Introducción: en el proceso del diagnóstico neuropsicológico, los instrumentos de tamizaje cognitivo, son una herramienta útil en la identificación de cambios mentales del sujeto, en momentos puntuales o a través del tiempo. Su uso se fundamenta en el análisis psicométrico. Objetivo: determinar el acuerdo inter e intra-observador en el MoCA test y el MMSE, aplicado por profesores y estudiantes en procesos de entrenamiento de tamización cognitiva. Materiales y métodos: a los estudiantes y profesores en entrenamiento en la puntuación del MoCA test y el MMSE, se les presentó un video en dos sesiones, con un intervalo de 5 meses, mostrando el desempeño de dos adultos mayores, respondiendo el MoCA test y el MMSE, previo consentimiento informado. Se compararon los puntajes dados en las dos sesiones por los sujetos en entrenamiento, con los de ellos mismos (intra-observador), usando el coeficiente de concordancia y correlación de Lin(rho) y con los del grupo restante (inter-observador) usando el coeficiente de correlación intra-clase (ICC). Resultados: participaron 46 evaluadores. Se encontró alta confiabilidad inter-observador para el MoCA (ICC=0.86), pero baja para el MMSE (ICC=0.24) y baja confiabilidad intra-observador tanto para el MoCA (rho paciente 1=0.012 y rho paciente 2=0.152) como para el MMSE (rho paciente 1=0.008 y rho paciente 2=0.012). Aunque los puntajes difirieron, las clasificaciones diagnósticas realizadas por los evaluadores fueron similares a las del patrón de oro. Conclusión: la correcta aplicación del test, requiere varios entrenamientos, y aunque hubo pocas diferencias entre los puntajes, los errores cuando se está cerca del punto de corte propuesto, aumentan el riesgo de sesgo.

Introduction: The instruments for screening cognitive functions, applied to subjects in clinical settings and research, are useful for determining if this person has any trouble in cognition or show changes in the time. The usefulness of these instruments is defined with the evaluation of their psychometrics properties. Objective: This study allows to determine the intra and inter-observer agreement, when the MoCA test and MMSE were applied by a group in training process Materials and methods: The study group who attended two training sessions, with an interval of 5 months, scored the MoCA test and MMSE, from two patients which were filmed responding the tests, previous informed consent signature. We compared how close were the scores of participants among themselves by concordance correlation coefficient of Lin (rho) and with those given from the others by intra-class correlation coefficients (ICC). Results: In total, 46 participants were included. Intra-rater reliability was high for MoCA test (ICC = 0.86), but it was poor for MMSE (ICC=0.24). Inter-rater was poor for MoCA test (rho patient 1= 0.012, rho patient 2= 0.152) and MMSE (rho patient 1 = 0.008, rho patient 2 = 0.012). Although the scores between participants and gold standard were different, the diagnoses were similar. Conclusion:The correct scoring of the test, requires several trainings to clinical and research groups, and although they can be found few differences between scores applied by non-expert personnel, if the scores mistakenly given, are close to the cut-of point proposed for each test, the bias increases.

Despite recent advances in the treatment of some forms of leishmaniasis, the available drugs are still far from ideal due to inefficacy, parasite resistance, toxicity and cost. The wide-spectrum antimicrobial activity of 2-nitrovinylfuran compounds has been described, as has their activity against Trichomonas vaginalis and other protozoa. Thus, the aim of this study was to test the antileishmanial activities of six 2-nitrovinylfurans in vitro and in a murine model of leishmaniasis. Minimum parasiticide concentration (MPC) and 50% inhibitory concentration (IC50) values for these compounds against the promastigotes of Leishmania amazonensis, Leishmania infantum and Leishmania braziliensis were determined, as were the efficacies of two selected compounds in an experimental model of cutaneous leishmaniasis (CL) caused by L. amazonensis in BALB/c mice. All of the compounds were active against the promastigotes of the three Leishmania species tested. IC50 and MPC values were in the ranges of 0.8-4.7 µM and 1.7-32 µM, respectively. The compounds 2-bromo-5-(2-bromo-2-nitrovinyl)-furan (furvina) and 2-bromo-5-(2-methyl-2-nitrovinyl)-furan (UC245) also reduced lesion growth in vivo at a magnitude comparable to or higher than that achieved by amphotericin B treatment. The results demonstrate the potential of this class of compounds as antileishmanial agents and support the clinical testing of Dermofural(r) (a furvina-containing antifungal ointment) for the treatment of CL.

Introduction: leishmaniasis is a parasitic disease caused by several species of the genus Leishmania. The disease presents in a great variety of clinical forms. Psoriatic leishmaniasis has occasionally been reported among the cutaneous forms of the disease. Diagnostic certainty of the condition is indispensable, given the toxicity and side effects of the first line drugs used to treat it. Several methods are applied to reach an accurate diagnosis. These range from identification of the parasite in the tissue to detection of its DNA. Methods: data from a clinical case with suspected leishmaniasis were collected and analyzed. Various laboratory methods were applied to define the diagnosis, among them direct parasitological examination, histopathological examination, culture and polymerase chain reaction (PCR-18S and PCR-hsp70). Results and discussion: the tests performed to confirm the diagnosis of leishmaniasis were all negative. Histological examination diagnosed the dermatological lesion as psoriasis. Conclusions: very few cases of psoriatic leishmaniasis have been reported in the scientific literature. Comprehensive examination of the case by various methods ruled out the presence of leishmaniasis, allowing the application of the appropriate treatment and thus avoiding the inappropriate use of antimonials.

Introducción: la leishmaniasis es una enfermedad parasitaria causada por varias especies del género Leishmania, que se presenta en formas clínicas muy diversas. Entre las formas cutáneas, se ha notificado ocasionalmente la leishmaniasis psoriasiforme. Es imprescindible tener la certeza diagnóstica de leishmaniasis dada la toxicidad y los efectos colaterales que tienen las drogas de primera línea para tratarla. Se emplean varios métodos para llegar al diagnóstico, como son desde la identificación del parásito en el tejido hasta la detección de su ADN. Métodos: se realizó la recopilación y análisis de la información de un caso clínico con supuesta leishmaniasis, y se aplicaron distintos métodos de laboratorio para definir el diagnóstico, tales como examen parasitológico directo, examen histopatológico, cultivo y reacción en cadena de la polimerasa (PCR-18S y PCR-hsp70). Resultados y discusión: los exámenes realizados para confirmar el diagnóstico de leishmaniasis fueron negativos. Al estudio histológico, la lesión dermatológica se concluye como psoriasis. Conclusiones: aunque la literatura científica notifica casos de leishmaniasis de tipo psoriasiforme estos son escasos. El estudio integral del caso por diferentes métodos descartó la supuesta leishmaniasis en este paciente, lo cual permitió la aplicación del tratamiento adecuado y evitó el uso indebido de antimoniales para su tratamiento.

Leishmania is a parasitic protozoon causing Leishmaniasis, disease with a varied clinic presentation and a wide world distribution. WHO considers it as a non-controlled and re-emergent disease and its transmission patterns have been affected in past years by the human action among other features. Its diagnosis change according to the clinical way of presentation and nowadays it is recommended the identification of infectious species like a very useful feature to prescribe a appropriate treatment, to perform the clinical monitoring and the most important in epidemiologic studies including the vector and or reservoirs study. The timely treatment is until now one of the few available control measures since despite the efforts performed there isn't a vaccine against this affection. The aim of present paper is to present a literature review including significant features of Leishmaniasis in the present international context.

Leishmania es un protozoo parásito causante de la leishmaniasis, enfermedad de variada presentación clínica y de amplia distribución mundial. La Organización Mundial de la Salud la considera una enfermedad re-emergente y no controlada, y sus patrones de transmisión se han visto afectados en los últimos años por la acción humana, entre otros aspectos. El diagnóstico varía de acuerdo con la forma clínica de presentación y actualmente se recomienda la identificación de la especie infectante como elemento de mucha utilidad para indicar un tratamiento adecuado, realizar el monitoreo clínico y como aspecto importante en estudios epidemiológicos, que incluyan el estudio de vectores y/o reservorios. El tratamiento oportuno es, hasta el momento, una de las pocas medidas de control disponibles, ya que a pesar de los esfuerzos realizados, no existe vacuna contra esta afección. En este trabajo se presenta una revisión de la literatura que incluye aspectos importantes de la leishmaniasis, en el contexto internacional actual.

Neste estudo descreve-se o efeito de um extrato metanólico de Echinaster echinophorus spp. no parasita Leishmania amazonensis. Em testes com as formas promastigotas e amastigotas, o IC50 do extrato foi 62,9 e 37,5 μg.mL-1, respectivamente. O extrato também tem toxicidade moderada em macrófagos de camundongos BALB/c. O tratamento de camundongos BALB/c infectados com L. amazonensis com doses diárias de 100 mg/kg/dia via intraperitoneal durante 15 dias mostrou-se relativamente efetivo no controle da infecção. Esta investigação confirma a importância de produtos naturais como fonte para a descoberta de fármacos com funções anti-Leishmania.

In this study, a methanolic extract from Echinaster (Othilia) echinophorus was evaluated for activity against Leishmania amazonensis. The extract showed activity against the promastigote and amastigote forms with IC50 values of 62.9 and 37.5 μg.mL-1 respectively. This extract showed a moderate toxicity on macrophages from BALB/c mice. A dose of 100 mg/kg/day was effective when administered during 15 days by intraperitoneal route to BALB/c mice infected experimentally.

Leishmaniasis is one of the most important parasitic infections, but current treatments are unsatisfactory due to their toxicity, cost and resistance. Therefore, the development of new antileishmanial compounds is imperative. Many people who live in endemic areas use plants as an alternative to treat the disease. In this paper, we characterised the essential oil from Piper auritum, evaluated its cytotoxicity and determined its antileishmanial activity. The chromatogram obtained by gas chromatography revealed 60 peaks and we found that safrole was the most abundant compound, composing 87% of the oil. The oil was active against the promastigotes of Leishmania major, Leishmania mexicana, Leishmania braziliensis and Leishmania donovani with a favourable selectivity index against peritoneal macrophages from BALB/c mice. The Piper-oil inhibited the growing of intracellular amastigotes of L. donovani with an IC50 value of 22.3 ± 1.8 μg/mL. This study demonstrates the usefulness of the essential oils as a promising alternative to treat leishmaniasis.

INTRODUCTION: the induction of heat shock proteins is a homeostatic mechanism that protects cells from the deleterious effects of thermal and other environmental stresses. In addition, they have important cell functions. The 20kDa heat shock protein in Leishmania amazonensis was recently reported. OBJECTIVE: to describe the methodology used for cloning of heat shock proteins, which allowed the study of some biological properties. METHODS: the hsp20 gene coding region was amplified by polymerase chain reaction using adequate primers. The amplified product was initially cloned in pCR2.1 vector (Invitrogen) and then in pET-28b vector (Novagen), to obtain recombinant protein. The same fragment was cloned also in the eukariote expression vector pcDNA3 (Invitrogen). The nucleotidic sequencing of the different clones was made, in order to verify their fidelity. RESULTS: the recombinant plasmids that encode HSP20 protein in Leishmania and allow obtaining massively recombinant protein and DNA were produced. CONCLUSIONS: both plasmids were useful to study some of the biological properties of this protein. This approach could be useful for similar research and represent a suitable methodological guideline.

INTRODUCCIÓN: la inducción de las proteínas de choque térmico constituyen un mecanismo homeostático que protege a las células del efecto destructivo del calor u otras condiciones de estrés ambiental, paralelamente, ellas cumplen importantes funciones celulares. La proteína de choque térmico de 20 kDa se reportó recientemente en Leishmania amazonensis. OBJETIVO: describir la metodología utilizada para realizar el clonaje de las proteínas de choque térmico, lo que permitió acometer estudios de algunas propiedades biológicas. MÉTODOS: la región codificante del gen hsp20 se amplificó mediante la reacción en cadena de la polimerasa con cebadores adecuados. El producto amplificado se clonó inicialmente en el vector pCR2.1 (Invitrogen) y después en el vector de expresión en procariotas pET-28b (Novagen), para obtener proteína recombinante. De manera paralela, el mismo fragmento se clonó en el vector de expresión en eucariotas pcDNA3 (Invitrogen) para obtener un posible preparado vacunal de ADN. Se realizó la secuenciación nucleotídica de los clones obtenidos, con la finalidad de verificar su fidelidad. RESULTADOS: se obtuvieron plásmidos recombinantes que codifican la HSP20 de Leishmania, y permiten la obtención de proteína recombinante y de ADN en forma masiva. CONCLUSIONES: ambos plásmidos fueron útiles para estudiar algunas de las propiedades biológicas de esta proteína. Este acercamiento puede ser de interés en otros trabajos de esta índole y constituir una guía metodológica.

BACKGROUND: leishmaniosis covers a group of infectious diseases caused by Leishmania genus hemoflagellate organisms. At present, this disease is prevailing in 88 tropical countries worldwide; it is estimated that 1.5 to 2 million new cases will occur every year. The control of the disease is deficient, mainly because there is no effective vaccine available. Although several drugs for treatment exist, all of them require parenteral way of administration, delayed and costly therapies and cause high toxicity. The tropical disease program of the World Health Organization considers plant research as an essential priority for Leishmania treatment. OBJECTIVE: to evaluate the antileishmanial action of extracts from Citrus limetta, Cucurbita maxima y Rhoeo spathacea. METHODS: aLeishmania amazonensis strain, the causative agent of cutaneous leishmaniosis, was used in this paper. The extract effect on the stagings of promastigot and amastigot of the parasite as well as on the peritoneal macrophages from BALB/c mouse was determined to assess its possible toxicity. RESULTS: Curcubita maxima y Rhoeo spathacea showed higher percentage of growth inhibition in promastigots. Good anti-leishmanial activity was also observed against amastigots, whereas its citotoxicity was moderate, with a selectivity index of 3. CONCLUSIONS: Curcubita maxima y Rhoeo spathacea extracts were the only ones that had anti-leishmanial activity.

INTRODUCCIÓN: la leishmaniosis comprende un grupo de enfermedades infecciosas causadas por organismos hemoflagelados del género Leishmania. Hoy día, esta enfermedad prevalece en 88 países tropicales alrededor del mundo y existe un estimado de 1,5 a 2 millones de nuevos casos por año. Su control es deficiente, principalmente al no contar con una vacuna eficaz. Aunque existen varios fármacos para su tratamiento, todos requieren una administración parenteral, tratamientos muy largos, costosos, y causan una alta toxicidad al organismo. El programa de enfermedades tropicales de la Organización Mundial de la Salud considera la investigación de plantas como una esencial prioridad para el tratamiento de Leishmania. OBJETIVO: evaluar la actividad antileishmanial de extractos provenientes de Citrus limetta, Cucurbita maxima y Rhoeo spathacea. MÉTODOS: en este trabajo se utilizó una cepa de Leishmania amazonensis agente causal de la leishmaniosis cutánea. Se determinó la actividad de los extractos frente a los estadios de promastigote y amastigote del parásito, así como frente a macrófagos peritoneales de ratón BALB/c, para valorar su posible toxicidad. RESULTADOS: los extractos provenientes de Curcubita maxima y Rhoeo spathacea fueron los que mostraron un mayor porcentaje de inhibición del crecimiento frente a promastigotes. Frente a amastigotes también mostraron una buena actividad antileishmanial, mientras que su citotoxicidad fue moderada con un índice de selectividad de 3. CONCLUSIONES: solo los extractos de Curcubita maxima y Rhoeo spathacea fueron los que mostraron una actividad antileishmanial.

Introducción. El análisis de la longitud de los fragmentos de restricción del producto amplificado y el estudio del ADN polimórfico amplificado al azar han demostrado ser herramientas útiles para la tipificación de Leishmania. Objetivos. Estudiar la utilidad de las técnicas moleculares para la identificación y tipificación de cepas de referencia de Leishmania spp. del Nuevo Mundo y valorar su aplicabilidad a muestras clínicas. Materiales y métodos. Se aplicó PCR para amplificar el gen que codifica la cisteíno-proteinasa B, y el análisis de la longitud de los fragmentos de restricción del producto amplificado utilizando ácido desoxirribonucleico de 16 cepas de referencia de Latinoamérica y de muestras clínicas de pacientes colombianos con leishmaniasis, y la técnica del ácido desoxirribonucleico polimórfico amplificado al azar utilizando ocho cepas de referencia. Se establecieron los patrones de bandas en cada caso. Resultados. Se obtuvo producto de amplificación en la PCR para Leishmania braziliensis, L. peruviana, L. panamensis y L. guyanensis. Para el resto, no fue posible amplificar el gen con los cebadores utilizados. La restricción mostró un patrón de bandas común para L. peruviana, L. guyanensis y L. panamensis, mientras L. braziliensis, presentaba un perfil individual único. El análisis de restricción del producto amplificado generó un patrón de bandas similar en los cinco pacientes estudiados, que se correspondía con el patrón generado por L. peruviana, L. guyanensis o L. panamensis. Mediante la amplificación al azar se obtuvieron patrones de bandas reproducibles con todas las cepas estudiadas, que posibilitaron la diferenciación. Se discuten las ventajas y limitaciones de ambos procederes. Conclusiones. El combinar ambas metodologías resultaría útil para identificar especies de importancia médica, tomando en cuenta sus ventajas y desventajas.

Introduction. The analysis of the PCR-restriction fragment length polymorphism and random amplified polymorphic DNA have been useful tools for Leishmania identification. Objectives. Molecular procedures were demonstrated for identification and typing of reference strains of New World Leishmania and their applicability was validated for clinical samples. Materials and methods. DNA was extracted from 16 reference strains of Latin American Leishmania as well as from clinical samples of leishmaniasis patients. A sequence coding for cysteine proteinase B was amplified by PCR and subjected to restriction fragment length polymorphism analysis. The enzyme used was Taq1. For eight of the reference strains, the random amplified polymorphic desoxyribonucleic acid technique (RAPD) was applied. Band patterns for Leishmania species differentiation were established each each method. The sample size of the clinical sample was of 5. Results. PCR products of the cysteine proteinase B gene were obtained for L. braziliensis, L. peruviana, L. panamensis and L. guyanensis. For the other species, L. mexicana, L. amazonensis, L. garnhami, L. lainsoni, L. chagasi, L. naiffi, no amplification occurred. The patterns of restriction fragments revealed band patterns in common for L. peruviana, L. guyanensis and L. panamensis, whereas L. braziliensis had a distinctive pattern. When human samples were examined, amplification occurred for all cases, and the profiles corresponded to the common profile of L. peruviana, L. guyanensis and L. panamensis. The RAPD technique demonstrated reproducible and distinctive patterns for each of the 8 reference strains, L. mexicana, L. amazonensis, L. garnhami, L. lainsoni, L. chagasi, L. naiffi, making possible to differentiate all them. The advantages and limitations of each procedure are discussed. Conclusions. The combination of RFP and RAPD methodologies provide useful tools to identify medical important species of Leishmania by recognizing DNA sequences characteristic of each species.

Até hoje não temos vacina contra a Leishmania e a quimioterapia é a indicação para o controle desta doença. Os remédios que hoje utilizamos são tóxicos e muito caros e além disso o resultado não é sempre o desejado. Por isso, uma terapia de combinação é a melhor opção. Este trabalho mostra que o óleo de essência de C. ambrosioides tem atividade sinérgica junto com a pentamidina sobre os promastigotas de L. amazonensis, diferente do resultado da combinação de antimônio de meglumine e anfotericina B e o óleo de essência.

To date, there are no vaccines against Leishmania, and chemotherapy remains the mainstay for the control of leishmaniasis. The drugs of choice used for leishmaniasis therapy are significantly toxic, expensive and with a growing frequency of refractory infections. Because of these limitations, a combination therapy is the better hope. This work demonstrates that the essential oil from Chenopodium ambrosioides shows a synergic activity after incubation in conjunction with pentamidine against promastigotes of Leishmania amazonensis. However, an indifferent effect has been found for combinations of meglumine antimoniate or amphotericin B and the essential oil.

The optimization of the PCR conditions for amplification of the gene coding for the 70kDa (HSp70) heat shock protein as well as the analysis of the restriction fragment length polymorphism (RFLP) were carried out. DNA from a reference strain of Leishmania mexicana was used as template. Analytical sensitivity and specificity, and reproducibility of PCR using DNA from L. mexicana, L.amazonensis, L. guyanensis and L. lainsoni were determined. A 1.3 kp band was obtained, which confirmed gene amplification. The band patterns derived from Haelll enzyme digestion allowed differentiating several species. L. guyanensis and L. lainsoni were different from each other, while L. mexicana and L. amazonensis, which shared a common pattern, were different from the other two species. Analytical sensitivity and specificity were adequate. The enzymatic restriction of the PCR product made it possible to differentiate Leishmania spp. from T. cruzi. The feasibility of identifying and typifying species from the American continent through PCR-RFLP/Hsp70 and of using enzymatic restriction of amplified product to distinguish Leishmania spp. from Trypanosoma cruzi was shown. This was the first step in implementing these molecular methods in the reference laboratory of the Institute.

Se realizó la estandarización de las condiciones de amplificación del gen que codifica para la proteína de choque térmico de 70 kDa (Hsp70) de Leishmania mediante la reacción en cadena de la polimerasa (PCR-Hsp70), así como el análisis posterior de la longitud de los fragmentos de restricción (RFLP) del producto amplificado, utilizando como molde ADN puro de una cepa de referencia de Leishmania mexicana. Se estudió la sensibilidad y especificidad analíticas de la PCR, así como la reproducibilidad, utilizando ADN de L. mexicana, L. amazonensis, L. guyanensis y L. lainsoni. Se obtuvo una banda de 1,3 Kpb, demostrándose la amplificación del gen que codifica para la Hsp70. Los patrones de bandas obtenidos tras la digestión enzimática, utilizando la enzima Hae III, permitieron establecer diferencias entre las especies estudiadas: L. guyanensis y L. lainsoni se diferencian entre sí y estas a su vez de L. mexicana y L. amazonensis, que mostraron un patrón de bandas común. La sensibilidad y especificidad analíticas de la técnica fueron adecuadas. Se demostró la factibilidad de identificar y tipificar especies del continente americano mediante la PCR-RFLP/Hsp70, y de utilizar la restricción enzimática del producto amplificado para distinguir entre Leishmania spp. y Trypanosoma cruzi, dándose un primer paso en el establecimiento de estos métodos moleculares en el laboratorio de referencia del instituto.

The infective capacity of Leishmania amazonensis promastigotes was evaluated after they were treated with a series of 10 thiadiazine derivatives. The parasites were incubated for 24 hours with 1 or 0,1 mg/ml of each compound and then, peritoneal macrophages from BALB/c mice were infected in cultures. All the compounds managed to reduce the infective capacity of parasites by more than 50%. T10 product exhibited the highest effect since it reduced infection by 92,8%.

Se evaluó la capacidad infectiva de promastigotes de Leishmania amazonensis al ser tratados con una serie de 10 derivados de la tiadiazina. Los parásitos fueron incubados durante 24 h con 1 o 0,1 mg/mL de cada compuesto y posteriormente, se infectaron macrófagos peritoneales de ratones BALB/c en cultivos. Todos los compuestos causaron una reducción de la capacidad infectiva de los parásitos mayor que 50 %. El producto T1O fue el que causó un mayor efecto, disminuyendo la infección en 92,8 % de infección.

Current therapy for leishmaniasis is not satisfactory. We describe the in vitro antiproliferative effects of new thiadiazine derivatives against Leishmania amazonensis. The compounds were found to be active against the amastigote form of the parasite, inhibiting parasite growing, from 10 to 89%, at a concentration of 100 ng/ml. This activity suggests that thiadiazine derivatives could be considered as potential antileishmanial compounds.

A genomic library of Leishaminia amazonensis was built through a pcDNA3 vector, with expression promoter in eukaryot cells, to contribute to the application of immunization technology with nucleic acids in leishmaniasis. To show the expression genomic library in the muscles of mice immunized with it, the indirect immunofluoresce technique was used. A mix of sera with high antileishmania titers from an area where L.braziliensis infection is predominant was used as primary antibody. A library of 80% recombinant clones was obtained. Antigen determinant expression was confirmed in immunized BALB/c mice´s muscles, according to the results of immunofluorescence testing.

Se construyó una biblioteca genómica de Leishmania amazonensis mediante el vector pcDNA3, con promotor de expresión en células eucariotas, con el objetivo de contribuir a la aplicación de la tecnología de inmunización con ácidos nucleicos en la leishmaniosis. Para demostrar la expresión de la genoteca en el músculo de ratones inmunizados con esta, se realizó la técnica de inmunofluorescencia indirecta. Como anticuerpo primario se utilizó una mezcla de sueros con alto título antileishmania, de una zona donde predomina la infección con L. braziliensis. Se obtuvo una biblioteca con 80 % de clones recombinantes. Se demostró la expresión de determinantes antigénicos en el músculo de ratones BALB/c inmunizados, según resultados de la inmunofluorescencia.