Laboratory detection of artemisinin-resistant Plasmodium falciparum

Abstract

Conventional 48-h in vitro susceptibility tests have low sensitivity in identifying artemisinin-resistant Plasmodium falciparum, defined phenotypically by low in vivo parasite clearance rates. We hypothesized originally that this discrepancy was explained by a loss of ring-stage susceptibility and so developed a simple field-adapted 24-h trophozoite maturation inhibition (TMI) assay focusing on the ring stage and compared it to the standard 48-h schizont maturation inhibition (WHO) test. In Pailin, western Cambodia, where artemisinin-resistant P. falciparum is prevalent, the TMI test mean (95% confidence interval) 50% inhibitory concentration (IC50) for artesunate was 6.8 (5.2 to 8.3) ng/ml compared with 1.5 (1.2 to 1.8) ng/ml for the standard 48-h WHO test (P = 0.001). TMI IC50s correlated significantly with the in vivo responses to artesunate (parasite clearance time [r = 0.44, P = 0.001] and parasite clearance half-life [r = 0.46, P = 0.001]), whereas the standard 48-h test values did not. On continuous culture of two resistant isolates, the artemisinin-resistant phenotype was lost after 6 weeks (IC50s fell from 10 and 12 ng/ml to 2.7 and 3 ng/ml, respectively). Slow parasite clearance in falciparum malaria in western Cambodia results from reduced ring-stage susceptibility.

FIG 1

Correlation between 50% inhibitory concentration (IC₅₀) from TMI test and in vivo responses. (A) IC₅₀ plotted against parasite clearance time (PCT) for the first of two consecutive negative blood films; (B) IC₅₀ plotted against parasite clearance half-life.

FIG 2

Correlation between 50% inhibitory concentration (IC₅₀) from WHO test and in vivo responses. (A) IC₅₀ plotted against parasite clearance time (PCT) for the first of two consecutive negative blood films; (B) IC₅₀ plotted against parasite clearance half-life.

FIG 3

Stability of resistant phenotype. Data are presented as means and standard deviations (SD).