Utility evaluation of two molecular methods for Leptospira spp. typing in human serum samples.

Most of the available genotyping methods were applied and evaluated in Leptospira isolates and only few of them in a relevant sample size of blood specimens but not of sera. The objective of this study was to evaluate the utility of one partial 16S rRNA gene sequencing assay (16S rRNA) and an optimized. Multilocus sequence typing scheme (MLST) for Leptospira typing directly in serum samples. Confirmed leptospirosis patients (n = 228) from Argentina (2005-2016) were randomly included. Septicemic-phase serum samples (n = 228) were studied by two genotyping methods. Available immune-phase serum samples of the included patients (n = 159) were studied by MAT to compare serological and molecular results. In culture-proven cases (n = 8), genotyping results between clinical samples and isolates were compared. Typing success rate (TSR) was 21.9% for 16S rRNA and 11.4% for MLST (full allelic profile) and a positive trend in both TSR during the study period was observed. Two species (L. interrogans and L. borgpertesenii) were identified by both methods and MLST assigned 8 different STs. The probable serogroups identified by MLST were coincident with the presumptive infecting serogroups identified by MAT, but with different frequencies. The three serogroups (Canicola, Sejroe and Icterohaemorrhagiae) most frequently identified by MAT were also genotyped by MLST. Typing results via 16S rRNA and MLST in clinical samples and isolates of culture-proven cases, were consistent except for one case. Performance of partial 16S rRNA gene sequencing assay and the optimized MLST scheme directly in sera may increase and improve the knowledge about species and serogroups causing human leptospirosis, especially in countries with low rates of culture sample collection or Leptospira isolation.

Seroprevalence of leptospiral antibodies in rodents from riverside communities of Santa Fe, Argentina.

BACKGROUND: Leptospirosis is a zoonotic disease that can be transmitted by contact with the urine of infected mammals. Rodents play a mayor role in the transmission of leptospires to humans. The province of Santa Fe reports the greatest number of cases in Argentina. Yet, in this region, there are still knowledge gaps regarding the diversity of rodent species that may be hosts of pathogenic leptospires. The aims of this study were to evaluate the presence of leptospiral antibodies in rodents from three riverside communities of Santa Fe, and to identify factors associated with leptospiral infection. METHODOLOGY/PRINCIPAL FINDINGS: Each community was divided into three environmental settings based on the level of human disturbance, and sampled during two springs (Sep-Oct 2014 and 2015) and one autumn (Mar-Apr 2015). Serum samples of captured sigmodontine and murine rodents were tested for leptospiral antibodies by enzyme-linked immunosorbent assay (ELISA), and microagglutination test (MAT) was used to assess the infecting serovar in seropositive individuals. Factors influencing seropositivity were analyzed using logistic regression models. We caught 119 rodents, of which 101 serums were suitable for analysis. Most frequently trapped species were Scapteromys aquaticus, Akodon azarae and Oligoryzomys spp., with seroprevalences of 41.3%, 42.9% and 55% respectively. Seropositivity was higher in individuals with an average body condition score and in those that were sexually mature, but in the latter the differences were marginally significant. CONCLUSIONS/SIGNIFICANCE: Our results suggest that native rodents may be playing a role in the environmental circulation of pathogenic leptospires and provide relevant information for public health policies in the area.

Leptospirosis humana en Argentina: un esquema de análisis, 2014 / Human Leptospirosis in Argentina: An Analysis Scheme, 2014

INTRODUCCIÓN: En general, sólo existen estimaciones sobre el número de casos de leptospirosis en las Américas. La fuente más común son los Ministerios de Salud, tanto nacionales como provinciales, que proporcionan datos útiles sobre tendencias de incidencia de la leptospirosis, identificación de brotes y efectos de intervenciones gubernamentales. OBJETIVOS: Conocer la incidencia de casos de leptospirosis detectados por laboratorio durante 2014 en Argentina y generar un esquema de análisis de bases de datos de diferentes organismos nacionales para que sea repetido y difundido anualmente. MÉTODOS: Se analizaron las bases de datos del Sistema de Vigilancia Laboratorial (SIVILA)y del Instituto Nacional de Enfermedades Respiratorias (INER), teniendo en cuenta los casos notificados en 2014, según la fecha de inicio de síntomas. RESULTADOS: La incidencia de casos confirmados de leptospirosis en 2014 fue de 0,32/100 000 habitantes, mayormente detectados en las provincias de Santa Fe, Buenos Aires y Entre Ríos. Los serogrupos circulantes más prevalentes fueron Icterohaemorrhagiae, Sejroe y Canicola. CONCLUSIONES: Se resalta la utilidad de este tipo de estudios, que incentivan la búsqueda de casos, notificación y vigilancia de la leptospirosis, tanto para mejorar el conocimiento de la incidencia de la enfermedad y su distribución como para orientar las acciones hacia los lugares de mayor riesgo del país.

INTRODUCTION: In general, there are only estimates of the number of cases in the Americas. This data is commonly obtained by both national and provincial Ministries of Health which, despite data limitations, provide useful information on leptospirosis incidence trends, outbreak identification and effects of government interventions. OBJECTIVES: To know the incidence of leptospirosis cases detected by laboratory during 2014 in Argentina, and to generate an analysis scheme of databases from different national organisms to be repeated and diffused annually. METHODS: The SIVILA and INER databases were analyzed, taking into account the cases reported in 2014, according to the date of onset of symptoms. RESULTS: The incidence of confirmed leptospirosis cases in 2014 was 0.32/100 000 persons, mostly detected in Santa Fe, Buenos Aires and Entre Ríos provinces. The most prevalent circulating serogroups were Icterohaemorrhagiae, Sejroe and Canicola. CONCLUSIONS: These studies motivate the suspicion, notification and surveillance of leptospirosis and are useful both to improve the knowledge of the incidence of cases and their distribution, as well as to guide actions towards the most risky places in the country.

Isolation and clinical sample typing of human leptospirosis cases in Argentina.

Leptospira typing is carried out using isolated strains. Because of difficulties in obtaining them, direct identification of infective Leptospira in clinical samples is a high priority. Multilocus sequence typing (MLST) proved highly discriminatory for seven pathogenic species of Leptospira, allowing isolate characterization and robust assignment to species, in addition to phylogenetic evidence for the relatedness between species. In this study we characterized Leptospira strains circulating in Argentina, using typing methods applied to human clinical samples and isolates. Phylogenetic studies based on 16S ribosomal RNA gene sequences enabled typing of 8 isolates (6 Leptospira interrogans, one Leptospira wolffii and one Leptospira broomii) and 58 out of 85 (68.2%) clinical samples (55 L. interrogans, 2 Leptospira meyeri, and one Leptospira kirschneri). MLST results for the L. interrogans isolates indicated that five were probably Canicola serogroup (ST37) and one was probably Icterohaemorrhagiae serogroup (ST17). Eleven clinical samples (21.6%), provided MLST interpretable data: five were probably Pyrogenes serogroup (ST13), four Sejroe (ST20), one Autumnalis (ST22) and one Canicola (ST37). To the best of our knowledge this study is the first report of the use of an MLST typing scheme with seven loci to identify Leptospira directly from clinical samples in Argentina. The use of clinical samples presents the advantage of the possibility of knowing the infecting strain without resorting to isolates. This study also allowed, for the first time, the characterization of isolates of intermediate pathogenicity species (L. wolffii and L. broomii) from symptomatic patients.

Diagnóstico de leptospirosis humana: evaluación de la aglutinación macroscópica en diferentes etapas de la enfermedad

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.

[Human leptospirosis diagnosis:macroscopic agglutination test evaluation in different stages of the disease]. / Diagnóstico de leptospirosis humana: evaluación de la aglutinación macroscópica en diferentes etapas de la enfermedad.

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.