1 in vivo therapeutic efficacy of chloroquine alone or in combination
TRANSCRIPT
1
In vivo therapeutic efficacy of chloroquine alone or in combination with 1
primaquine in vivax malaria in Kolkata, West Bengal, India and polymorphism 2
in pvmdr1 and pvcrt-0 genes 3
Swagata Gangulya, Pabitra Sahaa, Subhasish K. Guhaa, Sonali Dasa, Dilip K. Beraa, 4
Asit Biswasb, Pratip K. Kundua, Bibhuti Sahaa , Krishnangshu Raya, and Ardhendu 5
K. Majia,*. 6
a Calcutta School of Tropical Medicine, 108, C. R. Avenue, Kolkata 700 073, India. 7
bDepartment of Health & Family Welfare, Government of West Bengal, Swasthya 8
Bhawan, Salt Lake City, Kolkata-700092, India 9
Email addresses of Authors: 10
Swagata Ganguly: [email protected] 11
Pabitra Saha: [email protected] 12
Subhasish K. Guha: [email protected] 13
Sonali Das: [email protected] 14
Dilip K. Bera: [email protected] 15
Asit Biswas: [email protected] 16
Pratip K. Kundu: [email protected] 17
Bibhuti Saha: [email protected] 18
Krishnangshu Ray: [email protected] 19
20
21
22
23
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Copyright © 2012, American Society for Microbiology. All Rights Reserved.Antimicrob. Agents Chemother. doi:10.1128/AAC.02050-12 AAC Accepts, published online ahead of print on 21 December 2012
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* Corresponding author: 25
Name: Ardhendu Kumar Maji 26
Address: Calcutta School of Tropical Medicine, 27
108, C. R. Avenue, Kolkata - 700 073, 28
West Bengal, India 29
E mail: [email protected] 30
Phone No: +91 9433630077 31
Fax: +91 3322414915 32
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Running Title: Efficacy of CQ in vivax malaria in Kolkata, India 34
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Key word: 36
P. vivax 37
Chloroquine resistant 38
pvmdr1 39
pvcrt-0 40
Malaria 41
India 42
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Abstract: 48
Plasmodium vivax malaria though benign, has now become a matter of concern due 49
to recent reports of life threatening severity and development of parasite resistance 50
against different antimalarial drugs. The magnitude of the problem is still 51
undetermined. The present study was undertaken to determine the in vivo efficacy of 52
chloroquine and chloroquine plus primaquine in P. vivax malaria in Kolkata and 53
polymorphisms in pvmdr1 and pvcrt-0 genes. 54
A total of 250 patients with P. vivax monoinfection were recruited and randomised 55
into two groups A and B, treated with chloroquine and chloroquine plus primaquine 56
respectively and followed up for 42 days as per WHO protocol (2009). Data were 57
analysed using per protocol analyses. We assessed polymorphisms of pvmdr1 and 58
pvcrt-0 genes by DNA sequencing method. 59
Out of 250 recruited patients, 204 cases completed 42 days follow up period, 101 in 60
group A and 103 in group B. In group A, PCR uncorrected efficacy of CQ was 99% 61
(95% CI 0.944-1.00) and in group B all cases were classified as ACPR. Day 3 62
positivity was observed in 11 (5.3%) cases. No specific mutation pattern was 63
recorded in pvcrt-0 gene. Eight non-synonymous mutations were found in pvmdr1 64
gene, of which three were new. Y976F mutation was not detected in any isolate. 65
Chloroquine either alone or in combination with primaquine is still effective against P. 66
vivax malaria in the study area. 67
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1. Introduction 71
The burden of malaria caused by Plasmodium vivax has been greatly under-72
appreciated both in terms of its clinical spectrum and incidence of disease (1,2). P. 73
vivax is the most widely distributed cause of malaria in the world with approximately 74
2.6 billion people are at risk and ten countries including India are at highest risk of 75
infection (3,4,5,6). 76
Plasmodium vivax infections have been associated with mild symptoms such as 77
fever, headache, fatigue, chills, and musculoskeletal pain, in particular paroxysms. 78
Recently, however, severe complications, including renal failure, jaundice, acute 79
respiratory distress syndrome, cerebral malaria, seizures, anaemia, 80
hyperparasitaemia, thrombocytopenia, pulmonary edema, splenic rupture and death, 81
have been reported in exclusive association with P. vivax (7,8). The situation is 82
further complicated by the emergence of resistant power of the parasite against 83
chloroquine. 84
In most of the world chloroquine (CQ) remains the first line of treatment for patients 85
with vivax malaria. In India, CQ has been replaced by Artemisinin Combination 86
Therapy (ACT), a combination of Artesunate plus Sulphadoxine-Pyrimethamine in 87
2010 for P. falciparum malaria but in case of P. vivax malaria CQ remains the first 88
line agent along with primaquine (PQ, 0.25 mg/kg base) for 14 days under 89
supervision or by detecting G6PD level. The first case of P. vivax resistance to CQ 90
was reported in 1989, from Papua New Guinea (9). A higher rate of CQ resistant P. 91
vivax malaria is reported from different regions of Indonesia which exceeded 50% 92
(10,11,12,13,14). Further sporadic cases were subsequently observed in the 93
Philippines, Myanmar, Vietnam, Colombia, Guyana, and Turkey (15). Similar reports 94
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are also available from Madagascar (16) and Ethiopia (17). Despite these reports, it 95
remains difficult to estimate the worldwide prevalence of P. vivax resistance to CQ. 96
In India, first case of CQ resistant P. vivax malaria was reported from Assam in 1995 97
(18) and then in Bombay (19) and Gujarat (20). 98
The molecular mechanisms underlying CQ resistance in P. vivax malaria remain 99
unknown and may involve multigenic loci, but two genes orthologous to the pfmdr1 100
and pfcrt genes, which encode putative transporters, pvmdr1 (21) and pvcrt-0 (22), 101
have been suspected as possible genetic markers of CQ resistance. 102
Information about prevalence of CQ resistance P. vivax malaria and distribution of 103
possible genetic markers of CQ resistance in India are very poor particularly from 104
North-East India. The present study was designed to study the therapeutic efficacy 105
of chloroquine and chloroquine plus primaquine in P. vivax malaria and 106
polymorphisms of pvmdr-1 and pvcrt-0 genes associated with it. 107
2. Materials and methods 108
The study was a randomized, double-arm, open-label, interventional trial for 109
evaluation of clinical and parasitological responses of CQ and CQ plus PQ for 110
treatment of uncomplicated P. vivax malaria, based on the therapeutic efficacy 111
protocols "Methods for surveillance of antimalarial drug efficacy" of WHO 2009 (23). 112
2.1 Study site 113
The study was conducted in the Malaria Clinic attached with Protozoology Unit, 114
Calcutta School of Tropical Medicine during December 2011 – August 2012. In 115
Kolkata, vivax malaria is perennial, and falciparum malaria is seasonal with a peak 116
during August to December each year. Anopheles stephensi is the principal vector. 117
P. falciparum and P. vivax are the two predominant species with an incidence of 118
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almost 1:1. The study was approved by the Institutional Ethics Committee of the 119
Calcutta School of Tropical Medicine. 120
2.2 Patient recruitment and follow-up 121
All patients attending the clinic were screened for presence of malaria parasite by 122
examining thick and thin smears of peripheral blood followed by Giemsa staining, to 123
identify patients who might meet the inclusion criteria as per WHO protocol (WHO, 124
2009) (23). A total of 250 patients with P. vivax mono-infection, confirmed by 125
presence of negative HRP-II test, aged above 6 months were included in the study. 126
Inclusion criteria included a parasitaemia of 1000–100000 parasites/μl blood, 127
absence of severe disease, without any anti-malarial treatment during the preceding 128
four weeks, and no history of hepatic or kidney diseases. Written informed consent 129
for study participation was obtained from all patients or their guardians. 130
On enrolment, each patient was clinically examined thoroughly. A questionnaire 131
documenting fever, body weight, past history of malaria etc. was also completed. An 132
amount of 3 - 5 ml of blood was collected from each patient for bio-chemical and 133
molecular biological studies. 134
All recruited patients were requested to return for follow-up on days 1, 2, 3, 7, 14, 21, 135
28, 35 and 42 after initiation of treatment and were examined both clinically and 136
parasitologically. Members of the study team paid home visits to patients who 137
missed the scheduled clinic visits for clinical examination and collection of blood 138
samples for parasitological assessment. 139
2.3 Treatment 140
Before commencement of the treatment, G6PD level of all recruited patients were 141
determined qualitatively using G6PDH Hemopak kit (Apin Diagnostics & Chemicals, 142
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Vadodara, India). All the recruited patients were treated with standard dose of CQ 143
(25 mg/kg of base over 3 days) in group A and CQ (25 mg/kg of base over 3 days) 144
plus PQ (0.25 mg/kg daily for 14 days) in group B and followed-up for 42 days. 145
Patients were directly observed for 30 minutes after treatment, and the dose was re-146
administered, if vomiting occurred. Patients who repeatedly vomited their first dose 147
of study medication were excluded from the study. In the present study, PQ therapy 148
was supervised for first 7 days and then again on Day 14 by examining the empty 149
blister packs. 150
2.4 Study end points and statistical analysis 151
Therapeutic outcomes were classified according to WHO (WHO, 2009) (23) 152
guidelines into early treatment failure (ETF), late clinical failure (LCF), late 153
parasitological failure (LPF) and adequate clinical and parasitological response 154
(ACPR). Therapeutic efficacy was determined by per protocol method using WHO 155
software. The data was entered into a standard data entry programme designed by 156
Global Malaria Programme and analyzed by Kaplan-Meier survival curve according 157
to WHO standard procedures (http://www.who.int/malaria/resistance). The 95% 158
confidence interval was calculated by Dimension Research calculator (http: 159
//www.dimensionresearch. com/resources/ resources overview.html). 160
2.5 LABORATORY EXAMINATION 161
2.5.1 Blood smear for malaria diagnosis and parasite count 162
Giemsa-stained thick smear slides were read independently by two microscopists 163
and diagnosed as negative on initial review, if no parasites were seen in 100 oil 164
immersion fields. The number of parasites per 200 WBC was counted. Assuming a 165
WBC count to be 8000/μL, parasitaemia was calculated and expressed as per μL of 166
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blood. 167
2.5.2 Rapid Diagnostic Test 168
All microscopically positive P. vivax cases were screened for P. vivax specific pLDH 169
and HRP-II (SD Bio Standard Diagnostics Pvt. Ltd., Gurgaon, India) to detect any 170
mixed infection. 171
2.5.3 Preparation of DNA template from blood samples 172
Genomic DNA of P. vivax from all blood samples collected into EDTA coated vials 173
was extracted by using QIAamp DNA blood Kit (Qiagen, Hilden, Germany), following 174
the manufacturer’s instructions, with minor modifications - the incubation time with 175
proteinase K was increased to 20 minutes at 560C, to improve the yield of the 176
extraction. Extracted parasite genomic DNA of all the samples were preserved at 177
-200C and an aliquot was used as the DNA source for further study. 178
2.5.4 Genus and species specific PCR 179
To ensure P. vivax mono-infection genus and specific PCR method was applied as 180
described elsewhere (24). 181
2.5.5 Sequencing of pvmdr 1 and pvcrt-0 gene 182
The complete DNA sequence of pvmdr1 gene is 4606-basepairs (bp) and is located 183
on chromosome 10. The full pvmdr1 gene was amplified by polymerase chain 184
reaction (PCR). Six pairs of different primers, as described earlier (25), were used for 185
PCR amplification. All the PCR reactions were performed in total volume of 35 µl. 186
The reaction mixture consisted of 3 µl of genomic DNA, 0·3 µM of each primer pair, 187
0·2 mM of each deoxynucleotide triphosphate (dATP, dTTP, dGTP, dCTP), 2·5 mM 188
MgCl2, PCR buffer, and 2 unit of Taq DNA Polymerase (Perkin Elmer, Branchburg, 189
NJ, USA). Amplification was performed using a Veriti 96 well Thermal Cycler (Perkin 190
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Elmer, Branchburg, NJ, USA) under the following conditions: initial denaturation at 191
950C for 10 minutes, followed by 40 cycles of denaturation at 940C for 40 seconds, 192
annealing at 550C (570C for primer 1 & 2) for 1 minute, and extension at 720C for 2 193
minutes and a final extension period at 720C for 10 minutes. 194
The complete DNA sequence of pvcrt-0 gene is 4281 bp and is located on 195
chromosome 1. The gene contains 14 exons and 13 introns. Amplification of full 196
length pvcrt-0 gene by PCR was performed with similar PCR mixture as for pvmdr1 197
and five pairs of synthetic oligonucleotides, as described earlier (25). PCR was 198
performed under the following conditions: 950C for 10 minutes and 45 cycles of 950C 199
for 40 seconds, 550C (570C for primer 3 & 4 and 590C for primer 1) for 1 minute, 200
720C for 2 minutes and final extension at 720C for 10 minutes. 201
The quality and concentration of PCR products were ascertained by agarose gel 202
electrophoresis. They were subsequently purified using a commercial Kit (QIAquick 203
PCR Purification Kit; Qiagen) and used as template for sequencing. Sequencing 204
reactions were carried out with the ABI Prism Big Dye Terminator cycle sequencing 205
ready reaction kit on a 3730 XL genetic analyzer (Perkin Elmer, Branchburg, NJ, 206
USA). 207
The sequences were analyzed using the free software Bioedit Sequence Alignment 208
Editor version 7·0·5·2. The sequences were then aligned with the pvmdr1 (GenBank 209
Acc. No. AY618622) and pvcrt-0 (GenBank Acc. No. AF314649) of the Sal 1 using 210
the online sequence alignment tool ClustalW (available at: http: 211
//www.ebi.ac.uk/clustalw). 212
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3. Result: 215
3.1 Study population 216
During the study period, 7439 patients, attending the Malaria Clinic presenting with 217
fever were examined for presence of malaria parasite of whom 1695 were positive 218
for malaria (148 P. falciparum, 1544 P. vivax, and 3 mixed infections of both P. 219
falciparum and P. vivax). Of the 1544 P. vivax positive cases, 250 patients were 220
enrolled for the study. All the recruited patients were normal for the G6PD test. Their 221
baseline characteristics are described in Table -1. 222
3.2 In vivo drug efficacy 223
Total 250 recruited patients were divided into 2 study groups – A and B, each group 224
comprising of 125 patients. Patients in study arm A received only chloroquine and 225
those in study group B received both chloroquine and primaquine. 226
In study arm A, 24 patients were lost to follow-up, 101patients completed the CQ 227
treatment course as well as the 42 day follow-up schedule and reached one of the 228
study end-points. In the present study arm there was one case of LCF on day 42 and 229
rest 100 (99%, 95% CI 0·96-1·00) patients were classified as ACPR (Figure 1). 230
Subsequently we could not classify the LCF case as either relapse or re-infection as 231
we were unable to amplify the parasite DNA of day 42 sample for pvcsp and pvmsp-232
3α genotypes. 233
In study group B, 21 patients were lost to follow-up and 104 patients completed the 234
CQ treatment course as well as the 42 day follow-up schedule thus reaching one of 235
the study end-points. In this study arm, one case was classified as ETF, but 236
subsequently after parasite DNA amplification by PCR it was found to be a case of 237
mixed infection of both P. vivax and P. falciparum. Hence this case was excluded 238
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from the study. Remaining 103 (100%, 95% CI 0·96-1·00) patients were classified as 239
ACPR. There was no case of therapeutic failure (Figure1). 240
In all subjects, fever subsided by Day 2. In group A parasite was cleared in 63 241
(57.8%), 34 (31.2%) and 6 (5.5%) cases by Day1, 2 and 3 respectively. In group B it 242
was 66 (60%), 24 (21.8%) and 5 (4.5%) cases by Day1, 2 and 3 respectively. No 243
significant difference was observed on day 1 (Z = 1.74, P = 0.08), day 2 (Z = 0.465, 244
P = 0.641) and day 3 (Z = 0.337, P = 0.735) positivity in both the study groups. But in 245
eleven cases (6 in group A and 5 in group B) parasite was detected in peripheral 246
blood smears on Day 4 which was cleared on Day5 without any additional anti-247
malarial drug. Ten patients with long parasite clearance were adults, age ranging 248
from 20-60 years and 1 patient belonged to the age group 5-15 years. 249
During the entire study, no significant adverse side effects were recorded apart from 250
headache (n = 55), nausea (n = 34), vomiting (n = 4), abdominal pain (n = 8), 251
diarrhoea (n = 6), pruritis (n = 15) during the course of medication and were treated 252
symptomatically. 253
3.3 Polymorphism of pvcrt-0 and pvmdr1 gene 254
As we did not find any significant proportion of therapeutic failure cases, we 255
attempted to sequence 10% of the total isolates including those eleven cases with 256
long parasite clearance time for pvcrt-0 and pvmdr1 genes. 257
Sequences of pvcrt-0 gene of all 25 isolates were of mutant type. But no specific 258
pattern of polymorphism was detected. Four synonymous (L122L, P565P, Q782Q 259
and K807K) and four non-synonymous mutations (N706P, P1201L, R1224P and 260
R1278K) were detected. Among the synonymous mutations, L122L, P565P, and 261
K807K were located in exon 1, 4 and 7 respectively and Q782Q in intron 6. Among 262
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the non-synonymous mutations N706P, P1201L, were located in intron 5 and 10 263
respectively, and R1224P, R1278K were located in exon 11 and 12 respectively 264
(Table 2). 265
The pvmdr1 gene was fully sequenced in 25 isolates, among them 5 (20%) isolates 266
were of wild type. Four synonymous (K68K, G172G, L310L, L697L) and eight non-267
synonymous (R88Q, A296V, E478G, S513R, G698S, M908L, T958M and F1076L) 268
point mutations were recorded. However, the Y976F substitution, thought to be 269
involved in CQ resistance in P. vivax, was not recorded in the present study. Five 270
different haplotypes with two [G478S698 (2, 8%)], three [(S698L908M598 (7, 28.0%), 271
L908M958L1076 (4, 16.0%)] and four [Q88V296L908M958 (3, 12.0%), Q88R513L908M958 (4, 272
16.0%)] amino acid substitutions were identified (Table 2). Distribution of point 273
mutations in pvcrt-0 and pvmdr1 and haplotypes of pvmdr1 were equally prevalent 274
among the cases with long parasite clearance time and other cases. 275
4. Discussion 276
In vivo studies remain the gold standard for assessment of efficacy of different anti-277
malarial drugs. However there are some operational difficulties associated with the 278
follow-up of recruited patients for 42 days and differentiation of ‘recrudescence from 279
‘re-infection’ in endemic areas. In case of P. falciparum, msp 1, msp 2 and glurp 280
genotyping can address the problem (26). Relapse in P. vivax malaria caused by 281
hypnozoite reactivation makes interpretation of recurrences in drug efficacy trials 282
complicated. Genotyping cannot distinguish relapse from re-infection when the 283
parasites causing the relapse arise from a dormant subset of the inoculated 284
sporozoites that caused the primary infection. P. vivax parasites causing primary 285
infections as well as relapses have been compared using molecular markers pvcs 286
and pvmsp1, and pvmsp1 alone, respectively (27,28). 287
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In the present study one patient treated with chloroquine returned with fever and his 288
peripheral blood smear showed P. vivax infection on day 42. We could not classify it 289
either as re-infection or recrudescence or relapse as we were unable to amplify the 290
parasite DNA for pvcsp and pvmsp-3α genotyping. In the other study group, patients 291
treated with chloroquine plus primaquine, all cases were categorised as ACPR. So, 292
in Kolkata CQ alone was 99% and CQ plus PQ was 100% effective in P. vivax 293
malaria. Similar observation was also made during 1998-2001 (29) and 2003-2004 294
(30). So during past 15 years efficacy of CQ remained unchanged in P. vivax malaria 295
but in case of P. falciparum malaria CQ resistance level reached to 76.3% (31). 296
However, day 3 parasite positivity was noted in 11 (5.3%) patients. Ten of them were 297
in adult group and one in 5-15 years. The mean Day 0 parasite count of those eleven 298
patients was 10825 (Range: 2200-31680; SD: ±7946.7), which was higher than 299
those patients whose parasite cleared on Day 3 with mean Day 0 parasite count was 300
4210 (Range: 1000-25600; SD: ±3961.9). All eleven cases were classified as ACPR 301
as per WHO 2009 protocol (23). Perhaps, long parasite clearance time is an 302
indication of diminished sensitivity of the parasite to chloroquine alone as well as 303
chloroquine plus primaquine. G6PD is an important issue related to P. vivax malaria 304
Mediterranean type of G6PD deficiency significantly protects P. vivax infection 305
among Afghan refugees in Pakistan (32). Similar report is also available for the 306
G6PD Mahidol variant from South East Asia (33). 307
Molecular markers seem to be useful for monitoring the drug resistance of malarial 308
parasites. Two genes pfcrt and pfmdr1 were found to be associated with efficacy of 309
CQ in P. falciparum malaria. The K76T mutation in the pfcrt gene is known to be 310
involved in CQ resistance. But little information is available regarding the possible 311
relationship between the pvcrt-0 and pvmdr1 genes and CQ resistance. Only a few 312
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studies have been carried out and associations between treatment failure and non-313
synonymous mutations in isolates obtained before treatment have not yet been 314
clearly established (21,34,35). However, the Y976F substitution in the pvmdr1 gene 315
is thought to be involved in CQ resistance in P. vivax (25,36). Suwanarusk et al., 316
(2007) observed that the geometric mean 50% inhibitory concentration of CQ was 317
significantly higher in P. vivax isolates carrying the Y976F mutation than in isolates 318
with the wild-type allele. 319
In the present study no definite mutation pattern was noticed in pvmdr1 gene. Out of 320
25 isolates studied, 13 isolates were of wild type. Four synonymous and eight non 321
synonymous mutations were observed in pvmdr1 gene, three of which (R88Q, 322
A296V and E478G) were not identified previously. But no mutation at codon 976 was 323
observed. In pvcrt-0, four non-synonymous mutations were found, two ((N706P and 324
P1201L) in intron 5 and 10 and two (R1224P andR1278K) in exon 11 and 12. 325
Frequent multiple mutant pvmdr1 haplotypes (quadruple, sextuple and septuple) 326
were reported from Madagascar, but none of the mutant haplotypes was found to be 327
associated with CQ resistant P. vivax malaria (16). In the present study, only double 328
[G478S698 (8.0%)], triple [(S698L908M598 (28.0%), L908M958L1076 (16.0%)] and quadruple 329
[Q88V296L908M958 (12.0%), Q88R513L908M958 (16.0%)] mutant haplotypes along with 330
wild type allele (20.0%) were observed. Though the sample size was small but the 331
absence of Y976F point mutation and multiple mutant haplotypes (sextuple or 332
septuple) of pvmdr1 along with persistence of significant proportion of wild type gene 333
justified our in vivo therapeutic outcomes. 334
In the study area, CQ alone and CQ plus PQ (99% and 100%) were effective against 335
P. vivax malaria. Day 3 parasite positivity in 11 (5.3%) cases needs further 336
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evaluation for development of resistance of the parasite against the drugs. Neither 337
definite pattern of polymorphisms of pvcrt-0 and pvmdr1 nor mutation at pvmdr1 338
Y976F was observed. Hence, it is difficult to correlate any association of genetic 339
marker with therapeutic outcomes. Periodical monitoring may elucidate the changing 340
pattern of the susceptibility of the parasite to CQ and CQ plus PQ. 341
Clinical trial registration: The study protocol was registered in CTRI (Clinical trial 342
registry-India) of Indian council of Medical Research. Registration 343
No.CTRI/2011/09/002031 344
Conflicts of interest: The authors have no conflicts of interest concerning the work 345
reported in this paper. 346
Authors' contributions: AKM and SKG conceptualized and designed the study 347
protocol; SG, PS, SD,BS, PKK, DKB and AB performed the clinical assessment and 348
the in-vivo therapeutic efficacy study; SG and PS performed the PCR and 349
sequencing analysis and interpretation of data; AKM,SG, SKG, PS, BS, KR and DKB 350
drafted the manuscript. All authors read and approved the final manuscript. 351
Acknowledgements: We are grateful to the Department of Health and Family 352
Welfare, Government of West Bengal, India for funding the project. Special thanks to 353
all patients who participated in this study. We are grateful to the Director, Calcutta 354
School of Tropical Medicine, for his kind permission to publish the data. 355
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Table 1: Baseline Characteristics of the Study Patients 506
Characteristics CQ (n = 125) CQ + PQ (n = 125)
Sex: no. (%) Male Female
110 (88) 15 (12)
113 (90.4) 12 (9.6)
Weight: kg Mean Range SD 95% CI
49.3 11-87 ±12.703 47.07-51.53
49.38 16-95 ±13.56 41-51.76
Age category: no. (%) 5-15 Adult
26 (20.8) 99 (79.2)
24 (19.2) 101 (80.8)
Age: year Mean Range SD 95% CI
25.21 5-65 ±12.86 22.96-27.47
25.16 6-60 ±12.82 22.91-27.41
Temperature: 0 C Mean Range SD 95% CI
37.79 37.5-39.0 ±0.28 37.74-37.84
37.72 37.5-38.5 ±0.17 37.69-37.75
Haemoglobin: g/dl Mean Range SD 95% CI
12.61 6.8-20 ±1.95 12.27-12.95
12.60 7.6-20.0 ± 2.07 12.24-12.97
Parasite count: no/µl Mean Range SD 95% CI
4744 1000-31680 ±4595 3938-5549
4285 1000-25600 ±4214 3543-5027
507 508 509 510
511
512
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Table 2: Mutation profile of pvmdr1 and pvcrt-0 gene in study isolates 513
Candidate Gene Occurrence of mutation in study population
n % 95% CI pvmdr 1 (n = 25) Synonymous Mutation K68K G172G L310L L697L Non-synonymous Mutation R88Q A296V E478G S513R G698S M908L T958M F1076L Haplotypes Wild type Double mutants G478S698 Triple mutants
S698L908M958
L908M958L1076 Quadruple mutants Q88V296L908M958
Q88R513L908M958
2 4 3 5 7 3 2 4 9 18 18 4 5 2 7 4 3 4
8.0 16.0 12.0 20.0 28·0 12.0 8.0 16.0 36.0 72.0 72.0 16.0 20.0 8.0 28.0 16.0 12.0 16.0
0 – 18.63 1.63 – 30.37 0 - 24.74 4.32 – 35.68 10.4 – 45.6 0 - 24.74 0 – 18.63 1.63 – 30.37 17.18 – 54.82 54.4 – 89.6 54.4 – 89.6 1.63 – 30.37 10.4 – 45.6 0 – 18.63 10.4 – 45.6 1.63 – 30.37 0 - 24.74 1.63 – 30.37
Pvcrt-0 (n = 25) Synonymous Mutation L122L P565P Q782Q K807K Non-synonymous Mutation N706P P1201L R1224P R1278K
4 12 3 4 23 3 3 24
16.0 48.0 12.0 16.0 92.0 12.0 12.0 96.0
1.63 – 30.37 28.42 – 67.58 0 - 24.74 1.63 – 30.37 81.37 – 102.63 0 - 24.74 0 - 24.74 88.32 – 103.68
* Non-synonymous mutations are shown in bold faces 514
515
516
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Figure: 1 CONSORT FLOW CHART 517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
* WTH: Withdrawn, LFU: Loss to Follow-up 533
Total Screened:7439
Negative Cases 5744
P.vivax 1544
Total Enrolled n = 250
P.falciparum
148 Mixed Infection
3
CQ n = 125
CQ + PQ n = 125
Evaluable by Day 42 n = 101
Evaluable by Day 42 n = 103
WTH: 0
LFU: 24
WTH: 0
LFU: 21
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