Of all 348 patients, 91.3% responded clinically and parasitically adequately to the treatment until day 42. Adverse events were expectedly mild to moderate with low prevalence. Abdominal pain and diarrhoea were the most frequent symptoms.
The PCR-corrected failure rate showed incidence rates of 3.8% for new infections and 5.1% for recrudescences at day 42. No early treatment failure occurred. Compared to a trial in 2006, these data show a slight increase in recurrent parasitaemia as none had been detected until day 42 .
Other Ethiopian studies detected also high cure rates but most had a follow-up period of 28 days only [19–23]. In this study, the PCR-corrected cure rate per protocol at day 28 was 99.4% (95% CI 98.5-100.0). This result emphasizes the need for follow-up periods of at least 42 days. Most recrudescences occurred after day 28. The recrudescence rate in children ≤5 years was surprisingly high with 9.4%. None of the children with recrudescences had diarrhoea or vomiting at admission or during therapy. Apart from limitations discussed in the next paragraph, the weight-adapted drug dose is non-linear but stepwise. Two of the five children with LTF had calculated drug doses of less than 2 mg/kg and 12 mg/kg body weight, respectively. Furthermore, weight-adapted linear or non-linear doses might not correlate with plasma drug levels in children in general.
Unfortunately, there are several limitations concerning this study. Drug levels were not tested and only the first drug intake was observed. As absorption is fat-dependent, fatty food was provided and patients were precisely instructed how and when to take the rest of the medication. However, intake was not directly controlled and absorption individually varies, also impaired by symptoms like diarrhoea . Therefore, recrudescences and also the prolonged parasite presence could be due to insufficient drug levels. The advantage of this study is the closer approach to a real life situation.
Another limitation is the method of genotyping. Differentiation of recrudescences and re-infections by PCR is unprecise, especially in low to moderate transmission areas with a limited diversity of strains . Similar strains, defined as recrusdescence, could be new infections instead. The recrudescence rate given might be lower in reality.
Artemether has a very short half-life of about one hour. Lumefantrine has a half-life of three to six days and is able to clear the remaining parasites and to prevent recurrent parasitaemia [39, 40]. The first recrudescence was observed early at day 18. However, due to limitations mentioned above, other reasons than strain resistance might have caused recrudescences. New infections seemed to be efficiently prevented in the first four weeks.
Six patients showed prolonged presence of parasites detectable still at day 2 and day 3, the calculated half-life time was prolonged. As stated above, this could have been caused by low drug levels. Another theory is the hypothesis of a dormant non-responding parasite stage causing delayed susceptibility to drugs [41, 42]. This might be responsible for late clearance rates, though genetic analysis revealed a heritable trait at the Cambodia–Thailand border indicating changes in parasite genetics and a potential for spread . Controlled ACT studies with measurement of drug levels and observed drug intake should be conducted in Africa to examine drug efficacy and clearance rates.
The low number of recrudescences and prolonged parasite presence (>48 hrs) impaired multivariate analysis. Risk factors for these outcomes could not be determined. There was strong evidence for an association of both outcomes but the result has to be considered with caution as the sample size is low. However, an association of recrudescence and prolonged parasite presence (72 hrs) was described recently . Parasite clearance data from 18,699 patients with uncomplicated falciparum malaria were analysed. Blood smear result on day 3 was reported as a good predictor of subsequent treatment failure in low- and moderate-transmission areas. The risk of recrudescence was <5% if parasitaemia was cleared before day 3, patients with parasitaemia on day 3 on the other hand had a significantly higher risk in low-transmission areas (12%) and in moderate-transmission areas (34%) to develop recrudescences. Less pronounced but still significant was the association of parasitaemia at day 2 and recrudescences in moderate transmission areas.
This association was observed in the underlying study. Recurrent parasitaemia occurred in two patients with parasites at day 2 (P = 0.01). Prevalence of patients with parasites at day 3 was 0.6% (2/348) only. Even if adequate drug levels could be assumed, tolerance to artemether would not be suspected. Artemisinin resistance seems highly unlikely if prevalence of patients with parasites at day 3 is <3% .
Further analysis indicated a correlation of (prolonged) presence of gametocytes with prolonged parasitaemia and recrudescences. Sample size was low as mentioned but associations seemed plausible. Gametocytes have more opportunities to develop if parasite presence is prolonged and sub-microscopic level as with recrudescences seem to suffice for gametocyte production.
Interestingly, gametocytaemia was more frequent in season 2008 than 2009. The broad introduction of ACT in this region might have influenced the gametocyte carriage in the population. ACT clears gametocytes better than S/P, the previous first-line drug. ACT is supposed to destroy the early stages of gametocytes [16, 43]. The carrier reservoir might be reduced since introduction of ACT.
A random selection of samples showed very high levels of gametocytaemia with QT-NASBA technique compared to microscopy. Therefore, higher submicroscopic levels have to be assumed on recruitment as well as after treatment. Recent studies showed a difference of factor 3–6 between microscopic and submicroscopic gametocyte prevalence [44, 45]. Less than one gametocyte per μL is sufficient to infect a mosquito . Gametocytes detected by microscopy were rapidly reduced under AL in this study but 2.2% of carriers remained positive until day 7. A much higher proportion of submicroscopic gametocytaemia can be assumed. ACT seems to have less or no effect on mature gametocytes [16, 43]. Therefore, stage V gametocytes, if already developed at start of therapy, might circulate for two to three weeks. The reservoir is a permanent risk for further transmission and should be measured in clinical trials as well as in population-based studies with recently published methods .
Further, an additional drug for gametocyte clearance should be considered in the area. A single dose of primaquine, best given at day 8, might be sufficient for gametocyte clearance . Unfortunately, prevalence of G6PD enzyme deficiency is unknown. As tests are not yet available in this area, the application of primaquine remains ethically problematic, although single-dose treatment seems acceptable .
Interestingly, P. falciparum mono-infection occurred in only half of the malaria cases in the study area. Prevalence of mixed infections with P. vivax and infections with P. vivax alone was high and has increased over the last years [, pers comm with health centres in Jimma and surrounding]. In Serbo, 70% of the malaria patients were infected with P. vivax only. However, there was surprisingly no re-infection with P. vivax observed. One explanation for this and the very few re-infections with P. falciparum might be that new bed nets were given free of charge by the government to every patient visiting the health centres.
Plasmodium vivax infections diagnosed by PCR and afterwards excluded from analysis showed ACPR in all cases, no relapses occurred. Data on P. vivax and AL are unavailable in this area. Chloroquine is still first-line treatment of P. vivax. A recent study from another area from Ethiopia observed treatment with AL and CQ in vivax malaria. Recurrent parasitaemia rates were 19% and 8%, respectively, although, the evening dose of AL was not observed . Studies from Asia reported higher recurrence rates with AL compared to CQ or dihydro-artemisinin/piperaquine as well [48, 49]. However, relapses seem to occur in Ethiopia later than in Asia  and might be expected after day 42.