Sensitivity and specificity of some serological techniques by type of antigen, and evaluated population
Visceral leishmaniasis (VL) is a serious public health problem of great medical and veterinary importance. This disease is endemic in Brazil and in many other countries of Latin America, Asia, Africa and Europe (1). According to recent review (2), approximately 0.2 to 0.4 million cases of VL occur each year and although worldwide distributed, higher prevalence of the disease is concentrated in six countries, including India, Bangladesh, Sudan, South Sudan, Ethiopia and Brazil, that undertake for more than 90% of the cases. The clinical importance of VL resides in the severity of the disease that results in death of unrecognized cases and even for individuals with treatment access, death occurs in 10 to 20% of the cases [2-8].
Most of the VL cases are caused by the
The notion that dogs are the main urban domestic reservoir for this
Control strategies include performing accurate and early diagnosis of CVL to identify infected animals [19, 20]. CVL diagnosis is a difficult task since clinical signs of the disease in dogs can be confused with other diseases . In endemic areas, a large percentage of infected animals are asymptomatic or present low number of discrete signs. The role these animals play in parasite transmission is still largely unknown. Several diagnostic strategies have been implemented based on parasitological, serological or molecular methods in association with clinical and epidemiological parameters . Parasite culturing has been considered as gold standard for disease diagnosis [22, 23]. Although offering a high specificity since allows parasite identification, it offers very low sensitivity, besides it is laborious, time-consuming and largely dependent on the expertise of the observer [24, 25].
Serological tests are the most common diagnostic method employed for CVL diagnosis . Several serological methods have been implemented for diagnosis of CVL, including direct agglutination assay (DAT), enzyme linked immunoassay (ELISA) and indirect immunofluorescent antibody test (IFI) . However, most of these classical serological tests present important limitations for CVL diagnosis, including high consumption of time, and lack of sensitivity and specificity, mainly when animals present low antibody titers. This causes underestimation of disease, reflecting in failures in control measures, as well as the maintenance of infected untreated dogs in endemic areas [27, 28]. New methods based on immunochromatography have been implemented for serodiagnosis of CVL and have shown excellent results . These techniques offer several advantages since they are rapid tests easily performed even in field areas, and more specific since they use recombinant DNA technology that additionally facilitates reproducibility and large-scale production. These advantages result in better identification of infected dogs. However, the efficacy of immunochromatographic techniques for CVL diagnosis needs to be improved . In Brazil, a rapid test based in dual path platform (TR DPP®LVC - Biomanguinhos) had been recently implemented as screening test for CVL. This technique seems to be adequate to disease diagnosis in public health system. However, the TR DPP®LVC has shown an excellent performance identifying 98% of symptomatic dogs, it showed less efficacy for diagnosis of asymptomatic dogs (47%) . Since there is evidence that asymptomatic dogs can participate in natural transmission cycle of VL, new strategies should be implemented in order to improve CVL diagnosis [16, 32-34]. For serological diagnosis one strategy can be the development of rapid tests based on impregnation of multi-antigen that would offer more sensitivity, as well specificity.
Finally, it would be important to include more specific confirmatory tests for control strategies that can be advantageous to diagnose inconclusive cases. There is evidence that molecular diagnosis of
2. Importance of CVL diagnosis
Since the discovery of canine visceral leishmaniasis (CVL) in Tunisia by Nicolle & Comte (1908), several reports have shown that dog and man share a common etiologic agent. The notion that dog is the main reservoir of visceral leishmaniasis (VL) in urban centers  is supported by several evidences including the high cutaneous parasitism observed in dogs infected by
Some studies have shown a correlation between the presence of clinical signs in infected animals and transmissibility of the parasite to the vector and, consequently, a correlation with the occurrence of human cases [16, 32, 51]. In accordance with these studies, Travi et al. (2001) and Verçosa et al. (2008) showed that asymptomatic dogs did not transmit the parasite to the vector [38, 51]. There is not a consensus about this idea, since there is a wide variation in the rates of infectivity (70 to 90%) between asymptomatic and symptomatic dogs. Studies show that, regardless of the clinical presentation, any dog has the ability to transmit
3. Visceral leishmaniasis diagnosis in dogs
The diagnosis of VL in the dog must consider the association between clinical, laboratory and epidemiological data. As discussed above, clinical diagnosis is problematic and difficult for veterinarians to perform due to the great variability of clinical signs that
There are several laboratorial diagnosis methods for leishmaniasis: i) parasitological methods (detection of the parasite), ii) serological methods (detection of anti-
In spite of serological techniques such as enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence assay (IFAT) being the most widely used methods for the diagnosis of CVL  parasitological methods, such as direct examination of slides and isolation from tissue cultures, allow the parasite to be detected and can be used as confirmatory diagnostic methods for CVL . In recent decades, molecular techniques such as polymerase chain reaction (PCR) have been introduced for the diagnosis of CVL, exhibiting high sensitivity and specificity . These techniques detect the genetic material of the parasite, which can be used as confirmatory methods in cases of recently infected or asymptomatic animals, which tend not to be diagnosed serologically, and in most cases, do not show seroconversion, having a low parasite load [4, 60]. In a study conducted in Belo Horizonte-MG, a VL-endemic area in Brazil, among 1,443 dogs evaluated, 15.3% of them were seropositive, while 84.7% showed negative serology. Interestingly, among serologically negative dogs, 24.4% showed up as positive using the molecular diagnostic technique, and most of these (97.6%) would not be diagnosed, since they consist of asymptomatic dogs with negative serology .
3.1. Clinical diagnosis
Dogs from endemic areas considered resistant remain clinically normal and asymptomatic without exhibiting clinical signs. There is evidence that the parasites in these animals are effectively eliminated at the infection site [62, 63]. However, in susceptible animals, a large number of parasites are detected in infected tissues. In these animals, the presence of the parasite may occur in multiple organs, accompanied by a granulomatous inflammatory reaction and production of immune-mediated phenomena, probably responsible for the appearance of various types of clinical signs .
Initial clinical signs of CVL include: hypertrophy of the lymph nodes, changes in skin appendages such as onychogryphosis, swelling of the footpad, localized alopecia, skin ulcers and nasal and periocular dermatitis. Alopecia and non-pruritic exfoliative dermatitis can spread to other parts of the animal's body. Weight loss may also be present, as well as cachexia, anorexia and conjunctivitis. Internal organs such as spleen, liver, kidney and lymph nodes may also be affected, when kidney injuries are present may lead to the dogs death [13, 65]. Fever, apathy, diarrhea, epistaxis, intestinal bleeding, hepatosplenomegaly, hyperkeratosis, keratoconjunctivitis are also found in affected animals [66-68]. Some clinical signs are more frequent than others; skin lesions are the most frequent manifestations affecting approximately 50 to 90% of symptomatic dogs [4, 67, 69, 70], including non-pruritic exfoliative dermatitis, with or without alopecia, which can be generalized or localized to the muzzle, ears and limbs [67, 71, 72]. Other very common signs are weight loss, observed in 25 to 80% of CVL cases, including onychogryphosis in 30 to 75%, and ocular abnormalities in 16 to 24% . The most common clinical signs of VL in dogs are depicted in Figure 1.
In dogs with CVL, clinical-pathological changes may occur such as intestinal lesions, renal and hepatic abnormalities . The main biochemical laboratory findings from CVL are hyperglobulinemia, mainly due to increased production of antibodies, and hypoalbuminemia, attributed to chronic inflammation, as long as renal and hepatic failure . The result of these changes is a reduction in the albumin/globulin ratio and hyperproteinemia . Additionally, severe CVL is associated with changes in hematological parameters such as severe anemia and leukopenia, associated with lymphopenia, eosinopenia and monocytopenia [66, 74, 75]. Immune-mediated thrombocytopenia also occurs accounting for episodes of bleeding such as epistaxis, hematuria and hemorrhagic diarrhea .
Finally, nonspecific signs of illness that are mistaken for other diseases such as babesiosis, ehrlichiosis and canine trypanosomiasis also contribute to make CVL clinical diagnosis imprecise and difficult to perform .
3.2. Parasitological diagnosis
The detection by optical microscopy of the parasite by direct observation of stained smears from spleen aspirate, lymph node and bone marrow tissues has high specificity, allowing confirmation of CVL diagnosis [3, 53, 61, 77]. However, the sensitivity of this method is less than 30%, since the direct parasite identification may be limited, especially in mildly and asymptomatic dogs that have low parasitic load, producing false negative results [3, 53, 61, 77].
Another method that can identify the parasite in tissues is the culturing of tissue fragments or aspirates, preferably in a biphasic medium , composed by Novy-MacNeal-Nicolle (NNN), or Tobie modified medium or United States Army Medical Research Units (USAMRU) as solid phase medium and, most often, Schneider as liquid phase medium. This parasitological diagnostic method offers high specificity allowing isolation and characterization of parasites, as well as determination of which species and/or variants are circulating in endemic areas . However, the culturing consists of an indirect test, because when the parasites are isolated from various tissues, they are present in amastigote form and during cultivation they transform into the promastigote form. This process may be impaired as a result of parasite death due to a failure of temperature-control during transport of the tissue sample, or contamination during collection or cultivation . Additionally, a culturing is time consuming and may take up to 4 weeks of observation for definitive diagnosis [13, 79]. Furthermore, specific media for promastigote isolation are not easily obtained, being a technique restricted to specialized laboratories [70, 80], in which the outcome also depends on the experience of the observer [24, 25]. Although culturing offers greater sensitivity compared to direct viewing of amastigotes in tissue , it still remains at very low levels.
In summary, parasitological techniques have high specificity but low sensitivity, especially for the detection of dogs, recently infected, asymptomatic or those presenting low parasite load. In addition, the need for skilled personnel and the long delays to obtain the results prevent parasitological techniques to be used in epidemiological surveys [4, 23, 61, 81-84].
3.3. Serological diagnosis
Serological tests are based on the presence of specific humoral immune responses against the pathogen or purified fraction or recombinant proteins of the pathogen. These tests allow detection of immunoglobulin (IgG) levels, thus becoming an essential tool for the diagnosis of CVL. These methods are simple to carry out and therefore they are frequently used to determine the prevalence of leishmaniasis in epidemiological studies .
A wide variety of serological methods are available for CVL diagnosis, presenting variations in sensitivity and specificity. The performance of these diagnostic techniques varies depending on the type of antigen used and the detection of anti-
The most commonly employed serological tests for the diagnosis of CVL, including ELISA, indirect immunofluorescence test (IFAT), and direct agglutination test (DAT), uses parasite or crude extract of
Despite the practicality and simplicity of serological tests, they do not have 100% sensitivity because some dogs, especially those that are resistant or in the early stages of the disease, have negative results. Thus, the results of such tests should be evaluated carefully, always associating test results with epidemiological history, clinical state of the animal, and the result of a more specific diagnostic test . In addition, since titers of anti-
IFAT is a test in which anti-immunoglobulin antibodies labeled with fluorochromes react with parasites immobilized in a slide. IFAT is a laborious technique that presents difficulties for both standardization and interpretation of the results Therefore, detection of antigen-antibody reaction by fluorescence microscopy depends on the observer experience, compromising reproducibility of this test in different laboratories. Thus, it is not considered a simple and practical technique for evaluating a large number of canine sera . In spite of these limitations, it is still being used as a diagnostic method for mass screening of infected dogs . This method varies in its performance, with sensitivity ranging from 68 to 100% and specificity of 60 to 90% [5, 88-90].
In a study evaluating IFAT for the diagnosis of CVL, the efficacy of the test was evaluated using 254 sera from infected and uninfected dogs and sera from animals with other parasitic diseases. The authors observed low sensitivity (72%) and specificity (52%), as well as cross-reactions when sera from dogs infected with other pathologies, such as
The direct agglutination test (DAT) is an alternative method for the diagnosis of VL, first described in 1975 and adapted for the diagnosis of human and canine infection in the late 1980s [93, 94]. DAT is a method that uses whole stained promastigotes as antigen, either in suspension or freeze-dried . The advantage of this test lies in its low cost when compared with other tests . However, this test is not desirable for screening large numbers of samples, since it is a laborious procedure, due to the production process for crude
Changes to the DAT protocol have been proposed by Gómez-Ochoa
For various reasons, ELISA tests based on whole parasites or crude lysate of parasite antigens for the diagnosis of CVL do not provide satisfactory results, as follows: i) it is a laborious technique, which leads to a delay in the delivery of results and, consequently, the implementation of treatment or the removal of infected dogs from endemic areas [68, 99]; ii) leads to the appearance of cross-reactions with sera from individuals infected with other
A study using 234 domesticated dogs in an endemic area for CVL assessed the efficacy of ELISA, IFAT and DAT for the diagnosis of CVL. In this study, dogs were also parasitologically evaluated for identification of
Using sera from dogs with CVL, a comparison of an ELISA test using crude soluble antigen of
Thus, the search for tests with higher sensitivity and specificity for dogs with a variety of conditions became necessary for control of CVL, which would lead to a reduction of errors in actions taken for treatment or control. In countries that adopt culling of seropositive dogs as a control measure, low sensitivity of diagnostic tests can lead to the maintenance of dogs that transmit disease and lack of specificity can result in unnecessary culling of healthy dogs. The identification of new proteins of
Another way to overcome the obstacles of ELISA based on whole parasites or crude parasite antigen was the development of ELISA tests based on parasite fractions such as that using the parasite surface molecule, fucose-mannose ligand antigen (FML). The FML-based ELISA showed a high sensitivity, which was similar in detecting either oligosymptomatic (90%) or symptomatic (90%) dogs. Regarding specificity, ELISA using crude parasite antigen for the diagnosis of oligosymptomatic dogs was superior, achieving 100% in comparison to FML-based ELISA that was 93.3%. However, for symptomatic dogs the specificity of the FML-based ELISA showed similar results of 96.7% compared to that obtained by ELISA based on crude parasite antigen (93.3%) .
Other ELISA assays based on recombinant antigens such as rA2 from
Interestingly, the association of the recombinant proteins enhanced test performance both for detection of symptomatic and asymptomatic infected dogs. Indeed, using IFAT as the gold standard, ELISA based on the mix of rK9, rK26 and rK39 from
The combination of these findings reinforces the notion that the use of multiple antigens in diagnostic tests enhances test performance and the need to search for new antigens that may compose a diagnostic test able to better diagnose asymptomatic dogs.
New recombinant proteins are being evaluated. Faria
Another study evaluated the performance of the ELISA based on another recombinant antigens of
In summary, most studies using ELISA suggest that in comparison to tests based on crude antigen, those based on recombinant antigens improves accuracy, increasing sensitivity and specificity for the diagnosis of symptomatic dogs. Although improved, test accuracy is still low for the detection of asymptomatic animals.
Recently, rapid immunodiagnostic tests have begun to be employed as routine laboratory tests for detection of diseases such as leishmaniasis. The recombinant antigens of the parasite are impregnated onto nitrocellulose membranes and serum samples are applied in the rapid test platform. Antigens impregnated in nitrocellulose membranes are recognized by specific immunoglobulin present in the serum of infected individuals. This reaction is revealed by the interaction of protein A coupled to colloidal gold particles, with the Fc portion of the immunoglobulins associated with the recombinant antigens. The use of immunochromatographic assays as diagnostic methods has the main advantages of being rapid, completed in around 15 minutes, easy to carry out and can dispense with the need for equipment to read the results . Furthermore, these tests are easily stored, and test supplies and samples do not need to be maintained at low temperatures and can it even be performed at the place of collection. These tests are already widely used to detect HIV  and H1N1  infection. For the diagnosis of CVL and human VL, among the tested and commercially available recombinant proteins, the most widely used for composing immunochromatographic tests is the recombinant protein rK39. This protein contains repetitive sequences of 39 amino acids from a protein related to kinesin of kinetoplast from
Recently, a meta-analysis was performed in order to broadly assess the performance of rapid tests using rK39 as the antigen in the diagnosis of CVL. The combined analysis of 16 studies using rapid tests based on rK39 offered a sensitivity of 86.7% (95% CI: 76.9–92.8%) for the detection of clinical disease and 59.3% (95% CI: 37.9–77.6%) for identification of
||Rapid test||rK39||47||50||26||A: 52.9
||EIE® - LVC||25||16||11||72.0||87.5|
|IFI® - LVC||25||16||11||68.0||87.5|
||EIE® - LVC||234*||20||20||96.0||100|
|IFI® - LVC||234*||20||20||72.0||100|
||EIE® - LVC||305*||0||0||100||96.6|
|IFI® - LVC||305*||0||0||22.2||97.0|
||EIE® - LVC||39||39||39||100||68.0|
|IFI® - LVC||39||39||39||100||70.5|
|DPP® - LVC||rK28||39||39||39||100||97.5|
||DPP® - LVC||rK28||120||59||11||A: 47.0
Efforts have been made to improve the efficacy of rapid tests by developing more sensitive and specific method that could be used in mass screening for the diagnosis of CVL. An alternative proposal is to use a mixture of recombinant proteins or chimeric proteins. The protein rK28 chimeric for the relevant epitopes of three antigens, rK9, rK26 and rK39 [87, 108] that showed promising efficient results in an ELISA based test , was recently used to compose a new rapid test in DPP format. This format consists of a double track platform that offers greater sensitivity and specificity . In addition, this rapid test has advantages over previously used serological methods due to greater precision, simplified interpretation of the data, minimal use of sample volumes, and compatibility with different types of body fluids such as blood, serum, saliva, plasma and urine. In contrast to these advantages, recently Grimaldi et al (2012) showed that rK28-based DPP despite its high sensitivity (98%) and specificity (96%) towards sera from symptomatic dogs, showed low sensitivity of only 47% towards sera from dogs with no signs . With regard to sera from dogs with other diseases, the observed specificity was 96%, with false-positive reactions mainly for some sera of dogs infected with
In recent decades, due to advances in molecular biology techniques and reduced implementation costs, the polymerase chain reaction (PCR) began to be used in VL diagnosis [23, 126]. Its use has demonstrated superior results to those obtained by ELISA, IFA and culture in detecting animals infected with
PCR is a technique based on the principle of complementary bases pairing of the DNA molecule, allowing amplification and detection of a particular region of the target genome using a pair of specific oligonucleotide primers. The reaction can produce tens of billions of DNA fragments from a single molecule, and has high sensitivity small quantities of samples to be used. This type of PCR, hereafter referred as "conventional PCR" (cPCR) needs electrophoresis in agarose or polyacrylamide gels along with dyes such as ethidium bromide, SYBR Green or silver nitrate to view the amplified product. This approach is usually qualitative, with analysis of the presence or absence of bands, or semi-quantitative, when densitometry of bands is used in comparison with known standards. Since it uses qualitative or semi-quantitative analysis, it is imprecise and generates false negatives with some frequency.
A variant of cPCR called "quantitative real-time PCR" (qPCR) became popular in the 2000s. It uses a quantitative approach that allows real-time monitoring of the amplification of the target PCR fragment using fluorophores that bind to double stranded DNA or linked to probes. The most commonly used method is SYBR Green: fluorophore binds to double stranded DNA molecules produced during amplification of the target fragment, leading to the emission of fluorescence during the PCR. This method has the disadvantage of not being able to directly discriminate the amplification of nonspecific DNA fragments, which is usually solved by analyzing the dissociation curve. In contrast, the TaqMan method uses a probe containing between 13 and 30 nucleotides, specifically for the target sequence and combined with a fluorophore and a fluorescence inhibitor. During polymerization of the target fragment, DNA polymerase degrades the probe and fluorescence is emitted. The use of this technique enables an increase in the specificity of this method.
Various PCR-based protocols have been developed for the detection of parasite's DNA and CVL diagnosis. However, the methods used may vary with respect to several parameters, such as fluorophores, probes, target regions and tissue used for detection of target DNA (Table 2), making it difficult to do a comparative analysis between the different protocols. It is known that the sensitivity and specificity of PCR for detection of
The PCR protocol sensitivity is also affected by the type of tissue used in the detection of
The selection of target region in the parasite genome is important because the variation in the number of copies, depending on the region, influences the sensitivity for detecting the parasite's DNA and for quantification of parasite load. The highly conserved and repetitive regions are the most commonly employed, such as the gene for subunit ribosomal RNA (rRNA) or minicircle kinetoplast DNA (kDNA) [21, 23, 127, 140, 141], that has 40-200 copies per cell, while the kDNA minicircles have about 10,000 copies distributed among 10 different classes of sequences. Using this as a target region confers high sensitivity to PCR . For quantification of the parasitic load is recommended to normalize the amount of parasite gene amplification in relation to a constitutive gene derived from the host genome in order to correct distortions caused by errors in the DNA used in the PCR reaction .
|Ferreira et al. 2012||Syber
α pol DNA
|NI||Yes||Yes||ß - canine actin||(80) Infected dogs||Conjunctival swab, blood, bone marrow and skin||Comparative1,2||Skin > Bone marrow > Conjunctival swab > Blood|
||TaqMan kDNA||0.01 parasites/ reaction||Yes||Yes||18S eukaryotic rRNA||(51) Dogs||Bone marrow, conjunctival swab, lymph node, skin and spleen||Comparative1,2||Spleen > Blood > Lymph node > Skin > Bone marrow > Conjunctival swab|
|Belinchón-Lorenzo et al. 2013||TaqMan kDNA||0.0079 parasites/ reaction||Yes||Yes||18S eukaryotic rRNA||(28) Dogs||Blood, hair and lymph node||Comparative 2||Lymph node > Hair = Blood|
|Ferreira et al. 2013||Syber
α pol DNA
|NI||Yes||Yes||ß - canine actin||(62) CVL positive dogs||Conjunctival, nasal and ear swab, blood, Bone marrow and skin||Comparative
|Skin = Nasal swab and bone marrow > Conjunctival swab > Oral swab > Ear swab|
|Geisweid et al. 2013||Syber
|NI||Yes||No||Canine NCX1||(74) CVL suspected dogs||Conjunctival swab, blood, bone marrow and lymph node||Comparative 2||Bone marrow > Conjunctival Swab|
α pol DNA
|NI||Yes||No||G3PDH||(60) Seropositive dogs||Skin and spleen||Comparative
|Spleen > Skin|
|NI||No||No||---||(6) Treated dogs||Blood, lymph node and skin||Not comparative||---|
||TaqMan kDNA||0.001 parasites/ reaction||Yes||No||18S eukaryotic rRNA||(15) Dogs with clinical signs suggestive of CVL||Blood and bone marrow||Comparative1, 2||Bone marrow > Blood
||TaqMan kDNA||0.001 parasites/ reaction||Yes||Yes||18S eukaryotic rRNA||(6) Experimentally infected dogs||Blood, bone marrow, liver, lymph node, skin and spleen||Not comparative||---|
||Syber kDNA||7 parasites/ml||Yes||No||Canine GAPDH||(10) Symptomatic dogs naturally infected||Blood, bone marrow and urine||Comparative 2||Bone marrow > Blood
||TaqMan kDNA||0.001 parasites/ml||Yes||Yes||ß - actin||(18) Naturally infected treated dogs||Blood, lymph node and skin||Comparative 2||Lymph node > Skin > Blood
||TaqMan kDNA||NI||Yes||Yes||ß - actin||(56) Dogs||Blood and lymph node||Not comparative||---|
||Syber kDNA||0.1pg DNA/ml||Yes||Yes||ß -canine globin||(35) Dogs||Blood and bone marrow||Comparative2||Blood = Bone marrow|
|1 parasite /reaction||Yes||No||ß - canine actin||(12) Experimentally infected dogs||Blood, bone marrow, buffy coat, liver, lymph node, skin and spleen||Comparative1, 2||Spleen / Buffy coat / Liver / Lymph node / Bone marrow / Skin > Blood|
||TaqMan kDNA||0.03 parasite/ reaction||No||No||---||(88) Dogs||Conjunctival swab, Lymph node, bone marrow and blood||Comparative1||---|
||TaqMan kDNA||NI||No||No||---||(138) Dogs||Blood, conjunctival and oral swabs and lymph node||Comparative1||---|
||TaqMan kDNA||NI||Yes||No||18S eukaryotic rRNA||(22) Sick dogs||Main lacrimal gland, tarsal gland and nictitating membrane gland||Comparative1||---|
In a cytological study, Reis
Splenic collection, bone marrow and lymph node aspirates are considered invasive procedures  in addition to having an elevated cost compared to blood collection. Thus, it can be recommended to use samples obtained less invasively, such as blood and conjunctival swabs [136, 154, 155]. These samples are quick and easy to obtain, and it is low-cost compared to more invasive procedures, in addition to their higher acceptance by animal owners [132, 154, 155].
Some studies have shown that detection of parasites in the peripheral blood is less sensitive compared to other tissue samples such as spleen, bone marrow, lymph nodes and skin and tends to have variable parasitic load in accordance with the stage of infection [129, 141, 156]. However, depending on the technique and the target, blood can be used for detection of
According to Solano-Gallego
Among other less invasive sample types investigated, Solano-Gallego et al (2007) evaluated urine samples with qPCR technique, but the results described showed positivity only in dogs with severe renal injury . Naranjo et al. (2012) identified the presence of
|Elleviti – Torino, Italy||26.80*||---||63.00*||---|
|Scanelis - Toulouse, France||---||---||60.30*||---|
|Laboratoire d'Anatomie Pathologique Vétérinaire du Sud-Ouest – Toulouse, France||---||---||---||127.30*|
|Laboratório Veterinário INNO – Braga, Portugal||20.60*||54.40*||---||---|
|Instituto Nacional de Investigação Agrária e Veterinária, I.P. – Lisbõa, Portugal||28.00*||41.20*||---||---|
|Centro de Investigación y Análisis Biológicos – Madrid, Spain||13.60*||60.30*||73.70*||---|
|Texas Veterinary Medical Diagnostic Laboratory – San Antonio TX, USA||19.20||---||---||---|
|Cornell University - Ithaca NY, USA||22.50||60.00||---||---|
|Hermes Pardini - Belo Horizonte MG, Brazil||17.20*||60.20*||---||---|
|Análisis Biológicos– Chapecó SC, Brazil||9.40*||42.15*||72.25*||--|
|Laborlife - Rio de Janeiro RJ, Brazil||30.10*||77.40*||---||---|
Despite the high sensitivity and specificity, the use of molecular methods for the CVL diagnosis presents some limitations to its use in epidemiological surveys: i) it has higher costs than other techniques (Table 3) used in the CVL diagnosis, including reagent and equipment costs; ii) it presents relative complexity in its implementation, requiring personnel with training in the execution of PCR reactions. However, this method has advantages in terms of sensitivity and specificity when compared to other diagnostic techniques, which justify its use in confirming cases screened by serology [24, 132]. Particularly due to the possibility of quantifying target DNA, qPCR may be used to monitor the parasitic load of the animal during the experimental infection, or during and after treatment in countries where it is permitted [35-37, 162]. Compared with cPCR, qPCR enables a reduction in the probability of false positives resulting from amplification artifacts and greater speed in obtaining results, once electrophoresis is no longer performed .
In summary, detailed clinical evaluation complemented with highly sensitive test allows proper identification of infected dogs in an endemic area. Evidence shows that the use of a rapid serological test associated with a molecular diagnostic test with high specificity, such as qPCR, is required for identification of all infected dogs, both asymptomatic and symptomatic. On the other hand, for sick dogs a correct diagnosis is necessary either to perform dog culling in countries where this measure is used as a control strategy of VL or to define treatment. In this case, a detailed clinical evaluation should be associated with biochemistry and hematological tests to identify signs of renal and hepatic failure, in conjunction with a serological test to confirm animal clinical condition.
Desjeux P. Leishmaniasis. Nat Rev Microbiol. 2004;2(9):692.
Alvar J, Velez ID, Bern C, Herrero M, Desjeux P, Cano J, et al. Leishmaniasis worldwide and global estimates of its incidence. PLoS One. 2012;7(5):e35671.
Gomes YM, Paiva Cavalcanti M, Lira RA, Abath FG, Alves LC. Diagnosis of canine visceral leishmaniasis: biotechnological advances. Vet J. 2008;175(1):45-52.
Solano-Gallego L, Morell P, Arboix M, Alberola J, Ferrer L. Prevalence of Leishmania infantuminfection in dogs living in an area of canine leishmaniasis endemicity using PCR on several tissues and serology. J Clin Microbiol. 2001;39(2):560-3.
Ferreira Ede C, de Lana M, Carneiro M, Reis AB, Paes DV, da Silva ES, et al. Comparison of serological assays for the diagnosis of canine visceral leishmaniasis in animals presenting different clinical manifestations. Vet Parasitol. 2007;146(3-4):235-41.
Troncarelli MZ, Camargo JB, Machado JG, Lucheis SB, Langoni H. Leishmaniaspp. and/or Trypanosoma cruzidiagnosis in dogs from endemic and nonendemic areas for canine visceral leishmaniasis. Vet Parasitol. 2009;164(2-4):118-23.
Solca Mda S, Guedes CE, Nascimento EG, Oliveira GG, dos Santos WL, Fraga DB, et al. Qualitative and quantitative polymerase chain reaction (PCR) for detection of Leishmania in spleen samples from naturally infected dogs. Vet Parasitol. 2012;184(2-4):133-40.
Mary C, Faraut F, Lascombe L, Dumon H. Quantification of Leishmania infantumDNA by a real-time PCR assay with high sensitivity. J Clin Microbiol. 2004;42(11):5249-55.
Lainson R, Shaw JJ. Epidemiology and ecology of leishmaniasis in Latin-America. Nature. 1978;273(5664):595-600.
Killick-Kendrick R. The biology and control of phlebotomine sand flies. Clin Dermatol. 1999;17(3):279-89.
Kuhls K, Alam MZ, Cupolillo E, Ferreira GE, Mauricio IL, Oddone R, et al. Comparative microsatellite typing of new world Leishmania infantumreveals low heterogeneity among populations and its recent old world origin. PLoS Negl Trop Dis. 2011;5(6):e1155.
Mauricio IL, Stothard JR, Miles MA. The strange case of Leishmania chagasi. Parasitol Today. 2000;16(5):188-9.
Alvar J, Canavate C, Molina R, Moreno J, Nieto J. Canine leishmaniasis. Adv Parasitol. 2004;57:1-88.
Bevilacqua PD, Paixao HH, Modena CM, Castro MCPS. Urbanization of visceral leishmaniose in Belo Horizonte, Brazil. Arq Bras Med Vet Zoot. 2001;53:1-8.
Deane LM, Deane MP. Leishmaniose visceral urbana (no cão e no homem) em Sobral, Ceará. O Hospital 1955;47:113-28.
Molina R, Amela C, Nieto J, San-Andres M, Gonzalez F, Castillo JA, et al. Infectivity of dogs naturally infected with Leishmania infantumto colonized Phlebotomus perniciosus. Trans R Soc Trop Med Hyg. 1994;88(4):491-3.
Gramiccia M, Gradoni L. The current status of zoonotic leishmaniases and approaches to disease control. Int J Parasitol. 2005;35(11-12):1169-80.
Dye C. The logic of visceral leishmaniasis control. Am J Trop Med Hyg. 1996;55(2):125-30.
Coura-Vital W, Marques MJ, Veloso VM, Roatt BM, Aguiar-Soares RD, Reis LE, et al. Prevalence and factors associated with Leishmania infantuminfection of dogs from an urban area of Brazil as identified by molecular methods. PLoS Negl Trop Dis. 2011;5(8):e1291.
Podaliri Vulpiani M, Iannetti L, Paganico D, Iannino F, Ferri N. Methods of Control of the Leishmania infantumDog Reservoir: State of the Art. Vet Med Int. 2011;2011:215964.
Miro G, Cardoso L, Pennisi MG, Oliva G, Baneth G. Canine leishmaniosis--new concepts and insights on an expanding zoonosis: part two. Trends Parasitol. 2008;24(8):371-7.
Carvalho D, S.; OTMF, D.; BC, Z. MR. An enzyme-linked immunosorbent assay (ELISA) for the detection of IgM antibodies against Leishmania chagasi in dogs. Pesq Vet Bras 2009;29:120-4.
Sundar S, Rai M. Laboratory diagnosis of visceral leishmaniasis. Clinical and diagnostic laboratory immunology. 2002;9(5):951-8.
Moreira MA, Luvizotto MC, Garcia JF, Corbett CE, Laurenti MD. Comparison of parasitological, immunological and molecular methods for the diagnosis of leishmaniasis in dogs with different clinical signs. Vet Parasitol. 2007;145(3-4):245-52.
Ndao M. Diagnosis of parasitic diseases: old and new approaches. Interdiscip Perspect Infect Dis. 2009;2009:278246.
Morales-Yuste M, Morillas-Marquez F, Diaz-Saez V, Baron-Lopez S, Acedo-Sanchez C, Martin-Sanchez J. Epidemiological implications of the use of various methods for the diagnosis of canine leishmaniasis in dogs with different characteristics and in differing prevalence scenarios. Parasitol Res. 2012;111(1):155-64.
de Paula AA, da Silva AV, Fernandes O, Jansen AM. The use of immunoblot analysis in the diagnosis of canine visceral leishmaniasis in an endemic area of Rio de Janeiro. J Parasitol. 2003;89(4):832-6.
Almeida MA, Jesus EE, Sousa-Atta ML, Alves LC, Berne ME, Atta AM. Clinical and serological aspects of visceral leishmaniasis in northeast Brazilian dogs naturally infected with Leishmania chagasi. Vet Parasitol. 2005;127(3-4):227-32.
Porrozzi R, Santos da Costa MV, Teva A, Falqueto A, Ferreira AL, dos Santos CD, et al. Comparative evaluation of enzyme-linked immunosorbent assays based on crude and recombinant leishmanial antigens for serodiagnosis of symptomatic and asymptomatic Leishmania infantumvisceral infections in dogs. Clin Vaccine Immunol. 2007;14(5):544-8.
Quinnell RJ, Carson C, Reithinger R, Garcez LM, Courtenay O. Evaluation of rK39 rapid diagnostic tests for canine visceral leishmaniasis: longitudinal study and meta-analysis. PLoS Negl Trop Dis. 2013;7(1):e1992.
Grimaldi G, Jr., Teva A, Santos CB, Ferreira AL, Falqueto A. The effect of removing potentially infectious dogs on the numbers of canine Leishmania infantuminfections in an endemic area with high transmission rates. Am J Trop Med Hyg. 2012;86(6):966-71.
Courtenay O, Quinnell RJ, Garcez LM, Shaw JJ, Dye C. Infectiousness in a cohort of brazilian dogs: why culling fails to control visceral leishmaniasis in areas of high transmission. J Infect Dis. 2002;186(9):1314-20.
Michalsky EM, Rocha MF, da Rocha Lima AC, Franca-Silva JC, Pires MQ, Oliveira FS, et al. Infectivity of seropositive dogs, showing different clinical forms of leishmaniasis, to Lutzomyia longipalpis phlebotomine sand flies. Vet Parasitol. 2007;147(1-2):67-76.
Soares MR, de Mendonca IL, do Bonfim JM, Rodrigues JA, Werneck GL, Costa CH. Canine visceral leishmaniasis in Teresina, Brazil: Relationship between clinical features and infectivity for sand flies. Acta Trop. 2011;117(1):6-9.
Maia C, Campino L. Methods for diagnosis of canine leishmaniasis and immune response to infection. Vet Parasitol. 2008;158(4):274-87.
Pennisi MG, Reale S, Giudice SL, Masucci M, Caracappa S, Vitale M, et al. Real-time PCR in dogs treated for leishmaniasis with allopurinol. Veterinary research communications. 2005;29 Suppl 2:301-3.
Martinez V, Quilez J, Sanchez A, Roura X, Francino O, Altet L. Canine leishmaniasis: the key points for qPCR result interpretation. Parasit Vectors. 2011;4:57.
Travi BL, Tabares CJ, Cadena H, Ferro C, Osorio Y. Canine visceral leishmaniasis in Colombia: relationship between clinical and parasitologic status and infectivity for sand flies. Am J Trop Med Hyg. 2001;64(3-4):119-24.
Shang LM, Peng WP, Jin HT, Xu D, Zhong NN, Wang WL, et al. The prevalence of canine Leishmania infantuminfection in Sichuan Province, southwestern China detected by real time PCR. Parasit Vectors. 2011;4(1):173.
Athanasiou LV, Kontos VI, Saridomichelakis MN, Rallis TS, Diakou A. A cross-sectional sero-epidemiological study of canine leishmaniasis in Greek mainland. Acta Trop. 2012;122(3):291-5.
Pastor-Santiago JA, Chavez-Lopez S, Guzman-Bracho C, Flisser A, Olivo-Diaz A. American visceral leishmaniasis in Chiapas, Mexico. Am J Trop Med Hyg. 2012;86(1):108-14.
Berrahal F, Mary C, Roze M, Berenger A, Escoffier K, Lamouroux D, et al. Canine leishmaniasis: identification of asymptomatic carriers by polymerase chain reaction and immunoblotting. Am J Trop Med Hyg. 1996;55(3):273-7.
Quinnell RJ, Courtenay O, Davidson S, Garcez L, Lambson B, Ramos P, et al. Detection of Leishmania infantumby PCR, serology and cellular immune response in a cohort study of Brazilian dogs. Parasitology. 2001;122(Pt 3):253-61.
Lachaud L, Chabbert E, Dubessay P, Dereure J, Lamothe J, Dedet JP, et al. Value of two PCR methods for the diagnosis of canine visceral leishmaniasis and the detection of asymptomatic carriers. Parasitology. 2002;125(Pt 3):197-207.
Leontides LS, Saridomichelakis MN, Billinis C, Kontos V, Koutinas AF, Galatos AD, et al. A cross-sectional study of Leishmaniaspp. infection in clinically healthy dogs with polymerase chain reaction and serology in Greece. Vet Parasitol. 2002;109(1-2):19-27.
Lanotte G, Rioux JA, Perieres J, Vollhardt Y. [Ecology of leishmaniasis in the south of France. 10. Developmental stages and clinical characterization of canine leishmaniasis in relation to epidemiology. (author's transl]. Ann Parasitol Hum Comp. 1979;54(3):277-95.
Pozio E, Gradoni L, Bettini S, Gramiccia M. Leishmaniasis in Tuscany (Italy) V. Further isolation of Leishmania from Rattus rattus in the Province of Grosseto. Ann Trop Med Parasitol. 1981;75(4):393-5.
Moreno J, Alvar J. Canine leishmaniasis: epidemiological risk and the experimental model. Trends Parasitol. 2002;18(9):399-405.
Moreno J, Nieto J, Chamizo C, Gonzalez F, Blanco F, Barker DC, et al. The immune response and PBMC subsets in canine visceral leishmaniasis before, and after, chemotherapy. Vet Immunol Immunopathol. 1999;71(3-4):181-95.
Quinnell RJ, Courtenay O, Garcez LM, Kaye PM, Shaw MA, Dye C, et al. IgG subclass responses in a longitudinal study of canine visceral leishmaniasis. Vet Immunol Immunopathol. 2003;91(3-4):161-8.
Vercosa BL, Lemos CM, Mendonca IL, Silva SM, de Carvalho SM, Goto H, et al. Transmission potential, skin inflammatory response, and parasitism of symptomatic and asymptomatic dogs with visceral leishmaniasis. BMC Vet Res. 2008;4:45.
Guarga JL, Lucientes J, Peribanez MA, Molina R, Gracia MJ, Castillo JA. Experimental infection of Phlebotomus perniciosusand determination of the natural infection rates of Leishmania infantumin dogs. Acta Trop. 2000;77(2):203-7.
Miles MA, Vexenat JA, Furtado Campos JH, Fonseca de Castro JA. Canine leishmaniasis in Latin América: control strategies for visceral leishmaniasis. Canine Leishmaniasis: an update. 1999;Hoechst Roussel Vet.:46-53.
Moshfe A, Mohebali M, Edrissian G, Zarei Z, Akhoundi B, Kazemi B, et al. Canine visceral leishmaniasis: asymptomatic infected dogs as a source of L. infantuminfection. Acta Trop. 2009;112(2):101-5.
Banuls AL, Hide M, Prugnolle F. Leishmania and the leishmaniases: a parasite genetic update and advances in taxonomy, epidemiology and pathogenicity in humans. Adv Parasitol. 2007;64:1-109.
Grimaldi G, Jr., Tesh RB. Leishmaniases of the New World: current concepts and implications for future research. Clin Microbiol Rev. 1993;6(3):230-50.
Gontijo CM, Melo MN. Visceral Leishmaniasis in Brazil: current status, challenges and prospects. . Rev Bras Epidemiol 2004;7(3):338-49.
Ferrer L. International canine Leishmaniasis forum. Proceedings. 1999:6-10.
Srivastava P, Dayama A, Mehrotra S, Sundar S. Diagnosis of visceral leishmaniasis. Trans R Soc Trop Med Hyg. 2011;105(1):1-6.
Alves WA, Bevilacqua PD. Quality of diagnosis of canine visceral leishmaniasis in epidemiological surveys: an epidemic in Belo Horizonte, Minas Gerais, Brazil, 1993 – 1997. Cad Saúde Pública. 2004;20(1):259-65.
Barrouin-Melo SM, Larangeira DF, Trigo J, Aguiar PH, dos-Santos WL, Pontes-de-Carvalho L. Comparison between splenic and lymph node aspirations as sampling methods for the parasitological detection of Leishmania chagasi infection in dogs. Mem Inst Oswaldo Cruz. 2004;99(2):195-7.
Cabral M, O'Grady J, Alexander J. Demonstration of Leishmania specific cell mediated and humoral immunity in asymptomatic dogs. Parasite Immunol. 1992;14(5):531-9.
Solano-Gallego L, Llull J, Ramos G, Riera C, Arboix M, Alberola J, et al. The Ibizian hound presents a predominantly cellular immune response against natural Leishmania infection. Vet Parasitol. 2000;90(1-2):37-45.
Saridomichelakis MN. Advances in the pathogenesis of canine leishmaniosis: epidemiologic and diagnostic implications. Vet Dermatol. 2009;20(5-6):471-89.
Benderitter T, Casanova P, Nashkidachvili L, Quilici M. Glomerulonephritis in dogs with canine leishmaniasis. Ann Trop Med Parasitol. 1988;82(4):335-41.
Ciaramella P, Oliva G, Luna RD, Gradoni L, Ambrosio R, Cortese L, et al. A retrospective clinical study of canine leishmaniasis in 150 dogs naturally infected by Leishmania infantum. Vet Rec. 1997;141(21):539-43.
Koutinas AF, Polizopoulou ZS, Saridomichelakis MN, Argyriadis D, Fytianou A, Plevraki KG. Clinical considerations on canine visceral leishmaniasis in Greece: a retrospective study of 158 cases (1989-1996). J Am Anim Hosp Assoc. 1999;35(5):376-83.
Mendonça L, Alves LC, Faustino MAG, Vasconcelos JR. Clinical aspects of visceral Leishmania in naturally infected dogs in the of Teresina, Piauí. Revista Brasileira Parasitologia Veterinaria. 1998 8(1):23-5.
Shaw SE, Langton DA, Hillman TJ. Canine leishmaniosis in the United Kingdom: a zoonotic disease waiting for a vector? Vet Parasitol. 2009;163(4):281-5.
Solano-Gallego L, Miro G, Koutinas A, Cardoso L, Pennisi MG, Ferrer L, et al. LeishVet guidelines for the practical management of canine leishmaniosis. Parasit Vectors. 2011;4:86.
Ferrer L, Aisa MJ, Roura X, Portus M. Serological diagnosis and treatment of canine leishmaniasis. Vet Rec. 1995;136(20):514-6.
Ordeix L, Solano-Gallego L, Fondevila D, Ferrer L, Fondati A. Papular dermatitis due to Leishmaniaspp. infection in dogs with parasite-specific cellular immune responses. Vet Dermatol. 2005;16(3):187-91.
Baneth G, Koutinas AF, Solano-Gallego L, Bourdeau P, Ferrer L. Canine leishmaniosis - new concepts and insights on an expanding zoonosis: part one. Trends Parasitol. 2008;24(7):324-30.
Reis AB, Teixeira-Carvalho A, Giunchetti RC, Guerra LL, Carvalho MG, Mayrink W, et al. Phenotypic features of circulating leucocytes as immunological markers for clinical status and bone marrow parasite density in dogs naturally infected by Leishmania chagasi. Clin Exp Immunol. 2006;146(2):303-11.
Tropia de Abreu R, Carvalho M, Carneiro CM, Giunchetti RC, Teixeira-Carvalho A, Martins-Filho OA, et al. Influence of clinical status and parasite load on erythropoiesis and leucopoiesis in dogs naturally infected with Leishmania (Leishmania) chagasi. PLoS One. 2011;6(5):e18873.
Cortese L, Sica M, Piantedosi D, Ruggiero G, Pero ME, Terrazzano G, et al. Secondary immune-mediated thrombocytopenia in dogs naturally infected by Leishmania infantum. Vet Rec. 2009;164(25):778-82.
Reis AB, Teixeira-Carvalho A, Vale AM, Marques MJ, Giunchetti RC, Mayrink W, et al. Isotype patterns of immunoglobulins: hallmarks for clinical status and tissue parasite density in Brazilian dogs naturally infected by Leishmania (Leishmania) chagasi. Vet Immunol Immunopathol. 2006;112(3-4):102-16.
Palma G, Gutierrez Y. Laboratory diagnosis of Leishmania. Clin Lab Med. 1991;11(4):909-22.
de Almeida ME, Steurer FJ, Koru O, Herwaldt BL, Pieniazek NJ, da Silva AJ. Identification of Leishmaniaspp. by molecular amplification and DNA sequencing analysis of a fragment of rRNA internal transcribed spacer 2. J Clin Microbiol. 2011;49(9):3143-9.
Paltrinieri S, Ravicini S, Rossi G, Roura X. Serum concentrations of the derivatives of reactive oxygen metabolites (d-ROMs) in dogs with leishmaniosis. Vet J. 2010;186(3):393-5.
Fraga DB, Solca MS, Silva VM, Borja LS, Nascimento EG, Oliveira GG, et al. Temporal distribution of positive results of tests for detecting Leishmania infection in stray dogs of an endemic area of visceral leishmaniasis in the Brazilian tropics: a 13 years survey and association with human disease. Vet Parasitol. 2012;190(3-4):591-4.
da Silva RN, Amorim AC, Brandao RM, de Andrade HM, Yokoo M, Ribeiro ML, et al. Real-time PCR in clinical practice: a powerful tool for evaluating Leishmania chagasi loads in naturally infected dogs. Ann Trop Med Parasitol. 2010;104(2):137-43.
Pinelli E, Killick-Kendrick R, Wagenaar J, Bernadina W, del Real G, Ruitenberg J. Cellular and humoral immune responses in dogs experimentally and naturally infected with Leishmania infantum. Infect Immun. 1994;62(1):229-35.
Dos-Santos WL, Jesus EE, Paranhos-Silva M, Pereira AM, Santos JC, Baleeiro CO, et al. Associations among immunological, parasitological and clinical parameters in canine visceral leishmaniasis: Emaciation, spleen parasitism, specific antibodies and leishmanin skin test reaction. Vet Immunol Immunopathol. 2008;123(3-4):251-9.
Ferrer L. Clinical aspects of canine leishmaniasis. International Canine Leishmaniasis forum, Barcelona. 1999:6-10.
da Silva ES, van der Meide WF, Schoone GJ, Gontijo CM, Schallig HD, Brazil RP. Diagnosis of canine leishmaniasis in the endemic area of Belo Horizonte, Minas Gerais, Brazil by parasite, antibody and DNA detection assays. Vet Res Commun. 2006;30(6):637-43.
Boarino A, Scalone A, Gradoni L, Ferroglio E, Vitale F, Zanatta R, et al. Development of recombinant chimeric antigen expressing immunodominant B epitopes of Leishmania infantumfor serodiagnosis of visceral leishmaniasis. Clin Diagn Lab Immunol. 2005;12(5):647-53.
Lira RA, Cavalcanti MP, Nakazawa M, Ferreira AG, Silva ED, Abath FG, et al. Canine visceral leishmaniosis: a comparative analysis of the EIE-leishmaniose-visceral-canina-Bio-Manguinhos and the IFI-leishmaniose-visceral-canina-Bio-Manguinhos kits. Vet Parasitol. 2006;137(1-2):11-6.
Ferroglio E, Centaro E, Mignone W, Trisciuoglio A. Evaluation of an ELISA rapid device for the serological diagnosis of Leishmania infantuminfection in dog as compared with immunofluorescence assay and Western blot. Vet Parasitol. 2007;144(1-2):162-6.
Alves AS, Mouta-Confort E, Figueiredo FB, Oliveira RV, Schubach AO, Madeira MF. Evaluation of serological cross-reactivity between canine visceral leishmaniasis and natural infection by Trypanosoma caninum. Res Vet Sci. 2012;93(3):1329-33.
Reithinger R, Davies CR. Canine leishmaniasis: novel strategies for control. Trends Parasitol. 2002;18(7):289-90.
Figueiredo MM, Moura EP, Costa MM, Ribeiro VM, Michalick MS, Tafuri WL, et al. Histopathological and parasitological investigations of ear healthy skin of dogs naturally and experimentally infected with Leishmania (Leishmania) chagasi. Histol Histopathol. 2010;25(7):877-87.
el Harith A, Slappendel RJ, Reiter I, van Knapen F, de Korte P, Huigen E, et al. Application of a direct agglutination test for detection of specific anti-Leishmania antibodies in the canine reservoir. J Clin Microbiol. 1989;27(10):2252-7.
el Safi SH, Evans DA. A comparison of the direct agglutination test and enzyme-linked immunosorbent assay in the sero-diagnosis of leishmaniasis in the Sudan. Trans R Soc Trop Med Hyg. 1989;83(3):334-7.
Babakhan L, Mohebali M, Akhoundi B, Edrissian GH, Keshavarz H. Rapid detection of Leishmania infantuminfection in dogs: a comparative study using fast agglutination screening test (FAST) and direct agglutination test (DAT) in Iran. Parasitol Res. 2009;105(3):717-20.
Schallig HD, Canto-Cavalheiro M, da Silva ES. Evaluation of the direct agglutination test and the rK39 dipstick test for the sero-diagnosis of visceral leishmaniasis. Mem Inst Oswaldo Cruz. 2002;97(7):1015-8.
Sundar S, Maurya R, Singh RK, Bharti K, Chakravarty J, Parekh A, et al. Rapid, noninvasive diagnosis of visceral leishmaniasis in India: comparison of two immunochromatographic strip tests for detection of anti-K39 antibody. J Clin Microbiol. 2006;44(1):251-3.
Mohammadiha A, Haghighi A, Mohebali M, Mahdian R, Abadi AR, Zarei Z, et al. Canine visceral leishmaniasis: a comparative study of real-time PCR, conventional PCR, and direct agglutination on sera for the detection of Leishmania infantuminfection. Vet Parasitol. 2013;192(1-3):83-90.
Moreira ED, Jr., Mendes de Souza VM, Sreenivasan M, Nascimento EG, Pontes de Carvalho L. Assessment of an optimized dog-culling program in the dynamics of canine Leishmania transmission. Vet Parasitol. 2004;122(4):245-52.
Kar K. Serodiagnosis of leishmaniasis. Crit Rev Microbiol. 1995;21(2):123-52.
Candido TC, Perri SH, Gerzoschkwitz Tde O, Luvizotto MC, de Lima VM. Comparative evaluation of enzyme-linked immunosorbent assay based on crude and purified antigen in the diagnosis of canine visceral leishmaniasis in symptomatic and oligosymptomatic dogs. Vet Parasitol. 2008;157(3-4):175-81.
Coelho EA, Ramirez L, Costa MA, Coelho VT, Martins VT, Chavez-Fumagalli MA, et al. Specific serodiagnosis of canine visceral leishmaniasis using Leishmania species ribosomal protein extracts. Clin Vaccine Immunol. 2009;16(12):1774-80.
Silva DA, Madeira MF, Teixeira AC, de Souza CM, Figueiredo FB. Laboratory tests performed on Leishmania seroreactive dogs euthanized by the leishmaniasis control program. Vet Parasitol. 2011;179(1-3):257-61.
Nolan TJ, Herman R. Effects of long-term in vitro cultivation on Leishmania donovani promastigotes. J Protozool. 1985;32(1):70-5.
Faria AR, Costa MM, Giusta MS, Grimaldi G, Jr., Penido ML, Gazzinelli RT, et al. High-throughput analysis of synthetic peptides for the immunodiagnosis of canine visceral leishmaniasis. PLoS Negl Trop Dis. 2011;5(9):e1310.
Oliveira GG, Magalhaes FB, Teixeira MC, Pereira AM, Pinheiro CG, Santos LR, et al. Characterization of novel Leishmania infantumrecombinant proteins encoded by genes from five families with distinct capacities for serodiagnosis of canine and human visceral leishmaniasis. Am J Trop Med Hyg. 2011;85(6):1025-34.
Scalone A, De Luna R, Oliva G, Baldi L, Satta G, Vesco G, et al. Evaluation of the Leishmania recombinant K39 antigen as a diagnostic marker for canine leishmaniasis and validation of a standardized enzyme-linked immunosorbent assay. Vet Parasitol. 2002;104(4):275-85.
Rosati S, Ortoffi M, Profiti M, Mannelli A, Mignone W, Bollo E, et al. Prokaryotic expression and antigenic characterization of three recombinant Leishmania antigens for serological diagnosis of canine leishmaniasis. Clin Diagn Lab Immunol. 2003;10(6):1153-6.
de Souza CM, Silva ED, Ano Bom AP, Bastos RC, Nascimento HJ, da Silva Junior JG. Evaluation of an ELISA for canine leishmaniasis immunodiagnostic using recombinant proteins. Parasite Immunol. 2012;34(1):1-7.
de Lima VM, Fattori KR, Michelin Ade F, da Silveira Neto L, Vasconcelos Rde O. Comparison between ELISA using total antigen and immunochromatography with antigen rK39 in the diagnosis of canine visceral leishmaniasis. Vet Parasitol. 2010;173(3-4):330-3.
Vuylsteke B, Semde G, Sika L, Crucitti T, Ettiegne Traore V, Buve A, et al. HIV and STI prevalence among female sex workers in Cote d'Ivoire: why targeted prevention programs should be continued and strengthened. PLoS One. 2012;7(3):e32627.
Wu W, Huang L, Mendez S. A live Leishmania majorvaccine containing CpG motifs induces the de novo generation of Th17 cells in C57BL/6 mice. Eur J Immunol. 2010;40(9):2517-27.
Badaro R, Benson D, Eulalio MC, Freire M, Cunha S, Netto EM, et al. rK39: a cloned antigen of Leishmania chagasi that predicts active visceral leishmaniasis. J Infect Dis. 1996;173(3):758-61.
Burns JM, Jr., Shreffler WG, Benson DR, Ghalib HW, Badaro R, Reed SG. Molecular characterization of a kinesin-related antigen of Leishmania chagasi that detects specific antibody in African and American visceral leishmaniasis. Proc Natl Acad Sci U S A. 1993;90(2):775-9.
de Carvalho LP, Soto M, Jeronimo S, Dondji B, Bacellar O, Luz V, et al. Characterization of the immune response to Leishmania infantumrecombinant antigens. Microbes Infect. 2003;5(1):7-12.
Ferroglio E, Zanet S, Mignone W, Poggi M, Trisciuoglio A, Bianciardi P. Evaluation of a rapid device for serological diagnosis of Leishmania infantuminfection in dogs as an alternative to immunofluorescence assay and Western blotting. Clin Vaccine Immunol. 2013;20(5):657-9.
Kumar D, Khanal B, Tiwary P, Mudavath SL, Tiwary NK, Singh R, et al. Comparative evaluation of blood and serum samples in rapid immunochromatographic tests for visceral leishmaniasis. J Clin Microbiol. 2013;51(12):3955-9.
Lemos EM, Laurenti MD, Moreira MA, Reis AB, Giunchetti RC, Raychaudhuri S, et al. Canine visceral leishmaniasis: performance of a rapid diagnostic test (Kalazar Detect) in dogs with and without signs of the disease. Acta Trop. 2008;107(2):205-7.
Mettler M, Grimm F, Capelli G, Camp H, Deplazes P. Evaluation of enzyme-linked immunosorbent assays, an immunofluorescent-antibody test, and two rapid tests (immunochromatographic-dipstick and gel tests) for serological diagnosis of symptomatic and asymptomatic Leishmania infections in dogs. J Clin Microbiol. 2005;43(11):5515-9.
Otranto D, Paradies P, Sasanelli M, Spinelli R, Brandonisio O. Rapid immunochromatographic test for serodiagnosis of canine leishmaniasis. J Clin Microbiol. 2004;42(6):2769-70.
Otranto D, Paradies P, Sasanelli M, Leone N, de Caprariis D, Chirico J, et al. Recombinant K39 dipstick immunochromatographic test: a new tool for the serodiagnosis of canine leishmaniasis. J Vet Diagn Invest. 2005;17(1):32-7.
Mohebali M, Khamesipour A, Mobedi I, Zarei Z, Hashemi-Fesharki R. Double-blind randomized efficacy field trial of alum precipitated autoclaved Leishmania majorvaccine mixed with BCG against canine visceral leishmaniasis in Meshkin-Shahr district, I.R. Iran. Vaccine. 2004;22(29-30):4097-100.
Reithinger R, Quinnell RJ, Alexander B, Davies CR. Rapid detection of Leishmania infantuminfection in dogs: comparative study using an immunochromatographic dipstick test, enzyme-linked immunosorbent assay, and PCR. J Clin Microbiol. 2002;40(7):2352-6.
Costa MM, Penido M, dos Santos MS, Doro D, de Freitas E, Michalick MS, et al. Improved canine and human visceral leishmaniasis immunodiagnosis using combinations of synthetic peptides in enzyme-linked immunosorbent assay. PLoS Negl Trop Dis. 2012;6(5):e1622.
Pattabhi S, Whittle J, Mohamath R, El-Safi S, Moulton GG, Guderian JA, et al. Design, development and evaluation of rK28-based point-of-care tests for improving rapid diagnosis of visceral leishmaniasis. PLoS Negl Trop Dis. 2010;4(9).
de Paiva Cavalcanti M, Felinto de Brito ME, de Souza WV, de Miranda Gomes Y, Abath FG. The development of a real-time PCR assay for the quantification of Leishmania infantumDNA in canine blood. Vet J. 2009;182(2):356-8.
Bastien P, Procop GW, Reischl U. Quantitative real-time PCR is not more sensitive than "conventional" PCR. J Clin Microbiol. 2008;46(6):1897-900.
Lachaud L, Chabbert E, Dubessay P, Reynes J, Lamothe J, Bastien P. Comparison of various sample preparation methods for PCR diagnosis of visceral leishmaniasis using peripheral blood. J Clin Microbiol. 2001;39(2):613-7.
Fisa R, Riera C, Gallego M, Manubens J, Portus M. Nested PCR for diagnosis of canine leishmaniosis in peripheral blood, lymph node and bone marrow aspirates. Vet Parasitol. 2001;99(2):105-11.
Francino O, Altet L, Sanchez-Robert E, Rodriguez A, Solano-Gallego L, Alberola J, et al. Advantages of real-time PCR assay for diagnosis and monitoring of canine leishmaniosis. Vet Parasitol. 2006;137(3-4):214-21.
Galletti E, Bonilauri P, Bardasi L, Fontana MC, Ramini M, Renzi M, et al. Development of a minor groove binding probe based real-time PCR for the diagnosis and quantification of Leishmania infantumin dog specimens. Res Vet Sci. 2011;91(2):243-5.
Maia C, Ramada J, Cristovao JM, Goncalves L, Campino L. Diagnosis of canine leishmaniasis: conventional and molecular techniques using different tissues. Vet J. 2009;179(1):142-4.
Manna L, Vitale F, Reale S, Caracappa S, Pavone LM, Morte RD, et al. Comparison of different tissue sampling for PCR-based diagnosis and follow-up of canine visceral leishmaniosis. Vet Parasitol. 2004;125(3-4):251-62.
Ferreira Sde A, Almeida GG, Silva Sde O, Vogas GP, Fujiwara RT, de Andrade AS, et al. Nasal, oral and ear swabs for canine visceral leishmaniasis diagnosis: new practical approaches for detection of Leishmania infantumDNA. PLoS Negl Trop Dis. 2013;7(4):e2150.
Belinchon-Lorenzo S, Iniesta V, Parejo JC, Fernandez-Cotrina J, Munoz-Madrid R, Soto M, et al. Detection of Leishmania infantumkinetoplast minicircle DNA by Real Time PCR in hair of dogs with leishmaniosis. Vet Parasitol. 2013;192(1-3):43-50.
Lombardo G, Pennisi MG, Lupo T, Migliazzo A, Capri A, Solano-Gallego L. Detection of Leishmania infantumDNA by real-time PCR in canine oral and conjunctival swabs and comparison with other diagnostic techniques. Vet Parasitol. 2012;184(1):10-7.
Quaresma PF, Murta SM, Ferreira Ede C, da Rocha-Lima AC, Xavier AA, Gontijo CM. Molecular diagnosis of canine visceral leishmaniasis: identification of Leishmania species by PCR-RFLP and quantification of parasite DNA by real-time PCR. Acta Trop. 2009;111(3):289-94.
Andrade HM, de Toledo Vde P, Marques MJ, Franca Silva JC, Tafuri WL, Mayrink W, et al. Leishmania (Leishmania) chagasiis not vertically transmitted in dogs. Vet Parasitol. 2002;103(1-2):71-81.
Ferreira Sde A, Ituassu LT, de Melo MN, de Andrade AS. Evaluation of the conjunctival swab for canine visceral leishmaniasis diagnosis by PCR-hybridization in Minas Gerais State, Brazil. Vet Parasitol. 2008;152(3-4):257-63.
Antinori S, Calattini S, Longhi E, Bestetti G, Piolini R, Magni C, et al. Clinical use of polymerase chain reaction performed on peripheral blood and bone marrow samples for the diagnosis and monitoring of visceral leishmaniasis in HIV-infected and HIV-uninfected patients: a single-center, 8-year experience in Italy and review of the literature. Clin Infect Dis. 2007;44(12):1602-10.
Reale S, Maxia L, Vitale F, Glorioso NS, Caracappa S, Vesco G. Detection of Leishmania infantumin dogs by PCR with lymph node aspirates and blood. J Clin Microbiol. 1999;37(9):2931-5.
Lachaud L, Marchergui-Hammami S, Chabbert E, Dereure J, Dedet JP, Bastien P. Comparison of six PCR methods using peripheral blood for detection of canine visceral leishmaniasis. J Clin Microbiol. 2002;40(1):210-5.
Rodriguez-Cortes A, Ojeda A, Lopez-Fuertes L, Timon M, Altet L, Solano-Gallego L, et al. A long term experimental study of canine visceral leishmaniasis. International journal for parasitology. 2007;37(6):683-93.
Manna L, Gravino AE, Picillo E, Decaro N, Buonavoglia C. LeishmaniaDNA quantification by real-time PCR in naturally infected dogs treated with miltefosine. Ann NY Acad Sci. 2008;1149:358-60.
Manna L, Reale S, Vitale F, Gravino AE. Evidence for a relationship between Leishmaniaload and clinical manifestations. Res Vet Sci. 2009;87(1):76-8.
Maia C, Nunes M, Cristovao J, Campino L. Experimental canine leishmaniasis: clinical, parasitological and serological follow-up. Acta Trop. 2010;116(3):193-9.
Naranjo C, Fondevila D, Altet L, Francino O, Rios J, Roura X, et al. Evaluation of the presence of Leishmaniaspp. by real-time PCR in the lacrimal glands of dogs with leishmaniosis. Vet J. 2011.
Saldarriaga OA, Travi BL, Park W, Perez LE, Melby PC. Immunogenicity of a multicomponent DNA vaccine against visceral leishmaniasis in dogs. Vaccine. 2006;24(11):1928-40.
Reis LE, Coura-Vital W, Roatt BM, Bouillet LE, Ker HG, Fortes de Brito RC, et al. Molecular diagnosis of canine visceral leishmaniasis: a comparative study of three methods using skin and spleen from dogs with natural Leishmania infantuminfection. Vet Parasitol. 2013;197(3-4):498-503.
Leveille R, Partington BP, Biller DS, Miyabayashi T. Complications after ultrasound-guided biopsy of abdominal structures in dogs and cats: 246 cases (1984-1991). Journal of the American Veterinary Medical Association. 1993;203(3):413-5.
Barrouin-Melo SM, Larangeira DF, de Andrade Filho FA, Trigo J, Juliao FS, Franke CR, et al. Can spleen aspirations be safely used for the parasitological diagnosis of canine visceral leishmaniosis? A study on assymptomatic and polysymptomatic animals. Vet J. 2006;171(2):331-9.
Watson AT, Penninck D, Knoll JS, Keating JH, Sutherland-Smith J. Safety and correlation of test results of combined ultrasound-guided fine-needle aspiration and needle core biopsy of the canine spleen. Veterinary radiology & ultrasound : the official journal of the American College of Veterinary Radiology and the International Veterinary Radiology Association. 2011;52(3):317-22.
Carvalho D, Oliveira TMFS, Baldani CD, Machado RZ. An enzyme-linked immunosorbent assay (ELISA) for the detection of IgM antibodies against Leishmania chagasi in dogs. Pesq Vet Bras 2009;29:120-4.
Aoun O, Mary C, Roqueplo C, Marie JL, Terrier O, Levieuge A, et al. Canine leishmaniasis in south-east of France: screening of Leishmania infantumantibodies (western blotting, ELISA) and parasitaemia levels by PCR quantification. Vet Parasitol. 2009;166(1-2):27-31.
de Almeida Ferreira S, Leite RS, Ituassu LT, Almeida GG, Souza DM, Fujiwara RT, et al. Canine skin and conjunctival swab samples for the detection and quantification of Leishmania infantumDNA in an endemic urban area in Brazil. PLoS Negl Trop Dis. 2012;6(4):e1596.
Di Muccio T, Veronesi F, Antognoni MT, Onofri A, Piergili Fioretti D, Gramiccia M. Diagnostic value of conjunctival swab sampling associated with nested PCR for different categories of dogs naturally exposed to Leishmania infantuminfection. J Clin Microbiol. 2012;50(8):2651-9.
Nasereddin A, Ereqat S, Azmi K, Baneth G, Jaffe CL, Abdeen Z. Serological survey with PCR validation for canine visceral leishmaniasis in northern Palestine. J Parasitol. 2006;92(1):178-83.
Strauss-Ayali D, Jaffe CL, Burshtain O, Gonen L, Baneth G. Polymerase chain reaction using noninvasively obtained samples, for the detection of Leishmania infantumDNA in dogs. J Infect Dis. 2004;189(9):1729-33.
Leite RS, Ferreira Sde A, Ituassu LT, de Melo MN, de Andrade AS. PCR diagnosis of visceral leishmaniasis in asymptomatic dogs using conjunctival swab samples. Vet Parasitol. 2010;170(3-4):201-6.
Solano-Gallego L, Rodriguez-Cortes A, Trotta M, Zampieron C, Razia L, Furlanello T, et al. Detection of Leishmania infantumDNA by fret-based real-time PCR in urine from dogs with natural clinical leishmaniosis. Vet Parasitol. 2007;147(3-4):315-9.
Naranjo C, Fondevila D, Altet L, Francino O, Rios J, Roura X, et al. Evaluation of the presence of Leishmaniaspp. by real-time PCR in the lacrimal glands of dogs with leishmaniosis. Vet J. 2012;193(1):168-73.
Manna L, Reale S, Picillo E, Vitale F, Gravino AE. Urine sampling for real-time polymerase chain reaction based diagnosis of canine leishmaniasis. J Vet Diagn Invest. 2008;20(1):64-7.
Rolao N, Cortes S, Rodrigues OR, Campino L. Quantification of Leishmania infantumparasites in tissue biopsies by real-time polymerase chain reaction and polymerase chain reaction-enzyme-linked immunosorbent assay. J Parasitol. 2004;90(5):1150-4.