Reader technique as a source of variability in determining malaria parasite density by microscopy

Results obtained by 24–27 expert malaria microscopists, who had independently read 895 slides from 35 donors, were analysed to understand how reader technique contributes to discrepancy in measurements of parasite density over a wide range of densities.Among these 35 donations, standard deviations ranged from 30% to 250% of the mean parasite density and the percent discrepancy was inversely correlated with the mean parasite density. The number of white blood cells indexed and whether parasites were counted in the thick film or thin film were shown to significantly contribute to discrepancy amongst microscopists.Errors in microscopy measurements are not widely appreciated or addressed but have serious consequences for efficacy trials, including possibly abandoning promising vaccine candidates.Microscopy has been used to detect malaria parasites in the blood of infected patients since Laveran first identified the parasites in 1880 [1]. Microscopic examination of blood is the most affordable, accessible, widely used and reliable technique for diagnosis of malaria infection. Although molecular techniques for quantifying parasites have made significant progress in recent years, microscopy remains the primary technique for quantification of parasites. Microscopy is routinely relied upon as a primary endpoint measurement for epidemiological studies, intervention studies, and clinical trials. Despite the critical importance of microscopy for the study and treatment of malaria, little effort has been made to precisely determine and distinguish sources of error in microscopic diagnosis and quantification of parasitaemia or to evaluate the impact of this error on endpoint measurements.Like all detection methods, microscopy is an imperfect technique. However, unlike other methods, such as PCR and immunochromatographic assays, it relies heavily upon the judgment and experience of the individual user. This was noted at least as early as 1930, when Knowles and White reported on th