Pfmdr1 copy number and arteminisin derivatives combination therapy failure in falciparum malaria in Cambodia

Abstract

Background: The combination of artesunate and mefloquine was introduced as the national first-line treatment for Plasmodium falciparum malaria in Cambodia in 2000. However, recent clinical trials performed at the Thai-Cambodian border have pointed to the declining efficacy of both artesunate-mefloquine and artemether-lumefantrine. Since pfmdr1 modulates susceptibility to mefloquine and artemisinin derivatives, the aim of this study was to assess the link between pfmdr1 copy number, in vitro susceptibility to individual drugs and treatment failure to combination therapy.

Methods: Blood samples were collected from P. falciparum-infected patients enrolled in two in vivo efficacy studies in north-western Cambodia: 135 patients were treated with artemether-lumefantrine (AL group) in Sampovloun in 2002 and 2003, and 140 patients with artesunate-mefloquine (AM group) in Sampovloun and Veal Veng in 2003 and 2004. At enrollment, the in vitro IC50 was tested and the strains were genotyped for pfmdr1 copy number by real-time PCR.

Results: The pfmdr1 copy number was analysed for 115 isolates in the AM group, and for 109 isolates in the AL group. Parasites with increased pfmdr1 copy number had significantly reduced in vitro susceptibility to mefloquine, lumefantrine and artesunate. There was no association between pfmdr1 polymorphisms and in vitro susceptibilities. In the patients treated with AM, the mean pfmdr1copy number was lower in subjects with adequate clinical and parasitological response compared to those who experienced late treatment failure (n = 112, p < 0.001). This was not observed in the patients treated with AL (n = 96, p = 0.364). The presence of three or more copies of pfmdr1 were associated with recrudescence in artesunate-mefloquine treated patients (hazard ratio (HR) = 7.80 [95%CI: 2.09-29.10], N = 115), p = 0.002) but not with recrudescence in artemether-lumefantrine treated patients (HR = 1.03 [95%CI: 0.24-4.44], N = 109, p = 0.969).

Conclusion: This study shows that pfmdr1 copy number is a molecular marker of AM treatment failure in falciparum malaria on the Thai-Cambodian border. However, while it is associated with increased IC50 for lumefantrine, pfmdr1 copy number is not associated with AL treatment failure in the area, suggesting involvement of other molecular mechanisms in AL treatment failures in Cambodia.

Figure 1

Number of specimens analyzed for in vitro drug sensitivity, pfmdr1 polymorphism and pfmdr1 copy number according to the treatment group. N: Number. ^#Mey Bouth Denis et al, December 2006, Tropical Medicine and International Health [8]. *Mey Bouth Denis et al, September 2006, Tropical Medicine and International Health [9].

Figure 2

Comparison of pfmdr1 copy number and clinical failure outcome. Artesunate-mefloquine: ACPR (N = 103, copy number ≥ 2 = 20); LTF (N = 9, copy number ≥ 2 = 7); * p < 0.001. Artemether-lumefantrine: ACPR (N = 76, copy number ≥ 2 = 8); LTF (N = 20, copy number ≥ 2 = 4), p = 0.257 (the analysis was tested by Wilcoxon rank-sum (Mann-Whitney) test). The black horizontal lines and number represent geometric mean pfmdr1 copy number.

Figure 3

The relationship between pfmdr1 copy number (A, B) and time to recrudescence (the relationship was estimated using survival analysis).