1

Evaluation of a prototype flow cytometry test for serodiagnosis of canine visceral 1

leishmaniasis 2

3

Henrique Gama Kera,b

, Wendel Coura-Vitala,c

, Rodrigo Dian de Oliveira Aguiar-Soaresa,b

, Bruno 4

Mendes Roatta,b

, Nádia das Dores Moreiraa,b

, Cláudia Martins Carneiroa,b

, Evandro Marques 5

Machadob, Andréa Teixeira-Carvalho

d, Olindo Assis Martins-Filho

d, Rodolfo Cordeiro 6

Giunchettie, Márcio Sobreira Silva Araújo

d, Eduardo Antonio Ferraz Coelho

f, Denise da Silveira 7

Lemosg, Alexandre Barbosa Reis

a,b#

8

9

a Laboratório de Pesquisas Clínicas, Programa de Pós Graduação em Ciências Farmacêuticas, 10

Universidade Federal de Ouro Preto, Ouro Preto, Minas Gerais, Brazil 11

b Laboratório de Imunopatologia, Núcleo de Pesquisas em Ciências Biológicas, Universidade 12

Federal de Ouro Preto, Ouro Preto, Minas Gerais, Brazil 13

c Pós-Graduação em Infectologia e Medicina Tropical, Faculdade de Medicina, Universidade 14

Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil 15

d Departamento de Morfologia, Instituto de Ciências Biológicas, Universidade Federal de Minas 16

Gerais, Belo Horizonte, Minas Gerais, Brazil 17

e Laboratório de Biomarcadores de Diagnóstico e Monitoração, Centro de Pesquisas Renè 18

Rachou - FIOCRUZ, Belo Horizonte, Minas Gerais, Brazil 19

f Laboratório de Biotecnologia Aplicada ao Estudo das Leishmanioses, Universidade Federal de 20

Minas Gerais, Belo Horizonte, Minas Gerais, Brazil 21

g Laboratório de Imunoparasitologia, Núcleo de Pesquisas em Ciências Biológicas, Universidade 22

Federal de Ouro Preto, Ouro Preto, Minas Gerais, Brazil 23

CVI Accepts, published online ahead of print on 9 October 2013Clin. Vaccine Immunol. doi:10.1128/CVI.00575-13Copyright © 2013, American Society for Microbiology. All Rights Reserved.

on February 6, 2019 by guest

http://cvi.asm.org/

Dow

nloaded from

2

#Corresponding author: Alexandre Barbosa Reis, Laboratório de Pesquisas Clínicas, Programa 24

de Pós Graduação em Ciências Farmacêuticas, Escola de Farmácia, Morro do Cruzeiro, 25

Universidade Federal de Ouro Preto, Ouro Preto, Minas Gerais, CEP 35400-000, Brasil. 26

E-mail address: alexreis@nupeb.ufop.br (A.B. Reis) 27

Tel.: +55 21 31 3559 1036. 28

29

30

on February 6, 2019 by guest

http://cvi.asm.org/

Dow

nloaded from

3

Abstract 31

Diagnosing canine visceral leishmaniasis (CVL) is a critical challenge since conventional 32

immunoserological tests still present some deficiencies. The current study evaluated a prototype 33

flow cytometry serology test in a broad range of serum samples using antigens and fluorescent 34

antibodies that had been stored for 1 year at 4°C. Non-infected control dogs and Leishmania 35

infantum–infected dogs were tested and the prototype test showed excellent performance in 36

differentiating these groups with high sensitivity, specificity, positive and negative predictive 37

values, and accuracy (100% in all analyses). When the CVL group was evaluated according to 38

the dogs’ clinical status, the prototype test had an outstanding accuracy in all groups with 39

positive serology (asymptomatic-II, oligosymptomatic, symptomatic). However, in dogs which 40

present positive results by PCR-RFLP but negative by conventional serology (asymptomatic-I), 41

it was not observed. Additionally, sera from 40 dogs immunized with different vaccines 42

(Leishmune®

, Leish-Tec®

, LBSap) did not present serological reactivity in the prototype test. 43

Eighty-eight dogs infected with other pathogens (Trypanosoma cruzi, Leishmania braziliensis, 44

Ehrlichia canis, and Babesia canis) were used to determine cross-reactivity and specificity, and 45

the prototype presented high performance particularly in dogs with B. canis and E. canis (100% 46

and 93.3% specificity, respectively). In conclusion, our data reinforce the prototype’s potential 47

for use as a commercial kit and highlight its outstanding performance even after storage for 1 48

year at 4°C. Moreover, the prototype test efficiently provided accurate CVL serodiagnosis, with 49

an absence of false-positive results in vaccinated dogs and minor cross-reactivity against other 50

canine pathogens. 51

Running title: Prototype flow cytometry test for CVL serodiagnosis. 52

53

on February 6, 2019 by guest

http://cvi.asm.org/

Dow

nloaded from

4

INTRODUCTION 54

Canine visceral leishmaniasis (CVL) is considered one of the most important canine 55

protozoan diseases of zoonotic concern (1). Various Phlebotomus spp. and Lutzomyia spp. 56

sandflies are potential vectors for the pathogenic agent Leishmania infantum (2). In some 57

European, Asian, African, and American countries, the infection rates in dogs are associated with 58

the risk of human disease (3-5). In Brazil, the Ministry of Health, through the Visceral 59

Leishmaniasis Control and Surveillance Program (VLCSP), has instituted specific measures to 60

reduce morbidity and case-fatality rates, including treatment of human cases, vector control, and 61

uniquely in the world, sacrificing all seropositive infected dogs and prohibiting the treatment of 62

CVL cases (6). 63

During the last decade, the criteria for eliminating infected animals were based on 64

enzyme-linked immunosorbent assays (ELISAs) used for screening and indirect 65

immunofluorescence antibody test (IFAT) for confirmatory diagnosis of CVL (6-7). These tests 66

may lead to false-positive results due to cross-reactivity with other parasitic diseases is well 67

known in the literature (8-9).Recently, this approach was modified, and testing is now based on 68

Dual-Path Platform (DPP®

) for screening and ELISA for confirmation (10). However, Grimaldi 69

et al. (11) evaluated the DPP®

test for the serodiagnosis of CVL and showed that it does not 70

perform well in detecting asymptomatic dogs from endemic areas of canine disease. 71

It has already been shown that the Leishmune®

vaccination may leads to seroconversion 72

in healthy dogs (10). The vaccination of dogs has increasingly become a common practice in 73

endemic areas in Brazil, and recently, besides the Leishmune®

, Leish-Tec®

vaccine is available 74

for commercialization, and new candidates such as LBSap are being studied (12-15). Therefore, 75

it became an important problem for the surveillance programs of control that employs 76

on February 6, 2019 by guest

http://cvi.asm.org/

Dow

nloaded from

5

conventional methodologies in seroepidemiological surveys, because it could lead an 77

unnecessary euthanasia of healthy dogs. Nevertheless, it is still understudied the role of 78

vaccination in the diagnostic of CVL. 79

Because serological methods still represent the most realistic and applicable tool for 80

epidemiological surveys and for CVL diagnosis, the development of novel serological tests and 81

the validation of alternative methodologies are urgent. Toward these ends, several studies have 82

focused on applying flow cytometry technology to leishmaniasis serological analysis in human 83

and canine diseases (16-20). 84

Alongside the good performance of flow cytometry–based methodologies in serological 85

approaches, we recently developed a protocol for antigenic preparation and optimal antigen 86

preservation conditions, which improved the quality and efficiency of the antigen for a long 87

period, allowing routine use of this tool for laboratory CVL diagnosis (21).The goal of the 88

present study was to evaluate a prototype test based on a flow cytometry serology for CVL 89

diagnosis, using antigens and conjugate antibodies that were stored for 1 year at 4°C. For this 90

purpose, we conducted serological analysis on a broad range of serum samples obtained from L. 91

infantum–infected dogs presenting different clinical statuses, dogs vaccinated against visceral 92

leishmaniasis, and dogs infected with other important canine pathogens. 93

94

MATERIAL AND METHODS 95

96

Study Animals 97

Sera obtained from 278 mongrel dogs of either gender were used (Figure 1). Seventy sera 98

from non-infected dogs were included as a control group, and it is composed by a subset of 99

on February 6, 2019 by guest

http://cvi.asm.org/

Dow

nloaded from

6

control dogs from kennel (n = 30) born in the animal facility of Federal University of Ouro Preto, 100

and also by control dogs from endemic area (n = 40) from a cross-sectional study conducted in 101

2008 in the endemic area of Belo Horizonte (22). They were characterized by presenting 102

parasitological and PCR-RFLP negative results for L. infantum and seronegative by IFAT and 103

ELISA for Leishmania spp. 104

The CVL group (n = 80) was determined according the dogs’ serological reactivity in 105

ELISA and IFAT assays, and also by PCR-RFLP result. The PCR-RFLP was previously 106

performed in buffy coat from blood samples, according to Coura-Vital et al. (22). The CVL 107

group was divided into four subgroups according to the clinical status as proposed by Mancianti 108

et al. (19), and reviewed by Coura-Vital et al. (20). The asymptomatic dog group was composed 109

by two subsets: asymptomatic-I (n = 20) and asymptomatic-II ( n = 20); oligosymptomatic, (n = 110

21) and symptomatic (n = 19). The asymptomatic -I dogs were seronegative by IFAT and ELISA 111

but positive in PCR-RFLP molecular assay. The last three groups (asymptomatic-II, 112

oligosymptomatic and symptomatic) were characterized by presenting two positive serological 113

tests (IFAT and ELISA). 114

In addition to the groups already described, the study also used 40 mongrel adult dogs of 115

either gender, maintained at the kennel of Federal University Ouro Preto, Minas Gerais State, 116

Brazil, that were submitted to vaccination with two commercial vaccines Leishmune®

(n = 12) 117

and Leish-Tec®

(n = 16), as well as a potential candidate, LBSap (n = 12). All animals have 118

received three doses of vaccines used in this study, with an interval of 21 days between each 119

dose. The immunization was conducted according to the manufacturer´s instructions of the 120

commercial vaccines, and also by the proposed protocol for the LBSap candidate (15). 121

To further characterize the degree of cross-reactivity and specificity by flow cytometry 122

on February 6, 2019 by guest

http://cvi.asm.org/

Dow

nloaded from

7

serology, we also investigated 88 serum samples from dogs naturally infected by L. braziliensis 123

(n = 30), dogs experimentally infected with Trypanosoma cruzi (n = 18), and dogs with common 124

tick-borne infections such as Ehrlichia canis (n = 30) and Babesia canis (n = 10). These samples 125

composed the serum bank of the Clinical Research Laboratory of Pharmacy School from Federal 126

University of Ouro Preto and were kindly provided by different laboratories. Each infection was 127

previously characterized by presenting specific serology (ELISA) and PCR positive results, and 128

samples were PCR negative for L. infantum. 129

130

Sample Collection 131

Peripheral blood samples were collected by intravenous puncture in the radial vein of the 132

dogs using disposable 5 mL syringes and placed into vacuum vials (Vacuette, Campinas, SP, 133

Brazil). The serum obtained was stored at -20°C in 1.8 mL sterile cryogenic vials (Sarstedt, 134

Newton, NC, USA) until required for the assay. 135

For the bone marrow culture, dogs were sedated with an intravenous dose (8 mg/kg 136

bodyweight) of sodium thiopental (Thionembutal®

; Abbott Laboratories, São Paulo, Brazil), and 137

bone marrow fluid was removed from the ventral region of the sternum or from the iliac crest 138

using a sterile syringe. Then, bone marrow aspirate was transferred to sterile tubes containing 139

Novy-MacNeal-Nicolle-liver infusion tryptose medium (NNN-LIT) supplemented with 10% 140

FBS (23). 141

Dogs reagents for ELISA and IFAT were euthanized by Zoonotic Disease Control Center 142

of the Belo Horizonte, Minas Gerais, Brazil. After the euthanasia, the biopsies of the ear skin and 143

spleen were collected using a sterile scalpel. The tissue fragments were placed onto microscope 144

slides and stained with Giemsa for parasitological exam. 145

on February 6, 2019 by guest

http://cvi.asm.org/

Dow

nloaded from

8

The study was approved by the Committees of Ethics in Animal Experimentation of the 146

Universidade Federal de Ouro Preto (protocol no. 083/2007). 147

148

Design of flow cytometry prototype test 149

The prototype test described is registered at the Instituto Nacional da Propriedade 150

Industrial (Brazil) under patent number BR 1020120047420 deposited on 2 March 2012. The 151

antigen preparation and reaction conditions were as previously described by Ker et al. (21). 152

For this experiment the L. infantum antigen preserved in formaldehyde 0.5% and also the 153

IgG labeled antibody had been stored at 4°C for 1 year. Briefly, antigen suspensions (5.0 × 105 154

parasites/well) were incubated at 37°C for 30 min in the presence of 50 µL of diluted serum 155

samples at 1:4,096 dilution using a 96-well U-bottom plate (BD FalconTM

). Following 156

incubation, the parasite suspension was washed twice with 150 µL of PBS with 3% FBS (1,000 157

× g, 10 min, 4°C) and re-incubated in the dark for 30 min at 37°C in the presence of 50 µL of 158

previously diluted 1:1,000 anti-canine IgG FITC-labeled antibody (Bethyl Laboratories Inc., 159

Montgomery, TX, cat #A40-105F). After incubation (37°C, 30 min) and being washed twice 160

with 150 µL of PBS with 3% FBS (1,000 × g, 10 min, 4°C), the stained parasites were fixed with 161

FACS fix solution and maintained for at least 30 min at 4°C in the dark, prior to flow cytometric 162

data acquisition. In all plates, an internal control was included for all experiments to monitor 163

nonspecific binding in which the parasites were incubated in the absence of dog serum, but in the 164

presence of the FITC-labeled secondary reagent. Flow cytometric measurements were performed 165

on a FACScan flow cytometer (Becton Dickinson, San Jose, CA) interfaced to an Apple 166

FACStation, and the Cell-QuestTM

software package was used for data acquisition and storage. 167

The analysis was performed in FlowJo®

software (FlowJo, Ashland, OR). The IgG reactivity was 168

on February 6, 2019 by guest

http://cvi.asm.org/

Dow

nloaded from

9

expressed as the percentage of positive fluorescent parasites, and the cut-off value was obtained 169

through receiver operating characteristic curve according to Ker et al. (21) 170

171

Gold standard 172

Two parasitological methods were used as the gold standard for diagnosis: amastigotes 173

investigation on tissue smears of skin and spleen in Giemsa-stained slides and examination of 174

promastigote forms in bone marrow culture. 175

176

Statistical analysis 177

The data analyses were conducted using Stata software (version 11.0, Stata Corporation, 178

College Station, TX), and the flow cytometry serology performance was assessed by percentage. 179

The evaluation of the prototype test was estimated by the sensitivity, specificity, positive 180

predictive value (PPV), negative predictive value (NPV), and accuracy, using the results of 181

parasitological tests as the reference standard (at the 95% confidence interval). The overall 182

performance of the prototype was calculated using the non-infected control dogs as truly 183

negative and dogs with positive parasitological exams to L. infantum as truly positive. Moreover, 184

the groups of animals infected with other pathogens were used as negative samples for individual 185

calculations of specificity. 186

187

RESULTS 188

The prototype of flow cytometry serology presented high performance to discriminate non-189

infected from L. infantum–infected dogs with different clinical forms 190

The performance evaluation of the prototype flow cytometry serology test in CVL 191

on February 6, 2019 by guest

http://cvi.asm.org/

Dow

nloaded from

10

diagnosis is shown in Figure 1. We observed that 58/80 (72.5%) of CVL dogs had a positive 192

result and none of the dogs in the control group showed reactivity (Fig. 2A). To assess the 193

performance of flow cytometry serology in the diagnosing different clinical statuses, dogs 194

classified as asymptomatic I, asymptomatic II, oligosymptomatic, and symptomatic were 195

analyzed. Positive results were observed in 1/20 (5%), 18/19 (94.7%), 20/21 (95.2%), and 19/20 196

(95%) respectively (Fig. 2B). 197

198

The prototype flow cytometry serology showed a high capacity to discriminate reactivity of 199

Leishmania-vaccinated dogs and minimizes cross-reactivity with other canine pathogens 200

201

Herein, we performed an analysis of serologic reactivity in serum of dogs vaccinated with 202

two commercialized vaccines in Brazil (Leishmune®

and Leish-Tec®

) and also for a potential 203

vaccine candidate against CVL (LBSap). Our data demonstrated that none of vaccinated dogs 204

presented seroreactivity in the prototype flow cytometry serology test (Fig. 3A). 205

The prototype test showed a middle performance of cross-reactivity when sera of dogs 206

infected with L. braziliensis (6/30; 20%) and T. cruzi (7/18, 38.9%) were tested. Furthermore, 207

dogs infected with E. canis showed low cross-reactivity (2/30; 6.6%) and B. canis samples not 208

presented false-positive results (Fig. 3B). 209

The prototype test presented outstanding performance indices in the serological diagnosis of 210

CVL 211

This study included an analysis of sensitivity, specificity, predictive values, and accuracy 212

using 36 of the 80 dogs with CVL as references presenting positive parasitological exams; these 213

dogs were considered confirmed positive cases. The 70 dogs from the control group showed 214

on February 6, 2019 by guest

http://cvi.asm.org/

Dow

nloaded from

11

negative results in parasitological exams and were considered confirmed negative cases. Data 215

analysis demonstrated that the prototype test presented high sensitivity (100%), specificity 216

(100%), PPV (100%), and NPV (100%). Furthermore, the analysis of accuracy confirmed an 217

excellent performance of the prototype test (100%) in CVL diagnosis (Table 1). 218

To assess the performance indices of the flow cytometry serology prototype in animals 219

infected with other pathogens, animals infected with T. cruzi, L. braziliensis, E. canis, and B. 220

canis were evaluated. The specificity obtained for T. cruzi serum samples was the lowest in all 221

groups assessed (55.6%), followed by L. braziliensis–infected samples (80%). The prototype test 222

had high specificity for E. canis and B. canis samples, with 93.3% and 100%, respectively. 223

Furthermore, the NPVs were 100% for all groups. The PPVs were 85.7%, 81.8%, 94.7% and 224

100%, and the accuracy was 90.9%, 85.2%, 97%, and 100% for T. cruzi, L. braziliensis, E. canis, 225

and B. canis, respectively, confirming the excellent performance of the prototype test (Table 1). 226

227

DISCUSSION 228

Serological testing has been a basic and essential tool to diagnose and control many 229

infectious diseases (24). Flow cytometry is becoming an increasingly useful tool in both health 230

care and research laboratories because it is a rapid, accurate, and reproducible method of analysis 231

(25). Although there still exists a substantial cost regarding the operational support in 232

experiments involving flow cytometry, it was recently described that the creation of Shared 233

Resource Laboratory model, could enhance the scope and quality of scientific research that 234

applies the flow cytometry based methodologies (26-27). In the same context, through Oswaldo 235

Cruz Foundation, the Brazilian government implemented the Network Technology Platforms 236

Program for Technological Development in Health Supplies to enhance the research perspectives 237

on February 6, 2019 by guest

http://cvi.asm.org/

Dow

nloaded from

12

in flow cytometry approaches, and it would be suitable for diagnostic services in public health. 238

Thereby, this platform model offers new perspectives for the use of the flow cytometer facility as 239

a diagnostic tool to neglected tropical diseases such as visceral leishmaniasis. 240

In previous works that employed L. infantum antigens prepared just before the serological 241

reaction, it was observed that the flow cytometry serology presented outstanding performance in 242

CVL diagnosis (19, 28). In the current study, using a standard antigen preparation, we observed 243

excellent performance for the prototype test, which had high sensitivity (100%) and specificity 244

(100%) for detecting IgG in CVL. Moreover, our data demonstrate high PPV (100%) and NPV 245

(100%), indicating that the prototype flow cytometry test is highly reliable for detecting positive 246

CVL samples and also for excluding CVL in non-infected dogs. The high accuracy (100%) 247

observed in this prototype test point toward precise diagnosis. Thereby, our results reinforce the 248

flow cytometry serology assay as being a very useful tool for CVL diagnosis. 249

Tested in different CVL groups, our data demonstrated that the prototype flow cytometry 250

serology has an outstanding performance to identify asymptomatic II, oligosymptomatic as well 251

as the symptomatic dogs. These findings certify that the prototype test employing the current 252

conditions was capable to provide an excellent performance in CVL diagnosis, even after one 253

year of storage of both antigen preparation and IgG labeled antibody. However, we observed that 254

only one dog from the asymptomatic I group was detected. These animals have high prevalence 255

and incidence in endemic areas, and are not detected by either conventional serology (22, 29) or 256

flow cytometry serology as demonstrated in the current study. We believe that the low sensitivity 257

observed in detected this group is due to the immunological profile shown by this dogs that are 258

characterized by having low Ig antibodies production (IgG, IgG1, IgG2, IgM, IgA and IgE) 259

which occurs at early stages of the infection (10, 18, 28). During those periods, B-lymphocytes 260

on February 6, 2019 by guest

http://cvi.asm.org/

Dow

nloaded from

13

do not secrete polyclonal antibodies, and consequently, serological methods are less sensitive at 261

this stage of the infection (30-31). Moreover, it has been observed that, these dogs are more 262

likely to seroconvert compared to PCR negative (32). 263

Vaccines against CVL have been promoted as an important tool and a cost-effective 264

strategy for controlling the disease (33). Thus, knowledge about the performance of diagnostic 265

methods to vaccinated dog is urgently needed to avoid false-positive reactions, which can lead to 266

unnecessary euthanasia of noninfected dogs. Andrade et al. (19) described the ability of flow 267

cytometry serology to exclude seroreactivity from Leishmune®

vaccinated dogs. Extending this 268

research, we investigated the performance of a prototype flow cytometry test in dogs vaccinated 269

with Leishmune®

, Leish-tec®

and also in LBSap vaccine (14-15). Novel findings obtained in the 270

present study showed that the flow cytometry serology prototype had an extraordinary 271

performance with regard to excluding reactivity in animals vaccinated with commercial vaccines, 272

as well as in dogs immunized with a potential candidate vaccine. 273

Different pathogens, but from the same family such as Trypanossomatidae (Leishmania 274

spp and T. cruzi) shares a similarity antigenic repertory of epitopes that can lead to cross-reactive 275

antibodies in immunodiagnosis tests. The use of conventional serological methods in CVL 276

diagnosis may lead to cross-reactivity with other canine infections, mainly in dogs infected with 277

T. cruzi, L. braziliensis, E. canis, or B. canis (7-9). Herein, despite the flow cytometry serology 278

prototype test exhibit the lowest specificity in T. cruzi and L. braziliensis samples, the results 279

obtained in this study was superior than those observed for others serological tests which 280

assessed cross-reactivity of these pathogens using conventional methods (8, 34). Nevertheless, in 281

a previous flow cytometry serology study, Andrade et al. (19) verified that a higher dilution 282

(1:8192) of serum can reduce the cross-reactivity in dogs infected with T. cruzi or L. braziliensis 283

on February 6, 2019 by guest

http://cvi.asm.org/

Dow

nloaded from

14

with no change in the CVL diagnostic performance. 284

With regard to canine tick-borne infections, ehrlichiosis and babesiosis are highly 285

prevalent in Brazil and represent a challenge to veterinarians and public health workers (35). 286

Considering that these vector-borne diseases affect dogs’ concomitant with CVL in endemic 287

areas, we analyzed for the first time the cross-reactivity of the flow cytometry serology test in 288

dogs naturally infected with B. canis and E. canis. The results demonstrated high specificity, 289

predictive values, and accuracy, emphasizing the excellent performance of the flow cytometry 290

prototype in CVL diagnostics, even if animals were infected with those diseases. 291

The performance of diagnostic tests is greatly limited by the antigen in the technique. In 292

this study, we show that the conservation of L. infantum antigens employing a cheap preservative 293

in a controlled storage temperature ensures the efficiency of the antigen applied in the flow 294

cytometry prototype test for a long period. These particularities show the robustness of the 295

reagent employed in the antigen preservation, and points to a potential commercial perspective of 296

the prototype. In this context, beyond the excellent performance in CVL diagnosis and the ability 297

to discriminate immunized dogs, and with minor cross-reactivity, our findings strengthen the 298

usefulness of flow cytometry serology as a wider scale assay for CVL diagnosis, especially in 299

endemic areas with potential co-infections and vaccinated animals. Thus, as prospects, to 300

validate this test, we intend to test the prototype in large number of dogs of an urban endemic 301

area of Brazil. 302

303

on February 6, 2019 by guest

http://cvi.asm.org/

Dow

nloaded from

15

Acknowledgements 304

This work was supported by Fundação de Amparo à Pesquisa do Estado de Minas Gerais, Brazil 305

(FAPEMIG Grant: CBB – APQ-3073-4.01/07), Programa de Pesquisa para o SUS 306

(PPSUS/MS/CNPq/FAPEMIG/SES-MG/Grant CBB-APQ-00356-10), Conselho Nacional de 307

Pesquisa (CNPq Grant: 472554/2007-7), and Departamento de Ciência e Tecnologia do 308

Ministério da Saúde (DECIT/MS/CNPq/BR/Grant: 576062/2008-1). A.B.R., C.M.C., A.T.C., 309

O.A.M.F., and R.C.G. thank Conselho Nacional de Desenvolvimento Científico e Tecnológico 310

(CNPq) and W.C.V., N.D.M., and D.S.L. thank CAPES/PNPD for fellowships. 311

312

on February 6, 2019 by guest

http://cvi.asm.org/

Dow

nloaded from

16

References 313

1. Baneth G, Koutinas AF, Solano-Gallego L, Bourdeau P, Ferrer L. 2008. Canine 314

leishmaniosis - new concepts and insights on an expanding zoonosis: part one. Trends 315

Parasitol 24:324-330. 316

2. Killick-Kendrick R. 1999. The biology and control of phlebotomine sand flies. Clin 317

Dermatol 17:279-289. 318

3. Nunes CM, Pires MM, da Silva KM, Assis FD, Goncalves Filho J, Perri SH. 2010. 319

Relationship between dog culling and incidence of human visceral leishmaniasis in an 320

endemic area. Vet Parasitol 170:131-133. 321

4. Gavgani AS, Mohite H, Edrissian GH, Mohebali M, Davies CR. 2002. Domestic dog 322

ownership in Iran is a risk factor for human infection with Leishmania infantum. Am J 323

Trop Med Hyg 67:511-515. 324

5. Faye B, Banuls AL, Bucheton B, Dione MM, Bassanganam O, Hide M, Dereure J, 325

Choisy M, Ndiaye JL, Konate O, Claire M, Senghor MW, Faye MN, Sy I, Niang AA, 326

Molez JF, Victoir K, Marty P, Delaunay P, Knecht R, Mellul S, Diedhiou S, Gaye O. 327

2010. Canine visceral leishmaniasis caused by Leishmania infantum in Senegal: risk of 328

emergence in humans? Microbes Infect. 329

6. Ministério da Saúde. 2006. Manual de vigilância e controle da leishmaniose visceral, 330

1th ed. Secretaria de Vigilância em Saúde, Brasília. Available at: 331

http://portal.saude.gov.br/portal/arquivos/pdf/manual_leish_visceral2006.pdf Accessed 332

December 8. 333

7. Lira RA, Cavalcanti MP, Nakazawa M, Ferreira AG, Silva ED, Abath FG, Alves 334

LC, Souza WV, Gomes YM. 2006. Canine visceral leishmaniosis: a comparative 335

on February 6, 2019 by guest

http://cvi.asm.org/

Dow

nloaded from

17

analysis of the EIE-leishmaniose-visceral-canina-Bio-Manguinhos and the IFI-336

leishmaniose-visceral-canina-Bio-Manguinhos kits. Vet Parasitol 137:11-16. 337

8. Ferreira EC, de Lana M, Carneiro M, Reis AB, Paes DV, da Silva ES, Schallig H, 338

Gontijo CM. 2007. Comparison of serological assays for the diagnosis of canine visceral 339

leishmaniasis in animals presenting different clinical manifestations. Vet Parasitol 340

146:235-241. 341

9. Oliveira TM, Furuta PI, de Carvalho D, Machado RZ. 2008. A study of cross-342

reactivity in serum samples from dogs positive for Leishmania spp., Babesia canis and 343

Ehrlichia canis in enzyme-linked immunosorbent assay and indirect fluorescent antibody 344

test. Rev Bras Parasitol Vet 17:7-11. 345

10. Ministério da Saúde. 2011. Esclarecimento sobre substituição do protocolo diagnóstico 346

da leihsmaniose visceral canina; Nota técnica conjunta nº 01/2011 - CGDT-347

CGLAB/DEVIT/SVS/MS; . 348

11. Grimaldi G, Jr., Teva A, Ferreira AL, dos Santos CB, Pinto IS, de-Azevedo CT, 349

Falqueto A. 2012. Evaluation of a novel chromatographic immunoassay based on Dual-350

Path Platform technology (DPP(R) CVL rapid test) for the serodiagnosis of canine 351

visceral leishmaniasis. Trans R Soc Trop Med Hyg 106:54-59. 352

12. Borja-Cabrera GP, Santos FN, Bauer FS, Parra LE, Menz I, Morgado AA, Soares 353

IS, Batista LM, Palatnik-de-Sousa CB. 2008. Immunogenicity assay of the Leishmune 354

vaccine against canine visceral leishmaniasis in Brazil. Vaccine 26:4991-4997. 355

13. Fernandes AP, Costa MM, Coelho EA, Michalick MS, de Freitas E, Melo MN, Luiz 356

Tafuri W, Resende Dde M, Hermont V, Abrantes Cde F, Gazzinelli RT. 2008. 357

on February 6, 2019 by guest

http://cvi.asm.org/

Dow

nloaded from

18

Protective immunity against challenge with Leishmania (Leishmania) chagasi in beagle 358

dogs vaccinated with recombinant A2 protein. Vaccine 26:5888-5895. 359

14. Giunchetti RC, Correa-Oliveira R, Martins-Filho OA, Teixeira-Carvalho A, Roatt 360

BM, de Oliveira Aguiar-Soares RD, de Souza JV, das Dores Moreira N, Malaquias 361

LC, Mota e Castro LL, de Lana M, Reis AB. 2007. Immunogenicity of a killed 362

Leishmania vaccine with saponin adjuvant in dogs. Vaccine 25:7674-7686. 363

15. Roatt BM, Aguiar-Soares RD, Vitoriano-Souza J, Coura-Vital W, Braga SL, 364

Correa-Oliveira R, Martins-Filho OA, Teixeira-Carvalho A, de Lana M, Gontijo 365

NF, Marques MJ, Giunchetti RC, Reis AB. 2012. Performance of LBSap Vaccine after 366

Intradermal Challenge with L. infantum and Saliva of Lu. longipalpis: Immunogenicity 367

and Parasitological Evaluation. PLoS One 7:e49780. 368

16. Rocha RD, Gontijo CM, Eloi-Santos SM, Teixeira Carvalho A, Correa-Oliveira R, 369

Marques MJ, Genaro O, Mayrink W, Martins-Filho OA. 2002. [Anti-live Leishmania 370

(Viannia) braziliensis promastigote antibodies, detected by flow cytometry, to identify 371

active infection in american cutaneous leishmaniasis]. Rev Soc Bras Med Trop 35:551-372

562. 373

17. Pissinate JF, Gomes IT, Peruhype-Magalhaes V, Dietze R, Martins-Filho OA, 374

Lemos EM. 2008. Upgrading the flow-cytometric analysis of anti-Leishmania 375

immunoglobulins for the diagnosis of American tegumentary leishmaniasis. J Immunol 376

Methods 336:193-202. 377

18. Garcia LM, Coelho-Dos-Reis JG, Peruhype-Magalhaes V, Teixeira-Carvalho A, 378

Rocha RD, Araujo MS, Gomes IT, Carvalho SF, Dietze R, Lemos EM, Andrade 379

MC, Martins-Filho OA. 2009. Anti-fixed Leishmania chagasi promastigotes IgG 380

on February 6, 2019 by guest

http://cvi.asm.org/

Dow

nloaded from

19

antibodies detected by flow cytometry (FC-AFPA-IgG) as a tool for serodiagnosis and 381

for post-therapeutic cure assessment in American visceral leishmaniasis. J Immunol 382

Methods 350:36-45. 383

19. Andrade RA, Silva Araujo MS, Reis AB, Gontijo CM, Vianna LR, Mayrink W, 384

Martins-Filho OA. 2009. Advances in flow cytometric serology for canine visceral 385

leishmaniasis: diagnostic applications when distinct clinical forms, vaccination and other 386

canine pathogens become a challenge. Vet Immunol Immunopathol 128:79-86. 387

20. Andrade RA, Reis AB, Gontijo CM, Braga LB, Rocha RD, Araujo MS, Vianna LR, 388

Martins-Filho OA. 2007. Clinical value of anti-Leishmania (Leishmania) chagasi IgG 389

titers detected by flow cytometry to distinguish infected from vaccinated dogs. Vet 390

Immunol Immunopathol 116:85-97. 391

21. Ker HG, Dian de Oliveira Aguiar-Soares R, Mendes Roatt B, das Dores Moreira N, 392

Coura-Vital W, Martins Carneiro C, Teixeira-Carvalho A, Martins-Filho OA, 393

Cordeiro Giunchetti R, da Silveira-Lemos D, Barbosa Reis A. 2013. Effect of the 394

preservative and temperature conditions on the stability of Leishmania infantum 395

promastigotes antigens applied in a flow cytometry diagnostic method for canine visceral 396

leishmaniasis. Diagn Microbiol Infect Dis 76:470-476. 397

22. Coura-Vital W, Marques MJ, Veloso VM, Roatt BM, Aguiar-Soares RD, Reis LE, 398

Braga SL, Morais MH, Reis AB, Carneiro M. 2011. Prevalence and factors associated 399

with Leishmania infantum infection of dogs from an urban area of Brazil as identified by 400

molecular methods. PLoS Negl Trop Dis 5:e1291. 401

23. Camargo EP. 1964. Growth and Differentiation in Trypanosoma Cruzi. I. Origin of 402

Metacyclic Trypanosomes in Liquid Media. Rev Inst Med Trop Sao Paulo 6:93-100. 403

on February 6, 2019 by guest

http://cvi.asm.org/

Dow

nloaded from

20

24. Center of Disease Control. 1999, posting date. Achievements in Public Health, 1900-404

1999: Control of Infectious Diseases. [Online.] 405

25. Jaroszeski MJ, Radcliff G. 1999. Fundamentals of flow cytometry. Mol Biotechnol 406

11:37-53. 407

26. Moore J, Roederer M. 2009. The flow cytometry shared resource laboratory: best 408

practices to assure a high-quality, cost-effective partnership with biomedical research 409

laboratories. Cytometry A 75:643-649. 410

27. Monti F, Rosetti M, Masperi P, Tommasini N, Dorizzi RM. 2012. Shared resource 411

laboratories: impact of new design criteria to consolidate flow cytometry diagnostic 412

service. Int J Lab Hematol 34:533-540. 413

28. Carvalho Neta AV, Rocha RDR, Gontijo CMF, Reis AB, Martins-Filho O A. 2006. 414

Citometria de fluxo no diagnóstico da leishmaniose visceral canina [Flow cytometry used 415

in canine visceral leishmaniasis diagnosis]. Arq. Bras. Med. Vet. Zootec. 58:480-488. 416

29. Coura-Vital W, Reis AB, Reis LE, Braga SL, Roatt BM, Aguiar-Soares RD, 417

Marques MJ, Veloso VM, Carneiro M. 2013. Canine visceral leishmaniasis: Incidence 418

and risk factors for infection in a cohort study in Brazil. Vet Parasitol In press. 419

30. Oliveira GG, Santoro F, Sadigursky M. 1993. The subclinical form of experimental 420

visceral leishmaniasis in dogs. Mem Inst Oswaldo Cruz 88:243-248. 421

31. Coura-Vital W, Marques MJ, Giunchetti RC, Teixeira-Carvalho A, Moreira ND, 422

Vitoriano-Souza J, Vieira PM, Carneiro CM, Correa-Oliveira R, Martins-Filho OA, 423

Carneiro M, Reis AB. 2011. Humoral and cellular immune responses in dogs with 424

inapparent natural Leishmania infantum infection. Vet J 190:e43-47. 425

on February 6, 2019 by guest

http://cvi.asm.org/

Dow

nloaded from

21

32. Coura-Vital W, Reis AB, Fausto MA, Leal GGdA, Marques MJ, Veloso VM, 426

Carneiro M. 2013. Risk Factors for Seroconversion by Leishmania infantum in a Cohort 427

of Dogs from an Endemic Area of Brazil. PLoS One 8:e71833. 428

33. Gramiccia M, Gradoni L. 2005. The current status of zoonotic leishmaniases and 429

approaches to disease control. Int J Parasitol 35:1169-1180. 430

34. da Costa CA, Genaro O, de Lana M, Magalhaes PA, Dias M, Michalick MS, Melo 431

MN, da Costa RT, Magalhaes-Rocha NM, Mayrink W. 1991. [Canine visceral 432

leishmaniasis: evaluation of the serologic method used in epidemiologic studies]. Rev 433

Soc Bras Med Trop 24:21-25. 434

35. Dantas-Torres F. 2008. Canine vector-borne diseases in Brazil. Parasit Vectors 1:25. 435

436

437

438 on February 6, 2019 by guest

http://cvi.asm.org/

Dow

nloaded from

22

Legends: 439

FIG 1 Experimental design employed in a prototype flow cytometry serological testing. The 440

control dogs, includes control dogs from kennel (CDK) and control dogs from endemic 441

area (CDA). The L. infantum infected dogs were stratified according their statuses as 442

asymptomatic-I dogs (AD-I), asymptomatic-II dogs (AD-II), oligosymptomatic dogs 443

(OD), symptomatic dogs (SD). Vaccinated dogs and dogs infected with other pathogens 444

constitute the other two subsets. 445

446

FIG 2 Flow cytometry serology employing antigens and IgG labeled antibody stored for 1 year 447

to discriminate non-infected control dogs from L. infantum–infected dogs presenting 448

different clinical forms. The results are expressed as percentage of positive fluorescent 449

parasites (PPFP) for individual samples, at serum dilution 1:4,096 from noninfected 450

control dogs from kennel (CDK = ), control dogs from endemic area (CDA = ), and 451

L. infantum–infected dogs (CVL = ) (A). The CVL dogs were stratified according their 452

clinical statuses as asymptomatic dogs I (AD-I = ), asymptomatic dogs II (AD-II = ), 453

oligosymptomatic dogs I (OD = ), symptomatic dogs I (SD = ) (B). The dotted line 454

represents the cut-off between negative and positive results. 455

FIG 3 Flow cytometry serology employing antigens and IgG labeled antibody stored for 1 year 456

to discriminate reactivity of Leishmania-vaccinated dogs and also cross-reactivity with 457

other canine pathogens. The results are expressed as percentage of positive fluorescent 458

parasites (PPFP) for individual samples, at serum dilution 1:4,096 from vaccinated dogs 459

with, Leishmune®

( ), Leish-Tec®

( ) and LbSap ( ) (A). Dogs with other relevant 460

pathogens were tested as represented: L. braziliensis ( ), T. cruzi ( ), E. canis ( ), and 461

on February 6, 2019 by guest

http://cvi.asm.org/

Dow

nloaded from

23

B. canis ( ) (B). The dotted line represents the cut-off between negative and positive 462

results. 463

464

Table 1 Performance indices of flow cytometry serology for detection of anti-Leishmania IgG 465

antibodies in canine sera. 466

on February 6, 2019 by guest

http://cvi.asm.org/

Dow

nloaded from

Total samples (n=278)

FIGURE 1

Total samples (n=278)

Control Dogs (n=70)

CDK (n=30)

AD-I (n=20)

Control Dogs (n=70)CDA (n=40)

L. infantum infected dogs

(n=80)

AD-II (n=20)

OD (n=21)

SD (n=19)

Vaccinated dogs (n=40)

Leishmune® (n=12)

Leish-Tec® (n=16)

LBSap (n=12)LBSap (n=12)

D i f t d ith th

L. braziliensis (n=30)

T. cruzi (n=18)Dogs infected with other

pathogens (n=88)

( )

E. canis (n=30)

B. canis (n=10)

on February 6, 2019 by guest

http://cvi.asm.org/

Dow

nloaded from

FIGURE 2

100

A B

FP

60

80

PP

20

40

0

CDK CDA CVL AD-I AD-II OD SD

CVL

on February 6, 2019 by guest

http://cvi.asm.org/

Dow

nloaded from

FIGURE 3

A

100

60

80

20

40

PP

FP

0

LBSapLeishmune® Leish-Tec®

B

100

60

80

PP

FP

20

40

P

L. braziliensis T. cruzi E. canis B. canis

0

on February 6, 2019 by guest

http://cvi.asm.org/

Dow

nloaded from

Samples Sensitivity (%)

(95% CI)

Specificity (%)

(95% CI)

PPV (%)

(95% CI)

NPV (%)

(95% CI)

Accuracy (%)

(95% CI)

CVL 100.0 (90.4-100.0) 100.0 (83.3-99.4) 100.0 (86.2-99.5) 100.0 (88.3-100.0) 100.0 (91.9-99.7)

L. braziliensis --- 80.0 (62.7-90.5) 85.7 (72.2-93.3) 100.0 (86.2-100.0) 90.9 (81.6-95.8)

T. cruzi --- 55.6 (33.7-75.5) 81.8 (68.1-90.5) 100.0 (72.3-100.0) 85.2 (73.4-92.3)

B. canis --- 100.0 (70.1-100.0) 100.0 (90.4-100.0) 100.0 (70.1-100.0) 100.0 (92.1-100.0)

E. canis --- 93.3 (78.7-98.2) 94.7 (82.7-98.5) 100.0 (87.9-100.0) 97.0 (89.6-99.2)

on February 6, 2019 by guest

http://cvi.asm.org/

Dow

nloaded from