RESUMEN
Despite the clear need to control visceral leishmaniasis (VL), the existing diagnostic tests have serious shortcomings. Here, we introduce an innovative approach to directly identify Leishmania infantum antigens produced in vivo in humans with VL. We combined reverse-phase high-performance liquid chromatography (RP-HPLC) with mass spectrometry and categorized three distinct L. infantum proteins presumably produced in bone marrow/spleen/liver and excreted in the urine of patients with VL. The genes coding for these proteins (L. infantum iron superoxide dismutase, NCBI accession number XP_001467866.1; L. infantum tryparedoxin, NCBI accession number XP_001466642.1; and L. infantum nuclear transport factor 2, NCBI accession number XP_001463738.1) were cloned, and the recombinant molecules were produced in Escherichia coli. Antibodies to these proteins were produced in rabbits and chickens and were used to develop a capture enzyme-linked immunosorbent assay (ELISA) designed to detect these L. infantum antigens in the urine of VL patients. Specificity of the antibodies was confirmed by a Western blot analysis using both recombinant proteins and whole parasite extract. Importantly, a urinary antigen detection assay assembled with pairs of antibodies specific for each of these antigens identified 17 of 19 patients with VL. These results indicate that an improved antigen detection assay based on L. infantum proteins present in the urine of patients with VL may represent an important new strategy for the development of a specific and accurate diagnostic test that has the potential to both distinguish active VL from asymptomatic infection and serve as an important tool to monitor therapy efficacy.
Asunto(s)
Anticuerpos Antiprotozoarios , Antígenos de Protozoos/análisis , Técnicas de Laboratorio Clínico/métodos , Leishmania infantum/química , Leishmaniasis Visceral/diagnóstico , Proteínas Protozoarias/análisis , Orina/química , Adolescente , Adulto , Animales , Anticuerpos Antiprotozoarios/aislamiento & purificación , Antígenos de Protozoos/genética , Antígenos de Protozoos/aislamiento & purificación , Western Blotting , Pollos , Niño , Preescolar , Cromatografía Líquida de Alta Presión , Clonación Molecular , Escherichia coli/genética , Femenino , Humanos , Leishmania infantum/genética , Masculino , Espectrometría de Masas , Parasitología/métodos , Proteínas Protozoarias/genética , Proteínas Protozoarias/aislamiento & purificación , Conejos , Orina/parasitología , Adulto JovenAsunto(s)
Antígenos de Protozoos/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Leishmania donovani/inmunología , Leishmaniasis Cutánea/diagnóstico , Leishmaniasis Visceral/diagnóstico , Antígenos de Protozoos/sangre , Antígenos de Protozoos/aislamiento & purificación , Brasil , Estudios de Casos y Controles , Humanos , India , Leishmania donovani/aislamiento & purificación , Leishmaniasis Cutánea/sangre , Leishmaniasis Cutánea/epidemiología , Leishmaniasis Cutánea/inmunología , Leishmaniasis Visceral/sangre , Leishmaniasis Visceral/epidemiología , Leishmaniasis Visceral/inmunología , Sensibilidad y EspecificidadRESUMEN
The current schedule of the Brazilian Soccer Championship may not give players enough recovery time between games. This could increase the chances of muscle damage and impaired performance. We hypothesized that plasma creatine kinase (CK) activity could be a reliable indirect marker of muscle overload in soccer players, so we sought to identify the reference values for upper limits of CK activity during a real-life elite competition. This study analyzed changes in plasma CK activity in 128 professional soccer players at different times during the Brazilian Championship. The upper limits of the 97.5th and 90th percentiles determined for CK activity were 1.338U/L and 975U/L, respectively, markedly higher than values previously reported in the literature. We also evaluated a team monthly throughout the Championship. The upper limit of the 90th percentile, 975U/L, was taken as the decision limit. Six players showing plasma CK values higher than this were asked to decrease their training for 1 week. These players presented lower CK values afterwards. Only one player with a CK value higher than the decision limit (1800U/L 1 day before a game) played on the field and was unfortunately injured during the game. The CK activity in all the other players showed a significant decrease over the course of the Championship, and the values became more homogeneous at the end. The results presented here suggest that plasma CK upper limit values can be used as a practical alternative for early detection of muscle overload in competing soccer players.