In this study, the Dutch and Belgian experience with the use of IV artesunate on a named patient basis is presented, the largest case series of patients with severe malaria in Europe. Mortality was low and parasite clearance was rapid. Late onset haemolysis was observed, mostly short lived, which confirms recent findings in travellers with severe malaria treated with IV artesunate .
In this study, probably all patients treated with artesunate in the Netherlands were captured. The Netherlands is a small country, the professional groups are well organized and there is little chance that patients would have been treated outside our scope. For Belgium, this was different, only patients from two Belgian hospitals were traceable. There is no indication of a selection bias towards the use of artesunate or quinine. Once artesunate was available in hospitals, this became the drug of choice.
A named patient programme cannot substitute for prospective clinical research. Nevertheless, this type of pharmacovigilance studies may capture more information than a prospective clinical trial, especially rare and late onset events. All treating hospitals are large medical centres with high quality care, accredited laboratories and close follow up procedures, ensuring a high reliability and quality of the data.
A limitation of this retrospective study is the heterogeneity of patient characteristics and therapeutic interventions: Twenty-five patients received quinine or other anti-malarial agents before artesunate, while 17 received exchange transfusion or erythrocytapheresis. The decision to treat with IV artesunate is only part of the total routine case management. In Belgium, criteria for administrating IV artesunate are restricted to patients with 'very severe malaria' and vomiting patients intolerant of IV quinine. In the Dutch malaria treatment guidelines ('StichtingWerkgroepAntibioticabeleid' (SWAB), the Dutch Working Party on Antibiotics Policy; http://www.swab.nl), artesunate is treatment of choice in severe malaria, however, when not available, quinine should be administered instead or until artesunate is available. The availability of artesunate increased rapidly in the last few years (seven out of 90 Dutch hospitals had artesunate in stock in 2008, 44 in 2010 (distribution records)), while pre-treatment with quinine has decreased in the same period.
Although the contribution of exchange transfusion has been disputed, it accelerates parasite clearance during quinine treatment . Its benefit during artesunate treatment needs further study. Nonetheless, Dutch SWAB guidelines consider exchange transfusion in patients with > 10% parasitaemia or severe illness with organ failure. In Belgium, erythrocytapheresis was the standard procedure in patients with very high parasitaemia (> 15%) until IV artesunate became available. Since then, it has been largely abandoned.
The cure and survival rates among patients with severe malaria in the present study were high. The death of the two patients was not directly related to malaria and/or the treatment with artesunate. Although sample size of the present study is small and although it is inappropriate to infer the evidence from endemic regions [2, 3] and the treatment outcome of quinine in French malaria patients to the present population, it confirms what has been stated before; IV artesunate is an excellent drug for severe malaria, also under the resourceful conditions of case management in the Netherlands and Belgium.
It has been discussed whether the evidence from endemic regions can be generalized to the European population. Patients characteristics, clinical manifestations and supportive care may differ greatly between developing and industrialized countries . It has also been debated whether this would justify or even require a trial comparing artesunate and quinine for European patients. Our current view is that severe malaria is such a rare disease in Europe that sufficient sample sizes cannot be obtained. Furthermore, the question remains whether European physicians would be willing to participate in such a trial now that artesunate is recommended as the treatment of choice for severe malaria by WHO .
Severe post-treatment haemolysis occurred unexpectedly in seven patients. This was also observed in 24% of patients treated with IV artesunate in another study  in which post-treatment haemolysis was associated with higher doses of artesunate and longer treatment periods. This was not observed in the present study. Four patients received AL as a consolidation treatment. Artemether but also lumefantrine or quinine may induce haemolysis ('blackwater fever') [13, 14].
Surprisingly, in the SEAQUAMAT and AQUAMAT trials [2, 3], over 3000 patients were treated with IV artesunate, even with higher cumulative doses of artesunate than in the present study, and often also with AL, but no haemolysis was reported. These trials were not designed to capture this late onset event.
In acute falciparum malaria, anaemia and prolonged haemolysis may persist for weeks after elimination of parasites, regardless the treatment given [15, 16]. Other factors could also have contributed to the haemolysis. For example, one patient was heterozygous for glucose-6-phosphate dehydrogenase (G6PD) deficiency. Three patients received exchange transfusion in conjunction with anti-malarial treatment. Furthermore, both positive (3 times) and negative (3 times) direct Coombs test results were observed. Anti-erythrocyte antibodies can be secondary to malignancies, autoimmune disorders, transfusion reactions and also to drugs  and is rather common after malaria . Drug induced haemolysis with a negative direct Coombs test has also been documented .
Since physicians were not alerted to this phenomenon, additional investigations were not performed. It is therefore difficult to assess whether exposure to artesunate was the sole cause. Moreover, the causality of artesunate administration is not easily established by using adverse drug reaction scales, mainly because scores on these scales are largely affected by the relationship in time between drug administration and onset of the adverse event . The haemolysis started long after complete clearance of artesunate . There are several effects of artesunate on cellular biology, including suppressive effects on erythropoiesis and angiogenesis . Recently Berdelle and colleagues demonstrated that artesunate has mutagenic potential . Whether these effects extend to a-nuclear erythrocytes is not known. Another explanation of late onset hemolysis could be a reduced survival of 'pitted' infected erythrocytes [24, 25]. This would explain the fact that in this study haemolysis was only seen in patients with very high parasitaemia (11-37%). This is refuted by the fact that not all patients with high parasitaemia develop late onset haemolysis. Moreover, a similar type of hemolysis was also observed in a patient with non-hyperparasitaemic non-severe malaria, treated with AP followed by AL (unpublished data). Whatever the mechanism, a weekly follow up for 4 weeks with Hb measurement and, if not improving, also other parameters of haemolysis, should be performed. More importantly, as the majority of parasites is cleared within 24 hours, and in light of the possible role of artesunate in the development of late onset haemolytic anaemia, the authors would like to recommend to limit treatment to the period that IV treatment is deemed necessary. In practice this is no longer than 48 hours. Treatment should be followed by an adequate and full course of oral anti-malarial treatment.
There is still no marketing authorization for an artesunate product on the European market. This is due to the fact that it is challenging to develop a suitable formulation that meets the requirements of GMP. Several companies are currently working on GMP-conform IV artesunate formulations, while others work on semi-synthetic production of artemisinins [26, 27]. Importing drugs into Europe is subject to strict regulations, which ensure that patients would never receive a non-properly tested and released product. There are several companies involved in importing artesunate into Europe.
Conclusions and recommendation
The excellent efficacy of IV artesunate for severe imported malaria in industrialised countries supports the efforts to make this drug available throughout Europe. The roll-out of IV artesunate should be closely monitored by a pharmacovigilance programme, such as was set up for this study. Meanwhile, increased safeguards with respect to haematological abnormalities should be instituted in the follow-up period after anti-malarial treatment at least once weekly until 4 weeks after initiation of therapy. The fast clearance of parasites warrants the reduction of the length of treatment with artesunate. The authors would, therefore, recommend that treatment with artesunate should be limited to the period that IV treatment is required and should be followed by a full oral course of an appropriate anti-malarial drug.