Bennett, Hayley M

Berriman, Matthew

Chiodini, Peter L

Gkrania-Klotsas, Effrossyni

Kaur, Harparkash

Lansdell, Paul

Lynn, Roderick

Muwanguzi, Julian

Nolder, Debbie

Otto, Thomas D

Patel, Trupti A

Sanders, Mandy

Sutherland, Colin J

Tucker, Julie

van Schalkwyk, Donelly A

English

We present case histories of four patients treated with artemether-lumefantrine for falciparum malaria in UK hospitals in 2015 to 2016. Each subsequently presented with recurrent symptoms and Plasmodium falciparum parasitemia within 6 weeks of treatment with no intervening travel to countries where malaria is endemic. Parasite isolates, all of African origin, harbored variants at some candidate resistance loci. No evidence of pfk13-mediated artemisinin resistance was found. Vigilance for signs of unsatisfactory antimalarial efficacy among imported cases of malaria is recommended

Apollo (Cambridge)

pfk13-Independent Treatment Failure inFour Imported Cases of Plasmodiumfalciparum Malaria Treated withArtemether-Lumefantrine in the UnitedKingdomColin J. Sutherland,a,b Paul Lansdell,a Mandy Sanders,c Julian Muwanguzi,bDonelly A. van Schalkwyk,b Harparkash Kaur,b Debbie Nolder,a Julie Tucker,aHayley M. Bennett,c Thomas D. Otto,c Matthew Berriman,c Trupti A. Patel,dRoderick Lynn,e Effrossyni Gkrania-Klotsas,f Peter L. Chiodinia,dPublic Health England Malaria Reference Laboratory, London School of Hygiene & Tropical Medicine, London,United Kingdoma; Department of Immunology & Infection, Faculty of Infectious & Tropical Diseases, LondonSchool of Hygiene & Tropical Medicine, London, United Kingdomb; Wellcome Trust Sanger Institute, Hinxton,United Kingdomc; Hospital for Tropical Diseases, London, United Kingdomd; Haematology Department,Addenbrooke's Hospital, Cambridge University Hospitals NHS Foundation Trust, Cambridge, United Kingdome;Infectious Diseases Department, Addenbrooke's Hospital, Cambridge University Hospitals NHS FoundationTrust, Cambridge, United KingdomfABSTRACT We present case histories of four patients treated with artemether-lumefantrine for falciparum malaria in UK hospitals in 2015 to 2016. Each subse-quently presented with recurrent symptoms and Plasmodium falciparum parasitemiawithin 6 weeks of treatment with no intervening travel to countries where malaria isendemic. Parasite isolates, all of African origin, harbored variants at some candidateresistance loci. No evidence of pfk13-mediated artemisinin resistance was found. Vig-ilance for signs of unsatisfactory antimalarial efﬁcacy among imported cases of ma-laria is recommended.KEYWORDS imported malaria, treatment failure, parasite genotyping, antimalarialagents, drug resistance mechanismsTreatment of Plasmodium falciparum malaria has beneﬁted from the availability ofartemisinin-based combination therapies (ACT) (1). In Asia, reduced artemisininsusceptibility mediated by speciﬁc mutations in the parasite pfk13 locus may indicatea threat to ACT efﬁcacy (2, 3). However, these speciﬁc pfk13 variants have not been seenin Africa (2–4), where variable ACT efﬁcacy appears to be independent of pfk13polymorphism but may be linked to multilocus genotypes encompassing pfcrt, pfmdr1,pfap2mu, and pfubp1 (5–9).Artemether-lumefantrine (AL) is the recommended treatment for imported cases offalciparum malaria in the UK (1) and is the most widely used ACT in Africa, the originof most UK infections (10). Between October 2015 and February 2016, four cases ofrecurrent P. falciparum malaria in AL-treated patients occurring 17 to 43 days after theoriginal episode were reported to the Malaria Reference Laboratory (MRL) (Table 1) bytwo UK hospitals. Brief case reports and genotyping of parasites isolated from bothepisodes in each patient are presented.This work was undertaken under the statutory remit of the MRL set out by the UKDepartment of Health, which explicitly requires surveillance for evidence of emergingdrug resistance among UK malaria cases, as previously described (10). All tests per-formed were formally requested by physicians directly responsible for patient care. AllReceived 7 November 2016 Returned formodiﬁcation 11 December 2016 Accepted29 December 2016Accepted manuscript posted online 30January 2017Citation Sutherland CJ, Lansdell P, Sanders M,Muwanguzi J, van Schalkwyk DA, Kaur H,Nolder D, Tucker J, Bennett HM, Otto TD,Berriman M, Patel TA, Lynn R, Gkrania-Klotsas E,Chiodini PL. 2017. pfk13-independenttreatment failure in four imported cases ofPlasmodium falciparummalaria treated withartemether-lumefantrine in the UnitedKingdom. Antimicrob Agents Chemother61:e02382-16. https://doi.org/10.1128/AAC.02382-16.Copyright © 2017 Sutherland et al. This is anopen-access article distributed under the termsof the Creative Commons Attribution 4.0International license.Address correspondence to Colin J. Sutherland,Colin.sutherland@lshtm.ac.uk, or DebbieNolder, Debbie.nolder@lshtm.ac.uk.CLINICAL THERAPEUTICScrossmMarch 2017 Volume 61 Issue 3 e02382-16 aac.asm.org 1Antimicrobial Agents and ChemotherapyTABLE1GenotypingdatafromP.falciparumisolatestakenfromprimaryandsecondarysymptomaticmalariaepisodesinfourUKpatientsPatientSexandage(yrs)TravelEpisode1Episode2(elapsedtimesincepreviousepisode)Mutation(s)orhaplotypeWGSfclonalitypfk13bpfcrtcpfmdr1pfap2mupfubp11Maleover65AngolaAugust2015Lys189ThrCVIETTyr184PhedSer160AsnWT43daysLys189ThrCVIETTyr184PheSer160AsnWT2Femaleunder21UgandaJanuary2016WTCVIETWTWTGlu1525Gln28days(cultured)aWTCVIETWTWTGlu1525Gln3Maleover21LiberiaFebruary2016WTGly112GluCVIETCVMNKTyr184Pheins_Lys233,eins_Asn233WT2clones17daysGly112GluCVMNKTyr184Pheins_Lys233WT1clone4Femaleunder21UgandaJanuary2016Lys189ThrCVIETWTins_Asn226,ins_Asn326Thr1500Ile34daysLys189ThrCVIETWTins_Asn226,ins_Asn326Thr1500IleaParasitesfromthesecondmalariaepisodeofpatient2weresuccessfullyestablishedininvitrocultureforestimationofdrugsusceptibility.bThenomenclatureusedshowsthewild-type(WT)(reference)aminoacidencodedatthecodonofinterest,followedbytheaminoacidencodedbytheobservedvariant.Mutationsinthepfk13locuspreviouslyassociatedwithreducedsusceptibilitytoartemisinininvitroandinvivointheMekongregionoccurbetweencodon440andthe3=endofthecodingregion.c Thenomenclatureusedshowsthesingle-letteraminoacidcodeforcodons72to76,stronglyassociatedwithchloroquineresistance.TheCVMNKhaplotypehasbeenassociatedwithALtreatmentfailuresinKenya(5).dThesinglemutationTyrtoPheatcodon184identiﬁestheNFDgenotypeatcodons86,184,and1246inthepfmdr1locus,previouslyassociatedwithALfailureinTanzaniaandKenya(5,8).e ins,insertionofoneaminoacid,relativetothereferencesequence,afterthecodonshown.f Whole-genomesequencingresultsforbothparasiteisolatesfrompatient3.Intheprimaryepisode,heterozygosity(variantallelefrequenciesofbetween5%and95%oftotalreads)wasseeninover4,000loci.About50%ofreadsatinformativepositionscomprisedvariantsalsopresentinthemonoclonalrecrudescentepisode(17dayslater).Sutherland et al. Antimicrobial Agents and ChemotherapyMarch 2017 Volume 61 Issue 3 e02382-16 aac.asm.org 2patient identiﬁers were removed from the data prior to extraction from the PublicHealth England (PHE) MRL database. Other than those required for the primary diag-nosis of malaria, no additional blood samples were taken from patients. Patient 2 gavewritten informed consent for culture adaption of parasites from her blood samples,under UK National Research Ethics Service Approval 07/Q0505/60, granted to C. J.Sutherland.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 parasitemiafollowing travel in Angola. He was treated with AL in the hospital, and all doses wereobserved. The ﬁrst dose was consumed without food, and the patient vomited once 3h later. All but one of the subsequent AL doses were consumed with food, and thepatient was conﬁrmed negative for parasitemia prior to discharge. Symptoms recurredmore than 1 month later, and the patient presented with 0.02% P. falciparum para-sitemia, conﬁrmed by PCR and microscopy at the MRL. The patient responded well toadministration of 4 tablets of 100/250 mg atovaquone/proguanil daily for 3 days. Thepfmdr1 Tyr184Phe and pfap2mu Ser160Asn variants observed have both been associ-ated with recurrent parasitemia in AL-treated African patients (5, 8).Patient 2. A Ugandan female under 21 years of age was admitted to the Hospitalfor Tropical Diseases, London (HTD), with acute P. falciparum hyperparasitemia esti-mated at more than 30% in early 2016 following a visit to Uganda. She responded wellto initial treatment with intravenous (i.v.) artesunate. Parasitemia of 0.07% was re-corded 60 h after admission, and the patient was discharged 3 days later, with arecorded parasitemia level of 0.0001%, on a full course of oral AL. Full compliance wasreported under supervision of staff members at her place of learning. Symptomsrecurred 4 weeks later. The patient was readmitted to HTD with 2.7% P. falciparumparasitemia, received 2 days i.v. artesunate as an inpatient until the parasitemia levelfell 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 LondonSchool of Hygiene & Tropical Medicine (LSHTM), and standard 48-h 50% effectiveconcentration (EC50) estimates were derived for chloroquine (102.6 nM), dihydroarte-misinin (2.8 nM), and lumefantrine (38.7 nM) as previously described (11), with theresults indicating full sensitivity to AL components in vitro. The genotypes of bothparasite 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 associatedwith reduced susceptibility to ACT in vivo in one study (8). The recurrent parasitemiaoccurring 28 days after ﬁrst presentation may reﬂect incomplete clearance of theoriginal hyperparasitemia by the treatment received.Patient 3. A male over 21 years of age presented to CUH with 0.4% P. falciparumparasitemia in early 2016, after travel to Liberia. He was treated with AL and waseventually discharged but returned with recurrent symptoms and 0.02% parasitemia 17days later. He was successfully treated with atovaquone-proguanil. Genotyping iden-tiﬁed mixed genotypes at both pfcrt and pfap2mu loci in the isolate from the primaryepisode but not in that from the recurrent episode (Table 1). DNA extracted fromthe patient after both episodes was sent to the Wellcome Trust Sanger Institute forwhole-genome sequencing (WGS). Preliminary analysis strongly suggests that therecurrent infection which occurred 17 days after AL treatment comprised a singleparasite clone, whereas the original parasite population was multiclonal (Table 1).The recurrent isolate harbored the previously identiﬁed CVMNK and NFD haplotypes ofpfcrt 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 bioanalyticallaboratory of LSHTM conﬁrmed that the AL tablets used to treat this patient (NovartisRiamet [lot XO131; expiration date, December 2017]) met the speciﬁed InternationalPharmacopeia tolerance limits (12).Treatment Failure in Imported Malaria Antimicrobial Agents and ChemotherapyMarch 2017 Volume 61 Issue 3 e02382-16 aac.asm.org 3Patient 4. A female under 21 years of age who presented as febrile to the HTD inearly 2016 following travel to Uganda was diagnosed with 0.08% P. falciparum para-sitemia and treated with oral AL. The patient was discharged to complete the treatmentcourse at home and reported full compliance with all six doses. She experiencedrecurrent symptoms 1 month later, presenting to HTD with conﬁrmed 0.3% P. falcip-arum parasitemia, and was successfully treated with 7 days of oral quinine (10 mg/kgof body weight every 8 h) and doxycycline (200 mg daily). Data from the two episodesappeared identical at the parasite loci evaluated. Novel mutations at codons 226 and326 of pfap2mu and codon 1500 of pfubp1 are of unknown signiﬁcance, but, as this isclearly a recrudescent parasite genotype, both variants should be further evaluated infuture studies.Treatment failure cannot be unequivocally ascribed to parasite resistance in these fourpatients, although three harbored parasites with variant alleles of loci previously linked toreduced susceptibility to artemisinin or lumefantrine (Table 1). Our ﬁndings would havebeen strengthened by observing treatment for all patients to ensure full regimen compli-ance, and by measurement of lumefantrine blood levels at day 7, to rule out any problemswith malabsorption (1), as this can result in failure to clear parasites (13). None of the fourpatients harbored mutations in the propeller-encoding domain of the pfk13 locus and thusdid 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 haveevolved pfk13-independent mechanisms for posttreatment survival in vivo. This suggeststhat, in countries where malaria is not endemic and in which imported malaria cases fromAfrica are seen, vigilance is required to protect the efﬁcacy of our ﬁrst-line ACT. Studies incountries where malaria is endemic are required to further validate potential geneticmarkers for monitoring in African parasite populations.Artemisinins are very quickly metabolized and cleared from circulation, such thatonly a single full parasite life cycle is exposed to the highly potent action of thesecompounds under conditions of a 3-day regimen. The current practice of 3 days oftreatment may therefore be insufﬁcient to guarantee clearance of parasite genotypesunder ACT pressure for a decade in many parts of Africa (14). The use of two sequentialrounds of ACT, such as AL followed by 3 days of either artesunate-amodiaquine ordihydroartemisinin-piperaquine, would provide 6 days of treatment with artemisininwith two longer-half-life partner drugs (14, 15). This approach should also be consid-ered a potential strategy should increasing numbers of treatment failures be observedafter administration of standard ACT regimens for imported malaria cases in countrieswhere malaria is not endemic. In the majority of cases, ACT retains its efﬁcacy againstP. falciparum of African origin, but the cases presented here suggest that this favorablesituation may deteriorate in the future.ACKNOWLEDGMENTSP.L.C. is supported by the National Institute for Health Research University CollegeLondon Hospitals Biomedical Research Centre. All of us declare that we have noconﬂicting interests. This work was supported by funding from Public Health Englandto the Malaria Reference Laboratory at LSHTM. Genome sequencing was funded byWellcome Trust grant WT089051 to the Sanger Institute.REFERENCES1. Lalloo DG, Shingadia D, Bell DJ, Beeching NJ, Whitty CJ, Chiodini PL; PHEAdvisory Committee on Malaria Prevention in UK Travellers. 2016. UKmalaria treatment guidelines. J Infect 72:635–649. https://doi.org/10.1016/j.jinf.2016.02.001.2. Ashley EA, Dhorda M, Fairhurst RM, Amaratunga C, Lim P, Suon S, SrengS, Anderson JM, Mao S, Sam B, Sopha C, Chuor CM, Nguon C, Sovann-aroth S, Pukrittayakamee S, Jittamala P, Chotivanich K, Chutasmit K,Suchatsoonthorn C, Runcharoen R, Hien TT, Thuy-Nhien NT, Thanh NV,Phu NH, Htut Y, Han KT, Aye KH, Mokuolu OA, Olaosebikan RR, FolaranmiOO, Mayxay M, Khanthavong M, Hongvanthong B, Newton PN, Onyam-boko MA, Fanello CI, Tshefu AK, Mishra N, Valecha N, Phyo AP, Nosten F,Yi P, Tripura R, Borrmann S, Bashraheil M, Peshu J, Faiz MA, Ghose A,Hossain MA, Samad R, et al. 2014. Spread of artemisinin resistance inPlasmodium falciparum malaria. 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pfk13-Independent Treatment Failure in Four Imported Cases of Plasmodium falciparum Malaria Treated with Artemether-Lumefantrine in the United Kingdom.

https://www.repository.cam.ac.uk/bitstream/1810/294939/1/pfk13-Independent%20Treatment%20Failure%20in%20Four%20Imported%20Cases%20of%20Plasmodium%20falciparum%20Malaria%20Treated%20with%20Artemether-Lumefantrine%20in%20the%20United%20Kingdom.pdf

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

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

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