Misclassification of Plasmodium infections by conventional microscopy and the impact of remedial training on the proficiency of laboratory technicians in species identification

Peter Obare,David Dosoo,Ken Lilley,Seth Owusu-Agyei,Fred Binka,Bernhards Ogutu,Christine Adhiambo,James Sande Odera,Jacob Johnson,Mohammed Adams,Elizabeth Wanja

doi:10.1186/1475-2875-12-113

Abstract

BackgroundMalaria diagnosis is largely dependent on the demonstration of parasites in stained blood films by conventional microscopy. Accurate identification of the infecting Plasmodium species relies on detailed examination of parasite morphological characteristics, such as size, shape, pigment granules, besides the size and shape of the parasitized red blood cells and presence of cell inclusions. This work explores misclassifications of four Plasmodium species by conventional microscopy relative to the proficiency of microscopists and morphological characteristics of the parasites on Giemsa-stained blood films.Case descriptionTen-day malaria microscopy remedial courses on parasite detection, species identification and parasite counting were conducted for public health and research laboratory personnel. Proficiency in species identification was assessed at the start (pre) and the end (post) of each course using known blood films of Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale and Plasmodium vivax infections with densities ranging from 1,000 to 30,000 parasites/μL. Outcomes were categorized as false negative, positive without speciation, P. falciparum, P. malariae, P. ovale, P. vivax and mixed infections.Discussion and evaluationReported findings are based on 1,878 P. falciparum, 483 P. malariae, 581 P. ovale and 438 P. vivax cumulative results collated from 2008 to 2010 remedial courses. Pre-training false negative and positive misclassifications without speciation were significantly lower on P. falciparum infections compared to non-falciparum infections (p < 0.0001). Post-training misclassifications decreased significantly compared to pre- training misclassifications which in turn led to significant improvements in the identification of the four species. However, P. falciparum infections were highly misclassified as mixed infections, P. ovale misclassified as P. vivax and P. vivax similarly misclassified as P. ovale (p < 0.05).ConclusionThese findings suggest that the misclassification of malaria species could be a common occurrence especially where non-falciparum infections are involved due to lack of requisite skills in microscopic diagnosis and variations in morphological characteristics within and between Plasmodium species. Remedial training might improve reliability of conventional light microscopy with respect to differentiation of Plasmodium infections.

Highlights

Malaria diagnosis is largely dependent on the demonstration of parasites in stained blood films by conventional microscopy
These findings suggest that the misclassification of malaria species could be a common occurrence especially where non-falciparum infections are involved due to lack of requisite skills in microscopic diagnosis and variations in morphological characteristics within and between Plasmodium species
Misclassifications on P. falciparum infections Pre-training misclassification of P. falciparum infections as positive without speciation (PS) was significantly higher compared to misclassification as false negative (FN), misclassification as P. ovale, misclassification as P. vivax in addition to misclassification as mixed infections (MX) (p < 0.0001)

Summary

Introduction

Malaria diagnosis is largely dependent on the demonstration of parasites in stained blood films by conventional microscopy. Accurate identification of the infecting Plasmodium species relies on detailed examination of parasite morphological characteristics, such as size, shape, pigment granules, besides the size and shape of the parasitized red blood cells and presence of cell inclusions. Malaria diagnosis in most settings is largely dependent on the demonstration of parasites in stained blood films by conventional microscopy This enables parasite detection, identification of Plasmodium species and estimation of parasite densities. Accurate identification of the infecting Plasmodium species relies on detailed examination of parasite morphological characteristics such as size, shape, pigmentation, besides the size and shape of the parasitized red blood cells and the inclusions therein [1,2]. Since Plasmodium species differ with respect to their biology, clinical symptoms, and treatment requirements [5,6]; accurate identification of Plasmodium infections is of critical importance with regard to formulation and deployment of effective intervention strategies in endemic regions [6]

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