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. © 2009 Lim et al; licensee BioMed Central Ltd.; AuthorsConclusions:This study shows that pfmdr1 copy number is a molecular marker of AM treatment failure in falciparum malaria on the Cambodia-Thailand border. However, while it is associated with increased IC50 for lumefantrine, pfmdr1 copy number is not associated with AL [artemether-lumefantrine] treatment failure in the area, suggesting involvement of other factors or molecular mechanisms in AL treatment failures in Cambodia.; Results:10 in the AM group and 23 in the Riamet group were classified as recrudescence. All failures were LTFs. There were 96 (81%) ACPR and 23 (19%) LTF in Riamet group and 123 (92.5%) ACPR and 10 (7.5%) LTF in the AM group. Pfmdr1 copy number was lower in the ACPR group compared to the LTF group (n=112) for AM but not for Riamet (n=96). In the AM group (N=115), crude hazard ratio for increased copy number (≥3 copies compared to <3 copies) was 7.80. There was no association between increased copy number and treatment failure in the Riamet group.; AdverseEffects:No adverse events were mentioned.; FreeText:The therapeutic response was classified as early treatment failure (ETF), late clinical failure (LCF), late parasitological failure (LPF), late treatment failure (LTF=LCF + LPF), and adequate clinical and parasitological response (ACPR). Tests: parasite DNA genotyping of merozoite surface protein (msp) 1, msp2 and glurp to determine recurrence and re-infection; and Pfmdr1 copy number by real-time polymerase chain reaction.; Indications:135 patients with falciparum malaria.; Patients:275 inpatients, aged over 6 years. Riamet group: n=135. AM group: n=140. Follow-up: 28 days.; TypeofStudy:An open study assessing the link between pfmdr1 copy number and clinical response outcome in patients treated with Riamet and artesunate plus mefloquine (AM) for falciparum malaria in Cambodia.; DosageDuration:Artemether 20 mg/kg plus lumefantrine 120 mg/kg bid (=40/240 mg/kg daily) at 0 and 8 hours on the first day, and morning and late afternoon on the following 2 days; as tablets. Duration was 3 days.; ComparativeDrug:Artesunate was given at 4 mg/kg daily bid (morning and evening on day 0), and 4 mg/kg single dose on day 1 and 2 with a maximum adult dose of 600 mg in total. Mefloquine was given at 25 mg/kg daily bid (morning and evening on day 0), the maximum dose was limited to 1500 mg. Duration was 3 days.
