RESUMEN
Visceral Leishmaniasis and HIV-AIDS coinfection (VL/HIV) is considered a life-threatening pathology when undiagnosed and untreated, due to the immunosuppression caused by both diseases. Serological tests largely used for the VL diagnosis include the direct agglutination test (DAT), ELISA and immunochromatographic (ICT) assays. For VL diagnosis in HIV infections, different studies have shown that the use of the DAT assay facilitates the VL diagnosis in co-infected patients, since the performance of the most widely used ELISA and ICT tests, based on the recombinant protein rK39, are much less efficient in HIV co-infections. In this scenario, alternative recombinant antigens may help the development of new serological diagnostic methods which may improve the VL diagnosis for the co-infection cases. This work aimed to evaluate the use of the recombinant Lci2 antigen, related to, but antigenically more diverse than rK39, for VL diagnosis in co-infected sera through ELISA assays. A direct comparison between recombinant Lci2 and rK39 was thus carried out. The two proteins were first tested using indirect ELISA with sera from VL afflicted individuals and healthy controls, with similar performances. They were then tested with two different sets of VL/HIV co-infected cases and a significant drop in performance, for one of these groups, was observed for rK39 (32% sensitivity), but not for Lci2 (98% sensitivity). In fact, an almost perfect agreement (Kappa: 0.93) between the Lci2 ELISA and DAT was observed for the coinfected VL/HIV patients. Lci2 then has the potential to be used as a new tool for the VL diagnosis of VL/HIV co-infections.
Asunto(s)
Anticuerpos Antiprotozoarios/aislamiento & purificación , Infecciones por VIH/genética , Leishmania infantum/aislamiento & purificación , Leishmaniasis Visceral/diagnóstico , Proteínas Recombinantes/aislamiento & purificación , Pruebas de Aglutinación , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Coinfección/diagnóstico , Coinfección/genética , Coinfección/parasitología , Ensayo de Inmunoadsorción Enzimática , VIH/patogenicidad , Infecciones por VIH/complicaciones , Infecciones por VIH/parasitología , Infecciones por VIH/virología , Humanos , Leishmania infantum/genética , Leishmania infantum/patogenicidad , Leishmaniasis Visceral/genética , Leishmaniasis Visceral/parasitología , Leishmaniasis Visceral/virología , Proteínas Protozoarias/inmunología , Proteínas Recombinantes/genéticaRESUMEN
BACKGROUND: Visceral leishmaniasis (VL) is a major neglected disease, potentially fatal, whose control is still impaired by inefficient and/or expensive treatment and diagnostic methods. The most promising approach for VL diagnosis uses serological assays with recombinant proteins, since they are more efficient and easier to perform. Tests developed for the human form of the disease, however, have not been shown to be efficient for its diagnosis in the canine host, the major reservoir for the American VL. METHODOLOGY/PRINCIPAL FINDINGS: Here, we describe a systematic approach aimed at the production of a new chimeric protein potentially able to be used for both human and canine VL diagnosis and based both on in silico gene design and experimental data. Starting from the previous identification of Leishmania infantum recombinant antigens efficient for the diagnosis of either human or canine VL, three of the best performing antigens were selected (Lci2, Lci3 and Lci12). After a preliminary evaluation validating the chimeric approach, DNA fragments encoding predicted antigenic regions from each protein, enriched with repeats, were joined in various combinations to generate a total of seventeen chimeric genes optimized for prokaryotic expression. These were assessed for optimal expression and purification yield, with four chimeric proteins being efficiently produced. Their diagnostic potential was then evaluated through ELISA assays with sera from VL afflicted humans and dogs. After two rounds of gene design, the results showed high levels of sensitivity for the best chimeric protein, named Q5, in humans (82%) and dogs (100%) with 100% specificity in comparison with healthy controls. A single non-specific reaction was seen with serum from individuals with tegumentary leishmaniasis. CONCLUSION: The newly described chimeric protein is potentially useful for the detection of both humans and dogs afflicted with VL, with its use in rapid tests necessary for validation as a new diagnostic tool.
Asunto(s)
Enfermedades de los Perros/diagnóstico , Leishmaniasis Visceral/veterinaria , Pruebas Serológicas/veterinaria , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/química , Enfermedades de los Perros/sangre , Perros , Escherichia coli/metabolismo , Regulación de la Expresión Génica , Humanos , Leishmaniasis Visceral/sangre , Leishmaniasis Visceral/diagnóstico , Proteínas Protozoarias/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sensibilidad y Especificidad , Pruebas Serológicas/métodos , TranscriptomaRESUMEN
Current strategies for the control of zoonotic visceral leishmaniasis (VL) rely on its efficient diagnosis in both human and canine hosts. The most promising and cost effective approach is based on serologic assays with recombinant proteins. However, no single antigen has been found so far which can be effectively used to detect the disease in both dogs and humans. In previous works, we identified Leishmania infantum antigens with potential for the serodiagnosis of VL. Here, we aimed to expand the panel of the available antigens for VL diagnosis through another screening of a genomic expression library. Seven different protein-coding gene fragments were identified, five of which encoding proteins which have not been previously studied in Leishmania and rich in repetitive motifs. Poly-histidine tagged polypeptides were generated from six genes and evaluated for their potential for diagnosis of VL by ELISA (Enzyme Linked ImmunoSorbent Assay) with sera from infected humans and dogs. None of those was valid for the detection of human VL (26-52% sensitivity) although their performance was increased in the canine sera (48-91% sensitivity), with one polypeptide useful for the diagnosis of canine leishmaniasis. Next, we assayed a mixture of three antigens, found to be best for human or canine VL, among 13 identified through different screenings. This "Mix" resulted in similar levels of sensitivity for both human (84%) and canine (88%) sera. With improvements, this validates the use of multiple proteins, including antigens identified here, as components of a single system for the diagnosis of both forms of leishmaniasis.