RESUMO
Leptospirosis is a neglected disease causing severe infections in humans and animals. Due in part to misdiagnosis, this infectious disease results in nearly 60,000 deaths per year around the globe. This study represents the first effort to describe the diversity of pathogenic Leptospira in Cuba based on whole-genome sequencing. We have collected nineteen whole-blood samples from patients that were diagnosed as having leptospirosis between 2008 and 2012 in Cuba. In addition, we have enhanced our sample set by three historical strains that were used for the development of a human vaccine in 1990s. The Leptospira strains were grown and serotyped by the microscopic agglutination test, and the draft genomes were generated by NGS (Illumina). Subsequently, the core genomes were analyzed and compared to the genetic data available from other Caribbean islands and countries in Central America. Core genome Multi-locus Sequence Typing (cgMLST) revealed four different core genome clonal groups (cgCGs), with the highest number of samples belonging to L. interrogans, followed by L. borgpetersenii and L. kirschneri. All cgCGs that were found in Cuba have been also identified from multiple origins across the globe, except in neighbor countries and Central America. Serotyping divided the samples into the serogroups Canicola, Ballum and Pomona. The most frequent cgCGs, cgCG28, associated with serogroup Canicola, and cgCG15, associated with serogroup Ballum, have also been identified from samples isolated from dogs, rodents, and pigs; suggesting that these hosts represent the major source of human infection in Cuba. The vaccine strains did not significantly differ from the recent patient isolates. However, the increasing prevalence of samples belonging to the serogroup Ballum combined with the fact that the available vaccine in Cuba represents inactivated Leptospira belonging to serogroups other than Ballum, should be a valuable information for the National and Regional Leptospirosis Control Programs.
Assuntos
Leptospira/genética , Leptospirose/microbiologia , Animais , América Central , Cuba/epidemiologia , Cães , Variação Genética , Genoma Bacteriano , Humanos , Leptospira/classificação , Leptospira/isolamento & purificação , Leptospirose/epidemiologia , Tipagem de Sequências Multilocus , Filogenia , Sorogrupo , Sorotipagem , Suínos , Índias Ocidentais , Sequenciamento Completo do Genoma , Zoonoses/epidemiologia , Zoonoses/microbiologiaRESUMO
This study describes the development and application of a new PCR assay for the specific detection of pathogenic leptospires and its comparison with a previously reported PCR protocol. New primers were designed for PCR optimization and evaluation in artificially-infected paraffin-embedded tissues. PCR was then applied to post-mortem, paraffin-embedded samples, followed by amplicon sequencing. The PCR was more efficient than the reported protocol, allowing the amplification of expected DNA fragment from the artificially infected samples and from 44% of the post-mortem samples. The sequences of PCR amplicons from different patients showed >99% homology with pathogenic leptospires DNA sequences. The applicability of a highly sensitive and specific tool to screen histological specimens for the detection of pathogenic Leptospira spp. would facilitate a better assessment of the prevalence and epidemiology of leptospirosis, which constitutes a health problem in many countries.
Assuntos
Primers do DNA/genética , DNA Bacteriano/genética , Leptospira/genética , Leptospirose/diagnóstico , Humanos , Inclusão em Parafina , Reação em Cadeia da Polimerase , Índice de Gravidade de Doença , Fixação de TecidosRESUMO
This study describes the development and application of a new PCR assay for the specific detection of pathogenic leptospires and its comparison with a previously reported PCR protocol. New primers were designed for PCR optimization and evaluation in artificially-infected paraffin-embedded tissues. PCR was then applied to post-mortem, paraffin-embedded samples, followed by amplicon sequencing. The PCR was more efficient than the reported protocol, allowing the amplification of expected DNA fragment from the artificially infected samples and from 44% of the post-mortem samples. The sequences of PCR amplicons from different patients showed >99% homology with pathogenic leptospires DNA sequences. The applicability of a highly sensitive and specific tool to screen histological specimens for the detection of pathogenic Leptospira spp. would facilitate a better assessment of the prevalence and epidemiology of leptospirosis, which constitutes a health problem in many countries.
El presente estudio describe el desarrollo y aplicación de un nuevo ensayo de PCR para la detección específica de leptospiras patógenas y su comparación con un protocolo reportado previamente. Se diseñaron nuevos cebadores para la optimización y evaluación de la PCR en tejidos embebidos en parafina infectados artificialmente. La PCR se aplicó además a muestras de tejidos embebidos en parafina y se realizó la secuenciación del amplicón resultante. La PCR diseñada fue más eficiente que el protocolo reportado, permitiendo la amplificación del fragmento de ADN esperado en las muestras infectadas artificialmente y del 44% de las muestras post mortem. Se secuenciaron 10 amplicones provenientes de pacientes diferentes. La aplicabilidad de una herramienta altamente sensible y específica en la búsqueda de leptospiras patógenas en especímenes histopatológicos podría facilitar una mejor valoración de la prevalencia y la epidemiología de la leptospirosis, la que constituye un problema de salud en disímiles países.
Assuntos
Humanos , Primers do DNA/genética , DNA Bacteriano/genética , Leptospira/genética , Leptospirose/diagnóstico , Inclusão em Parafina , Reação em Cadeia da Polimerase , Índice de Gravidade de Doença , Fixação de TecidosRESUMO
Durante las dos últimas décadas se ha observado a nivel mundial un alza en el número de casos de leptospirosis humana, frecuentemente asociada a desastres naturales. En Cuba, esta enfermedad tiene un comportamiento endemo epidémico, y estacional. Las características tropicales del país, el clima, la orografía, la red fluvial natural y la artificial, las extensas áreas agrícolas, y los regímenes lluviosos en determinadas épocas del año, han propiciado un aumento en el número de casos de esta enfermedad. En el presente artículo se hace una revisión documental del tema y se propone una guía para el control y prevención de esta zoonosis en la atención primaria de salud, y así brindarle al Médico de Familia elementos importantes para su trabajo diario en la comunidad.
For two last decades it was observed a rise in cases of human leptospirosis frequently associated to natural disasters. In Cuba, this condition has a endemic, epidemic, and seasonal behaviour. Tropical features of our country, climate, orography, natural and artificial fluvial network, extensive agricultural areas, and rainy regimes en determined times of year, have propitiate a increase in case-number of this disease. In present paper a documentary review of this topic was made and we propose a guide for control and prevention of this zoonosis in health primary care, and thus provide to Family Physician with significant components for his daily practice in community.
Assuntos
Humanos , Leptospirose/prevenção & controleRESUMO
Se aplicó un método de la reacción en cadena de la polimerasa para la detección temprana de Leptospiras spp. en hemocultivos procedentes de pacientes con sospecha de leptospirosis humana. Los métodos moleculares, y en particular la amplificación del ADN por la reacción en cadena de la polimerasa y sus variantes, constituyen herramientas muy útiles y específicas, utilizadas en la actualidad con esta finalidad. Se lograron identificar por la reacción en cadena de la polimerasa Leptospiras spp. en 41,3 por ciento (33/80) de los hemocultivos a los 7 d de incubación, los que también se clasificaron como positivos por los métodos convencionales. Sin embargo, hubo 20 por ciento (16/80) en los que también por este método se lograron identificar Leptospiras spp., pero por los métodos convencionales resultaron ser negativos. De los hemocultivos 38,7 por ciento (31/80) resultó negativo tanto por la reacción en cadena de la polimerasa como por los métodos convencionales. El uso del método de la reacción en cadena de la polimerasa a partir de hemocultivos, es una alternativa valiosa para la detección temprana y el diagnóstico rápido de Leptospiras spp.
The PCR method for the early detection of Leptospiras spp. in hemocultures from patients suspected of human leptospiros was applied. Molecular methods and, particularly, the amplification of DNA by PCR, and its variants, are very useful and specific tools used nowdays to this end. Leptospiras spp. were identied by means of PCR in 41.3 percent(33/80) of the hemocultures at 7 days of incubation.They were also classified as positive by the conventional methods. However, it was also possible to identify Leptospiras spp. in 20 percent (16/80) by this method, but they proved to be negative by the conventional methods. Of the hemocultures, 38.7 percent (31/80) yielded negative by PCR and by the conventional methods. The use of PCR starting from hemocultures is a valuable alternative for the early detection and rapid diagnosis of Leptospiras spp
Assuntos
Leptospira/enzimologia , Reação em Cadeia da Polimerase/métodosRESUMO
By using the monoclonal antibodies methodology we have classified Cuban serovars of leptospires isolated from blood culture of confirmed leptospirosis patients. The serogroups Pomona and Canicola were the most predominant found in our study. Pomona, Mozdok and Canicola serovars were the most prevalent types in these serogroups. Our study has essentially presented a validation of a monoclonal antibody method for the serotyping of Cuban leptospire isolates.
Se clasificaron, mediante la metodología de los anticuerpos monoclonales, los serovares cubanos de Leptospira aislados en hemocultivos de pacientes con leptospirosis confirmada. Los serogrupos Pomona y Canicola fueron los predominantes. Los serovares Pomona, Mozdok y Canicola resultaron los mßs frecuentes dentro de esos serogrupos. Se presentó la validación del método de anticuerpos monoclonales para la tipificación serológica de las leptopiras aisladas en Cuba.
Assuntos
Humanos , Anticorpos Monoclonais , LeptospiroseRESUMO
Se evaluaron por vez primera en Cuba las tecnologías serológicas rápidas para realizar la pesquisa y confirmación de la leptospirosis humana, que mundialmente estßn disponibles. Su reconocimiento clínico resulta difícil, el diagnóstico etiológico rápido es de vital importancia Se obtuvieron valores de sensibilidad y especificidad superiores a 90 por ciento en los sistemas confirmatorios y de 85 por ciento en el de pesquisa. De los pacientes graves estudiados por estas tecnologías 50 por ciento fue positivo, además se confirmaron 203 casos pertenecientes a 4 brotes epidémicos, así como 12 enfermos vacunados con vaxSPIRAL®, demostrándose la eficacia de la vacuna (78,1 por ciento). Se desarrolló y aplicó un sistema látex para pesquisa rápida de leptospirosis (LeptoCuba), con excelente sensibilidad, especificidad, reproducibilidad y estabilidad. La aplicación de nuevas tecnologías a la pesquisa y confirmación rápida de la enfermedad, permitió incrementar la positividad y calidad del diagnóstico en el período 2002-2006, fortaleciéndose la vigilancia microbiológica en el país.
For the first time in Cuba the rapid serologic technologies available worldwide were evaluated for the screening and confirmation of human leptospirosis. As its clinical recognition is difficult, the fast etiological diagnosis is of vital importance. Sensitivity and specificity values higher than 90 percent were obtained in the confirmatory systems, and of 85 percent in the screening systems. Of the severe patients studied by these technologies 50 percent were positive. 203 cases corresponding to 4 epidemic outbreaks were confirmed, as well as 12 sick persons vaccinated with vaxSPIRAL®, which demonstrates the vaccine effectiveness (78.1 percent). We developed and applied a latex system for a rapid screening of leptospirosis (LeptoCuba), with an excellent sensitivity, specificity, reproducibility, and stability. The application of new technologies for the screening and fast confirmation of the disease allowed to increase the positivity and quality of the diagnosis from 2000 to 2006, strengthening the microbiological surveillance in the country
Assuntos
Humanos , Leptospirose/diagnóstico , Testes SorológicosRESUMO
Se realizó una caracterización de la epidemia ocurrida en la provincia Guantßnamo durante los meses de octubre y noviembre de 2005, así como de las actividades desarrolladas para su control. Se comprobó que existían factores propicios para su desarrollo, esto trajo consigo que a partir de la semana estadística 42 se notificara un alza en el número de atenciones médicas con cuadros clínicos y elementos epidemiológicos. Se notificaron un total de 885 casos sospechosos, 61 fueron confirmados microbiológicamente para una positividad de 6,9 por ciento. Los municipios con mayor riesgo fueron Niceto Pérez, la ciudad de Guantßnamo y Manuel Tames. Se llevaron a cabo de forma inmediata una serie de actividades con el apoyo multisectorial e interdisciplinario de distintas organizaciones, que permitieron controlar la situación en un lapso menor de un mes. El estudio de este brote demostró que la leptospirosis continúa siendo un problema de salud en Cuba y que para su control se necesitan unificar esfuerzos y recursos.
A characterization of the epidemic occurred in Guantßnamo province during October and November 2005, and as well as of the activities developed for its control was made. The existence of favourable factors was confirmed, and as a result from the 42nd statistical week on there was an increase in the number of medical attentions with clinical pictures and epidemiologic elements. 885 suspected cases were notified, 61 were microbiologically confirmed, accounting for a positivity of 6.9 percent. The municipalities with the highest risk were Niceto Pérez, Guantßnamo city, and Manuel Tames. Some activities were immediately carried out, with the multisectorial and interdisciplinary support from different organizations, allowing the control of the situation in less than a month. The study of this outbreak showed that leptospirosis is still a health problem in Cuba, and that it is necessary to join efforts and resources to control it.
Assuntos
Humanos , Leptospirose/epidemiologiaRESUMO
A characterization of the epidemic occurred in Guantánamo province during October and November 2005, and as well as of the activities developed for its control was made. The existence of favourable factors was confirmed, and as a result from the 42nd statistical week on there was an increase in the number of medical attentions with clinical pictures and epidemiologic elements. 885 suspected cases were notified, 61 were microbiologically confirmed, accounting for a positivity of 69%. The municipalities with the highest risk were Niceto Pérez, Guantánamo city, and Manuel Tames. Some activities were immediately carried out, with the multisectorial and interdisciplinary support from different organizations, allowing the control of the situation in less than a month. The study of this outbreak showed that leptospirosis is still a health problem in Cuba, and that it is necessary to join efforts and resources to control it.
Assuntos
Surtos de Doenças , Leptospirose/epidemiologia , Criação de Animais Domésticos , Animais , Cuba/epidemiologia , Vetores de Doenças , Doenças do Cão/epidemiologia , Doenças do Cão/microbiologia , Cães/microbiologia , Doxiciclina/uso terapêutico , Doenças das Cabras/epidemiologia , Doenças das Cabras/microbiologia , Cabras , Promoção da Saúde , Doenças dos Cavalos/epidemiologia , Doenças dos Cavalos/microbiologia , Cavalos , Humanos , Higiene , Incidência , Leptospirose/diagnóstico , Leptospirose/tratamento farmacológico , Leptospirose/prevenção & controle , Leptospirose/transmissão , Leptospirose/veterinária , Doenças Profissionais/epidemiologia , Doenças Profissionais/microbiologia , Vigilância da População , Roedores/microbiologia , Rodenticidas , Ovinos , Doenças dos Ovinos/epidemiologia , Doenças dos Ovinos/microbiologia , Suínos , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/microbiologia , ZoonosesRESUMO
Clinical samples from 293 patients with suspicion of leptospirosis were studied for the microbiologic confirmation of these events, in October-December 2005, when there were 2 outbreaks in humans, in Cuba. Sera samples of patients in acute phase and during convalescence, as well as hemocultures performed before the beginning of the antibiotic therapy, were analyzed. Conventional techniques (passive hemagglutination test and serogroups hemagglutination), and advanced or fast diagnosis techniques (Lepto tek Dri-Dot, Lepto-Cuba, Latex-India, Lepto tek Lateral Flow) were used for serologic diagnosis. In outbreak 1, 26 % of the studied cases were confirmed by serological tests (22/84), and 25% (5/20) through hemocultures; whereas in outbreak 11, 48 of the 162 studied cases (30%) were serologically confirmed, and it was possible to obtain isolation of leptospires in 6 of the 27 processed cases (22%). The main serovariants found by serology were Canicola, Ballum, Icterohaemorrhagiae, and Pomona. The rapid diagnosis methods were useful screening tools in the most severe cases or at pediatric ages. The two epidemiologic events were caused by pathogenic leptospires infection, which contributed to the adoption of hygienic-sanitary measures in both provinces.
Assuntos
Testes de Aglutinação/métodos , Doenças Transmissíveis Emergentes/epidemiologia , Surtos de Doenças , Leptospirose/epidemiologia , Adulto , Anticorpos Antibacterianos/sangue , Bacteriemia/microbiologia , Técnicas Bacteriológicas , Criança , Doenças Transmissíveis Emergentes/diagnóstico , Doenças Transmissíveis Emergentes/microbiologia , Cuba/epidemiologia , Testes de Hemaglutinação , Humanos , Testes de Fixação do Látex , Leptospira interrogans/classificação , Leptospira interrogans/crescimento & desenvolvimento , Leptospira interrogans/imunologia , Leptospira interrogans/isolamento & purificação , Leptospirose/sangue , Leptospirose/diagnóstico , Leptospirose/microbiologia , Estudos Soroepidemiológicos , Fatores de TempoRESUMO
For the first time in Cuba the rapid serologic technologies available worldwide were evaluated for the screening and confirmation of human leptospirosis. As its clinical recognition is difficult, the fast etiological diagnosis is of vital importance. Sensitivity and specificity values higher than 90 % were obtained in the confirmatory systems, and of 85 % in the screening systems. Of the severe patients studied by these technologies 50 % were positive. 203 cases corresponding to 4 epidemic outbreaks were confirmed, as well as 12 sick persons vaccinated with vaxSPIRAL, which demonstrates the vaccine effectiveness (78.1%). We developed and applied a latex system for a rapid screening of leptospirosis (LeptoCuba), with an excellent sensitivity, specificity, reproducibility, and stability. The application of new technologies for the screening and fast confirmation of the disease allowed to increase the positivity and quality of the diagnosis from 2000 to 2006, strengthening the microbiological surveillance in the country.
Assuntos
Leptospirose/diagnóstico , Kit de Reagentes para Diagnóstico , Testes Sorológicos/tendências , Doenças dos Animais/diagnóstico , Animais , Anticorpos Antibacterianos/sangue , Vacinas Bacterianas , Ensaios Clínicos como Assunto , Cuba/epidemiologia , Surtos de Doenças , Testes de Hemaglutinação , Humanos , Imunoglobulina M/sangue , Testes de Fixação do Látex , Leptospira/classificação , Leptospira/imunologia , Leptospirose/epidemiologia , Leptospirose/prevenção & controle , Leptospirose/veterinária , Reprodutibilidade dos Testes , População Rural , Sensibilidade e Especificidade , Estudos Soroepidemiológicos , Sorotipagem , Fatores de Tempo , População Urbana , VacinaçãoRESUMO
By using the monoclonal antibodies methodology we have classified Cuban serovars of leptospires isolated from blood culture of confirmed leptospirosis patients. The serogroups Pomona and Canicola were the most predominant found in our study. Pomona, Mozdok and Canicola serovars were the most prevalent types in these serogroups. Our study has essentially presented a validation of a monoclonal antibody method for the serotyping of Cuban leptospire isolates.
Assuntos
Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Antígenos de Bactérias/sangue , Bacteriemia/microbiologia , Leptospira interrogans/classificação , Leptospirose/microbiologia , Sorotipagem/métodos , Antígenos de Bactérias/imunologia , Bacteriemia/epidemiologia , Cuba/epidemiologia , Humanos , Leptospira interrogans/imunologia , Leptospira interrogans/isolamento & purificação , Leptospira interrogans serovar canicola/imunologia , Leptospira interrogans serovar canicola/isolamento & purificação , Leptospira interrogans serovar icterohaemorrhagiae/imunologia , Leptospira interrogans serovar icterohaemorrhagiae/isolamento & purificação , Leptospirose/epidemiologia , Especificidade da EspécieRESUMO
The PCR method for the early detection of Leplospiras spp. in hemocultures from patients suspected of human leptospiros was applied. Molecular methods and, particularly, the amplification of DNA by PCR, and its variants, are very useful and specific tools used nowdays to this end. Leptospiras spp. were identied by means of PCR in 41.3 % (33/80) of the hemocultures at 7 days of incubation. They were also classified as positive by the conventional methods. However, it was also possible to identify Leptospiras spp. in 20% (16/80) by this method, but they proved to be negative by the conventional methods. Of the hemocultures, 38.7% (31/80) yielded negative by PCR and by the conventional methods. The use of PCR starting from hemocultures is a valuable alternative for the early detection and rapid diagnosis of Leptospiras spp.
Assuntos
Bacteriemia/diagnóstico , Leptospira/isolamento & purificação , Leptospirose/diagnóstico , Reação em Cadeia da Polimerase/métodos , Bacteriemia/microbiologia , Diagnóstico Precoce , Reações Falso-Negativas , Humanos , Leptospira/genética , Leptospirose/sangue , Leptospirose/microbiologia , Reprodutibilidade dos TestesRESUMO
Se aplicó un sistema ELISA cuantitativo para la detección de anticuerpos IgG en respuesta a la vacuna cubana contra la leptospirosis humana (vax-SPIRAL), a sueros de 930 voluntarios, 483 inmunizados con vax-SPIRAL y 447 con una vacuna contra la hepatitis B. Fueron estudiadas las muestras tomadas antes de comenzar el estudio, a los 21 d de aplicada la segunda dosis y al cabo del año. Se pudo observar una alta seroprevalencia de anticuerpos a los serovares vacunales antes de comenzar el estudio, un incremento al doble del valor inicial de la respuesta a los 21 d de la segunda dosis de la vacuna antileptospirósica en 45 por ciento de los individuos, existiendo diferencias marcadamente significativas (p= 0,000000) entre ambos grupos. Se detectó respuesta a los 3 serovares leptospirales de forma similar. Al cabo del año los niveles de anticuerpos caen, sin embargo, hay 23,1 por ciento de individuos que logran duplicar su nivel de anticuerpos respecto al momento inicial, manteniéndose la diferencia significativa con el grupo que recibió la vacuna contra hepatitis B. Se recomendó la continuación de la aplicación de este inmunógeno cubano a los principales grupos de riesgo
Assuntos
Humanos , Adulto , Anticorpos Anti-Idiotípicos , Imunoglobulina G , Leptospira , Leptospirose , Vacinas , Ensaio de Imunoadsorção EnzimáticaRESUMO
A method to determine the minimum inhibitory concentration for leptospiras was developed, since there is not a standard method to measure it at the international level. Reference strains from the pathogenic complex L. interrogans and L. biflexa were used against penicillin, cyprofloxacine, chloramphenicol, rifampicine and tetracycline. The minimum inhibitory concentration was defined as the lowest concentration of antibiotic where it was observed the inhibition of the bacterial mobility by direct examination in dark field. Ranges for penicillin were from 0.095 to 152 microg/mL, for tetracycline from 0.156 to 3.13 microg/mL, for chloramphenicol, from 0.08 to 12.52 microg/mL, for rifampicine from 0.08 to 1.56 microg/mL, and for cyprofloxacine from 0.15 to 2.4 microg/mL. The antibiotics that showed the lowest values were cyprofloxacine, rifampicine and tetracycline, whereas the most elevated value was obtained against chloramphenicol and penicillin. The strains from the serogroups circulating more frequently in Cuba were used in this research. This study will allow in a near future to determine the antimicrobial susceptibility in autochthonous strains isolated from patients with Leptospirosis at the national level.
Assuntos
Antibacterianos/farmacologia , Leptospira/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Cloranfenicol/farmacologia , Ciprofloxacina/farmacologia , Cuba , Previsões , Humanos , Leptospira interrogans/efeitos dos fármacos , Leptospira interrogans serovar canicola/efeitos dos fármacos , Leptospira interrogans serovar pomona/efeitos dos fármacos , Leptospirose/microbiologia , Penicilinas/farmacologia , Rifampina/farmacologia , Tetraciclina/farmacologiaRESUMO
A quantitative ELISA for the detection of IgG antibodies in response to the Cuban vaccine against the human leptospirosis (vax-SPIRAL) was applied to 930 volunteers'sera, 483 immunized with vax-SPIRAL and 447 with a vaccine against hepatitis B. Samples were taken before beginning the study, 21 days after the second dose and a year later. A high seroprevalence of antibodies to the vaccine serovars was observed before beginning the study. The initial value of the response doubled at 21 days of the second dose in 45 % of the individuals. There were markedly significant differences (p=0,000000) between both groups. A similar response to the three leptospiral serovars was found. After the year the levels of antibodies decreased; however, 23.1% of the individuals were able to duplicate their level of antibodies regarding the initial moment, and the significant difference with the group that received the vaccine against hepatitis B still existed. The application of this Cuban immunogen amomg the main risk groups was recommended.
Assuntos
Anticorpos Antibacterianos/sangue , Vacinas Bacterianas/imunologia , Ensaio de Imunoadsorção Enzimática , Imunoglobulina G/sangue , Leptospira interrogans/imunologia , Leptospirose/prevenção & controle , Vacinas Bacterianas/administração & dosagem , Distribuição de Qui-Quadrado , Intervalos de Confiança , Cuba , Seguimentos , Vacinas contra Hepatite B/administração & dosagem , Vacinas contra Hepatite B/imunologia , Humanos , Leptospira interrogans serovar canicola/imunologia , Leptospirose/imunologia , Fatores de TempoRESUMO
OBJECTIVES: To assess the sensitivity, specificity, reproducibility, and stability of five latex agglutination systems for detecting antibodies against leptospira in human and animal sera, by using the Leptospira serotypes that are most widely prevalent in Cuba. METHODS: We performed an analytic and descriptive study with 706 human sera (65 tested positive for antibodies against leptospira with microagglutination (MAT) and hemagglutination (HA) techniques; 156 sera that tested negative with MAT and HA); 485 sera from 424 patients who had clinical or epidemiologic signs of leptospirosis; and 29 animal sera (16 from equines, 6 from bovines, 5 from porcines, 1 from a canine, and 1 from an ovine). All of the samples were tested with five latex conjugates made from whole cells of Leptospira interrogans, specifically the four serogroups that circulated most widely in Cuba from 2002 to 2004. The cells obtained from cultured cell lines yielded four specific conjugates (latex-canicola, latex-icterohemorrhagiae, latexpomona, and latex-sejroe), as well as one latex conjugate made from a combination of all four serogroups in equal quantities (latex-pool). In addition, samples were tested with the commercial latex agglutination Lepto Tek Tri Dot (bioMeriuex, France) kit. The stability and reproducibility of the latex conjugates were assessed through monthly controls over a period of 6 months with positive and negative sera. RESULTS: Of the systems that were assessed, the best combination of sensitivity and specificity was obtained with the latex-Pool conjugate (93,8% and 90,4%, respectively). The best combination of positive and negative predictive values was seen with the latex-Sejroe conjugate (90,9% and 95,8%), respectively), followed by the latex-Pool conjugate (94.2% and 96.6%, respectively). The positive and negative predictive values of the Lepto Tek Dri Dot commercial system were 78.5% and 88.4%, respectively. Among the 137 patient samples that tested positive for one of the serotypes when MAT was used, latex conjugates succeeded in correctly identifying 107 (78.1%), whereas the latex-Pool conjugate detected 116 (84.7%) positive sera. When animal sera were tested, the latex-Pool conjugate detected the greatest number of positive serum samples and showed the greatest concordance with MAT (93.1%). The conjugates studied showed good stability and reproducibility. CONCLUSIONS: Latex conjugates made from whole cells of the most widely circulating leptospira in Cuba showed a degree of concordance with MAT that was similar to or better than that seen with the Lepto Tek Dri Dot commercial system, both in human and animal sera. We recommend more widespread use of the latex-Pool conjugate in Cuba in the initial screening for antibodies against leptospira.
Assuntos
Anticorpos Antibacterianos/sangue , Testes de Fixação do Látex/métodos , Leptospira interrogans/imunologia , Leptospirose/imunologia , Animais , Antígenos de Bactérias/imunologia , Bovinos , Cuba , Cães , Humanos , Leptospira interrogans/classificação , Leptospirose/classificação , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Sorotipagem , Ovinos , SuínosRESUMO
OBJETIVOS: Evaluar la sensibilidad, la especificidad, la reproducibilidad y la estabilidad de cinco sistemas de aglutinación con látex diseñados para detectar anticuerpos contra leptospira en sueros de humanos y de animales, basados en los serogrupos de Leptospirade mayor circulación en Cuba. MÉTODOS: Se realizó un estudio analítico descriptivo con 706 sueros humanos (65 sueros positivos a anticuerpos contra leptospira mediante microaglutinación (MAT) y hemaglutinación (HA); 156 sueros negativos, según MAT y HA; 485 sueros de 424 pacientes con signos clínicos o epidemiológicos de leptospirosis); y 29 sueros de animales (16 equinos, 6 bovinos, 5 porcinos, 1 canino y 1 ovino). Todas las muestras se evaluaron con cinco conjugados de látex con células enteras de Leptospira interrogans de los cuatro serogrupos de mayor circulación en Cuba en el período entre 2002 y 2004. Con las células obtenidas de los cultivos celulares de cepas tipo se obtuvieron cuatro conjugados específicos (látex-Canicola, látex-Icterohaemorrhagiae, látex-Pomona y látex-Sejroe) y un conjugado de látex con la mezcla de las células de esos cuatro serogrupos a partes iguales (látex-Pool). Adicionalmente, las muestras se evaluaron con el sistema comercial de aglutinación con látex Lepto Tek Dri Dot (bioMerieux, Francia). La estabilidad y la reproducibilidad de los conjugados de látex se evaluaron mediante controles mensuales durante 6 meses con sueros positivos y negativos. RESULTADOS: De los sistemas evaluados, la mejor combinación de sensibilidad y especificidad se observó con el conjugado látex-Pool (93,8 por ciento y 90,4 por ciento, respectivamente). La mejor combinación de valores predictivos positivos y negativos se observó con el conjugado látex-Sejroe (90,9 por ciento y 95,8 por ciento, respectivamente), seguido del conjugado látex-Pool (94,2 por ciento y 96,6 por ciento, respectivamente). Los valores predictivos positivo y negativo del sistema comercial Lepto Tek Dri Dot fueron 78,5 por ciento y 88,4 por ciento, respectivamente. De las 137 muestras de pacientes positivas a alguno de los serogrupos estudiados según MAT, los conjugados de látex lograron identificar correctamente 107 (78,1 por ciento), mientras que el conjugado látex-Pool detectó como positivos 116 sueros (84,7 por ciento). Al evaluar los sueros de animales, el conjugado látex-Pool detectó...
Assuntos
Látex , Leptospira interrogans , Leptospirose , CubaRESUMO
Se estudió la seroconversión en 408 individuos vacunados y 135 placebo, incluidos en 2 ensayos clínicos de la vacuna cubana contra la leptospirosis humana. Se estudiaron 2 dosis vacunales y 5 esquemas. Seroconvirtieron de 38 vacunados (Fase I), 11 (29 por ciento) por MAT y 12 (32 por ciento) por ELISA, y de 33 placebo, 2 (6 por ciento) y 3( 9 por ciento) respectivamente. En la Fase II de 68 vacunados (dosis de 0,25 mL) y de 65 (dosis de 0,50 mL), seroconvirtieron 21 (31 por ciento) y 16 (25 por ciento) por ELISA respectivamente, por MAT 9 (13 por ciento) y 7 (11 por ciento) individuos fueron positivos. No hubo diferencias significativas entre las dosis utilizadas. La seroconversión por MAT, en 237 individuos vacunados con diferentes esquemas, fue de 22,4 por ciento, no existiendo diferencias significativas entre estos. En la mayoría de los individuos reactivos, se encontraron niveles de anticuerpos al menos a una de las cepas vacunales. Se recomendó, buscar y evaluar otros métodos para demostrar in vivo, el nivel de protección de esta vacuna
Assuntos
Humanos , Masculino , Feminino , Ensaios Clínicos como Assunto , Leptospirose , Vacinas , Ensaio de Imunoadsorção Enzimática , Testes Sorológicos/métodosRESUMO
OBJECTIVES: To evaluate the efficacy of vax-SPIRAL, a Cuban vaccine against leptospirosis, and to provide additional information concerning the safety of this vaccine (which was developed by Cuba's Finlay Institute). METHODS: This phase III efficacy trial of vax-SPIRAL was controlled, randomized, and double blind. The control vaccine used for the trial was Heberbiovac-HB (Heber Biotec, Cuba), a recombinant hepatitis B vaccine. The randomization unit for allocating persons to the study group or the control group were 523 family physician offices in the selected municipalities. The study covered the entire population of males and females from 20 to 64 years old who voluntarily agreed to participate, from the municipalities of Ranchuelo, Quemado, Santo Domingo, Encrucijada, Corralillo, Cifuentes, and Camajuaní, which are in the province of Villa Clara, in the central region of Cuba. The vaccinations were given in the physicians' offices between February and July 1998, with an interval of 6 weeks between the two doses. The follow-up period was 12 months. A case was considered positive if a person who had received the two doses of the vaccine became ill with leptospirosis more than 21 days after receiving the second dose, with the diagnosis confirmed through serological and microbiological methods. We calculated the efficacy of the vaccine and the relative risk of becoming ill with leptospirosis after the vaccination. For the safety study, two persons were chosen at random from among the individuals vaccinated at the office of each physician participating in the study. Follow-up of local and systemic adverse reactions was carried out by the family physicians during the seven days after the application of each dose. The level of statistical significance was set at 0.05. RESULTS: A total of 101 832 persons were vaccinated, with 50 354 of them (49.4%) receiving the leptospirosis vaccine and 51 478 of them (50.6%) receiving the control vaccine. The efficacy of the vax-SPIRAL vaccine was 78.1% (95% confidence interval (CI): 59.2% to 88.3%), and the relative risk of becoming ill with leptospirosis after receiving the leptospirosis vaccine was 0.22 (95% CI: 0.12 to 0.41). General discomfort was the most frequent systemic adverse reaction, and mild spontaneous pain at the injection site was the most frequent local effect. The local and systemic adverse reactions were both more frequent in the study group than in the control group (P = 0.003). There were no serious adverse events. CONCLUSION: The vax-SPIRAL vaccine proved to be safe and efficacious for leptospirosis control. The vaccine is recommended for use in preventing this disease among groups at risk of contracting it.