in vitro susceptibility of plasmodium v ivax to...

#Address correspondence to Adriana Pabón. Universidad de Antioquia, UdeA Calle 70 No. 52-21, Medellín, Colombia Tel: +574 219 6490. Fax: +574 219 6487. e-mail: [email protected]

In vitro Susceptibility of Plasmodium Vivax to antimalarials in Colombia 1

2

Diana Fernández1, César Segura1, Margarita Arboleda2, Giovanny Garavito3, Silvia Blair1, 3

Adriana Pabón1,4# 4

5

6

1 Grupo Malaria, Facultad de Medicina, Universidad de Antioquia, Medellín, Colombia. 7

2 Instituto Colombiano de Medicina Tropical, Universidad CES, Apartadó, Colombia. 8

3 Departamento de Farmacia, Facultad de Ciencias, Universidad Nacional de Colombia. 9

4 Programa de Biología, Facultad de Ciencias Básicas, Universidad del Atlántico, Barranquilla 10

Colombia. 11

12

13

Running title: Susceptibility of Colombian Plasmodium vivax isolates 14

Abstract word count: 211 15

Text word count: 3170 16

17

AAC Accepts, published online ahead of print on 11 August 2014Antimicrob. Agents Chemother. doi:10.1128/AAC.03191-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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ABSTRACT 18

The In vitro susceptibility of 30 isolates of Plasmodium vivax to a number of antimalarials 19

(chloroquine, mefloquine, amodiaquine, quinine and artesunate) was evaluated. The isolates 20

came from the malaria-endemic region of Urabá in Colombia, and were evaluated by the 21

schizont maturation test. The IC50 was found to be 0.6 nM (CI 95%: 0.3 – 1.0nM) for 22

artesunate, 8.5nM (CI 95%: 5.6 – 13.0nM) for amodiaquine, 23.3nM (CI 95%: 12.4 – 44.1nM) 23

for chloroquine, 55.6nM (CI 95%: 36.8 – 84.1nM) for mefloquine and 115.3nM (CI 95%: 57.7 24

– 230.5nM) for quinine. The isolates were classified according to whether the initial parasites 25

were mature or immature trophozoites (Tfz). It was found that the IC50 values for chloroquine 26

and artesunate were significantly different in the two aforementioned groups (p<0.001). The 27

IC50 of CQ and AS was higher in the isolates from mature Tfz (CQ: 39.3 nM vs 17 nM, AS: 1.4 28

nM vs 0.3 nM) and 10% of the isolates showed lower susceptibility to one of the antimalarial, 29

13.3% to two antimalarial and 3.3% over three antimalarial drugs. It should be highlighted that 30

despite the extensive use of chloroquine in Colombia, P. vivax continues to be susceptible to 31

antimalarials. This is the first report, to our knowledge, showing in vitro susceptibility of P. 32

vivax isolates to antimalarials in Colombia. 33

34

Key words: Plasmodium vivax, in vitro susceptibility, Chloroquine, amodiaquine, mefloquine, 35

quinine and artesunate antimalarials 36

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INTRODUCTION 38

Plasmodium vivax is responsible for more than 50% of malaria cases worldwide and is 39

prevalent in Southeast Asia, the Western Pacific, Central and South America. P. vivax malaria 40

is known for causing relapses (1) and in recent years complicated clinical conditions have 41

been reported for this type of malaria that are similar to those found for P. falciparum. Indeed, 42

the aforementioned two species coexist in many parts of the world (2). In Colombia 85% of 43

the territory is suitable for the transmission of Plasmodium spp., and according to an 44

epidemiological report from the National Health Institute of Colombia, 53,963 cases of malaria 45

were reported in 2012, of which 40,314 (74.7%) corresponded to P. vivax malaria (3). 46

47

The therapeutic failure of chloroquine to treat P. vivax malaria has been reported in different 48

regions around the world. The first case of therapeutic failure was reported in Papua New 49

Guinea in 1989 (4, 5), and since then cases have also been recorded in Asia, principally in 50

Indonesia, Malaysia, Myanmar, India, The Philippines, Vietnam, Korea and Thailand. Cases 51

have also been reported in Ethiopia in Africa (6-12) and in Guyana, Brazil, Venezuela, Peru 52

and Colombia in South America (13-17). In Colombia, whereas one study reports the 53

therapeutic failure of chloroquine in 11% (3/27) of the patients evaluated (17), there are other 54

studies that report a 100% efficacy of this antimalarial (14, 18). Therefore, the In vitro 55

susceptibility profile of P. vivax to antimalarials must be determined. 56


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This work investigated In vitro susceptibility of fresh isolates of P. vivax from the region of 57

Urabá (Antioquia, Colombia) to chloroquine, mefloquine, amodiaquine, quinine and 58

artesunate using the schizont maturation test. 59

60

MATERIALS AND METHODS 61

Pre-dosing of plates with antimalarials: Seven serial dilutions of the antimalarials 62

chloroquine (CQ) (Sigma R-6628), mefloquine (MQ) (Sigma R-2319), amodiaquine (AQ) 63

(Sigma R-2799), quinine (Qn) (Sigma R-0132) and artesunate (Sigma R-3731) were pre-64

dosed. Each antimalarial plate was pre-dosed in triplicate, in a range of: 1.646 nM –1.200nM; 65

12.5nM – 800nM; 2.34nM – 150nM; 3.7nM – 2.700nM and 0.05nM – 40nM for CQ, MQ, AQ, 66

Qn and AS respectively (4, 19-22). The pre-dosed plates were stored at 4°C until they were 67

used. The quality control of the pre-dosing stage was carried out with the NF54 strain of P. 68

falciparum (sensitive to all known antimalarials) (23) and parasitaemia was recorded through 69

the radioisotope method (24). 70

P. vivax isolates: Between September 2010 and November 2012, at the malaria diagnosis 71

stations in the endemic municipalities of Turbo and Apartadó (Urabá, Antioquia, Colombia), 72

patients were received with the following inclusion criteria: any age and gender, women who 73

were not pregnant, single P. vivax infections (according to thick smear test and confirmed by 74

the rapid diagnostic test SD Bio Line malaria Ag Pf/Pan Standard diaagnostiscs Inc®, Ref. 75

05FK66) and parasitaemia > 2000 parasites/µL of blood. Additionally, the following inclusion 76


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criteria were applied: diagnosis of uncomplicated malaria according to the WHO criteria 77

adapted for Colombia (25), patients who hadn’t taken antimalarials the month before the test, 78

and patients who didn’t have chloroquine in their urine (according to the Saker-Solomons 79

method). Patients who fulfilled the criteria and were willing to take part signed an informed 80

consent previously approved by the Ethics Committee of the Universidad de Antioquia 81

through act number 012 of the 18th June 2009. In the case of minors, the consent was signed 82

by a parent or guardian over the age of 18. 83

Collecting and processing samples: In order to evaluate the in vitro susceptibility of P. 84

vivax to antimalarials, 10 mL of venous blood was taken from each patient and put in a 85

vacutainer tube with heparin. The samples were processed within four hours after collected 86

from the patients. Additionally, 200 µL of blood was poured onto filter paper (Ref: Whatman 87

#3) to confirm the P. vivax monoinfection in the analysed samples using PCR (26). 88

In the thick smear test, 200 asexual parasites were counted in all of the different parasitic 89

stages: rings or immature trophozoites (Tfz imm), amoeboid trophozoites (Tfz ameob), mature 90

trophozoites (Tfz mat), pre-schizonts (pre-sch) and mature schizonts (Sch mat) (27). 91

According to the initial parasitic stages, the isolates can be divided into two groups. Group 1 92

consists of isolates with more than 133 immature Tfz (rings + amoeboid Tfz) out of 200 93

asexual parasites, and Group 2 consists of isolates that do not fulfil the previous criterion and 94

present > 133 mature Tfz out of 200 asexual parasites (21, 27). 95


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Assay of in vitro susceptibility of P. vivax to antimalarials using the schizont 96

maturation method: Before the assay, the leukocytes in a 10 mL sample of heparinized 97

blood were eliminated during two rounds of filtration using cellulose columns, in accordance 98

with the method described by Rusell, et al, 2003 (22). The brand of cellulose (Sigma Aldrich, 99

S6288) used was different to that employed in Rusell´s method, but it had similar 100

characteristics. 101

The parasitized erythrocytes, recovered during the second filtration, were centrifuged at 1,500 102

rpm for 5 minutes. The supernatant liquid was then removed and 800 µL of the parasitized 103

erythrocytes was used to prepare a suspension at a hematocrit of 2% in McCoy’s 5A medium 104

(Sigma Aldrich, M4892), supplemented with 25% AB+ of human serum. 200 µL of this 105

suspension was poured into each well of the pre-dosed plates, which were incubated at 37°C 106

in an atmosphere of 5% CO2, 5% O2 and 90% N2 until 40% of the initial parasitic stages of 107

each isolate matured into schizonts in the drug-free control wells. After incubation, the 108

supernatant was removed from each well and the thick smear test was performed (28). To 109

obtain readings from the tests, 200 asexual parasites differentiated by their parasitic stages 110

were counted as previously described. 111

Inhibition percentages and inhibitory concentration (IC50): The inhibition percentage of 112

schizont maturation in the treatment was calculated by relating the number of schizonts 113

present in the controls (greater than 40%) with the number of schizonts in the treatments. 114

Calculations were made of the mean or median, standard deviation and coefficient of 115

variation of the schizont inhibition percentages obtained in three replicas of the antimalarial 116


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concentrations evaluated. The IC50s were estimated using a dose-response regression model 117

from the drug-free controls by the GraphPad Prism version 5.0 program. The IC50s that were 118

within the concentration range evaluated for each antimalarial, and that had an r2 > 9.0, were 119

classified as valid. The IC50 of each antimalarial was correlated with the antimalarial group to 120

which it belonged (aminoquinolines and artemisinins) in order to find overlapping 121

susceptibility. 122

Statistical analysis: The statistical analysis was carried out using the GraphPad Prism 123

version 5.0 software. The Kruskal-Wallis test was applied to determine the non-parametric 124

comparisons, and the Fisher test was employed to calculate statistical significance. The non-125

parametric correlation analysis was carried out using the Spearman (rs) correlation model. 126

127

RESULTS 128

Of the 68 P. vivax monoinfection isolates (confirmed by PCR) that fulfilled the inclusion 129

criteria, 30 (44.1%) achieved a schizont maturation of >40%. The mean initial parasitaemia 130

was 9853.3 parasites/µL of blood (CI 95%: 7640.7–17494.0). The isolates took an average of 131

28.8 hours (CI 95%: 25.7–31.9 hours) to reach a schizont maturation and each isolate 132

reached an average maturation of 54% (CI95%: 46 - 100). Of the evaluated isolates, 15 133

(50%) were classified in Group 1, with more than 133 immature tfz out of 200 asexual 134

parasites and a mean initial parasitaemia of 12,605 parasites/µL of blood [CI 95%: 8,985 – 135

16,226]. The other 15 (50%) were classified in Group 2, with more than 133 mature tfz out of 136


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200 asexual parasites and a mean initial parasitaemia of 6,949 parasites/µL of blood [CI95%: 137

4,749 – 9,150]. The parasitaemia was significantly different in the two groups (p value <0.05). 138

In Group 1, 66.7% (10/15) of the isolates achieved schizont maturation after 30 hours, 20% 139

(3/15) reached this stage after 40 hours and 13.3% (2/15) achieved maturation after 48 hours 140

and in Group 2, 73.3% (11/15) achieved maturation after 30 hours and the rest reached this 141

stage after 20 hours. The majority of cultures in both groups had a maturation time of 30 142

hours. The maturation time was significantly higher in the isolates of Group 1 than those of 143

Group 2 (p value < 0.0001) (Table 1). 144

In vitro susceptibility of P. vivax to antimalarials 145

The number of valid tests and the IC50 found for each antimalarial can be seen in Table 2. 146

Due to the fact that only 30 isolates matured successfully, the IC50 medians were analysed 147

based on the predominance of the initial parasitic stages, as shown in Table 3. The maximum 148

and minimum values of IC50 were found for CQ (2.5nM – 1109.0nM), MQ (10.6nM – 522.3 149

nM), AQ (2.7nM – 105.2nM), Qn (4.0nM – 1398.0nM) and AS (0.1nM – 10.7nM) (Figure 1). 150

151

Correlation of the IC50s of the different antimalarials in P. vivax from Colombia 152

The overlapping susceptibility of the 30 isolates of P. vivax to all of the antimalarials evaluated 153

is shown in Table 4. The susceptibility of the antimalarials CQ, MQ, AQ and Qn of the 154

quinoline group were compared with AS (a derivative of artemisinin). A positive correlation 155

was found between the CQ and the MQ with AS (p value < 0.001) (Table 4). 156


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DISCUSSION 157

This study analysed the susceptibility of 30 Colombian isolates of P. vivax to different 158

antimalarials. Chloroquine is a drug of special interest as it corresponds to a first-line 159

treatment for P. vivax malaria in Colombia (25). Given the difficulty of maintaining P. vivax in 160

continuous cultures (29-31), an in vitro short-term growth method was used which consisted 161

of maintaining the parasites in culture for periods of time of less than 48 hours until they 162

reached their schizont maturation (32). This method has helped to determine the in vitro 163

susceptibility of P. vivax to antimalarials in different geographic regions of Africa and Asia (20-164

22, 33-35). 165

166

The IC50s observed in the isolates from the region of Urabá, which is a low malaria-endemic 167

area for South America but a high endemic area for Colombia, were low for all of the 168

antimalarials. However, there were isolates that presented IC50 values for CQ that were 47.6 169

times higher than the mean IC50 (23.3 nM). Four out of 30 (13.3%) of the isolates presented 170

IC50 values greater than 100 nM, which is similar to what has been found in Asian countries 171

like Thailand, Indonesia, Myanmar, Papua New Guinea and Brazil (36) (Table 5). 172

It is important to note that the schizont maturation method gives variables like the level of 173

maturity of the parasitic stages at the beginning of the assay. These variables influence the 174

maturation time of the schizonts and the reproducibility of the results. This is one of the 175

possible reasons that the IC50 values of CQ and AS were significantly different (p<0001) in the 176


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two groups established according to initial parasitic stages, as has been reported previously 177

in the literature. Despite the differences found, the mean IC50 values for all of the antimalarials 178

were observed within the range of susceptibility, suggesting that the Colombian isolates were 179

susceptible even when they were cultured from mature trophozoites (21, 37). 180

Whereas in the case of P. falciparum the mature trophozoite is considered to be the target 181

stage of CQ, for P. vivax this target is not recognised. It is believed that each species has 182

characteristics that can explain the difference in susceptibility of each parasitic stage to 183

chloroquine (21, 37). During studies that monitor the resistance of P. vivax to antimalarials, it 184

is important to take into account the target parasite form. In this study, the cultures that began 185

with a predominance of mature forms (Group 2) presented significantly higher IC50 values 186

than those that began with immature forms (Group 1). 187

When determining the susceptibility of P. vivax to antimalarials, the monitoring of schizont 188

maturation is fundamental in order to avoid taking premature readings which would cause a 189

smaller percentage schizonts to be recorded than expected. However, an excess in 190

maturation time would cause the schizonts to rupture and their numbers would be reduced 191

(21). 192

The positive correlation observed between the susceptibility of the isolates to the quinolines 193

CQ, MQ, AQ, Qn and AS (a derivative of artemisinins) suggests a common mode of action, 194

as proposed by other authors (21, 27). Artesunate is a first-line drug for the treatment of P. 195

falciparum malaria in Colombia (25). This study shows that in Colombia AS is also effective 196


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against P. vivax, as an IC50 of 0.6 nM (CI 95%: 0.3 – 1.0nM) is presented. Studies carried out 197

between 2004 and 2009 in different regions of Asia, like Thailand and Indonesia, report an 198

IC50 of < 10 nM for the effect of artesunate against P. vivax. Such results suggest that 199

derivatives of artemisinins have been effective against this type of parasite. However, in 200

Thailand in the year 2011, an IC50 of 32.6 nM was found for artesunate and in South América, 201

in Brazilian region in the year 2014, an IC50 of 21.0 nM was found for this antimalarial which 202

suggests a susceptibility loss of P. vivax to this antimalarial in two different geographic 203

regions (Table 5) (9, 19, 21, 22, 36, 38). In this study, when the isolates were classified 204

according to the initial parasitic stages, IC50s for AS of 0.3 nM in Group 1 and 1.4 nM in Group 205

2 were found (Table 3). Although these values are significantly different, they are below 10 206

nM, which indicates that this antimalarial is effective against P. vivax in Colombia. With 207

respect to MQ, in this study an IC50 of 55.6 nM (Table 2) was found, which differs from the 208

values reported from 2008 to 2011 in Thailand, Indonesia and China, and from the values 209

reported for Thailand and Korea in 2004 and 2009 respectively (19, 33). In the case of AQ, an 210

IC50 of 8.5 nM (Table 2) was recorded, which is similar to the values reported in different 211

regions of Asia (IC50 < 30 nM) between the years of 2004 and 2008 (Table 5). (19, 22, 39). 212

This suggests that AQ is effective against P. vivax in the various geographical regions studied 213

in Asia and South America. Finally, the Qn in this study had an IC50 of 115.3 nM, which was 214

almost three times below the values reported in the literature (393.4 nM) (Table 5). However, 215

it should be noted that there are very few reports of quinine being evaluated in vitro against P. 216

vivax. More monitoring of this antimalarial in different geographical regions would help to 217

establish its affectivity. 218


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Given the difficulty of maintaining an in vitro culture of P. vivax (29-31), resistance studies 219

have been complimented with genetic markers. One example of a genetic marker is the 220

pvmdr-1 gene that encodes for the protein associated to multiple drug resistance. The 221

presence of mutations is associated to the loss of in vitro susceptibility of P. vivax to the 222

antimalarials. Furthermore, the mutations in the pvdhfr and pvdhpr genes of P. Vivax that 223

encode for the dihydrofolate reductase and dihydropteroate synthase proteins respectively 224

have shown to be associated with a loss of susceptibility to sulfadoxine/pyrimethamine (40). 225

In the year 2008, Suwanarusk R et al. evaluated how in vitro susceptibility is associated with 226

the presence of mutations in the pvmdr-1 gene (39). They found a significantly high IC50 (78.6 227

nM) for mefloquine and an increase in the number of copies of the pvmdr-1 gene compared 228

with isolates which had a low IC50 (38 nM) and a single copy of the gene. The presence of the 229

Y976F mutation of the pvmdr-1 gene has been associated with a greater loss of susceptibility 230

to CQ (IC50: 154 nM) compared to the isolations without mutation (IC50: 34 nM), but with a 231

greater susceptibility to artesunate (IC50 mutant: 1.8 nM vs native: 9.5 nM) and mefloquine 232

(IC50 mutant: 14 nM vs wild: 121 nM). 233

234

In Central America polymorphisms have been found in the pvmdr-1 and pvdhfr genes that 235

may be related to the loss of susceptibility of P. vivax to antimalarials. However, there is no 236

evidence from in vitro susceptibility studies that support this association (41). In South 237

America, only isolated cases of the therapeutic failure to CQ for the treatment of P. vivax 238


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malaria have been documented and no data on CQ drug levels to ensure therapeutic levels 239

were informed. Also there are few reports of in vitro susceptibility of P. vivax to antimalarials. 240

However, ongoing clinical efficacy monitoring would be important in addition to integrated in 241

vitro susceptibility and molecular markers studies. For example, in Amazonian region of 242

Brazil, in the year 2013, Chehuan et al., reported that 12 out of 112 (10.7%) isolates with IC50 243

> 100 nM where considered resistant to CQ, while 3 out of 47 (6.4%) where considered 244

resistant to MQ. The same study, it was shown that Amazonian P. vivax strains with both CQ 245

and MQ resistance may be common; and a non-synonymous mutation at Pvdhps codon 382 246

(S→C) was associated to in vitro susceptibility to CQ, thus further studies should be done to 247

confirm this observation (42). On the other hand, in the year 2014, Aguiar et al, found similar 248

IC50´s to those reported to CQ and MQ in this region however, the IC50´s to AS observed in 249

this work for Colombia was twenty times lower to that reported in Brazil (36) (Table 5). 250

251

In conclusion, this study showed that the Colombian isolates of P. vivax continue to be 252

susceptible to all of the antimalarials evaluated, and it is important to note that this is the first 253

report that has investigated the effect of the IC50s of antimalarials against P. vivax in 254

Colombia. However, the presence of isolates with IC50 values above 100 nM for CQ, MQ and 255

Qn suggests the need of monitoring periodically the therapeutic response to antimalarials and 256

the evaluation of antimalarial drugs levels in blood and importantly will be associate the IC50s 257

in vitro of antimalarials in P. vivax and their tendency to increase with an eventual therapeutic 258


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failure. It is also important to search for specific mutations in genetic markers in accordance 259

with the results reported by authors in Asia (10, 20, 21, 37, 39, 43-45). 260

261

ACKNOWLEDGEMENTS 262

The authors wish to thank Colciencias (code 111549326137, RC 488-2009) and the 263

Universidad de Antioquia, Colombia (CODI-Sustainability strategy 2013-2014 UdeA). The 264

authors would like to thank to the Instituto Colombiano de Medicina Tropical (ICMT), 265

Apartadó, for providing the conditions necessary to carry out the field work, and the hospitals 266

Antonio Roldán Betancur of the Municipality of Apartadó and Francisco Valderrama of the 267

Municipality of Turbo for their support in acquiring the patients. Furthermore, the authors wish 268

to thank: Dr. Omar Triana for his advice on the methodology, Gonzalo Alvarez for his help 269

with the statistics, the microbiologist and bioanalyst Gabriel Vélez for his support with the 270

experimental activities carried out in the laboratory at Apartadó and with the inclusion of 271

patients, the microbiologists and bioanalysts Claudia Patiño and Alexandra Ríos for their help 272

with the inclusion of patients and their support with field activities in Turbo, the Bacteriologist 273

Luz Yaned Usuga for acquiring patients in Apartadó, and the investigative assistant Fernando 274

Méndez for his support with the field activities. 275

276

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421


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FIGURES AND TABLES 422

Figure1. Geometric mean of the IC50 of antimalarials in P. Vivax isolates, Urabá, 423

Colombia. 424

425

426

427


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Table 1. Parasitic stages, maturation time and the percentage of schizonts obtained by the 428

isolate groups. 429

430

Characteristics of the

isolates pre and post

culture

Isolates Group 1 a

(n=15)

Isolates Group 2 b

(n=15) F - test

Mean (CI 95%) Mean CI 95%

Immature Tfz (a+Ta)* 172.2 (161.1 – 183.3) 18.33 (0.0 – 38.5) <0.0001

Mature Tfz* 23.3 (13.8 – 32.9) 174.7 (151.4 – 198.1) <0.0001

Pre- schizonts* 3.0 (0.7 – 5.3) 1.4 (0.2 – 2.6) 0.19

Schizonts* 1.5 (0.2 – 2.8) 5.5 (0.0 – 14.1) 0.32

Maturation time

(hours) 34.9 (31.0 – 38.8) 22.7 (20.1 – 25.2) <0.0001

% Schizonts post

maturation 59.9 (46.5 – 76.4) 48 (39.2 – 56.8) 0.12

* Count of differential stages at the beginning of the assay carried out with 200 asexual 431

parasites. aGroup 1: Isolates with predominance of immature trophozoites. bGroup 2: Isolates 432

with predominance of mature trophozoites. 433

434


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Table 2. IC50 of the antimalarials evaluated in the isolates of P. vivax, Urabá, Colombia 435

436

Antimalarial N° of valid assays* (%) IC50** (CI95%)

Chloroquine 30 (100) 23.3 (12.4 – 44.1)

mefloquine 28 (93.3) 55.6 (36.8 – 84.0)

Amodiaquine 26 (86.7) 8.5 (5.6 – 13.0)

Quinine 28 (93.3) 115.3 (57.7 – 230.5)

Artesunate 26 (86.7) 0.6 (0.3 – 1.0)

*Number of valid assays with IC50s over total number of assays (n=30). 437

**IC50: Geometric mean of the inhibitory concentration in nM. CI 95%: Confidence interval 438

95%.439


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Table 3. Comparison of the IC50 medians of each antimalarial based on initial parasitic stages 440

in the P. Vivax isolates. 441

442

Antimalarial

Group 1* Group 2* F – test

(n) Median of IC50**

(IQR) (n)

Median of IC50**

(IQR) P value

Chloroquine 15 17.0 (3.2 – 59.8) 15 39.3 (11.5 – 73.7) <0.0001

Mefloquine 13 24.1 (17.3 – 58.8) 15 72.2 (37.4 – 169.5) 0.644

Amodiaquine 12 3.7 (3.0 – 31.6) 14 7.2 (4.6 – 17.5) 0.199

Quinine 14 65.0 (24.8 – 946.2) 14 209.2 (53.5 – 378.3) 0.301

Artesunate 12 0.3 (0.1 – 1.0) 14 1.4 (0.2 – 2.0) <0.0001

IQR: Interquartile range 25% - 75%. 443

*Isolates with valid IC50 values for each antimalarial. F – test: Analysis of statistical 444

significance. 445

**Analysis of the median of the concentration that inhibits 50% of the maturation of the 446

schizonts, in nM. 447

448


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Table 4. Overlapping susceptibility of P. vivax among the different antimalarials. 449

450

COMBINATIONS Mefloquine Amodiaquine Quinine Artesunate

Chloroquine 0.557 ** 0.489 * 0.457 * 0.689 ***

Mefloquine -- 0.456 * 0.564 ** 0.639 ***

Amodiaquine -- -- 0.475 * 0.414 *

Quinine -- -- -- 0.483 *

Spearman correlation coefficient (rs). Correlations are shown of the in vitro susceptibility of the 451

Colombian isolates of P. vivax to all of the antimalarials evaluated. Statistical significance is 452

designated as: p<0.001 (***); p<0.01 (**); and p<0.05 (*). A positive correlation among the 453

antimalarials evaluated can be seen. 454


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Table 5. Susceptibility profile of the P. vivax isolates to antimalarials in different geographical regions. 455

456

Geometric mean of IC50 (nM) of antimalarials reported in the literature

Year Region Successful

assays CQ MQ AQ AS Qn Reference

1995 Thailand 57 360.5* -- -- -- -- (10)

2002 Thailand 121 50.3** -- -- -- -- (27)

2003 Thailand 34 96.5 -- -- -- -- (22)

2004 Thailand 20 96.9 335.9 30.1 1.3 393.4 (19)

2007 Thailand 81 46.8 -- -- -- --

(42) Indonesia 145 312* -- -- -- --

2007 Myanmar 2

15

>100*

<100 -- -- -- -- (20)

2008 Thailand ND 12+ -- -- -- --

(36) ND 415++ -- -- -- --

2008 Indonesia 216 295* 12.7 15.4 1.31 -- (21)

55.2+ 7.9+ 16.6+ -- --


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2812++ 12.5++ 24.2++ -- --

2008 Thailand 65 36.7 111 34 8.3 --

(38) Indonesia 85 114* 9.87 13.7 1.4 --

2009 Korea

Tailandia

50

24

75.6

96.9

103.2

335.9

--

--

1.5

1.3

75.4

393.4 (33)

2011 Thailand 34 167.2* 139.4 -- 32.6 -- (43)

2011 China 42 12.6 11.2 -- 0.3 -- (37)

2014 Brazil 32 32 57 -- 21 -- (36)

This

study Colombia 30 23.3 55.6 8.5 0.6 115.3

457

ND: No data; S: Susceptible; --: Not analyzed in the study. *: The authors suggest loss of susceptibility to 458

chloroquine when the IC50 is greater than 100 nM. +: Ring stage. ++: Trophozoite mature stage. 459

460


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Figure1. Geometric mean of the IC50 of antimalarials in P. Vivax isolates, Urabá, Colombia.


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#Address correspondence to Adriana Pabón. Universidad de Antioquia, UdeA Calle 70 No. 52-21, Medellín, Colombia Tel: +574 219 6490. Fax: +574 219 6487. e-mail: [email protected]

Table 1. Parasitic stages, maturation time and the percentage of schizonts obtained by the 1

isolate groups. 2

3

Characteristics of the

isolates pre and post

culture

Isolates Group 1 a

(n=15)

Isolates Group 2 b

(n=15) F - test

Mean (CI 95%) Mean CI 95%

Immature Tfz (a+Ta)* 172.2 (161.1 – 183.3) 18.33 (0.0 – 38.5) <0.0001

Mature Tfz* 23.3 (13.8 – 32.9) 174.7 (151.4 – 198.1) <0.0001

Pre- schizonts* 3.0 (0.7 – 5.3) 1.4 (0.2 – 2.6) 0.19

Schizonts* 1.5 (0.2 – 2.8) 5.5 (0.0 – 14.1) 0.32

Maturation time

(hours) 34.9 (31.0 – 38.8) 22.7 (20.1 – 25.2) <0.0001

% Schizonts post

maturation 59.9 (46.5 – 76.4) 48 (39.2 – 56.8) 0.12

* Count of differential stages at the beginning of the assay carried out with 200 asexual 4 parasites. aGroup 1: Isolates with predominance of immature trophozoites. bGroup 2: Isolates 5 with predominance of mature trophozoites. 6

7


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Table 2. IC50 of the antimalarials evaluated in the isolates of P. vivax, Urabá, Colombia 8

9

Antimalarial N° of valid assays* (%) IC50** (CI95%)

Chloroquine 30 (100) 23.3 (12.4 – 44.1)

mefloquine 28 (93.3) 55.6 (36.8 – 84.0)

Amodiaquine 26 (86.7) 8.5 (5.6 – 13.0)

Quinine 28 (93.3) 115.3 (57.7 – 230.5)

Artesunate 26 (86.7) 0.6 (0.3 – 1.0)

*Number of valid assays with IC50s over total number of assays (n=30). 10 **IC50: Geometric mean of the inhibitory concentration in nM. CI 95%: Confidence interval 11 95%.12


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Table 3. Comparison of the IC50 medians of each antimalarial based on initial parasitic stages 13 in the P. Vivax isolates. 14 15

Antimalarial

Group 1* Group 2* F – test

(n) Median of IC50**

(IQR) (n)

Median of IC50**

(IQR) P value

Chloroquine 15 17.0 (3.2 – 59.8) 15 39.3 (11.5 – 73.7) <0.0001

Mefloquine 13 24.1 (17.3 – 58.8) 15 72.2 (37.4 – 169.5) 0.644

Amodiaquine 12 3.7 (3.0 – 31.6) 14 7.2 (4.6 – 17.5) 0.199

Quinine 14 65.0 (24.8 – 946.2) 14 209.2 (53.5 – 378.3) 0.301

Artesunate 12 0.3 (0.1 – 1.0) 14 1.4 (0.2 – 2.0) <0.0001

IQR: Interquartile range 25% - 75%. 16 *Isolates with valid IC50 values for each antimalarial. F – test: Analysis of statistical 17 significance. 18 **Analysis of the median of the concentration that inhibits 50% of the maturation of the 19 schizonts, in nM. 20

21


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Table 4. Overlapping susceptibility of P. vivax among the different antimalarials. 22

23

COMBINATIONS Mefloquine Amodiaquine Quinine Artesunate

Chloroquine 0.557 ** 0.489 * 0.457 * 0.689 ***

Mefloquine -- 0.456 * 0.564 ** 0.639 ***

Amodiaquine -- -- 0.475 * 0.414 *

Quinine -- -- -- 0.483 *

Spearman correlation coefficient (rs). Correlations are shown of the in vitro susceptibility of the 24 Colombian isolates of P. vivax to all of the antimalarials evaluated. Statistical significance is 25 designated as: p<0.001 (***); p<0.01 (**); and p<0.05 (*). A positive correlation among the 26 antimalarials evaluated can be seen. 27


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Table 5. Susceptibility profile of the P. vivax isolates to antimalarials in different geographical regions. 28

29

Geometric mean of IC50 (nM) of antimalarials reported in the literature

Year Region Successful

assays CQ MQ AQ AS Qn Reference

1995 Thailand 57 360.5* -- -- -- -- (10)

2002 Thailand 121 50.3** -- -- -- -- (27)

2003 Thailand 34 96.5 -- -- -- -- (22)

2004 Thailand 20 96.9 335.9 30.1 1.3 393.4 (19)

2007 Thailand 81 46.8 -- -- -- --

(42) Indonesia 145 312* -- -- -- --

2007 Myanmar 2

15

>100*

<100 -- -- -- -- (20)

2008 Thailand ND 12+ -- -- -- --

(36) ND 415++ -- -- -- --

2008 Indonesia 216 295* 12.7 15.4 1.31 -- (21)

55.2+ 7.9+ 16.6+ -- --


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2812++ 12.5++ 24.2++ -- --

2008 Thailand 65 36.7 111 34 8.3 --

(38) Indonesia 85 114* 9.87 13.7 1.4 --

2009 Korea

Tailandia

50

24

75.6

96.9

103.2

335.9

--

--

1.5

1.3

75.4

393.4 (33)

2011 Thailand 34 167.2* 139.4 -- 32.6 -- (43)

2011 China 42 12.6 11.2 -- 0.3 -- (37)

2014 Brazil 32 32 57 -- 21 -- (36)

This

study Colombia 30 23.3 55.6 8.5 0.6 115.3

30 ND: No data; S: Susceptible; --: Not analyzed in the study. *: The authors suggest loss of susceptibility to 31 chloroquine when the IC50 is greater than 100 nM. +: Ring stage. ++: Trophozoite mature stage. 32 33


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