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

Development and validation of an ELISA for the detection of leptospire-specific antibodies in rodents

An enzyme-linked immunosorbent assay (ELISA) for the detection of IgG antibodies in rodents was developed and validated with the microscopic agglutination test (MAT) and leptospiral cultures. Sonicated antigen from cultures of serovars tarassovi and pyrogenes was used. As conjugate, a combination of anti-rat and anti-hamster IgG labeled with peroxidase was used. The optimal cut-off point was determined by plotting the sensitivity and specificity for various cut-off point values by means of receiver operating characteristic (ROC) curve. Concordance between ELISA and each of the MAT titers was measured by kappa (kappa). Proportions of positive results were compared by means of McNemar's test. Total 214 rodents were trapped, but only 117 could be processed by the three techniques (culture, ELISA, MAT; 1:20, 1:40, 1:50) and used for statistical analysis. Although, MAT titers in rodents infected with the serogroup Ballum tended to be lower than those infected with the serogroup Icterohaemorrhagiae, all (20/20) were ELISA-positive and almost all (19/20) were MAT-positive.The percentage of positive results obtained by ELISA, 47.0% exceeded significantly the 40.2% obtained by MAT (1:50). Difference between ELISA and MAT (1:40) was not significant and no differences were observed between ELISA and MAT (1:20). Agreement, specificity, sensitivity and the consequent area under the ROC curve between ELISA and MAT were higher as MAT cut-off points were lowered, being optimal at 1:20. The fact that differences between ELISA and MAT were significant at 1:50, but not at 1:40 or 1:20, supports the suggestion that lower MAT titers should be considered positive in rodents. The ELISA developed to detect leptospire-specific antibodies had optimal sensitivity and specificity in relation to MAT and it is concluded that it may constitute a very useful indicator for epidemiological purposes of past or present leptospiral infection in rodents

Enzyme-linked immunosorbent assay to diagnose human leptospirosis: a meta-analysis of the published literature

We report an evaluation of the accuracy of ELISA for the detection of Leptospira-specific antibodies in humans. Eighty-eight studies published in 35 articles met all inclusion criteria and were submitted to meta-analysis. Pooled sensitivity and specificity were 0·779 (95% CI 0·770-0·789) and 0·913 (95% CI 0·908-0·917), respectively, and the area under the curve was 0·964. Heterogeneity across studies was statistically significant, but none of the sources of heterogeneity (disease stage, antigen used, antibody detected) could fully explain this finding. Although the convalescent stage of disease was significantly associated with higher diagnostic accuracy, IgM ELISA was the best choice, regardless of the stage of disease. Negative ELISAs (IgG or IgM) applied in the acute phase do not rule out leptospirosis due to the possibility of false-negative results. In this case it is advisable to request a second blood sample or to apply a direct method for leptospiral DNA

Associations between leptospiral infection and seropositivity in rodents and environmental characteristics in Argentina

Fil: Vanasco, Bibiana N. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Respiratorias; Argentina.Fil: Sequeira, María Delfina. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Respiratorias; Argentina.Fil: Sequeira, Gabriel. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias; Argentina.Fil: Tarabla, Héctor D. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias; Argentina.Our objective was to look for associations between leptospiral infection in rodents and selected environmental and rodent characteristics in Santa Fe, Argentina. Rodents (n = 214) were trapped alive from January 1998 to December 1999 in three environmental settings. Kidneys from 118 rodents were cultured and serum samples from 201 were processed by enzyme-linked immunosorbent assay (ELISA). Logistic regression was performed with ELISA seropositivity as the dependent variable and rodent characteristics were offered as independent variables. Overall prevalence of positive ELISA reactions was 42% (84/201). In urban areas, leptospiral isolations belonged to the Ballum serogroup; in natural corridors, they belonged to the Icterohaemorragiae serogroup. M. musculus (house mouse) was the most-frequently captured species and the predominant one in urban areas. Most isolates and seropositivity results were obtained on this species. Adults and subadults had higher seroprevalences than juvenile rodents. Oligoryzomys flavescens had higher seroprevalence than Akodon azarae, Mus musculus, Rattus rattus and Rattus norvegicus

Comparison of three diagnostic techniques for the detection of leptospires in the kidneys of wild house mice (Mus musculus)

Forty-one wild house mice (Mus musculus) were trapped in an urban area, near railways, in Santa Fe city, Argentina. Both kidneys from each mouse were removed for bacteriological and histological examination. One kidney was inoculated into Fletcher semi-solid medium and isolates were serologically typed. The other kidney was microscopically examined after hematoxylin-eosin, silver impregnation and immunohistochemical stains. Leptospires, all of them belonging to the Ballum serogroup, were isolated from 16 (39%) out of 41 samples. The presence of the agent was recorded in 18 (44%) and in 19 (46%) out of 41 silver impregnated and immunohistochemically stained samples respectively. Additionally, leptospires were detected in high number on the apical surface of epithelial cells and in the lumen of medullary tubules and they were less frequently seen on the apical surface of epithelial cells or in the lumen of the cortical tubules, which represents an unusual finding in carrier animals. Microscopic lesions consisting of focal mononuclear interstitial nephritis, glomerular shrinkage and desquamation of tubular epithelial cells were observed in 13 of 19 infected and in 10 of 22 non-infected mice; differences in presence of lesions between infected and non-infected animals were not statistically significant (P=0,14). The three techniques, culture, silver impregnation and immunohistochemistry, had a high agreement (k³0.85) and no significant differences between them were detected (P>0.05). In addition, an unusual location of leptospires in kidneys of carrier animals was reported, but a relationship between lesions and presence of leptospires could not be established.Foram capturados 41 camundongos (Mus musculus) na região urbana, próximo à ferrovia da cidade de Santa Fé, Argentina. Os rins de cada animal capturado foram removidos para estudos bacteriológicos e histológicos. Um dos rins foi imerso em meio semi-sólido de Fletcher para isolamento de leptospiras, as quais foram serologicamente tipificadas. O outro rim foi microscopicamente examinado por coloração de cortes histológicos pela hematoxilina-eosina, impregnação pela prata e imunohistoquímica. Leptospiras pertencentes ao serogrupo Ballum foram isoladas em 16 (39%) das 41 amostras availadas. A presença do agente foi observada em 18 (44%) e 19 (46%) das 41 amostras avaliadas por impregnação pela prata e imunohistoquímica, respectivamente. Leptospiras foram detectadas em grande numero na superfície apical das células epiteliais e no lumen dos túbulos medulares e foram menos frequentemente encontradas na superficie apical de células epiteliais ou no lúmen dos túbulos corticais, o que é considerado achado raro em animais portadores. Lesões microscópicas consistindo de nefrite mononuclear intersticial focal, atrofia glomerular e descamação das células tubulares epiteliais foram observadas em 13 dos 19 animais infectados e em 10 dos 22 animais não infectados. Não houve diferença estatisticamente significativa entre presença de lesões em animais infectados e não infectados (P=0,14). As três técnicas empregadas, isolamento, impregnação pela prata e imunohistoquímica, apresentaram alta concordância (k³0,85) e não apresentaram diferenças estatisticamente significativas (P>0,05). Esse trabalho descreve a presença incomum de leptospira em rins de animais portadores, porém com esse estudo não foi possível estabelecer uma relação entre lesões e presença de leptospira.Fil: Rossetti, Carlos A. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina.Fil: Vanasco, Bibiana N. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Respiratorias Dr. Emilio Coni; Argentina.Fil: Pini, Noemí. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Virales Humanas Dr. Julio Maiztegui; Argentina.Fil: Carfagnini, Julio C. Universidad de Buenos Aires. Área de Patología Básica; Argentina

Response from N.B. Vanasco, M.D. Sequeira, G. Sequeira and H.D. Tarabla to H. Leblebicioglu and M. Sunbul

Fil: Vanasco, Bibiana N. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Respiratorias; Argentina.Fil: Sequeira, María Delfina. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Respiratorias; Argentina.Fil: Sequeira, Gabriel. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Respiratorias; Argentina.Fil: Tarabla, Héctor D. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Respiratorias; Argentina.We are in complete agreement that “PCR may be a valuable method for the detection of leptospires in animals for comparative studies” (Merien et al., 1995; Sunbul et al., 2001). However, in our paper, culture, inmunohistochemical and silver-stained (Warthin-Starry) results were discussed only in relationship to the high seroprevalence found in O. flavescens. Reasons for negative culture results in this species and A. azarae (that, by the way, are not “rats”) were just hypotheses, as it was clearly stated in our paper. Moreover, our aim was not only to find relationships between infection and environmental and rodent characteristics, but also to explore risk factors for past and current infections measured by means of antibody response

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 (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%), con respecto a la primera (68,1%), y descendió en la tercera (78,8%). La especificidad aumentó gradualmente desde el 96,3% en la primera etapa hasta el 100% 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 particular, para adelantar el diagnóstico de la enfermedad aguda.Objective. To develop a solid-phase enzyme immunoassay (ELISA) for genusspecific 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 (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.Fil: Vanasco, Norma B. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Respiratorias Dr. Emilio Coni; Argentina.Fil: Lottersberger, Javier. Uni-versidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina.Fil: Schmeling, María F. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Respiratorias Dr. Emilio Coni; Argentina.Fil: Gardner, Ian A. University of California. Department of Medicine and Epidemiology; Estados Unidos.Fil: Tarabla, Héctor D. Instituto Nacional de Tecnología Agropecuaria (INTA); Argentina

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 (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%), con respecto a la primera (68,1%), y descendió en la tercera (78,8%). La especificidad aumentó gradualmente desde el 96,3% en la primera etapa hasta el 100% 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 particular, para adelantar el diagnóstico de la enfermedad aguda.Objective. To develop a solid-phase enzyme immunoassay (ELISA) for genusspecific 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 (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.Fil: Vanasco, Norma B. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Respiratorias Dr. Emilio Coni; Argentina.Fil: Lottersberger, Javier. Uni-versidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina.Fil: Schmeling, María F. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Respiratorias Dr. Emilio Coni; Argentina.Fil: Gardner, Ian A. University of California. Department of Medicine and Epidemiology; Estados Unidos.Fil: Tarabla, Héctor D. Instituto Nacional de Tecnología Agropecuaria (INTA); Argentina