Accuracy of quantitative polymerase chain reaction in samples of frozen and paraffin-embedded healthy skin for the diagnosis of canine visceral leishmaniasis

Campos, Monique Paiva; Madeira, M.F.; Silva, D.A.; Solcà, Manuela da Silva; Espíndola, Otávio de Melo; Júnior, Artur Augusto Velho Mendes; Figueiredo, Fabiano Borges

doi:10.1590/1678-4162-9053

Cited by 12 publications

(20 citation statements)

References 28 publications

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“…These samples were obtained from previous investigations and were collected in several T . cruzi endemic settings from Brazil [27–29]. Combined analysis of panel 1 and panel 2 was denominated as “Merged.” In addition to T .…”

Section: Methodsmentioning

confidence: 99%

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

Leony

Freitas

Del-Rei³

et al. 2019

PLoS Negl Trop Dis

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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.

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Section: Methodsmentioning

confidence: 99%

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

Leony

Freitas

Del-Rei³

et al. 2019

PLoS Negl Trop Dis

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“…( 27 ) and the quantitative polymerase chain reaction (qPCR) according to the protocol of Ref. ( 23 ) in the BM samples.…”

Section: Methodsmentioning

confidence: 99%

“…Considering that the main screening and confirmatory tests of canine infection in Brazil are serological, it is possible that the antibody responses elicited by the vaccine interfere with the diagnosis, leading to the inability to distinguish between vaccinated and naturally infected animals. We have recently demonstrated that in a non-endemic area, and up to 1 year after Leish-Tec ® immunization, vaccinated dogs remained negative in the DPP/EIE serological protocol used by the BMH ( 22 , 23 ). On the other hand, the high degree of serological response in rA2 ELISAs observed in previous studies with sera of CVL animals ( 19 – 21 ) led us to evaluate the potential role of this protein for diagnosis, focusing on possible differences in the responses elicited against immunodominant linear B cell epitopes in a group of Leish-Tec ® vaccinated dogs and in two groups of CVL animals (symptomatic and asymptomatic), naturally infected by L. infantum .…”

Section: Introductionmentioning

confidence: 99%

Leishmania infantum Virulence Factor A2 Protein: Linear B-Cell Epitope Mapping and Identification of Three Main Linear B-Cell Epitopes in Vaccinated and Naturally Infected Dogs

et al. 2018

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In Brazil, canine visceral leishmaniasis (CVL) is caused by Leishmania infantum, presenting a broad spectrum of clinical manifestations. Dogs are the main parasite reservoir in urban areas and canine cases precede human infection. Currently, A2 protein based Leish-Tec® vaccine is the only vaccine commercially available against CVL in Brazil. Considering that the main screening and confirmatory tests of canine infection are serological, it is possible that the antibody response elicited after vaccination interfere with diagnosis, leading to the inability to distinguish between vaccinated and infected animals. In order to identify the specific B-cell response induced after vaccination, A2 protein sequence was screened for main linear B-cell epitopes using in silico prediction (Bepipred) and immunological confirmation by ELISA. Three amino acid sequences were described as potential B-cell epitopes (SV11-SAEPHKAAVDV, PP16-PQSVGPLSVGPQSVGP, and VQ34-VGPLSVGPQSVGPLSVGPLSVGPQAVGPLSVGPQ). Specific IgG ELISAs were performed in sera of 12 immunized dogs living in non-endemic areas, followed for up to 1 year after immunization. The results were compared with those obtained in a group of 10 symptomatic and 10 asymptomatic CVL dogs. All predicted epitopes were confirmed as linear B-cell epitopes broadly recognized by sera from studied dogs. Total IgG ELISAs demonstrated distinct patterns of response between peptides in the immunized and CVL groups. VQ34 peptide was recognized by the majority of sera from vaccinated and symptomatic dogs, and increases after vaccination. PP16 induced low levels of specific IgG that increased 1 year after immunization. Interestingly, a low frequency of reactivity was found against SV11 in naturally infected dogs (symptomatic and asymptomatic), while 83.3% of vaccinated dogs presented positive responses 1 year after immunization. The two animals in the vaccinated group that did not respond to SV11 1 year after immunization presented positive serology both 30 days and 6 months after immunization. In summary, we identified three main linear B-cell epitopes in A2 based vaccine. Moreover, the humoral response against SV11 presented marked differences between infected and Leish-Tec vaccinated dogs, and should be further investigated, in large trials, to confirm its potential as a serological marker able to distinguish between infected and vaccinated dogs.

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“…Preservation of nucleic acid and retrieval from paraffin-embedded tissues (PET) 9,12,25,32,46 and evaluation of alternatives to formalin fixation are areas also being evaluated actively. 25 In general, the length of time that tissues are exposed to formalin has a measurable deleterious effect on the retrievability of DNA and an even more profound effect on retrievability of RNA, with an optimal time found to be 12–24 h. 12 In the same study, slower processing times for paraffin embedding resulted in improved RT-rtPCR performance.…”

Section: Preanalytical: Sample Acquisition and Transportmentioning

confidence: 99%

Best practices for performance of real-time PCR assays in veterinary diagnostic laboratories

et al. 2020

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The exquisite sensitivity of in vitro amplification assays such as real-time polymerase chain reaction (rtPCR) requires the establishment of thorough and robust laboratory practices. To this end, an American Association of Veterinary Laboratory Diagnosticians (AAVLD) committee of subject matter experts was convened to develop a set of best practices for performance of nucleic acid amplification assays. Consensus advice for the performance of preanalytical, analytical, and postanalytical steps is presented here, along with a review of supporting literature.

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Accuracy of quantitative polymerase chain reaction in samples of frozen and paraffin-embedded healthy skin for the diagnosis of canine visceral leishmaniasis

Abstract: Accuracy of quantitative polymerase chain reaction in samples of frozen and paraffinembedded healthy skin for the diagnosis of canine visceral leishmaniasis

Cited by 12 publications

References 28 publications

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

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

Leishmania infantum Virulence Factor A2 Protein: Linear B-Cell Epitope Mapping and Identification of Three Main Linear B-Cell Epitopes in Vaccinated and Naturally Infected Dogs

Best practices for performance of real-time PCR assays in veterinary diagnostic laboratories

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