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PURPOSE: The current diagnostic methods for leptospirosis diagnosis are technically complex and expensive, with limited applicability to specialized laboratories. Furthermore, they lack diagnostic accuracy in the acute stage of the disease, which coincides with a period when antibiotics are highly effective. New simple and accurate tests are mandatory to decentralize and improve diagnosis. Here, we introduced a new lateral flow immunoassay (Lepto-LF) for human leptospirosis. METHODS: We conducted a double-blinded assay using 104 serum samples from patients with confirmed or discarded diagnosis for leptospirosis. The diagnostic performance of Lepto-LF was estimated across different ranges of days from onset of symptoms (dpo), considering the diagnostic algorithm as reference standard. Additionally, it was compared with the screening methods enzyme-linked immunosorbent assay (IgM-ELISA) and the slide agglutination test using temperature-resistant antigen (SATR). RESULTS: Lepto-LF exhibited perfect diagnostic performance with a Youden´s index J = 1 from 6 dpo in the acute phase. IgM-ELISA gave slightly lower accuracy with J = 0.91 and 95.5% of both sensitivity and specificity; while SATR showed a markedly inferior yield (J = 0.41, sensitivity = 95.5%, specificity = 45.5%). The performances remained consistent in the convalescence phase of the disease (> 10 dpo). CONCLUSION: Lepto-LF was found to be a reliable test for simple, rapid and early diagnosis of leptospirosis, resulting a promising tool for decentralizing leptospirosis diagnosis and enabling timely treatment of patients. In addition, Lepto-LF may be employed as confirmatory test, especially in remote areas and vulnerable contexts where the standard MAT is not available.
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Leptospirosis is a disease caused by pathogenic spirochetes of the genus Leptospira, transmitted by wild and domestic animals. Rodents play a fundamental role in the transmission cycle of this zoonosis but the function of reptiles is unknown. For example, crocodilians could play an important role in the transmission of this disease by living in ideal environments (bodies of shallow water and high temperatures) for the colonization of this bacterium. However, few studies have documented the presence of zoonotic diseases in caiman populations. Our objective was to assess the prevalence of antibodies to leptospira and the presence of Leptospira spp. in wild and captive Caiman latirostris. Blood samples were taken from 45 individuals (20 wild and 25 captive). Before extraction, we cleaned each caiman's neck in order to prevent contamination of samples. We determined the presence of antibodies in serum by microscopic agglutination test (MAT) and polymerase chain reaction (PCR) to detect DNA of the bacteria. We excluded 9 of the 45 samples analyzed by MAT because 5 had lipemic serum and 4 were contaminated (colonized by other organisms). Of the 36 caimans studied by microscopic agglutination test (MAT), 56% (20/36) were considered reactive (titers ≥50). In 74% (14/19) of captive samples and 35% (6/17) of wild samples, antibodies to leptospira were detected by MAT. The serogroup with highest occurrence was Pyrogenes (85%, n = 17/20), presenting coagglutinations with Icterohaemorrhagiae (25%, n = 5/20). One sample from a captive animal was positive for PCR, and we could not isolate leptospires because of agar contamination. Of the 45 blood agar media, 17.8% were contaminated and the rest were negative. This work determined the presence of Leptospira spp. in one caiman and a high prevalence of antibodies in captive caiman relative to wild individuals.
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OBJECTIVE: To evaluate if the use of the 19 Leptospira strains panel suggested by the International Leptospirosis Society of World Health Organization for microagglutination allows confirmation of more cases that the 12 strains panel used in Argentina. MATERIALS AND METHODS: Cross-sectional observational study. We studied 441 serum samples corresponding to Argentinean patients with suspected leptospirosis derived during from July to December, 2009 and from January to October, 2013. RESULTS: The same number of positive samples was obtained using the MAT with the 19 or 12 strains. In six cases a serovar of the expanded collection was presumably infecting, but always coagglutinated with strains of the reduced panel. CONCLUSION: In Argentina, the diagnosis of leptospirosis by MAT could be made using the reduced 12 strains panel, obtaining the same result in case detection as using the 19 strains panel. Additional information provided by the use of all strains could be the presumably infecting serogroup.
Assuntos
Testes de Aglutinação/normas , Leptospira/classificação , Leptospirose/diagnóstico , Argentina/epidemiologia , Estudos Transversais , Humanos , Leptospirose/epidemiologia , Leptospirose/microbiologia , SorogrupoRESUMO
Objetivo. Evaluar si el uso del panel de 19 cepas de leptospiras, sugerido por la Sociedad Internacional de Leptospirosis para la microaglutinación (MAT, por sus siglas en inglés), permite mayor confirmación de casos que el de 12 cepas. Material y métodos. Estudio observacional de corte transversal. Se estudiaron 441 muestras de sueros de pacientes de Argentina, derivadas para el diagnóstico de leptospirosis en los periodos de julio de 2009 a diciembre de 2010 y enero a octubre de 2013. Resultados. Se obtuvo el mismo resultado con el panel reducido que con el ampliado. En seis casos resultó presumiblemente infectante algún serovar del panel ampliado, aunque siempre coaglutinando con cepas del reducido. Conclusión. En Argentina, el diagnóstico de leptospirosis por MAT podría continuar realizándose con el panel reducido, lo que reduciría el costo y tiempo de diagnóstico. La información adicional que aportaría el panel ampliado está relacionada con la epidemiología, mediante un mejor conocimiento del serogrupo presumiblemente infectante.
Objective. To evaluate if the use of the 19 Leptospira strains panel suggested by the International Leptospirosis Society of World Health Organization for microagglutination allows confirmation of more cases that the 12 strains panel used in Argentina. Materials and methods. Cross-sectional observational study. We studied 441 serum samples corresponding to Argentinean patients with suspected leptospirosis derived during from July to December, 2009 and from January to October, 2013. Results. The same number of positive samples was obtained using the MAT with the 19 or 12 strains. In six cases a serovar of the expanded collection was presumably infecting, but always coagglutinated with strains of the reduced panel. Conclusion. In Argentina, the diagnosis of leptospirosis by MAT could be made using the reduced 12 strains panel, obtaining the same result in case detection as using the 19 strains panel. Additional information provided by the use of all strains could be the presumably infecting serogroup.
Assuntos
Humanos , Testes de Aglutinação/normas , Leptospira/classificação , Leptospirose/diagnóstico , Argentina/epidemiologia , Estudos Transversais , Sorogrupo , Leptospirose/microbiologia , Leptospirose/epidemiologiaRESUMO
La leptospirosis es la zoonosis de mayor prevalencia mundial. Santa Fe y Entre Ríos concentran la mayoría de casos en Argentina. OBJETIVO: Implementar y describir un sistema de vigilancia intensificada de casos de leptospirosis en Santa Fe y Entre Ríos. MÉTODOS: La investigación, desarrollada desde enero de 2012 hasta marzo de 2013, implicó la intervención en sistemas y servicios de salud, así como el fortalecimiento de la red de laboratorios para el diagnóstico específico y aislamiento de leptospiras. La información obtenida a partir de las fichas clínico-epidemiológicas de los casos confirmados fue analizada y presentada mediante medidas de resumen. La vigilancia intensificada se implementó en siete hospitales estratégicos. RESULTADOS: Ingresaron 183 pacientes. Se confirmaron 24 casos (13%): 10 por MAT, 9 por PCR en tiempo real y 5 por ambos métodos. Se obtuvieron 3 aislamientos (sero grupo Canicola). Fallecieron 4 pacientes con hemorragia pulmonar sumada a compromiso renal, hepático y/o plaquetopenia. CONCLUSIONES: La vigilancia intensificada permitió obtener aislamientos humanos, ratificó el valor de la MAT, evidenció la utilidad de PCR en tiempo real para diagnóstico precoz y corroboró la dificultad de obtener segundas muestras. La presentación clínica evidenció una elevada mortalidad global y una alta frecuencia de compromiso respiratorio asociado a disfunciones orgánicas múltiples de aparición precoz...
Leptospirosis is the most prevalent zoonosis worldwide. In Argentina, Santa Fe and Entre Ríos are the provinces where most cases occur. OBJECTIVE:To implement and describe a system of enhanced surveillance of leptospirosis in Santa Fe and Entre Ríos. METHODS: The enhanced surveillance was carried out from January 2012 to March 2013. The study involved the intervention in health services and systems as well as the laboratory network strengthening for specific diagnosis and leptospira isolation. The information collected from clinical epidemiological records of confirmed cases was analyzed and presented through summary measures. The surveillance was implemented in seven strategic hospitals. RESULTS: A total of 183 patients were enrolled, and 24 cases (13%)were confirmed: 10 through MAT, 9 through real time PCR and 5 through both methods. It was possible to obtain 3 isolates (Canicola serogroup). 4 patients died with pulmonary hemorrhage coupled with renal impairment, hepaticand/ or thrombocytopenia. CONCLUSIONS: The enhanced surveillance allowed to obtain human isolates, confirmed the diagnostic value of MAT, showed the utility of real time PCR for early diagnosis and the difficulties to obtain second samples. The clinical presentation revealed a high global mortality and a high frequency of respiratory involvement related to multiple organ dysfunctions from early stages...
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Humanos , Diagnóstico Precoce , Serviços de Vigilância Epidemiológica , Planos de Sistemas de Saúde , Leptospirose/epidemiologia , Monitoramento Epidemiológico/organização & administraçãoRESUMO
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.
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Ensaio de Imunoadsorção Enzimática , Leptospirose/diagnóstico , Doença Aguda , Adolescente , Adulto , Idoso , Testes de Aglutinação/métodos , Argentina , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Fatores de TempoRESUMO
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...