Experimental Parasitology 199 (2019) 9–16

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Experimental Parasitology
journal homepage: www.elsevier.com/locate/yexpr

The diagnosis of canine visceral leishmaniasis in Brazil: Confronting old T

problems
Rômulo Pessoa-e-Silvaa, Victor Vaitkevicius-Antãoa, Thiago André Santos de Andradeb,
Anny Caroliny de Oliveira Silvaa, Gilsan Aparecida de Oliveirac,
Lays Adrianne Mendonça Trajano-Silvad, Eiji Kevin Nakasone Nakasoneb,
Milena de Paiva-Cavalcantia,∗
a
Aggeu Magalhães Institute, Av. Moraes Rego, Cidade Universitária, CEP 50670-420, Recife-PE, Brazil
b
Federal Rural University of Pernambuco (UFRPE), R. Manuel de Medeiros, s/n - Dois Irmãos, CEP 52171-900, Recife-PE, Brazil
c
CESMAC University Center, R. da Harmônia - Farol, CEP 57081-350, Maceió-AL, Brazil
d
University of São Paulo (USP), Av. Bandeirantes, CEP 14049-900, Ribeirão Preto, São Paulo, SP, Brazil

ARTICLE INFO ABSTRACT

Keywords: In Brazil, the main strategy adopted to contain Visceral Leishmaniasis (VL) is the controversial culling of dogs
Visceral leishmaniasis with reagent serology for Canine VL (CVL). Despite there are studies showing that significant reduction of human
Dog cases has not been observed, as well as there are works demonstrating the occurrence of false-positive results in
Diagnosis the confirmatory test, the protocol has been maintained. Researches that can reinforce the existence and per-
sistence of this problem, as well as bring concrete alternatives are pivotal. In this context, the aim of this work
was to evaluate and compare the serological, molecular and parasitological methods employed for CVL detection
in Brazil, in dogs with diverse clinical profiles, from two endemic areas of Pernambuco state. Comparisons
among TR-DPP®, EIE-LVC and qPCR (animals from Goiana-PE: 91) demonstrated that agreements varied from
‘poor’ to ‘moderate’ (kappa = 0.162–0.442), and a triple agreement occurred in 61.36% (54/88) of the samples.
The highest percentage of agreement was obtained between TR-DPP® and EIE-LVC within the polysymptomatic
group (93.33%; 14/15). Of the 34 dogs with reagent serology from Caruaru-PE, 17 (50%) and 29 dogs (85.29%)
were positive for qPCR and parasitological exam, respectively. By comparing serology, qPCR and parasitological
exam, the lowest percentage of agreements were obtained within the asymptomatic group (40%–72.72%). It was
possible to observe that the percentage of agreement tended to decrease according to the absence of clinical
manifestations in the dogs. Thus, from the fact that all diagnostic tools evaluated have their limitations, it is very
important to be careful before to propose an alternative set of diagnostic criteria. Besides, the answer for better
results in the control of CVL may not be in the choose of the best set of diagnostic tools, but it may be in the
strategy of culling itself. In this context, it is very important to invest in alternative control measures, such as
mass vaccination and treatment of dogs, thus reducing the transmission to the vector and helping to avoid new
canine and human cases.

1. Introduction number of infected individuals is higher than in any other country from
Latin America, and the victims are normally those with lower incomes
The zoonotic visceral leishmaniasis (VL) is a neglected and severe from rural and peri-urban areas (Romero and Boelaert, 2010; Brasil,
disease caused by protozoan parasites (Kinetoplastida: 2014). In the biological cycle of VL, domestic dogs are considered a key
Trypanosomatidae) of the genus Leishmania (Elmahallawy et al., 2014), element, since the infected animals may harbor in their dermis a high
and it has been reported in several areas around the world, including number of macrophages infected with amastigote forms, thereby be-
South America and Southern Europe (Dujardin, 2006). In Brazil, the coming infective to the vector (Neves, 2016). The proximity to the

Corresponding author.
∗

E-mail addresses: romulops12@gmail.com (R. Pessoa-e-Silva), victorvaitkeantao@gmail.com (V. Vaitkevicius-Antão),

thiago.biovet@gmail.com (T.A.S. de Andrade), annycaroliny.os@gmail.com (A.C. de Oliveira Silva), gilsanaraujo@gmail.com (G.A. de Oliveira),
laystrajano@gmail.com (L.A.M. Trajano-Silva), eijitus@gmail.com (E.K.N. Nakasone), mp@cpqam.fiocruz.br (M. de Paiva-Cavalcanti).

https://doi.org/10.1016/j.exppara.2019.02.012
Received 1 November 2018; Received in revised form 1 January 2019; Accepted 19 February 2019
Available online 20 February 2019
0014-4894/ © 2019 Elsevier Inc. All rights reserved.
R. Pessoa-e-Silva, et al.
human environment increases their epidemiological importance
(Gramiccia and Gradoni, 2005).
The parasitic specie commonly associated with Canine VL (CVL) is
L. (Leishmania) infantum (syn. L. (L.) chagasi). The CVL is endemic in
more than 70 countries, being reported in Africa, Asia, Southern
Europe, South America, Central America, and even in the South of the
United States (Petersen and Barr, 2009; Kaszak et al., 2015). Clinical
signs such as onychogryphosis, splenomegaly, lymphadenomegaly,
dermatitis and keratoconjunctivitis may occur in different stages of the
disease. Renal injury is commonly associated to death in CVL (Solano-
Gallego et al., 2009; Sousa et al., 2016). Nevertheless, dogs generally
develop subclinical infection, which makes it more difficult to detect
(Hosein et al., 2017).
For the controlling of the CVL, the detection, quantification and
mapping of infected dogs through active searches is an important step
for the strategic direction of control measures (Travi et al., 2018).
Commonly, serological tests for the detection of anti-Leishmania anti-
bodies such as the Direct Agglutination Test (DAT) and the Im-
munochromatographic Rapid Test (ICT) are employed to perform the
screening during these searches due to their practicality, low cost and
high sensitivity (Adams et al., 2012; Da Silva et al., 2013). The ICTs are
broadly used in field and in clinics since provide very fast results, but
the accuracy varies according to the antigen employed: by evaluating
six different ICTs for CVL, sensitivity varied from 63.6% to 98%, and
specificity varied from 60% to 100% (Otranto et al., 2004; Athanasiou
et al., 2014; Solano-Gallego et al., 2014; De Mendonça et al., 2017). The
Enzyme-linked Immunosorbent Assay (ELISA) is another broadly em-
ployed tool, but just as the ICTs, its sensitivity depends on the antigen
employed (Gomes et al., 2008; Srivastava et al., 2011). ELISA allows the
screening of a large number of samples and may use a combination of
multiple antigens to increase specificity and sensitivity (Soto et al.,
1998; Travi et al., 2018). In Brazil, according to the protocol estab-
lished by the Ministry of Health for CVL diagnosis, the ICT is used as
screening tool, and the ELISA as confirmatory test (Coura-Vital et al.,
2014).
Despite their advantages, it is important to mention that serological
diagnostic methods have important limitations, which impair the de-
finitive diagnosis of L. infantum infection, especially in asymptomatic
dogs (Fraga et al., 2016; Solano-Gallego et al., 2017). Immunological
tests may present cross-reactivity with other trypanosomatids such as
Trypanosoma cruzi, with protozoa from other families such as Tox-
oplasma gondii, or even with the bacteria Ehrlichia canis (Zanette et al.,
2014), and they may not to differentiate naturally infected and vacci-
nated dogs (Solano-Gallego et al., 2017).
Molecular methods have the potential to overcome the hindrances
of serological procedures, but they are still relatively expensive.
Therefore, they are still not entirely feasible for field research
(Magalhães et al., 2017; Singh and Sundar, 2015). Possibility of false-
negative results associated with losses in the extraction protocol, pre-
sence of Taq polymerase inhibitors or incorrect storage still exists;
nonetheless, the use of endogenous controls has minimized this oc-
currence in routine (Gonçalves-de-Albuquerque et al., 2014). An in-
teresting fact is that Polymerase Chain Reaction (PCR) and its variations
can be used in addition to other tests and, together to clinical aspects,
bring an even greater reliability to the final report (Colombo et al.,
2015).
Lastly, there is also the parasitological exam. The technique is based
on the microscopical demonstration of amastigote forms in the bone
marrow biopsy, lymph node aspirate or lesion scarification (World
Health Organization, 2010), and it is still considered a gold standard
technique in the diagnosis of CVL, with a specificity of 100%
(Saridomichelakis et al., 2005; Sousa et al., 2017). However, the sen-
sitivity is normally reduced, varying according to the expertise of the
microscopist and degree of parasitism (asymptomatics and oligo-
symptomatics tend to have less parasites) (Moreira et al., 2007). Be-
sides, biopsy and aspiration are invasive techniques.
10
Experimental Parasitology 199 (2019) 9–16
Significant divergences among the results given by reference tech-
niques for CVL diagnosis have been reported by several authors (Gomes
et al., 2008; Nunes et al., 2015), and this has been a current topic of
debate. These studies indicate an urgent necessity to establish a reliable
set of criteria to determine CVL, aiming to minimize the false negatives
and especially the false-positive results, thus avoiding the erroneous
euthanasia of dogs, adopted in Brazil as a control measure for VL.
Studies that may strengthen the proof of the existence of this problem
inside of dog populations of different regions are pivotal.
Considering that all diagnostic methods have their advantages, but
also significative limitations, in this work we compared different
techniques (serological, molecular and parasitological) employed for
CVL detection in Brazil for different purposes, tested/performed in dogs
with diverse clinical profiles, from two endemic areas of Pernambuco
state. From the results, we demonstrate if the establishment of a reliable
set of criteria to CVL diagnosis at different situations is a feasible goal.
2. Material and methods
2.1. Sampling
A sampling of convenience was adopted (Reis, 2003). Blood samples
were collected from dogs coming from endemic areas for VL in Per-
nambuco state (PE), Brazil. The field searches were performed in a rural
area from Goiana city, located in the northern part of the state of
Pernambuco (Lat. 07° 33′38″ S, Lon. 35° 00′9″ W). Some samples were
also collected from suspected animals belonging to the Unidade de
Controle de Zoonoses (UCZ), located in Caruaru city (PE) (Lat. 08° 16′53″
S, Lon. 35° 58′25″ W) (Fig. 1).
2.2. Clinical evaluation and sample collection
The owners of the animals were invited to participate of the re-
search, and after the acceptance, they signed an informed consent form,
authorizing the use of the material collected for scientific purposes.
For each animal, the clinical status was assessed and then the
findings were recorded on a specific form, prior to sample collection.
The following signals/symptoms were investigated: weight loss, hepa-
tosplenomegaly, lymphadenomegaly, ulcerative/seborrheic dermatitis,
generalized or localized alopecia, onychogryphosis, cardiorespiratory
changes, mucosal bleeding/secretions and neurological changes. From
these findings, each animal was classified as asymptomatic (no signals/
symptoms suggestive of CVL), oligosymptomatic (one to three clinical
signs, for example dogs with lymphoid adenopathy, weight loss and
opaque hair), and polysymptomatic (more than three signals/symp-
toms) (Queiroz et al., 2011).
After clinical evaluation, peripheral blood collection was performed
by venipuncture using a 10 mL syringe coupled to a 25 × 7 mm needle,
and two different collection tubes (5 mL): one serum tube and one tube
with ethylenediaminetetraacetic acid – EDTA (Vacuette®, SP, BR). A
cotton soaked with alcohol 70% was used for asepsis. Each sample was
unequivocally identified and properly transported to the laboratory,
where they were aliquoted and stored in freezer (−20 °C) until the
Fig. 1. Dog samples were collected from two cities in Pernambuco state,
Caruaru and Goiana, which are highlighted in the map.
R. Pessoa-e-Silva, et al.
processing.
For animals whose collection procedure included bone marrow and/
or lymph node aspiration, atropine was applied at a dose of 0.044 mg/
kg as preanesthetic medication (PAM). After 5–10 min of latency, and
after observation of a discrete tachycardia, dissociative anesthesia was
started with intramuscular application of the combination of xylazine
(ANASEDAN; Ceva/Paulínia, SP, BR - 1 mg/kg) and ketamine (Cetamin;
Syntec/Santana de Parnaíba, SP, BR - 15 mg/kg). After five minutes of
latency (Mansoni, 2008), bone marrow puncture at the crest of the
sternum bone (20 mL syringe, 25 × 7 mm needle) and/or popliteal
lymph node puncture (10 mL syringe, 25 × 7 mm needle) were started.
Only animals that presented hypertrophy of the popliteal lymph node
were assigned to aspiration puncture. Throughout the procedure, a
veterinarian monitored the interdigital and eyelid reflexes, as well as
the heart rate of the animals. Only animals from the UCZ in Caruaru-PE
were submitted to the bone marrow and/or lymph node aspiration, due
to the existence of appropriate structural conditions.
2.3. Laboratorial diagnosis
2.3.1. Serological tests for CVL
The search for anti-Leishmania antibodies in serum was performed
using two different procedures. Firstly, all dog's samples were tested by
a rapid immunochromatographic test - Dual Path Platform (TR-DPP®
CVL, Rio de Janeiro, RJ, BR), performed according to the manufac-
turer's instructions (Bio-Manguinhos/FIOCRUZ). All samples were to-
tally thawed and vigorously homogenized before the test. Afterwards,
serum samples from all dogs were also tested by the ELISA (Enzyme
immunoassay – Canine Visceral Leishmaniasis/enzima imunoensaio-
Leishmaniose Visceral Canina – EIE-LVC, Rio de Janeiro, RJ, BR), fol-
lowing the manufacturer's instructions (Bio-Manguinhos/FIOCRUZ).
The cut-off was calculated from the values of optical density (OD) ob-
tained from the triplicates of negative controls (NC):
(NC1+NC2+NC3/3) x 2. The “grey zone” establishes the ‘in-
determinate’ samples and it was calculated as follows: cut-off x 1.2. The
TR-DPP® and the EIE-LVC are the kits adopted by the Brazilian Ministry
of Health to determine the positivity of dogs in the active searches of
CVL (screening and confirmatory) (Brasil, 2014).
2.3.2. Molecular test for CVL
The DNA extraction from whole blood in EDTA tubes was performed
using the QIAamp® DNA Blood Mini Kit (QIAGEN® Sample and Assay
Technologies, Germantown, MD, USA), according to the manufacturer's
instructions. The DNA was stored in microcentrifuge tubes (1.5 mL) at
−20 °C until the molecular assays.
A real-time quantitative PCR (qPCR) based on the amplification of L.
infantum kinetoplast minicircle DNA (kDNA) was performed, and for
this, the set LINF 1B was employed, according to the protocol stan-
dardized by Paiva-Cavalcanti et al. (2009). The primers detect a frag-
ment of 132 base pairs (bp) from the kDNA of the L. donovani Complex.
For quantification of the parasite load, a standard curve obtained from
L. infantum (syn. L. chagasi) reference DNA (MHOM/BR/1974/PP75)
was prepared (1:10; from 1 fg to 1 × 106fg per reaction). The final re-
action volume of 25 μL was constituted of 12,5 μL of SYBR® Green
Master Mix 1× (Applied Biosystems™, CA, USA) and 2 μL of extracted
template. A total of 3 pmol of each primer, Linf.1–23F (5′-TCCCAAAC
TTTTCTGGTCCT-3′) and Linf.1–154R (5′-TTACACCAACCCCCAGT
TTC-3′) was used.
All samples were tested in duplicate, and the PCR program was run
for 40 cycles, in the following cycling conditions: 95 °C/15s, 60 °C/
1min. A no template control (NTC) was added in duplicate in all re-
actions. As criteria of positivity of the qPCR assay, the amplification
curve had to surpass the threshold before cycle 36, as established by
Trajano-Silva et al. (2017). The quality assurance of each sample was
achieved by mammalian G3PD constitutive gene amplification, em-
ploying primers G1F (5′-ATCTTCCAGGAGCGAGATCCC-3′) and G1R (
11
Experimental Parasitology 199 (2019) 9–16
5′-AGGGATGACCTTGCCCAC-3′) (Gonçalves et al., 2012). All experi-
ments were performed using the ABI Prism 7500 (Applied Biosystems®,
CA, USA) equipment. The software ABI Prism 7500 SDS was used for
the analysis, interpretation, and registration of results.
2.3.3. Parasitological examination
After collection of material from bone marrow and/or lymph node,
four to six slide smears were prepared, per animal. The slides were
stained by Giemsa (GCC Diagnostics, Deeside, UK) or by Fast Panoptic
(Laborclin, Alta Floresta, MT, BR), and the reading was performed
under an optical microscope at 1,000× magnification. The reading of
the smears was done by experienced microscopists.
2.3.4. Serological examination for Chagas Disease (CD)
An examination for CD was done to point out possible cases among
the dogs of the rural region from Goiana-PE. The search for anti-
Trypanosoma cruzi antibodies in serum was performed by an immuno-
fluorescence antibody test (IFAT). Epimastigotes of T. cruzi (Bio-
Manguinhos/FIOCRUZ) were employed for sensitization of slides. The
sera were initially diluted in phosphate-buffered saline (PBS) – pH 7.2
in the proportion of 1:40. Positive and negative sera were used in each
slide as controls. Each hole of the sensitized slides received 15 μL of the
dilutions. Afterwards, the slides were incubated during 30 min at 37 °C
under humidity. After three washings using PBS (5 min in each wash),
the slides were dried. Anti-Dog IgG antibodies conjugated with the
Fluorescein Isothiocyanate (FITC) (SIGMA®, MO, USA) were diluted in
Evans Blue Dye 0.04% (1:300), and then 15 μL of this mixture was
added in each hole of the slides. After the incubation (30 min, 37 °C
under humidity), slides were again washed three times using PBS
(5 min in each wash). Finally, for the reading, buffered glycerol plus
coverslips were added in the surface of the slides. The reading was
performed at an immunofluorescence microscope, under 40× magni-
fication. For those animals that presented a positive result in the dilu-
tion of 1:40 and were also positive in the serology for CVL, a serial
dilution was performed (factor 2) for confirmation of cross-reaction
(Trindade et al., 2015). In this case, CD was suggested if there was
maintenance of positivity until the dilution rate of 1:320 or further.
2.4. Data analysis
Techniques of descriptive statistics were used by means of absolute
and percentage distributions. Concordance between methods was
evaluated by applying kappa (k) coefficient with Confidence Interval
(CI) set at 95%, and judged as k = 0.0, no agreement; 0.0 ≤ k ≤ 0.20,
poor; 0.21 ≤ k ≤ 0.40, fair; 0.41 ≤ k ≤ 0.60, moderate;
0.61 ≤ k ≤ 0.80, good; and 0.81 ≤ k ≤ 1.00, very good agreement
between tests, according to Cohen (1960). All analyses were performed
using the BioEstat (version 5.0; Mamirauá/CNPq, Belém, PA, BR).
2.5. Ethical considerations
All collection procedures and diagnostic tests performed for the
construction of this study, including the informed consent form, are
inserted in the project entitled “evaluation of the cellular immune re-
sponse in dogs (ex vivo) against new antigens (chimeras) of Leishmania
spp.”. It was submitted and approved by the Ethics Committee on
Animal Use (CEUA) from the Aggeu Magalhães Institute (IAM/
FIOCRUZ) (protocol 76/2014).
3. Results
3.1. Sampling and clinical classification
A total of 91 dogs of different breeds, ages and sizes from a rural
area of Goiana city were included in the study. The active searches
occurred between January and September 2017. Of the total, 60
R. Pessoa-e-Silva, et al. Experimental Parasitology 199 (2019) 9–16

In the ELISA, 14 (15.38%) presented as reagents. The three animals

Fig. 2. Records of the active searches of dogs in a rural area from Goiana and
dogs collected by the UCZ in Caruaru, Pernambuco. (A): whole blood collection
performed in domiciled dog; (B): animal presenting cachexia, in addition to
localized type alopecia, in the anterior and posterior limbs; (C): dog with evi-
dent onychogryphosis; (D): animal presenting cachexia and onychogryphosis;
(E): dog showing ulcerated lesion at the tip of the ear; (F): dog presenting a
hepatosplenomegaly clearly perceptible to palpation.
(65.93%) presented at least one clinical sign indicative of CVL (see
topic 2.2), and from these, 15 (25%) presented more than three sug-
gestive clinical signals. The distribution was as follows: 15 poly-
symtomatics (POLY) (16.48%), 45 oligosymptomatics (OLIGO)
(49.45%), and 31 asymptomatics (ASY) (34.07%). The most common
clinical alterations were lymphadenomegaly (95%), cachexia/thinness
(68.33%) and alopecia (43.33%).
In Caruaru city, 34 animals of different breeds, ages and sizes were
included in the study, all collected for the UCZ between February 2017
and July 2018, and all having positive TR-DPP® and ELISA. The dis-
tribution was as follows: 19 POLY (55.88%), 4 OLIGO (11.76%), and 11
ASY (32.36%). The bone marrow and/or lymph node aspiration was
performed in all these animals.
Fig. 2 shows some records of the searches performed in field
(Goiana), as well as some animals from the UCZ in Caruaru.
3.2. Laboratorial diagnosis and data analysis
3.2.1. Animals from Goiana-PE
From 91 animals, 34 (37.36%) had a reagent result in the TR-DPP®.
with ‘indeterminate’ results (3.29%) in the ELISA were not considered
in the statistical analyzes. In the qPCR, 14 (15.38%) had a positive
result (parasite load: 0.26 fg – 69.42 fg; x̄ = 6.38 fg). All negative re-
sults were confirmed by the amplification of the G3PD constitutive gene
(quality control). In relation to the serological test for CD, the IFAT
showed positivity in the dilution of 1:40 in 16 serum samples (17.58%);
however, 13 of these 16 samples had reactivity in TR-DPP® plus ELISA,
and only two of these 13 samples maintained a positivity until the di-
lution of 1:320 (1:320 and 1:2560). Thus, five dogs (5.49%) were
considered in fact positive in the IFAT for CD, being two of them po-
tentially co-infected with CVL.
The agreements between the techniques: TR-DPP® and ELISA: 68
(77.27%), with k = 0.442 (moderate); TR-DPP® and qPCR: 58
(65.91%), with k = 0.162 (poor); ELISA and qPCR: 70 (79.55%), with
k = 0.236 (fair). By comparing the TR-DPP®, ELISA and qPCR, there
was agreement in the results of 54 animals (61.36%), being five positive
and 49 negative results. All these results are summarized in Table 1. In
the context of the ICT as a screening test, it is noteworthy that of the 57
no reagent samples in the TR-DPP®, one was reagent in the ELISA and
six were positive in the qPCR, totalizing seven (12.28%) disagreements.
Besides, eight of the 13 TR-DPP® plus ELISA reagent samples were ne-
gative in the qPCR, that is, 61.54% of disagreement.
In relation to the clinical status, it was possible to observe that
among the polysymptomatic dogs, the highest percentage agreement
obtained was between TR-DPP® and ELISA (93.33%); for both oligo-
symptomatic and asymptomatic groups, the agreement was higher be-
tween ELISA and qPCR (79.54% and 82.76%, respectively). Between
TR-DPP® and qPCR, the percentage agreement was near to 65% in the
three clinical groups (Table 1).
3.2.2. Animals from the UCZ, Caruaru-PE
The TR-DPP® and the ELISA were previously performed by the
‘Laboratório de Saúde Pública da IV Regional de Saúde’, in Caruaru, and
all 34 animals had reagent results for both serological tests. The animals
were clinically classified: POLY = 19; OLIGO = 4; ASY = 11. In the
qPCR assay, 17 animals (50%) were positive (parasite load: 0,76 fg –
478.45 fg; x̄ = 71.98 fg). There was no amplification of the G3PD gene
in one negative sample on CVL qPCR, thus it was discarded from the
statistical analyses. In the parasitological examination, 33 bone marrow
aspirations and one lymph node puncture were performed, and 29 dogs
(85.29%) had positive results (Table 2). The IFAT for CD was not per-
formed with the samples obtained in Caruaru city.
By comparing the results of qPCR and parasitological investigation,
there were 18 agreements (54.55%), being 16 positive and two negative
results; k = 0.078 (poor). Taking into account the clinical status, the
absolute and percentage agreements were as follows: POLY = 12
(63.16%); OLIGO = 2 (50%); ASY = 4 (40%). Between qPCR and ser-
ology: POLY = 11 (57.89%); OLIGO = 2 (50%); ASY = 4 (40%).

Table 1
Agreements obtained between the diagnostic tools applied for canine visceral leishmaniasis detection in dogs from Goiana city, Pernambuco-BR.
Clinical status TR-DPP®/ELISA Percentage TR-DPP®/qPCR Percentage ELISA/qPCR Percentage TR-DPP®/ Percentage
agreement agreement agreement ELISA/ agreement
qPCR

+ – + – + – + –

Polysymptomatic (15) 5 9 93.33 3 7 66.66 3 8 73.33 3 7 66.66

Oligosymptomatic (44) 7 27 77.27 4 25 65.91 2 33 79.54 2 25 61.36
Asymptomatic (29) 1 19 68.96 1 18 65.52 0 24 82.76 0 17 58.62

Total (88) 13 55 77.27 8 50 65.91 5 65 79.55 5 49 61.36

Kappa 0.442 0.162 0.236 –
SE = 0.094 SE = 0.098 SE = 0.130
CI = 0.257–0.626 CI = −0.029−0.354 CI = −0.020−0.491

SE: standard error; CI: confidence interval. The animals with ‘indeterminate’ results in the ELISA were not considered in the statistical analyzes.

12
R. Pessoa-e-Silva, et al.
Table 2
Agreements obtained between the diagnostic tools applied for canine visceral leishmaniasis detection in dogs from UCZ-Caruaru, Pernambuco-BR.
Clinical status TR-DPP®+ ELISA Percentage Serology/qPCR Percentage
agreement agreement
+ – + –
Polysymptomatic (19) 19 0 – 11 0 57.89
Oligosymptomatic (4) 4 0 – 2 0 50.00
Asymptomatic (11/10) 11 0 – 4 0 40.00
Total (34/33) 34 0 - 17 0 51.52
Kappa – –
SE: standard error; CI: confidence interval. The animal which presented negative for the endogenous control (G3PD) was not considered in the analyzes. One sample
with a negative result for the quality control (G3PD) was excluded in the analyses involving the qPCR.
Between serology and parasitological investigation: POLY = 18
(94.74%); OLIGO = 3 (75%); ASY = 8 (72.72%). In the three com-
parisons performed between the diagnostic procedures, the lowest
percentage agreement obtained was with the asymptomatic animals
(Table 2).
4. Discussion
One of the challenges to control visceral leishmaniasis is the re-
duction of the number of dogs participating in the biological cycle of
the disease. The high parasite load that a dog may harbor in its skin
propitiates the infection of phlebotomine sand flies, which in turn may
infect other dogs or the humans (Borja et al., 2016; Travi et al., 2018).
The Brazilian Ministry of Health orientates and recommends the culling
of dogs with reagent serology, independently of the clinical status of the
animal, and using two methods with the same diagnostic principle: the
search for anti-Leishmania antibodies in blood/serum (Brasil, 2014).
The occurrence of false-positive results due to cross-reactions with
other phylogenetically related microorganisms is widely addressed
(Baneth and Aroch, 2006; Ferreira et al., 2007; Eloy et al., 2008). False-
negative results in asymptomatic dogs are also reported, since they may
present no detectable increase in anti-Leishmania antibody levels (De
Mendonça et al., 2017).
In this context, this work aimed to compare different diagnostic
protocols commonly used for CVL detection in different situations: re-
searches, clinical/veterinary purposes and for the control program in
Brazil. The comparisons were performed by evaluating/testing samples
of clinically heterogeneous dogs from endemic areas from Pernambuco
state, and evaluating the possibility to indicate alternative set of criteria
for a more reliable confirmation of infection.
The percentage agreement between the TR-DPP® and the EIE-LVC
was 77.27%, with a k index considered moderate (Table 1). Considering
the specificity and the sensitivity of the enzyme immunoassay (94.54%
and 91.76%, respectively) and of the TR-DPP® (93.8%–97.9%, and
89.7%–93.1%, respectively) informed by the manufacturer (Bio-Man-
guinhos/FIOCRUZ/MS), the agreement obtained was lower than the
expected, especially when we consider the number of reagent samples
in the TR-DPP® and in the EIE-LVC (34 and 14/91, respectively). Fur-
thermore, some works obtained different specificities and sensitivities
of both serological tests. As example, De Mendonça et al. (2017) eval-
uated the specificity of the TR-DPP® in samples from non-infected ani-
mals of two cities belonging to different states (57 dogs from Teresina,
Piauí-BR and 100 dogs from Vitória, Espírito Santo-BR). Whereas the
specificity of the TR-DPP® found in Vitória was 98%, in Teresina it was
60%. The authors suggest that this variation may be associated with
differences in the prevalence of the disease: the higher is the pre-
valence, the lower is the test performance. The theory proposed by the
authors was that “when the prevalence of the disease increases, im-
mune dogs generate false-positive results and decrease the serological
13
Experimental Parasitology 199 (2019) 9–16
Serology/ Percentage qPCR/Parasitological Percentage
Parasitological agreement agreement
+ – + –
18 0 94.74 11 1 63.16
3 0 75.00 2 0 50.00
8 0 72.72 3 1 40.00
29 0 85.29 16 2 54.55
– 0.078
SE = 0.110
CI = −0.138−0.294
specificity, and thus, the ROC curve area and likelihood ratio” (De
Mendonça et al., 2017). This may also explain at least in part our data,
since the rural area in Goiana city explored for this research presented a
high serological prevalence (EIE-LVC: 15.4%). From this information,
an important question arises: can a test with reduced efficacy in areas
with a high prevalence of VL be used for diagnostic screening?
The sensitivities found for EIE-LVC in literature vary from 72% to
100% (Alves et al., 2012; Lira et al., 2006; De Mendonça et al., 2017).
However, in relation to the specificity, the data among the works are
more dissimilar. Corroborating with the manufacturer, Figueiredo et al.
(2010) evaluated the sensitivity and specificity of the same ELISA using
serum samples from dogs submitted to parasitological examination
(with specie confirmation by isoenzyme electrophoresis), and the spe-
cificity and sensitivity obtained were 100% and 96.6%, respectively. In
contrast, using serum samples from dogs submitted to parasitological
and immunological tests, Ferreira et al. (2007) have found a specificity
and a sensitivity of 52% and 95%, respectively. According to the au-
thors, the EIE-LVC presented cross-reactivity with sera from dogs ex-
perimentally infected with T. cruzi, Leishmania braziliensis and E. canis.
Alves et al. (2012) have found a specificity of 68% for EIE-LVC, and
their data suggest cross-reactivity with samples from animals infected
with Trypanosoma caninum. In the rural area of Goiana city, five dogs
were identified as serologically reagents in the IFAT for CD, and two of
them had suggestive co-infection. Nevertheless, 13 samples that were
reagent in the dilution between 1:40 and 1:160 were also reagent in the
EIE-LVC, and seven of these 13 were negative in the qPCR, confirming
the absence of L. infantum DNA. Therefore, in this case we cannot rule
out possible cross-reactions in the ELISA with dogs infected with T.
cruzi. Thus, the high sensitivity of TR-DPP® and variable specificity,
combined with the possibility of cross-reactions in the EIE-LVC re-
inforce the concern about erroneous euthanasia of dogs following the
scheme proposed: ICT as screening and EIE-LVC as confirmatory.
One out of 57 no reagent samples in the TR-DPP® was reagent in the
EIE-LVC, and six of these 57 samples were positive in the qPCR. Despite
it represented only 12.28% of the analyzed samples, this is an important
result when we consider it in the context of the control program of CVL,
in which hundreds to thousands of animals are tested monthly, in some
Brazilian states. Besides, eight of the 13 TR-DPP® + EIE-LVC reagent
samples had a negative result in the qPCR.
In short, when compared to the serology, the data obtained by the
molecular tool point out the possible occurrence of a significative
number of false-negative and false-positive results going on in the ac-
tive searches. The primer set employed in this work for qPCR assays was
developed by Paiva-Cavalcanti et al. (2009) and it has, according to the
authors, 100% of sensitivity and 83.33% of specificity (by using whole
blood from dogs). The set was able to detect the equivalent of 0.07
parasites per reaction. Several works have employed this set for dif-
ferent applications (Gonçalves et al., 2012; Pessoa-e-Silva et al., 2016;
Trajano-Silva et al., 2017). Reinforcing the high accuracy of the qPCR
R. Pessoa-e-Silva, et al.
but using other primer set, Ramos et al. (2012) demonstrated that the
tool was able to detect DNA from L. infantum in 100% (35/35) of the
naturally infected dogs, using lymph node aspirate and biopsy from
bone marrow and spleen as samples. The result showed that qPCR was
superior to the conventional PCR, which detected the DNA only in 40%
(14/35) of the animals, using the same samples. Nonetheless, it is im-
portant to be alert about limitations that persist in the molecular di-
agnosis (even in qPCR), especially false-negative results due to in-
correct storage or Taq-polymerase inhibitors.
In relation to the dogs obtained from the UCZ-Caruaru, all of them
presented a reagent serology, since they were animals collected for the
euthanasia procedure (by the Municipal Health Secretariat – Caruaru-
PE). Even using a highly sensitive PCR-based method, the set was able
to detect DNA from L. infantum only in 50% (17/34) of the samples
tested (Table 2). In this case, there is the possibility to have occurred
small losses during the extraction process that were not enough to in-
terfere in the amplification of the endogenous control (G3PD), but that
may have carried out some samples to a false-negative result, since the
DNA from the parasite is in a much smaller amount (Mumy and Findlay,
2004; Gonçalves-de-Albuquerque et al., 2014; Lebuhn et al., 2016). The
fact that only 16 out of 29 animals with a positive parasitological exam
were positive in the qPCR reinforces this possibility. Furthermore, the
percentage agreement obtained between serology and the para-
sitological was good (85.29%; 29/34) (Table 2). To overcome this
limitation, alternative samples which normally present higher parasite
loads may be used, such as bone marrow or lymph node aspirate
(Martínez et al., 2011; Solcà et al., 2014), however they are not feasible
for active searches. Alternatively, a quality control to verify the oc-
currence of small losses during the extraction process may be employed,
thus reducing the issuing of false-negative results (Gonçalves-de-
Albuquerque et al., 2014). Furthermore, of the five negative results in
the parasitological exam, one was positive in the qPCR, reinforcing that
despite the high specificity of the bone marrow examination, the sen-
sitivity is variable and depends on several factors (Sundar and Rai,
2002).
Taking into consideration the clinical status, it was possible to ob-
serve that among the polysymptomatic animals from Goiana city, the
highest percentage agreement obtained was between the TR-DPP® and
the EIE-LVC (93.33%; 14/15) (Table 1). Since the high levels of cir-
culating antibodies are associated with the development of several
clinical manifestations in dogs with CVL, it is expected a greater
agreement between serological tests when applied for CVL diagnosis in
symptomatic animals. These animals normally have higher parasite
loads (hence, a greater number of antigenic epitopes), thus leading to
an increased production of anti-Leishmania antibodies (Fernandez-Perez
et al., 2003; Solano-Gallego et al., 2014). Considering a triple con-
cordance between the results of the three techniques (TR-DPP®, EIE-
LVC and qPCR), the higher percentage agreement was obtained with
the polysymptomatic group (66.66%; 10/15) (Table 1). Considering
now only the dogs with positivity for at least one test, it was observed
that 16.67% (2/12) of the asymptomatic, 47.37% (9/19) of the oligo-
symptomatic, and 62.5% (5/8) of the polysymptomatic animals had
positivity in two or more techniques. These data clearly demonstrate
the clinical status influencing the serological and molecular results.
As expected, the greater agreement between serological and para-
sitological methods was obtained with polysymptomatic dogs (94.74%;
18/19). The same occurred between the qPCR and the parasitological
method (63.16%; 12/19) (Table 2). Da Silva et al. (2017) reported that
the clinical score of sick dogs increased according to the positivity
obtained with serological (ELISA) and parasitological tests, thus cor-
roborating with our findings. In the context of the bone marrow/lymph
node examination, a higher parasite load reflects in the number of
amastigote forms found in these tissues, thus elevating the probability
of being found; in the context of the qPCR, the reasoning is similar,
since the higher is the parasite load, the higher is the circulating
Leishmania DNA.
14
Experimental Parasitology 199 (2019) 9–16
Fig. 3. Proposal of a set of actions to be performed for the accurate confirma-
tion of visceral leishmaniasis in dogs, at different situations, and also for the
actual control of the disease. ICT: immunochromatographic test.
In this work, the discrepancies in the number of dogs inserted in
each clinical status is a point that may be optimized afterwards.
Another point to take in consideration was the lack of animals with
negative serology and with parasitological examination. In this case,
the field conditions did not allow to perform the bone marrow/lymph
node puncture in the field searches. Nevertheless, by analyzing our
data, it was possible to observe that there is the possibility of the oc-
currence of false-negative and false-positive results, in a significative
number, in the serological protocol employed by the Brazilian control
program of CVL. Despite the adoption of a PCR or a qPCR as con-
firmatory test would require a high initial financial investment by the
laboratories (especially the qPCR), the implementation of a highly
sensitive and specific molecular test would significantly decrease the
chances of unnecessary culling, and all equipment acquired could be
explored by the laboratory for the diagnosis of other diseases or for
other control programs.
An important fact that needs to be considered is that the culling of
serologically positive dogs does not have an impact on the reduction of
human cases (Romero and Boelaert, 2010; Costa et al., 2013; Ribeiro
et al., 2018). Research has shown several domestic, wild and synan-
thropic animals positive for VL (Quaresma et al., 2011); will culling the
animals be the right way to go? In the face of the advances in the ve-
terinary therapeutics and vaccinology against VL, old paradigms need
to be overcome. The VL needs to be faced in a broader way; the natural
history of the disease cannot be overlooked.
In conclusion, it was possible to observe that there was no ‘good’ or
‘excellent’ agreement between none of the diagnostic tools analyzed,
and the percentage agreement tended to decrease according to the
absence of clinical manifestations in the dogs. From our data, we in-
tended to establish alternative sets of criteria, for different purposes
(Fig. 3). Nevertheless, it is important to be aware that molecular pro-
tocols and parasitological search may also have significant limitations,
and their applicability as well as their requirement must be extensively
evaluated, before their employment in a control program, or in re-
search, or for clinical/veterinary purposes. Besides, it is important to
comment that maybe the answer for better results in the control pro-
gram is not in the choose of the tools for detection of CVL, but it may be
in the strategy of culling itself. The applicability of this strategy must be
rigorously reanalyzed and discussed. In this context, it is very important
to invest in alternative control measures, such as mass vaccination and
treatment of dogs, thus reducing the transmission to the vector and
helping to avoid new canine and human cases.
Competing interests
The authors declare that they have no competing interest.
R. Pessoa-e-Silva, et al.
Declarations of interest
None.
Acknowledgements
The authors would like to thank the Municipal Health Secretariat
from Caruaru-PE and the Unidade de Controle de Zoonoses (UCZ-
Caruaru) by the structural support for the sample collection and ex-
amination of dogs. We also thank the Program of Technological
Development in Health Supplies (PDTIS/FIOCRUZ) for allowing us to
use their facilities. The reagents for the molecular assays as well as the
material for sample collection were acquired through the financial
support of the Fundação de Amparo à Ciência e Tecnologia de
Pernambuco (FACEPE) - Programa de Excelência em Pesquisa (PROEP)
[APQ:1558-2.13/15].
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