RESUMEN
BACKGROUND: There is little information about the frequency of Leishmania infection in asymptomatic people living with HIV (PLWH) and about the performance of laboratory diagnostic methods in coinfected patients in Latin America. The main objective of this study is to evaluate the frequency of Leishmania spp. infection in HIV-infected patients living in an urban area in Brazil. METHODS: To detect Leishmania infection, diagnostic tests were performed to detect anti-Leishmania antibodies (ELISA using Leptomonas seymouri antigens; ELISA using rK39 antigens; ELISA using rK28 antigens; indirect fluorescent-antibody test (IFAT); direct agglutination test (DAT)) and Leishmania DNA (polymerase chain reaction (PCR) with the target genes kDNA and ITS-1). RESULTS: The frequency of at least one positive test was 15%. For ELISA using Leptomonas antigens and IFAT, there was an association between CD4+ T lymphocyte counts and test positivity, with a higher positivity of these tests in more immunosuppressed patients (CD4+ T cell count < 200/mm3). CONCLUSIONS: According to our data, there was a high prevalence of Leishmania spp. infections in this population living with HIV. Although there is the possibility of cross-reaction, some tests that are considered highly specific for the diagnosis of Leishmania infection were positive. There was also an association between the positivity of some tests studied and lower values of CD4+ T lymphocytes.
Asunto(s)
Coinfección/epidemiología , Infecciones por VIH/epidemiología , VIH , Leishmania/genética , Leishmania/inmunología , Leishmaniasis Visceral/diagnóstico , Leishmaniasis Visceral/epidemiología , Adulto , Anciano , Anciano de 80 o más Años , Pruebas de Aglutinación , Animales , Brasil/epidemiología , Recuento de Linfocito CD4 , Estudios de Cohortes , Coinfección/virología , Estudios Transversales , ADN de Cinetoplasto/genética , Pruebas Diagnósticas de Rutina/métodos , Ensayo de Inmunoadsorción Enzimática , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Infecciones por VIH/virología , Humanos , Leishmaniasis Visceral/parasitología , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Prevalencia , Adulto JovenRESUMEN
The serologic assay is an important tool in the diagnosis of leishmaniasis. One of the most commonly used tests is enzyme-linked immunosorbent assay (ELISA). Since total Leishmania promastigotes are used as antigen in the routine assay, false-positive reactions are frequent due to cross-reaction with sera from other diseases, mainly Chagas' disease. Therefore, an antigen that determines less cross-reactivity has been pursued for the serodiagnosis of leishmaniasis. In the present study we analyzed the use of recombinant Leishmania infantum heat shock protein (Hsp) 83 in ELISA for the serodiagnosis of cutaneous (N = 12) and mucocutaneous leishmaniasis (N = 14) and we observed the presence of anti-L. infantum Hsp 83 antibodies in all samples as well as anti-Leishmania total antigen antibodies. When cross-reactivity was tested, chronic Chagas' disease patients (N = 10) did not show any reactivity. Therefore, we consider this L. infantum Hsp 83 to be a good antigen for routine use for serodiagnosis of tegumentary leishmaniasis.
Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/sangre , Proteínas de Choque Térmico , Leishmania infantum/inmunología , Leishmaniasis Cutánea/diagnóstico , Proteínas Protozoarias , Animales , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Estudios de Casos y Controles , Enfermedad de Chagas/diagnóstico , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Epítopos , Técnica del Anticuerpo Fluorescente , Humanos , Pruebas Serológicas/métodosRESUMEN
The serologic assay is an important tool in the diagnosis of leishmaniasis. One of the most commonly used tests is enzyme-linked immunosorbent assay (ELISA). Since total Leishmania promastigotes are used as antigen in the routine assay, false-positive reactions are frequent due to cross-reaction with sera from other diseases, mainly Chagas' disease. Therefore, an antigen that determines less cross-reactivity has been pursued for the serodiagnosis of leishmaniasis. In the present study we analyzed the use of recombinant Leishmania infantum heat shock protein (Hsp) 83 in ELISA for the serodiagnosis of cutaneous (N = 12) and mucocutaneous leishmaniasis (N = 14) and we observed the presence of anti-L. infantum Hsp 83 antibodies in all samples as well as anti-Leishmania total antigen antibodies. When cross-reactivity was tested, chronic Chagas' disease patients (N = 10) did not show any reactivity. Therefore, we consider this L. infantum Hsp 83 to be a good antigen for routine use for serodiagnosis of tegumentary leishmaniasis.
Asunto(s)
Humanos , Animales , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/sangre , Proteínas de Choque Térmico , Leishmania infantum/inmunología , Leishmaniasis Cutánea/diagnóstico , Proteínas Protozoarias , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Estudios de Casos y Controles , Reacciones Cruzadas , Enfermedad de Chagas/diagnóstico , Ensayo de Inmunoadsorción Enzimática , Epítopos , Técnica del Anticuerpo Fluorescente , Pruebas Serológicas/métodosRESUMEN
This paper deals with the analysis of 10 batches of L.major-like and L. (V.) braziliensis antigens added or not of a proteases inhibitor evaluated by means of an IgG-ELISA on three consecutive days using positive standard sera from patients with diagnosis of American Leishmaniasis previously tested for the presence of IgG antibodies by means of ELISA. The statistical analysis showed that for L. (V.) braziliensis the PMSF-containing antigen did not show any difference among batches or days of testing; the L. (V.) braziliensis antigen without PMSF showed statistical significance for differences among batches and a two-way ANOVA showed significant differences between antigens. L.major-like antigen prepared with or without PMSF showed differences among batches; all 3 days of testing displayed differences for the PMSF antigen but only for days 1 and 2 for the antigen without inhibitor. A two-way ANOVA showed differences among batches of the antigens but not for antigens with and without the protein inhibitor. According to the statistical analysis the L.major-like antigen added or not of PMSF has shown that it is the choice antigen for mucocutaneous leishmaniasis serology.
Asunto(s)
Antígenos de Protozoos/análisis , Inmunoglobulina G/análisis , Leishmania braziliensis/inmunología , Leishmania major/inmunología , Inhibidores de Proteasas/inmunología , Compuestos de Tosilo/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Niño , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reproducibilidad de los ResultadosRESUMEN
The influence of time and temperature on the storage of an alkaline antigen of L. major-like and L.(V.) braziliensis promastigotes added or not of a proteases inhibitor (PMSF) was evaluated by means of an IgG-ELISA. Antibodies in assays using L. major-like antigen stored at -20 degrees C for 6 months had a statistically lower geometric mean titer (GMT) and different 95% confidence interval limits (CL) than antigens stored otherwise, as assessed by the [quot ]t[quot ] statistic. The PMSFL. major-like antigen after storage for 6 months at a temperature of 4 degrees C had the same GMT and 95% CL displayed at time zero as well as when storage for 4 and 6 months at -20 degrees C. Significant differences were not found when L.(V.) braziliensis antigens were stored at times and temperatures mentioned; the PMSF antigen stored for 2 months at -70 degrees C resulted in a lower serum GMT and 95% CL than any other, as assessed by the [quot ]t[quot ] statistic. Antigen performance did not show any statistical difference associated to the addition of PMSF within the same species; the largest difference between antigens was that between PMSF-L. (V.) braziliensis and L. major-like without PMSF.
A influência do tempo e temperatura de estocagem de antígenos alcalinos de promastigotas de L. major-like e L. (V.) braziliensis adicionados ou não de um inibidor de proteases foi avaliada por meio de reações de IgG-ELISA. A reação que empregava o antígeno de L. major-like estocado por 6 meses a -20oC mostrou que médias geométricas dos títulos (MGT)e intervalos de confiança 95% (IC 95%) eram estatisticamente inferiores àquelas obtidas com antígenos estocados em outros intervalos de tempo, medido pela estatística "t". O antígeno PMSF-L. major-like depois de 6 meses de estocagem à temperatura de 4oC tinha a mesma MGT e IC 95% do tempo zero assim como quando ele foi estocado a -20oC por 4 e 6 meses. Não foram observadas diferenças estatisticamente diferentes com os antígenos de L. (V.) braziliensis estocados nas mesmas condições de tempo e temperatura exceto o antígeno PMSF estocado por 2 meses a -70oC que apresentou MGT e IC 95% inferiores a quaisquer outras como aferido pela estatísitca "t". Quando comparados os desempenhos dos antígenos não houve direrenças estatisticamente significantes entre a adição ou não de PMSF para qualquer dos parasitas. A análise do cruzamento entre antígenos mostrou que a maior diferença netre eles foi a do contraste entre L. (V.) braziliensis adicionado de PMSF e L. major-like sem adição de PMSF.
Asunto(s)
Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Niño , Femenino , Humanos , Masculino , Persona de Mediana Edad , Antígenos de Protozoos/aislamiento & purificación , Leishmania braziliensis/inmunología , Leishmania major/inmunología , Antígenos de Protozoos , Preservación Biológica , Temperatura , Factores de Tiempo , Técnicas para Inmunoenzimas/estadística & datos numéricosRESUMEN
The influence of time and temperature on the storage of an alkaline antigen of L. major-like and L.(V.) braziliensis promastigotes added or not of a proteases inhibitor (PMSF) was evaluated by means of an IgG-ELISA. Antibodies in assays using L. major-like antigen stored at -20 degrees C for 6 months had a statistically lower geometric mean titer (GMT) and different 95% confidence interval limits (CL) than antigens stored otherwise, as assessed by the "t" statistic. The PMSFL. major-like antigen after storage for 6 months at a temperature of 4 degrees C had the same GMT and 95% CL displayed at time zero as well as when storage for 4 and 6 months at -20 degrees C. Significant differences were not found when L.(V.) braziliensis antigens were stored at times and temperatures mentioned; the PMSF antigen stored for 2 months at -70 degrees C resulted in a lower serum GMT and 95% CL than any other, as assessed by the "t" statistic. Antigen performance did not show any statistical difference associated to the addition of PMSF within the same species; the largest difference between antigens was that between PMSF-L. (V.) braziliensis and L. major-like without PMSF.
Asunto(s)
Antígenos de Protozoos/aislamiento & purificación , Leishmania braziliensis/inmunología , Leishmania major/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Niño , Femenino , Humanos , Técnicas para Inmunoenzimas/estadística & datos numéricos , Masculino , Persona de Mediana Edad , Preservación Biológica , Temperatura , Factores de TiempoRESUMEN
A slide hemagglutination test, here called SHAT, which is practical and economical for seroepidemiological surveys was standardized. This is an improved modification of the rapid hemagglutination test (RHA) which utilizes a short-lived reagent prepared with fresh blood cells. The reagent and conditions of the test were considerably modified and, most importantly, an alkaline-solubilized Trypanosoma cruzi epimastigote antigen reagent is proposed. The stability of the SHAT reagent was at least one year at 4 degrees C, in an appropriate liquid suspension. The SHAT was applied to 71 serum samples from patients with Chagas' disease and from 235 clinically healthy blood donors. Sensitivity, specificity and positive and negative predictive values for the selected cutoff titer corresponding to 1:4 dilution were 0.972 (0.903-0.992), 0.983 (0.957-0.993), 0.945 (0.867-0.979) and 0.991 (0.969-0.998), respectively. These values were comparable to those found for the RHA, immunofluorescence (IFT), indirect hemagglutination (IHAT) and complement fixation (CFT) tests. These data suggest that the SHAT should be useful for seroepidemiological surveys conducted at public health laboratories in developing countries.
Asunto(s)
Enfermedad de Chagas/diagnóstico , Pruebas de Hemaglutinación/métodos , Humanos , Valor Predictivo de las Pruebas , Sensibilidad y Especificidad , Estudios SeroepidemiológicosRESUMEN
A slide hemagglutination test, here called SHAT, which is practical and economical for seroepidemiological surveys was standardized. This is an improved modification of the rapid hemagglutination test (RHA) which utilizes a short-lived reagent prepared with fresh blood cells. The reagent and conditions of the test were considerably modified and, most importantly, an alkaline-solubilized Trypanosoma cruzi epimastigote antigen reagent is proposed. The stability of the SHAT reagent was at least one year at 4 degrees Celsius, in an appropriate liquid suspension. The SHAT was applied to 71 serum samples from patients with Chagas' disease and from 235 clinically healthy blood donors. Sensitivity, specificity and positive and negative predictive values for the selected cutoff titer corresponding to 1:4 dilution were 0.972 (0.903-0.992)9 O.983 (O.957-0.993) 0.945 (O.867-0.979) and O.991 (O.969-0.998), respectively. These values were comparable to those found for the RHA, immunofluorescence (IFT), indirect hemagglutination (IHAT) and complement fixation (CFT) tests. These data suggest that the SHAT should be useful for seroepidemiological surveys conducted at public health laboratories in developing countries.
Asunto(s)
Humanos , Enfermedad de Chagas/diagnóstico , Pruebas de Hemaglutinación/métodos , Valor Predictivo de las Pruebas , Sensibilidad y Especificidad , Estudios SeroepidemiológicosRESUMEN
The performance of an antigen of L. major-like promastigotes for the serological diagnosis of mucocutaneous leishmaniasis in the IgG-immunofluorescent test was compared to that of an antigen of L.braziliensis braziliensis. Each antigen was used to test two hundred and twenty-four sera of etiologies such as mucocutaneous leishmaniasis, deep mycoses, toxoplasmosis, malaria. Chagas' disease, visceral leishmaniasis, anti-nuclear factor, schistosomiasis, rheumatoid factor and normal controls. Agreement between responses to each antigen was high: 77.2% of leishmaniases sera agreed on a positive or a negative result to both antigens and 91.1% of control sera. Cross reactivity was restricted to Chagas' disease sera, visceral leishmaniasis, anti-nuclear factor and paracoccidioidomycosis. The quantitative response of leishmaniasis and Chagas' disease sera to both antigens was evaluated by a linear regression; although the y-intercept and the slope were different for each antigen, neither was better than the other in the disclosure of anti-Leishmania antibodies. In the case of Chagas' disease sera the L.major-like antigen was better than L.b.braziliensis' to disclose cross-reacting antibodies.
Asunto(s)
Antígenos de Protozoos/sangre , Inmunoglobulina G/inmunología , Leishmania braziliensis/inmunología , Leishmania tropica/inmunología , Leishmaniasis Cutánea/diagnóstico , Animales , Técnica del Anticuerpo Fluorescente , Humanos , Valor Predictivo de las Pruebas , Análisis de RegresiónRESUMEN
Diagnostic performance indexes of sensitivity, specificity, positive predictive value and efficiency were determined for dot-ELISA and IgG-ELISA tests in 340 leishmaniasis sera. Sensitivity of the dot-ELISA was significantly lower than IgG-ELISA's; the two tests had indexes of specificity and positive predictive value of the same magnitude. Seventy-eight sera gave a negative dot-ELISA test result and a positive IgG-ELISA test result. When sera were classified according to different criteria as how to interpret this diversity, the kappa statistic did not corroborate the classification indicating that the two tests display a substantial strength of agreement. The results presented indicate that performance indexes accrued in a survey where variables are well known may be extrapolated to other population studies if the disease presents itself as highly prevalent (due to a selection bias or not) and may be expected to discriminate a disease status among test positives.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Leishmaniasis Cutánea/diagnóstico , Humanos , Leishmaniasis Cutánea/epidemiología , Prevalencia , Sensibilidad y EspecificidadRESUMEN
A total of 341 sera were screened for anti-Leishmania IgA, IgG, and IgM antibodies by immunofluorescent (IF) tests and enzyme immunoassay (ELISA). Altogether, 292 of the sera originated from patients with clinically as well as parasitologically diagnosed (positive lesion imprint or the Montenegro skin test) cutaneous leishmaniasis; 49 of the sera were from controls from the same base population. In terms of diagnostic performance, the ELISAs for IgG and IgM yielded indices of diagnostic utility, and the positive predictive value for the IgG-ELISA was 94.6%. A remarkably high specificity (100%) was obtained with the IgA-IF test, but its sensitivity was very low.