pfk13-Independent Treatment Failure in Four Imported Cases of Plasmodium falciparum Malaria Treated with Artemether-Lumefantrine in the United Kingdom

Patient 1.A male over 65 years of age presented to Addenbrooke's Hospital, Cambridge University Hospital (CUH), UK, in 2015 with 1.4% P. falciparum parasitemia following travel in Angola. He was treated with AL in the hospital, and all doses were observed. The first dose was consumed without food, and the patient vomited once 3 h later. All but one of the subsequent AL doses were consumed with food, and the patient was confirmed negative for parasitemia prior to discharge. Symptoms recurred more than 1 month later, and the patient presented with 0.02% P. falciparum parasitemia, confirmed by PCR and microscopy at the MRL. The patient responded well to administration of 4 tablets of 100/250 mg atovaquone/proguanil daily for 3 days. The pfmdr1 Tyr184Phe and pfap2mu Ser160Asn variants observed have both been associated with recurrent parasitemia in AL-treated African patients (5, 8).

Patient 2.A Ugandan female under 21 years of age was admitted to the Hospital for Tropical Diseases, London (HTD), with acute P. falciparum hyperparasitemia estimated at more than 30% in early 2016 following a visit to Uganda. She responded well to initial treatment with intravenous (i.v.) artesunate. Parasitemia of 0.07% was recorded 60 h after admission, and the patient was discharged 3 days later, with a recorded parasitemia level of 0.0001%, on a full course of oral AL. Full compliance was reported under supervision of staff members at her place of learning. Symptoms recurred 4 weeks later. The patient was readmitted to HTD with 2.7% P. falciparum parasitemia, received 2 days i.v. artesunate as an inpatient until the parasitemia level fell below 0.0001%, and was discharged on a full course of AL plus 7 days of doxycycline (200 mg daily). This recurrent isolate was established in in vitro culture at the London School of Hygiene & Tropical Medicine (LSHTM), and standard 48-h 50% effective concentration (EC₅₀) estimates were derived for chloroquine (102.6 nM), dihydroartemisinin (2.8 nM), and lumefantrine (38.7 nM) as previously described (11), with the results indicating full sensitivity to AL components in vitro. The genotypes of both parasite isolates were consistent with AL sensitivity at pfk13, pfcrt, pfmdr1, and pfap2mu (Table 1). However, the Glu-to-Gln change at codon 1525 of pfubp1 was associated with reduced susceptibility to ACT in vivo in one study (8). The recurrent parasitemia occurring 28 days after first presentation may reflect incomplete clearance of the original hyperparasitemia by the treatment received.

Patient 3.A male over 21 years of age presented to CUH with 0.4% P. falciparum parasitemia in early 2016, after travel to Liberia. He was treated with AL and was eventually discharged but returned with recurrent symptoms and 0.02% parasitemia 17 days later. He was successfully treated with atovaquone-proguanil. Genotyping identified mixed genotypes at both pfcrt and pfap2mu loci in the isolate from the primary episode but not in that from the recurrent episode (Table 1). DNA extracted from the patient after both episodes was sent to the Wellcome Trust Sanger Institute for whole-genome sequencing (WGS). Preliminary analysis strongly suggests that the recurrent infection which occurred 17 days after AL treatment comprised a single parasite clone, whereas the original parasite population was multiclonal (Table 1). The recurrent isolate harbored the previously identified CVMNK and NFD haplotypes of pfcrt and pfmdr1, known to be selected in parasites persisting after AL treatment (5, 8). The impact of the ins_Lys233 variant of pfap2mu and that of the novel pfk13 non-propeller region mutation Gly112Glu require further study. Analysis in the bioanalytical laboratory of LSHTM confirmed that the AL tablets used to treat this patient (Novartis Riamet [lot XO131; expiration date, December 2017]) met the specified International Pharmacopeia tolerance limits (12).

Patient 4.A female under 21 years of age who presented as febrile to the HTD in early 2016 following travel to Uganda was diagnosed with 0.08% P. falciparum parasitemia and treated with oral AL. The patient was discharged to complete the treatment course at home and reported full compliance with all six doses. She experienced recurrent symptoms 1 month later, presenting to HTD with confirmed 0.3% P. falciparum parasitemia, and was successfully treated with 7 days of oral quinine (10 mg/kg of body weight every 8 h) and doxycycline (200 mg daily). Data from the two episodes appeared identical at the parasite loci evaluated. Novel mutations at codons 226 and 326 of pfap2mu and codon 1500 of pfubp1 are of unknown significance, but, as this is clearly a recrudescent parasite genotype, both variants should be further evaluated in future studies.

Treatment failure cannot be unequivocally ascribed to parasite resistance in these four patients, although three harbored parasites with variant alleles of loci previously linked to reduced susceptibility to artemisinin or lumefantrine (Table 1). Our findings would have been strengthened by observing treatment for all patients to ensure full regimen compliance, and by measurement of lumefantrine blood levels at day 7, to rule out any problems with malabsorption (1), as this can result in failure to clear parasites (13). None of the four patients harbored mutations in the propeller-encoding domain of the pfk13 locus and thus did not exhibit reduced artemisinin susceptibility as seen in the Greater Mekong Region (2–4). The parasites that we describe may represent adaptive African genotypes that have evolved pfk13-independent mechanisms for posttreatment survival in vivo. This suggests that, in countries where malaria is not endemic and in which imported malaria cases from Africa are seen, vigilance is required to protect the efficacy of our first-line ACT. Studies in countries where malaria is endemic are required to further validate potential genetic markers for monitoring in African parasite populations.

Artemisinins are very quickly metabolized and cleared from circulation, such that only a single full parasite life cycle is exposed to the highly potent action of these compounds under conditions of a 3-day regimen. The current practice of 3 days of treatment may therefore be insufficient to guarantee clearance of parasite genotypes under ACT pressure for a decade in many parts of Africa (14). The use of two sequential rounds of ACT, such as AL followed by 3 days of either artesunate-amodiaquine or dihydroartemisinin-piperaquine, would provide 6 days of treatment with artemisinin with two longer-half-life partner drugs (14, 15). This approach should also be considered a potential strategy should increasing numbers of treatment failures be observed after administration of standard ACT regimens for imported malaria cases in countries where malaria is not endemic. In the majority of cases, ACT retains its efficacy against P. falciparum of African origin, but the cases presented here suggest that this favorable situation may deteriorate in the future.