Pyronaridine exhibited a high level of in vitro activity against isolates of P. falciparum from infected Gabonese subjects in this study. Fifty percent effective concentrations were 2.0 and 1.9 nmol/l, which is consistent with previously published data from studies with laboratory strains and field isolates obtained by a different drug sensitivity assay (isotopic hypoxanthine assay) [15, 16].
Median pyronaridine concentrations of 9.3 nmol/l (EC99 in HRP-2 assay) and 9.0 nmol/l (cut-off concentration in WHO microtest) inhibited growth of P. falciparum in vitro. In a recent pharmacokinetic assessment of a fixed-dose pyronaridine artesunate combination in African children, the mean maximal plasma concentrations of pyronaridine were 115 nmol/l in patients . Earliest reappearances of parasites in peripheral blood in this study were observed on day 21 (one patient) and day 28 (three patients) after treatment. Interestingly, the patient with parasite reappearance on day 21 exhibited a pyronaridine plasma level of 9.9 nmol/l at that time. In polynomial regression analysis of pooled plasma levels of all patients, mean pyronaridine blood concentrations fell below levels of 9 nmol/l on day 22. First reappearances in vivo therefore occurred only after plasma levels fell below concentrations similar to cut-off concentrations obtained in this in vitro study.
Although the extrapolation of in vitro drug sensitivity assessments to in vivo data should always be cautious , in vitro results of the present study matched closely clinical findings of this clinical trial on pyronaridine-artesunate combination therapy.
The relationship between in vitro activity of pyronaridine and chloroquine against P. falciparum has been discussed controversially in previous reports and contradictory conclusions concerning cross-resistance have been drawn from studies, which found a significant correlation between pyronaridine and chloroquine in vitro activity and differences in activity of pyronaridine against chloroquine resistant and sensitive strains [15, 16, 18, 19]. In the present study, in vitro response to chloroquine was poor in all tested isolates, yet parasites proved to be highly susceptible to pyronaridine. There was no statistically significant correlation of the in vitro activities of the two drugs against P. falciparum. These in vitro findings and the recently reported high cure rates of three days pyronaridine-artesunate combination therapy provide convincing evidence against a clinically significant cross resistance of pyronaridine and chloroquine in vivo. The results underline the usefulness of pyronaridine for the treatment of patients in regions affected by P. falciparum resistant to chloroquine [9, 11].
Since the first in vitro drug susceptibility assessment in Lambaréné in 1992 , high levels of resistance against chloroquine have been observed in vitro and in vivo [3–5]. Similar to previous studies in vitro response to chloroquine was poor in this study. EC50 levels were comparable to the last assessment in 2002 , despite a decrease in drug pressure due to a change of national treatment policies to artemisinin-based combination therapy in 2003. Artesunate was highly active against P. falciparum in the present assessment. EC50 levels were similar to recent in vitro and in vivo findings at the study site and in neighbouring countries [20–22]. One isolate showed a diminished sensitivity to artesunate with schizont maturation up to the highest concentration of 85.8 nmol/l and a corresponding EC50 level of 19.9 nmol/l. Another isolate had a comparably high EC50 value of 10.1 nmol/l and a respective cut-off concentration in the WHO microtest of 85.8 nmol/l. These findings support recent in vitro susceptibility assessments by Cojean et al , reporting on 6 out of 397 African P. falciparum isolates with dihydroartemisinin EC50 levels above 10 nmol/l and a maximum EC50 of 31.8 nmol/l in one isolate. Especially in the light of increasing availability and use of artemisinins in many parts of Africa, the importance of close surveillance for susceptibility as well as strict deployment of artemisinins exclusively in combination therapy with effective partner drugs cannot be overemphasized.
As in previous reports from Lambaréné, susceptibility to quinine was high in this study. Mean EC50 (204 and 272 nmol/l in HRP-2 assay and WHO microtest, respectively) were comparable to results obtained in 2002 (286 nmol/l) . Despite its widespread use, quinine remains a highly effective anti-malarial in Gabon, especially as drug of choice for parenteral treatment of hospitalized patients.
Comparative assessment of the HRP-2 assay and WHO microtest was performed in order to establish baseline data for the use of the ELISA based assay in Lambaréné in the future. Results obtained by the HRP-2 assay closely paralleled those obtained by the WHO microtest despite methodological problems in this study due to a deficient batch of culture medium. Considering its reduced labour-intensity and good reproducibility the HRP-2 assay can, therefore, be recommended for follow up of anti-malarial drug susceptibility testing and will replace the WHO microtest in future assessments at our study site.