José Rodríguez

Eficacia y seguridad de una vacuna contra la leptospirosis humana en Cuba

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 Cubas 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 Camajuani, 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.

Sistema de aglutinación con látex para el diagnóstico rápido de la leptospirosis en Cuba

OBJETIVOS: Evaluar la sensibilidad, la especificidad, la reproducibilidad y la estabilidad de cinco sistemas de aglutinacion con latex disenados para detectar anticuerpos contra leptospira en sueros de humanos y de animales, basados en los serogrupos de Leptospirade mayor circulacion en Cuba. METODOS: Se realizo un estudio analitico descriptivo con 706 sueros humanos (65 sueros positivos a anticuerpos contra leptospira mediante microaglutinacion (MAT) y hemaglutinacion (HA); 156 sueros negativos, segun MAT y HA; 485 sueros de 424 pacientes con signos clinicos o epidemiologicos 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 latex con celulas enteras de Leptospira interrogans de los cuatro serogrupos de mayor circulacion en Cuba en el periodo entre 2002 y 2004. Con las celulas obtenidas de los cultivos celulares de cepas tipo se obtuvieron cuatro conjugados especificos (latex-Canicola, latex-Icterohaemorrhagiae, latex-Pomona y latex-Sejroe) y un conjugado de latex con la mezcla de las celulas de esos cuatro serogrupos a partes iguales (latex-Pool). Adicionalmente, las muestras se evaluaron con el sistema comercial de aglutinacion con latex Lepto Tek Dri Dot (bioMerieux, Francia). La estabilidad y la reproducibilidad de los conjugados de latex se evaluaron mediante controles mensuales durante 6 meses con sueros positivos y negativos. RESULTADOS: De los sistemas evaluados, la mejor combinacion de sensibilidad y especificidad se observo con el conjugado latex-Pool (93,8% y 90,4%, respectivamente). La mejor combinacion de valores predictivos positivos y negativos se observo con el conjugado latex-Sejroe (90,9% y 95,8%, respectivamente), seguido del conjugado latex-Pool (94,2% y 96,6%, respectivamente). Los valores predictivos positivo y negativo del sistema comercial Lepto Tek Dri Dot fueron 78,5% y 88,4%, respectivamente. De las 137 muestras de pacientes positivas a alguno de los serogrupos estudiados segun MAT, los conjugados de latex lograron identificar correctamente 107 (78,1%), mientras que el conjugado latex-Pool detecto como positivos 116 sueros (84,7%). Al evaluar los sueros de animales, el conjugado latex-Pool detecto como positivos el mayor numero de sueros y tuvo la mayor coincidencia con MAT (93,1%). Se observo una adecuada estabilidad y reproducibilidad de los conjugados estudiados. CONCLUSIONES: Los conjugados de latex con celulas enteras de leptospira de los serogrupos de mayor circulacion en Cuba demostraron un grado de coincidencia con MAT similar o superior al observado con el sistema comercial Lepto Tek Dri Dot, tanto en sueros de humanos como de animales. Se recomienda extender el uso del conjugado latex-Pool en Cuba para el tamizaje inicial de anticuerpos contra leptospira.

Evaluation of a modified culture medium for Borrelia burgdorferi sensu lato

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.

Detection of leptospires from infected urine and tissue samples in vitro by modified Fontana silver stain

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.

486 Therapeutic Effect and Security in Asthmatics Adult Patients Treated with Dermatophagoides Pteronyssinus Allergen Sublingual Immunotherapy.

Background The specific active immunotherapy, employing vaccine of allergen of mite is a treatment considered as effective for the respiratory allergy and asthma. The sublingual route has minor risk of systematises reactions. The objective of this study was to determine the therapeutic effect and security of sublingual immunotherapy (ITSL) employing the standard vaccine VALERGEN-DP (BIOCEN, CUBA) in a population of asthmatic Cuban patients. Methods A phase II Clinical Trials double blind, placebo controlled in a total of 40 adult patients with mild or moderate asthma and specific sensibility preponderant to this mite. Half of patients received drops by sublingual route with growing doses up to 2000 UB. Results The treatment was effective in the reduction of clinical symptoms and medication intake as compared to conventional treatment in control group. The cutaneous sensibility to this mite was significant reduced, increasing in 1.9 log; the amount of necessary allergen to provoke a positive Prick Test. An improvement of the lung function was observed with a significant reduction (P < 0.05) of expiratory pick flow variability. The frequency of local reactions were only 0.58% of administration. Conclusions The VALERGEN-DP vaccine is an effective treatment and profitable against asthma in our population and guarantee its generalization in the Allergy Services of our health system.

485 Therapeutic Effect and Safety of Tropical Mite Allergen Vaccines by Subcutaneous Route in Allergic Asthmatics Patients

Background Allergen-specific subcutaneous immunotherapy (SCIT) is presently recognized as a biological response modifier, as it is the only available treatment able to influence the natural course of allergic disease. Extensive clinical evidence supports its efficacy. Safety concerns are related to the risks of anaphylactic reactions during treatment. Standardization of allergen vaccines in terms of allergenic activity allows a more precise control over the administered doses and can be, therefore, very relevant for both efficacy and safety of SCIT. House Dust Mites (HDM), particularly Dermatophagoides pteronyssinus (Dp), Dermatophagoides siboney (Ds) and Blomia tropicalis (Bt) have been described as very relevant allergen sources in Cuba, with a strong association to respiratory allergy symptoms. Objective To asses the efficacy and safety of standardized allergen vaccines of these 3 mite species (Valergen, Biocen, Cuba) in Cuban asthmatic patients. Methods Three Double-Blind Placebo-Controlled clinical trials were performed in 40 patients each, showing asthmatic symptoms and positive predominant Skin Prick Test (SPT) to each mite, respectively. Half of patients received the active treatment consisting of subcutaneous injections with increasing doses, up to 6000 BU. Results The total 1 year cumulative dose was 63035 BU, in an average of 20.5 injections. The treatment was effective in the reduction of clinical symptoms (up to 32%, 95%CI: 28-36%; P = 0.0006) and medication intake (23%, 95%CI:18-28%), as compared to control treatment. The skin sensitivity to the allergens decreased significantly (P = 0.0001), with regard to the beginning of the treatment. The allergen amount needed to induce a positive SPT increased 297-fold. An improvement of the lung function was observed, expressed in a modest Peak-Expiratory-Flow increase (P < 0.05) and reduction of PEF daily variability. SIT was considered effective in 71% of patients. The frequency of local adverse reactions was 2.4 % of injections. Conclusions The results indicates that immunotherapy, using standardized House Dust Mite vaccines, including tropical species, is effective and safe for the control and amelioration of the asthma in our population.

487 safety of Sublingual Immunotherapy with Standardized Vaccines of Domestic Mites

Background Allergen-specific immunotherapy consists of administering gradually increasing doses of the allergen, to which the patient is sensitized, aiming at achieving tolerance to it and decreasing clinical symptoms. The sublingual immunotherapy (SLIT) was introduced as an alternative to subcutaneous route. Its use is being increased in the world and in Cuba, using standardized vaccines owing to greater safety. The objective of this study was to determine the safety of sublingual standardized vaccines of 3 domestic mite species (Valergen, Cuba) and its adverse events in allergic patients from the Calixto García University Hospital in Havana, as well as the frequency of its prescription. Methods Descriptive and cross sectional study design, which included 130 patients with treatment of SLIT with VALERGEN-DP (Dermatophagoides pteronyssinus), VALERGEN-DS (D. siboney) and VALERGEN-BT (Blomia tropicalis) (BIOCEN, Cuba), who attended the Allergy Service in the period January-September 2010. Age distribution: mean 19.6 years (range 1–75), 40.7 % was younger than 18 years. Results The multiallergen vaccine was the type of vaccine most used (63.8%). The most common allergen was D. pteronyssinus followed by B. tropicalis. 71.55% of administered allergens vaccines were in maintenance phase. We found 4 adverse events (3.1% of patients), all local, mild, and not requiring treatment or change of vaccination dosing schedule. Conclusions The Valergen vaccines by sublingual route are safe and well tolerated in Cuban allergic patients.