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

Flow Cytometry as a Tool for Quality Control of Fluorescent Conjugates Used in Immunoassays

Submitted by Sandra Infurna ([email protected]) on 2017-03-28T15:46:20Z No. of bitstreams: 1 alvaro_bertho_etal_IOC_2016.pdf: 2901056 bytes, checksum: 7d5255b1b013920484bc8b0918988719 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2017-03-28T16:01:38Z (GMT) No. of bitstreams: 1 alvaro_bertho_etal_IOC_2016.pdf: 2901056 bytes, checksum: 7d5255b1b013920484bc8b0918988719 (MD5)Made available in DSpace on 2017-03-28T16:01:38Z (GMT). No. of bitstreams: 1 alvaro_bertho_etal_IOC_2016.pdf: 2901056 bytes, checksum: 7d5255b1b013920484bc8b0918988719 (MD5) Previous issue date: 2016Fundação Oswaldo Cruz. Instituto de Tecnologia Imunobiológica - Farmanguinhos. Laboratório de Tecnologia de Diagnóstico. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Tecnologia Imunobiológica - Farmanguinhos. Laboratório de Tecnologia de Diagnóstico. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Tecnologia Imunobiológica - Farmanguinhos. Laboratório de Tecnologia de Diagnóstico. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Tecnologia Imunobiológica - Farmanguinhos. Laboratório de Tecnologia de Diagnóstico. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunoparasitologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunologia Clínica. Rio de Janeiro, RJ. Brasil.The use of antibodies in immunodiagnostic kits generally implies the conjugation of these proteins with other molecules such as chromophores or fluorochromes. The development of more sensitive quality control procedures than spectrophotometry is essential to assure the use of better fluorescent conjugates since the fluorescent conjugates are critical reagents for a variety of immunodiagnostic kits. In this article, we demonstrate a new flow cytometric protocol to evaluate conjugates by molecules of equivalent soluble fluorochromes (MESF) and by traditional flow cytometric analysis. We have coupled microspheres with anti-IgG-PE and anti-HBSAg-PE conjugates from distinct manufactures and/or different lots and evaluated by flow cytometry. Their fluorescence intensities were followed for a period of 18 months. Our results showed that there was a great difference in the fluorescence intensities between the conjugates studied. The differences were observed between manufactures and lots from both anti-IgG-PE and anti-HBSAg-PE conjugates. Coefficients of variation (CVs) showed that this parameter can be used to determine better coupling conditions, such as homogenous coupling. The MESF analysis, as well as geometric mean evaluation by traditional flow cytometry, showed a decrease in the values for all conjugates during the study and were indispensable tools to validate the results of stability tests. Our data demonstrated the feasibility of the flow cytometric method as a standard quality control of immunoassay kits

EVIDENCE OF Leishmania (Leishmania) infantum INFECTION IN DOGS FROM JUIZ DE FORA, MINAS GERAIS STATE, BRAZIL, BASED ON IMMUNOCHROMATOGRAPHIC DUAL-PATH PLATFORM (DPP®) AND PCR ASSAYS

In Brazil, domestic dogs are branded as the primary reservoir for zoonotic visceral leishmaniasis, due to the clear positive correlation observed between human and canine infection rates. This study aimed to carry out a serological survey of canine visceral leishmaniasis (CVL) in dogs housed at a public kennel in the municipality of Juiz de Fora, Minas Gerais State, Brazil, using the immunochromatographic TR DPP&#174; CVL rapid test. Additionally, conventional and/or real time PCR assay was used to detect and confirm L. infantum infection in the DPP positive dogs only. Of the 400 dogs studied, most did not present clinical signs for CVL (p < 0.05), and fifteen (3.8%) were seropositive in the DPP test. There was no statistically significant difference between the DPP seropositive dogs and the clinical signs of the disease (p > 0.05). Both conventional and real time PCR tests confirmed L. infantum infection in nine (75.0%) of the twelve DPP seropositive dogs that remained alive during the follow-up period. This study is the first seroepidemiologic survey of CVL held in the city of Juiz de Fora, and the results reinforce the idea that this disease is currently in a process of expansion and urbanization in Brazil. Furthermore, this study highlights the use of the DPP test as an alternative for diagnosing CVL in large and mid-sized cities, due to its ease of implementation

FITC and PE detection after anti-IgG-FITC staining.

<p>FITC and PE detection after anti-IgG-FITC staining.</p

Conjugate stability study during 18 months.

<p>A: A1, A2, A3, A4 and MESF bead #4 fluorescence intensities during the months of analysis. B: B1, B2, C, D and MESF bead #4 fluorescence intensities during the months of analysis. C: Conjugates histograms of PE-fluorescence intensity (X axis) vs. count (Y axis) on the first month (black peak) and last month of analysis (white peak). MESF bead #4 was included as an example of the MESF kit used to control instrument performance during the 18 months of evaluation. The A4 conjugate was coupled later than the other conjugates and its intensities evaluation started at month 4.</p

Staining profile of anti-IgG-PE conjugates.

<p>A: Representative histograms of PE-fluorescence intensity (X axis) vs. count (Y axis). 0.5 μg/mL = white peak and 5 μg/mL = black peak. B: Coefficient of variation of 0.5 μg/mL (white bars) and 5 μg/mL (black bars). Data expressed as the means of CVs from of three tests ± standard deviation. T test was performed for group comparison. An asterisk indicates a significantly different value assumed at p≤0.05.</p

Evaluation of the best conjugate concentration and dilution.

<p>A: Anti-IgG-PE concentration test (0.5, 1, 2.5 and 5 μg/mL). * = statistically different value when compared to the lowest concentration (p≤0.05). A1- black bars, A4- dashed bars and B2- white bars. B: Anti-HBSAg-PE dilution test (1:3000, 1:1000 and 1:300). C- black bars and D- dashed bars. One way ANOVA was performed for group comparison * = statistically different value among concentrations (p≤0.05). Data are expressed as mean of three geometric means ± standard deviation.</p