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Publication Open Access FlexiChip package: an universal microarray with a dedicated analysis software for high-thoughput SNPs detection linked to anti-malarial drug resistance.(2009-10-15) Steenkeste, Nicolas; Dillies, Marie-Agnès; Khim, Nimol; Sismeiro, Odile; Chy, Sophy; Lim, Pharath; Crameri, Andreas; Bouchier, Christiane; Mercereau-Puijalon, Odile; Beck, Hans-Peter; Mallika Imwong; มัลลิกา อิ่มวงศ์; Dondorp, Arjen M.; Socheat, Duong; Rogier, Christophe; Coppée, Jean- Yves; Ariey, Frédéric; Steenkeste, Nicolas; Mahidol University. Faculty of Tropical Medicineof practical methods enabling high throughput analysis. Here a new Zip-code array is described, called FlexiChip, linked to a dedicated software program, which largely overcomes this problem. METHODS: Previously published microarray probes detecting singlePublication Open Access Persistent Plasmodium falciparum and Plasmodium vivax infections in a western Cambodian population: implications for prevention, treatment and elimination strategies(2016) Rupam Tripura; Peto, Thomas J.; Jeremy Chalk; Lee, Sue J.; Pasathorn Sirithiranont; Chea Nguon; Mehul Dhorda; Seidlein, Lorenz von; Maude, Richard J.; Day, Nicholas P. J.; Mallika Imwong; White, Nicholas J.; Dondorp, Arjen M.; Mahidol University. Faculty of Tropical Medicine. Mahidol-Oxford Tropical Medicine Research UnitBackground: Subclinical Plasmodium parasitaemia is an important reservoir for the transmission and persistence of malaria, particularly in low transmission areas. Methods: Using ultrasensitive quantitative PCR (uPCR) for the detection of parasitaemia, the entire population of three Cambodian villages in Pailin province were followed for 1 year at three-monthly intervals. A cohort of adult participants found initially to have asymptomatic malaria parasitaemia was followed monthly over the same period. Results: The initial cross sectional survey in June 2013 (M0) of 1447 asymptomatic residents found that 32 (2.2 %) had Plasmodium falciparum, 48 (3.3 %) had P. vivax, 4 (0.3 %) had mixed infections and in 142/1447 (9.8 %) malaria was detected but there was insufficient DNA to identify the species (Plasmodium. species). Polymorphisms in the ‘K13-propeller’ associated with reduced susceptibility to artemisinin derivatives (C580Y) were found in 17/32 (51 %) P. falciparum strains. Monthly follow-up without treatment of 24 adult participants with asymptomatic mono or mixed P. falciparum infections found that 3/24 (13 %) remained parasitaemic for 2–4 months, whereas the remaining 21/24 (87 %) participants had cleared their parasitaemia after 1 month. In contrast, 12/34 (35 %) adult participants with P. vivax mono-infection at M0 had malaria parasites (P. vivax or P. sp.) during four or more of the following 11 monthly surveys. Conclusions: This longitudinal survey in a low transmission setting shows limited duration of P. falciparum carriage, but prolonged carriage of P. vivax infections. Radical treatment of P. vivax infections by 8-aminoquinoline regimens may be required to eliminate all malaria from Cambodia. Trial registration ClinicalTrials.gov NCT01872702Publication Open Access Parasite clearance rates in Upper Myanmar indicate a distinctive artemisinin resistance phenotype: a therapeutic efficacy study(2016) Tun, Kyaw Myo; Atthanee Jeeyapant; Mallika Imwong; Min Thein; Sai Soe Moe Aung; Hlaing, Tin Maung; Prayoon Yuentrakul; Cholrawee Promnarate; Mehul Dhorda; Woodrow, Charles J.; Dondorp, Arjen M.; Ashley, Elizabeth A.; Smithuis, Frank M.; White, Nicholas J.; Day, Nicholas P. J.; Mahidol University. Faculty of Tropical Medicine. Mahidol-Oxford Tropical Medicine Research UnitBackground: Artemisinin resistance in Plasmodium falciparum extends across Southeast Asia where it is associated with worsening partner drug resistance and a decline in the efficacy of frontline artemisinin-based combination therapy. Dihydroartemisinin-piperaquine (DP) is an essential component of preventive and curative treatment in the region, but its therapeutic efficacy has fallen in Cambodia. Methods: A prospective clinical and parasitological evaluation of DP was conducted at two sites in Upper Myanmar between August 2013 and December 2014, enrolling 116 patients with acute uncomplicated falciparum malaria. Patients received DP orally for 3 days together with primaquine 0.25 mg/kg on admission. Parasite clearance half-lives based on 6 hourly blood smears, and day 42 therapeutic responses were assessed as well as parasite K13 genotypes. Results: Median parasite clearance half-life was prolonged, and clearance half-life was greater than 5 h in 21 % of patients. Delayed parasite clearance was significantly associated with mutations in the propeller region of the parasite k13 gene. The k13 F446I mutation was found in 25.4 % of infections and was associated with a median clearance halflife of 4.7 h compared with 2.7 h for infections without k13 mutations (p < 0.001). There were no failures after 42 days of follow-up, although 18 % of patients had persistent parasitaemia on day 3. Conclusion: The dominant k13 mutation observed in Upper Myanmar, F446I, appears to be associated with an intermediate rate of parasite clearance compared to other common mutations described elsewhere in the Greater Mekong Subregion. Discerning this phenotype requires relatively detailed clearance measurements, highlighting the importance of methodology in assessing artemisinin resistance.Publication Open Access Establishing research priorities for malaria elimination in the context of the emergency response to artemisinin resistance framework‑the Cambodian approach(2016) Canavatม Sara E.; Lawfordม Harriet L. S.; Fatunmbi, Bayo S.; Dysoley Lek; Narann Top‑Samphor; Rithea Leang; Dondorp, Arjen M.; Rekol Huy; Kazadi, Walter M.; Mahidol University. Faculty of Tropical Medicine. Department of Clinical Tropical MedicineBackground: Countries of the greater Mekong subregion have made a transition from malaria control to an aim for falciparum and vivax malaria elimination. The elimination of falciparum malaria will have to be achieved against a background of increasing artemisinin and multi-drug resistance. This ambitious goal requires an operational research (OR) agenda that addresses the dynamic challenges encountered on the path to elimination, which will need to be flexible and developed in close relation with the cambodian national programme for parasitology, entomology and malaria control (CNM). In Cambodia, a number of meetings with stakeholders were convened by the CNM and emergency response to artemisinin resistance (ERAR) hub, producing an initial list of priority OR topics. The process and outcome of these meetings are described, which could serve as a template for other countries in the region. Methods: A landscaping exercise was conducted to gather all past, on-going and planned malaria focussed OR activities conducted by the cambodian research consortium in Cambodia and categorized according to research theme. The six themes included (1) malaria epidemiology, surveillance and response, (2) malaria case management, (3) malaria vector control, (4) malaria behavioural issues, (5) malaria clinical studies, and (6) other vector-borne diseases (dengue, neglected tropical diseases, soil-transmitted helminths). The different themes were discussed in small focus groups, which made an initial prioritization list which was then presented to a plenary group for further discussion. This produced a list of research questions ranked according to priority. Results: OR priorities produced by the thematic groups were discussed in the plenary meeting and given a priority score by group voting. A list of 17 OR questions were developed, finalized and listed, which included questions on surveillance, active case detection and treatment efficacy. Conclusion: This paper describes ERAR’s work on supporting Cambodia’s transition to malaria elimination by identifying national operational research priorities. ERAR has initiated and currently plays a critical role in the development of country specific research agendas for malaria elimination. The first example of this has been the described exercise in Cambodia, which could serve a template for setting OR priorities in the wider region.Publication Open Access A population survey of the glucose-6-phosphate dehydrogenase (G6PD) 563C>T (Mediterranean) mutation in Afghanistan(2014-02-21) Natsuda Jamornthanyawat; นาถสุดา จามรธัญญวาท; Awab, Ghulam R.; Naowarat Tanomsing; เนาวรัตน์ ถนอมสิงห์; Sasithon Pukrittayakamee; ศศิธร ผู้กฤตยาคามี; Yamin, Fazel; Dondorp, Arjen M.; Day, Nicholas P. J.; White, Nicholas J.; Woodrow, Charles J.; Mallika Imwong; มัลลิกา อิ่มวงศ์; Mallika Imwong; Mahidol University. Faculty of Tropical Medicine. Department of Clinical Tropical Medicine; Mahidol University. Faculty of Tropical Medicine. Mahidol-Oxford Tropical Medicine Research Unit (MORU); Mahidol University. Faculty of Tropical Medicine. Department of Molecular Tropical Medicine and GeneticsGlucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzyme defect and an important problem in areas with Plasmodium vivax infection because of the risk of haemolysis following administration of primaquine to treat the liver forms of the parasite. We undertook a genotypic survey of 713 male individuals across nine provinces of Afghanistan in which malaria is found, four in the north and five in the east. RFLP typing at nucleotide position 563 detected 40 individuals with the Mediterranean mutation 563C>T, an overall prevalence of 5.6%. This varied according to self-reported ethnicity, with prevalence in the Pashtun/Pashai group of 33/369 (8.9%) compared to 7/344 individuals in the rest of the population (2.0%; p<0.001, Chi-squared test). Multivariate analysis of ethnicity and geographical location indicated an adjusted odds ratio of 3.50 (95% CI 1.36-9.02) for the Pashtun/Pashai group, while location showed only a trend towards higher prevalence in eastern provinces (adjusted odds ratio = 1.73, 0.73-4.13). Testing of known polymorphic markers (1311C>T in exon 11, and C93T in intron XI) in a subset of 82 individuals wild-type at C563 revealed a mixture of 3 haplotypes in the background population and was consistent with data from the 1000 Genomes Project and published studies. By comparison individuals with G6PD deficiency showed a highly skewed haplotype distribution, with 95% showing the CT haplotype, a finding consistent with relatively recent appearance and positive selection of the Mediterranean variant in Afghanistan. Overall, the data confirm that the Mediterranean variant of G6PD is common in many ethnic groups in Afghanistan, indicating that screening for G6PD deficiency is required in all individuals before radical treatment of P. vivax with primaquine.Publication Open Access Geographic distribution of amino acid mutations in DHFR and DHPS in Plasmodium vivax isolates from Lao PDR, India and Colombia(2016) Naowarat Saralamba; Supatchara Nakeesathit; Mayfong Mayxay; Newton, Paul N.; Lyda Osorio; Kim, Jung‑Ryong; White, Nicholas J.; Day, Nicholas P. J.; Dondorp, Arjen M.; Mallika Imwong; Mahidol University. Faculty of Tropical Medicine. Department of Molecular Tropical Medicine and GeneticsBackground: Non-synonymous mutations in dhfr and dhps genes in Plasmodium vivax are associated with sulfadoxine– pyrimethamine (SP) resistance. The present study aimed to assess the prevalence of point mutations in P. vivax dhfr (pvdhfr) and P. vivax dhps (pvdhps) genes in three countries: Lao PDR, India and Colombia. Methods: Samples from 203 microscopically diagnosed vivax malaria were collected from the three countries. Five codons at positions 13, 57, 58, 61, and 117 of pvdhfr and two codons at positions 383 and 553 of pvdhps were examined by polymerase chain reaction-restriction fragment length polymorphism methodology. Results: The largest number of 58R/117 N double mutations in pvdhfr was observed in Colombia (94.3 %), while the corresponding wild-type amino acids were found at high frequencies in Lao PDR during 2001–2004 (57.8 %). Size polymorphism analysis of the tandem repeats within pvdhfr revealed that 74.3 % of all the isolates carried the type B variant. Eighty-nine per cent of all the isolates examined carried wild-type pvdhps A383 and A553. Conclusions: Although SP is not generally used to treat P. vivax infections, mutations in dhfr and dhps that confer antifolate resistance in P. vivax are common. The data strongly suggest that, when used primarily to treat falciparum malaria, SP can exert a substantial selective pressure on P. vivax populations, and this can lead to point mutations in dhfr and dhps. Accurate data on the global geographic distribution of dhfr and dhps genotypes should help to inform anti-malarial drug-use policies.Publication Open Access Genetic variability of Plasmodium malariae dihydropteroate synthase (dhps) in four Asian countries.(2014-04-03) Naowarat Tanomsing; เนาวรัตน์ ถนอมสิงห์; Mayxay, Mayfong; Newton, Paul N.; Nosten, Francois; Dolecek, Christiane; Hien, Tran Tinh; White, Nicholas J.; Day, Nicholas P. J.; Dondorp, Arjen M.; Mallika Imwong; มัลลิกา อิ่มวงศ์; Mallika Imwong; Mahidol University. Faculty of Tropical Medicine. Department of Molecular Tropical Medicine and Genetics; Mahidol University. Faculty of Tropical Medicine. Mahidol-Oxford Tropical Medicine Research Unit; Mahidol University. Faculty of Tropical Medicine. Shoklo Malaria Research Unit, Mahidol-Oxford Tropical Medicine Research Uni.The dihydropteroate synthase (dhps) genes of 44 P. malariae strains from four Asian countries were isolated. Only a limited number of polymorphisms were observed. Comparison with homologous mutations in other Plasmodium species showed that these polymorphisms are unlikely to be associated with sulfadoxine resistance.Publication Open Access Clinical trials of artesunate plus sulfadoxine‑pyrimethamine for Plasmodium falciparum malaria in Afghanistan: maintained efficacy a decade after introduction(2016) Ghulam Rahim Awab; Mallika Imwong; Sasithon Pukrittayakamee; Fazel Alim; Warunee Hanpithakpong; Joel Tarning; Dondorp, Arjen M.; Day, Nicholas P. J.; White, Nicholas J.; Woodrow, Charles J.; Mahidol University. Faculty of Tropical Medicine. Mahidol-Oxford Tropical Medicine Research Unit (MORU)Background: Combination therapy with artesunate plus sulfadoxine-pyrimethamine (SP) was adopted as recommended treatment for Plasmodium falciparum infection in Afghanistan in 2003. Methods: A series of prospective clinical studies examining the efficacy of artesunate plus sulfadoxine-pyrimethamine (AS + SP) against P. falciparum were undertaken in sentinel sites in Afghanistan from 2007 to 2014, accompanied by relevant molecular studies. The first study was a randomized trial of AS + SP versus dihydroartemisinin-piperaquine, while two subsequent studies were standard therapeutic efficacy studies of AS + SP. Results: Three hundred and three patients were enrolled across four provinces in the north and east of the country. Curative efficacy was high in all the trials, with an adequate clinical and parasitological response (ACPR) of more than 95 % in all groups and trial stages. Genotyping for drug-resistance alleles at dhfr indicated fixation of the S108 N mutation and a prevalence of the C59R mutation of approximately 95 % across all sites. Other mutations in dhfr and dhps remained rare or absent entirely, although five isolates from the first trial carried the dhps triple mutant SGEGA haplotype. In the last study undertaken in 2012–2014 the K13 artemisinin resistance marker was examined; only two of 60 successfully sequenced samples carried a K13-propeller mutation. Conclusions: These data confirm maintained efficacy 10 years after introduction of artesunate plus SP as combination treatment of P. falciparum in Afghanistan. The molecular data indicate that despite a substantial fall in incidence, resistance has not developed to artemisinins, or intensified to the ACT partner drug components. Trial Registration http://www.clinicaltrials.gov/ct NCT00682578, NCT01115439 and NCT01707199Publication Open Access Asymptomatic Plasmodium infections in 18 villages of southern Savannakhet Province, Lao PDR (Laos)(2016) Koukeo Phommasone; Bipin Adhikari; Henriques, Gisela; Tiengkham Pongvongsa; Panom Phongmany; Seidlein, Lorenz von; White, Nicholas J.; Day, Nicholas P. J.; Dondorp, Arjen M.; Newton, Paul N.; Mallika Imwong; Mayfong Mayxay; Mahidol University. Faculty of Tropical Medicine. Mahidol Oxford Tropical Medicine Research Unit (MORU)Background: A large fraction of Plasmodium infections do not cause clinical signs and symptoms of disease and persist at densities in blood that are not detectable by microscopy or rapid diagnostic tests. These infections may be critical as a transmission reservoir in areas of low malaria endemicity. Understanding the epidemiology of these infections would be helpful for malaria elimination. Methods: A cross-sectional survey was conducted in Thapangthong and Nong Districts of Savannakhet Province, Lao PDR, to determine the prevalence of parasitaemia. A total of 888 blood samples were collected from afebrile volunteers aged ≥15 years in 18 villages during March and July 2015. Plasmodium infections were diagnosed by rapid diagnostic tests (RDT) and high volume, ultra-sensitive quantitative polymerase chain reaction (uPCR). Results: uPCR detected Plasmodium infections in 175 of 888 samples (20 %). The species distribution was Plasmodium falciparum 3.6 % (32/888), Plasmodium vivax 11.1 % (99/888), mixed infections with P. falciparum and P. vivax 1.6 % (14/888) and Plasmodium of undetermined species 3.4 % (30/888). RDT identified only 2 % (18/888) positive cases. Using uPCR as reference, the sensitivity and specificity of RDTs were 28 and 100 %, respectively, in detecting P. falciparum infections, and 3 and 99 % in detecting asymptomatic P. vivax infections. The K13 kelch propeller domain C580Y mutation, associated with reduced susceptibility to artemisinin derivatives, was found in 75 % (12/18) of P. falciparum isolates from Thapangthong and in 7 % (2/28) from Nong (p < 0.001). In a multivariate analysis, males were more likely to have P. vivax infections [adjusted odds ratio (aOR) 4.76 (95 % CI 2.84–8.00)] while older villagers were at lower risk for parasitaemia [aOR for increasing age 0.98 (95 % CI 0.96–0.99)]. Conclusion: There is a high prevalence of asymptomatic Plasmodium infections in southern Savannakhet. Artemisinin- resistant P. falciparum strains form an increasing proportion of the parasite population in Thapangthong District and are already present in the more remote Nong District. This worrying trend has wider implications for Laos and could reverse the gains achieved by the successful control of malaria in Laos and the Greater Mekong Sub-region (GMS). Rapid elimination of P. falciparum has to be a top priority in Laos as well as in the wider GMS.Publication Open Access Sequence variation does not confound the measurement of plasma PfHRP2 concentration in African children presenting with severe malaria(2012-08-16) Thiranut Ramutton; Hendriksen, Ilse CE; Mwanga-Amumpaire, Juliet; Mtove, George; Olaosebikan, Rasaq; Tshefu, Antoinette K.; Onyamboko, Marie A.; Karema, Corine; Maitland, Kathryn; Gomes, Ermelinda; Gesase, Samwel; Reyburn, Hugh; Kamolrat Silamut; Kesinee Chotivanich; เกศินี โชติวานิช; Kamoltip Promnares; Fanello, Caterina I.; Seidlein, Lorenz von; Day, Nicholas P.J.; White, Nicholas J.; Dondorp, Arjen M.; Mallika Imwong; มัลลิกา อิ่มวงศ์; Woodrow, Charles J.; Woodrow, Charles J.; Mahidol University. Faculty of Tropical Medicine. Department of Clinical Tropical Medicine.; Mahidol University. Faculty of Tropical Medicine. Department of Molecular Tropical Medicine and Genetics.; Mahidol University. Faculty of Tropical Medicine. Mahidol-Oxford Tropical Medicine Research Unit.BACKGROUND: Plasmodium falciparum histidine-rich protein PFHRP2 measurement is used widely for diagnosis, and more recently for severity assessment in falciparum malaria. The Pfhrp2 gene is highly polymorphic, with deletion of the entire gene reported in both laboratory and field isolates. These issues potentially confound the interpretation of PFHRP2 measurements. METHODS: Studies designed to detect deletion of Pfhrp2 and its paralog Pfhrp3 were undertaken with samples from patients in seven countries contributing to the largest hospital-based severe malaria trial (AQUAMAT). The quantitative relationship between sequence polymorphism and PFHRP2 plasma concentration was examined in samples from selected sites in Mozambique and Tanzania. RESULTS: There was no evidence for deletion of either Pfhrp2 or Pfhrp3 in the 77 samples with lowest PFHRP2 plasma concentrations across the seven countries. Pfhrp2 sequence diversity was very high with no haplotypes shared among 66 samples sequenced. There was no correlation between Pfhrp2 sequence length or repeat type and PFHRP2 plasma concentration. CONCLUSIONS: These findings indicate that sequence polymorphism is not a significant cause of variation in PFHRP2 concentration in plasma samples from African children. This justifies the further development of plasma PFHRP2 concentration as a method for assessing African children who may have severe falciparum malaria. The data also add to the existing evidence base supporting the use of rapid diagnostic tests based on PFHRP2 detection.