Diagnosis Of Canine Visceral Leishmaniasis Research Articles

High-resolution mapping of linear epitopes from LiNTPDase2: Advancing leishmaniasis detection using optimized protein and peptide antigens

Visceral Leishmaniasis, caused by Leishmania infantum, is a tropical neglected disease and the most dangerous form of Leishmaniasis. It occurs zoonotically, with domestic transmission posing risks to humans as dogs have high susceptibility and are natural reservoirs of the parasite. Given their epidemiological role, improvements are needed in diagnosing Canine Visceral Leishmaniasis (CVL). Thus, we mapped linear epitopes from the rLiNTPDase2 antigen through peptide microarray and identified six positive epitopes. Validation through peptide ELISA revealed three promising peptides with accuracies of 78.6%, 85.92%, and 79.59%. Their combination yielded 97.58% accuracy. Negative epitopes were also found, which interacted with CVL-negative and Chagas Disease positive samples. Their removal from the rLiNTPDase2 sequence resulted in the rNT2.neg, which obtained enhanced specificity over rLiNTPDase2. The rNT2.neg validation achieved 87.50% sensitivity, 90.55% specificity, and 93.5% accuracy within 127 CVL-positive and 96 CVL-negative samples. Therefore, three peptides and rNT2.neg show significant promise for CVL diagnosis.

Comparison of SensPERT® Leishmania rapid test with two other immunochromatographic tests for the diagnosis of canine visceral leishmaniasis in Brazil.

In urban environments, dogs serve as the primary reservoir for visceral leishmaniasis (VL). Rapidly diagnosing canine VL through tests enables early treatment and a favorable prognosis. This study aimed to assess the diagnostic performance of the SensPERT® Leishmania test kit (Dechra®), Alere® Leishmaniasis Ac test kit, and the rapid test dual path platform (TR-DPP®) Bio-Manguinhos in detecting VL. 30 serum samples from reactive VL dogs and 30 serum samples from healthy dogs were employed for assessing the sensitivity and specificity variation between SensPERT® Leishmania test kit, Alere® Leishmaniasis Ac test kit, and rapid test dual platform - TR-DPP®. The SensPERT® Leishmania test outperformed Alere® and TR-DPP® in terms of sensitivity, specificity, positive and negative predictive values and demonstrated near-perfect concordance with Alere® and substantial concurrence with TR-DPP®. The SensPERT® Leishmania rapid test proved to be a promising test in the detection of VL in dogs.

Synthetic peptides-based SPR biosensor evaluation towards canine Visceral leishmaniasis diagnosis: A simple and effective approach

A vital component of surface plasmon resonance (SPR) biosensors is the recognition element. It generates the sensor selectivity towards the target biomarker and plays an important role in defining the sensor’s analytical sensitivity. In serological diagnostics, recombinant proteins (purified antigens) have presented better selectivity compared to soluble crude antigens in SPR immunosensors. However, recombinant DNA technology to obtain proteins is only explored by specialized laboratories, and commercial recombinant proteins still have a high cost. Thus, with the aim of developing a much simpler and lower-cost method, we investigated the prospect of using synthetic peptides as recognition elements for constructing an SPR biosensor. Two synthetic peptides, named PEP13 and PEP16, were tested for the serological diagnosis of Canine Visceral Leishmaniasis (CVL), an endemic neglected disease that affects humans and dogs worldwide. Between the two peptides tested, PEP13 was more sensitive when evaluating its responses against purified antibodies in buffer solution (LOD = 1.05 nmol L-1), and it also discriminated the response better when applied in diluted serum samples of infected dogs compared to diluted healthy dogs’ samples. For this reason, the PEP13 immunosensor was used to analyze canine serum samples, precisely identifying all the positive (n = 7) and negative (n = 7) CVL cases (p = 0.00136) in less than 12 min. In brief, this study explored promising biostructures through a simple and fast methodology for serological diagnosis, addressing the suitability of synthetic peptides for use in biosensors in the urgent field of neglected diseases.

Synthetic Peptides Selected by Immunoinformatics as Potential Tools for the Specific Diagnosis of Canine Visceral Leishmaniasis.

Diagnosing canine visceral leishmaniasis (CVL) in Brazil faces challenges due to the limitations regarding the sensitivity and specificity of the current diagnostic protocol. Therefore, it is urgent to map new antigens or enhance the existing ones for future diagnostic techniques. Immunoinformatic tools are promising in the identification of new potential epitopes or antigen candidates. In this study, we evaluated peptides selected by epitope prediction for CVL serodiagnosis in ELISA assays. Ten B-cell epitopes were immunogenic in silico, but two peptides (peptides No. 45 and No. 48) showed the best performance in vitro. The selected peptides, both individually and in combination, were highly diagnostically accurate, with sensitivities ranging from 86.4% to 100% and with a specificity of approximately 90%. We observed that the combination of peptides showed better performance when compared to peptide alone, by detecting all asymptomatic dogs, showing lower cross-reactivity in sera from dogs with other canine infections, and did not detect vaccinated animals. Moreover, our data indicate the potential use of immunoinformatic tools associated with ELISA assays for the selection and evaluation of potential new targets, such as peptides, applied to the diagnosis of CVL.

Visceral leishmaniasis seroprevalence and associated sociocultural factors in an endemic area of Sergipe, northeastern Brazil

The present study aimed to investigate the presence of anti-Leishmania antibodies in samples of domiciled dogs and associate them with epidemiological indicators in the city of Lagarto, Sergipe. The diagnosis of Canine Visceral Leishmaniasis (CVL) was carried out in 755 dogs, where the rapid two-way platform immunochromatographic test (TR-DPP) (Fiocruz/Biomanguinhos) and the serological test of Enzyme-Linked Immunosorbent Assay (ELISA) were applied. The results showed that all homes with seroreactive dogs were close to abandoned land, squares and/or green areas. There was a significant association between seroactive animals and type of shelter (p=0.008); 54.8% had an indoor shelter and 45.2% of the dogs had a shelter outside their homes. The disease is geographically distributed in 14 (73.68%) of the 19 sample areas in the municipality. Of these, five (26.31%) had cases of Visceral Leishmaniasis in dogs and humans. Eight cases of Human Visceral Leishmaniasis (HVL) were reported during the years 2017 to 2020 in the municipality. Dogs that are located in areas of greatest risk impact the maintenance of natural foci of Leishmaniasis transmission to human and animal hosts in areas endemic for Visceral Leishmaniasis. Such results are essential to assist epidemiological surveillance in the municipality of Lagarto-SE with the implementation of control measures aimed at the insect vector of the disease, the sand fly, to prevent human and canine cases.

Efficiency of rLb6H recombinant protein from Leishmania (Viannia) braziliensis for the detection of canine visceral leishmaniasis

Introduction: Visceral leishmaniasis (VL) is a serious endemic disease in many tropical and subtropical countries, with a strong incidence in Brazil. The disease is transmitted by the bite of infected female phlebotomine sandflies, with dogs being the main urban reservoirs of the parasite. The diverse clinical profile and the long incubation period are challenges for the diagnosis of canine visceral leishmaniasis (CVL). Recombinant proteins from Leishmania spp. have been studied as antigens that can increase the accuracy of serological tests. Objective: To evaluate the diagnostic performance of the recombinant protein rLb6H, from Leishmania braziliensis, in comparison to the reference antigens rK39 and rK28, from L. donovani, prioritizing the identification of subclinical infected dogs. Material and Methods: Serum IgG reactivity to rLb6H, rK28, and rK39 recombinant proteins was assessed in dogs with previously parasitological confirmation of CVL, subdivided according to their clinical status, using immunoenzymatic assay (ELISA). Diagnostic accuracy of each ELISA was evaluated by receiver operating characteristic (ROC) curve analysis. Results: While all antigens showed a better performance in detecting CVL in symptomatic dogs (SD), detection of CVL in the oligosymptomatic (OD) and asymptomatic (AD) groups was lower, but rLb6H achieved high sensitivity for asymptomatic CVL. Interestingly, the most reactive CVL samples to rK28 were barely detected by rLb6H, while the less reactive to rK28, mostly from the AD group, presented higher reactivity to rLb6H. Conclusion: The recombinant protein rLb6H showed utility in the detection of asymptomatic CVL, displaying a complementary reactivity to rK39 and rK28. Thus, these results suggest that rLb6H could be incorporated into multi-antigen strategies, to increase diagnostic accuracy of CVL.

Evaluación clínica y resultados falsos negativos para leishmaniasis visceral canina: Revisión

Canine Visceral Leishmaniasis (CVL) is one of the forms of the disease caused by the protozoan of the genus Leishmania spp., being classified as one of the main zoonoses of public health interest. As we still present diagnostic methods with low sensitivity to the causative agent of CVL, this literature review aimed to identify laboratory tests and methods that contribute to a correct diagnosis, reducing the chances of false negatives or false positives in the Veterinarian’s routine. Through this literature review, it was possible to observe that the PCR test proved to be 100% reliable in the evaluated studies, whereas the parasitological technique, classified as the gold standard, will depend on certain factors, such as precision, the intensity of the parasitism, the nature of the biological material obtained, the duration of the slide analysis and the expertise of the specialists involved, for the diagnosis of CVL.

Diagnostic Potential for the Detection of Canine Visceral Leishmaniasis of an ELISA Assay Based on the Q5 Recombinant Protein: A Large-Scale and Comparative Evaluation Using Canine Sera with a Positive Diagnosis from the Dual-Path-Platform (DPP) Test.

Dogs are considered the major domestic reservoir for human visceral leishmaniasis, a serious disease caused by the Leishmania infantum parasite. Diagnosis of canine visceral leishmaniasis (CVL) is critical for disease control, with several methods currently available. Among the serological tests, the DPP rapid test and the EIE-LVC, more commonly used in Brazil, are associated with variable sensitivity and specificity. Research with novel recombinant proteins such as the ELISA with the recombinant chimeric protein Q5 may therefore improve the CVL diagnosis. This study aimed to evaluate the true diagnostic potential of Q5 in an ELISA assay using a large number of CVL-suspected sera (406) with a previous positive diagnosis based on the rapid DPP test. Sera from the DPP-positive dogs, also assessed with the EIE-LVC test, were compared with sera from healthy dogs (n = 46) and used for ELISA tests using the recombinant Q5. The resulting data as well as the correlation with the clinical signs and the environmental characteristics of the animals were analyzed using Medal and GraphPad Prism 8.0. Overall, similar levels of lower sensitivity (67-68%) were seen for both the commercial EIE-LVC test and the Q5 ELISA when all assessed sera were considered, but a much greater sensitivity (92%) was seen for those samples from symptomatic dogs only. In contrast, many negative results were observed for the DPP-positive sera from asymptomatic dogs or those with no clinical information available. A selection of those sera were tested yet again in new ELISA assays using a second batch of the recombinant Q5, purified under milder denaturing conditions, as well as using another recombinant protein (Lci13). The results reveal a higher-than-expected incidence of likely false-positive results for DPP, reinforcing the need for other recombinant proteins, such as the chimeric Q5, to be investigated as possible alternatives to the currently used CVL diagnostic methods.

Canine visceral leishmaniasis diagnosis by UV spectroscopy of blood serum and machine learning algorithms

Visceral leishmaniasis (VL) is a zoonotic disease caused by the protozoan Leishmania infantum, and dogs are considered the main urban hosts for future disease transmission. The first and most effective control against the spread of disease relies on identifying infected animals, followed by their treatment or sacrifice, to reduce the protozoan reservoirs. Despite the availability of various diagnostic tests for VL in dogs the development of a quick and accurate diagnosis is essential from a public health and ethical point of view. Here we analyze the use of UV–Vis spectroscopy as an alternative diagnostic method for VL diagnosis by using the antigen-antibody interaction in canine blood serum and machine learning algorithms. The main UV spectra in the 220 to 280 nm range exhibit nine electronic absorption bands, but no significative difference could be identified between the positive and negative group spectra. Finally, UV pre-proceed spectra by SNV (standard normal variate) were submitted to principal component analysis followed by Linear SVM algorithm, the prediction model was tested in a leave-one-out cross-validation and external validation test reaching an overall accuracy of 75%.

Immunoproteomics approach for the discovery of antigens applied to the diagnosis of canine visceral leishmaniasis

In the present study, an immunoproteomic approach using Leishmania infantum parasites isolated from naturally infected dogs from an endemic region of the disease, was carried out to identify new antigens to be used in the diagnosis of canine visceral leishmaniasis (CVL). Protein extracts, obtained from parasites isolated from asymptomatic (CanLA) and symptomatic (CanLS) dogs, were used to perform the two-dimensional gels. Western Blotting assays were carried out by employing a pool of sera from dogs with visceral leishmaniasis (CanLA or CanLS), healthy dogs from an endemic area, or dogs with similar diseases associated with cross-reactions (babesiosis and ehrlichiosis). With these results, it was possible to exclude the spots that showed a cross-reactivity of the sera from groups of healthy dogs, and those with babesiosis or ehrlichiosis. Taken together, 20 proteins were identified, 15 of which have already been described in the literature and 5 of which are hypothetical. An immunogenomic screen strategy was applied to identify conserved linear B-cell epitopes in the identified hypothetical proteins. Two peptides were synthesized and tested in ELISA experiments as a proof of concept for the validation of our immunoproteomics findings. The results demonstrated that the antigens presented sensitivity and specificity values ranging from 81.93% to 97.59% and 78.14 to 85.12%, respectively. As a comparative antigen, a preparation of a Leishmania extract showed sensitivity and specificity values of 75.90% and 74.88%, respectively. The present study was able to identify proteins capable of being used for the serodiagnosis of canine visceral leishmaniasis.

Validation of an immunochromatographic assay kit based on colored latex particles for the identification of the canine visceral leishmaniasis

Visceral leishmaniasis is a zoonotic infectious disease with a severe impact on humans and animals. Infection is transmitted by phlebotomine sand flies. The dogs are main reservoir for human infection. A rapid and accurate diagnosis of canine visceral leishmaniasis is essential for an efficient surveillance program. The aim of this study was to assess the performance of a rapid immunochromatographic strip test based on functionalized colored particles and a new recombinant antigenic protein, as a visual “in situ” method for the diagnosis of canine visceral leishmaniasis. The results were evaluated using an in-house ELISA assay with the same antigen. Both tests produced concordant results and the immunochromatographic strip test showed good diagnostic sensitivity (98%) and specificity (95%). Finally, meta-analysis was used to compare the sensitivity and specificity of the here developed test with the results of commercial immunochromatographic strip tests obtained from literature.

Sensitivity of PCR in conjunctival swab samples for the diagnosis of canine visceral leishmaniasis: a systematic review and meta-analysis.

To compare the sensitivity of conjunctival swab (CS) and conventional samples (blood, spleen, liver, lymphoid and cutaneous tissue) in the diagnosis of canine visceral leishmaniasis (CVL) by polymerase chain reaction (PCR), a systematic review and meta-analysis was carried out using PubMed, Science Direct, Scopus, Web of Science, VHL/BVS (Virtual Health Library), CAPES, and Scielo databases. Articles published from 2002 to 2022 were considered and the review was updated in Jul 2023. From the total of 371 identified studies, 8 met all the eligibility criteria and were included in this review. Data from 658 CVL-positive dogs and 2541 PCR results were considered. Using a random effect model, data on the sensitivity of the test was compared between intervention (CS samples) and comparison (all the other samples) groups. Overall, the use of CS in the PCR diagnosis of CVL produced 12% higher sensitivity (p=0.013) in the test than all the other samples in combination. The animals' clinical condition did not influence (p>0.142) this overall result. However, when CS was individually compared to each of the conventional samples, the consistent result was observed (p=0.012) only in the CS versus bone marrow comparison. Given their rapid acquisition, minimal invasiveness, and lower cost relative to conventional samples, CS samples present a promising alternative for the molecular diagnosis of CVL.

Development of Immunological Assays Based on Leishmania donovani Antigen for Diagnosis of Canine Visceral Leishmaniasis and Their Multicenter Evaluation in Brazil and Italy.

Canine visceral leishmaniasis (CVL) due to Leishmania infantum infection is a zoonotic disease prevalent in the areas of South America and the Mediterranean. Infected dogs as reservoirs can contribute to disease transmission and can be a scourge to public health. Therefore, early diagnosis of infected dogs may play a pivotal role in circumscribing disease progression. Invasive tissue aspiration and insufficient serological methods impair a single assay for prompt CVL diagnosis. In the present study, we aimed to evaluate the potential of Leishmania donovani isolated membrane protein, LAg, for the diagnosis of CVL through immunological assays. Initially, enzyme-linked immunosorbent assay was done with Brazilian dog sera to evaluate the performance of LAg in diagnosing CVL and found sensitivity and specificity of 92.50% and 95%, respectively. The study further confirmed the diagnostic efficacy of LAg in a dipstick format. The dipstick test of canine sera from three centers in Brazil and one center in Italy collectively showed sensitivity values in the range of 53.33% to 100% in recognizing symptomatic dogs and specificity values between 75% and 100% to rule out healthy dogs. Moreover, a rapid immunochromatographic test was developed and optimized using LAg. This test was able to identify 94.73% of CVL of Brazilian origin with specificity of 97.29%. The current results highlight the reactive potential of the L. donovani antigen, LAg, for L. infantum CVL diagnosis and support our previous findings, which suggest the utility of LAg for the diagnosis of both L. donovani and L. infantum human VL in a variety of endemic regions. LAg as a diagnostic candidate may be employed to identify comprehensive CVL cases in epidemiological areas.

Humoral and Cellular Immune Response in Asymptomatic Dogs with Visceral Leishmaniasis: A Review.

Visceral leishmaniasis is one of the deadliest parasitic diseases in the world and affects both humans and dogs. The host immune response to Leishmania infection plays a critical role in the evolution of canine visceral leishmaniasis (CVL) and consequently in the manifestation of clinical signs. The asymptomatic form of the disease is a major concern in the diagnosis of CVL and in the transmission control of Leishmania infection. Asymptomatic dogs are found in large proportions in endemic areas and are an unquantifiable source of infection. The present review analyzes the possible relationship between the activation of the antigen-specific immune response of the host and resistance or susceptibility to CVL. The review focuses on works that address the characterization of the humoral and cellular immune response profile, at both the functional and phenotypic levels, in infected dogs. Most studies relate the absence of clinical symptomatology to an increased proliferative response and a Th1 cytokine profile. Despite the numerous findings pointing to a differential immune response in asymptomatic dogs, the contradictory results reported in this review highlight the importance of establishing a precise clinical classification of the disease, performing more longitudinal studies, and including a higher number of animals in trials.

Leishmaniasis visceral canina como diagnóstico diferencial a la hemoparasitosis transmitida por garrapatas: reporte de un caso

Canine Visceral Leishmaniasis (CVL) can serve as a differential diagnosis for Canine Ehrlichiosis and Canine Babesiosis, tick-borne hemoparasitosis, given the similarity of clinical manifestations and the urbanization of the CVL vector. In this case report, we point out the importance of having the region where the dog lives in its anamnesis. The treated dog came from the region of Itu-SP and presented only unilateral epistaxis as a clinical manifestation, plus non-regenerative anemia, lymphadenomegaly, and splenomegaly. The first suspicion was infection with Babesia or Ehrlichia canis. The lack of successful treatment and having information about the dog's origin led to the correct CVL diagnosis. The canine transmission has been expanding among municipalities in São Paulo macroregion, and the transmitting mosquito has been recorded in the municipality of Itu.

Bayesian latent class models for identifying canine visceral leishmaniosis using diagnostic tests in the absence of a gold standard.

Like many infectious diseases, there is no practical gold standard for diagnosing clinical visceral leishmaniasis (VL). Latent class modeling has been proposed to estimate a latent gold standard for identifying disease. These proposed models for VL have leveraged information from diagnostic tests with dichotomous serological and PCR assays, but have not employed continuous diagnostic test information. In this paper, we employ Bayesian latent class models to improve the identification of canine visceral leishmaniasis using the dichotomous PCR assay and the Dual Path Platform (DPP) serology test. The DPP test has historically been used as a dichotomous assay, but can also yield numerical information via the DPP reader. Using data collected from a cohort of hunting dogs across the United States, which were identified as having either negative or symptomatic disease, we evaluate the impact of including numerical DPP reader information as a proxy for immune response. We find that inclusion of DPP reader information allows us to illustrate changes in immune response as a function of age. Utilization of continuous DPP reader information can improve the correct discrimination between individuals that are negative for disease and those with clinical VL. These models provide a promising avenue for diagnostic testing in contexts with multiple, imperfect diagnostic tests. Specifically, they can easily be applied to human visceral leishmaniasis when diagnostic test results are available. Also, appropriate diagnosis of canine visceral leishmaniasis has important consequences for curtailing spread of disease to humans.

Avaliações histológica e imunoistoquímica da medula óssea do fêmur e do manúbrio esternal de cães reagentes para leishmaniose aos testes DPP® e ELISA

Abstract As the bone marrow is one of the most organs affected by canine visceral leishmaniasis (CVL), samples from this are frequently taken for parasitological tests, with occurrence of myelodysplastic changes, with consequent anemia, leukopenia, and thrombocytopenia. Thus, this study aimed to investigate the histological and immunohistochemical changes in the bone marrow of the femur and sternal manubrium of dogs reactive for leishmaniasis by DPP® and ELISA tests. For this, thirteen canines from the epidemiological routine for CVL carried out by the Directorate of Zoonosis Surveillance of Goiânia (DVZ), GO, Brazil, were subjected to anatomopathological examination. 46.2% of bone marrow samples from the femur showed a higher proportion of the red series, and 53.9% of bone marrow of the sternal manubrium evidenced a higher proportion of the red series. Also, there were varied macrophage hyperplasia, hemosiderosis, and megakaryocytic emperipolesis. Amastigote forms of Leishmania spp. in the bone marrow of the femur and sternal manubrium to histopathological and immunohistochemical evaluations were observed, with good agreement them, but without difference in the parasite intensity between the bone marrow of these anatomical sites. It was concluded that bone marrow of the femur and sternal manubrium of dogs reactive for leishmaniasis by DPP® and ELISA tests has histological changes resulting from the disease, regardless of the parasite presence or intensity, with macrophage hyperplasia, hemosiderosis, and emperipolesis being the main medullary changes in these animals. Also, the bone marrow of the femur and sternal manubrium are useful anatomical sites for the diagnosis of CVL by direct methods.

Assessment of a New Antigen Detection Test for the Diagnosis of Canine Visceral Leishmaniasis.

Canine visceral leishmaniasis (CVL) is a serious zoonotic disease in Brazil and Southern Europe. CVL is primarily caused by Leishmania infantum and its diagnosis relies largely on detection of parasites in bone marrow or lymph node aspirates by microscopic observation of the parasites in stained smears, parasite culture, or polymerase chain reaction (PCR). Serological tests exist but they do not distinguish active disease from simple exposure to parasite antigens. Here, we have assessed the utility of a new monoclonal antibody--based antigen (protein) detection test for the diagnosis of CVL. The test was positive in 70% of beagle dogs experimentally infected with L. infantum. In contrast, culture of the parasites from bone marrow aspirates was positive in only 40% of the infected animals. These preliminary results suggest that this antigen detection test, which we have recently described for the diagnosis of human VL, has the potential to be a useful diagnostic tool for CVL.

Potential of recombinant LiHyQ, a novel Leishmania infantum protein, for the diagnosis of canine visceral leishmaniasis and as a diagnostic and prognostic marker for human leishmaniasis and human immunodeficiency virus co-infection: A preliminary study

Laboratory diagnosis of leishmaniasis shows variable efficacy in detecting infected mammalian hosts and there is a need to identify suitable antigens to improve the accuracy of diagnostic tests. In the present study, a L. infantum hypothetical protein called LiHyQ was evaluated for the diagnosis of tegumentary (TL) and visceral (VL) leishmaniasis using canine and human samples. A collection of dog sera (n=155) were tested and contained samples from asymptomatic (n=20) and symptomatic (n=25) VL animals, from healthy dogs living in endemic (n=25) or non-endemic (n=25) areas of disease, from Leish-Tec® vaccinated dogs (n=20) or from dogs infected with Ehrlichia canis (n=15), Babesia canis (n=10) and Trypanosoma cruzi (n=15). Sensitivity (Se), Specificity (Sp), Positive Predictive Value (PPV) and Negative Predictive Value (NPV) of 100% were observed for rLiHyQ with these samples, whereas the Se, Sp, PPV and NPV values with L. infantum Soluble Leishmania Antigen (SLA) preparation were 60.0%, 99.0%, 96.0% and 86.0%, respectively. A collection of human sera (n=305) were tested and contained samples from TL (n=50) and VL (n=40) patients, from VL/HIV co-infected patients (n=35), from patients infected with HIV alone (n=30), Chagas Disease (n=30), malaria (n=10), tuberculosis (n=10), paracoccidioidomycosis (n=15), leprosy (n=30) or aspergillosis (n=15); and from healthy subjects (n=40). Se, Sp, PPV and NPV values of 100% were observed for rLiHyQ with these samples, whereas the Se, Sp, PPV and NPV values with SLA were 58.0%, 76.0%, 50.0% and 82.0%, respectively. The antibody reactivity against the protein was compared with commercial kits, and the kappa index varied from 0.95 to 1.00 for rLiHyQ, and of 0.55 to 0.82 for the kits. In addition, the serological follow-up of treated patients showed a significant reduction in rLiHyQ-specific IgG antibody levels. All canine and human samples were tested at the same time using the same reagents, in order to reduce experimental variation and interference in data interpretation. In conclusion, our preliminary data suggest a diagnostic and prognostic role for rLiHyQ against leishmaniasis.

A new strategy for canine visceral leishmaniasis diagnosis based on FTIR spectroscopy and machine learning

Visceral leishmaniasis is a neglected disease caused by protozoan parasites of the genus Leishmania. The successful control of the disease depends on its accurate and early diagnosis, which is usually made by combining clinical symptoms with laboratory tests such as serological, parasitological, and molecular tests. However, early diagnosis based on serological tests may exhibit low accuracy due to lack of specificity caused by cross-reactivities with other pathogens, and sensitivity issues related, among other reasons, to disease stage, leading to misdiagnosis. In this study was investigated the use of mid-infrared spectroscopy and multivariate analysis to perform a fast, accurate, and easy canine visceral leishmaniasis diagnosis. Canine blood sera of 20 noninfected, 20 Leishmania infantum, and eight Trypanosoma evansi infected dogs were studied. The data demonstrate that principal component analysis with machine learning algorithms achieved an overall accuracy above 85% in the diagnosis.