In vivo monitoring of dihydroartemisinin-piperaquine sensitivity in Plasmodium falciparum along the China-Myanmar border of Yunnan Province, China from 2007 to 2013

China and Myanmar share 2,185 kilometre border. There are 19 counties of China and fives special regions of Myanmar on the border. Populations includes 4,687, 896 residents of 19 counties of Yunnan Province of China and 586,000 residents of the five Special Regions of Myanmar. Due to the complex emergency situation in the five Special Regions of Myanmar, where fell outside the coverage of national malaria control efforts supported by Myanmar’s Ministry of Health, effective access of malaria interventions could only be achieved from China. In March, 2008, the baseline survey of evaluation indicator for the sixth grant to China of the Global Fund to fight AIDS, Tuberculosis and Malaria showed that the parasite prevalence rate was 13.6% (761/5585, 95% CI: 12.7–14.6%), and the proportion of P. falciparum among slides with parasites was 38.1% (290/761, 95% CI: 34.6–41.7%) in the five Special Regions of Myanmar. In eliminating settings, malaria predominantly occurs in border areas and imported cases tend to represent a majority of recorded cases [19]. In order to recruit malaria patients for the study, surveillance was conducted in Yingjiang of Dehong Prefecture in 2007, Menglian of Puer Prefecture in 2009, in both Tengchong of Baoshan Prefecture and Yingjiang of Dehong Prefecture from 2010 to 2013 (Figure 1).

Patients whose axillary temperature was ≥ 37.5°C or with a history of fever during the previous 24 hours were diagnosed by using microscopy of thick and thin blood smears. Patients with mono-infections of P. falciparum were recruited to this study, after obtaining full informed consent. Only patients older than one year, body weight ≥ 5 kg, presenting with parasite density 500–100,000 parasites per μl were enrolled into the study. Imported malaria was identified as patients who had travelled from endemic areas of Myanmar within one month and were diagnosed as malaria in China [13]. Patients were excluded from the study if any of the following criteria were present: (1) positive pregnancy test or breastfeeding; (2) complicated malaria; (3) having taken any anti-malarial, tetracycline and sulphonamides derivatives drugs within the past seven days; (4) history of hypersensitivity to any of the study drugs; (5) presence of febrile conditions due to diseases other than malaria (e.g. measles, acute lower respiratory tract infection, severe diarrhoea with dehydration) or other known underlying chronic or severe diseases (e.g. cardiac, renal and hepatic diseases, HIV/AIDS); (6) over 60 years; (7) presence of severe malnutrition (defined as a child whose growth standard is below –3 z-score, has symmetrical oedema involving at least the feet or has a mid-upper arm circumference < 110 mm); and, (8) unable to follow-up [20,21].

DAPQ was manufactured by Zhejiang Holley Nanhu Pharmaceutical Co. Ltd, and provided by the West Pacific Office of WHO (WPRO/WHO). The drug quality was controlled by WPRO/WHO too. The batch numbers of DAPQ were 600807 (manufactured on 30 Aug 2007), 460909 (23 Sep 2009) and 400911 (20 Sep 2011). Each tablet contains 40 mg base dihydroartemisinin and 320 mg piperaquine phosphate. DAPQ was given once a day for three days and the dosing were based on the recommendation of WPRO/WHO. For convenient administration, the doses were calculated into tablets for different categories of kg body weight ranges (Table 1). The treatment intake was observed while the patients visited the hospital as requested for the first three days. Patients of late clinical failure and late parasitological failure were subsequently given a total dose of artemisinin-naphthoquine 24.5 mg/kg (naphthoquine 7 mg/kg and artemisinin 17.5 mg/kg), once a day for three days [22].

Parasite microscopy was conducted and axillary temperatures were measured on admission and every 8–12 hours in the first three days. Patients were asked to visit the hospital and further parasitological examinations were performed on day 7, 14, 21, 28, 35 and 42. In a case of patient had not visited the hospital, a researcher actively visited the patient home to prepare blood smears. Malaria blood films were stained with Giemsa, and slides were examined by two independent microscopists and considered negative if no parasites were seen after examination of 200 oil-immersion fields in a thick blood film. Parasite clearance was defined as no asexual parasite per 500 white blood cells being detected in two continuously microscopic examinations with an 8–12 hr interval. Fever clearance was defined as axillary temperatures <37.1°C in duration of 24 hours. Parasites were counted per 500 white blood cells. The number of parasites was calculated as per μl of blood by the level of 8,000 of leukocyte per μl [23]. A filter paper dried blood spot (about 100ul blood) was prepared on admission, day 7, 14, 21, 28, 35 and 42, day of treatment failure or at any other unscheduled visit, and subsequently stored in plastic zip bags containing silica gel dessicant. Polymerase chain reaction (PCR) was performed respectively to distinguish between reinfection and recurrence of asexual parasites of blood stages, to confirm the Plasmodium species or to detect mixed infection. DNA extraction and genotype analysis were conducted based on investigation of the three polymorphic genetic markers msp1, msp2, and glurp, according to WHO recommended procedures [24]. Recrudescence was defined as at least one identical allele for each of the three markers in the pre-treatment and post-treatment samples. New infections were diagnosed when all alleles for at least one of the markers differed between the two samples. Cases with new infection were excluded from the analysis [25]. In case of failure after day 7, patients whose PCR results were unknown were excluded from the analysis too [11].

Treatment outcome was categorized based on the WHO definitions for early treatment failure (ETF), late clinical failure (LCF), late parasitological failure (LPF), and adequate clinical and parasitological response (ACPR). The ETF definition was to conform to any one of the criteria: (1) danger signs or severe malaria on day 1, 2 or 3, in the presence of parasitaemia; (2) parasitaemia on day 2 higher than on day 0, irrespective of axillary temperature; (3) parasitaemia on day 3 with axillary temperature ≥ 37.5°C; and, (4) parasitaemia on day 3 ≥ 25% of count on day 0. The LCF definition was to satisfy any one of the criteria: (1) danger signs or severe malaria in the presence of parasitaemia on any day between day 4 and day 42 in patients who did not previously meet any of the criteria of ETF; and, (2) presence of parasitaemia on any day between day 4 and day 42 with axillary temperature ≥ 37.5°C in patients who did not previously meet any of the criteria of ETF. LPF definition is to satisfy presence of parasitaemia on any day between day 7 and day 42 with axillary temperature < 37.5°C in patients who did not previously meet any criteria of ETF or LCF. ACPR definition was to satisfy absence of parasitaemia on day 42, irrespective of axillary temperature, in patients who did not previously meet any criteria of ETF, LCF or LPF [20,21].

The data were by double independent data entry and analysed by the Kaplan-Meier method [22,26]. The patients of loss to follow-up and withdrawal from the study were not involved into the analysis. Mean fever and parasite clearance time were compared by covariance through Epi Info 6.04 [22,26]. The proportions of ETF, LCF, LPF and ACPR were, respectively, compared by Chi-square test for trend of quantitative data [27]. The treatment outcome was assessed on the basis of parasite clearance from the blood.

According to the Helsinki Declaration, ethical approval for the study was granted by the Ethics Committee of Yunnan Institute of Parasitic Diseases, China. The purpose of the study was explained and then approval was sought from patients and their caretakers. Informed written consent was obtained from patient or carers of Child patients. All results were kept confidential and were unlinked to any identifying information.