RESUMEN
The diagnosis of tegumentary leishmaniasis (TL) is hampered by variable sensitivity and/or specificity of the tests. Serological assays are suitable to diagnose visceral leishmaniasis (VL); however, they present low performance for the detection of TL cases. Additionally, blood collection to obtain patient serum represents a challenge, as it is an invasive and uncomfortable procedure, requiring laboratorial infrastructure and trained professionals. In this context, the present study proposed to evaluate patient urine to detect TL, given that this analyte has proven to be effective in ELISA experiments for the detection of VL cases. For this, a Leishmania protein called LiHyV, two specific B-cell epitopes derived from protein amino acid sequence, and a Leishmania antigenic extract (SLA) were used as antigens. A total of 215 paired urine and serum samples were evaluated, and results showed that, when serum was employed as an analyte, rLiHyV, Peptide1, Peptide2, and SLA presented a sensitivity of 85%, 29%, 58%, and 31%, respectively, and a specificity of 97.5%, 98%, 100%, and 97.5%, respectively, in the diagnosis of TL. When urine was used, rLiHyV, Peptide1, Peptide2, and SLA presented a sensitivity of 95%, 74%, 67%, and 52%, respectively, and a specificity of 100%, 99%, 98%, and 86%, respectively. In conclusion, preliminary data suggest that urine could be considered as an alternative biological sample for the detection of TL cases.
RESUMEN
The diagnosis if leprosy is difficult, as it requires clinical expertise and sensitive laboratory tests. In this study, we develop a serological test for leprosy by using bioinformatics tools to identify specific B-cell epitopes from Mycobacterium leprae hypothetical proteins, which were used to construct a recombinant chimeric protein, M1. The synthetic peptides were obtained and showed good reactivity to detect leprosy patients, although the M1 chimera have showed sensitivity (Se) and specificity (Sp) values higher than 90.0% to diagnose both paucibacillary (PB) and multibacillary (MB) leprosy patients, but not those developing tegumentary or visceral leishmaniasis, tuberculosis, Chagas disease, malaria, histoplasmosis and aspergillosis, in ELISA experiments. Using sera from household contacts, values for Se and Sp were 100% and 65.3%, respectively. In conclusion, our proof-of-concept study has generated data that suggest that a new recombinant protein could be developed into a diagnostic antigen for leprosy.
Asunto(s)
Antígenos Bacterianos , Proteínas Bacterianas , Epítopos de Linfocito B , Lepra , Mycobacterium leprae , Sensibilidad y Especificidad , Humanos , Mycobacterium leprae/inmunología , Mycobacterium leprae/genética , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito B/genética , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/genética , Lepra/diagnóstico , Lepra/inmunología , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/genética , Ensayo de Inmunoadsorción Enzimática/métodos , Adulto , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Masculino , Femenino , Pruebas Serológicas/métodos , Biología Computacional/métodos , Persona de Mediana Edad , Adulto Joven , AdolescenteRESUMEN
Leprosy diagnosis is difficult due to the clinical similarity with other infectious diseases, and laboratory tests presents problems related to sensitivity and/or specificity. In this study, we used bioinformatics to assess Mycobacterium leprae proteins and formulated a chimeric protein that was tested as a diagnostic marker for the disease. The amino acid sequences from ML0008, ML0126, ML0308, ML1057, ML2028, ML2038, ML2498 proteins were evaluated, and the B-cell epitopes QASVAYPATSYADFRAHNHWWNGP, SLQRSISPNSYNTARVDP and QLLGQTADVAGAAKSGPVQPMGDRGSVSPVGQ were considered M. leprae-specific and used to construct the gene encoding the recombinant antigen. The gene was constructed, the recombinant protein was expressed, purified and tested in ELISA using 252 sera, which contained samples from multibacillary (MB) or paucibacillary (PB) leprosy patients, from their household contacts and healthy individuals, as well as from patients with Chagas disease, visceral and tegumentary leishmaniases (VL/TL), malaria, tuberculosis, and HIV. Sensitivity (Se) and specificity (Sp) for MB and PB samples compared to sera from both healthy subjects and individuals with cross-reactive diseases were 100%. The Se value for MB and PB samples compared to sera from household contacts was 100%, but Sp was 64%. In conclusion, data suggest that this protein could be considered in future studies for leprosy diagnosis.