ABSTRACT
OBJETIVO: Evaluar la aglutinación macroscópica con antígeno termorresistente (TR) como tamiz diagnóstico de leptospirosis humana en diferentes etapas de la enfermedad. MATERIAL Y MÉTODOS:La definición de casos se basó en la microaglutinación (MAT), recuento de leucocitos y neutrofilia. Se incluyeron 218 casos confirmados y 242 no casos. Cada muestra del banco de sueros del laboratorio del Instituto Nacional de Enfermedades Respiratorias de Santa Fe, Argentina, de 2008 a 2010, se clasificó según días de evolución en tres etapas: primera (<10 días), segunda (10- 25 días) y tercera (>25 días). RESULTADOS: La sensibilidad hallada fue: 71.1, 93.4 y 95.6% para etapas 1, 2 y 3 respectivamente. La especificidad varió de 79.0 a 69.2%. La variabilidad intra e interoperador fue moderada. CONCLUSIÓN: La variabilidad del TR, su baja sensibilidad en la primera etapa y baja especificidad en todas las etapas de la enfermedad sugieren que sería indispensable la incorporación de nuevos métodos diagnósticos de tamiz para la detección precoz de casos en nuestro país, y países donde aún se apliquen este tipo de métodos.
OBJECTIVE: To evaluate the macroscopic agglutination test using Temperature Resistant (TR) antigen as a screening test for the diagnosis of human leptospirosis in different stages of the disease. MATERIALS AND METHODS: The criteria for case definition were based on the results of the microscopic agglutination test (MAT), leukocyte counts and neutrophilia, resulting 218 confirmed cases and 242 non- cases. Each sample was classified according to the days of the disease progression in three stages: first (<10 days), second (10 - 25 days) and third (> 25 days). The design was cross-sectional observational. RESULTS: TR sensitivity was 71,1% on stage 1. 93.4% on stage 2 and 95.6% on stage 3. The specificity at different stages ranged from 79.0 to 69.2%. Intra and inter-operator variability was moderate. CONCLUSION: TR variability, low sensitivity in the first stage and low specificity found in all stages of the disease, suggest that it is essential to incorporate new diagnostic methods to screen for early detection of cases in our country and in countries that still apply such methods.
Subject(s)
Humans , Agglutination Tests , Leptospirosis/diagnosis , Antigens, Bacterial/immunology , Blood Banks , Blood Preservation , Cross-Sectional Studies , Cryopreservation , Disease Progression , Early Diagnosis , Leptospira/immunology , Leptospirosis/blood , Leukocyte Count , Mass Screening/methods , Observer Variation , Predictive Value of Tests , Sensitivity and SpecificityABSTRACT
OBJECTIVE: To evaluate the macroscopic agglutination test using Temperature Resistant (TR) antigen as a screening test for the diagnosis of human leptospirosis in different stages of the disease. MATERIALS AND METHODS: The criteria for case definition were based on the results of the microscopic agglutination test (MAT), leukocyte counts and neutrophilia, resulting 218 confirmed cases and 242 non- cases. Each sample was classified according to the days of the disease progression in three stages: first (<10 days), second (10 - 25 days) and third (> 25 days). The design was cross-sectional observational. RESULTS: TR sensitivity was 71,1% on stage 1. 93.4% on stage 2 and 95.6% on stage 3. The specificity at different stages ranged from 79.0 to 69.2%. Intra and inter-operator variability was moderate. CONCLUSION: TR variability, low sensitivity in the first stage and low specificity found in all stages of the disease, suggest that it is essential to incorporate new diagnostic methods to screen for early detection of cases in our country and in countries that still apply such methods.
Subject(s)
Agglutination Tests , Leptospirosis/diagnosis , Antigens, Bacterial/immunology , Blood Banks , Blood Preservation , Cross-Sectional Studies , Cryopreservation , Disease Progression , Early Diagnosis , Humans , Leptospira/immunology , Leptospirosis/blood , Leukocyte Count , Mass Screening/methods , Observer Variation , Predictive Value of Tests , Sensitivity and SpecificityABSTRACT
AIMS: To identify LipL32 epitopes and to evaluate their capability to recognize specific antibodies using ELISA. METHODS AND RESULTS: Epitope mapping by means of a library of overlapping peptide fragments prepared by simultaneous and parallel solid phase peptide synthesis on derivatized cellulose membranes (SPOT synthesis) was carried out. Eighty-seven overlapping decapentapeptides corresponding to the complete sequence of LipL32 were synthesized. According to spot-image intensities, the most reactive sequences were localized in regions 151-177 (sequence AAKAKPVQKLDDDDDGDDTYKEERHNK) and 181-204 (sequence LTRIKIPNPPKSFDDLKNIDTKKL). Two peptides (P1 and P2) corresponding to these sequences were synthesized, and their reactivity evaluated using ELISA test. CONCLUSIONS: Epitope identification and analysis suggested the existence of two antigenic regions within LipL32. These LipL32 reactive regions were highly conserved among antigenically variants of Leptospira spp. isolates. Peptides containing these regions (P1 and P2) showed a good capability for anti-leptospiral antibody recognition. SIGNIFICANCE AND IMPACT OF THE STUDY: This finding could have potential relevance not only for serodiagnosis but also as a starting point for the characterization of targets for vaccine design.
Subject(s)
Epitope Mapping , Leptospira/immunology , Amino Acid Sequence , Enzyme-Linked Immunosorbent Assay , Humans , Leptospira/chemistry , Leptospirosis/immunology , Leptospirosis/microbiology , Molecular Sequence Data , Peptides/chemical synthesis , Peptides/chemistry , Peptides/immunologyABSTRACT
OBJECTIVE: To develop a solid-phase enzyme immunoassay (ELISA) for genus-specific immunoglobulin G (IgG) determination with leptospirosis and to evaluate the ELISA in different stages of the disease. METHODS: A total of 1,077 serum samples from 812 patients with suspected leptospirosis were analyzed. The samples had come from diagnoses done in the laboratory of the National Institute of Respiratory Diseases (Instituto Nacional de Enfermedades Respiratorias), in the city of Santa Fe, Argentina, between 1999 and 2005. Included in the study were 182 confirmed cases (267 samples), 167 negative cases (293 samples), and 40 probable cases (60 samples) (based on case definitions based on the results from the microscopic agglutination test (MAT), leukocyte counts, and neutrophilia values). Each sample was classified, according to the days of the natural history of disease, into one of three stages: first (< 10 days), second (10-25 days), or third (> 25 days). The antigen used in the ELISA was an extract of a mixture of pyrogenes and tarassovi serovars cultivated in a liquid medium, treated with ultrasound, and immobilized by adsorption on polystyrene plates. As a secondary antibody, a peroxidase-conjugated goat anti-human IgG monoclonal antibody was used. The cutoff value, sensitivity, and specificity of the ELISA were determined using the definitions of confirmed cases and of negatives cases as the standard. In order to determine the optimal cutoff value, the area under the receiver operating characteristic curve was calculated. RESULTS: The sensitivity of the evaluated test was much higher in the second stage (93.2%) than in either the first stage (68.1%) or the third stage (78.8%). The specificity increased gradually from 96.3% in the first stage to 100% in the third stage. CONCLUSIONS: Our results indicate that this ELISA test can be a very useful complement to the MAT for the diagnosis of leptospirosis in all the stages and, in particular, in order to diagnose acute disease sooner.
Subject(s)
Enzyme-Linked Immunosorbent Assay , Leptospirosis/diagnosis , Acute Disease , Adolescent , Adult , Aged , Agglutination Tests/methods , Argentina , Child , Child, Preschool , Enzyme-Linked Immunosorbent Assay/methods , Female , Humans , Infant , Male , Middle Aged , Sensitivity and Specificity , Time FactorsABSTRACT
OBJETIVO: Desarrollar un enzimoinmunoensayo en fase sólida (ELISA) para la determinación de inmunoglobulinas G (IgG) (específico de género) y evaluarlo en diferentes etapas de la enfermedad. MÉTODOS: Se analizaron 1 077 muestras séricas de 812 pacientes con sospecha de leptospirosis derivadas al laboratorio del Instituto Nacional de Enfermedades Respiratorias (INER) de la ciudad de Santa Fe, Argentina, entre 1999 y 2005. A partir de un criterio de definición de casos basado en los resultados de la microaglutinación (MAT) y del recuento de leucocitos, y de los valores de neutrofilia, se incluyeron en el estudio 182 casos confirmados (267 muestras), 167 casos negativos (293 muestras) y 40 casos probables (60 muestras). Cada muestra se clasificó según el tiempo de evolución de la enfermedad en tres etapas: primera (< 10 días), segunda (10-25 días) y tercera (> 25 días). En el ELISA, se utilizó como antígeno un extracto de una mezcla de las serovariedades Pyrogenes y Tarassovi cultivadas en medio líquido, tratado con ultrasonidos e inmovilizado por adsorción en placas de poliestireno. Como anticuerpo secundario se empleó un anticuerpo monoclonal de cabra anti-IgG humana conjugado con peroxidasa. El valor de corte, la sensibilidad y la especificidad del ELISA se determinaron utilizando como patrón la definición de casos. Para determinar el valor de corte óptimo se calculó el área bajo la curva de eficacia diagnóstica (curva ROC). RESULTADOS: La sensibilidad de la prueba evaluada aumentó considerablemente en la segunda etapa (93,2 por ciento), con respecto a la primera (68,1 por ciento), y descendió en la tercera (78,8 por ciento). La especificidad aumentó gradualmente desde el 96,3 por ciento en la primera etapa hasta el 100 por ciento en la tercera. CONCLUSIONES: Los resultados obtenidos indican que esta prueba de ELISA puede ser de gran utilidad como complemento de la MAT para el diagnóstico de la leptospirosis en todas las etapas y, en...
OBJECTIVE: To develop a solid-phase enzyme immunoassay (ELISA) for genus-specific immunoglobulin G (IgG) determination with leptospirosis and to evaluate the ELISA in different stages of the disease. METHODS: A total of 1 077 serum samples from 812 patients with suspected leptospirosis were analyzed. The samples had come from diagnoses done in the laboratory of the National Institute of Respiratory Diseases (Instituto Nacional de Enfermedades Respiratorias), in the city of Santa Fe, Argentina, between 1999 and 2005. Included in the study were 182 confirmed cases (267 samples), 167 negative cases (293 samples), and 40 probable cases (60 samples) (based on case definitions based on the results from the microscopic agglutination test (MAT), leukocyte counts, and neutrophilia values). Each sample was classified, according to the days of the natural history of disease, into one of three stages: first (< 10 days), second (10-25 days), or third (> 25 days). The antigen used in the ELISA was an extract of a mixture of pyrogenes and tarassovi serovars cultivated in a liquid medium, treated with ultrasound, and immobilized by adsorption on polystyrene plates. As a secondary antibody, a peroxidase-conjugated goat anti-human IgG monoclonal antibody was used. The cutoff value, sensitivity, and specificity of the ELISA were determined using the definitions of confirmed cases and of negatives cases as the standard. In order to determine the optimal cutoff value, the area under the receiver operating characteristic curve was calculated. RESULTS: The sensitivity of the evaluated test was much higher in the second stage (93.2 percent) than in either the first stage (68.1 percent) or the third stage (78.8 percent). The specificity increased gradually from 96.3 percent in the first stage to 100 percent in the third stage. CONCLUSIONS: Our results indicate that this ELISA test can be a very useful complement to the MAT for the diagnosis of leptospirosis in...
Subject(s)
Adolescent , Adult , Aged , Child , Child, Preschool , Female , Humans , Infant , Male , Middle Aged , Enzyme-Linked Immunosorbent Assay , Leptospirosis/diagnosis , Acute Disease , Agglutination Tests/methods , Argentina , Enzyme-Linked Immunosorbent Assay/methods , Sensitivity and Specificity , Time FactorsABSTRACT
The study of interactions between biological molecules and model membranes is essential for the understanding of a number of physiological mechanisms involved in viral infections and dissemination. In this paper, the analysis of the interaction between a peptide from the p24 protein of Human Immunodeficiency Virus type 1 (HIV-1) and a phospholipid monolayer has pointed to a cooperative response in which very small amounts of peptide p24-1 (e.g. 0.05 mol%) can lead to measurable effects. Monolayer surface pressure and surface potential isotherms were affected for peptide concentrations as low as 0.05 mol%, with saturation at 0.5 mol%. The expansion effect from p24-1 is confirmed by changes in morphology of the monolayers using Brewster angle microscopy. Even though p24-1 is disordered in aqueous solutions, the interaction with dipalmitoyl phosphatidylcholine (DPPC) causes it to adopt an alpha-helix structure, as shown by circular dichroism (CD) data for multilamellar vesicles (MLV). The expansion of the phospholipid monolayer in a cooperative way may imply that p24-1 has potential antiviral activity, by participating in the cell rupture, with no need of specific receptors in the membrane.
Subject(s)
1,2-Dipalmitoylphosphatidylcholine/chemistry , HIV Core Protein p24/chemistry , HIV-1/chemistry , Peptides/chemistry , Phosphatidylglycerols/chemistry , 1,2-Dipalmitoylphosphatidylcholine/metabolism , Amino Acid Sequence , Circular Dichroism , HIV Core Protein p24/metabolism , Humans , Molecular Sequence Data , Phosphatidylglycerols/metabolism , Protein Structure, SecondaryABSTRACT
We studied the interaction of the peptide AAMQMLKETINEEAAEWDRVHPVHAGPIA from the HIV-1 p24 protein in the presence of SDS (anionic) and CTABr (cationic) micelles at pH 7.0 by circular dichroism, fluorescence, and electron spin resonance (ESR). The micelles induced secondary structure as well as a blue shift in the tryptophan fluorescence emission, indicating an interaction between the peptide and the micelles. However, different contents of secondary structure elements were found when the peptide interacts with SDS or CTABr micelles. Steady-state anisotropy indicates a constraint on the rotational mobility of the tryptophan residue of the peptide upon interaction with micelles. ESR studies pointed to different locations for the peptide in either micelle. Our results suggested that at least part of the peptide might be located at the hydrophobic core of the CTABr micelles, probably at the C-terminal region, while it is more inserted into the SDS micelles.
