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Mornitoring Antimalarial Drug Efficacy In

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MONITORING ANTIMALARIAL DRUG EFFICACY IN THE GREATER MEKONG SUBREGION: AN OVERVIEW OF IN VIVO RESULTS FROM 2008 TO 2010 Abstract. In vivo Therapeutic Efficacy Studies (TES) have been routinely conducted in the Greater Mekong Subregion (GMS) for decades. Results from the last 10 years have contributed to update national antimalarial drug policies, to identify hotspots of multi-drug resistance and from 2008 onwards, to stimulate ambitious multi-country programs and innovative research projects to contain and eliminate artemisinin resistant Plasmodium falciparum strains in the subregion. This paper describes the results of TES of first-line antimalarials in six countries of the GMS from 2008-2010 using the WHO in vivo standard protocol. A total of 91 studies were conducted at 32 sentinel sites testing dihydroartemisinin-piperaquine (DHA-PIP), artesunate+mefloquine (A+M), and artemether-lumefantrine (AL) against P. falciparum malaria, as well as chloroquine and DHA-PIP against P. vivax. Overall, artemisinin-based combination therapies (ACTs) remained efficacious against falciparum malaria with some exceptions. The 42-day adequate clinical and parasitological response (ACPR) for DHA-PIP dropped significantly to 73% (95% CI 53-87) in 2010 in the same hotspot area of western Cambodia known to harbor artemisinin resistant P. falciparum strains. Because P. falciparum sensitivity to artemisinin is a major concern, especially on the Cambodia-Thailand border, attempts were also made to strengthen the monitoring of parasite clearance time elsewhere in the region and globally. The proportion of patients still blood-smear positive on Day 3 above 10% is considered a proxy indicator to strongly suspect the appearance of falciparum resistance to artesunate. This has led to substantial extra measures to confirm the suspicion and eventually set up interventions to eliminate artemisinin resistant parasites. Notably, increasing proportions (>10%) of Day 3 positives among falciparum malaria patients treated with DHA-PIP have been observed in western Cambodia, Myanmar, Viet Nam and China from 2008. Percent Day 3 parasitemia associated with A+M has increased along the Thailand-Myanmar border to surpass 10% at several sites, adding to the known pool of sites with ‘suspected’ artemisinin resistance in the GMS. Chloroquine remains highly effective against P. vivax except for northeastern and north-central Cambodia. TES results from this subregional-wide monitoring of antimalarial efficacy have influenced the changes of 1st line drugs against both P. falciparum and P. vivax in Cambodia, against P. falciparum in selected areas in Thailand, and pinpointed hotspot areas elsewhere that should be closely monitored in order to take action in a timely manner.

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INTRODUCTION The Greater Mekong Subregion (GMS), composed of Cambodia, China (Yunnan Province), Lao PDR, Myanmar, Thailand and Viet Nam, is known for multi-drug resistant malaria with the Cambodia-Thailand border, in particular, as the epicenter of antimalarial resistance (Singhasivanon et al, 1999). Since 1999, the Roll Back Malaria Mekong project has taken several steps to advance multi-country collaboration aimed at in vivo monitoring by Therapeutic Efficacy Studies (TES) of first line antimalarial drugs used in the Subregion. In January 2007, a WHO informal consultation in Cambodia extensively reviewed available data and acknowledged the decreased efficacy of artemisinin-based combination therapies (ACTs) against P. falciparum on the Cambodia-Thailand border (WHO, 2007a). Recommendations were made to better document drug resistance in the GMS through intensifying TES as part of routine activities of National Malaria Control Programmes (NMCP) and through in-depth research studies by recognized academic institutions (WHO, 2009c). The primary objective of the TES was to evaluate the therapeutic efficacy of the first- and second-line antimalarials as recommended by national programs. Specifically we evaluated the efficacy of ACTs against P. falciparum, and chloroquine and DHA-PIP against P. vivax. The secondary objective was to detect foci of artemisinin resistance by assessing the proportion of patients with delayed parasite clearance time, especially the presence of parasites on blood smear 3 days after treatment initiation (so-called “Day 3 parasitemia”) considered as a proxy indicator of suspected P. falciparum resistance to artemisinins. To coordinate these collaborative efforts, WHO was tasked to revitalize the “Mekong Malaria Therapeutic Efficacy Study Network” encompassing the six GMS countries. These countries have all been using ACTs or artemisinin monotherapies for uncomplicated falciparum malaria cases for over a decade. Through WHO’s leadership, the in vivo Network, which includes collaboration between national malaria program managers and academic and government researchers, endorsed a single TES protocol across the Subregion. As a result, this standardized approach has led to progressive improvements in data quality, provided needed evidence for policy makers to update drug policies, and allowed for comparison of data across countries especially at border areas. In this article, we describe the 91 studies conducted under the Subregional TES Network at 32 sentinel sites across the GMS, from 2008 to 2010 (Some sites began their studies in 2010 but did not complete patient recruitment until 2011). The findings and implications for antimalarial drug policy changes and artemisinin resistance containment operations are discussed.

MATERIALS AND METHODS Two successive workshops were held by the WHO Mekong Malaria Programme (MMP), 202

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one in Phuket, Thailand in 2007 and the other in Mandalay, Myanmar in 2009 and were attended by key personnel responsible for TES in the six GMS countries. Study sites were identified and all principal investigators (PI) familiarized with the WHO standard TES protocol (WHO, 2007b; WHO, 2009d) with a follow-up schedule of either 28 days or 42 days. In addition to the general WHO guidelines, the countries agreed on standard outcomes and methodology for data management and analysis, drugs to be monitored, sentinel sites, informed consent-form template (in English), and common supervision including training and reporting mechanisms. Each country had the informed consent translated into local language(s). Subsequently, at the national level, PIs and WHO staff finalized the individual country proposals, processed for approval by the national ethics committee, then by the WHO Ethics Review Committee (ERC) before the requested funds were officially released. All studies were registered with the Australia-New Zealand Clinical Trial Registration website (ANZCTR). Supervisory and monitoring visits on site were conducted at least once a year by WHO MMP staff and several times a year by WHO national staff in conjunction with TES training workshops and microscopy refresher courses. Study design The aim of these one-arm TES was to evaluate the clinical and parasitological responses of uncomplicated P. falciparum and P. vivax infections to antimalarial drugs given under direct observed therapy. Patients were enrolled according to pre-determined criteria (see Study participants below), treated on site with the first-line drug according to the corresponding national treatment guidelines, and monitored through clinical and laboratory examinations during 28 or 42 days depending on the drug given (see Anti-malaria drugs and follow-up procedures below). The same drug was intended to be tested every other year at each sentinel site, but there were some exceptions for more or less frequent studies. Sentinel sites The TES were conducted from 2008 to 2010 at 32 sentinel sites across the six GMS countries: Cambodia (4), China (3), Lao PDR (3), Myanmar (8), Thailand (9), and Viet Nam (5) (Figs 1 and 2). The sentinel sites were chosen on the basis of high malaria burden, risk of drug resistance development as well as availability of local health facilities and staff for case finding, treatment and follow-up. Most of them are located along international borders. Study participants Inclusion and exclusion criteria as per WHO standard protocol are presented in Box 1 (WHO, 2009a). Briefly we enrolled patients who were >2 years old with mono-infection of P. falciparum or P. vivax. Patients were enrolled at a remote village/commune health station, a district health center or a government malaria clinic. Often, project staff also conducted active Vol 44 (Supplement 1) 2013

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Trat

Myanmar Thailand Yunnan PRC Lao PDR Cambodia Viet Nam

Fig 1–Location of sentinel sites for therapeutic efficacy studies (TES) in the Greater Mekong Subregion (GMS), 2008-2010.

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Sentinel sites (2008-2010)

Sentinel Sites (2008-2010) 1. Cambodia 1.1 Pailin

1. Cambodia 1.1 Pailin

1.2 Pursat 1.3 Preah Vihear

Kachin

1.4 Rattanakiri

2. Yunnan, China

Tenchong Yingjiang

Sagaing

1.2 Pursat 2.1 Menglian 2.2 Yingjiang 1.3 Preah Vihear

Menglian

2.3 Tengchong

East Shan

1.4 Rattanakiri 3. Lao PDR 3.1 Luang Prabang

Rakhine

2. Yunnan, China

Luang Prabang Mae Hong Son

3.2 Khammoune

3.3 Attapeu 2.1 Menglian

Bago

4. Myanmar

2.2 Yingjiang 4.1 Kachin

Kayin Tak Khammoune

4.2 Eastern Shan 2.3 Tengchong

Mon

4.3 Rakhine

2.4 Jiangsu4.4 Province Sagaing

Kanchanaburi

4.5 Kayin

3. Lao PDR

4.6 Bago

Ubon Ratchathani

Preah Vihear Chantaburi Pailin Ratchaburi Trat Pursat

4.7 Mon 3.1 Luang Prabang 4.8 Tanintharyi

3.2 Khammoune 5. Thailand

Taninthary

3.3 Attapeu 5.1 Mae Hong Son 5.2 Tak

Ranong

4. Myanmar 5.3 Kanchanaburi 4.1 Kachin

5.4 Ratchaburi 5.5 Ubon Ratchathani

Chanthaburi 4.2 Eastern 5.6 Shan 5.7 Ranong

Yala

4.3 Rakhine5.8 Yala

4 4 Sagaing5.9 Trat (previous site) 6. Viet Nam

6.1 Quang Tri 6.2 Ninh Thuan 6.3 Gia Lai

Areas of suspected resistance to artemisinins (where more than 10% of enrolled patients still have parasites 72-hours after onset of treatment) Areas of confirmed resistance to artemisinins

6.4 Dak Nong 6.5 Binh Phuoc

Fig 2–Location of “hotspots” in the GMS where artemisinin resistance has been either “suspected” or “confirmed” from 2008 to 2010.

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case surveys in surrounding villages when passive case detection at a local health center gave insufficient yield for the required sample size. Due to the generally low malaria incidence in the Subregion (except in Myanmar and some locations in Cambodia), Day 0 parasite density cut-offs for inclusion of falciparum cases have been modified to include ≥100 parasites/µl blood, and patients with <500 parasites/µl blood must be febrile at enrolment. Similarly, the upper limit of parasitemia for inclusion has been raised to 250,000 parasites/µl blood, and the patients must still be without any other signs of severe malaria. Previously in the Subregion, Day 0 parasitemia of up to 150,000/µl was allowed due to difficulty in finding eligible cases. Box 1 Inclusion and exclusion criteria used by the Mekong TES Network during 2008-2010. Inclusion criteria • • • • • • • •

age between 2 to 60 years old; mono-infection with P. falciparum or P. vivax detected by microscopy; P. falciparum parasitemia 500-250,000 asexual forms/µl; (see discussion pertaining to modified entry criteria) P. vivax parasitemia ≥ 250 asexuals/µl; presence of axillary temperature ≥ 37.5ºC or history of fever during the past 24 hour; ability to swallow oral medication; ability and willingness to comply with the study protocol for the duration of the study and to comply with the study visit schedule; and informed consent from the patient or from a parent or guardian in the case of children.

Exclusion criteria • • •



• • • •

206

presence of general danger signs in children aged under 5 years or signs of severe falciparum malaria; mixed or mono-infection with another Plasmodium species detected by microscopy; presence of severe malnutrition (defined as a child whose growth standard is below –3 z-score, has symmetrical edema involving at least the feet or has a mid-upper arm circumference < 110 mm); presence of febrile conditions due to diseases other than malaria (eg measles, acute lower respiratory tract infection, severe diarrhea with dehydration) or other known underlying chronic or severe diseases (eg cardiac, renal and hepatic diseases, HIV/ AIDS); regular medication which may interfere with antimalarial pharmacokinetics; history of hypersensitivity reactions or contraindications to any of the medicine(s) being tested or used as alternative treatment(s); unmarried female age 12-18 years old; and positive pregnancy test or breastfeeding.

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Sample size Assuming a treatment failure rate of 10% with a study precision of 10% and the desired confidence level of 95%, the minimum sample size was calculated to be 50 patients per study (ie per an individual drug at a specific site for a specific year). In practice, we targeted at enrolling 60 or more patients in order to account for withdrawals and loss-to-follow-ups. Anti-malarial drugs and TES follow-up procedures P. falciparum treatment. GMS countries rely on various first-line ACT regimens. China and Viet Nam have used artesunate monotherapy for decades but currently promote DHA-PIP. Myanmar began using either DHA-PIP, A+M, or AL from 2002 depending on donor source. AL alone has been promoted in Lao PDR since 2002. Cambodia has recently switched from A+M to DHA-PIP countrywide except in Pailin where Atovaquone-Proguanil (Malarone®, Glaxo SmithKline, UK) was adopted in mid-2012. Loose tablets of artesunate and mefloquine given together have been the practice for Thailand, in selected locations since 1995, and countrywide since 2005. One exception is for Chantaburi and Trat Provinces on the southeastern border with Cambodia, where Atovaquone-Proguanil has been used since 2009 (see also Chapter 7) as a part of the artemisinin resistance containment operation. P. vivax treatment. Chloroquine was tested in all countries except Lao PDR because of its low incidence of vivax malaria. Cambodia added a TES of DHA-PIP for P. vivax in 2010. Under the P. vivax section of the protocol, primaquine was expected to be given after Day 28 as an anti-relapse drug, with either a 14-day or 8-week regimen depending on the G6PD status of the patient. All drugs used in the studies met the international pharmacopoeia standards. Drugs used in the TES in Cambodia, China, Myanmar and Lao PDR were provided directly by WHO after QA testing in recognized reference laboratories. The National Institute of Drug Quality Control of Viet Nam (NIDQC) in Hanoi (a WHO Drug Quality Collaborating Center) provided QA lot testing for artesunate and DHA-PIP used in the TES in Viet Nam and Thailand. Certificates of passed batch analysis were obtained for all lots. After initial treatment on Day 0, all patients were asked to return to receive subsequent doses (until Day 2 for ACTs and chloroquine, and Day 28 for primaquine), and for clinical assessment and blood film examination per protocol on Days 1, 2, 3, 7, 14, 21and 28 (or including Days 35 and 42 for those receiving A+M and DHA-PIP). China, Myanmar and Viet Nam initially could not implement the 42-Day follow-up scheme of DHA-PIP therapy for logistic reasons. Patients were also instructed to return on any other days if they experienced fever or other clinical symptoms suggestive of malaria or other diseases. Patients showing recurrent P. falciparum infection on follow-up received rescue treatment as per national guidelines (artemether+ACT in China;

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iv artemether+mefloquine in Cambodia; quinine sulfate+ doxycycline in Lao PDR, Thailand and Viet Nam; and artesunate+doxycycline in Myanmar). During all follow-up visits, each patient was routinely asked about any prior or newly developed symptoms. When clinically indicated, patients were further evaluated and treated appropriately. All adverse events were clearly documented on a special case report form. Laboratory procedures Parasite enumeration. Giemsa-stained thick and thin blood films were examined at the magnification of 1,000x to identify the parasite species and to determine the parasite density (Parasite density = number of asexual parasites in 200 (1,000x) microscopic fields/number of white blood cells multiplied by an average white blood cell density of 8,000 per µl. When the number of asexual parasites was less than 10 per 200 white blood cells in follow-up smears, counting continued for at least 500 white blood cells). A blood slide was declared negative when no asexual parasites were detected against 1,000 white blood cells counted across the thick smear. The presence of gametocytes was recorded, but was not accounted for in the analysis. In the case of suspicious malaria species, the thin film was examined. Patients found to have mixed species infections were excluded. Two qualified microscopists (Level 2 or above) read all slides independently and parasite densities were calculated using the average of the two counts. Blood smears with significant discordant results (difference between the two microscopists in species diagnosis and/or with >50% difference in parasite densities) were re-checked by a third, independent microscopist blinded to the initial readings, and parasite density calculated by getting the average of the two closest counts. As part of the quality control procedures for China, Cambodia, Thailand and Viet Nam, the third in-country (Level 1 or Level 2) microscopist independently re-read selected slides of their respective countries. Additionally, WHO certified Level 1 expert microscopist provided external cross-checking as the final validation of slides from China, Myanmar and Thailand. Differentiating P. falciparum recrudescence from re-infection. In order to differentiate recrudescence, which can be due to drug resistance, from a newly acquired infection, finger-prick blood samples collected as dry blood spots on filter paper were processed for msp1, msp2 and glurp genotyping using standardized PCR techniques (WHO, 2008b). PCR was carried out in-country except for Lao PDR. Mahidol-Oxford Research Unit (MORU) in Bangkok, which serves as the regional reference lab validating country PCR results as needed, performed the genotyping of samples from Lao PDR (Mekong Molecular Surveillance Network). All results presented here are PCR-corrected, meaning that only recrudescent cases were accounted for in the ACPR calculation. Chloroquine blood levels. To determine if the blood concentration of chloroquine was at an 208

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adequate level in patients with vivax infection showing recurrence, 100 µl blood was collected by finger-prick on Days 0, 7, 28 and day of recurrence on filter paper. However, due to technical inconsistencies in blood sampling and storage, pharmacokinetic assay results of some countries were difficult to interpret (no results are included in this paper). Classification of therapeutic outcomes and assessment of study results Treatment outcomes were assessed on the basis of parasitological and clinical findings according to the latest WHO guidelines (WHO, 2009a) (Box 2). In brief, the patients were classified as having early treatment failure (ETF), late clinical failure (LCF), late parasitological failure (LPF), or an adequate clinical and parasitological response (ACPR). ACPR is the absence of parasitemia on Day 28 (or Day 42), irrespective of axillary temperature, in patients who did not previously meet any of the criteria of early treatment failure, late clinical failure or late parasitological failure. Patients initially enrolled for P. falciparum study who were later diagnosed with vivax malaria during the follow-up or on slide validation were not included in the (P. falciparum) ACPR analysis and vice versa for those initially enrolled as P. vivax cases. Patients who withdrew or were lost to follow-up anytime between D0 to D3 were excluded from the analysis. In P. falciparum, the proportion of patients with PCR-confirmed recrudescence was used to estimate the therapeutic failure. Patients were further removed from the analysis if PCR results indicated that the failure was due to reinfection or if PCR results were inconclusive. Therefore, a PCR-corrected ACPR could be higher than the uncorrected ACPR. Box 2 Classification of therapeutic outcomes The study outcomes were classified on the basis of an assessment of the parasitological and clinical outcome of antimalarial treatment according to the latest WHO guidelines (WHO 2009a). All patients were classified as having early treatment failure (ETF), late clinical failure (LCF), late parasitological failure (LPF), or an adequate clinical and parasitological response (ACPR) based on the following determination: •

ETF: presence of danger signs or severe malaria on Days 1, 2 or 3 with evidence of parasitemia; parasitemia on Day 2 higher than on D0, irrespective of axillary temperature; parasitemia on D3 with axillary temperature ≥37.5ºC; and parasitemia on D3 ≥25% of count on D0.



LCF: danger signs or severe malaria in the presence of parasitemia on any day between Day 4 and Day 28 (or till Day 42) in patients who did not previously meet any of the criteria of early treatment failure; presence of parasitemia on any day between Day 4 and Day 28 (Day 42) with axillary temperature ≥37.5ºC (or history of fever) in

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LPF: presence of parasitemia on any day between Day 7 and Day 28 (Day 42) with axillary temperature <37.5ºC in patients who did not previously meet any of the criteria of early treatment failure or late clinical failure.



ACPR: absence of parasitemia on Day 28 (or Day 42), irrespective of axillary temperature, in patients who did not previously meet any of the criteria of early treatment failure, late clinical failure or late parasitological failure.

Parasite clearance in ACT-treated P. falciparum patients In the absence of molecular markers for artemisinin resistance or any established in vitro artemisinin resistance threshold, the monitoring of ACT efficacy should be supplemented with parasite clearance as measured by “Day 3 parasitemia.” Day 3 parasitemia is defined as the percentage of P. falciparum patients who remain blood smear positive for asexual blood stage parasites on Day 3 (ideally 72 hours) following the initial dose of ACTs (WHO, 2012c). Day 3 parasitemia is the best available parasitological marker of artemisinin sensitivity although its interpretation is complex and influenced by multiple factors, some unknown. Based on the WHO’s ‘working definition’ of artemisinin resistance, ≥ 10% Day 3 parasitemia is considered an early warning signal of ‘suspected’ artemisinin resistance at a given location. That information can be collected practically along with TES conducted in routine monitoring and is also of use to facilitate classification of tiers to plan artemisinin resistance containment operations (WHO, 2011b). Data management and analysis After study completion, data were independently double-entered by the individual country team into Microsoft Excel spread-sheets according to standard procedures and stored under confidential control of the country’s Principal Investigator. Data were analyzed by two methods: the Kaplan-Meier intent-to-treat method and per-protocol method analysis (WHO, 2009b). The primary outcomes representing therapeutic efficacy endpoints were the 28-day (for AL) or preferably 42-day (for A+M and DHA-PIP) PCR-corrected cure rates of each study using Kaplan Meir-survival analysis. Secondary outcomes were the percentages of P. falciparum patients whose Day 3 blood smears remained positive for asexual falciparum parasites. As a supplement, the positive and negative predictive values for treatment failure of Day 3 parasitemia, in a few selected sites, are also calculated. Data analysis was conducted using Intercooled Stata 8.0 software (Stata Corporation, College Station, TX, SN: 198049310). Therapeutic outcome estimates as represented by

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Table 1 Patient characteristics by malaria species and country. Country

Cambodia n=540

China n=127

Lao PDR n=62

Myanmar n=1,099

Thailand n=698

Viet Nam n=306

P. falciparum Males (%) 361 (66.9) 81 (63.8) 48 (77.4) 798 (72.6) 493 (70.6) 211 (69.0) Adults: >15 yrs (%) 355 (65.7) 100 (78.8) 35 (56.5) 860 (78.3) 629 (90.1) 208 (68.0) Day 0 fever ≥37.5 C (%) 501 (92.8) 115 (90.6) 60 (96.8) 767 (69.8) 633 (90.7) 292 (95.4) Day 0 parasite count (µl-1)           Geometric mean 15,328 5,060 6,595 8,267 14,464 11,892 (range)

(506-233,379) (80-127,600) (317-136,597) (100-124,030) (119-243,750) (643-187,686) n=388

n=76

n=552

n=220

n=111

P. vivax Males (%) 279 (71.9) 50 (65.8) 411 (74.4) 158 (71.8) 62 (55.9) Adults (%) 257 (66.2) 56 (73.7) 413 (74.8) 176 (80.0) 49 (44.1) Day 0 fever ≥37.5 C (%) 367 (94.6) 46 (60.5) 410 (74.3) 189 (85.9) 107 (96.4) Day 0 parasite count (µl-1)           Geometric mean 4,149 4,381 4,319 4,341 4,313 (range) (259-73,214) (225-46,227)   (261-166,471) (120-49,672) (117-66,852)

ACPRs, prevalence of Day 3 parasitemia (%) and predictive values (positive and negative) of Day 3 parasitemia are reported with 95% confidence intervals (binomial exact). Differences of ACPRs and % Day 3 parasitemia across years were compared using Pearson chi-square test. 

RESULTS Patient characteristics (Table 1) The majority of the patients were male (>70%) and adults 16-70 years old. Lao PDR had the largest proportion of children ≤15 years old (44%). Day 0 geometric mean parasite densities for P. falciparum ranged from 5,060 in China to 15,328/µl blood in Cambodia, and for P. vivax from 4,149 to 4,381/µl in Cambodia and China, respectively. Most patients were febrile (axillary temperature ≥37.5ºC) at the time of enrolment (>80% in both the P. falciparum and P. vivax groups). In Lao PDR in 2009, there were too few patients with P. falciparum but more patients matching entry criteria were enrolled in 2010 and data collection continued for almost a year into 2011 in Khammoune and Luang Prabang. The sample size, however, remained small despite active case finding effort in surrounding villages, which were experiencing very low transmission. In China, sentinel sites were located close to the Myanmar border and at two of the three sites

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Table 2 PCR-corrected therapeutic efficacy results for P. falciparum by drug, country/site and year of study, 2008-2010. Year Drug/Country Site 2008 2009 2010 n (Total 28-day 42-day n (Total 28-day 42-day n (Total 28-day 42-day analysed) ACPR ACPR analysed) ACPR ACPR analysed) ACPR ACPR (95% CI) (95% CI) (95% CI) (95% CI) (95% CI) (95% CI) DHA-PIP Cambodia Pailin 49 98% 100% 38 92% 92% 29 76% 73% (89.1-99.9) (91.6-100) (78.6-98.3) (78.1-98.3) (56.5-89.7) (52.8-87.3)   Pursat 76 100% 100%       54 92.6% 88.7% (95.2-100) (95.3-100) (82.1-97 9) (77-95.7)   Preah Vihear       59 100% 100% (93.9-100) (93.9-100)         Rattanakiri       54 100% 100% 60 100% 100% (93.5-100) (93.4-100) (94-100) (93.9-100) China Tenchong             19 100% (83.2-100)     Yingjiang             22 95.5% (77.2-99.9)     Menglian       71 100% (94.9-100)         Myanmar Kawthaung       80 97.4%   57 94.7% (90.8-99.7) (85.4-98.9)     Rakhine             80 100% (95.5-100)     Kachin             57 98.2% (90.6-99.9)     Eastern Shan             51 100% (93-100)     Bago       72 100% (95-100)           Mon             75 98.7% (92.8-99.9)   Viet Nam Binh Phuoc       46 97.8% (90.9-99.9)           Dak Nong       37 100% (90.5-100)           Gia Lai   48 100%       60 95% 94.8% (93.9-100) (87.1-99) (85.6-98.9) Quang Tri 66 100% 15 100% 100% (94.6-100) (78.2-100) (78.2-100) A+M Cambodia Preah Vihear       46 100% 97.9% (92.3-100) (88.5-99.9)       Thailand Mae Hong Son 38 100% 100% 27 96.40% 92.3% (90.7-100) (90.7-100) (74.9-99.1)         Tak 62 98.4% 96.8% 53 89.1% 90.4% 39 92.5% 92.3% (88.8-99.6) (78.9-96.8) (79.1-98.4)

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Table 2 (Continued). Year Drug/Country Site 2008 2009 2010 n (Total 28-day 42-day n (Total 28-day 42-day n (Total 28-day 42-day analysed) ACPR ACPR analysed) ACPR ACPR analysed) ACPR ACPR (95% CI) (95% CI) (95% CI) (95% CI) (95% CI) (95% CI)   Kanchanaburi       53 94.6% 92.5% (81.8-97.9)         Ratchaburi 50 100% 100%       44 100% 100% (92.9-100) (92.9-100) (91.9-100) (91.9-100)   Ranong 35 100% 97.15 47 90% 87.2% 47 93.6% 91.5% (85.1-99.9) (74.3-95.2) (79.6-97.6)   Yala 47 100% 100%       52 98.1% 98.1% (92.5-100) (92.5-100) (89.7-99.9)   Ubon Ratcha- 45 (100% (100% thani 92.1-100) 92.1-100)             AL Cambodia Rattanakiri             62 94.9% 88.7% (85.9-98.9) (78.9-95.3) Lao PDR Khammoune       24 95.8%   26 100% (78.9-99.9) (86.3-100)     Luang Prabang             12 91.7% (61.5-99.8)   Myanmar Kawthaung       80 93.8%   84 94% (86-97.9) (86.7-98)     Rakhine             70 100% (94.6-100)     Kachin             59 100% (93.9-100)     Sagaing       73 98.6% (92.6-99.9)           Eastern Shan             50 100% (92.7-100)     Bago       86 98.8% (93.7-99.9)           Kayin             66 97% (89.2-99.6)

almost all patients contracted malaria in Myanmar. China also experienced difficulty in reaching the requested sample size especially in 2009 and 2010. Therapeutic efficacy of ACTs against P. falciparum (Table 2) Efficacy of DHA-PIP (Fig 3a). Over the 3-year study period, DHA-PIP remained highly efficacious (42-day ACPR>98%) against P. falciparum in Rattanakiri and Preah Vihear Provinces of Cambodia (Rithea et al, 2013). However, in Pailin, the 42-day ACPR decreased significantly (p=0.001) from 100% (95%CI 91.6-100, n=49) in 2008, to 92% (95% CI 78.1-98.3, n=38) in Vol 44 (Supplement 1) 2013

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2009, and 73% (95% CI 52.8-87.3, n=29) in 2010, although the 2010 sample size was rather small. In Pursat, just southeast of Pailin, 42-day ACPR also significantly declined from 100% (95% CI 95.3-100, n=76) in 2008 to 89% (95%CI 77.0-95.7, n=54) in 2010 (p=0.003). In Myanmar, TES were carried out at six sites across the countries during 2009-2010. All showed 28-day ACPRs to be consistently >96% indicating that the drug remained highly efficacious. In 2010, results of small samples (<30 at each site) from China showed >95% ACPR in Tengchong and Yingjiang. Similarly, in Viet Nam, the 28-day ACPRs for all 4 sites were 100% or approaching 100%. Two ETFs were recorded in Gia Lai, Viet Nam in 2010. All other cases who failed DHA-PIP were LPF or LCF. Efficacy of A+M (Fig 3b). A+M therapeutic efficacies are available for Cambodia (1 site) and Thailand (7 sites) and all are based on 42-day follow-up. The ACPR was 100% in Preah Vihear (north-central Cambodia) in 2009. In Thailand, the 2008 ACPR at each of the six sentinel sites was >96%. The ACPRs remained consistently >98% in Ratchaburi and Yala through 2010. However, there was a consistent decline for 2008 compared to 2010 at 3 of the 4 sites along the Thai-Myanmar border, ie Mae Hong Son: 100% to 92% (95% CI 75.0-99.1, n=27), Tak: 97% (95% CI 88.8-100, n=62) to 92% (95% CI 79.198.4, n=39), and Ranong: 97% (95% CI 85.1-99.9, n=35) to 87.2% (95% CI 74.3-95.2, n=47). Overall, the 2010 ACPR for these three border sites was slightly above 90% (pooled data). The other site along the same border, Kanchanaburi, only had a single data point with an ACPR of 92.5% (95% CI 81.8-97.9, n=53) in 2009. Preliminary 2011 data indicated a further drop at this site to <90% ACPR (WHO, 2012 b). Efficacy of AL (Fig 3c). The 28-day ACPR was 95% (95% CI 85.9-98.9) for Rattanakiri (north eastern Cambodia) in 2010. In Myanmar, seven TES for AL (28-day follow-up) were conducted during 2009-2010. ACPRs at two sites in 2009 and four sites in 2010 ranged from 94% to 100%. Lower ACPRs were noted for Kawthuang bordering the southwestern border of Thailand in both years; it was 94% (95% CI 86.0-97.9) in 2009 and 94% (95% CI 86.7-98) in 2010. One case in Bago developed ETF in 2009. In Lao PDR, low malaria endemicity delayed study completion. Enrolment had to be extended and results included data from the 2011 malaria season. Based on small sample sizes (Ns<30 per site per year), the 28-day ACPRs were 92% (95% CI 61.5-99.8, n=12) in Luang Prabang in 2011 and 98% (95% CI 81.9-100, n=49) in Khammoune in 2010 and 2011. Parasite clearance as indicated by Day 3-positive parasitemia (%) (Table 3) For DHA-PIP, the most striking finding came from Pailin, western Cambodia, in the area 214

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Therapeutic efficacy of DHA-PIP, 2008-2010

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Therapeutic efficacy of DHA-PIP, with 42-day follow-up, by site, Cambodia, 2008-2010

100

100

95

95

90

90

85

85

80

80

75

75

70

70

Cambodia Pailin

Cambodia Pursat

Cambodia/ East

2008

China

2009

Myanmar

CAM Pailin

Viet Nam

CAM Pursat

2008

2010

2009

CAM/Elsewhere

2010

Therapeutic efficacy of artesunate+mefloquine Cambodia and Thailand, 2008-2010

b 100

90

80

70

ar

on

ihe

M

gS

Pv

CA

e

A TH

A

TH

n Ho

Ma

ri

k Ta n

A TH

ri

Ka

ng

bu

bu

no

ha

na

a ch

A

tc Ra

TH

2008

2009

A

Ra

TH

A TH

on

la Ya

A TH

Ub

2010

Therapeutic efficacy of artemether-lumefrantine by country and site, 2008-2010

c 100 95 90 85 80 75 70

iri

M

/E

an

hu

R MM

t+ es /W

R

MM

g

as

No

oP

La

CA

t

rth

DR

ak

an

tt Ra

t aw

R

K

MM

89% for 42-d FU 2008

2009

2010

*28-day FU

Fig 3–a-c Bar graphs depicting the therapeutic efficacy of DHA-PIP, A+M and AL by sentinel site and/or country, 2008-2010. (28-day follow-up, otherwise indicated). Vol 44 (Supplement 1) 2013

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where artemisinin resistance emerged. There, Day 3 parasitemia rose from 25.5% (95%CI 15.6-41.0, n=53) in 2008 to 45% (95% CI 26.4-64.3, n=29) in 2010 (Fig 4a). In Myanmar, Day 3 parasitemia began to increase to about 20% at two sites near the Thai border (Kawthaung and Mon State) during 2009-2010. Also at two sites in Viet Nam and one in China, the proportions of Day 3 parasitemia went up slightly above 10% during 2009-2010. In Viet Nam, one-year data from Binh Phuoc (southern Viet Nam) in 2009 showed 15% (95% CI 7.2-26.9, n=46). In Gia Lai (also south-central Viet Nam) there was a significant increase from 0% (95% CI 0-6.1, n=48) in 2008 to 11% (95% CI 4.6-21.6, n= 60) in 2010 (p=0.008). In Thailand, an increase in the percentage of Day 3 parasitemia associated with A+M was observed along the Myanmar border (Fig 4b). In 2008 it was 0% in both Tak and Ranong Provinces, but went up significantly to 9.1% (95% CI 2.5-21.7, n=39, p=0.009) and 5.4% (95% CI 1.1-14.9, n=47, p=0.005), respectively, in 2010. A single data point from Kanchanaburi, also located on the Myanmar border south of Tak and north of Ranong, showed 25% (95% CI 13.8-38.3, n=53) in 2009. In western Cambodia, Day 3 parasitemia >10% associated with A+M therapy had been documented since before 2008. The 2009 data from Preah Vihear (north-central Cambodia) showed 0% Day 3 parasitemia indicating that such a delayed parasite clearance may so far be confined to the western part of the country. At the sites where AL data were available, Day 3 parasitemia was much less common (Fig 4c). In Lao PDR, all patients cleared parasites by Day 3. In Myanmar, the Day 3 parasitemia for all sites were <10% and in Rattanakiri (northeastern Cambodia), it was 3.6% in 2010. However, it should be noted that in western Cambodia, where AL was not tested during 2008-2010, Day 3 parasitemia has exceeded 10% in studies done between 2002 to 2004 (Denis et al, 2006b). Inclusion of patients with initially high-density parasitemia (above 150,000/µl blood) (Table 4) Inclusion of patients with Day 0 parasitemia above 100,000/µl was allowed in the TES conducted in the Mekong countries. As malaria incidence dropped further, more cases with high parasitemia were enrolled. The effects of including patients with Day 0 parasitemia >150,000/µl up to 250,000/µl were explored. The inclusion only minimally changed the ACPR estimates and the differences in % Day 3 positive parasitemia were negligible. Table 4 shows comparison of the results with and without the inclusion for selected sites in Thailand and Cambodia, countries that were most affected. Efficacy of chloroquine and DHA-PIP against P. vivax (Fig 5) In Cambodia, while chloroquine showed >95% ACPR for P. vivax in the western border, ACPRs were documented at less than 90% in 2008-2009 in the northeastern province of Rattanakiri (83%, 95% CI 68.6-92.2) and Preah Vihear (88%, 95% CI 77.4-95.2) (Rithea et al, 2013). 216

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Kachin

79 57 72 80 75 57 19.0% 22.8% 4.2% 0 22.5% 3.5% (11-29) (12.7-35.8) (0.9-11.7) (0.0-4.5) (13.9-33) (0.4-12.1) 0.587 - - - -

2009

Rakhine

2010

76 54 7.8% 7.4% (2.8-15.6) (2.1-17.9) 0.984

2008

Total analysis: 42day Day 3 % parasitemia (95% CI) p-value

2011

Bago

29 45.0% (26.4-64.3)

2010

2009

Kawthaung

38 33.0% (19.1-50.2) 0.261

Veal Veng, Pursat

CAMBODIA

Year

Site

49 25.5% (15.6-41)

2009

2008

Year

Total analysis: 42day Day 3 % parasitemia (95% CI) p-value

Pailin

Site

Country

DHA-PIP

2010

2008

2010

Quang Tri

2008

2010

19 5.3% (0.1-26) -

2010

Tengchong

2009 46 37 15.2% 0 (7.2-26.9) (0.0-9.5) - -

2009

Binh Phuoc Dak Nong

22 13.6% (2.9-34.9) -

2010

Gia Lai

71 0 (0.0-5.1) -

2009

Yingjiang

48 60 0 11.3% (0-6.1) (4.6-21.6) 0.008

59 5.1% (1.1-14.1) -

2009

Preah Vihear Menglian

51 66 15 2.0% 0 0 (0.1-10.4) (0.0-5.4) (0-21.8) - NA

East Shan

54 60 0 0 (0.0.-6.6) (0.0-5.9) NA

2009

Rattanakiri

CHINA

Day 3 parasitemia in DHA-PIP treated P. falciparum cases per site/country and year of study.

Table 3a

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218

2009

2010

2009

MYANMAR

AL

2010

2009

2010

LAO PDR 2011

86 73 10.2% 0 (4.8-18.5) (0.0-4.7) - -

70 0 (0.0-5.1) -

Table 3c

66 4.5% (0.9-12.7) -

59 1.7% (0.0-8.9) -

50 2% (0.1-10.6) -

24 0 (0.0-14.20

THAILAND

A+M

2008

2009

2010

2008

2009

2010

2008

2009

2008

2010

2008

2010

2008

2009

Ubon Rat- Kancha- chathani naburi

Total analysis: 42day 62 53 39 35 47 47 38 27 47 52 50 44 45 53 Day 3 % parasitemia 0 14.3% 9.1% 0 17% 5.4% 4.8% 9.1% 0 0 0 4.5% 0 24.5% (95% CI) (0.0-5.5) (6.4-26.2) (2.5-21.7) (0-7.5) (7.6-30.8) (1.1-14.9) (0.6-16.2) (1.9-24.3) (0.0-7.5) (0.0-6.8) (0.0-7.1) (0.6-16.2) (0.0-7.9) (13.8-38.3) p-value 0.009 0.005 0.521 NA 0.128 - -

Year

Rattanakiri

2010

CAMBODIA

46 0 (0.0-7.3) -

2009

Preah Vihear

CAMBODIA

26 12 56 0 0 3.2% (0.0-13.7) (0.0-26.5) (0.4-12.3) NA - -

Day 3 parasitemia in A+M treated P. falciparum cases per site/country and year of study.

80 84 6.3% 8.3% (2-13.9) (3.4-16.4) 0.609

Site Tak Ranong Mae Hong Son Yala Ratchaburi

Country

Total analysis: 28day Day 3 % parasitemia (95% CI) p-value

Site Kawthaung Bago Sagaing Kayin Rakhine Kachin East Shan Khammoune Kahmmoune Luang Prabang

Year

Country

Day 3 parasitemia in Artemether-Lumefantrine (AL) treated P. falciparum cases per site/country and year of study.

Table 3b

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2010

2008

2009

2010

2008

2010

2009

2010

2008

Ubon Rat- chathani

2009

Kanchanaburi

2009

2010

2008

2009

2010

2008

2010

2009

2010

2008

Ubon Rat- chathani

2009

Kanchanaburi

Total analysis 76 54 54 60 59 35 47 47 50 44 45 53 Day 3 % parasitemia 6 (7.8%) 4 (7.4%) 0 0 3 (5%) 0 8 (17%) 3 (5.4%) 0 2 (4.5%) 0 13 (24.5%) (95% CI) (2.8-15.6) (2.1-17.9) (0.0-6.6) (0.0-5.9) (1.1-14.1) (0-7.5) (7.6-30.8) (1.1-14.9) (0.0-7.1) (0.6-16.2) (0.0-7.9) (13.8-38.3) p-value 0.989 NA - 0.005 0.128 -                       PCR-corrected ACPR 100% 88.7% 100% 100% 100% 97.1% 87.2% 91.5% 100% 100% 100% 92.5% (95% CI) (95.3-100) (77-95.7) (93.4-100) (93.9-100) (93.9-100) (85.1-99.9) (74.3-95.2) (79.6-97.6) (92.9-100) (91.9-100) (92.1-100) (81.8-97.9) p-value 0.003 NA - 0.283 NA - -

2008

Year

Thailand (A+M) Preah Ranong Ratchaburi Vihear

Cambodia (DHA-PIP)

Site Veal Veng, Pursat Rattanakiri

Country (Drug)

Excluding patients with Day 0 parasitemia >250,000/cumm

Total analysis 75 48 52 58 57 32 47 47 49 44 45 53 Day 3 % parasitemia 5 (6.4%) 2 (2.4%) 0 0 3 (5%) 0 8 (17%) 3 (5.4%) 0 2 (4.5%) 0 13 (24.5%) (95% CI) (2.2-14.9) (0.5-14.3) (0.0-6.8) (0.0-6.2) (1.0-13.9) (0-10.9) (7.6-30.8) (1.1-14.9) (0.0-7.3) (0.6-15.5) (0.0-7.9) (12.9-36.4) p-value 0.704 NA - 0.024 0.221 -                       PCR-corrected ACPR 100% 88% 100% 100% 100% 96.9% 87.2% 91.5% 100% 100% 100% 92.3% (95% CI) (95-100) (74.8-95.3) (93.2-100) (93.8-100) (93.9-100) (85.1-99.9) (74.3-95.2) (79.6-97.6) (92.7-100) (91.9-100) (92.1-100) (81.5-98) p-value 0.003 NA - 0.329 NA - -

2008

Year

Thailand (A+M) Preah Ranong Ratchaburi Vihear

Cambodia (DHA-PIP)

Site Veal Veng, Pursat Rattanakiri

Country (Drug)

Excluding patients with Day 0 parasitemia >150,000

Comparison of Day 3 and ACPR outcome in relation to initial parasitemia in selected sites.

Table 4

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b

c

PCT of DHA-PIP by country and site, 2008-2010*

% Day 3 parasitemia for patients treated with artesunate+mefloquine, Cambodia and Thailand, 2008-2010

% Day 3 parasitemia for patients treated with AL* by Country/Site, 2008-2010

Fig 4–a-c Bar graphs depicting Day 3 parasitemia in TES patients treated with DHA-PIP, A+M and AL by sentinel site and/or country, 2008-2010. 220

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Therapeutic efficacy of chloroquine against P. vivax by country and site, 2008-2010* 100

95

90

85

VTN Quang Tri

VTN Nin Thuan

VTN Binh Phuoc

THA Yala

THA Chanthaburi

THA Kan

THA MHS

MMR Mon

MMR Bago

MMR East Shan

MMR Kachin

MMR Rakhine

MMR Sagaing

MMR Kawthuang

CAM Rattanakiri

CAM Preah Vihear

CAM Pailin

75

CHN Yingjiang

80

Fig 5–ACPR of chloroquine against P. vivax by country/site, 2008-2010.

The therapeutic efficacy of chloroquine in Myanmar remained above 90% at all sites (Sagaing Division, Rakhine, Mon, Kachin and Eastern Shan states) except Kawthaung (Thanintharyi Region) in the south bordering Thailand, where the Day 28 ACPR was 80% (95% CI 68.2-89.4) in 2009 and 88% (95% CI 77.8-94.7) in 2010. In Thailand, chloroquine was assessed against P. vivax in Chanthaburi and Yala in 2009 and in Kanchanaburi and Mae Hong Son in 2010. The therapeutic efficacy remained above 90% at all of the four sites. All treatment failures were LPF and the study participants were asymptomatic. Comparing results from the 4 sites, patients from Chanthaburi showed the highest level of parasitemia on admission and the slowest parasite clearance, but there was no clear evidence of P. vivax resistance to chloroquine from this site (Congpuong et al, 2011), or any other site in Thailand. In Viet Nam, chloroquine demonstrated generally high efficacy against vivax malaria. An ACPR of 100% was documented in Binh Phuoc in 2009 and in Quang Tri in 2010. However, in Ninh Thuan it was 88% (95% CI 77.2-94.5) in 2009. Data was available from only one site in China; the ACPR was 96% (95%CI 86.0-100, n=45) in Yingjiang in 2009. Due to low incidence of P. vivax, TES were not conducted in Lao PDR during 2008-2010.

DISCUSSION In 2001, the WHO recommended ACTs as first-line drugs (WHO, 2001), and in 2006, urged Vol 44 (Supplement 1) 2013

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member states to “cease the provision in both the public and private sectors of oral artemisinin monotherapy” (WHO, 2010b). Compliance with these recommendations by the National Malaria Control Programs (NMCPs) in the six countries of the GMS has evolved slowly (Chapter 7). As the result of documented high-level falciparum resistance to mefloquine in the border areas, the Thai NMCP decided to implement a two-day artesunate-mefloquine combination (A+M) regimen in 1995 in selected locations only (Wongsrichanalai et al, 2004) which in retrospect might have contributed to the increasing tolerance of falciparum to artemisinins (WHO, 2007b). The country adopted the two-day A+M regimen nationwide in 2005 and then modified the regimen to three-day in accordance with WHO recommendations in 2008 (Chapter 7). In Cambodia, A+M has been used to manage falciparum infections since 2000, but the national program decided to shift to dihydroartemisinin-piperaquine (DHA-PIP) in selected western provinces in 2009 as the result of increasing failure rate of A+M on the border with Thailand. From 2010, DHA-PIP has been made the first-line therapy for the management both P. falciparum and P. vivax infections following evidence of increased A+M treatment failures in western border provinces and chloroquine treatment failures in vivax infections in the eastern part of Cambodia (Chapter 7). However, the actual implementation of the policy took place in 2012. In Myanmar the initial policy to use A+M as the first line drug against P. falciparum was set in 2002, then DHA-PIP and AL were added in 2005, thus the country operated on multiple first-line antimalarial drugs depending on funding sources (Chapter 7). After more than two decades of successful countrywide implementation of a 5 to 7-day artemisinin monotherapy regimen, national programs in Viet Nam and China decided to shift to an ACT, DHA-PIP in 2009, to manage uncomplicated falciparum infections (Thanh et al, 2010; Cawthorne and Schapira, 2010; Cawthorne et al, 2010). Viet Nam also used CV8 (a fixed combination tablet composed of DHA, PIP, trimethoprim and primaquine) nationwide up to the commune level from 2003 to 2008 (WHO, 2005a). In China, three other ACTs are currently part of the first-line drug policy namely: artesunate-amodiaquine in blister pack, co-formulated single dose naphthoquine-artemisinin and artemisinin-piperaquine (also co-formulated) mainly for trade and export purposes, but actually using only DHA-PIP against falciparum infections in the country (Chapter 7). In 2001, the national program in Lao PDR decided to shift from chloroquine + sulfadoxinepyrimethamine to artemether-lumefantrine (AL) to manage both falciparum and non-falciparum infections. Countrywide implementation of that policy started only in 2005 with Global Fund to Fight AIDS, TB, and Malaria (GFATM) support. Monitoring the therapeutic efficacy of AL started in 2003 at four sentinel sites across the country. To date, as per in vivo TES network results, which have been confirmed by similar studies conducted in Lao PDR, AL remains highly effica-

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cious countrywide (Mayxay et al, 2012b). With the scaling up use of ACTs as first line antimalarial drug in the region, the importance of carefully and systematically tracking the therapeutic efficacy of ACTs in each country and in the GMS has been taken seriously by programme managers with the technical support of WHO and development partners (WHO, 2007b). The Mekong in vivo therapeutic efficacy study Network jointly managed by NMCPs and WHO has contributed to provide policy makers with early warning signals of decreasing efficacy of ACTs across the GMS. This was acknowledged during a strategic technical consultation in Cambodia in 2007 (WHO, 2007a) which was the basis to further strengthen the capacity of the TES Network engaging country principal investigators to operate common good clinical and laboratory practices (WHO, 2010b; WHO, 2012b). All GMS countries have gradually changed their first line national drug policies to follow WHO recommendations and are actually promoting free or at-cost ACTs from government-run health stations with progressive involvement of private providers. The scaled-up implementation of new and more expensive drug policies in remote places and as part of development projects attracting migrants remains a serious challenge which is discussed in Chapter 4 & 7. It includes the actual implementation of the ban of artemisinin monotherapies and various unknown cocktail regimens and sub-standard medicines still widely available in some locations (Newton et al, 2008; Nayar et al, 2012). Prior to 2008, ACTs have remained highly efficacious in the GMS with the notable exception of western Cambodia and southeastern Thailand, where A+M efficacy (42-day follow-up) significantly dropped below 90% (Denis et al, 2006; Vijaykadga et al, 2006). Emerging falciparum resistance to selected ACTs on the Cambodia-Thailand border became a source of regional and global concern for policy makers and researchers triggering additional research and development partners’ interest (WHO, 2007a). Interpretation of results from in vivo TES of combination artemisinin derivatives with different partner drugs, with different but synergistic modes of action is not as straightforward as it was with other previous antimalarials used in malaria control (WHO, 2007a). The rising A+M therapeutic failure rate observed in selected locations in Thailand could be the consequence of the pre-existing mefloquine resistance in the country (Wongsrichanalai et al, 2004). Drug pressure on mefloquine has been considerable over the last decades especially in Thailand where mefloquine has been used as monotherapy or with sulfadoxine-pyrimethamine from 1986 at different regimens in different locations (15 or 25 mg/kg in one or 2-day regimens) and later in combination with artesunate (loose tablets) in selected locations and then countrywide from 2005 (Vijaykadga et al, 2006). Thus the progressive decline in A+M therapeutic efficacy is a major concern not only for Thailand, but in the whole Subregion and globally since falciparum response to artemisinins (which have been under pressure) is seriously declining in selected Vol 44 (Supplement 1) 2013

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locations (Wongsrichanalai and Meshnick, 2008; Rogers et al, 2009; WHO, 2010b). As a result, the Thai NMCP continues to monitor A+M and is actively searching for suitable safe alternatives to A+M in collaboration with national and international academic and research institutions. Treatment failures observed from 2008 to 2010 with DHA-PIP in Pailin and Pursat have quickly reached unacceptable levels. This is in the same area of western Cambodia and eastern Thailand that resistance of P. falciparum to other antimalarials has previously emerged (Singhasivanon et al, 1999). The recent results are of serious concern and could be related to emerging falciparum resistance to piperaquine, a partner drug which shares chemical structure similarities with chloroquine and had been used as monotherapy previously (Yang et al, 1997; Davis et al, 2005). The long history of mefloquine and piperaquine being used as monotherapies in the region is well known (Yang et al, 2007) as well as the unregulated availability and use of DHA-PIP in some locations in Cambodia and the northern part of Myanmar bordering China for the past decades (Cawthorne and Schapira, 2010). That context raises concern on the expected long-term effectiveness of DHA-PIP as a first line drug in the GMS. Determination of parasite clearance time as measured by percent Day 3 parasitemia (ideally 72-hours after initiation of treatment) is so far considered as a surrogate marker to identify suspected hotspots of P. falciparum resistance to artemisinins (Stepniewska et al, 2010). This data can be captured in sentinel sites from quality TES managed by national programs. Unfortunately, the number of sentinel sites per country is limited due to the number of skilled staff, funds, and mandatory protocol requirements necessary to implement TES standards. Following field experiences from NGOs in Cambodia, one suggested option to intensify artemisinin resistance monitoring was to limit individual follow-up of each patient only until Day 3 in additional sites (WHO, 2011a). However, even if considered far less complicated than the full TES study design, these “Day 3 simplified studies” still require a highly demanding protocol in terms of entry criteria, adherence to treatment, quality microscopy, quality drugs, accurate follow-up, adequate data management, and mandatory ethical prerequisites. To avoid multiplication of false alerts and further investigations to cross check results, WHO recommended that such studies should be conducted by research institutions in collaboration with field partners (WHO, 2012c). To date there is no established relationship between prolonged parasite clearance and ACT therapeutic failures in P. falciparum. Percent Day 3 parasitemia has a low positive predictive value for 28-day (and likely so for 42-day) treatment failures with A+M in Thailand; however, the negative predictive value is consistently high, meaning that patients who cleared parasites before Day 3 are likely to be fully cured (Vijaykadga et al, 2012). Results from selected sites in recent TES confirm these conclusions. A negative predictive value of 99.4% and 92.3% in Tak and Pursat, respectively, but only 72% in Pailin, as may be expected from other resistance data, have been found. 224

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Between 2001 and 2007, the proportion of patients with parasites on Day 3 associated with either AL or A+M has been progressively exceeding 10% in the western part of Cambodia, including the provinces of Pailin, Battambang, and Kampot. Across the border in Trat and Chanthaburi Provinces of Thailand, the proportion of slow-clearing patients under A+M therapy has increased in 2001 to exceed 10% in 2010. From TES, the parasite clearance time (PCT) reached 3.7 days in 2007 in Trat Province as compared to 2.0 days in 2003 (Vijaykadga, BVBD, 2006, personal communications). These findings, in addition to in vivo and in vitro studies conducted across the region, have mobilized the international community and highlighted the urgent need to better understand the situation, build additional evidence, and address the emerging artemisinin resistant threat first on the Cambodia-Thailand border and further in the region and globally (WHO, 2007a; 2008a; 2011b; Chapter 7) The emerging foci of delayed parasite clearance associated with DHA-PIP in eastern Myanmar deserve attention and action. Almost 20% of patients treated with DHA-PIP remained positive on Day 3 in Kawthaung (Tanintharyi region bordering the province of Ranong in Thailand) and in Mon State. These observations from the TES led to a 7-day artesunate monotherapy study with additional pharmacokinetic investigations in 2011. Results demonstrated a high percentage of patients remaining parasite positive on Day 3 (27%, 95% CI 15.6-41) but no single patient with LTF was detected during the 28-day follow-up. Pharmacokinetic results showed an appropriate blood level of the drug and could not explain the delayed parasite clearance (WHO, 2012b). Thus the area remains classified as a ‘suspected foci’ for artemisinin resistance (Fig 2) as per WHO classification (WHO, 2011b). The proportion of DHA-PIP treated patients remaining with parasites on Day 3 in some locations in Viet Nam also raises concerns that these area are becoming a suspected hotspot of reduced artemisinin sensitivity either de novo or extended from the Cambodia-Thailand border. Recent studies in Viet Nam showed that DHA-PIP remained highly efficacious from a therapeutic viewpoint but demonstrated longer parasite clearance times (Hien et al, 2012). The proportion of patients treated with AL who remained positive on Day 3 was below 10% at all locations with the exception of Bago Region, Myanmar, where that proportion was only slightly above the threshold in 2009. Further investigations are needed to confirm those findings. However, AL appears to remain one of the most powerful ACTs in the GMS except in western Cambodian provinces (Denis et al, 2006a). The actual implication of the delayed parasite clearance time (which is partly explained by the parasite’s heritable trait) under ACTs in the Subregion and elsewhere remain to be fully understood. Meanwhile, the increasing number of patients still documented with falciparum parasites on Day 3 with DHA-PIP in Cambodia, Viet Nam and Myanmar, and with A+M in Thailand is of concern. These data underscore the importance of continued monitoring of ACTs by Vol 44 (Supplement 1) 2013

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national malaria programs alongside national, regional and global research institutions. Small sample sizes, due to the declining number of malaria cases in the Subregion, might have contributed to less precise outcome estimates. In some countries, the study duration extended to two malaria seasons in an effort to meet the required sample size. Studies were planned to take place during the peak transmission season, but start dates were sometimes delayed by the ethical approval process, logistic hurdles, and fund transfer mechanisms. As a result, the study duration varied depending on the pace of patient recruitment at individual sites. Challenges in identifying an adequate number of patients also led to including a small number of cases with parasitemia levels above 100,000/µl and below 1,000/µl. However, as per TES results, inclusion of additional patients with these entry criteria did not significantly change ACPRs or the proportion of patients still positive at Day 3. The Mekong in vivo TES Network has significantly contributed to addressing the knowledge gap by increasing the pool of valuable information in the GMS. Information obtained from studies conducted in a standardized way across Mekong countries have already proven beneficial to countries in the programmatic use of antimalarials and strategic planning such as the recent policy change decisions made in Cambodia and Thailand. With WHO technical assistance, complemented by strong financial support from the President’s Malaria Initiative (PMI) and the GFATM, the Mekong Network has also created a leading partnership and commitment among the six countries in the GMS to work collaboratively. Funding support from national governments and direct participation by local public health personnel further enhanced the awareness of the program managers of the need to continue assessing the efficacy of firstline antimalarial medicines and to increase the capacity of national programs to perform quality research studies. Ultimately the multi-country TES results have helped to define geographical areas or ‘tiers’ in order to plan and advocate for specific artemisinin resistance containment interventions (WHO, 2011b). The TES results of chloroquine indicated that vivax infections are properly managed by chloroquine in the GMS with the exception of eastern Cambodia and southern Myanmar where ACPRs were less than 90%. This triggered a policy shift to DHA-PIP in Cambodia. Subsequent results in 2010 from all sites in eastern and western Cambodia showed a 100% efficacy with DHA-PIP. The situation in Myanmar also deserves further investigations. Continuous TES monitoring of chloroquine for P. vivax is being planned in all countries except Lao PDR and Cambodia. In conclusion, results presented here consolidate the latest situation of GMS countries in their ongoing efforts to track drug resistant malaria. Standardized in vivo TES have been implemented across multiple settings in the GMS. That approach makes possible a comprehensive picture of the resistance status of ACTs and allows comparison of therapeutic efficacy results of individual ACTs in various and even remote geographical locations. 226

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ACTs have been widely used in the GMS since 1995 and in other areas from 2002 (Delacollette et al, 2009). ACTs are largely acknowledged as the cornerstone of modern-day malaria control efforts and globally considered as the most effective treatments to manage uncomplicated falciparum malaria. Overall in the GMS, the therapeutic efficacy of ACTs remains satisfactory and has contributed to accelerating substantial gains in reducing malaria incidence and malaria deaths in the subregion during the last decade (Chapter 2). However, TES results from some sentinel sites presented here are raising concern over possible extension of artemisinin resistance development. To date, only two locations in the world have been documented as ‘confirmed’ artemisinin resistance hotspots based on WHO working definition (WHO, 2011a,b). Both of them are in the GMS, namely the western border of Cambodia and Thailand, and in Tak Province of northwestern Thailand bordering Myanmar (Noedl et al, 2008; Dondorp et al, 2009; Phyo et al, 2012). These confirmations have triggered extra specific containment interventions in those locations (Chapter 7). Public health policy makers and malaria researchers should take the issue seriously in order to protect gains reached so far towards malaria elimination and to mitigate the extension of resistance beyond the Mekong Subregion. Successful TES require good team work, a well-planned study scheme, attention to details, and stringent QA/QC processes to ensure accuracy of data to inform policy-makers and sustain funding support. Improvement has been noticeable as compared to previous studies in the Subregion and national programs have realized the importance of running high quality TES sentinel sites.

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