A multicentric evaluation of the recombinant Leishmania infantum antigen-based immunochromatographic assay for the serodiagnosis of canine visceral leishmaniasis.

Background: Visceral leishmaniasis (VL) is a serious public health challenge in Brazil and dogs are considered to be the main urban reservoir of the causative agent. The culling of animals to control VL in some countries makes the accurate diagnosis of canine VL (CVL) essential. Recombinant antigens rLci1A and rLci2B were selected from a cDNA library of Leishmania infantum amastigotes due to their strong potential as candidates in diagnostic testing for CVL. The present multicentric study aimed to evaluate the sensitivity of a prototype test using these antigens (DPPrLci1A/rLci2B) against 154 sera obtained from symptomatic dogs within three endemic areas of VL in Brazil. The specificity was evaluated using 40 serum samples from negative dogs and dogs infected with other pathogens. Sensitivity and specificity rates of DPP rLci1A/rLci2B prototype were compared to rates from other diagnostic tests currently in use by the Brazilian Ministry of Health, including DPP?LVC, EIE?LVC. Findings: DPP rLci1A/rLci2B prototype offered similar performance to that offered by DPP?LVC rapid test, as follows: sensitivity of 87% (CI 81?91) and 88% (CI 82?93) and specificity of 100% (CI 91?100) and 97% (CI 87?100), respectively for DPP rLci1A/rLci2B and DPP?LVC. When results of these two tests were considered concomitantly, sensitivity increased to 93.5% (CI 89?96). Conclusions: The recombinant antigens rLci1A and rLci2B represent promising candidates for use in a multi-antigen rapid test for CVL. The inclusion of novel antigens to the DPP rLci1A/rLci2B prototype model could offer additionally enhanced sensitivity to detect animals infected by L. infantum

Performance of recombinant chimeric proteins in the serological diagnosis of Trypanosoma cruzi infection in dogs.

Background: Dogs are considered sentinels in areas of Trypanosoma cruzi transmission risk to humans. ELISA is generally the method of choice for diagnosing T. cruzi exposure in dogs, but its performance substantially depends on the antigenic matrix employed. In previous studies, our group has developed four chimeric antigens (IBMP-8.1, 8.2, 8.3, and 8.4) and evaluated their potential for diagnosing T. cruzi exposure in humans. For human sera, these chimeric antigens presented superior diagnostic performances as compared to commercial tests available in Brazil, Spain, and Argentina. Therefore, in this study we have evaluated the potential of these antigenic proteins for detection of anti-T. cruzi IgG antibodies in dog sera. Methodology/Principal findings: The IBMP-ELISA assays were optimized by checkerboard titration. Subsequently, the diagnostic potential was validated through analysis of ROC curves and the performance of the tests was determined using double entry tables. Cross-reactivity was also evaluated for babesiosis, ehrlichiosis, dirofilariosis, anaplasmosis, and visceral leishmaniasis. Best performance was shown by IBMP-8.3 and IBMP-8.4, although all four antigens demonstrated a high diagnostic performance with 46 positive and 149 negative samples tested. IBMP-8.3 demonstrated 100% sensitivity, followed by IBMP-8.4 (96.7?100%), IBMP-8.2 (73.3?87.5%), and IBMP-8.1 (50?100%). The highest specificities were achieved with IBMP-8.2 (100%) and IBMP-8.4 (100%), followed by IBMP-8.3 (96.7?97.5%) and IBMP 8.1 (89.1?100%). Conclusions/Significance: The use of chimeric antigenic matrices in immunoassays for anti-T. cruzi IgG antibody detection in sera of infected dogs was shown to be a promising tool for veterinary diagnosis and epidemiological studies. The chimeric antigens used in this work allowed also to overcome the common hurdles related to serodiagnosis of T. cruzi infection, especially regarding variation of efficiency parameters according to different strains and cross-reactivity with other infectious diseases

Multiplex flow cytometry serology to diagnosis of canine visceral leishmaniasis.

An accurate diagnosis of visceral leishmaniasis is an essential tool for control of the disease. While serologic methods are very useful, these conventional methodologies still present limitations in terms of sensitivity and specificity. The use of flow cytometry is a worldwide trend in the development of high-performance diagnostic methods. Herein, we describe a new flow cytometry serology test, characterized by the employment of the Cytometric Bead Array microspheres A4 and E4 coated with the recombinant antigens rLci1A and rLci2B respectively, to improve the serodiagnosis of canine visceral leishmaniasis. The tests were conducted in a wide variety of sera groups (n = 140), where the diagnostics development would be optimized accounting not just the ability to identify infected dogs with different clinical status, but also to exclude cross-reaction and differentiate vaccinated dogs from dogs infected. Serological testing of the antigenic system A4?rLci1A showed a sensitivity of 90.0% and specificity of 75%, while the E4?rLci2B testing demonstrated a sensitivity of 95.0% and specificity of 82.5%. The use of a multiplex assay of A4?rLci1A and E4?rLci2B, resulted in a diagnostic improvement, with a sensitivity of 95.0% and specificity of 91.2%. Our results show that this novel flow cytometry serology test is a viable tool for sensitive and specific serodiagnosis. Notably, the combination of distinct antigenic systems allows us to test for antibodies to multiple recombinant antigens from a single serum sample. This benefit emphasizes the importance of this methodology as an alternative in the serological diagnosis