Thick blood film examination for Plasmodium falciparum malaria has reduced sensitivity and underestimates parasite density

Abstract

Background: Thick blood films are routinely used to diagnose Plasmodium falciparum malaria. Here, they were used to diagnose volunteers exposed to experimental malaria challenge.

Methods: The frequency with which blood films were positive at given parasite densities measured by PCR were analysed. The poisson distribution was used to calculate the theoretical likelihood of diagnosis. Further in vitro studies used serial dilutions to prepare thick films from malaria cultures at known parasitaemia.

Results: Even in expert hands, thick blood films were considerably less sensitive than might have been expected from the parasite numbers measured by quantitative PCR. In vitro work showed that thick films prepared from malaria cultures at known parasitaemia consistently underestimated parasite densities.

Conclusion: It appears large numbers of parasites are lost during staining. This limits their sensitivity, and leads to erroneous estimates of parasite density.

Figure 1

At each serial dilution (x axis), parasite densities seen by PCR (open circles) and parasite densities seen by thick blood film examination (filled circles) are both plotted on the y axis. The PCR readings are the result of a single experiment, the thick film readings are the results of two experiments. The solid line (least squares regression line for PCR results against densities known from serial dilution) is given by y = 0.98x + 0.07 (95% CI -0.12 to 0.27). The dotted line (regression line for thick film densities against serial dilution, ignoring the outlier) is given by y = 0.78x - 1.5 (95% CI -0.61 to -2.4) (y = 0.81x - 1.26, 95% CI - 0.07 to -2.5 including the outlier). Densities measured by thick film are therefore approximately 1 log lower than those calculated by serial dilution, whereas PCR readings match the serial dilution more closely.