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

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.

External quality assessment in resource–limited countries

“…found 71.4% sensitivity and 47.3% specificity of routine blood slides in 12 urban public health facilities in Dar es Salaam, Tanzania ( 12 ). Misclassification of malaria species occurs from lack of skills in microscopic diagnosis ( 13 ). Improving and monitoring malaria-related test performance in peripheral laboratories on a countrywide basis is achieved through training and supervision but programmes must be sustained by national commitment ( 14 ).…”

Parasitological correlates of Plasmodium ovale curtisi and Plasmodium ovale wallikeri infection

“…Prevalent in West and East Africa and the Asia–Pacific [1], P. ovale infections can be challenging to diagnose in part due to the typical presentation of clinically mild disease and low parasite burden [2]. Consequently, conventional diagnostic tools, such as microscopy of blood films and rapid diagnostic tests (RDT), have limited performance in detecting P. ovale infection [3–5], often resulting in the use of molecular techniques to confirm the diagnosis [5]. …”

Sequence-Based Optimization of a Quantitative Real-Time PCR Assay for Detection of Plasmodium ovale and Plasmodium malariae

“…Diagnoses of P. malariae and P. ovale based on microscopy present challenges due to the technical expertise required, the typically low parasitemias associated with these species, and the similar morphological features to other Plasmodium species (1). PCR assays may be either genus or species specific and do not require extensive training to perform; however, significant laboratory infrastructure is required, thus limiting their use to resourced settings (3,4).…”

“…here are five Plasmodium species which are known to cause human malaria, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi (1). Although P. malariae and P. ovale occur less frequently among human populations, they are widely distributed and their diagnosis has proven challenging due to their relatively low parasitemia and the morphology of blood stages, as well as the poor sensitivity of rapid diagnostic tests in non-falciparum species (1,2).…”

“…Although P. malariae and P. ovale occur less frequently among human populations, they are widely distributed and their diagnosis has proven challenging due to their relatively low parasitemia and the morphology of blood stages, as well as the poor sensitivity of rapid diagnostic tests in non-falciparum species (1,2).…”