Pilot assessment of the sensitivity of the malaria thin film.

BACKGROUND: Malaria microscopy remains the reference standard for malaria diagnosis in clinical trials (drug and vaccine), new diagnostic evaluation, as well as in clinical care in much of the world today. It is known that microscopy is an imperfect gold standard, and that very low false positive rates can dramatically lower protective efficacy estimates in malaria prevention trials. Although new methods are now available, including malaria rapid diagnostic tests and PCR, neither is as yet validated in the clinical trial setting and both have limitations. Surprisingly, the sensitivity of thin smears is not well established and thin smears are not commonly used in the developing world. METHODS: Malaria thick and thin films were collected in the lowlands of Western Kenya. All had density determined by four readings with two methods, as well as species identified. Thirty-six with low density parasitaemia had the thin smear read by five independent microscopists, two were expert and three were qualified. Microscopists read the entire thin film. For the first 10 parasites seen, they reported the species, appearance, time, field number, and red blood cells in the field. Total parasites, total fields, and total time to examine the smear were also recorded. RESULTS: Median parasitaemia was 201 parasites/mul, mean 1,090 +/- 2,195, range 6-11,124 parasites/mul for the 36 smears evaluated. The data revealed a density dependent increase in sensitivity, with 100% sensitivity achieved at >200 parasites/mul for experts and >500 parasites/mul for qualified readers. Thin film readings confirmed parasitaemia 74% of the time by experts, and 65% of the time for qualified microscopists. The 95th percentile for time to detect parasitaemia was 15 minutes for experts, 17 minutes for qualified microscopists. This decreased to 4-10 minutes for experts at densities of > 200 parasites/mul. Additionally, substantial discordance for species identification was observed. CONCLUSION: The thin film is sensitive enough to be a useful tool to confirm malaria diagnosis in study subjects in some settings. Specificity of the thin film and its utility for confirming thick film or other diagnostic test results should be assessed further.

Systematic comparison of two methods to measure parasite density from malaria blood smears.

This study was designed to directly compare the accuracy, reproducibility, and efficiency of three methods commonly used to measure blood-stage malaria parasite density from Giemsa-stained blood films. Parasites and white blood cells (WBCs) were counted in 154 thick films by two independent microscopists. Forty-six slides were read by counting parasitized red blood cells (RBCs) in the thin film. Using these same slides, parasites were again counted by two independent microscopists using an ocular grid. Overall, parasite densities were significantly lower and discrepancy between readers was higher when using the grid method compared to the WBC method, but there was no difference when compared to the RBC method. When one reader who had difficulty with the grid method was excluded, the discrepancy between readers was equivalent for the three methods. Densities and discrepancy between readers were indistinguishable when parasites were counted until 200 or 500 WBCs. Counting beyond 200 WBCs may not significantly improve parasite density measurements. Using an ocular grid directly measures parasites per volume rather than using a WBC per microliter conversion factor and eliminates the need to switch from the thick film to the thin film for high parasitemias. However, significant differences in densities measured by the grid method and the WBC method need to be evaluated.