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resistensi Dihydroartemisinin-piperaquine di Plasmodium falciparum malaria di Kamboja:


studi prospektif kohort multisite

Chanaki Amaratunga *, Pharath Lim *, Seila Suon, Sokunthea Sreng, Sivanna Mao, Chantha Sopha, Baramey Sam, Dalin Dek, Vorleak Coba, Roberto Amato, Daniel
Blessborn, Lijiang Lagu, Gregory S Tullo, Michael P Fay, Jennifer M Anderson, joel Tarning, Rick M Fairhurst

Ringkasan
Lancet Infect Dis 2016
Latar Belakang resistensi artemisinin di Plasmodium falciparum mengancam untuk mengurangi keampuhan e FFI terapi kombinasi artemisinin (ACT), sehingga
mengurangi global yang e ff orts untuk menghilangkan malaria. kegagalan pengobatan baru-baru ini dengan dihydroartemisinin-piperaquine, saat pertama-line ACT di Diterbitkan On line 7 Januari

2016 http://dx.doi.org/10.1016/
Kamboja, menunjukkan bahwa resistensi piperaquine dapat muncul di negeri ini. Kami menjelajahi hubungan antara resistensi artemisinin dan kegagalan piperaquine
dihydroartemisinin-, dan berusaha untuk con fi rm kehadiran piperaquine-tahan P falciparum infeksi di Kamboja.
S1473-3099 (15) 00.487-9 See Secara
online / Komentar
http://dx.doi.org/10.1016/

S1473-3099 (15) 00.525-3 *


metode Dalam penelitian kohort prospektif ini, kami terdaftar pasien berusia 2-65 tahun dengan tidak rumit P falciparum Kontribusi sama

malaria di tiga provinsi Kamboja: Pursat, Preah Vihear, dan Ratanakiri. Peserta diberi kursus 3 hari standar dihydroartemisinin-piperaquine. parasit Laboratorium Malaria dan Penelitian Vektor ( C

darah kepadatan perifer diukur sampai parasit dibersihkan dan kemudian mingguan untuk 63 hari. Hasil utama adalah yg timbul P falciparum Amaratunga PhD, P Lim PhD, GS Tullo BS, JM
Anderson PhD, RM Fairhurst PhD), dan
parasitemia dalam 63 hari. Kami mengukur konsentrasi piperaquine plasma pada awal, 7 hari, dan hari luapan baru. Kami menilai fenotipik dan biostatistik Penelitian Cabang

spidol genotipe resistensi obat pada isolat parasit. Penelitian ini terdaftar dengan ClinicalTrials.gov, nomor NCT01736319.

(MP Fay PhD), Institut Nasional Alergi dan


Penyakit Infeksi, National Institutes of
Health, Rockville, MD, USA; Pusat Nasional
temuan Antara 4 September 2012, dan 31 Desember 2013, kami terdaftar 241 peserta. Di Pursat, di mana resistensi artemisinin sudah mengakar, 37 (46%) dari 81 pasien untuk Parasitologi, Entomologi, dan Malaria
memiliki parasit luapan baru. Di Preah Vihear, di mana resistensi artemisinin yang muncul, sepuluh (16%) dari 63 pasien memiliki luapan baru dan di Ratanakiri, di mana Control, Phnom Penh, Kamboja

resistensi artemisinin jarang, satu (2%) dari 60 pasien lakukan. Pasien dengan yg timbul P falciparum infeksi lebih mungkin untuk memiliki terdeteksi piperaquine
konsentrasi plasma pada awal dibandingkan dengan pasien non-yg timbul, tapi tidak di ff er secara
(P Lim, S Suon MD, S Sreng, D Dek BA, V

signifikan di usia, kepadatan parasit awal, atau piperaquine konsentrasi plasma pada 7 hari. parasit yg timbul memiliki prevalensi lebih tinggi dari kelch13 mutasi,
Coba BA); Rumah Sakit Rujukan Sampov
piperaquine lebih tinggi 50% konsentrasi penghambatan (IC nilai-nilai, dan menurunkan saya IC fl oquine nilai-nilai; tidak memiliki beberapa pfmdr1 eksemplar,
50) 50
Meas, Pursat, Kamboja ( S Mao MD); Rumah

penanda genetik saya fl oquine perlawanan. Sakit Rujukan Makara 16, Preah Vihear,
Kamboja ( C Sopha MD);

Rumah Sakit Rujukan Ratanakiri,

Interpretasi kegagalan Dihydroartemisinin-piperaquine disebabkan oleh kedua artemisinin dan piperaquine perlawanan, dan umumnya terjadi di tempat-tempat Ratanakiri, Kamboja

(B Sam MD); Wellcome Trust Sanger


dihydroartemisinin-piperaquine telah digunakan di sektor swasta. Di Kamboja, artesunat ditambah saya fl oquine mungkin menjadi pilihan yang layak untuk mengobati
Institute, Hinxton, UK
kegagalan dihydroartemisinin-piperaquine, dan lebih e ff efektif pertama-line ACT di daerah di mana kegagalan dihydroartemisinin-piperaquine yang umum.
(R Amato PhD); Medical Research Council
Penggunaan primakuin dosis rendah tunggal untuk menghilangkan beredar gametosit diperlukan di daerah di mana artemisinin dan ACT resistensi adalah lazim. Pusat Genomics dan Kesehatan Global ( R

Amato), Pusat Kedokteran Tropis dan

Kesehatan Global, Nuffield Department of

Medicine ( D Blessborn PhD, L Lagu PhD, J

Tarning PhD),
pendanaan Institut Nasional Alergi dan Penyakit Infeksi.
University of Oxford, Oxford, UK; dan

pengantar resistensi artemisinin di Kamboja 7-11 dan di seluruh daratan Asia Tenggara 10-12 mengancam
Mahidol-Oxford Tropical Medicine

Research Unit Fakultas Kedokteran


Terapi-kombinasi artemisinin penggunaan ampuh, short-acting artemisinin e FFI keampuhan dari dihydroartemisinin-piperaquine dan semua terapi
Tropis, Universitas Mahidol, Bangkok,
dan kurang kuat, long-acting obat-adalah mitra direkomendasikan di kombinasi artemisinin lainnya. 13 Bahaya ini timbul karena sebagai lebih Thailand

seluruh dunia untuk pengobatan Plasmodium falciparum malaria. Dihydroartemisininparasitmenjadi-piperaquine,resisten terhadap artemisinin, lebih parasit perlu
1 (D Blessborn, L Lagu, J Tarning)

salah satu dari beberapa terapi kombinasi artemisinin masih e ff efektif dihilangkan dengan obat mitra tunggal; Oleh karena itu, mereka lebih Korespondensi ke: Dr Rick M Fairhurst,
Laboratorium Malaria dan Vector
melawan multidrugresistant P falciparum di Asia tenggara, diadopsi mungkin untuk secara spontan mengembangkan resistensi genetik
Penelitian, Institut Nasional Alergi dan
sebagai lini pertama pengobatan antimalaria di Kamboja pada tahun terhadap obat mitra piperaquine dan lainnya. bukti awal untuk
Penyakit Infeksi, National Institutes of
2008. Beberapa studi sebelumnya 2-4 mendokumentasikan keamanan yang pengembangan ini telah disediakan oleh tiga studi yang menunjukkan Health, 12735 Twinbrook Parkway,

sangat baik dan tolerabilitas dihydroartemisinin-piperaquine di Kamboja, penurunan e FFI keampuhan dari dihydroartemisinin-piperaquine Room 3E-10A, Rockville, MD 20.852,

serta e FFI keampuhan dari 96-98% setelah 28 hari atau 63 hari di USA

provinsi Kamboja Oddar Meancheay, Siem Reap, Pursat, dan Kratie. 3-6 Namun, segera setelah -nya
penyebaran cepat penyebaran luas di Kamboja barat. Dalam sebuah penelitian
rfairhurst@niaid.nih.gov
2008-10, 14 e FFI keampuhan dari dihydroartemisinin-
piperaquine setelah 42 hari adalah 75% di Pailin dan 89% di

www.thelancet.com/infection Dipublikasikan secara online 7 Januari 2016 http://dx.doi.org/10.1016/S1473-3099(15)00487-9 1


Artikel

Panel: Penelitian dalam konteks

Bukti sebelum penelitian ini usia pasien, kepadatan parasit awal yang lebih tinggi, atau piperaquine tinggi konsentrasi

Kami mencari PubMed menggunakan istilah “dihydroartemisinin”, “piperaquine”, “e FFI plasma pada 7 hari. Sebaliknya, parasit yg timbul memiliki lebih banyak kelch13 mutasi dan IC

keampuhan”, dan “Kamboja” tanpa tanggal atau bahasa pembatasan pada 5 Juni 2015. Kami piperaquine tinggi 50 nilai-nilai, menunjukkan bahwa kegagalan dihydroartemisinin-piperaquine

teridentifikasi 13 artikel, enam di antaranya adalah uji klinis asli dari e FFI keampuhan dari adalah karena kedua artemisinin dan resistance piperaquine. Parasit yg timbul juga telah

dihydroartemisinin-piperaquine untuk pengobatan tanpa komplikasi mengurangi saya fl oquine IC 50 nilai-nilai dan beberapa salinan kurangnya pfmdr1, penanda
genetik untuk saya fl oquine perlawanan.

Plasmodium falciparum malaria di Kamboja. Tiga studi 2001-05 menunjukkan bahwa e FFI

keampuhan adalah 96-98% sebelum dihydroartemisinin-piperaquine secara luas digunakan.

Tiga studi kemudian melaporkan berkurang e FFI keampuhan (46-89%) di 2008-13, setelah
Implikasi dari semua bukti yang tersedia Dihydroartemisinin-piperaquine gagal cepat
dihydroartemisinin-piperaquine menjadi banyak digunakan. kegagalan pengobatan telah
di empat provinsi Kamboja barat (Pailin, Pursat, Oddar Meanchey, dan Preah Vihear),
dikaitkan dengan parasit kelch13 mutasi, yang berhubungan dengan resistensi artemisinin.
dan berhubungan dengan resistensi parasit untuk kedua turunan artemisinin dan
Semua tiga dari penelitian kemudian tidak menemukan hubungan antara kegagalan
piperaquine. Bukti perlawanan piperaquine di P falciparum harus segera e ff orts
pengobatan dan piperaquine tinggi in-vitro IC 50 nilai-nilai (ukuran parasit kerentanan terhadap
untuk memetakan fenotipe ini di Kamboja dan negara-negara Asia tenggara lainnya,
piperaquine). Peran resistensi piperaquine in-vivo kegagalan pengobatan dinilai belum
untuk menjelaskan mekanisme molekuler, dan menemukan obat baru yang
memadai.
menghindari resistensi piperaquine. Artesunat ditambah saya fl oquine harus diuji
sebagai fi terapi pertama-line di mana kegagalan dihydroartemisinin-piperaquine
telah didokumentasikan, dan juga sebagai pengobatan penyelamatan atas kegagalan

nilai tambah penelitian ini dihydroartemisinin-piperaquine di Kamboja. uji klinis harus dilakukan dari rejimen
tiga-obat dihydroartemisinin-piperaquine ditambah saya fl oquine.
Temuan kami menunjukkan bahwa pengobatan dihydroartemisinin-piperaquine
gagal di Pursat dan Preah Vihear, di mana resistensi artemisinin adalah lazim,
namun tetap sangat e FFI cacious di Ratanakiri mana resistensi artemisinin jarang
terjadi. kegagalan pengobatan tidak dikaitkan dengan yang lebih tua

Pursat, tapi 100% di Preah Vihear dan Ratanakiri di utara dan timur kegagalan dihydroartemisinin-piperaquine, mengidentifikasi dan memvalidasi
Kamboja. Karena kegagalan dihydroartemisinin-piperaquine ditemukan penanda genetik untuk digunakan dalam program pengawasan besar, dan
tidak terkait dengan piperaquine 50% konsentrasi penghambatan (IC 50) mempelajari mekanisme molekuler nya. Kami melakukan studi kohort untuk
dalam penelitian ini, dan konsentrasi piperaquine plasma pada 7 hari mengidentifikasi piperaquine tahan

tidak diukur, resistensi piperaquine di Pailin dan Pursat tidak bisa con fi P falciparum infeksi di Kamboja. Kami menduga bahwa infeksi
rmed. Munculnya resistensi piperaquine juga di FFI kultus untuk tersebut akan dikaitkan dengan resistensi artemisinin, 19 kegagalan
berdamai dengan penurunan bersamaan di IC piperaquine 50 nilai-nilai dihydroartemisinin-piperaquine, paparan piperaquine memadai,
di Pailin dan Pursat. 14 dan menurun
kerentanan P falciparum isolat untuk piperaquine in vitro. Kami juga
Dalam sebuah penelitian tahun 2013, 15,16 e FFI keampuhan dari mendalilkan bahwa piperaquine dihydroartemisinin- akan gagal lebih
dihydroartemisinin-piperaquine setelah 42 hari di sering di daerah di mana resistensi artemisinin adalah lazim dari mana
Oddar Meancheay adalah 46%. Meskipun pasien dengan luapan baru atau ia muncul. Oleh karena itu kami membandingkan keampuhan e FFI dari
penyembuhan harus eksposur mirip dengan piperaquine dalam penelitian ini, IC dihydroartemisinin-piperaquine untuk pengobatan tanpa komplikasi P
piperaquine 50 nilai untuk parasit yg timbul tidak lebih tinggi dari orang-orang falciparum malaria di Pursat, Preah Vihear, dan Ratanakiri, di mana
untuk parasit non-yg timbul. Mengingat hasil ini, resistensi piperaquine di prevalensi kelch13
provinsi ini juga tidak bisa con fi rmed. Dalam sebuah penelitian 2011-13, 17

mutasi-penanda genetik untuk ketahanan artemisinin di Kamboja dan tempat


proporsi infeksi yg timbul oleh 42 hari setelah dihidro artemisinin-piperaquine lain di Asia tenggara 9,10 -were 76%, 21%, dan 4%, masing-masing, di 2011-12. 10
mengobati ment lebih tinggi di Kamboja barat (15%) dibandingkan di Kamboja Kami juga membandingkan prevalensi kelch13 mutasi, konsentrasi piperaquine
timur (3%). Pasien dengan luapan baru atau penyembuhan dalam penelitian ini plasma setelah 7 hari, dan IC piperaquine in-vitro 50 nilai antara non-yg timbul
memiliki eksposur serupa dengan piperaquine dan parasit dilakukan dengan IC dan infeksi yg timbul
piperaquine yang sama 50 nilai-nilai. Dalam pandangan temuan tersebut dan untuk menyelidiki adanya parasit piperaquine-tahan.
kurangnya penanda genetik, resistensi piperaquine di
Kamboja barat belum con fi rmed, meskipun meningkatkan IC piperaquine 50

nilai-nilai
di Kamboja utara menyarankan bahwa mungkin akan muncul. 18
metode
Studi desain dan peserta
Kurangnya bukti yang jelas resistensi piperaquine di Kamboja menghalangi Untuk calon ini penelitian kohort, kami merekrut pasien dari rumah sakit
e ff orts untuk mendefinisikan perannya dalam rujukan provinsi dan kesehatan kabupaten

2 www.thelancet.com/infection Dipublikasikan secara online 7 Januari 2016 http://dx.doi.org/10.1016/S1473-3099(15)00487-9


Artikel

pusat di Pursat, Preah Vihear, dan provinsi Ratanakiri, Kamboja. dua tablet anak; 9-10 kg, tiga tablet anak; 11-20 kg, satu tablet dewasa;
Pasien yang memenuhi syarat jika mereka berusia 2-65 tahun dan 21-30 kg, dua tablet dewasa; 31-40 kg, tiga tablet dewasa;
memiliki akut, tidak rumit P falciparum > 40 kg, empat tablet dewasa) per rekomendasi pabrik. Pasien
malaria (tidak termasuk infeksi campuran dengan spesies non- kemudian dipantau setiap hari untuk resolusi demam dan pembersihan
falciparum), kepadatan parasit tidak lebih dari 200 000 parasit per uL, parasitemia. Pasien yang dikembangkan P vivax Infeksi (dengan atau
dan demam (suhu timpani ≥37 · 5 ° C) atau demam dalam 24 jam. tanpa gejala malaria) yang berjarak 63 hari dirawat dengan tablet Duo-
Kriteria eksklusi utama adalah pengobatan gejala hadir dengan Cotecxin seperti dijelaskan di atas. sampel plasma diangkut di atas es
antimalaria pada minggu sebelumnya, kehamilan atau menyusui, dan kering untuk Departemen Farmakologi Klinik, Mahidol-Oxford Unit
hematokrit <25%. Penelitian Kedokteran Tropis di Bangkok, Thailand. Laboratorium
terakreditasi sesuai dengan ISO15189 dan ISO15190, dan berpartisipasi
protokol ini disetujui oleh Komite Kamboja Nasional Etik dalam WorldWide antimalaria Perlawanan Jaringan kontrol kualitas dan
Penelitian Kesehatan dan Institut Nasional Alergi dan Penyakit jaminan pro fi program pengujian efisiensi. 20 Konsentrasi Piperaquine
Infeksi review kelembagaan dewan. Pasien atau orang tua dari diukur dengan metode divalidasi. 21 For more on the WorldWide

anak-anak muda dari 18 tahun diberikan persetujuan tertulis. Antimalarial Resistance Network see
http://www. wwarn.org/toolkit/qaqc

Prosedur sampel kontrol kualitas (4 · 5 ng / mL, 20 ng / mL, dan 400 ng / mL)


Pasien dirawat di rumah sakit untuk perawatan diawasi dan menunjukkan intra-hari dan antar-hari variabilities di bawah 10% selama
pemantauan untuk resolusi parasitemia. Tepat sebelum pemberian pengukuran obat sampel penelitian. Batas bawah kuantifikasi (LLOQ) adalah
dosis pertama pengobatan pada 0 jam, kepadatan parasit awal diukur 1 · 5 ng / mL; batas bawah deteksi (LLOD) adalah 0 · 375 ng / mL. Nilai di
dalam LMS darah fi tebal. Semua pasien kemudian dirawat di 0 h, 24 bawah batas-batas ini diperhitungkan sebagai LLOQ / 2 atau LLOD / 2,
jam, dan 48 jam dengan tablet Duo-Cotecxin (Holley Farmasi, Beijing, masing-masing, sebelum analisis statistik. Kami melakukan genotipe untuk

Cina), masing-masing berisi dihydroartemisinin 40 mg dan piperaquine pfmdr1 dan X5R menyalin nomor 22

320 mg, menurut berat badan (<10 kg,


setengah tablet ; 10-19 kg, satu tablet; 20-29 kg, satu setengah dan kelch13 baling-baling dan pfcrt mutasi. 23 Dalam 168 sampel yang
tablet, 30-39 kg, dua tablet; ≥40 kg, tiga tablet) per rekomendasi kelch13 genotipe tidak tersedia, domain baling-baling kelch13 itu penguat
manu facturer ini. ed oleh bersarang PCR dengan yang dijelaskan sebelumnya 9 primer (K13-1
maju 5 '- cggagtgaccaaatctggga-3 '
dan K13-4 membalikkan

Untuk pasien dengan kepadatan parasit dari 10 000 situs para per uL 5 '- gggaatctggtggtaacagc-3 ' untuk reaksi utama, dan K13-N1 maju 5 '- gccaagctgccattcatttg-
3 ' dan K13-N1 membalikkan 5 '- gccttgttgaaagaagcaga-3 '
atau lebih di screening, kami mengukur kepadatan parasit pada 0 jam,
untuk sekunder
2 jam, 4 jam, 6 jam, 8 jam, 12 jam, dan setiap 6 jam setelah itu sampai
tiga darah berturut-turut LMS fi tidak menunjukkan parasitemia (yaitu, reaksi), dengan beberapa kation modi fi untuk PCR kondisi. 1 uL DNA adalah
penguat ed dengan 0 · 2 umol / L masing-masing primer, 0 · trifosfat 2
tidak ada parasit cincin-tahap yang diamati setelah 500 leukosit
mmol / L deoxynucleoside (Bioline USA; Taunton, MA, USA), 1 · 6 mmol / L
diperiksa dengan mikroskop). Untuk pasien dengan kepadatan parasit
MgCl
awal kurang dari 10 000 parasit per uL, kami mengukur kepadatan
2,

parasit setiap 24 jam sampai satu darah fi lm tidak menunjukkan dan 0 · 25 U PerfectTaqTM DNA polimerase (5 PRIME; Gaithersburg,

parasitemia. Pada 7 hari dan kemudian mingguan untuk 63 hari, kami MD, USA) sesuai dengan program bersepeda berikut: 4 menit pada 94 °
C, 35 siklus 30 s pada 94 ° C, 1 menit pada 58 ° C, 1 menit pada 72 ° C,
mengukur suhu tubuh, gejala malaria Ulasan, dan mengambil sampel
dan 4 menit pada 72 ° C. Untuk PCR nested, 1 · 5 uL produk PCR primer
darah jari-tusukan ke layar untuk parasitemia berulang menggunakan
penguat ed bawah kondisi yang sama, kecuali dengan 1 · 2 mmol / L
tes diagnostik cepat (First Response; Premier Medical Corporation,
MgCl 2 dan 0 · 375 U PerfectTaq, dan anil selama 1 menit pada 60 ° C.
Nani Daman, India ) dan mikroskop. kepadatan parasit diukur dalam
produk PCR yang dimurnikan dari 2% gel agarose dan diurutkan oleh
sampel dengan parasitemia terdeteksi.
Macrogen (Rockville, MD, USA). Urutan dianalisis dengan DNASTAR
Lasergene. Itu kelch13 urutan garis parasit 3D7 digunakan sebagai

referensi (nomor aksesi


XM_001350122.1) untuk menemukan tunggal nucleotide
Pasien yang dikembangkan asimtomatik P falciparum parasitemia
polymorphisms in clinical isolates. For recurrent
atau rumit P falciparum malaria (dengan atau tanpa co-insiden Plasmodium
infections, PCR genotyping was done, with msp1, msp2,
vivax parasitemia) dalam 63 hari dirawat di rumah sakit untuk pengobatan
and glurp as genetic markers to distinguish recrudescence from a newly acquired
oral diawasi pada 0 jam, 24 jam, dan 48 jam dengan artesunat (4 mg / kg;
infection. 24 In brief, DNA samples extracted from 200 μL of whole blood were
Guilin Farmasi, Shanghai, Cina) ditambah tablet Malarone (GlaxoSmithKlein;
assessed for polymorphisms in these genes by nested PCR. 25 Genomic DNA
Hanover, PA, USA ), masing-masing berisi atovakuon 250 mg dan
samples from the HB3 and 3D7 parasite lines were used as controls. According to
proguanil 100 mg (di tablet dewasa) atau atovaquone 62 · 5 mg dan
WHO recommendations, 26
proguanil 25 mg (di tablet anak), menurut berat badan (5-8 kg,

www.thelancet.com/infection Dipublikasikan secara online 7 Januari 2016 http://dx.doi.org/10.1016/S1473-3099(15)00487-9 3


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recurrent episodes were classifi ed as recrudescences if all terikat jenis familywise tingkat saya kesalahan; 32 oleh karena itu, tidak ada
msp1, msp2, and glurp alel hadir pada saat kambuh juga hadir sebelum kebutuhan untuk menyesuaikan nilai p. analisis survival mendekati waktu untuk
pengobatan. Dalam semua kasus lain, mereka dianggap infeksi baru. kambuh atau menyensor pada saat pengambilan sampel darah dan
Kami melakukan in-vitro pengujian kerentanan obat untuk parasit baru menggunakan perkiraan Kaplan-Meier dan uji log-rank (Mantel-Cox) (GraphPad
yang diperoleh dari peserta dengan cara metode 72-h standar Prism 6). Untuk analisis survival PCR-dikoreksi, reinfections dan mereka yang kita
menggunakan SYBR Hijau I noda. 22 Kami IC dihitung 50 nilai-nilai dengan tidak dapat mengklasifikasikan sebagai luapan baru atau reinfeksi disensor. Untuk
menggunakan software IVART 27 untuk muat data konsentrasi- membandingkan IC piperaquine 50 nilai dengan yang sesuai konsentrasi plasma,
hambatan. standar obat antimalaria disediakan oleh WorldWide kami menggunakan dipasangkan
antimalaria Perlawanan Jaringan, kecuali untuk piperaquine (Sigma;
Steinheim, Jerman). t menguji CI pada nilai-nilai log-berubah. Untuk membandingkan IC
piperaquine 50 nilai untuk dipasangkan isolat awal dan yg timbul, kami
menggunakan uji Wilcoxon signed-rank (GraphPad Prism 6). Parasit
izin paruh adalah ukuran tingkat parasit izin berasal dari segmen linier
hasil dari kurva log parasitemia-waktu (izin parasit paruh = log e 2 dibagi
Hasil primer adalah P falciparum luapan baru dalam 63 hari dari mulai dengan tingkat clearance parasit). Kami dianggap nilai p kurang dari 0 ·
pengobatan dihydroartemisinin-piperaquine. Hasil sekunder adalah 05 sebagai signifikan.
piperaquine konsentrasi plasma pada 7 hari dan hari luapan baru;
parasit izin paruh; 28-30 proporsi pasien dengan parasit izin paruh lebih
dari 5 jam; 10 dan proporsi pasien dengan parasitemia terdeteksi oleh Peran sumber pendanaan
mikroskop pada 72 h. 31
Penyandang dana penelitian tidak memiliki peran dalam desain penelitian, pengumpulan data,

analisis data, interpretasi data, atau penulisan laporan. Penulis yang sesuai memiliki akses penuh

ke semua data dalam penelitian dan memiliki tanggung jawab nal fi untuk keputusan untuk

mengirimkan untuk publikasi.

Analisis statistik
Untuk menganalisis data kategori, kami menggunakan uji Fisher (R versi
3.1.2). Untuk data kuantitatif, kami menggunakan uji Kruskal-Wallis (untuk hasil
membandingkan tiga situs) atau Mann-Whitney Antara 4 September 2012, dan 31 Desember 2013, kami disaring 6209 duals
uji (untuk perbandingan dua situs; indivi menyajikan dengan gejala yang konsisten dengan malaria untuk
GraphPad Prism 6). Jika tes keseluruhan antara ketiga situs itu kelayakan (tabel 1). Kami terdaftar 241 (tabel 2). Kebanyakan pasien adalah
signifikan, hal ini diikuti oleh tiga tes membandingkan pasang situs. laki-laki dan usia rata-rata adalah 24 tahun. Sebagian besar pasien di Pursat
Ketika empat tes ini diterapkan dengan cara ini dengan tingkat fi dan Preah Vihear adalah laki-laki, lebih tua, dan memiliki berat badan lebih
signifikansi yang sama, tidak ada penyesuaian untuk beberapa besar daripada mereka yang Ratanakiri (tabel 2). hematokrit rata-rata
parisons com perlu adalah 39% dan secara signifikan lebih tinggi pada pasien di Pursat
daripada di Ratanakiri. Kepadatan parasit rata-rata adalah 12 249 sel per uL,

PreRatanakirihVihear
dan tidak di ff er antara tiga situs. 11% pasien memiliki gametocytaemia saat
Pursat
pendaftaran, dengan secara signifikan lebih di Pursat daripada di Ratanakiri

disaring 3063 1580 1566 (tabel 2). pasien Lainnya di Pursat memiliki konsentrasi piperaquine

Negatif 2485 1337


terdeteksi dan lebih tinggi daripada mereka yang Ratanakiri. Konsentrasi
1351
piperaquine relatif di tiga lokasi sejajar dengan jumlah pasien dikeluarkan
Positif 578 229 229
dari penelitian kami karena sebelumnya menggunakan terapi kombinasi
pv positif 280 141 58
artemisinin di sektor swasta (Tabel 1). 29 pasien disensor dalam analisis
Pv dan Pf positif 63 11 35
survival karena mereka mangkir-up (n = 18), menarik diri dari penelitian (n =
Pf positif 235 77 136
2), atau dikembangkan P vivax parasitemia antara 42 hari dan 63 hari yang
Sebelumnya ACT penggunaan 59 0 7
diperlukan penafsiran dengan dihydroartemisinin-piperaquine (n = 9; tabel
malaria berat 3 4 8
3). Dari 29 pasien tersebut, 23 berasal dari Pursat, mencerminkan insiden
Hematokrit <25% 4 1 2
yang lebih tinggi dari P vivax malaria dan emigrasi dari provinsi ini selama
Hamil 2 0 1
penelitian. Di antara 212 pasien yang diikuti sampai dengan 63 hari, proporsi
Menyusui 1 0 2
mereka yang berulang P falciparum Infeksi di ff ered secara signifikan oleh
Menolak 4 1 12
situs, dengan yang paling di Pursat dan sedikit di Ratanakiri (tabel 3). infeksi
Pf density >200 000 parasites per μL 20 6 4
berulang yang terdeteksi antara 14 hari dan 63 hari (median 28 hari).
Previous enrolment 2 0 0

Cannot follow up 30 0 34

Enrolled 110 65 66

Pv= Plasmodium vivax. Pf= Plasmodium falciparum. ACT=artemisinin combination therapy.

Table 1: Characteristics of screened patients

4 www.thelancet.com/infection Published online January 7, 2016 http://dx.doi.org/10.1016/S1473-3099(15)00487-9


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All sites Pursat Preah Vihear Ratanakiri p value

Patients (n) 241 110 65 66

Male participants (n, %) 183 (76%) 93 (85%) 51 (78%) 39 (59%) 0·0008*

Median age (IQR; years) 24 (18–32) 24 (19·75–33) 28 (20–33) 19 (11·75–31) 0·0005†

Weight (kg) 48 (14) 51 (9) 50 (13) 39 (16) <0·0001†

Haematocrit (%) 39·15 (4·95) 39·66 (5·06) 39·55 (4·29) 37·88 (5·22) 0·037†

Median parasite density (IQR; parasites per μL) 12 249 (2042–43 893) 11 159 (2260–40 074) 15 212 (1694–47 180) 12 504 (1859–51 926) 0·65†

Gametocytaemia at 0 h (n, %)‡ 26 (11%) 19 (17%) 6 (9%) 1 (2%) 0·0022*

Median gametocyte density (IQR; 32 (16–99) 32 (32–95) 16 (15·75–107) 1163 (1163–1163) 0·20§
gametocytes per μL)

Detectable piperaquine at 0 h (n, %)¶ 97 (40%) 70 (64%) 9 (14%) 18 (27%) <0·0001*

Piperaquine plasma concentration (ng/mL) 8·05 (22·95) 14·43 (29·38) 1·53 (5·20) 3·79 (18·37) <0·0001†

Data are mean (SD) unless otherwise stated. p values in the table are for diff erence between all three sites. *Calculated with Fisher’s exact test. The proportion of males was signifi cantly lower in Ratanakiri than in
Pursat (p<0·001) and than in Preah Vihear (p=0·023), with no signifi cant diff erence between Pursat and Preah Vihear (p=0·31); gametocytaemia was more common in Pursat than in Ratanakiri (p=0·0010), and with no
signifi cant diff erence between Preah Vihear and Ratanakiri (p=0·062) or between Pursat and Preah Vihear (p=0·18); and detectable piperaquine concentration before treatment was more common in Pursat than in
Preah Vihear (p<0·0001) and than in Ratanakiri (p<0·0001), with no signifi cant diff erence between Ratanakiri and Preah Vihear (p=0·083). †Calculated with the Kruskal-Wallis test; Mann-Whitney tests indicate that: age
was lower in Ratanakiri than in Pursat (p=0·0005) and Preah Vihear (p=0·0023), with no signifi cant diff erence between Pursat and Preah Vihear (p=0·71); weight was lower in Ratanakiri than in Pursat (p<0·0001) and
Preah Vihear (p<0·0001), with no signifi cant diff erence between Pursat and Preah Vihear (p=0·63); haematocrit was higher in Pursat than in Ratanakiri (p=0·012), with no signifi cant diff erence between Pursat and
Preah Vihear (p=0·52) or between Ratanakiri and Preah Vihear (p=0·075); and piperaquine concentration was lower in Ratanakiri than in Pursat (p<0·0001) and lower in Pursat than in Preah Vihear (p<0·0001), with no
signifi cant diff erence between Ratanakiri and Preah Vihear (p=0·083). ‡One patient in Pursat (at 24 h) and three patients in Preah Vihear (at 24 h, 72 h, and 78 h) developed gametocytaemia. §Calculated with the
Mann-Whitney test. ¶Piperaquine plasma concentration at enrolment was not measured for one patient in Ratanakiri.

Table 2: Baseline characteristics of patients

All sites Pursat Preah Vihear Ratanakiri p value

Patients (n) 241 110 65 66

Piperaquine plasma concentration at 7 days 67·34 (51·84) 71·63 (53·56) 73·00 (51·70) 54·98 (47·91) 0·0059†
(ng/mL)*

Dose-normalised piperaquine plasma 3·58 (2·70) 3·86 (2·86) 3·96 (2·81) 2·77 (2·15) 0·0013†
concentration at day 7 (ng/mL per dose)

Recurrent Plasmodium falciparum infection by day 63 56/212 (26%) 43/87 (49%) 11/64 (17%) 2/62 (3%) <0·0001§
(n, %)‡

Patients with fever (≥37·5 o C) at day of recurrent 43/56 (77%) 35/43 (81%) 6/11 (55%) 2/2 (100%) 0·15§
infection (n, %)

Median day of recurrent P falciparum 28 (21–38) 28 (21–35) 35 (21–39) 51 (39–63) 0·098†


infections by day 63 (IQR)

Median parasite density (IQR; parasites per μL) 1508 (234·5–5895) 1263 (186–4691) 3400 (288–6772) 9857 (609–19 104) 0·51†

Effi cacy

Without PCR correction (95% CI, %) 75·8 (69·7–80·8) 58 (47·7–66·9) 83·1 (71·5–90·2) 96·8 (87·7–99·2) <0·0001¶

With PCR correction (95% CI, %) 79·2 (73·3–83·9) 63·2 (52·8–71·8) 84·6 (73·3–91·4) 98·4 (89·2–99·8) <0·0001¶

Data are mean (SD) unless otherwise stated. p values in the table are for diff erence between all three sites. *Not measured for 21 patients because of missed visit (16 in Pursat, one in Preah Vihear, three in Ratanakiri) or low sample quantity

(one in Ratanakiri). †Calculated with the Kruskal-Wallis test; Mann-Whitney tests indicate that absolute piperaquine plasma concentrations at day 7 were signifi cantly lower in Ratanakiri than in Pursat (p=0·0020) and than in Preah Vihear

(p=0·012), with no signifi cant diff erence between Pursat and Preah Vihear (p=0·97); and that normalised piperaquine plasma concentrations at day 7 were also signifi cantly lower in Ratanakiri than in Pursat (p=0·0004) and than in Preah

Vihear (p=0·0038), with no signifi cant diff erence between Pursat and Preah Vihear (p=0·91). ‡The denominator excludes patients who were lost to follow-up (n=18), withdrew themselves from the study (n=2), or developed Plasmodium vivax

parasitaemia between 42 days and 63 days that required retreatment with dihydroartemisinin–piperaquine (n=9). §Calculated with Fisher’s exact test; recurrence was higher in Pursat than in Preah Vihear (p=0·0001) and Ratanakiri (p<0·0001),

and higher in Preah Vihear than in Ratanakiri (p=0·016); these eff ects remained signifi cant after dose-normalisation. ¶Calculated with the log-rank (Mantel-Cox) test.

Table 3: Follow-up of patients

Neither the day nor parasite density of recurrent infections diff ered indeterminate. The effi cacy of dihydro arte misinin– piperaquine with
signifi cantly between sites. More than three quarters of patients with PCR correction also diff ered signifi cantly by site, being greatest in
recurrent P falciparum infection were febrile (table 3), and all cleared Ratanakiri (fi gure, table 3). Piperaquine concentrations at 7 days
their parasitaemia within 72 h of receiving Malarone. PCR correction were signifi cantly higher in patients in Pursat and Preah Vihear than
identifi ed seven recurrent parasitaemias as reinfections and one as in Ratanakiri (table 3). These diff erences were still signifi cant

www.thelancet.com/infection Published online January 7, 2016 http://dx.doi.org/10.1016/S1473-3099(15)00487-9 5


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after correcting for each individual’s dose of piperaquine. At the time non-recrudescent infections (table 5). A larger proportion of patients with
of recrudescence, mean piperaquine con centration was 22·6 ng/mL recrudescence were male, and had detectable and higher piperaquine
(SD 35·5). Piperaquine con cen trations correlated signifi cantly with plasma concentrations at the time of enrolment than those with no
the day of recrudescence (Spearman’s r=–0·40, p=0·005; appendix p recrudescence, but their age, initial parasite density, total piper aquine dose,
See Online for appendix 1). The parasite clearance half-life was signifi cantly longer in Pursat and piperaquine plasma con cen tration at 7 days did not diff er signifi cantly
than in Preah Vihear or Ratanakiri (table 4). The time to 90% (but not (table 5, appendix p 2). Compared with non-recrudescent parasites,
50%) parasite clearance was also signifi cantly longer in Pursat than in recrudescent parasites had higher chloroquine, piper aquine, and
Preah Vihear and Ratanakiri (table 4). The proportions of patients with atovaquone IC 50 values; similar artesunate, dihydroartemisinin, quinine, and
parasite clearance half-life longer than 5 h or detectable parasitaemia pyronaridine IC 50 values; and lower mefl oquine IC 50 values (table 5,
at 72 h were signifi cantly greater in Pursat than in Preah Vihear, and appendix p 3). These data are consistent with observations 7,8 that artemisinin
greater in Preah Vihear than in Ratanakiri. The presence of a resistance is not associated with increased artesunate or dihydro artemisinin
nonsynonymous single nucleotide polymorphism in kelch13 after IC 50 values. Recrudescent parasites had piperaquine IC 50 values (geometric
position 440 was higher in Pursat than in Preah Vihear, and higher in mean 64·6 ng/mL) that were 3·85-times (95% CI 2·70–5·47) higher than the
Preah Vihear than in Ratanakiri (table 3). To investigate patient and corresponding patients’ piperaquine plasma con centrations (16·8 ng/mL,
parasite factors associated with dihydroartemisinin–piperaquine n=30) at the time of recrudescence, suggesting that they were resistant to
failure, we compared piperaquine. Piperaquine IC 50 values did not diff er between paired initial
and recrudescent isolates (p=0·13, n=23), suggesting that piperaquine
resistance did not arise within patients during the study.

the characteristics of recrudescent and

100
(%)

80

Signifi cantly more recrudescent parasites carried


o

e
c

r
f

o
b

b
P

y
r

t
i
l
i

60
kelch13 mutations than did non-recrudescent parasites (table 5). None
of 48 recrudescent parasites had multiple
40
pfmdr1 copies, compared with 11% of non-recrudescent parasites (table 5).
Ratanakiri Preah
Although multiple chromosome 5 region ( X5r) copies and the pfcrt
20
Vihear Pursat cys101phe mutation have been associated with in-vitro piperaquine
resistance, 33 multiple X5r copies were not associated with recrudescence or
0

0 7 14 21 28 35 42 49 56 63 piperaquine IC 50 values in our study, and pfcrt cys101phe was not present in
Time (days) any sample.
Number at risk
Ratanakiri 66 66 66 65 65 64 62 62 61 60
Preah Vihear 65 65 65 64 62 60 56 56 55 54
Pursat 110 110 106 100 83 75 67 57 53 48
Discussion
The intensive spread of artemisinin resistance in Cambodia 7–10 is
Figure: Kaplan-Meier curves showing effi cacy of dihydroartemisinin–piperaquine with
PCR correction for reinfection rapidly threatening to reduce the effi cacy of

All sites (n=110) Pursat (n=41) Preah Vihear (n=35) Ratanakiri (n=34) p value

Parasite clearance half-life >5 h (n, %) 41/110 (37%) 27/41 (66%) 13/35 (37%) 1/34 (3%) <0·0001*

Positive for parasitaemia at 72 h (n, %) 35/110 (32%) 25/41 (61%) 9/35 (26%) 1/34 (3%) <0·0001*

Median parasite clearance half-life (IQR; h) 3·38 (2·24–6·78) 6·07 (4·20–7·52) 2·99 (1·98–7·01) 2·43 (2·03–3·22) <0·0001†

Median time to 50% parasite clearance (IQR; h) 7·35 (5·42–11·6) 8·26 (6·13–13·4) 7·17 (4·14–11·0) 6·60 (5·32–11·3) 0·24†

Median time to 90% parasite clearance (IQR; h) 16·6 (11·0–24·4) 22·9 (16·2–29·7) 15·5 (10·4–23·4) 12·4 (10·5–17·0) <0·0001†

Nonsynonymous SNPs in kelch13 after position 440‡ 111/238 (47%) 82/107 (77%) 22/65 (34%) 7/66 (11%) <0·0001*

p values in the table are for diff erence between all three sites. Time to 50% parasite clearance was not determined for two patients in Pursat, one patient in Preah Vihear, and three patients in Ratanakiri. Time to 90%
parasite clearance was not determined for one patient in Ratanakiri. SNP=single nucleotide polymorphism. *Calculated with Fishers’ exact test; the proportion of patients with parasite clearance half-life >5 h was higher
in Pursat than in Preah Vihear (p=0·021), higher in Preah Vihear than in Ratanakiri (p=0·0006), and higher in Pursat than in Ratanakiri (p<0·0001); the proportion of patients still positive for parasitemia at 72 h was higher
in Pursat than in Preah Vihear (p=0·0027), higher in Preah Vihear than in Ratanakiri (p=0·013), and higher in Pursat than in Ratanakiri (p<0·0001); the proportion of parasites with a kelch13 mutation was higher in Pursat
than in Preah Vihear (p<0·0001), higher in Preah Vihear than in Ratanakiri (p=0·016), and higher in Pursat than in Ratanakiri (p<0·0001). †Calculated with Kruskal-Wallis test; Mann-Whitney tests indicate that half-life and
time to 90% parasite clearance were longer in Pursat than in Ratanakiri (p<0·0001 for both); half-life was longer in Pursat than in Preah Vihear (p=0·0077), with no signifi cant diff erence between Preah Vihear and
Ratanakiri (p=0·17); 90% parasite clearance was signifi cantly longer in Pursat than in Preah Vihear (p=0·0096), with no signifi cant diff erence between Preah Vihear and Ratanakiri (p=0·14). ‡The denominator excludes
missing and heterozygous genotypes.

Table 4: Parasite clearance

6 www.thelancet.com/infection Published online January 7, 2016 http://dx.doi.org/10.1016/S1473-3099(15)00487-9


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Recrudescence (n=48) No recrudescence (n=156) p value*

Male participants 42/48 (88%) 109/156 (70%) 0·046

Median age (IQR; years) 23·5 (20–32) 25 (16–33) 0·81

Median parasite concentration at 0 h (IQR; parasites per μL) 15 731 (3789–55 733) 11 316 (1547–41 594) 0·13

Gametocyte carriage at 0 h (n, %) 4/48 (8%) 9/156 (6%) 0·51

Detectable piperaquine at 0 h (n, %) 32/48 (67%) 40/155 (26%) <0·0001

Piperaquine plasma concentration at 0 h (ng/mL) 20·74 (35·56; n=48) 3·91 (15·52; n=155) <0·0001

Total piperaquine given (mg/kg) 55·47 (7·85) 57·40 (8·47) 0·059

Piperaquine plasma concentration on day 7 (ng/mL) 71·87 (42·06; n=45) 67·58 (55·59; n=148) 0·13

Dose-normalised piperaquine plasma concentration on day 7 (ng/mL per 3·93 (2·37; n=45) 3·56 (2·87; n=148) 0·11
dose)

kelch13 mutation (n/N, %) 41/46 (89%) 51/155 (33%) <0·0001

pfmdr1 copy number >1 (n/N, %) 0/48 (0%) 17/156 (11%) 0·014

X5r copy number >1 (n/N, %) 6/47 (13%) 10/156 (6%) 0·21

Chloroquine geometric mean IC 50 ( range; nmol/L) 625 (269–1084; n=29/46) 416 (19–1313; n=85/111) 0·0043

Quinine geometric mean IC 50 ( range; nmol/L) 240 (81–992; n=45/46) 255 (43–957) n=109/111 0·35

Mefl oquine geometric mean IC 50 ( range; nmol/L) 10 (2–52; n=45/46) 22 (3–70; n=104/111) <0·0001

Piperaquine geometric mean IC 50 ( range; nmol/L) 64† (17–136; n=32/46) 40 (8–185; n=104/111) 0·0002

Artesunate geometric mean IC 50 ( range; nmol/L) 3 (1–9; n=44/46) 3 (1–9; n=104/111) 0·41

Dihydroartemisinin geometric mean IC 50 ( range; nmol/L) 3 (1–6; n=45/46) 3 (1–8; n=107/111) 0·33

Atovaquone geometric mean IC 50 ( range; nmol/L) 1 (0–9; n=41/44) 0 (0–14; n=70/74) 0·0010

Pyronaridine geometric mean IC 50 ( range; nmol/L) 5 (1–17; n=41/44) 5 (1–15); n=71/74) 0·62

Data are mean (SD) unless otherwise stated. *Calculated with Fisher’s exact test for categorical variables and Mann-Whitney test for continuous variables. IC 50 data are for the total number of isolates with
interpretable data (numerator) out of the total number of isolates tested (denominator). †Equivalent to 64 ng/mL.

Table 5: Characteristics of patients and parasites, by recrudescence

all artemisinin combination therapies used in this country and in nearly four-times higher than piperaquine plasma concentrations at
bordering areas of Vietnam, Laos, and Thailand. This threat arises the time of recrudescence, strongly indicating that piperaquine
because more parasites survive exposure to the fast-acting resistance has emerged and spread in Cambodia. Surprisingly,
artemisinin component, increasing the chance that some of them will patients with recrudescence were much more likely to have
spontaneously develop genetic resistance to long-acting partner detectable and higher piperaquine plasma concentrations at the
drugs. In this study, dihydro artemisinin–piperaquine cured 63% of time of enrolment
patients in Pursat province, where artemisinin resistance is than were patients without
entrenched, 85% of patients in Preah Vihear province, where recrudescence, suggesting that they presented to our study with a
artemisinin resistance is emerging, and 98% of patients in Ratanakari recrudescent parasitaemia following an earlier dihydro
province, where artemisinin resistance is rare. The proportion of artemisinin–piperaquine failure in the private sector. This result is
patients cured paralleled the prevalence of kelch13 mutations (77% vs 34%reminiscent of a fi nding of detectable piperaquine plasma
concentrations in 15% of patients in Pursat in 2008, 37 and suggests
that intensifi ed eff orts are needed to discourage what appears to be
vs 11%). Treatment failures were not associated with patient age, initial a highly ineff ective approach of self-treatment in the private sector,
parasite density, or piperaquine plasma concentration at 7 days, and instead to encourage hospital admission for patients in areas
suggesting that they did not result from lower levels of age-dependent, where artemisinin combination therapy-resistant falciparum malaria is
parasite-clearing immunity, 34,35 higher parasite load, or lower plasma prevalent.
exposure to piperaquine. Although patients in Ratanakiri had signifi
cantly lower piperaquine concentrations at 7 days than those in other
provinces (probably due to the greater proportion of children, who Recrudescent parasites had signifi cantly lower
clear piperaquine more rapidly than do adults), 3,36 recrudescences in mefl oquine IC 50 values and all had only one copy of
Ratanakiri were rare. pfmdr1. This latter fi nding is consistent with that of a previous study 14 in
which 17 of 18 dihydroartemisinin– piperaquine failures in Pailin and
Pursat were also associated with one pfmdr1 copy. Together, all
Recrudescent parasites were almost three-times more likely to have available data suggest
kelch13 mutations than were nonrecrudescent parasites. Recrudescent that dihydroartemisinin–piperaquine failures are
parasites also had higher piperaquine IC 50 values than non- due to both artemisinin and piperaquine resistance. They also suggest
recrudescent parasites, and had piperaquine IC 50 values that were that artesunate plus mefl oquine should be tested as a front-line
artemisinin

www.thelancet.com/infection Published online January 7, 2016 http://dx.doi.org/10.1016/S1473-3099(15)00487-9 7


Articles

com real-time
with bination therapy
drug resistance
in areas data,
of Cambodia
identifi cation
whereand
dihydro
treatment of artemisinin–piperaquine failures have been documented, and also as
asymptomatic parasite carriers through community treatment
a salvage treatment for patients with dihydroartemisinin–piperaquine campaigns, and prevention of gametocyte transmission to
failures elsewhere in the country. Whether deamplifi cation of pfmdr1 andmosquitoes with single lowdose primaquine, are now needed more
increased sensitivity to mefl oquine is a result of the removal of mefl than ever if malaria elimination is to succeed in southeast Asia. oquine
selection pressure, the addition of piperaquine selection
pressure, or both, awaits further investigation. Given that piperaquine- Contributor
s
resistant parasites are highly susceptible to atovaquone and pyronaridine CA, MPF, and RMF designed the study. CA, PL, SSu, SSr, SM, CS, BS, DD, VT, RA, DB, LS, and
GST collected data. CA, PL, MPF, JT, and RMF analysed data. CA, PL, MPF, JT, and RMF
in vitro, artesunate plus atovaquone–proguanil or artesunate–
interpreted data and prepared the report. CA, PL, SSu, JMA, and RMF oversaw the project.
pyronaridine 38 might be eff ective alternatives for patients who cannot
take mefl oquine. Our study is the third report of poor clinical effi cacy of
Declaration of interests
dihydroartemisinin–piperaquine in Cambodia, and extends this fi nding We declare no competing interests.
to Preah Vihear. In Pursat, where the prevalence of mutant kelch13 alleles
Acknowledgment
has increased from 40% in 2003–04, 9 to 77% in 2012–13, the effi cacy of s
This work was funded by the Intramural Research Program of the National Institutes of Health,
dihydroartemisinin–piperaquine has decreased from 98% in 2005, 6 to National Institute of Allergy and Infectious Diseases. The Wellcome Trust-Mahidol University-
63% in 2012–13. These fi ndings, and the observation that piperaquine IC Oxford Tropical Medicine Research Programme is supported by the Wellcome Trust (grant
50 values have increased since dihydroartemisinin–piperaquine was widely 089275/Z/09/2). We thank all the patients and families for participating in this study; Na Vany,
Sdoeung Saray, Prum Phoeun, Kong Sam Ath, Lor Vanny, Koeut Savuth, Eam Teang, Chim Sokea,
used in 2010, 15,22 suggest that parasites resistant to artemisinin and
Yek Vanna, and Nov Nhet in Pursat, Chou Ponina, Mork Neang, Phan Vichea, Chhun Tang Kae, Soy
piperaquine are spreading rapidly in Cambodia, that the parasites most Sovann, Chhim Chon, Lem Vinh, Prom Nit, and Sit Samean in Preah Vihear, and Lok Vanthan,
sensitive to piperaquine are being eliminated, or both. Results from this Nhem Heng, Chan Tola, Kong Sochea, Chan Marann, Chheng Monivan, Tohart Eysa, Sor Bunleng,
study and two previous studies 10,16 have documented an increased and Norn Sophy in Ratanakiri, for screening, enrolling, and caring for patients; Vunsokserey Ou for
managing data; Pho Samphors, Math Hakim, Mam Sopheap, Ngin Sam Nang, and Ngan Ny for
gametocyte prevalence in patients with artemisininresistant parasites,
providing logistic support; Michelle Xiong (Guilin Pharmaceutical) for donating artesunate tablets;
suggesting that they have increased transmission potential. Whether this Chris Lourens (WorldWide Antimalarial Resistance Network) for providing antimalarial drugs for in-
fi nding is related to increased transmissibility of slow-clearing parasites vitro assays; and Khoy Dy, Chan Sokha, Khoy Bun Thanny, Koung Lo, Sim Sonlay, Hing
following dihydroartemisinin–piperaquine treatment in Kenya 39 requires Phansakunthea, Say Seuang, Char Meng Chuor, Robert Gwadz, and Thomas Wellems for
supporting this work.
further investigation. Studies are also needed to test whether single low-
dose primaquine 40

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t
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n www.thelancet.com/infection Published online January 7, 2016 http://dx.doi.org/10.1016/S1473-3099(15)00487-9
e
6,42 and ar
pyronaridine 38) are available, and because artemisinin resistance will
probably accelerate resistance to any partner drug, investigations of
alternative treatment approaches are urgently needed. These include
further clinical testing of new compounds; 43 frequent cycling between
combination therapies, which has tremendous logistic challenges;
deployment of multiple fi rst-line artemisinin combination therapies
simultaneously at the population level; treating patients sequentially
with two artemisinin combination therapies, such as
dihydroartemisinin–piperaquine followed by artesunate plus mefl
oquine; 44 using extended combination therapies, such as three doses of
artesunate followed by a full course of an artemisinin combination
therapy; 10 and introducing three-drug regimens such as dihydro
artemisinin– piperaquine plus mefl oquine (as is being tested in a
clinical trial; ClinicalTrials.gov number NCT02453308). Improvements in
the treatment of P falciparum malaria

8
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