Immunodiagnosis of Canine Visceral Leishmaniasis Using Mimotope Peptides Selected from Phage Displayed Combinatorial Libraries

Toledo-Machado, Christina Monerat; Ávila, Ricardo Andrez Machado de; Nguyen, Christophe; Granier, Claude; Bueno, Lilian Lacerda; Carneiro, Cláudia Martins; Menezes‐Souza, Daniel; Carneiro, Rubens Antônio; Chávez-Olórtegui, Carlos; Fujiwara, Ricardo Toshio

doi:10.1155/2015/401509

Cited by 10 publications

(15 citation statements)

References 38 publications

(47 reference statements)

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“…The use of synthetic peptides [30, 31] as antigens in diagnosis of canine leishmaniasis may limit cross reactivity. It would also circumvent reliance on parasite extracts, which are not easy to reproducibly produce, and thus may help assay standardization.…”

Section: Discussionmentioning

confidence: 99%

Epitope mapping of recombinant Leishmania donovani virulence factor A2 (recLdVFA2) and canine leishmaniasis diagnosis using a derived synthetic bi-epitope

Mendes¹,

Roma²,

Costal-Oliveira³

et al. 2017

PLoS Negl Trop Dis

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BackgroundLeishmaniasis is one of the most important zoonotic diseases spread in Latin America. Since many species are involved in dog infection with different clinical manifestations, the development of specific diagnostic tests is mandatory for more accurate disease control and vaccine strategies.Methodology/Principal findingsSeventy-five 15-mer peptides covering the sequence of recombinant Leishmania donovani virulence factor A2 (recLdVFA2) protein were prepared by Spot synthesis. Membrane-bound peptides immunoreactivity with sera from dogs immunized with recLdVFA2 and with a specific anti-recLdVFA2 monoclonal antibody allowed mapping of continuous B-cell epitopes. Five epitopes corresponding to the N-terminal region of recLdVFA2 (MKIRSVRPLVVLLVC, RSVRPLVVLLVCVAA, RPLVVLLVCVAAVLA, VVLLVCVAAVLALSA and LVCVAAVLALSASAE, region 1–28) and one located within the repetitive units (PLSVGPQAVGLSVG, regions 67–81 and 122–135) were identified. A 34-mer recLdVFA2-derived bi-epitope containing the sequence MKIRSVRPLVVLLVC linked to PLSVGPQAVGLSVG by a Gly-Gly spacer was chemically synthesized in its soluble form. The synthetic bi-epitope was used as antigen to coat ELISA plates and assayed with dog sera for in vitro diagnosis of canine visceral leishmaniasis (CVL). The assay proved to be highly sensitive (98%) and specific (99%).Conclusions/SignificanceOur work suggests that synthetic peptide-based ELISA strategy may be useful for the development of a sensitive and highly specific serodiagnosis for CVL or other parasitic diseases.

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Section: Discussionmentioning

confidence: 99%

Epitope mapping of recombinant Leishmania donovani virulence factor A2 (recLdVFA2) and canine leishmaniasis diagnosis using a derived synthetic bi-epitope

Mendes¹,

Roma²,

Costal-Oliveira³

et al. 2017

PLoS Negl Trop Dis

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“…Several studies demonstrate that promastigote antigens of Leishmania cross-react with sera showing positive reactions against antigens of malaria and Chagas disease ( Romero et al, 2009 , Pinedo-Cancino et al, 2013 , Toledo-Machado et al, 2015 ). In this study, sera positive for malaria and Chagas disease showed considerable cross reactivity in the p-ELISA while the rK39 ICT and IFAT did not.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of recombinant K39 antigen and various promastigote antigens in sero-diagnosis of visceral leishmaniasis in Bangladesh

Banu

Ahmed

Shamsuzzaman

et al. 2016

Parasite Epidemiology and Control

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BackgroundDefinitive diagnosis of visceral leishmaniasis (VL) by demonstrating parasites in tissue smears or by culture involves invasive procedures, technical expertise and adequate laboratory facilities. Endemic countries rely mainly on serological tests to diagnose VL. Currently, the immunochromatographic test incorporating the recombinant K39 antigen (rK39 ICT) is the reference test for rapid diagnosis of VL in the Indian subcontinent. The performance of serological tests using rK39 and other promastigote antigens can vary due to differences in antigen expression, the various hosts and environmental factors. To achieve elimination of VL, diagnostic accuracy will be necessary for active case detection especially in those who carry asymptomatic infections. We evaluated the performance of rK39 ICT, enzyme linked immunosorbent assay using mixed Leishmania promastigotes from different Leishmania species (p-ELISA) and indirect fluorescent antibody test (IFAT) utilizing whole promastigotes from the Leishmania donovani complex for sero-diagnosis of VL in Bangladesh.MethodsThe sensitivity of each serological test was evaluated on 155 patients who were diagnosed to have VL by microscopy and/or by culture methods. Test specificities were calculated on 706 healthy blood donors, 91 diagnostic sera from patients with a febrile illness and sera from patients positive for malaria (n = 91) and Chagas disease (n = 91). All statistical calculations were at 95% confidence intervals.ResultsThe sensitivities of rK39 ICT, p-ELISA and IFAT were 100%, 86.5% and 92.3%, respectively. All three serological methods had a pooled sensitivity of 82.6%. The specificities of rK39 ICT, p-ELISA and IFAT from combined control groups were 100%, 93.1% and 99.9%, respectively. The respective positive and negative predictive values of the tests were both 100% for rK39 ICT, 66.3% and 97.8% for p-ELISA and 99.3% and 98.8% for IFAT. The p-ELISA showed cross reactivity with 36.3% of sera positive for malaria and 28.6% of sera positive for Chagas disease while rK39 ICT and IFAT showed no cross reactivity.ConclusionThis study confirms the efficiency of rK39 ICT for rapid diagnosis of VL. The p-ELISA using mixed promastigote antigens did not perform well as a serological test for VL in Bangladesh. Due to high sensitivity and specificity of whole promastigote antigen of L. donovani complex utilized in IFAT, this test can be considered in combination with rK39 ICT to confirm VL diagnosis when clinical diagnosis cannot distinguish between other diseases.

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“…Due to the limited antileishmanial drugs available for treatmenf of VL, effective clinical management depend largely on early and unequivocal diagnosis. However, regarding the diagnosis of human cases, problems of cross-reactivity have also been described in serological tests, especially when it refers to individuals from areas where Chagas’ disease is also present [27]. These data indicate the urgent need to discover new antigens to improve the current serological tests for VL diagnosis.…”

Section: Discussionmentioning

confidence: 99%

“…In this sense, several antigens, as peptides, recombinant proteins and chimeric proteins were obtained to be used in VL and CVL diagnosis [21, 23]. Moreover, identification of reactive peptides on the surface of the phages and integration with immunoinformatics allows their use and/or the subsequent synthetic construction with a directed application for diagnosis [27, 28]. Thus, using the knowledge acquired in immunoinformatics for the discovery of targets for the diagnosis of infectious diseases [27, 29–31], and also for the limitations known for the serodiagnosis of VL, the objective to the present study was to identify and validate new antigens of L .…”

Section: Introductionmentioning

confidence: 99%

“…Moreover, identification of reactive peptides on the surface of the phages and integration with immunoinformatics allows their use and/or the subsequent synthetic construction with a directed application for diagnosis [27, 28]. Thus, using the knowledge acquired in immunoinformatics for the discovery of targets for the diagnosis of infectious diseases [27, 29–31], and also for the limitations known for the serodiagnosis of VL, the objective to the present study was to identify and validate new antigens of L . infantum for the development of a biotechnological product applied to the serological diagnosis of this disease.…”

Section: Introductionmentioning

confidence: 99%

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New antigens for the serological diagnosis of human visceral leishmaniasis identified by immunogenomic screening

Carvalho¹,

Mendes²,

Coelho³

et al. 2018

PLoS ONE

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Visceral leishmaniasis (VL) still represents a serious public health problem in Brazil due to the inefficiency of the control measures currently employed, that included early diagnosis and treatment of human cases, vector control, euthanasia of infected dogs and, recently approved in Brazil, treatment with Milteforam drug. Effective clinical management depend largely on early and unequivocal diagnosis, however, cross-reactivity have also been described in serological tests, especially when it refers to individuals from areas where Chagas’ disease is also present. Thus, to discover new antigens to improve the current serological tests for VL diagnosis is urgently needed. Here, we performed an immunogenomic screen strategy to identify conserved linear B-cell epitopes in the predicted L. infantum proteome using the following criteria: i) proteins expressed in the stages found in the vertebrate host, amastigote stage, and secreted/excreted, to guarantee greater exposure to the immune system; ii) divergent from proteins present in other infectious disease pathogens with incidence in endemic areas for VL, as T. cruzi; iii) highly antigenic to humans with different genetic backgrounds, independently of the clinical stage of the disease; iv) stable and adaptable to quality-control tests to guarantee reproducibility; v) using statistical analysis to determine a suitable sample size to evaluate accuracy of diagnostic tests established by receiver operating characteristic strategy. We selected six predicted linear B-cell epitopes from three proteins of L. infantum parasite. The results demonstrated that a mixture of peptides (Mix IV: peptides 3+6) were able to identify VL cases and simultaneously able to discriminate infections caused by T. cruzi parasite with high accuracy (100.00%) and perfect agreement (Kappa index = 1.000) with direct methods performed by laboratories in Brazil. The results also demonstrated that peptide-6, Mix III (peptides 2+6) and I (peptides 2+3+6) are potential antigens able to used in VL diagnosis, represented by high accuracy (Ac = 99.52%, 99.52% and 98.56%, respectively). This study represents an interesting strategy for discovery new antigens applied to serologic diagnosis which will contribute to the improvement of the diagnosis of VL and, consequently, may help in the prevention, control and treatment of the disease in endemic areas of Brazil.

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Immunodiagnosis of Canine Visceral Leishmaniasis Using Mimotope Peptides Selected from Phage Displayed Combinatorial Libraries

Cited by 10 publications

References 38 publications

Epitope mapping of recombinant Leishmania donovani virulence factor A2 (recLdVFA2) and canine leishmaniasis diagnosis using a derived synthetic bi-epitope

Epitope mapping of recombinant Leishmania donovani virulence factor A2 (recLdVFA2) and canine leishmaniasis diagnosis using a derived synthetic bi-epitope

Evaluation of recombinant K39 antigen and various promastigote antigens in sero-diagnosis of visceral leishmaniasis in Bangladesh

New antigens for the serological diagnosis of human visceral leishmaniasis identified by immunogenomic screening

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