In vivo monitoring of dihydroartemisinin-piperaquine sensitivity in Plasmodium falciparum along the China-Myanmar border of Yunnan Province, China from 2007 to 2013
Liu et al. Malaria Journal
In vivo monitoring of dihydroartemisinin- piperaquine sensitivity in Plasmodium falciparum along the China-Myanmar border of Yunnan Province, China from 2007 to 2013
Hui Liu 0
Heng-lin Yang 0
Lin-hua Tang 2
Xing-liang Li 0
Fang Huang 2
Jia-zhi Wang 1
Chun-fu Li 0
Heng-ye Wang 0
Ren-hua Nie 0
Xiang-rui Guo 3
Ying-xue Lin 3
Mei Li 2
Jian Wang 0
Jian-wei Xu 0
0 Yunnan Institute of Parasitic Diseases, Yunnan Provincial Center of Malaria Research, Yunnan Provincial Collaborative Innovation Center for Public Health and Disease Prevention and Control, Yunnan Provincial Key Laboratory of Vector-borne Diseases Control and Research , Puer 665000 , China
1 Tengchong County Center for Disease Control and Prevention , Tengchong 679100 , China
2 National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention , Shanghai 200025 , PR China
3 Yangjiang County Center for Disease Control and Prevention , Yingjiang 679300 , China
Background: Artemisinin-based combination therapy (ACT) is the recommended first-line treatment of falciparum malaria in all endemic countries. Artemisinin resistance in Plasmodium falciparum has been confirmed in the Greater Mekong subregion (GMS). Dihydroartemisinin-piperaquine (DAPQ) is the most commonly used ACT in China. To understand the DAPQ sensitivity of P. falciparum, DAPQ resistance was monitored in vivo along the China-Myanmar border from 2007 to 2013. Methods: Eligible patients with mono-infections of P. falciparum were recruited to this study after obtaining full informed consent. DAPQ tablets for different categories of kg body weight ranges were given once a day for three days. Patients were followed up for 42 days. Polymerase chain reaction (PCR) was conducted to distinguish between re-infection and recrudescence, to confirm the Plasmodium species. The data were entered and analysed by the Kaplan-Meier method. Treatment outcome was assessed according to the WHO recommended standards. Results: 243 patients were completed valid follow-up. The fever clearance time (FCT) and asexual parasite clearance times (APCT) were, respectively, 36.5 10.9 and 43.5 11.8 hours, and there was an increasing trend of both FCT (F = 268.41, P < 0.0001) and APCT (F = 88.6, P < 0.0001) from 2007 to 2013. Eight (3.3%, 95% confidence interval, 1.4-6.4%) patients present parasitaemia on day three after medication; however they were spontaneous cure on day four. 241 (99.2%; 95% CI, 97.1-99.9%) of the patients were adequate clinical and parasitological response (ACPR) and the proportions of ACPR had not changed significantly from 2007 to 2013 (X2 = 2.81, P = 0.7288). Conclusion: In terms of efficacy, DAPQ is still an effective treatment for falciparum malaria. DAPQ sensitivity in P. falciparum had not significantly changed along the China-Myanmar border of Yunnan Province, China. However more attentions should be given to becoming slower fever and parasite clearance.
Plasmodium falciparum; Dihydroartemisinin-piperaquine; In vivo test; Resistance; China-Myanmar border
Malaria remains one of the major global public health
problems . Intensive efforts in controlling malaria are
producing impressive results. The number of malaria
cases has fallen by more than half in 40% of malaria
endemic countries in the recent decade. Estimates suggest
that nearly 750,000 lives have been saved in Africa alone
thanks to malaria control measures .
Artemisininbased combination therapy is the recommended
firstline treatment of falciparum malaria in all endemic
countries. However, the emergence of artemisinin
resistance in malaria parasites is threatening malaria control
and elimination programmes. Artemisinin resistance in
Plasmodium falciparum has been identified and
confirmed in Cambodia, Thailand, Myanmar, and Vietnam
[2-10]. Other suspected foci have been identified in the
Greater Mekong Subregion (GMS), but are not yet
confirmed. The threat must be taken seriously. Resistance to
previous generations of anti-malarials spread rapidly
around the world, resulting in an increase in child
mortality and untold number of deaths . The
artemisininbased combination therapy (ACT) is the most potent
weapon in treating falciparum malaria  and no other
anti-malarial is available that offers the same level of
efficacy and tolerability. In order to contain artemisinin
resistance, the World Health Organization (WHO) Global
Malaria Programme launched the Global Plan for
Artemisinin Resistance Containment. Surveillance of
artemisinin sensitivity in P. falciparum is one of important
components to prevent the emergence of new foci of
resistance, as well as to limit the spread of resistance .
China has declared a national policy for malaria
elimination by 2020 [12,13]. ACT is the first-line therapy for
falciparum malaria and dihydroartemisinin-piperaquine
(DAPQ) is the most common anti-malarial drug in
China . Historically, population movement has
contributed to the spread of disease. Failure to consider this
factor contributed to failure in malaria eradication
campaigns in the 1950s and 1960s [15,16]. Resistance to
anti-malarial drugs has often threatened malaria
elimination efforts and historically has led to the short-term
resurgence of malaria incidences and deaths . With
the development of modern transportation, the world is
becoming smaller and smaller. China has the largest
population in the world and Chinese people move around
globally , especially to some parts of the GMS where
artemisinin resistance has been identified and confirmed.
The Yunnan Province is in southwestern China and
belongs to the GMS. The Chinese Myanmar border is one of
the very few remaining areas of malaria transmission in all
of China. In 2013, only 423 malaria cases, 81 (19.1%)
P. falciparum, were reported along the border in China,
and the annual parasite incidence was only 0.9 (95% CI,
0.81.0) per ten thousand. Monitoring artemisinin resistance
in Yunnan Province would contribute both to malaria
elimination in China and in containing global artemisinin
resistance. From 2007 to 2013, DAPQ resistance was
monitored in vivo to determine the dynamics of P. falciparum
sensitivity to DAPQ on the China- Myanmar border.
Surveillance sites and time
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 Myanmars 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:
12.714.6%), 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 . 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
Patients and recruiting criteria
Patients whose axillary temperature was 37.5C 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 l 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 .
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
Figure 1 Surveillance sites: Menglian, Tengchong and Yingjian in Yunnan Province of China and relative to neighbouring countries.
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].
Drug and administration
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
artemisininnaphthoquine 24.5 mg/kg (naphthoquine 7 mg/kg and
artemisinin 17.5 mg/kg), once a day for three days .
Laboratory and in vivo clinical monitoring
Parasite microscopy was conducted and axillary
temperatures were measured on admission and every 812 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
Table 1 Number of DAPQ tablets for each category of
body weight range
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
oilimmersion 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 812 hr interval. Fever clearance was
defined as axillary temperatures <37.1C in duration of
24 hours. Parasites were counted per 500 white blood
cells. The number of parasites was calculated as per l of
blood by the level of 8,000 of leukocyte per l . 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 . 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 . In
case of failure after day 7, patients whose PCR results were
unknown were excluded from the analysis too .
Classification standards for treatment outcome
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 37.5C;
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 37.5C 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 < 37.5C 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
Statistical analysis and resistance assessment
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 . 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.
A total of 290 falciparum malaria patients were recruited
in the study during 20072013 (the study was
interrupted in 2008 because of shortage of funding), 22(7.6%)
withdrew, 25(8.6%) were lost at follow-up, and 243
(83.8%) completed valid follow-up, of which 178 (72.3%)
were Burmese (Table 2). The fever clearance time (FCT)
and asexual parasite clearance times (APCT) were,
respectively, 36.5 10.9 and 43.5 11.8 hours. The results
of covariance analysis showed an increasing trend of
both FCT (F = 268.41, P < 0.0001) and APCT (F = 88.6,
P < 0.0001) (Table 3). Eight (3.3%, 95% confidence
interval, 1.46.4%) patients present parasitaemia on day 3
after medication, three in 2010, one in 2011, three in
2012 and one in 2013 (Figure 2); and then parasites were
spontaneously cleared by day 4. The results showed that
241 (99.2%; 95% CI, 97.199.9%) of the patients were
ACPR, one ETF in 2012 and one LCF in 2007, without
LPF (Table 3). The results of Chi-square test for trend of
quantitative data showed that percentages of ACPR had
not changed significantly from 2007 to 2013 (X2 = 2.81,
P = 0.7288) (Table 3). The ETF patient was cured
spontaneously by day four. The LCF patients responded well
to the three-day treatment regimen of
artemisininnaphthoquine tablets. The ETF and LCF cases were
imported from the neighbouring districts of Myanmar.
The PCR identified the LCF as recrudescence on day
28 in 2007, and did not identify any new infection of
Body temperature (C)
Parasite count (per ul)
Plasmodium falciparum resistance to artemisinin
derivatives in Southeast Asia threatens malaria control and
elimination activities worldwide [28,29]. The results of
the study showed that the FCT and APCT were
increasing from 2007 to 2013 (Table 3); 3.3% patients present
parasitaemia on day 3 after medication, but were
spontaneously cured by day 4 without treatment. The
principal pharmacodynamic advantage of using artemisinins is
that they accelerate parasite clearance by clearing young,
circulating, ring-stage parasites and preventing the further
maturation and sequestration of these parasites. This
effect accounts for the rapidity of the therapeutic response,
its lifesaving benefit in patients with severe malaria,
and the notable gametocytocidal activity of the drugs
[3,28-30]. The reduced susceptibility of ring-stage
parasites causes the slow parasite clearance [29-33]. The
markedly prolonged time to parasite clearance showed
a decline in the efficacy of DAPQ during the six years.
However, DAPQ was still highly efficacious for the
treatment of falciparum malaria on the China-Myanmar border
areas, presumably because of the efficacy of piperaquine
X2 = 2.61
X2 = 5.00
X2 = 2.81
Note: FCT = fever clearance times (hours), APCT = asexual parasite clearance times (hours); ETF = early treatment failure, LCF = late clinical failure, LPF = late parasitological failure,
ACPR = adequate clinical and parasitological response, and 95%CI = 95% confidence interval.
Figure 2 Number (%) of patients of parasitaemia presence on day 1, 2 and 3 after medication.
phosphate. 99.2% of the patients were ACPR and the
proportions of ACPR had not changed significantly from 2007
to 2013 (X2 = 2.81, P = 0.7288).
DAPQ sensitivity of P. falciparum had been monitored
on the China-Myanmar border. Sun et al. reported that
DAPQ was efficacious for falciparum malaria treatment
in 2006 , however Liu et al. reported a 97.0% of
cumulative success rate (CSR) and reduced DAPQ
sensitivity in P. falciparum of a trial in which the only eight
tablets of DAPQ (a dosage for >40 kg body weight) were
used for uncomplicated falciparum malaria treatment
. In the study by Liu et al., the technical limitation
was to not use PCR to distinguish between re-infection
and recrudescence, and they conduct their study in Wa
State of Myanmar, where P falciparum prevalence was
high during 20072008. The technical limitation and the
different regimen might result in the declined CSR, and
geographic differences might be one of explanations for
reported differences too. Huang et al. reported a 95.9%
(47/49) of artesunate CSR and a high frequency of
mutations in pfcrt, pfdhfr and pfdhps associated with
chloroquine and sulphadoxinepyrimethamine resistance and
no pfatp6 mutation in P. falciparum . Wang et al.
reported low levels of point mutations in pfmdr1 and
pfatp6 prevalence and no pfmdr1 gene amplification
detected . The results of the two molecular epidemiology
studies showed lack of molecular basis of artemisinin
resistance along China-Myanmar border. All these
investigations together were not enough to confirm the artemisinin
resistance in P. falciparum along the China-Myanmar
Several limitations should be considered while using
the results of study. Firstly, both total and yearly sample
size were limited by the difficulty in recruiting patients
because of low malaria incidence in eliminating settings,
only total 1011 falciparum malaria cases were reported
during 20102013. Secondly, the surveillance activity
was interrupted in 2008 because of shortage of funding.
Thirdly, the surveillance sites had to be changed in
order to recruit falciparum malaria patients, in Yingjiang
in 2007, Menglian in 2009, in both Tengchong and
Yingjiang from 2010 to 2013, however all the three
sites are on the China-Myanmar border. Fourthly, the
study did not use any pharmacokinetics (PK)
measurement to ascertain drug absorption and to characterize
the concentrationtime profile of drugs and the
relevant covariates (e.g. immunity, young age and drug
interactions), so it did not exclude confounding effect of
some variables that could influence APCT . Fifthly,
mutations of PF3D7_1343700 kelch propeller domain
(K13-propeller) are important determinants of
artemisinin resistance ; the study did not investigate mutant
K13-propeller alleles. According to the new working
definition of ACT or artesunate monotherapy resistance, the
suspected resistance is as evidenced by either 5%
K13propeller domain mutants or 10% of patients who are
parasitaemic on day three; and the confirmed resistance
should satisfy both 5% K13-propeller domain mutants
and 10% of patients with parasitaemia on day three, or
by the persistence of parasites for seven days, or by the
presence of parasites on day three and recrudescence
within 28/42 days [29,39]. The monitored results showed
that only 3.3% (8/243) of patients presented parasitaemia
by day three and only 0.8% (2/243) were ETF or LCF.
According to the new working definition DAPQ was still
sensitive in P. falciparum along the China-Myanmar border.
While artemisinin resistance was found on the western
border of Cambodia-Thailand and the western border of
Thailand-Myanmar , P. falciparum was still sensitive
to artemisinins on the China-Myanmar border. The
genetic diversity of malaria parasites and multiclonal
infections are correlated with transmission intensity as well
as the spread of anti-malarial resistance. Despite the
intensified control efforts and the decline of malaria prevalence
along China-Myanmar border, the parasite population size
and transmission intensity remained high enough to allow
effective genetic recombination of the parasites and
continued maintenance of genetic diversity in
ChinaMyanmar border area . Therefore, surveillance for
artemisinin resistance in P. falciparum should be
maintained and containment activities should be further
strengthened to monitor the spread of resistance,
define therapeutic and operational strategies to understand
its molecular basis and counter its impact [28,42]. In
terms of monitoring artemisinin resistance, treatment
failure of ACT is more direct and accurate , so it may be
more useful in artemisinin sensitivity surveillance.
Detection of drug efficacy is only the first step to
producing accessible and useful information for decision
makers. The translation of increased access to data on
health outcomes into usable evidence for rational policy
and planning requires a global coordination and
communication effort . In terms of containment measures,
a 97.5% of DAPQ CSR for P. falciparum treatment had
been reported in Hainan Province of China in 2004 
and then 100% in 2008 , P. falciparum has been
eliminated by using the strategy of early diagnosis and
treatment (EDT) with ACT . The emergence of resistance
to artesunate in P. falciparum, the strategy of EDT with
ACT has reduced malaria in the migrant population
living on the Thai-Myanmar border . The addition
of one dose of primaquine to ACT could help to
counter the spread of artemisinin resistance . Despite
the 8-aminoquinoline compound cause patients with
G6PD deficiency haemolysis, it can sterilize
gametocytes of P. falciparum. Lower doses of the
gametocytocide would be safer, might still be very effective for
In terms of efficacy, DAPQ is still effective for falciparum
malaria treatment. DAPQ sensitivity in P. falciparum had
not significantly changed along the China-Myanmar
border of Yunnan Province, China. However more
attentions should be given to becoming slower fever and
parasite clearance, surveillance for artemisinin
resistance in P. falciparum should be maintained and
containment measures are urgently needed.
The authors declare that they have no competing interests.
L-HT, H-LY, HL, FH and ML designed the study and developed the protocol.
J-WX and HL analysed and interpreted the data. HL organized and supervised
the study in field. L-HT, H-LY, FH and ML monitored the study in general. HL,
X-LL, J-ZW, C-FL, H-YW, R-HN, X-RG, Y-XL and JW conducted the study in
field, and entered the data. FH and M L performed the PCR test. J-WX and
HL wrote the first draft of the paper. All authors read and approved the
This study was supported by The WHO Mekong Malaria Programme (WP/10/
MVP/005837), the sixth (CHN-607-G09-M) and tenth (CHN-011-G15-M) grant
to China of the Global Fund to fight AIDS, Tuberculosis and Malaria (GFATM).
We thank all participants for their contribution of time and patience in the
study. We also thank staff of Yunnan Institute of Parasitic Diseases (YIPD) for
logistic support, and clinical and laboratory staff of Menglian, Tengchong
and Yangjiang County Center for Disease Control and Prevention for their
hard work. The opinions expressed are those of the authors and do not
necessarily reflect those of WHO, GFATM and YIPD.
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