosis of haemozoin by white blood cells which persist after the infection is resolved presents the potential for false positive diagnosis; therefore, the focus of this work is to identify a feature of the haemozoin signal to discriminate between infected red blood cells and haemozoin-containing white blood cells.    Methods  Conventional brightfield microscopy of thin film blood smears was used to analyse haemozoin absorbance signal in vitro. Cell type and parasite maturity were morphologically determined using colocalized DAPI staining. The ability of features to discriminate between infected red blood cells and haemozoin-containing white blood cells was evaluated using images of smears from subjects infected with two species of  Plasmodium ,  Plasmodium yoelii  and  Plasmodium falciparum . Discriminating features identified by blood smear microscopy were characterized in vivo in  P. yoelii -infected mice.    Results  Two features of the haemozoin signal, haemozoin diameter and normalized intensity difference, were identified as potential parameters to differentiate infected red blood cells and haemozoin-containing white blood cells. Classification performance was evaluated using the area under the receiver operating characteristic curve, with area under the curve values of 0.89 for the diameter parameter and 0.85 for the intensity parameter when assessed in  P. yoelii  samples. Similar results were obtained from  P. falciparum  blood smears, showing an AUC of 0.93 or greater for both classification features. For in vivo investigations, the intensity-based metric was the best classifier, with an AUC of 0.91.    Conclusions  This work demonstrates that size and intensity features of haemozoin absorbance signal collected by in vivo microscopy are effective classification metrics to discriminate infected red blood cells from haemozoin-containing white blood cells. This reduces the potential for false positive results associated with optical imaging strategies for in vivo diagnosis of malaria based on the endogenous biomarker haemozoin.    Electronic supplementary material  The online version of this article (10.1186/s12936-017-2096-1) contains supplementary material, which is available to authorized users.

Towards a needle-free diagnosis of malaria: in vivo identification and classification of red and white blood cells containing haemozoin