ABSTRACT Introduction: Hard ticks are hematophagous ectoparasites, vectors of emerging zoonotic pathogens. Until 2011, there was evidence of infection/exposure to Borrelia burgdorferi in humans in Cuba, but the circulation of other agents is unknown. Aim: implement new diagnostic tools, explore infection/exposure of humans to tick-borne pathogens, their presence in vectors and level of knowledge in medical personnel. Methods: Multiplex PCR to detect B. burgdorferi-Anaplasma spp.-Babesia spp., PCR for Borrelia spp., and artificial infection of ticks were implemented. Positivity to infection by B. burgdorferi (2011-2018) was described. Presence of antibodies against borrelias in blood donors from a low-exposure region, antibodies against other zoonotic agents in a population at risk, presence of these agents in ticks, as well as the level of knowledge about Lyme disease in doctors were explored. Results: IPK capabilities for molecular detection of tick-borne pathogens were strengthened. New serological evidence of B. burgdorferi infection was found in patients with suspicion of Lyme disease. Absence of specific antibodies against B. burgdorferi was demonstrated in blood donors. Evidence of antibodies to Anaplasma phagocytophilum, Ehrlichia chaffeensis and Babesia microti were found. The presence of Anaplasma spp., Babesia spp., Rickettsia amblyommii and Coxiella burnetii was demonstrated in ticks. Insufficient knowledge about Lyme disease was found in medical personnel. Conclusions: these results alert the national public health authorities and medical personnel about the risk of infection in individuals exposed to tick bites.

RESUMEN Introducción: Las garrapatas duras son ectoparásitos hematófagos, vectores de patógenos zoonóticos emergentes. Hasta 2011 en Cuba existían evidencias de infección/exposición a Borrelia burgdorferi en seres humanos, pero se desconoce sobre la circulación de otros agentes. Objetivo: implementar nuevas herramientas diagnósticas, explorar infección/exposición de seres humanos a patógenos transmitidos por garrapatas, presencia de estos en vectores y nivel de conocimientos en personal médico. Métodos: Se implementó PCR múltiple para detección de B. burgdorferi sl-Anaplasma spp. -Babesia spp., PCR para Borrelia spp., e infección artificial de garrapatas. Se describió la positividad a infección por B. burgdorferi (2011-2018), se exploró la presencia de anticuerpos contra borrelias en donantes de sangre de una región de baja exposición, anticuerpos contra otros agentes zoonóticos en población de riesgo, presencia de estos agentes en garrapatas, así como el nivel de conocimientos sobre enfermedad de Lyme en médicos. Resultados: Se fortalecieron capacidades del IPK para detección molecular de patógenos transmitidos por garrapatas. Se encontraron nuevas evidencias serológicas de infección por B. burgdorferi en pacientes con sospechas de enfermedad de Lyme. Se demostró ausencia de anticuerpos específicos contra B. burgdorferi en donantes de sangre. Se encontraron evidencias de anticuerpos contra Anaplasma phagocytophilum, Ehrlichia chaffeensis y Babesia microti. Se demostró presencia de Anaplasma spp., Babesia spp., Rickettsia amblyommii y Coxiella burnetii en garrapatas. Se constataron conocimientos insuficientes sobre enfermedad de Lyme en personal médico. Conclusiones: estos resultados alertan a las autoridades nacionales de salud pública y personal médico sobre el riesgo de infección en individuos expuestos a picaduras de garrapatas.

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.

Four variants of the potassium acetate procedure for DNA extraction from ixodid ticks at different stage of their life cycles were evaluated and compared with phenol-chloroform and ammonium hydroxide methods. The most rapid and most efficient variant was validated in the DNA extraction procedure from the engorged ticks collected from bovine, canine as well as from house ticks for the screening of Borrelia burgdorferi sensu lato, Anaplasma spp. and Babesia spp. The ammonium hydroxide procedure was used for non-engorged ticks. All the variants were efficient and allowed obtaining PCR-quality material according to the specific amplification of 16S rRNA gene fragment of the original tick. DNA extracted from the ticks under the study was tested by multiplex PCR for the screening of tick-borne pathogens. Anaplasma spp. and Babesia spp. amplification products were obtained from 29/48 extracts. Ammonium hydroxide protocol was not efficient for two extracts. Detection of amplification products from the PCR indicated that DNA had been successfully extracted. The potassium acetate procedure could be an alternative, rapid, and reliable method for DNA extraction from the ixodid ticks, mainly for poorly-resourced laboratories.

INTRODUÇÃO: O diagnóstico microbiológico da leptospirose inclui métodos bacteriológicos e sorológicos; os primeiros permitem a detecção direta de leptospiras e, à exceção do cultivo, são considerados como presuntivos, mas constituem ferramentas valiosas para o diagnóstico rápido, principalmente nos falecidos. OBJETIVO: Avaliar a coloração de prata Fontana modificada em amostras experimentalmente infectadas. MATERIAL E MÉTODOS: Urinas humanas e hamsters foram infectados experimentalmente com diferentes cepas de Leptospira interrogans sensu lato. Para a avaliação, foi utilizado meio de cultura líquido, culturas de leptospiras, urinas humanas experimentalmente infectadas e não infectadas, impressões clarificadas e não clarificadas e esfregaços de suspensões clarificadas e não clarificadas a partir de tecidos de hamsters infectados e não infectados para o experimento. A sensibilidade analítica do ensaio foi comparada com a microscopia de campo escuro e de cultura. Outras espécies de bactérias e fungos também foram utilizadas. RESULTADOS: A coloração de prata de Fontana modificada permitiu observar claramente e bem definida a estrutura helicoidal das leptospiras. Nas culturas destas e de urinas humanas infectadas, pôde-se observar até (1-10) × 10³ leptospiras/ml, sensibilidade superior à da microscopia de campo escuro e inferior à da cultura. Os melhores resultados nos tecidos foram obtidos em impressões clarificadas e a partir de esfregaços de suspensões não clarificadas. Estruturas morfológicas e tingidas compatíveis com leptospiras não foram observadas nas amostras livres destas. CONCLUSÃO: Esse procedimento permitiu diferenciar a morfologia característica das leptospiras. É um procedimento simples, fácil de realizar e com reagentes estáveis pelo o que a sua aplicação é sugerida.

INTRODUCTION: The microbiological diagnosis of leptospirosis comprises bacteriological and serological methods. The former ones allow the direct detection of leptospires and are considered presumptive with the exception of culture. Therefore, they constitute invaluable tools for rapid diagnosis, mainly in samples from deceased subjects. OBJECTIVE: To evaluate a modified Fontana silver staining method in experimentally infected samples. MATERIAL AND METHODS: Human and animal (hamster) urine samples were experimentally infected with different strains of Leptospira interrogans sensu lato. Liquid culture medium, leptospira cultures, experimentally infected and non-infected human urine samples, clarified and non-clarified imprints, and clarified and non-clarified suspension smears from tissues of experimentally infected and non-infected hamsters were applied for the ass essment of silver staining. The analytical sensitivity of the assay was compared with dark field microscopy and culture. Other bacterial and fungi species were also used. RESULTS: The modified Fontana silver staining allowed the accurate observation of the well-defined leptospire helical structure. On leptospire cultures from infected human samples, we could observe until (1-10) × 10³ leptospires/ml, higher sensitivity in comparison with direct dark field microscopy and lower in comparison with culture. The best results in tissues were obtained on clarified imprints and non-clarified suspension smears. Morphological and stainable structures compatible with leptospires were not observed in the samples without them. CONCLUSION: This procedure allowed differentiating the characteristic morphology of leptospires. As its application suggests, it consists of a simple and easily conducted procedure with stable reagents.

Borrelia burgdorferi sensu lato is the causative agent of Lyme disease, borreliosis not reported in Cuba but clinical and epidemiological suspicions exist since the 1980's, and there were no microbiological testing for confirmation. This led to perform a series of researches to provide scientific evidences about the presence of this agent in the country, including the evaluation and implementation of microbiological methods for the detection of this spirochete, confirmation of infection in clinical samples from patients with clinical and epidemiological suspicions, the estimated seroprevalence of antibodies against this agent in a population at risk and the molecular detection of borrelia in ticks of veterinary and medical importance. The microbiological tools evaluated (culture medium modified, methods of extracting genetic material from ticks for subsequent detection of B. burgdorferi sensu lato by molecular testing, and analytical sensitivity of two sets of primers) and implemented (specific serological tests) strengthened the IPK analytical capacity with novel methods for future researches. We found specific serological evidences of autochthonous infection by this organism in samples of individuals with clinical and epidemiological suspicion, and those exposed to tick bites. Genetic material of B. burgdorferi sensu lato was undetected in the analyzed ticks, what does not rule them out as potential carriers of the agent. In this paper we show evidence highly suggestive of infection with B. burgdorferi sensu lato and it is the first of its kind in the country.

Borrelia burgdorferi sensu lato causa la enfermedad de Lyme, una borreliosis no reportada en Cuba. Desde la década de 1980 se tenían sospechas clínicas y epidemiológicas, pero no había pruebas microbiológicas que lo confirmaran. Las investigaciones científicas sobre su presencia en el país incluyeron la evaluación e implementación de métodos microbiológicos para su detección, la confirmación de la infección en muestras clínicas de pacientes con sospechas clínico-epidemiológicas de esta enfermedad, la estimación de la seroprevalencia de anticuerpos contra este agente en una población de riesgo y la detección molecular de borrelias en garrapatas de importancia médico-veterinaria. Las herramientas microbiológicas evaluadas (medio de cultivo modificado, métodos de extracción de material genético de garrapatas para la posterior detección de B. burgdorferi sensu lato por pruebas moleculares, y la sensibilidad analítica de dos juegos de cebadores) e implementadas (pruebas serológicas específicas) fortalecieron la capacidad analítica del Instituto de Medicina Tropical Pedro Kourí, en Cuba, con nuevos métodos para futuras investigaciones. En las muestras de individuos con sospechas clínico-epidemiológicas y en los expuestos a mordeduras por garrapatas, se encontraron evidencias serológicas específicas de la infección autóctona por este microorganismo. No se detectó material genético de B. burgdorferi sensu lato en las garrapatas analizadas, lo que no descarta que sean posibles vectores del agente. Las evidencias de este estudio son altamente sugestivas y constituye el primero de su tipo en Cuba.

Lyme disease has not been officially reported in Cuba. However, clinical cases have been serologically reported. Seroprevalence survey of Borrelia burgdorferi sensu stricto antibodies in humans in the country has not been conducted. OBJECTIVE: To estimate the prevalence of borrelial antibodies in inhabitants of a village with historically high level of tick infestation. METHODS: Serum specimens from 247 persons randomly selected from the population of the village were examined by IgG Western blot using B31 strain for estimating the prevalence of antibodies profile. RESULTS: A seroprevalence value interval (95% CI) of 0.6%-7.2% was estimated for the studied population. The prevalent borrelial protein bands on immunoblots were 41, 72, 90/93, 34, 47, 60, 58, 56, 65/66 and 31 kDa in a decreasing order of significance. CONCLUSION: These results support the previous serological findings, suggesting the presence of this borreliosis in Cuba.

INTRODUCTION: at present, there are several tick-borne emerging pathogen species of medical and veterinary importance. Few studies on these agents and its diseases have been made in Cuba. OBJECTIVES: to determine the presence of some of these pathogens in Cuban ticks existing in the equine cattle. METHODS: ninety five ticks collected from domestic use horses were processed, preserved in alcohol and taxonomically identified according to the set classifications. Their DNA was extracted and subjected to several polymerase chain reactions with specific primers for microbial groups Borrelia burgdorferi sensu lato, Anaplasma-Ehrlichia, y Babesia-Theileria. Each of the products from polymerase chain reactions underwent reverse line blot hybridation using probes for each group as well as specific probes for the main species included in these groups. RESULTS: the studied ticks belonged to Dermacentor (Anocentor) nitens (60 %), Amblyomma cajennense (38 %) y Rhipicephalus (Boophilus) microplus (2 %). Seven Dermacentor (Anocentor) nitens ticks infested with Anaplasma/Ehrlichia bacteria were detected but the species in question could not be detected by the used probes. One of these ticks was also co-infested with Babesia bovis. CONCLUSIONS: it is suggested that a new species of Anaplasma o Ehrlichia, not reported in Cuba before now, is circulating, so studying a higher number of ticks is needed and new probes in reverse line blot hybridation or other methodologies must be incorporated to allow exactly determining the species that may affect the Cuban domestic horses at present.

INTRODUCCIÓN: en la actualidad son varias las especies de patógenos emergentes de importancia médica y veterinaria transmitidos por garrapatas. Los estudios sobre estos agentes y sus enfermedades han sido escasos en Cuba. OBJETIVOS: conocer la presencia de algunos de estos patógenos en garrapatas cubanas que afectan el ganado equino. MÉTODOS: se procesaron 95 garrapatas colectadas de caballos domésticos, conservadas en alcohol e identificadas taxonómicamente según claves convencionales. A cada una se le realizó extracción de ADN y posteriormente diferentes reacciones en cadena de la polimerasa utilizando cebadores específicos para los grupos microbianos Borrelia burgdorferi sensu lato, Anaplasma-Ehrlichia, y Babesia-Theileria. Cada uno de los productos de las reacciones en cadena de la polimerasa fue sometido a hibridaciones en línea reversa utilizando sondas para cada grupo en cuestión, así como específicas para las principales especies de estos. RESULTADOS: las garrapatas estudiadas pertenecían a las especies Dermacentor (Anocentor) nitens (60 %), Amblyomma cajennense (38 %) y Rhipicephalus (Boophilus) microplus (2 %). Se detectaron 7 garrapatas Dermacentor (Anocentor) nitens infectadas con bacterias del grupo Anaplasma/Ehrlichia, y no se pudo identificar la especie en cuestión con las sondas utilizadas. Una de estas garrapatas estaba además coinfectada con Babesia bovis. CONCLUSIONES: se sugiere la circulación de una nueva especie de Anaplasma o Ehrlichia no reportada antes en Cuba, por lo que se necesita estudiar un número mayor de garrapatas, así como la incorporación de nuevas sondas en la hibridación en línea reversa u otras metodologías que permitan conocer con exactitud las especies que pudiesen afectar hoy día los caballos domésticos.

The aim of the present study was to assess the possible use of a modified medium, prepared in the laboratory using the constituents of Barbour-Stonner-Kelly (BSK) medium and medium 199 as base, for the culture of Borrelia strains, comparing the growth of individual strains in this medium and in the BSK-H medium, and the protein profile and antigenic characteristics of Borrelia proteins expressed in these media. A qualitative evaluation of growth of Borrelia species was made with acceptable results (morphology and motility), but during a quantitative evaluation using the three main genospecies of Borrelia, the better results were obtained with a B. burgdorferi sensu stricto strain. The modified medium did not enable the growth of a B. afzelii strain. The protein profile and antigenic characteristic of the expressed proteins in the modified medium were studied with satisfactory results. These results suggest the modified medium as an alternative for the cultivation of Borrelia strains, with some limitations, in poorly-resourced laboratories.

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.

Se evaluaron por vez primera en Cuba las tecnologías serológicas rápidas para realizar la pesquisa y confirmación de la leptospirosis humana, que mundialmente están disponibles. Su reconocimiento clínico resulta difícil, el diagnóstico etiológico rápido es de vital importancia Se obtuvieron valores de sensibilidad y especificidad superiores a 90 % en los sistemas confirmatorios y de 85 % en el de pesquisa. De los pacientes graves estudiados por estas tecnologías 50 % 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 %). 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.

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 % (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.

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 % (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 % (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 % (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.

An agglutination latex of national production (leptospira-latex-IPK) was developed for the rapid diagnosis of human leptospira. The optimum working dilution of the thermoresistant antigen (TR) coupled to the latex particles was 1/80. Commerciable latex particles of 0.8 m were used. A sensitivity of 86.6 % and a specificity of 88.7 % were obtained. The index of coincidence between the leptospira-latex-IPK and the lepto dri dot (commercial set) was 85 %. On applying the statistical method X2, there were no significant differences between leptospira-latex-IPK, MAT (gold standard) and the passive hemagglutination. This way, it was obtained the first biological result of latex in Cuba that is useful for the rapid diagnosis of human leptospirosis, and that should be evaluated in practice to determine new parameters, such as stability and reproducibility.

Se elaboró un látex de aglutinación de producción nacional (leptospira - látex - IPK) para el diagnóstico rápido de la leptospirosis humana. La dilución óptima de trabajo del antígeno termorresistente (TR) acoplado a las partículas de látex fue de 1/80. Se emplearon partículas de látex comerciables de 0,8 µm. Se obtuvo una sensibilidad del 86,6 % y una especificidad del 88,7. El índice de coincidencia entre el leptospira - látex - IPK y el lepto dri dot (estuche comercial) fue de un 85 %. Al aplicar el método estadístico de X2, no existieron diferencias significativas entre el leptospira - látex-IPK, MAT (gold standard) y la hemaglutinación pasiva. Se obtuvo, de esta manera, el primer resultado biológico de látex en Cuba, útil en el diagnóstico rápido de la leptospirosis humana, el cual deberá ser evaluado en el terreno para determinar nuevos parámetros, entre ellos, la estabilidad y la reproducibilidad.

The Lepto Tek Lateral Flow technique (LF) in the serological and rapid diagnosis of severe cases with clinical and epidemiological suspicion of human leptospirosis was introduced in and applied by the National Laboratory of Leptospirosis Reference (NLLR). Conventional lab techniques (microagglutination MAT and indirect hemagglutination HA ) were used in the confirmation of cases under study, 40 serum samples corresponding to 33 patients suspected of human leptospirosis were studied. Of them, 21 samples proved to be positive by LF, accounting for 52.5 %. On comparatively applying the LF and HA techniques to 26 monosera and 7 pairs of serum, 11 and 5 positive cases were obtaind, respectively. The coincidence between them was 73 % on studying monosera and 71.0 on studying the pairs of serum. On applying MAT to 9 suspected cases, 7 of them yielded positive. The most frequently found serogroups were: Icterohaemorrhagiae and Canicola. Only 5 positive cases were detected by the 3 applied techniques. The coincidence between MAT and LF techniques was 89 % and that between HA and MAT was 100.0. The application of MAT and HA offered reliability to the obtained results and it was evidenced the high sensitivity of LF. The importance of the use of matched sera for the accurate diagnosis of this disease was demonstrated.

La técnica de Lepto tek lateral flow (LF) en el diagnóstico serológico y rápido de casos graves con sospecha clínica y epidemiológica de leptospirosis humana se introdujo y se aplicó en el Laboratorio Nacional de Referencia de Leptospiras (LNRL). Se emplearon las técnicas convencionales de laboratorio (microaglutinación [MAT] y hemaglutinación indirecta [HA]) en la confirmación de los casos en estudio. Se estudiaron 40 muestras de sueros correspondientes a 33 pacientes sospechosos de leptospirosis humana, de las cuales se obtuvo positividad en 21 de ellas mediante LF, que representa un 52,5 %. Al aplicar comparativamente las técnicas de LF y HA en 26 monosueros y 7 pares de sueros, se obtuvieron 11 y 5 casos positivos respectivamente. La coincidencia entre ambas fue de un 73 % al estudiar los monosueros y del 71,0 al estudiar los pares de sueros. Al aplicar la MAT en 9 casos sospechosos, resultaron 7 positivos. Los serogrupos más frecuentes encontrados fueron: icterohaemorrhagiae, y canicola. Se detectaron solo 5 casos positivos por las 3 técnicas aplicadas. La coincidencia entre las técnicas de MAT y LF fue del 89 % y la de HA y la MAT del 100,0. La aplicación de la MAT y la HA ofreció confiabilidad en los resultados obtenidos y se evidenció la gran sensibilidad del LF. Se demostró la importancia del uso de sueros pareados para el diagnóstico certero de esta enfermedad.

The effect of different alkalizing agents of urine on the isolation of leptospiras was studied. Better results were obtained on using the basic solution of NaOH 1N inoculated in urine one hour after being alkalized.

Se estudió el efecto de diferentes agentes alcalinizantes de la orina sobre el aislamiento de leptospiras. Se obtuvieron mejores resultados al emplear la solución básica de NaOH 1N y en la orina inoculada 1 h después de haber sido alcalinizada.