Presence of Leptospira spp. in Caiman latirostris (Crocodylia, Alligatoridae) populations in Santa Fe, Argentina.

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.

Clinical Evaluation of Rapid Diagnostic Test Kit Using the Polysaccharide as a Genus-Specific Diagnostic Antigen for Leptospirosis in Korea, Bulgaria, and Argentina.

Leptospirosis, a zoonotic disease that is caused by many serovars which are more than 200 in the world, is an emerging worldwide disease. Accurate and rapid diagnostic tests for leptospirosis are a critical step to diagnose the disease. There are some commercial kits available for diagnosis of leptospirosis, but the obscurity of a species- or genus-specific antigen of pathogenic Leptospira interrogans causes the reduced sensitivity and specificity. In this study, the polysaccharide derived from lipopolysaccharide (LPS) of nonpathogenic Leptospira biflexa serovar patoc was prepared, and the antigenicity was confirmed by immunoblot and enzyme linked immunosorbent assay (ELISA). The performance of the rapid diagnostic test (RDT) kit using the polysaccharide as a diagnostic antigen was evaluated in Korea, Bulgaria and Argentina. The sensitivity was 93.9%, 100%, and 81.0% and the specificity was 97.9%, 100%, and 95.4% in Korea (which is a rare region occurring with 2 serovars mostly), Bulgaria (epidemic region with 3 serovars chiefly) and Argentina (endemic region with 19 serovars mainly) respectively. These results indicate that this RDT is applicable for global diagnosis of leptospirosis. This rapid and effective diagnosis will be helpful for diagnosis and manage of leptospirosis to use and the polysaccharide of Leptospira may be called as genus specific antigen for diagnosis.

Vigilancia intensificada de Leptospirosis en Santa Fe y Entre Ríos (2012-2013) / Leptospirosis Enhanced Surveillance System in Santa Fe and Entre Rios (2012-2013)

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...

Epitope mapping of pathogenic Leptospira LipL32.

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.

[Diagnosis of leptospirosis: evaluation of a solid-phase enzyme immunoassay in different stages of the disease]. / Diagnóstico de leptospirosis: evaluación de un enzimoinmunoensayo en fase sólida en diferentes etapas de la enfermedad.

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.

Diagnóstico de leptospirosis: evaluación de un enzimoinmunoensayo en fase sólida en diferentes etapas de la enfermedad / Diagnosis of leptospirosis: evaluation of a solid-phase enzyme immunoassay in different stages of the disease

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...

Descripción de un brote de leptospirosis en la ciudad de Santa Fe, Argentina, marzo-abril de 1998 / Description of a leptospirosis outbreak in the city of Santa Fe, Argentina, March-April 1998

En marzo-abril de 1998 se identificó en un barrio de la ciudad de Santa Fe (Argentina) un brote de una enfermedad aguda caracterizada por fiebre, cefaleas y mialgias intensas. Se presentan los estudios realizados en relación con este brote y los intentos de identificación de la fuente y del modo de transmisión. Los hallazgos epidemiológicos, serológicos y clínicos indicaron que el agente causal fue Leptospira interrogans. Como prueba de tamizaje se aplicó la técnica de aglutinación macroscópica con antígeno termorresistente, seguida de la prueba de ELISA y, como prueba de confirmación, la aglutinación microscópica frente a 10 serovariedades de L. interrogans. Se estudiaron 32 individuos, 8 perros y 8 muestras de agua. Se registraron 12 casos confirmados, 2 probables y 18 negativos. En seis perros se demostró la existencia de infección y en las muestras de agua se detectó la presencia de espiroquetas móviles. Los sueros humanos reaccionaron con las serovariedades ballum, canicola, icterohaemorrhagiae y pyrogenes, y los caninos con ballum, canicola y pomona. La coaglutinación observada en todos los casos confirmados indica que se trató de casos agudos de leptospirosis, pero no permite identificar la serovariedad causal. Salvo en el caso índice, no se reconoció clínicamente la enfermedad. Varios hechos sugieren que la causa del brote fue la inundación pluvial de la zona estudiada. Los resultados de este estudio resaltan la necesidad de una vigilancia activa de la leptospirosis ante desastres naturales como las inundaciones