Evaluation of three recombinant proteins for the development of ELISA and immunochromatographic tests for visceral leishmaniasis serodiagnosis.

BACKGROUND: Visceral Leishmaniasis (VL) is an infectious disease that is a significant cause of death among infants aged under 1 year and the elderly in Brazil. Serodiagnosis is a mainstay of VL elimination programs; however, it has significant limitations due to low accuracy. OBJECTIVE: This study aimed to evaluate three recombinant Leishmania infantum proteins (rFc, rC9, and rA2) selected from previous proteomics and genomics analyses to develop enzyme-linked immunosorbent assay (ELISA) and immunochromatographic tests (ICT) for the serodiagnosis of human VL (HVL) and canine VL (CVL). METHODS: A total of 186 human (70 L. infantum-infected symptomatic, 20 other disease-infected, and 96 healthy) and 185 canine (82 L. infantum-infected symptomatic, 27 L. infantum-infected asymptomatic, and 76 healthy) sera samples were used for antibody detection. FINDINGS: Of the three proteins, rA2 (91.5% sensitivity and 87% specificity) and rC9 (95.7% sensitivity and 87.5% specificity) displayed the best performance in ELISA-HVL and ELISA-CVL, respectively. ICT-rA2 also displayed the best performance for HVL diagnosis (92.3% sensitivity and 88.0% specificity) and had high concordance with immunofluorescence antibody tests (IFAT), ELISA-rK39, IT-LEISH®, and ELISAEXT. ICT-rFc, ICT-rC9, and ICT-rA2 had sensitivities of 88.6%, 86.5%, and 87.0%, respectively, with specificity values of 84.0%, 92.0%, and 100%, respectively for CVL diagnosis. MAIN CONCLUSIONS: The three antigens selected by us are promising candidates for VL diagnosis regardless of the test format, although the antigen combinations and test parameters may warrant further optimisation.

Immunogenomic screening approach to identify new antigens for the serological diagnosis of chronic Chagas' disease.

Serological tests are preferentially used for the diagnosis of Chagas' disease (CD) during the chronic phase because of the low parasitemia and high anti-Trypanosoma cruzi antibody titers. However, the current methods showed several disadvantages, as contradictory or inconclusive results, mainly related to the characteristics of the antigens used, in general, crude or whole parasites, but also due to antigen production protocol and the experimental conditions used in serological tests. Thus, better-quality serological assays are urgently needed. Here, we performed a wide immunogenomic screen strategy to identify conserved linear B-cell epitopes in the predicted proteome based on genome sequence from T. cruzi strains to will be applied as synthetic peptides in the serodiagnosis of the chronic CD. Three B-cell epitopes derived from mucin-associated surface protein (MASP) family, expressed in both infective parasite stages, trypomastigote and amastigotes, conserved in T. cruzi strains, and highly divergent as compared with Leishmania spp. proteome, were selected for this study. The results demonstrated that synthetic peptide 2 and a mixture of peptides (Mix II: peptides 2 and 3) were able to identify all chronic CD cases, indeterminate or Chagas cardiomyopathy clinical presentation, and simultaneously able to discriminate infections caused by Leishmania parasites, with high accuracy (98.37 and 100.00%, respectively) and agreement (kappa index = 0.967 and 1.000, respectively) with direct methods as compared to current diagnostic pipeline performed by reference laboratories in Brazil. This study represents an interesting strategy for the discovery of new antigens applied to serologic diagnosis of infectious diseases and for the technological development of platforms for large-scale production of diagnostic tests.

Validation of the Dual-path Platform chromatographic immunoassay (DPP® CVL rapid test) for the serodiagnosis of canine visceral leishmaniasis.

BACKGROUND Visceral leishmaniasis is a major public health challenge in South America, and dogs are its main urban reservoir. OBJECTIVE Validation of the canine Dual-path Platform immunoassay for canine visceral leishmaniasis (DPP® CVL) for a sample set composed of 1446 dogs from different Brazilian endemic areas. METHODS A well-defined reference standard by means of parasitological culture, immunohistochemistry, and histopathology was used. Animals were classified as asymptomatic, oligosymptomatic, or symptomatic. Sensitivity and specificity were assessed as a single set and in clinical groups. A reproducibility assessment of the tests was conducted using the Kappa (κ) index at three different laboratories (A, B, and C). FINDINGS Overall, 89% sensitivity and 70% specificity were obtained for the entire sample set. Analysis of the clinical groups showed a gradual decrease in the sensitivity and an increase in the specificity with the reduction of clinical signs in the dogs that were assessed, reaching a sensitivity of 75% (42.8-94.5%) among asymptomatic dogs and lower specificity of 56% (46.2-66.3%) among symptomatic dogs. Inter-laboratory agreement was substantial (κAB= 0.778; κAC= 0.645; κCB= 0.711). MAIN CONCLUSIONS The test performance is somewhat dependent on canine symptomatology, but such influence was less evident than in previous studies. Favourable results for sensitivity and specificity can be obtained even in asymptomatic animals; however, caution is needed in these evaluations, and the results suggest that the immunochromatographic test may be further improved for better investigation in asymptomatic dogs. The results obtained confirm the usefulness of DPP® CVL for application in serological surveys.

Sensitivity and specificity of parallel or serial serological testing for detection of canine Leishmania infection.

In Brazil, human and canine visceral leishmaniasis (CVL) caused by Leishmania infantum has undergone urbanisation since 1980, constituting a public health problem, and serological tests are tools of choice for identifying infected dogs. Until recently, the Brazilian zoonoses control program recommended enzyme-linked immunosorbent assays (ELISA) and indirect immunofluorescence assays (IFA) as the screening and confirmatory methods, respectively, for the detection of canine infection. The purpose of this study was to estimate the accuracy of ELISA and IFA in parallel or serial combinations. The reference standard comprised the results of direct visualisation of parasites in histological sections, immunohistochemical test, or isolation of the parasite in culture. Samples from 98 cases and 1,327 noncases were included. Individually, both tests presented sensitivity of 91.8% and 90.8%, and specificity of 83.4 and 53.4%, for the ELISA and IFA, respectively. When tests were used in parallel combination, sensitivity attained 99.2%, while specificity dropped to 44.8%. When used in serial combination (ELISA followed by IFA), decreased sensitivity (83.3%) and increased specificity (92.5%) were observed. Serial testing approach improved specificity with moderate loss in sensitivity. This strategy could partially fulfill the needs of public health and dog owners for a more accurate diagnosis of CVL.

Correlations between tissue parasite load and common clinical signs in dogs naturally infected by Leishmania infantum.

qPCR is being used for the quantification of parasite load in different tissues of dogs infected by Leishmania infantum with or without clinical manifestations. It may be employed in the diagnosis, monitoring of the infection during treatment, and clinical studies for validation of vaccines. Aimed at enhancing the molecular diagnosis and the subsequent monitoring of the infection, this study evaluated the parasite load in several tissues from dogs infected by Leishmania infantum, showing different clinical status. Thus, the qPCR was performed on skin, conjunctival swab, popliteal lymph node, and bone marrow puncture samples taken from 65 dogs naturally infected by L. infantum. Dogs were divided into three groups per clinical score: group 1 (n = 12), included animals with zero points and no clinical manifestations of the disease; group 2 (n = 35), included animals with a score ranging from 1 to 5 points and moderate clinical manifestations; and group 3 (n = 18), included dogs with a score ranging from 6 to 11 points and intensive clinical manifestations. Another analysis was performed classifying the animals into two groups, considering the presence of, or lack of clinical signs of the disease. Analyses of these results showed that the skin was the tissue with a higher parasite load, followed by popliteal lymph node and bone marrow punctures, and conjunctival swab samples having the lowest loads. Furthermore, the skin was also the tissue with the highest parasite load when evaluating the groups individually. Animals in group 3, with intensive clinical manifestations, showed a higher parasite load in different tissues when compared to animals from groups 1 and 2. Finally, animals with clinical manifestations of the disease showed a higher parasite load when compared to dogs with no manifestations. The importance of the dog as a reservoir of L. infantum in nature is reinforced by the demonstration of skin having the highest amount of parasites/µL in this study's analysis, as well as the fact that skin is the main point of access to the parasite vector. Also, a strong and positive correlation between the intensity of clinical manifestations and the increase of parasite load in the skin was observed. In conclusion, skin was the tissue that was demonstrated to be the best option for the molecular diagnosis of L. infantum infection in dogs with varying clinical statuses used in this study.

Phase II validation study of the rK39 ELISA prototype for the diagnosis of canine visceral leishmaniasis in Brazil.

Dogs are the main reservoirs in the domestic transmission cycle of visceral leishmaniasis, and the diagnosis is essential for the effectiveness of the control measures recommended by the Brazilian Ministry of Health. We assessed the diagnostic performance of the ELISA-Vetlisa/BIOCLIN prototype with serum samples from 200 dogs, in triplicate, including symptomatic, oligosymptomatic, asymptomatic, and healthy dogs, originated by two distinct panels (A and B) characterized by parasitological tests as the reference standard. In this study, the prototype kit showed a 99% sensitivity (95%CI: 94.5-100.0) and a 100% specificity (95%CI: 96.4-100.0). The sensitivity of the prototype kit did not vary significantly with the clinical status of the dogs. Considering the final result classification (positive or negative), agreement between the results of repeated tests was almost perfect (kappa = 0.99; 95%CI: 0.98-1.00). ELISA-Vetlisa/BIOCLIN is a promising option for the serological diagnosis of canine visceral leishmaniasis in Brazil.

Comparative PCR-based diagnosis for the detection of Leishmania infantum in naturally infected dogs.

The applicability of molecular biology/PCR for canine visceral leishmaniasis diagnosis presents challenges, mainly due to the diversity of targets described. The objectives of this study were to compare the sensitivities and reliability of five targets (kDNA/120, kDNA/145, ITS1, hsp70/234 and hsp70/1300) in four different tissue samples (bone marrow, popliteal lymph node, skin and conjunctival swab). Sixty-five dogs (32 males and 33 females) naturally infected with Leishmania infantum and ten dogs without infection were examined. Dogs were characterized by serological and parasitological methods. The parasitological test was considered the gold standard for analysis. All tests presented high specificity 100% (95% CI 0.72-1), and variable sensitivity. The targets kDNA/145, ITS1, kDNA/120, hsp70/234 and hsp70/1300 detected 100% (65/65), 93.4% (61/65), 92.3% (60/65), 84.61% (55/65) and 72.3% (77/65) of positive animals respectively. The performance of PCR methods was analyzed in two different scenarios. The highest sensitivity value identified in all scenarios studied was kDNA/145. Our results suggest that popliteal lymph node and conjunctival swab samples, besides being less invasive collections, represent a good substratum for PCR-based diagnosis, and the target kDNA/145 is the best choice for detecting L. infantum DNA in naturally infected dogs.

Evidence of a sylvatic enzootic cycle of Leishmania infantum in the State of Amapá, Brazil.

INTRODUCTION: Leishmania infantum was considered to be absent from Amapá until 2017 when canine infection was detected. However, there is a lack of knowledge about which reservoir species are involved in transmission in this region. METHODS: Between 2014 and 2016, 86 samples from wild mammals and 74 from domestic dogs were collected in Wajãpi Indigenous Territory and were tested for the presence of deoxyribonucleic acid (DNA) of Leishmania. RESULTS: The DNA of Le. infantum was detected in two rodent samples, Dasyprocta sp. and Proechimys cuvieri. CONCLUSIONS: This is the first evidence characterizing a sylvatic transmission cycle of Le. infantum in the State of Amapá.

Performance of different serological tests in the diagnosis of natural infection by Leishmania infantum in dogs.

Visceral leishmaniasis (VL) is a zoonosis caused by the parasite Leishmania infantum and the dog is its main reservoir in rural and urban areas. The diagnosis of infection is mainly based on the presence of anti-Leishmania IgG antibodies in the serum of infected dogs. In this study, the sensitivity and specificity of qualitative rapid tests (RTs) dual path platform (DPP) Bio-Manguinhos, rapid enzyme-linked immunosorbent assay (ELISA) IDEXX, Kalazar Detect and ALERE, as well as quantitative ELISA Bio-Manguinhos and in-house indirect immunofluorescence assay (IFA) tests were analyzed in sera from infected and uninfected dogs. Serial dilutions of the in-house IFA were compared with RTs and ELISA Bio-Manguinhos. The results showed that none of the tests reached 100% sensitivity and specificity. There was no statistical difference between the analyzed RTs. The most sensitive test was the DPP Bio-Manguinhos (97.9%), while the rapid ELISA IDEXX showed higher specificity (100%). In the treatment setting of infected and/or diseased animals, quantitative tests for monitoring the evolution of antibody titers are required, which indicates the maintenance of in-house IFA in animal handling. Furthermore, we demonstrate that the RTs present higher sensitivity in serum samples with superior antibody titers obtained in the in-house IFA. However, the RTs exhibited false negatives in samples with low titers of antibodies. Among the RTs, only the DPP Bio-Manguinhos presented better performance in this situation. Therefore, the use of RTs for the diagnosis of VL in dogs with low titers of antibodies, such as asymptomatic, should be carefully evaluated.

Reliability of techniques used in the diagnosis of canine visceral leishmaniasis by the national control program in Brazil: A survey in an area of recent transmission.

One of the key components of the Brazilian Program for the Control of Visceral Leishmaniasis (PCLV) is the euthanasia of Leishmania-infected canine reservoirs, the detection of which depends on a screening procedure involving a Dual Path Platform® (DPP) immunoassay and a confirmatory enzyme-linked immunosorbent assay (ELISA). The aims of the present study were to evaluate the reliability of these techniques in a region of recent transmission of canine VL, to follow up the seroconversion 3-4 months after the initial diagnosis of DPP reactive but ELISA indeterminate or non-reactive dogs, and to identify the species of Leishmania in circulation in the area. Each animal was submitted to DPP under field conditions, performed by municipal health workers using peripheral blood (DPP-field), to DPP under laboratory conditions using serum (DPP-lab) and to ELISA using serum. The agreements between the tests were determined using McNemar's χ2 test, Cohen's kappa coefficient (k) at the 95% confidence interval and prevalence-adjusted bias-adjusted kappa (PABAK). Of the 1130 dogs examined, 74.2% were non-reactive in all three tests applied. Based on the PCLV positive-infection criterion, seroprevalence was 8.9% (101/1130) with 83.2% (84/101) of infected animals showing reactivity in all three tests while 7.8% (8/101) were reactive in DPP-field and ELISA and 8.9% (9/101) in DPP-lab and ELISA. The proportions of disagreements were substantial in all comparisons. Inter-rater reliability between DPP-field and ELISA (k=0.55; PABAK=0.78) and DPP-lab and ELISA (k=0.59; PABAK=0.81) were considered moderate, while that between DPP-field and DPP-lab (k=0.61; PABAK=0.79) was classified as marginally good. The proportion of seroconversions in DPP reactive animals that were initially ELISA indeterminate was significantly higher than in those that were DPP reactive but initially ELISA non-reactive. Restriction fragment length polymorphism analysis revealed the presence of Leishmania infantum, the etiologic agent of VL, in bone marrow samples from VL-infected animals. Our data showed that the techniques and protocols currently employed in the PCLV screening approach are not entirely reliable. Further consideration should be given to monitoring dogs with undetermined results in ELISA and a better training should be provided for health workers responsible for performing DPP tests applied under field conditions.

Performance of Alere™ immunochromathographic test for the diagnosis of canine visceral leishmaniasis.

The diagnosis and control of canine visceral leishmaniasis (CVL) has fundamental importance in the control of human visceral leishmaniasis. In this context, the immunochromathographic test (ICT) has emerged as a valuable diagnostic tool. Currently, in Brazil, the algorithm for the diagnosis of CVL uses Dual Path Platform (DPP) ICT as a screening test and ELISA as a confirmatory test. However, the DPP(®) ICT has been facing a problem of insufficient production to meet the national demand. Here, the Alere™ canine visceral leishmaniasis ICT was tested with serum samples from 159 dogs, including symptomatic, asymptomatic and healthy dogs, characterized by DPP(®) ICT and ELISA (panel A), or ELISA and Indirect Immunofluorescence Assay (IFA) (panel B), and parasitological examination as gold standard. In this study, the Alere™ ICT obtained 0.974 sensitivity for panel A and 0.854 sensitivity for panel B. The specificity was 1.0 for both panels. Thus, the Alere™ ICT has potential to be used for the serological diagnosis of CVL.

Phase II validation study of the rK39 ELISA prototype for the diagnosis of canine visceral leishmaniasis in Brazil / Estudo de validação da fase II do teste ELISA rK39 para o diagnóstico da leishmaniose visceral canina no Brasil / Estudio de validación de la fase II del test ELISA rK39 para el diagnóstico de la leishmaniasis visceral canina en Brasil

Abstract: Dogs are the main reservoirs in the domestic transmission cycle of visceral leishmaniasis, and the diagnosis is essential for the effectiveness of the control measures recommended by the Brazilian Ministry of Health. We assessed the diagnostic performance of the ELISA-Vetlisa/BIOCLIN prototype with serum samples from 200 dogs, in triplicate, including symptomatic, oligosymptomatic, asymptomatic, and healthy dogs, originated by two distinct panels (A and B) characterized by parasitological tests as the reference standard. In this study, the prototype kit showed a 99% sensitivity (95%CI: 94.5-100.0) and a 100% specificity (95%CI: 96.4-100.0). The sensitivity of the prototype kit did not vary significantly with the clinical status of the dogs. Considering the final result classification (positive or negative), agreement between the results of repeated tests was almost perfect (kappa = 0.99; 95%CI: 0.98-1.00). ELISA-Vetlisa/BIOCLIN is a promising option for the serological diagnosis of canine visceral leishmaniasis in Brazil.

Resumo: Os cães são os principais reservatórios do ciclo de transmissão domiciliar da leishmaniose visceral, e o diagnóstico é essencial para a efetividade das medidas de controle recomendadas pelo Ministério da Saúde. Os autores avaliam o desempenho diagnóstico do protótipo da ELISA-Vetlisa/BIOCLIN em amostras sorológicas de 200 cães, em triplicata, incluindo cães sintomáticos, oligossintomáticos e saudáveis, com dois painéis distintos (A e B) caracterizados por testes parasitológicos enquanto referência. No estudo, o kit-protótipo mostrou sensibilidade de 99% (IC95%: 94,5-100,0) e especificidade de 100% (IC95%: 96,4-100,0). A sensibilidade do kit-protótipo não variou de maneira significativa de acordo com o estado clínico dos cães. Considerando a classificação final dos resultados (positivo ou negativo), a concordância entre os resultados dos testes em triplicata foi quase perfeita (kappa = 0,99; IC95%: 0,98-1,00). O protótipo ELISA-Vetlisa/BIOCLIN tem o potencial de ser utilizada para o diagnóstico sorológico da leishmaniose visceral canina no Brasil.

Resumen: Los perros son los principales reservorios en el ciclo de transmisión doméstica de la leishmaniasis visceral, por ello el diagnóstico es esencial para la efectividad de las medidas de control recomendadas por el Ministerio de Salud de Brasil. Evaluamos el desempeño diagnóstico de ELISA-Vetlisa/BIOCLIN prototipo con muestras de sérum de 200 perros, en triplicado, incluyendo sintomático, oligosintomático, asintomático y perros sanos, originadas por dos paneles distintos (A y B), caracterizados por test parasitológicos como referencia estándar. En este estudio, el kit prototipo mostró un 99% de sensibilidad (IC95%: 94,5-100,0) y un 100% de especificidad (IC95%: 96,4-100,0). La sensibilidad del kit prototipo no varió significativamente con el estatus clínico de los perros. Considerando la clasificación final del resultado (positiva o negativa), el acuerdo entre los resultados de los tests repetidos fue casi perfecto (kappa = 0,99; IC95%: 0,98-1,00). ELISA-Vetlisa/BIOCLIN tiene potencial para ser usado para el diagnóstico serológico de la leishmaniasis visceral canina en Brasil.

Leishmania, Babesia and Ehrlichia in urban pet dogs: co-infection or cross-reaction in serological methods?

INTRODUCTION: The present study was designed to assess the occurrence of co-infection or cross-reaction in the serological techniques used for detecting the anti-Leishmania spp., -Babesia canis vogeli and -Ehrlichia canis antibodies in urban dogs from an area endemic to these parasites. METHODS: The serum samples from dogs were tested for the Babesia canis vogeli strain Belo Horizonte antigen and Ehrlichia canis strain São Paulo by immunofluorescence antibody test (IFAT) and by anti-Leishmania immunoglobulin G (IgG) antibody detection to assess Leishmania infection. We used the following four commercial kits for canine visceral leishmaniasis: ELISA, IFAT, Dual Path Platform (DPP) (Bio Manguinhos(r)/FIOCRUZ/MS) and a rK39 RDT (Kalazar Detect Canine Rapid Test; Inbios). RESULTS: Of 96 serum samples submitted to serological assays, 4 (4.2%) were positive for Leishmania as determined by ELISA; 12 (12.5%), by IFAT; 14 (14.6%) by rK39 RDT; and 20 (20.8%), by DPP. Antibodies against Ehrlichia and Babesia were detected in 23/96 (23.9%) and 30/96 (31.2%) samples, respectively. No significant association was identified between the results of tests for detecting Babesia or Ehrlichia and those for detecting Leishmania (p-value>0.05). CONCLUSIONS: In the present study, we demonstrated co-infection with Ehrlichia or Babesia and Leishmania in dogs from Minas Gerais (Brazil); we also found that the serological tests that were used did not cross-react.

Evidence of a sylvatic enzootic cycle of Leishmania infantum in the State of Amapá, Brazil

Abstract INTRODUCTION: Leishmania infantum was considered to be absent from Amapá until 2017 when canine infection was detected. However, there is a lack of knowledge about which reservoir species are involved in transmission in this region. METHODS: Between 2014 and 2016, 86 samples from wild mammals and 74 from domestic dogs were collected in Wajãpi Indigenous Territory and were tested for the presence of deoxyribonucleic acid (DNA) of Leishmania. RESULTS: The DNA of Le. infantum was detected in two rodent samples, Dasyprocta sp. and Proechimys cuvieri. CONCLUSIONS: This is the first evidence characterizing a sylvatic transmission cycle of Le. infantum in the State of Amapá.

Factors associated with the seroprevalence of leishmaniasis in dogs living around Atlantic Forest fragments.

Canine visceral leishmaniasis is an important zoonosis in Brazil. However, infection patterns are unknown in some scenarios such as rural settlements around Atlantic Forest fragments. Additionally, controversy remains over risk factors, and most identified patterns of infection in dogs have been found in urban areas. We conducted a cross-sectional epidemiological survey to assess the prevalence of leishmaniasis in dogs through three different serological tests, and interviews with owners to assess features of dogs and households around five Atlantic Forest remnants in southeastern Brazil. We used Generalized Linear Mixed Models and Chi-square tests to detect associations between prevalence and variables that might influence Leishmania infection, and a nearest neighbor dispersion analysis to assess clustering in the spatial distribution of seropositive dogs. Our findings showed an average prevalence of 20% (ranging from 10 to 32%) in dogs. Nearly 40% (ranging from 22 to 55%) of households had at least one seropositive dog. Some individual traits of dogs (height, sterilization, long fur, age class) were found to positively influence the prevalence, while some had negative influence (weight, body score, presence of ectoparasites). Environmental and management features (number of cats in the households, dogs with free-ranging behavior) also entered models as negative associations with seropositivity. Strong and consistent negative (protective) influences of the presence of chickens and pigs in dog seropositivity were detected. Spatial clustering of cases was detected in only one of the five study sites. The results showed that different risk factors than those found in urban areas may drive the prevalence of canine leishmaniasis in farm/forest interfaces, and that humans and wildlife risk infection in these areas. Domestic dog population limitation by gonadectomy, legal restriction of dog numbers per household and owner education are of the greatest importance for the control of visceral leishmaniasis in rural zones near forest fragments.

Evaluation of three recombinant proteins for the development of ELISA and immunochromatographic tests for visceral leishmaniasis serodiagnosis

BACKGROUND Visceral Leishmaniasis (VL) is an infectious disease that is a significant cause of death among infants aged under 1 year and the elderly in Brazil. Serodiagnosis is a mainstay of VL elimination programs; however, it has significant limitations due to low accuracy. OBJECTIVE This study aimed to evaluate three recombinant Leishmania infantum proteins (rFc, rC9, and rA2) selected from previous proteomics and genomics analyses to develop enzyme-linked immunosorbent assay (ELISA) and immunochromatographic tests (ICT) for the serodiagnosis of human VL (HVL) and canine VL (CVL). METHODS A total of 186 human (70 L. infantum-infected symptomatic, 20 other disease-infected, and 96 healthy) and 185 canine (82 L. infantum-infected symptomatic, 27 L. infantum-infected asymptomatic, and 76 healthy) sera samples were used for antibody detection. FINDINGS Of the three proteins, rA2 (91.5% sensitivity and 87% specificity) and rC9 (95.7% sensitivity and 87.5% specificity) displayed the best performance in ELISA-HVL and ELISA-CVL, respectively. ICT-rA2 also displayed the best performance for HVL diagnosis (92.3% sensitivity and 88.0% specificity) and had high concordance with immunofluorescence antibody tests (IFAT), ELISA-rK39, IT-LEISH®, and ELISAEXT. ICT-rFc, ICT-rC9, and ICT-rA2 had sensitivities of 88.6%, 86.5%, and 87.0%, respectively, with specificity values of 84.0%, 92.0%, and 100%, respectively for CVL diagnosis. MAIN CONCLUSIONS The three antigens selected by us are promising candidates for VL diagnosis regardless of the test format, although the antigen combinations and test parameters may warrant further optimisation.

Validation of the Dual-path Platform chromatographic immunoassay (DPP® CVL rapid test) for the serodiagnosis of canine visceral leishmaniasis

BACKGROUND Visceral leishmaniasis is a major public health challenge in South America, and dogs are its main urban reservoir. OBJECTIVE Validation of the canine Dual-path Platform immunoassay for canine visceral leishmaniasis (DPP® CVL) for a sample set composed of 1446 dogs from different Brazilian endemic areas. METHODS A well-defined reference standard by means of parasitological culture, immunohistochemistry, and histopathology was used. Animals were classified as asymptomatic, oligosymptomatic, or symptomatic. Sensitivity and specificity were assessed as a single set and in clinical groups. A reproducibility assessment of the tests was conducted using the Kappa (κ) index at three different laboratories (A, B, and C). FINDINGS Overall, 89% sensitivity and 70% specificity were obtained for the entire sample set. Analysis of the clinical groups showed a gradual decrease in the sensitivity and an increase in the specificity with the reduction of clinical signs in the dogs that were assessed, reaching a sensitivity of 75% (42.8-94.5%) among asymptomatic dogs and lower specificity of 56% (46.2-66.3%) among symptomatic dogs. Inter-laboratory agreement was substantial (κAB= 0.778; κAC= 0.645; κCB= 0.711). MAIN CONCLUSIONS The test performance is somewhat dependent on canine symptomatology, but such influence was less evident than in previous studies. Favourable results for sensitivity and specificity can be obtained even in asymptomatic animals; however, caution is needed in these evaluations, and the results suggest that the immunochromatographic test may be further improved for better investigation in asymptomatic dogs. The results obtained confirm the usefulness of DPP® CVL for application in serological surveys.

Sensitivity and specificity of parallel or serial serological testing for detection of canine Leishmania infection

In Brazil, human and canine visceral leishmaniasis (CVL) caused byLeishmania infantum has undergone urbanisation since 1980, constituting a public health problem, and serological tests are tools of choice for identifying infected dogs. Until recently, the Brazilian zoonoses control program recommended enzyme-linked immunosorbent assays (ELISA) and indirect immunofluorescence assays (IFA) as the screening and confirmatory methods, respectively, for the detection of canine infection. The purpose of this study was to estimate the accuracy of ELISA and IFA in parallel or serial combinations. The reference standard comprised the results of direct visualisation of parasites in histological sections, immunohistochemical test, or isolation of the parasite in culture. Samples from 98 cases and 1,327 noncases were included. Individually, both tests presented sensitivity of 91.8% and 90.8%, and specificity of 83.4 and 53.4%, for the ELISA and IFA, respectively. When tests were used in parallel combination, sensitivity attained 99.2%, while specificity dropped to 44.8%. When used in serial combination (ELISA followed by IFA), decreased sensitivity (83.3%) and increased specificity (92.5%) were observed. Serial testing approach improved specificity with moderate loss in sensitivity. This strategy could partially fulfill the needs of public health and dog owners for a more accurate diagnosis of CVL.