Comparative Efficacies of Artemisinin Combination Therapies in Plasmodium falciparum Malaria and Polymorphism of pfATPase6, pfcrt, pfdhfr, and pfdhps Genes in Tea Gardens of Jalpaiguri District, India

Abstract

In India, chloroquine has been replaced by a combination of artesunate and sulfadoxine-pyrimethamine (AS-SP) for uncomplicated P. falciparum malaria. Other available combinations, artemether-lumefantrine (AM-LF) and artesunate-mefloquine (AS-MQ), not included in the national program, are widely used by private practitioners. Little is known about the therapeutic efficacy of these artemisinin combinations and the prevalence of molecular markers associated with antimalarial drug resistance. A total of 157 patients with P. falciparum monoinfection were recruited and randomized into three study groups (AS-SP, AM-LF, and AS-MQ). All patients were followed up for 42 days to study the clinical and parasitological responses according to the WHO protocol (2009). We assessed the polymorphism of the pfATPase6, pfcrt, pfdhfr, and pfdhps genes by the DNA-sequencing method. The PCR-corrected therapeutic efficacies of AS-SP, AM-LF, and AS-MQ were 90.6% (95% confidence interval [CI], 0.793 to 0.969), 95.9% (95% CI, 0.860 to 0.995), and 100% (95% CI, 0.927 to 1.00), respectively. No specific mutational pattern was observed in the pfATPase6 gene. All isolates had a K76T mutation in the pfcrt gene. In the pfdhfr-pfdhps genotype, quadruple mutation was frequent, and quintuple mutation was documented in 6.3% of P. falciparum isolates. The significant failure rate of AS-SP (9.5%), although within the limit (10%) for drug policy change, was due to SP failure because of prevailing mutations in pfdhfr, I₅₁R₅₉N₁₀₈, with pfdhps, G₄₃₇ and/or E₅₄₀. The efficacy of this ACT needs periodic monitoring. Artemether-lumefantrine and artesunate-mefloquine are effective alternatives to the artesunate-sulfadoxine-pyrimethamine combination.

INTRODUCTION

Malaria remains one of the major global public health problems, with an estimated 225 million confirmed cases and 781,000 deaths in 2009 (34). Plasmodium falciparum is the most virulent infectious species of malaria parasite in tropical and subtropical countries. Chloroquine (CQ) has been in use as the major antimalarial drug for the past few decades in all areas where malaria is endemic. The widespread use of CQ has led to the emergence of CQ-resistant parasites. CQ resistance in P. falciparum malaria, first reported from Southeast Asia and South America in the late 1950s, has spread throughout the world. In India, CQ-resistant P. falciparum was first reported from the Diphu area of the Karbi Anglong district of Assam in 1973 (23), followed by its spread to various parts of the country. Due to the emergence of resistant malarial parasites, particularly P. falciparum against different antimalarial drugs, malaria treatment has moved into the era of artemisinin-based combination therapy (ACT). Recently, the National Vector Borne Disease Control Program (NVBDCP) of India has introduced ACT, a combination of artesunate-sulfadoxine-pyrimethamine (AS-SP), as the first-line agent to treat uncomplicated P. falciparum malaria. Two other ACTs consisting of artemether-lumefantrine (AM-LF) and artesunate-mefloquine (AS-MQ), not included in the national program but readily available in the market, are in frequent use by private practitioners.

In the late 20th century, candidate molecular markers for antimalarial drug resistance were identified by cloning and sequencing parasite genes homologous to those known to mediate resistance in other organisms (4, 5, 14). Based on differences in DNA sequence between sensitive and resistant parasite clones, point mutations (12, 13, 22) were evaluated for their association with in vitro resistant phenotypes. Molecular markers for resistance were assessed in clinical trials (6, 7, 17) to establish their association and ability to predict treatment failure in vivo.

In P. falciparum, two candidate genes have been found to be potentially associated with artemisinin resistance. Mutations and increased copy numbers of the pfmdr1 gene were associated with reduced in vitro susceptibility to the artemisinins (9, 24) and reduced in vivo clinical efficacy of AM-LF in Africa (25). P. falciparum ATPase6 was shown to be a target for the artemisinin drugs in Xenopus oocytes (10). A single amino acid change, L263E, in pfATPase6 was associated with resistance to artemisinin in this model (10, 28). Mutation of S769N in pfATPase6 of P. falciparum isolates from French Guiana was associated with decreased in vitro sensitivity to artemether (16). However, it is unclear whether mutations in pfATPase6 are associated with in vivo artemisinin resistance (1). The dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes are associated with SP resistance. The quintuple mutation (pfdhfr, I51, R59, N108, and pfdhps, G437 and E540) is considered a relevant molecular marker of SP treatment failure (17).

A recent in vivo therapeutic efficacy study showed a high rate of resistance of P. falciparum to different antimalarial drugs (CQ, 66%; SP, 16%; CQ-SP, 8%) in Jalpaiguri (20). Considering the SP resistance status in the study area and the frequent use of two other combinations of ACTs, the present study was designed to evaluate the in vivo efficacies of three different regimens of ACTs against P. falciparum malaria and polymorphism of the pfATPase6, pfcrt, pfdhfr, and pfdhps genes in tea gardens of Jalpaiguri District.

MATERIALS AND METHODS

Study site and design.

The study was performed at Turturi Primary Health Centre (PHC) under Alipurduar Block II of Jalpaiguri District, West Bengal, India, from November 2009 to February 2010, where the annual parasite index (API) (total number of slides positive for parasites in a year × 1,000/total population) in 2008 was 2.8. In Jalpaiguri, malaria transmission is seasonal, with a peak from September to January and predominance of P. falciparum malaria (78.07%; West Bengal Government data). The study area is surrounded by several tea gardens and inhabited by different tribal communities.

The study was a randomized, three-arm, open-label, prospective trial for evaluation of clinical and parasitological responses of three ACTs for treatment of uncomplicated P. falciparum malaria, based on the therapeutic efficacy protocols of the WHO (32). The study protocol was approved by the Institutional Ethics Committee of the Kolkata School of Tropical Medicine.

Patient screening and enrollment.

Febrile patients from surrounding tea gardens and local villages attending the outpatient department (OPD) of the Turturi PHC were screened for malarial parasites by examining Giemsa-stained thick and thin peripheral blood smears (PBS). All patients with confirmed P. falciparum monoinfection were informed about the study protocol and requested to participate in the study. Those who fulfilled the inclusion criteria according to the WHO protocol (32) were enrolled after written informed consent was obtained.

Inclusion criteria.

Patients over 6 months of age with P. falciparum monoinfection (asexual parasite count, 1,000 to 100,000/μl), an axillary temperature of ≥37.5°C or history of fever during the past 24 h, and ability to swallow oral medication were included in the study. Those having symptoms of severe falciparum malaria, a febrile condition due to diseases other than malaria, or history of regular medication that might interfere with antimalarial pharmacokinetics and pregnant women and lactating mothers were excluded.

Treatment.

The therapeutic efficacies of three different regimens of ACTs were evaluated in the present study. The tested ACTs were AS-SP, AM-LF, and AS-MQ. AS and SP were supplied by NVBDCP and AM-LF and MQ were procured from M/S Themis Medicare Limited, India. The recruited patients were randomized by using simple random sampling without replacement (SRSWR) into three study groups, each receiving one of the above-mentioned drug regimens. The study was designed in such a way that patients below 5 years of age were not enrolled in the AS-MQ study arm, as local health officials objected to enrolling them due to a paucity of safety data about mefloquine in Indian children. All the patients were treated according to WHO guidelines (31) for the treatment of malaria according to their body weight. In the AS-SP group, artesunate at 4 mg/kg body weight once daily for 3 days and a single dose of SP (25/1.25 mg base/kg body weight) on day 0 were administered. In the AM-LF group (coformulated tablets containing 20 mg of artemether and 120 mg of lumefantrine), a total of six doses were administered at 0, 8, 24, 36, 48, and 60 h. The numbers of tablets per dose were as follows: one tablet for 10 to 15 kg of body weight, two tablets for 15 to 25 kg, three for 25 to 35 kg, and four for those weighing over 35 kg. In the AS-MQ group, artesunate was administered in a dose of 4 mg/kg once daily for 3 days and mefloquine was dosed at 25 mg base/kg over 2 days on day 1 (15 mg/kg) and day 2 (10 mg/kg). All recruited patients were admitted to the PHC for the first 4 days for monitoring of the clinical and parasitological response to the treatment. Primaquine (PQ) in a single dose of 0.75 mg/kg was given on day 1. Study investigators directly observed and documented the administration of each dose of medication. In case of vomiting within 30 min of administration, a full dose was repeated.

Follow-up schedule.

The day, the patient was enrolled and received the first dose of medicine was designated day 0. Thereafter, the schedule called for clinical reassessment on days 1, 2, 3, 7, 14, 21, 28, 35, and 42. Patients were advised to return on any day during the follow-up period if symptoms recurred and advised not to wait for a scheduled visit day. During the follow-up visits, all patients were examined both clinically and parasitologically. In the event of occurrence of any complications, the patient was withdrawn from the study and treated according to the existing standard of care for that condition.

Study endpoints and statistical analysis.

Therapeutic outcomes were classified according to WHO guidelines (32) into early treatment failure (ETF), late clinical failure (LCF), late parasitological failure (LPF), and adequate clinical and parasitological response (ACPR). The data were entered into a standard data entry program designed by the Global Malaria Program and analyzed with a Kaplan-Meier survival curve according to WHO standard procedures (http://www.who.int/malaria/resistance). The free statistical software R (version 2.13.1) was used to calculate the Z-test value for the comparison of the efficacy of the three study arms, and Fisher's exact test was done to verify the correlation between dhfr-dhps haplotypes and the AS-SP treatment failure cases.

Laboratory examination. (i) Microscopic blood examination and parasite count.

To calculate the parasite count, the number of parasites per 200 white blood cells (WBCs) was determined by experienced microscopists using a light microscope. Assuming a WBC count to be 8,000/μl of blood, the parasitemia was calculated and expressed per μl of blood.

(ii) p-LDH test.

All microscopically confirmed P. falciparum-positive cases were screened for a P. vivax-specific p-LDH (Plasmodium lactate dehydrogenase) test (by using an rapid diagnostic test that contained monoclonal anti-P. falciparum HRP-II-specific and anti-P. vivax p-LDH-specific antibodies [M/S Tulip Group, Goa, India]) to detect any mixed infection.

(iii) DNA isolation and msp1 and msp2 genotyping assays.

The genomic DNA of the parasite was isolated from the blood samples collected on day 0 and in all episodes of malaria during the 42-day study period using a QIAamp DNA Blood Minikit (Qiagen, Valencia, CA) according to the manufacturer's instructions.

Genotyping of P. falciparum parasites was performed with a nested PCR assay based on the amplification of msp1 and msp2 as described in detail previously (11, 26). In the primary reaction, the oligonucleotide primers used spanned the entire genetic segments, i.e., block 2 for msp1 and block 3 for msp2. In the nested reaction, separate primer pairs targeted the respective allelic types of msp1 (K1, MAD20, and RO33) and msp2 (FC27 and 3D7).

The amplified products from the nested reaction were separated by electrophoresis on a 2% high-resolution agarose gel (SeaKem LE Agarose; Lonza, Rockland, ME) in 1× TBE buffer (100 mM Tris, 100 mM boric acid, 5 mM EDTA [pH 8.0]) and visualized with UV light following staining with ethidium bromide. The fragment size was estimated in relation to a 100-base-pair DNA ladder (Invitrogen Corporation, Carlsbad, CA) both by the naked eye and in a GelDoc Xr system with Quality One analysis software version 4.4.1 (Bio-Rad).

Before treatment, the mean multiplicity of infection (MOI) was calculated as the quotient of the total number of P. falciparum genotypes and the number of positive PCR samples for each marker msp1 and msp2 gene.

In patients with recurrent parasitemia, blood samples on day 0 and on the day of parasite reappearance were used to distinguish between recrudescence and new infection. A recrudescent infection was defined as one that showed a match in size of at least one allele for both the msp1 and msp2 genes on day 0 and on the day of recurrent parasitemia.

(iv) Polymorphism of the pfATPase6, pfcrt, pfdhfr, and pfdhps genes.

Five pairs of primers were used to span 4,068 bp of the P. falciparum pfATPase6 gene as described previously (15). A part of the pfcrt gene covering single-nucleotide polymorphisms (SNPs) at codons 72 to 76 was amplified as described previously (19).

Portions of the pfdhfr gene spanning codons 16, 51, 59, 108, and 164 and the pfdhps gene spanning codons 436, 437, 540, 581, and 613 on day 0 and the day of recurrence were amplified by two rounds of PCR using primers described previously (2, 3).

Direct sequencing of the PCR products was done on an ABI 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA). The sequences were analyzed using Bioedit software.

RESULTS

Enrollment and demographics.

A total of 2,544 patients with fever were screened for malaria parasites, 479 of which were positive for P. falciparum and 83 for P. vivax, while 21 had mixed infections. Among the 479 P. falciparum-positive cases, 157 subjects were enrolled in the study and randomized into three study arms. The demographic data and clinical parameters of the study groups are summarized in Table 1.

Table 1.

Baseline characteristics of the study patients

Characteristic	Value
	AS-SP (n = 53)	AM-LF (n = 52)	AS-MQ (n = 52)
Sex [no. (%)]
Male	31 (58.49)	27 (51.92)	32 (61.54)
Female	22 (41.51)	25 (48.08)	20 (38.46)
Weight (kg)
Mean	30.58	23.23	34.90
Range	09–65	06–52	14–60
SD	14.90	13.88	13.32
95% CI	26.56–34.59	19.46–27.00	31.28–38.52
Age category [no. (%)]
< 5 yr	06 (11.32)	19 (36.54)	0
5–15 yr	27 (50.94)	25 (48.08)	28 (53.85)
Adult	20 (37.34)	08 (15.38)	24 (46.15)
Age (yr)
Mean	16.45	10.62	17
Range	02–55	01–55	05–50
SD	14.18	10.33	10.75
95% CI	12.64–20.26	7.81–13.43	14.08–19.92
Temperature (°C)
Mean	38.00	37.96	37.9
Range	37.60–39.3	37.6–39.3	37.5–39.3
SD	0.45	0.40	0.36
95% CI	37.88–38.12	37.85–38.07	37.81–37.99
Hemoglobin (g/dl)
Mean	11.84	11.04	12.21
Range	9.9–15.0	8.5–14.0	9.0–14.8
SD	1.46	1.15	1.36
95% CI	11.45–12.23	10.73–11.35	11.84–12.58
Parasite count (no/μl)
Mean	15,371	14,538	12,380
Range	1,040–70,000	1,040–51,680	1,000–44,000
SD	15,402.25	12,375.19	10,266.47
95% CI	11,224–19,518	11,174–17,902	9,589–1,5171
Gametocytes on day 0 [no. (%)]	3 (5.66)	1 (1.92)	2 (3.85)

In vivo therapeutic efficacy outcomes.

Out of a total of 157 recruited patients, 4 withdrew from the study voluntarily, 1 was lost to follow-up, and 152 (53 in the AS-SP, 49 in the AM-LF, and 50 in the AS-MQ groups) completed the 42-day follow-up period (Fig. 1).

Fig 1.

Profile of patients screened, enrolled, and completing the study protocol.

Of the 53 subjects recruited in the AS-SP group, all completed the 42-day follow-up. The non-PCR-corrected incidence of failure (LPF+LCF) of AS-SP was 9.5% (5/53). msp1 and msp2 genotyping was done for 5 apparent therapeutic failures to differentiate between new infection and recrudescence, and all were classified as recrudescence. Therefore, the PCR-corrected cumulative incidence of failure of AS-SP for P. falciparum was 9.5% (95% confidence interval [CI], 0.04 to 0.212), and the therapeutic efficacy was 90.6% (95% CI, 0.793 to 0.969) (Table 2).

Table 2.

Therapeutic efficacies of three study arms among patients with uncomplicated malaria in Jalpaiguri, India

Clinical outcome	AS-SPa			AM-LFa			AS-MQa
	n	%	95% CI	n	%	95% CI	n	%	95% CI
Non-PCR corrected
Treatment failure	5	9.5	0.04–0.212	2	4.1	0.010–0.149	1	2.0	0.003–0.134
ETF	0	0	0.0–0.067	0	0	0.0–0.073	0	0	0.0–0.071
LCF	3	5.7	0.012–0.157	2	4.1	0.005–0.140	1	2.0	0.001–0.106
LPF	2	3.8	0.005–0.130	0	0	0.0–0.073	0	0	0.0–0.071
ACPR	48	90.6	0.793–0.969	47	95.9	0.860–0.995	49	98.0	0.894–0.999
PCR corrected
Treatment failure	5	9.5	0.04–0.212	2	4.1	0.010–0.149	0	0	0.0–0.073
ETF	0	0	0.0–0.067	0	0	0.0–0.073	0	0	0.0–0.073
LCF	3	5.7	0.012–0.157	2	4.1	0.005–0.140	0	0	0.0–0.073
LPF	2	3.8	0.005–0.130	0	0	0.0–0.073	0	0	0.0–0.073
ACPR	48	90.6	0.793–0.969	47	95.9	0.860–0.995	49	100	0.927–1.00

^aAS-SP, n = 53; AM-LF and AS-MQ, n = 52 for non-PCR corrected and n = 49 for PCR corrected.

Among the 52 patients in the AM-LF group, 3 withdrew before day 42 and 49 completed the study. The crude therapeutic efficacy of AM-LF was 95.9% (47/49), and crude therapeutic failure was 2 (4.1%). Based on msp1 and msp2 genotyping of the parasite on day 0 and the day of recurrence, the two cases were classified as recrudescence. The PCR-corrected 42-day cumulative incidence of failure of AM-LF therapy for P. falciparum was 4.1% (95% CI, 0.010 to 0.149), and therapeutic efficacy was 95.9% (95% CI, 0.860 to 0.995), which was higher than for the AS-SP group (Table 2).

In the AS-MQ study arm, out of 52 subjects, 1 withdrew voluntarily and 1 was lost to follow-up. Fifty patients completed the 42-day follow-up. One came back to the clinic with fever on day 26, and the ring form of P. falciparum was detected in a peripheral blood smear, but genotyping could not be done to differentiate between reinfection and recrudescence, as parasite DNA could not be amplified. This combination was 98% (95% CI, 0.894 to 0.999) effective, with failure to successfully PCR correct (Table 2). Importantly, no ETF was recorded in this group.

The statistical software R (version 2.13.1) was used for two sample tests for proportion. The difference in efficacy between AS-MQ (98%) and AS-SP (90.5%) was not statistically significant (Z = 1.61; P = 0.05). Similarly, no significant difference in efficacy was noted between AS-MQ (98%) versus AM-LF (95.9%) (Z = 0.604; P = 0.27) and AS-SP (90.5%) versus AM-LF (95.9%) (Z = 1.06; P = 0.14).

Fever and parasite clearance.

In 27 (50.9%), 25 (47.1%), and 1 (1.9%) recipients of AS-SP, fever subsided by days 1, 2, and 3, respectively, while parasite clearance took place in 27 (50.9%), 17 (32.1%), and 9 (16.9%) by days 1, 2, and 3, respectively.

In the AM-LF group, fever subsided in 18 (34.6%), 32 (61.5%), and 2 (3.9%) patients by days 1, 2, and 3, respectively, with parasite clearance in 8 (15.4%), 30 (57.7%), and 14 (26.9%) by days 1, 2, and 3, respectively.

Fever subsided in 23 (42.2%) and 29 (55.8%) participants by days 1 and 2, respectively, while the parasites were cleared in 20 (38.5%), 22 (42.3%), and 10 (19.2%) by days 1, 2, and 3, respectively, among patients who received AS-MQ.

Side effects.

Dizziness, headache, nausea, vomiting, and muscle pain were common in all the treatment arms, particularly among the patients who received AS-MQ (Table 3). These symptoms were treated symptomatically.

Table 3.

Side effects

Adverse effect	No. of patientsa
	AS-SP (n = 53)	AM-LF (n = 52)	AS-MQ (n = 52)
Muscle pain	33	41	45
Headache	26	22	43
Dizziness	12	16	23
Nausea	21	18	34
Vomiting	5	2	43
Anorexia	19	3	11
Diarrhea	1	0	2
Itching	7	0	0
Abdominal pain	2	1	9
Cough	3	2	2

^aNumber of patients having symptoms at least once during ACT treatment in Jalpaiguri.

Allelic diversity and MOI of P. falciparum.

Among the 157 samples from day 0, msp1 and msp2 genotyping was done successfully in 154 isolates. At the msp1 locus, 13 alleles of the K1 family, 7 of the MAD20 family, and 10 of the RO33 family ranging from 110 to 530 bp, 150 to 250 bp, and 160 to 410 bp, respectively, were detected. For the msp2 locus, 17 FC27 and 15 3D7 alleles ranging from 175 to 750 bp and 170 to 690 bp, respectively, were noted. The family distributions for K1, MAD20, and RO33 at msp1 were 41.9%, 17.9%, and 40.2%, respectively, while for FC27 and 3D7 at msp2 they were 49.0% and 51.0%, respectively. The frequencies of individual msp1 genotypes were low, with 16 genotypes less than 10% and 14 greater than 10%, while at msp2, 25 genotypes were less than 10% and 7 more than 10%. The mean MOI was 3.10 (Table 4).

Table 4.

Genetic diversity of msp1 and msp2 of 154 P. falciparum isolates (day 0) from Jalpaiguri District, India

Parameter	Value
	mspI				msp2			Total
	K1	MAD20	Ro33	Total	FC27	3D7	Total
No. of distinct bands	13	7	10	30	17	15	32	62
No. (%) of isolates having alleles of the corresponding family	136 (88.3)	90 (58.4)	150 (97.4)		154 (100)	148 (96.1)
% of bands assigned to the corresponding family	41.9	17.9	40.2		49.0	51.0
Mean MOI (±SD)	2.67 (±1.15)	1.73 (±0.92)	2.32 (±0.96)	2.30	3.94 (±1.49)	4.27 (±1.29)	4.10	3.10

Sequencing of full-length pfATPase6.

Sequencing of the full length of the pfATPase6 gene was done successfully in 142 isolates. It revealed only sporadic point mutations compared to the wild-type sequence. Mutations at codons L263E and S769N, which have been proposed to confer artemisinin resistance, were not detected. While nonsynonymous mutations were detected at codons E431K (19%), K649E (25.4%), and N683K (5.6%), synonymous mutations were noted at codons G468G (2.8%), N483N (4.9%), and I898I (26.8%) (Table 5).

Table 5.

Polymorphism of Pfcrt, PfATPase6, Pfdhfr and Pfdhps genes in study population

Candidate genea	Occurrence of mutation
	n	%	95% CI
Pfcrt (n = 139)
Haplotypes
S72V73M74N75T76	120	86.3	80.62–92.04
C72V73I74E75T76	19	13.7	7.96–19.38
PfATPase6 (n = 142)
Synonymous mutation
G468G	4	2.8	0.1–5.54
N483N	7	4.9	1.37–8.49
I898I	38	26.8	17.56–31.74
Nonsynonymous mutation
E431K	27	19.0	12.56–25.46
K649E	36	25.4	18.19–32.51
N683K	8	5.6	1.84–9.42
Pfdhfr (n = 138)
A16V	0	0	0–2.13
N51I	13	9.4	4.55–14.29
C59R	138	100.0	97.87–100.69
S108N	132	95.7	92.25–99.05
I164L	8	5.8	1.9–9.7
Genotype
A16I51R59S108I164	6	4.3	0.95–7.75
A16N51R59N108I164	117	84.8	78.79–90.77
A16N51R59N108L164	8	5.8	1.9–9.7
A16I51R59N108I164	7	5.1	1.41–8.73
Pfdhps (n = 126)
S436A	63	50.0	41.27–58.73
A437G	42	33.3	25.1–41.56
K540E	64	50.8	42.06–59.52
A581G	7	5.6	1.56–9.56
A613S	0	0	0–2.34
Genotype
S436A437K540A581A613	6	4.8	1.04–8.48
A436A437K540A581A613	7	5.6	1.56–9.56
S436G437K540A581A613	42	33.3	25.1–41.56
S436A437E540A581A613	8	6.3	2.09–10.61
S436A437K540G581A613	7	5.6	1.56–9.56
A436A437E540A581A613	56	44.4	35.76–53.12

^aMutated amino acids are in boldface.

Mutations in pfcrt, pfdhfr, and pfdhps.

The K76T mutation in the pfcrt gene was present in all the parasite strains (n = 139). The Southeast Asian haplotype (SVMNT) was predominant (86.3%) over the South American haplotype (CVIET) (13.7%) (Table 5).

The pfdhfr gene was sequenced successfully in 138 isolates that covered codons 16, 51, 59, 108, and 164. Mutations at codons C59R (100%) and S108N (95.7%) were more frequent than others. Four different haplotypes were recorded, among which A₁₆N₅₁R₅₉N₁₀₈I₁₆₄ was the most prevalent (84.8%) (mutated amino acids are in boldface) (Table 5).

Sequencing of the pfdhps gene was successful in 126 isolates that covered codons 436, 437, 540, 581, and 613. Six different pfdhps genotypes were noticed, of which the A₄₃₆A₄₃₇E₅₄₀A₅₈₁A₆₁₃ (44.4%) and S₄₃₆G₄₃₇K₅₄₀A₅₈₁A₆₁₃ (33.3%) haplotypes were common (Table 5). No changes were observed in the pfdhfr and pfdhps genotypes of pretreatment and recrudescent samples from five treatment failure cases in AS-SP recipients.

Association between pfdhfr and pfdhps haplotypes and therapeutic outcomes of three ACT regimens.

Sequencing of both the pfdhfr and pfdhps genes was done in 126 isolates. Quadruple mutations were found in 55 (43.6%), and quintuple mutations were recorded in only 8 (6. 3%) cases (Table 6). Although the sample size was small, a trend of association was observed between quadruple (pfdhfr, I₅₁R₅₉N₁₀₈, and pfdhps, G₄₃₇) (P = 0.0316) and quintuple (pfdhfr, I₅₁R₅₉N₁₀₈, and pfdhps, A₄₃₆E₅₄₀) haplotypes (P = 0.01107) with AS-SP resistance, as analyzed by Fisher's exact test. A triple pfdhfr mutation (I₅₁R₅₉N₁₀₈) was common in both the haplotypes. Thus, the pfdhfr I₅₁R₅₉N₁₀₈ triple mutant was significantly associated (P = 0.000198) with AS-SP resistance, along with either a pfdhps G₄₃₇ or E₅₄₀ mutation.

Table 6.

Association between dhfr-dhps mutations and in vivo therapeutic efficacy outcomes of three ACT regimens in the treatment of P. falciparum malaria in Jalpaiguri District, India

Pfdhfr-Pfdhps genotypea	No. of patients
	AS-SP (n = 43)		AM-LF (n = 41)		AS-MQ (n = 42)
	ACPR	TFb	ACPR	TF	ACPR	TF
Double mutant
A16N51R59N108I164-S436A437K540A581A613	2	0	3	0	1	0
Triple mutant
A16N51R59N108I164-A436A437K540A581A613	3	0	2	0	2	0
A16N51R59N108I164-S436G437K540A581A613	10	0	7	0	12	0
A16N51R59N108I164-S436A437E540A581A613	2	0	3	0	3	0
A16N51R59N108I164-S436A437K540G581A613	3	0	2	0	2	0
A16I51R59S108I164-S436G437K540A581A613	2	0	2	0	2	0
Quadruple mutant
A16I51R59N108I164-S436G437K540A581A613	1	2	1	0	1	0
A16N51R59N108I164-A436A437E540A581A613	14	0	19	0	15	0
A16N51R59N108L164-S436G437K540A581A613	1	0	0	0	1	0
Quintuple mutant
A16I51R59N108I164-A436A437E540A581A613	0	2	0	0	0	0
A16N51R59N108L164-A436A437E540A581A613	0	1	0	2	3	0

^aMutated amino acids are in boldface.

^bTF, treatment failure.

DISCUSSION

Artemisinin-based combination therapy has been recommended by WHO to treat uncomplicated P. falciparum cases in all countries where malaria is endemic (30, 31, 33), as artemisinin and its derivatives reduce most of the parasite biomass during their initial rapid action and an effective long-acting partner drug can usually eliminate the small number of remaining parasites. Five combinations are currently recommended: AM-LF, AS-MQ, AS-SP, dihydroartemisinin-piperaquine, and artesunate-amodiaquine (34). The combination AS-SP was introduced by the Government of India in 2010 as the first-line agent to treat all diagnosed uncomplicated P. falciparum malaria.

In the present study, a treatment failure rate of 9.5% (95% CI, 0.04 to 0.212) was recorded for AS-SP. A previous study showed that a significant proportion of P. falciparum malaria in the study area was resistant to SP alone (20), with a record of both ETF and LTF, but in the present study, not a single case of ETF was observed in any of the three study arms. Perhaps this was due to early clearance of the parasite by the artemisinin component of the combination therapy. Dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) are two essential enzymes in the folate biosynthesis pathway and are inhibited by pyrimethamine and sulfadoxine, respectively. The mutations at pfdhfr I₅₁R₅₉N₁₀₈ and pfdhps G₄₃₇ and E₅₄₀ are associated with SP treatment failure (17, 27). In the present study, a quadruple haplotype (pfdhfr I₅₁R₅₉N₁₀₈ and pfdhps G₄₃₇) and a quintuple haplotype (pfdhfr I₅₁R₅₉N₁₀₈ and pfdhps A₄₃₆E₅₄₀) were noted to be significantly associated with AS-SP failure (P = 0.0316 and P = 0.01107, respectively). In both the haplotypes, a triple pfdhfr I₅₁R₅₉N₁₀₈ mutation was commonly associated with AS-SP failure (P = 0.000198). However, it is not possible to postulate that the pfdhfr I₅₁R₅₉N₁₀₈ mutation alone was responsible for AS-SP failure, as we did not come across any isolate having this genotype exclusively without a pfdhps G₄₃₇ and/or E₅₄₀ mutation.

Currently, 11 countries are using AS-SP as a first- or second-line treatment. AS-SP treatment failure rates remained low in studies conducted in South American countries (Colombia, Ecuador, and coastal areas of Peru), the Middle East and Central Asia (Afghanistan, the Islamic Republic of Iran, Pakistan, Tajikistan, and Yemen), South Asia (India and Sri Lanka), and eastern Africa (Somalia and the Sudan), including countries in which AS-SP was used as a first-line treatment, where the median treatment failure rate was 0 to 1.5% (34). The high clinical efficacy of this combination may be partially due to the rarity of the pfdhfr and pfdhps quintuple mutant (36). In contrast, high failure rates of this combination have been observed in several African countries with high SP resistance.

AM-LF remains highly effective in most parts of the world, with the exception of Cambodia (34). In India, two cases of AM-LF-resistant P. falciparum were reported from Orissa (29). A treatment failure rate of 4.1% (95% CI, 0.010 to 0.149) was recorded in the present study with AM-LF.

The artesunate-mefloquine combination was first introduced in Thailand to treat P. falciparum malaria after the spread of mefloquine resistance. Presently, eight countries are using this combination as a first- or second-line treatment. High therapeutic efficacy of this combination has been recorded in different parts of the world, except in Cambodia and Thailand (35). No such record is available from India. In this study, only a single treatment failure was observed. However, it could not be determined whether it was due to reinfection or recrudescence, as we failed to genotype the strain.

As the pharmacokinetics and molecular markers associated with LF and MQ were not studied, the observed failure of P. falciparum cases treated with AM-LF (4.1%) could not be explained.

pfATPase6 has been suggested to be a specific target of the artemisinin drugs (10). Decreased in vitro sensitivity to artemether of field isolates of P. falciparum was reported from French Guiana (but not in Cambodia) and was found to be associated with the S769N mutation in the pfATPase6 gene (10, 16). In this study, no consistent pattern of pfATPase6 mutations was noted. Mutations at codons L263E and S769N, associated with artemisinin resistance, were not detected, although in vivo failure rates of 9.5% and 4.1% with AS-SP and AM-LF, respectively, were documented. Similar observations were made by Dondorp et al. (8) with in vivo artemether monotherapy.

Genotyping of day 0 samples showed that both the allelic variation and mean MOI of msp2 were higher than those of msp1 and that the frequencies of most of the alleles (41/62) were below 10%. The P. falciparum population of the study area was genetically diverse, with 30 msp1 and 32 msp2 alleles. As the frequencies of most of the msp1 and msp2 alleles were low, the probability of new infection with a genotype identical to that in pretreatment infection was very rare. In the present study, all 7 (5 in the AS-SP arm and 2 in the AM-LF arm) recurrent parasitemias were classified as recrudescence. We used the agarose gel electrophoresis method for genotyping. The method is limited by its inability to discriminate among DNA fragments with small differences in molecular weight. Recently, various researchers (18, 21) have used capillary-based genotyping assays, which have a highly accurate fragment-sizing capacity.

The PCR-corrected failure rate of the recommended ACT (AS-SP) in P. falciparum malaria was 9.5%, which was just below the cutoff level for drug policy change (10%) recommended by WHO. Considering the observed failure rate of AS-SP and its association with a quadruple haplotype (pfdhfr I₅₁R₅₉N₁₀₈ and pfdhps G₄₃₇) and a quintuple haplotype (Pfdhfr I₅₁R₅₉N₁₀₈ and Pfdhps A₄₃₆E₅₄₀) in the study area, the efficacy of this combination should be closely monitored periodically. The efficacies of the other two combinations were superior to that of AS-SP, which may attract policy planners. However, further studies are needed in other parts of the country to have a better understanding and knowledge of the resistance pattern of artemisinin combination therapy.