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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.
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#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: [email protected] (A.B. Reis) 27
Tel.: +55 21 31 3559 1036. 28
29
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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436
437
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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
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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
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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)
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FIGURE 2
100
A B
FP
60
80
PP
20
40
0
CDK CDA CVL AD-I AD-II OD SD
CVL
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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
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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)
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