Iwagami et al. Malar J (2018) 17:483 https://doi.org/10.1186/s12936-018-2625-6 Malaria Journal RESEARCH Open Access Heterogeneous distribution of k13 mutations in Plasmodium falciparum in Laos Moritoshi Iwagami1,2,3, Masami Nakatsu1,2, Phonepadith Khattignavong2,3, Pheovaly Soundala2,3, Sengdeuane Keomalaphet2,3, Lavy Lorpachan2,3, Phonepadith Xangsayalath2,4, Emilie Matsumoto‑Takahashi1,2, Virginie Pommelet3, Bouasy Hongvanthong2,5, Paul T. Brey2,3* and Shigeyuki Kano1,2,3* Abstract Background: The emergence and transnational spread of artemisinin resistance in Plasmodium falciparum in the Greater Mekong Sub-region (GMS) is a serious threat to malaria elimination in the region and could present a threat to malaria control in Africa. Recently, the Lao Government adopted the goal of malaria elimination by 2030, for which monitoring of artemisinin-resistant malaria within the country is indispensable. This study’s objectives were to assess the distribution of k13 mutations in Laos. Methods: Plasmodium falciparum isolates (n 1151) were collected from fve southern provinces in Laos between 2015 and 2016, and three isolates from the northernmost= province bordering China in 2017. Polymorphisms of the k13 gene and two fanking regions were analysed to estimate relationship among the isolates. Results: In the fve southern provinces, overall 55.5% of the isolates possessed artemisinin-resistant mutations of the k13 gene (C580Y, P574L, R539T, Y493H). The C580Y was the predominant mutation (87.2%). The frequencies of the k13 mutations were heterogeneous in the fve southern provinces, but with a clear tendency showing the highest frequency in the south (72.5%) and to a lower degree when moving northward (28.0%). The three isolates from the Lao–Chinese border also possessed the C580Y mutation. Analysis of the fanking loci demonstrated that these three isolates were genetically very close to resistant strains originating from western Cambodia. Conclusions: Artemisinin resistance was observed to be rapidly increasing and spreading northwards through Laos and has now reached the Chinese border. The Lao and Chinese governments, as well as the international community, should make dedicated eforts to contain the spread of k13 mutations within Laos and in the GMS. Keywords: Artemisinin-resistance, Laos, k13, Malaria, Plasmodium falciparum, Greater Mekong Sub-region Background challenges to overcome to achieve this goal. First, in Laos, Extensive eforts have reduced the burden of malaria in the artemisinin-based combination therapy (ACT) is the frst- Greater Mekong Sub-region (GMS) including Laos, also line treatment regimen for both Plasmodium falciparum known as the Lao People’s Democratic Republic (Lao PDR) and Plasmodium vivax [1]; however, therapeutic efcacy [1]. Recently, the Lao government adopted the goal of studies conducted in the southern part of Laos in 2013 eliminating malaria by 2030 [2]. However, there are many demonstrated that 2.2% (Salavan province), 22.2% (Cham- pasak province) [3] and 5.2% (Attapeu province) [4] of the patients treated with artemether–lumefantrine, one ACT, *Correspondence: [email protected]; [email protected] 1 Department of Tropical Medicine and Malaria, Research Institute, were still parasitaemic on day 3 after treatment. Second, National Centre for Global Health and Medicine, 1‑21‑1 Toyama, Shinjuku, mutations in a gene-encoding kelch-propeller domains in Tokyo 162‑8655, Japan chromosome 13 in P. falciparum (k13) are widely used as 3 Institut Pasteur du Laos, Ministry of Health, Sansenthai Road, Ban Kao‑Gnot, Sisattanak District, P.O. Box 3560, Vientiane, Lao People’s primary determinant of artemisinin-resistance [5, 6], and Democratic Republic this genetic marker enables the monitoring of the spread of Full list of author information is available at the end of the article artemisinin-resistance [7–10]. Te previous study showed © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Iwagami et al. Malar J (2018) 17:483 Page 2 of 10 an average of 20.0% (24/122) of P. falciparum isolates col- by microscopy or RDT. Socio-demographic and clinical lected in the southern provinces of Laos in 2013 (Savanna- data were also obtained using questionnaire forms. Te khet Champasak, Salavan) possessed artemisinin-resistant blood samples were sent to the NCGM for DNA analy- mutations in the k13 gene [11]. In another study, as much sis after obtaining authorization from the Lao Ministry of as 77.5% (86/111) of the isolates collected in Champasak Health. in 2014 possessed the C580Y mutation in the k13 gene, Te research proposal was reviewed and approved by which is one of the predominant artemisinin-resistant the National Ethics Committee for Health Research, mutations in the GMS [12]. Te rapid increase and distri- Ministry of Health, Lao PDR (No. 049 NIOPH/NECHR) bution of the artemisinin-resistant k13 mutations in Laos in 2014. Written informed consent was obtained from is of major concern. all the participants prior to interview and the collec- Blood samples were collected from October 2015 to tion of blood for the diagnosis of malaria. Te guardians April 2016 together with socio-demographic and clinical of child participants (< 18 years old) consented to their data from malaria patients who visited public healthcare participation. facilities in the fve southern provinces (Savannakhet, Salavan, Sekong, Attapeu, Champasak) where 96.5% of Genotyping malaria cases in Laos were reported in 2014 [13]. Blood DNA extraction and PCR for Plasmodium species iden- samples were also collected from malaria-suspected tifcation are shown in Additional fle 2. Mutations in the patients in Phongsaly, the northernmost province, k13 gene were examined by the k13 toolbox previously which is adjacent to China. In addition, the associations described [11]. When the k13 gene region was not ampli- between the k13 polymorphisms and demographic and fed by the primer sets in the toolbox, additional primer clinical profles were estimated. Tis survey provides the sets were designed and used (Additional fle 3). When latest status on increase and spread of the k13 polymor- the mutation was observed in the k13 gene in the sam- phisms in the southern and northern parts of Laos, which ple, the PCR amplifcation and DNA sequencing were is crucial for making and updating drug policy to accel- performed once again as an internal quality assessment. erate the elimination of falciparum malaria in the coun- Te sequencing results were analysed by ClustalW using try, as well as safeguarding against the further spread of the 3D7 kelch13 sequence as a reference (Accession: resistant malaria beyond the country’s borders. XM_001359122.1). Te samples with mixed alleles (wild type and mutated type) were considered to be mutated Methods isolates to estimate the frequency of the k13 mutations. Study design, sampling and oversight When two mutations were observed in the k13 gene from Investigators from National Centre for Global Health and a single patient, the PCR genotyping of two other gene Medicine (NCGM), Japan and Institut Pasteur du Laos regions (msp1 and msp2) encoding merozoite surface (IPL) designed the study based on malaria epidemiologi- proteins (MSP1 and MSP2) was performed [15], which cal data in 2013 summarized by the Centre of Malari- allowed to diferentiate “double mutations in the k13 ology, Parasitology and Entomology, Lao Ministry of gene” from “multiple clone infection”. Health. Tis study has three components: training of local Two fanking loci of the k13 gene, PF3D7_1337500 healthcare facilities staf, malaria patient blood sampling (K13_151) and PF3D7_1339700 (K13_159), were ampli- and data collection, and DNA analysis. Two-fve malaria fed and DNA sequences were analysed (Additional fle 3) high endemic districts were selected in each province [11]. Haplotypes were generated based on the combina- based on annual parasite incidence in 2013. A total of 163 tions of the two loci to assess dissemination of the k13 public healthcare facilities (hospitals, health centres and mutations (Additional fles 4, 5, 6). malaria sections in Provincial and District Health Ofces in the selected districts) were chosen for this study (Addi- Statistical analysis tional fle 1). Finger-prick blood samples were collected Data were analysed with Microsoft Excel. For bivari- from malaria patients (microscopic or rapid diagnostic ate analyses, the Chi square test and Fisher’s exact test test (RDT) positive) on flter paper (Whatman™ FTA™ were used to evaluate an association between variables Classic Cards, GE Healthcare Life Science, UK) [13, 14] and Plasmodium infection. P-values less than 0.05 were from the fve southern provinces from October 2015 considered statistically signifcant. Multivariate logistic through April 2016. Finger-prick blood samples were regression analyses adjusted for the efects of other varia- also collected from malaria suspected patients on flter bles were conducted to estimate the association between paper