Reduced in vitro susceptibility to artemisinin derivatives associated with multi-resistance in a traveller returning from South-East Asia

Decreased in vitro susceptibility to dihydroartemisinin (21.2 nM) and artesunate (16.3 nM) associated with decreased susceptibility or resistance to quinine (1131 nM), mefloquine (166 nM), lumefantrine (114 nM), pyronaridine (70.5 nM) and piperaquine (91.1 nM) is reported in a patient returning from South-East Asia after trekking along the Mekong from the south of Laos to the north of Thailand. Decreased in vitro susceptibility to artemisinin derivatives did not appear to be mediated by the number of copies of pfmdr1 or pfATPase6, pfcrt, pfmdr1 or pfmrp polymorphism. The high IC50 to mefloquine of this Asian isolate was not associated with pfmdr1 copy number. Pfnhe-1 microsatellite ms4760 showed a profile 7 (ms4760-7) with three repeats of DNNND and one repeat of DDDNHNDNHNN, which is associated with high quinine reduced susceptibility. The patient recovered in three days without relapse after treatment with the association of quinine and doxycycline. Decreased in vitro susceptibility to quinine and the delayed effect of doxycycline may both have contributed to the delayed parasite clearance time, D4 (0.5%) and D7 (0.004%). The in vitro data, with IC50 for dihydroartemisinin and artesunate were up to ten times those of the reference clone W2, which suggests that this isolate may be resistant to artemisinin derivatives, associated with a decreased susceptibility to quinine.


Background
Artemisinin-based combination therapy (ACT) is now recommended by the World Health Organization as firstline treatment of uncomplicated falciparum malaria in all areas in which malaria is endemic. However, recent reports from Cambodia of delayed parasite clearance after treatment by ACT have now been confirmed [1]. The resistant phenotype is not yet reflected by the results of conventional in vitro drug susceptibility assays. Parasites with slow clearance rate after ACT did not show in vitro decreased susceptibility [1]. In vitro decreased susceptibility to artemisinin derivatives was never or very rarely reported in Cambodia [1][2][3][4]. No molecular marker has been identified, which impedes surveillance studies to monitor the spread of artemisinin resistant phenotype. Decrease of in vitro susceptibility to dihydroartemisinin and artesunate, associated with reduced susceptibility to standard anti-malarials, such as quinine, mefloquine and lumefantrine, and new drugs, such as pyronaridine and piperaquine, is reported here.

Case presentation
A 52-year old female visited rural areas in Laos (Nov 9 to 12, 2009), Cambodia (Nov 12 to 29) and Thailand (Nov 29 to Dec 1). She took part in trekking along the Mekong from the south of Laos to the north of Thailand. The patient presented with fever since November 29 and was hospitalized (Dec 2) in intensive care unit (Centre Hospitalier Universitaire l'Archet, Nice, France) for complicated malaria (15% parasitaemia and altered consciousness). The patient used irregularly doxycycline (100 mg/day) as chemoprophylaxis. The patient was treated by intra-venous quinine chlorhydrate (25 mg/kg/day) and doxycycline (200 mg/day) for seven days. The patient recovered in three days without relapse and was discharged. Plasmodium falciparum parasites were identified at Day 0 (15%), D4 (0.5%) and D7 (0.004%) but were not detected at D43.

Methods
In vitro testing of drug susceptibility was performed by the standard 42-hour 3 H-hypoxanthine uptake inhibition method [5]. Susceptibility to dihydroartemisinin, artesunate, and ten standard or new anti-malarial drugs, ie chloroquine, quinine, mefloquine, lumefantrine, monodesethylamodiaquine (biologically active metabolite of amodiaquine), pyronaridine, piperaquine, atovaquone, doxycycline and pyrimethamine, was assessed. The laboratory-adapted clone W2, tested on the same day, was used as a reference. Isolates from imported malaria, tested on the same batch of plates, were used as comparators.
Polymorphisms of pfcrt, pfmdr1, pfmrp and pfnhe-1, involved in quinoline resistance, and in pfATPase6, postulated to be involved in artemisinin resistance, and the copy number of pfmdr1 were assessed [6].

Consent
Informed consent was not required as the sampling procedures and testing are part of the French national recommendations for the care and surveillance of malaria.
Mutations were not identified in pfmdr1, pfcrt and pfmrp genes. Only one copy of pfmdr1 was found. Two synonymous mutations were detected in pfATPase6 (N460N and I898I), which were previously described [11]. Pfnhe-1 microsatellite ms4760 showed a profile 7 (ms4760-7) with three repeats of DNNND and one repeat of DDDNHNDNHNN, which is associated with high quinine reduced susceptibility [12].

Conclusion
This isolate showed reduced susceptibility to artemisinin derivatives, but also to other ACT components commonly used in Asia or in clinical trials, such as mefloquine, lumefantrine, pyronaridine or piperaquine. Surprising, this isolate was susceptible in vitro to chloroquine and monodesethylamodiaquine. It was also susceptible to doxycycline. Plasmodium falciparum parasite with high IC 50 to artemisinin and artemether (20.1 nM and 21.4 nM, respectively), but with low IC 50 to dihydroartemisinin and artesunate (1.8 nM and 6.2 nM, respectively) was recently isolated in a traveller returning from Nigeria, who took artesunate prophylactically (two 50 mg tablets weekly for 4 weeks) [13]. Yet, recent clinical trials of oral artesunate monotherapy suggest that the loss of ACT efficacy might result from decreased efficacy of artemisinin derivatives [1,3]. The median parasite clearance time was 36 hours longer in patients from Western Cambodia, where the efficacy of ACT is decreasing [1]. Nevertheless, this phenomenon was not correlated with artemisinin derivatives IC 50 .
Only one copy of pfmdr1 was found. The high IC 50 to mefloquine of this Asian isolate was not associated with pfmdr1 copy number. Pfnhe-1 microsatellite ms4760 showed a profile 7 (ms4760-7) with three repeats of DNNND and one repeat of DDDNHNDNHNN, which is associated with high quinine reduced susceptibility [12]. Two repeats of DNNND were seen to be associated with high IC 50 in quinine clinical failure in traveller from Senegal [14].
The persistence of parasites seven days after the start of treatment with quinine chlorhydrate and doxycycline, but without clinical signs, is consistent with the high IC 50 and with the profile ms4760 of pfnhe-1. The action of doxycycline is delayed. There is a relationship between the amount and duration of exposure and the effect of doxycycline on the erythrocytic stages with an increased activity during the second cycle. Doxycycline has been shown to exert its effect during the first 48 h, but is only detectable in second-generation parasites near the 96 hour time point [15][16][17]. The French malaria consensus recommends quinine associated with doxycycline for Asian and South-American P. falciparum (and not only quinine as for African parasites). Clinical failure with quinine has been shown in a patient returning from French Guiana treated by quinine only [18]. The patient recovered in three days without relapse and left intensive care due to the treatment she received, the association of quinine and doxycycline, as recommended by the French malaria consensus. In vitro quinine IC 50 and pfnhe-1 ms4760 analysis suggest a reduced susceptibility to quinine. This decreased susceptibility to quinine and the delayed effect of doxycycline may both have contributed to the delayed parasite clearance time, D4 (0.5%) and D7 (0.004%).
Decreased in vitro susceptibility to artemisinin derivatives did not appear to be mediated by the number of copies of pfmdr1 or pfATPase6 polymorphism. The in vitro data, with IC 50 for dihydroartemisinin and artesunate up to ten times those of the reference clone W2 or the geometric mean of the other isolates, suggest that this isolate could be resistant to artemisinin derivatives, even if there is no evidence that this isolate was clinically resistant to ACT, associated with decreased susceptibility to quinine. And its association with the other in vitro decreased susceptibilities is alarming, especially with the components of ACT used in Asia.