This paper summarized some interesting aspects related with the microbiological investigations on human leptospirosis carried out in Cuba from 1959 to 2017. The role of the Cuban national reference laboratory on Spirochetes and Brucella in the microbiological surveillance, the confirmation of epidemic outbreaks and in clinical trials related on reactogenicity, immunogenicity, efficacy and effectiveness of the Cuban anti-leptospirosis vaxSPIRAL® vaccine is also assessed. All these results represent contributions to support the National Program of Prevention and Control of the disease in Cuba.

Obregón Fuentes AM (2017) Leptospira Interrogans Sensu Lato, Etiological Agent of a Zoonotic Disease with Impact on Public Health in Cuba. J Vet Med Res 4(7): 1099.

•    Leptospirosis
•    Laboratory surveillance
•    Epidemic outbreak
•    Vaxspiral® Vaccine
•    Typing
•    Scientific meetings

Human leptospirosis in Cuba was poorly described prior to 1959. In the period 1970-1980, a number of nationwide outbreaks occurred, associated with agricultural work in lowland areas infested by rodents, as well as in rivers, dams, canals and water reservoirs contaminated with livestock residues. In 1972, the first World Health Organization recommendation was received, its aim was to implement microbiological diagnosis, with the objective of establishing confirmatory techniques for leptospirosis. Four years later (1978), serological diagnosis by the Indirect Hemagglutination Test (HAT) was made available, in all the provinces of the country. In 1981, the Program for Prevention and Control of Leptospirosis was published and in 1983 anti-leptospiral immunization was initiated in the main risk groups with a Russian vaccine containing serovars from the Icterohaemorrhagiae, Pomona, Grippotyphosa and Hebdomadis serogroups [1]. One of the first descriptive studies was carried out with data available from the National Statistics Office of the Ministry of Public Health. It investigated 397 deaths of leptospirosis, from 1987 to 1993, and reported a sharp increase in morbidity rate, reaching a value of 1.03 / 100.000 inhabitants, the highest in over 50 years. The most affected population group was retirees, followed by production workers and agricultural workers [2].

A national action plan was therefore established in 1994, to reduce the morbidity caused by leptospirosis, with a national incidence rate of 25.6 / 100.000 [3]. In this context, the first Cuban vaccine against human leptospirosis (vaxSPIRAL®), was developed by the Vaccine Research and Production Center of the Finlay Institute. During the vaxSPIRAL® preclinical trial the formulation of vaccine adsorbed on aluminum hydroxide was shown in the hamster model to be safe, immunogenic and protective [4]. The effectiveness of vax-SPIRAL® was 97%. The clinical trial of phase IV was conducted in Holguín, using a prospective, quasi-experimental cohort study in groups at risk of leptospirosis, where 101.137 subjects were immunized with two doses of 0.5 mL, 6 weeks apart (cohort of vaccinates), aged between 15 and 65 and with permanent or temporary risk of contracting the disease, 16 881 participants were not immunized (unvaccinated cohort) [5].

The clinical trial of reactogenicity (Phase I) and immunogenicity (Phase II) of vax-SPIRAL® was undertaken with healthy volunteer groups from Havana. The Russian vaccine was used as a control. There were no serious adverse reactions and low titers of agglutinating antibodies against leptospires were found in the adult volunteers [6]. The vax-SPIRAL® efficacy was 78.1% (95% CI: 59.2 to 88.3) and the relative risk of contracting leptospirosis infection between vaccinated and unvaccinated individuals was 0.22 (95% CI: 0.12 to 0.41). This study of phase III was performed in a controlled, randomized, double-masked manner in Villa Clara using as a control the Heberbiovac-HB recombinant hepatitis B vaccine (Heber Biotec, Cuba) [7].

The sera from individuals who were vaccinated (vaxSPIRAL®) or received the placebo, from phases I and II were studied by the ELISA [8]. Results showed (phase I) that out of 38 vaccinees, 11 seroconverted (29%) by MAT and 12 (32%) by ELISA. Out of 33 subjects receiving the placebo, two (6%) and three (9%) seroconverted, respectively. In phase II, out of 68 persons vaccinated with a dose of 0.25 mL and 65 with a dose of 0.50 mL, the seroconversion rate was 21 (31%) and 16 (25%) by ELISA, and by MAT 9 (13%) and 7 (11%) respectively. In most reactive individuals, antibody levels were found by MAT against at least one of the vaccine strains. However, the level of agglutinating antibodies detected with respect to the vaccine-induced protection in sera could not be correlated in this investigation. Therefore, it is recommended to apply the passive immunization test to measure the level of protection of the vaccine [9]. Other research demonstrated a high seroprevalence of antibodies (by ELISA-IgG), against the vaccine serovars before the start of the study, and an increase to twice the initial value of the antibody response at 21 days after the second dose of the vaccine, in 45% of individuals, with markedly significant differences (p= 0.000000). Antibody levels declined after one year. However, 23.1% of subjects still showed levels that were double those before vaccination. Based on these results, it was decided to administer the vaccine to the main risk groups [10].

An outbreak of human leptospirosis occurred in 1997 (Villa Clara City).This situation was associated with predisposing epidemiological factors in the affected population. The infecting serogroups found were Ballum, Pomona, Canicola and Icterohaemorrhagiae. Sera from seven suspected cases and four severe patients were confirmed as leptospirosis by HAT and MAT. Two isolates were typed as Ballum. Two urine and one blood culture were positive by PCR [11].

From 1998 to the present, strict surveillance of human leptospirosis has been maintained. The national incidence is an average of 980 patients / year (8.8 / 100.000) with around 25 deaths was reported [12]. However, the occurrence (in 1999), of the hurricanes George and Mitch in some Cuban provinces, accompanied by significant rainfall and flooding resulted in new outbreaks, particularly in Pinar del Río, Las Tunas and Havana. The main cause of infection was contact with rodents and the failure to take protective precautions [3]. Another leptospirosis epidemic event occurred in Havana (2001), associated with the city’s lack of adequate sanitation. A descriptive analytical study was carried out on 240 suspected patients from IPK’ hospital, of which 33 were confirmed microbiologically. By typing, the Ballum serogroup was predominant, whereas serologically (MAT) Canicola, Sejroe and Icterohaemorrhagiae were the most frequent ones. The predominant clinical form was anicteric (73%), and the principal symptomatology observed were fever (88%), headache (73%), and myalgia (67%). Bilirubin and creatine phosphokinase (CPK) levels were elevated in 83.3% and 67% of cases, respectively values. Almost 73% of the confirmed were not related to occupations at risk [13].

After that outbreak there was a clear reduction in the leptospirosis morbidity rate (2.7 / 100.000) and again, between 2005-2006, the incidence rates increased (6 and 7.4 /100.000 respectively). The use of a warning system applied in the provinces of Guantánamo and Santiago de Cuba, detected a new epidemic in 2005, coincident with the significant increase in rainfall and floods caused by Hurricane Wilma. From 885 suspected cases, 61 (6.9%), were confirmed microbiologically [14]. Leptospira isolates were typed as Canicola, Ballum, Icterohaemorrhagiae and Pomona serovars [15].

From a study carried out in two municipalities of Havana city, a slight increase in lethality due to leptopirosis was observed, with case fatality reaching more than 46%. Concomitantly, the morbidity rate reached 0.54 / 105 mortality of 0.28 / 105 [14] [15].

Several laboratory techniques had been evaluated for the diagnosis of the disease since the 1980’s. One of the first studies was the validation of the HAT using sera from patients and personnel with occupational biological risk [16]. In the same year, leptospires were isolated from 32.7% of the waters, with three strains L. biflexa and three such as L. interrogans, serogroups Australis (2) and Icterohaemorrhagiae (1) [17]. The slide agglutination technique was used in 1990 and found 55% of seropositive cases [18]. In 1991, leptospiral antigens were produced for use in the counterimmunolectrophoresis technique, achieving a sensitivity of 82% and a specificity of 100% [19]. In the same year, antigens of L. interrogans were used in immunofluorescence (IFI) achieving a slightly greater sensitivity of 86.5% [20]. Another study from 1992 reported the use of three variants of the HAT, and it was observed that the variant with formalinized human blood of the O negative group had a sensitivity of 84%, a specificity of 98% with a good reproducibility and repeatability [21]. In 1993, a study of three variants of IFI reported a sensitivity of 80% [22]. However, a study from august 1993 to January 1994, on 500 presumptive patients with leptospirosis found only 28% serological positivity by HAT [23]. The same year the use of the ELISA system for the detection of total antibodies against leptospires was reported for the first time in Cuba, with a 65% concordance with MAT [8]. However, using a higher number of sera in 1995, a 100% agreement with MAT was reported [24]. The Cuban ELISA was applied (1996) to different sera groups and showed a concordance of 89% with HAT and 65% with MAT [25]. After that, a technical variant of MAT, using 1:10 as first dilution of the sera was developed. The sensitivity of MAT-V was of 78% and the specificity of 89%. The Ballum serogroup was prevalent in positive sera by MAT-V, supporting the use of this variant to detect patients with low levels against others leptospire serovars [26]. Shortly after (2004), variants of slide agglutination systems with specific leptospire serovars / latex particles were studied for the detection of specific antibodies in human and animal sera. The antigenic concentrations were adjusted between 1.4 and 1.5 X109 cells / mL. The Cuban latex-pool presented acceptable values of sensitivity (94%) and specificity (90%), and was reproducible and stable for 6 months [27]. The evaluation of the immunochromatographic lateral flow system showed a concordance of 98% with MAT [28,29]. A new home made immunochromatographic system was developed in 2015, using an antigen mixture of the five serogroups of leptospires predominant in the country. It showed a sensitivity of 96%, a specificity of 97.1% and a concordance with MAT of 96.7% [30]. More recently a commercial system of SD Leptospira ELISAIgM (BIO-LINE Standard Diagnostics, INC) was applied in 2011, which showed more than 90% agreement with MAT. The system enhanced the rapid diagnosis of human leptospirosis and active laboratory surveillance in all Cuban provinces [31].

The first molecular diagnoses for early detection of DNA from Leptospira spp in blood cultures from suspect patients was published in Cuba in 2007, showing high diagnostic specificity [32]. One year later (2008), this system was used for the postmortem diagnosis of leptospirosis using 171 fresh tissue samples from organs of 50 deceased suspects and 32% were confirmed. In this paper, the lung showed a positivity of the 25% (12 / 48); the liver of 25.6% (11 / 43); the kidney of 22.5% (9 / 40); the brain of 6.3% (1 / 16); the heart 7.1% (1 / 14); the spleen of 12.5% (1 / 8) and the muscle of 50% (1 / 2) [33]. After that, the PCR was optimized by adjusting the hybridization temperature and the concentration of primers. Standardized PCR with novel primers was specific and more sensitive than the reference PCR [34,35]. In addition, a comparison of DNA extraction methods for pathogenic leptospires (QIAmp DNA mini kit (QIAGEN, Germany), CLART HPV kit (GENOMICA, Spain) and Chelex-100), demonstrated that the last one was the most efficient for DNA extraction in clinical samples for use in PCR [36].

A basic leptospirosis research developed has involved the study of bacterial outer membrane proteins (OMP) as potential vaccine candidates. The Triton X-100 method was used for the extraction of these proteins from the Castellon 3 reference strain of the Ballum serogroup, Serovar Castellonis. Different concentrations were tested and the rates and times of the extraction were modified. The occurrence of cell lysis through PCR and zymography, as well as the presence of lipopolysaccharides by proteinase K assay, were explored in the protein extracts [37]. In 2015, an efficient method was described for the detection of protease and nuclease activity in leptospiral isolates. Extracts of ten strains of leptospires (nine pathogenic and one nonpathogenic) were evaluated and protease activity was observed with different behavior between the pathogenic strains and the non-pathogenic strain [38].

During this time frame, several studies have attempted to characterize the Cuban serovars of leptospires. One of the first reports was carried out in 1996, typing leptospiral strains from Holguin, where the Ballum, Canicola and Pomona serogroups predominated [39]. Later (2002), 204 strains of L. interrogans pathogenic complex, from different regions of the country were studied, showing that serogroups Ballum, Pomona, Canicola, Pyrogenes, Autumnalis and Bataviae were the most prevalent. These findings identified mice, pigs and dogs as the main reservoirs [40]. At the same time, 18 strains of Leptospira, isolated from three provinces of Cuba, were studied by serological and genetic methods [41].

A method to determine the minimum inhibitory concentration of a range of antibiotics against leptospires was developed in 2005. Reference strains of the pathogenic complex L. interrogans sensu lato and L. biflexa sensu lato were tested against penicillin, ciprofloxacin, chloramphenicol, rifampicin and tetracycline. The results showed lowest values for ciprofloxacin, rifampicin and tetracycline and the highest values with chloramphenicol and penicillin [42].

A new report (2006-2008) demonstrated as the predominant serogroups, Pomona (11) 42.3%, Canicola (6) 23.1%, Icterohamorrhagiae (5) 19.2 %, Ballum (2) 7.7%, Hebdomadis and Lousiana (1) 3.8% [43]. Sixteen of the isolates were then typed with monoclonal antibodies as Pomona Pomona, Pomona Mozdok and Canicola Canicola [44]. The most recent study in Cuba embraced a total of 79 isolates (from Las Tunas and Holguín provinces) where the predominant serovars were Ballum Guangdong, Ballum Arborea, Ballum Ballum, Pomona Pomona, Pomona Mozdok, Pomona Proechimis, Canicola Canicola, Icterohamorrhagiae Copenhageni and Icterohamorrhagiae Icterohamorrhagiae. Eleven untyped isolates were confirmed as pathogens by PCR based on the pathogen-specific lipL32 gene [45].

In summary, the main results of epidemiological data and laboratory surveillance of human leptospirosis in Cuba appeared in table 1. All them support the national program for prevention and control of leptospirosis and to a better understanding of the etiological agent and its circulation in the country.

Finally, since 2001 and every two years, the reference laboratory of Spirochetes and Brucella have made several scientific meetings [46-49], aimed at the theoretical - practical training of human resources on leptospirosis. More than 400 colleagues and experts from America, Europe, Asia and Australia have participated. Also consultancies have been received by ILS / WHO ‘experts in the leptospirosis field.

Table 1: Main results of epidemiological data and laboratory surveillance of human leptospirosis in Cuba (1959-2017).

I would like to thanks to Prof. Ben Adler, from Monash University, Australia for revision of these commentaries. The contribution of the authors cited in this commentary is also acknowledged.

1. Cruz de la Paz R, Rodríguez P, López C, Atienzar E, Abreus J, Aldama F. Reactogenicidad de la vacuna antileptospirósica en Cuba. Rev Cubana Hig Epidemiol. 1986; 24: 407-412.

2. Padilla Pérez O, Toledo H, Vidal I, Rodríguez I. Comportamiento de la mortalidad por leptospirosis en Cuba, 1987-1993. Rev Cubana Med Trop. 1998; 50: 61-66.

3. Cruz de la Paz R. Estrategia cubana para el control y prevención de la leptospirosis en Cuba. Rev Latinoamericana Microbiol. 2002; 44: 22.

4. González González M, Martínez R, Cruz R, Infante JF, González I, Baró M, et al. vax-SPIRAL®. Vacuna antileptospirósica trivalente para uso humano; investigación, desarrollo e impacto sobre la enfermedad en Cuba. Biotecnología Aplicada. 2004; 21.

5. Martínez Sánchez R, Pérez A, Baró M, Alvarez M, Menéndez J, Díaz M, et al. Evaluation of the effectiveness of a new vaccine against human leptospirosis among groups at risk. Rev Panam Salud Pública. 2000; 8: 385-392.

6. Martínez Sánchez R, Pérez A, Obregón AM, Rodriguez I, Baly A, Baró M, et al. Reactogenicidad e inmunogenicidad de la vacuna cubana inactivada trivalente contra la leptospirosis humana según diferentes esquemas. Rev Cubana Med Trop. 2002; 54: 37-43.

7. Martínez Sánchez R, Pérez A, Quiñones M, Cruz R, Alvarez A, Armesto M, et al. Efficacy and safety of a vaccine against human leptospirosis in Cuba. Rev Panam Salud Pública. 2004; 15: 249-255.

8. Obregón Fuentes AM, Otero A, Laferté J. ELISA for detection of human leptospirosis. Mem Del Inst FIO CRUZ. 1994; 365.

9. Obregón Fuentes AM, Martínez G, Martínez R, LLop A, Rodríguez I, Rodríguez J, et al. Respuesta serológica por ELISA y MAT en voluntarios cubanos vacunados con vax SPIRAL®. Rev Cubana Med Trop. 2004; 56: 148-151.

10. Rodríguez González I, Martínez R, Zamora Y, Rodríguez J, Fernández C, Obregón AM. Respuesta de anticuerpos IgG antileptospira en individuos inmunizados con vax-SPIRAL. Rev Cubana Med Trop. 2005; 57: 32-37.

11. Obregón Fuentes AM, Fernández C, Rodríguez I, Rodríguez J, Fernández N, Enrique G. Importancia de la confirmación microbiológica en un brote de leptospirosis humana en la ciudad de Villa Clara. Rev Cubana Med Trop. 2003; 55: 96-99.

12. González Arrebato J. Comportamiento de las zoonosis en Cuba [disertación]. Taller Nacional de Zoonosis. IPK. La Habana. 2017.

13. Hurtado Mola I. Caracterización clínica epidemiológica y de laboratorio en pacientes con sospecha de Leptospirosis ingresados en el IPK (enero-junio de 2002). [Tesis para optar por el grado académico de Master en Bacteriología Micología]. La Habana: IPK; 2003.

14. Berdasquera Corcho D, Rodríguez I, Obregón AM, Fernández C, Segura R, Bustabad E, et al. Leptospirosis humana en la provincia Guantánamo: reflexiones de una epidemia. Rev Cubana Med Trop. 2007; 59.Rodríguez González I, Fernández C, Obregón AM, Zamora Y, Rodríguez J, Rodríguez N, et al. Confirmación microbiológica de 2 brotes emergentes de leptospirosis humana en Cuba. Rev Cubana Med Trop. 2007; 59: 19-23.

15. Barrios Fajardo MM. Hemaglutinación. Su aplicación al diagnóstico de la Leptospirosis humana en el municipio Santiago de Cuba. La Habana: INHEM; 1986.

16. De Valle Fernández Y. Aislamiento y determinación de leptospiras en aguas. La Habana: INHEM. 1986.

17. Morales Casas G. Empleo de una técnica de aglutinación en lámina en el serodiagnóstico de la leptospirosis humana. La Habana: UH; 1990.

18. Obregón Fuentes AM, Herrera C, López C. Evaluación de la CIE en el diagnóstico serológico de la Leptospirosis humana. Rev Cubana Med Trop. 1994; 45: 67-70.

19. Gil Fernández MC. Empleo de antígenos de L. interrogans para el diagnóstico serológico de la leptospirosis por IFI. La Habana: IPK. 1991.

20. Obregón Fuentes AM, Martell M. Diagnóstico serológico de la Leptospirosis humana empleando 3 variantes de la HAT. Rev Cubana Med Trop. 1999; 51: 60-62.

21. Mora Llanos MM. Empleo de tres variantes de IFI para el diagnóstico de la leptospirosis humana. [Tesis para optar por el título de médico especialista de primer grado en Microbiología]. La Habana: IPK; 1993.

22. Tomas Abreu M. Diagnóstico sero-epidemiológico y clínico de la leptospirosis humana. [Tesis para optar por el título de médico especialista de primer grado en Microbiología]. La Habana: IPK; 1994.

23. Obregón Fuentes AM, Laferté J, Otero A. Aplicación del sistema ELISA indirecto para la detección de anticuerpos totales a leptospiras en diferentes grupos de estudio. Boletín de la Sociedad Venezolana de Microbiología. 1995; 14: 6-10.

24. Rodríguez González I. ELISA cuantitativo, útil para el diagnóstico de la leptospirosis humana y en la evaluación de una vacuna contra leptospiras. La Habana: UH. 1996.

25. Pérez Orphee M. Estudio de una variante de MAT para el diagnóstico de la leptospirosis humana. [Tesis para optar por el Grado Académico de Master en Bacteriología Micología]. La Habana: IPK; 2000.

26. Obregón Fuentes AM, Fernández C, Rodríguez I, Balbis Y, Martínez B, Rodríguez J. Sistema de aglutinación con látex para el diagnóstico rápido de la Leptospirosis humana en Cuba. Pan American J of Public Health. 2004; 16: 259-265.

27. Rodríguez González I, Fernández C, Llerena C, Victoria B, Rodríguez J, et al. Lepto dipstick: resultados de su aplicación al diagnóstico rápido de la leptospirosis humana. Rev Cubana Med Trop. 2002; 54: 44-47.

28. Obregón Fuentes AM, Fernández C, Martínez I, Llop A, Rodríguez I, Rodriguez J, et al. Sistemas serológicos rápidos utilizados para la pesquisa de leptospirosis humana en Cuba. Rev Cubana Med Trop. 2011; 63.

29. Pérez Elías Y, Obregón AM, Lemos G, Machado H, Rodríguez IC, Rodríguez J. Evaluación de un sistema inmunocromatográfico de flujo lateral para la pesquisa de la leptospirosis humana Rev. Cubana de Med Tropical. 2015; 67.

30. Obregón Fuentes AM, Fernández C, Rodríguez I, Rodríguez Y, Echevarría E, Rodríguez J, et al. Detección de anticuerpos IgM contra leptospiras por un sistema comercial ELISA-IgM. Rev Cubana Med Trop. 2013; 65: 202-210.

31. Zamora Martínez Y, Fernández C, Rodríguez I, Obregón AM, Rodríguez J, Rodríguez N. Método de la PCR en la detección temprana de Leptospira spp en hemocultivos de pacientes con sospecha de leptospirosis. Rev Cubana Med Trop. 2007; 59.

32. Rodríguez Olivera Y, Rodríguez I, Zamora Y, Rodríguez J, Valdés Y, Echevarría E. et al. Detección de ADN de leptospiras en tejidos frescos de fallecidos en Cuba, 2008-2011. Rev Cubana de Med Trop. 2013; 65: 211-222.

33. Noda Ramos AA, Rodríguez I, Rodríguez Y, Govin A, Fernández C, Obregón AM. High sensitive PCR methods for detection of pathogenic Leptospira spp. in paraffin-embedded tissues. Rev Inst Med Trop Sao Paulo. 2014; 56: 411-415.

34. Noda Ramos AA, Rodríguez I, Rodríguez Y, Govín A, Obregón AM. Evaluación de una PCR para la confirmación molecular de leptospirosis en fallecidos a partir de tejidos frescos. Rev Cubana Med Trop. 2014; 66: 447-452.

35. Noda Ramos AA, Rodríguez I. DNA isolation by Chelex-100: an efficient approach to consider in leptospirosis early stages. J Coastal Life Med. 2014; 2: 501-504. 36.Beltrán Lissabet JF. Extracción de proteínas de membrana externa de leptospiras con la utilización del Tritón X-100. La Habana: UH. 2012.

37. Tamayo Brito C. Establecimiento de técnicas de zimografía para el análisis de proteasas y nucleasas en Leptospira spp. La Habana: UH. 2015.

38. Saltarén Cobas A. Agrupación de cepas de leptospiras por el método de aglutinación microscópica con sueros hiperinmunes. IPK-CPHEM Holguín. [Tesis para optar por el grado académico de Master en Bacteriología Micología]. La Habana: IPK. 1996.

39. Rodríguez González I, Obregón AM, Rodríguez J, Fernández C, Arzola A, Victoria B. Caracterización serológica de cepas aisladas de pacientes con leptospirosis humana en Cuba. Rev Cubana Hig Epidemiol. 2002; 40: 11-15.

40. Victoria Martínez B, Fernández C, Rodríguez J, Obregón AM, Rodríguez I. Identificación de aislamientos de Leptospira por métodos serológicos y genéticos. Rev Cubana Med Trop. 2002; 54: 48-51.

41. Obregón Fuentes AM, Llanes R, Fernández C, Hernández I, Rodríguez J. Desarrollo de un método para determinar la concentración mínima inhibitoria en cepas de referencias de leptospiras. Rev Cubana Med Trop. 2005; 57: 11-16.

42. Zamora Martínez Y. Caracterización de cepas de leptospiras circulantes en Cuba en el periodo 2006-2008. [disertación]. Congreso Internacional 70 Aniversario IPK. Congreso Cubano de Microbiología y Parasitología. Palacio de Convenciones. La Habana. 2009.

43. Obregón Fuentes AM, Fernández C, Rodríguez I, Rodríguez J. The application of monoclonal antibody methodology as a tool for serotyping leptospira isolates in Cuba. Rev Cubana Med Trop. 2007; 59: 68-70.

44. Rodríguez González Y. Caracterización de cepas de leptospiras circulantes en Cuba en el periodo 2008-2014. Congreso Internacional “Espiroquetas Habana 2016“. Hotel Meliá Habana. La Habana. 2016.

45. Fernández Molina C, Obregón AM. Reunión Científica Internacional “Leptospirosis 2001”. Rev Cubana Med Trop. 2002; 54: 5-6.

46. Fernández Molina C, Obregón AM. Tercer Taller y Tercera Reunión Científica Internacional “Leptospirosis Habana 2006”. Rev Cubana Med Trop. 2007; 59: 5-7.

47. Fernández Molina C, Obregón AM. VI Taller Internacional “Leptospirosis abana 2012”. I Curso Pre-Congreso de “Borreliosis de Lyme: del vector a la enfermedad”. II Congreso Internacional de Leptospirosis, Sífilis y Borreliosis “Espiroquetas Habana 2012”. Rev Cubana Med Trop. 2013; 65: 146-148.

48. Obregón Fuentes AM, Rodríguez Y. IV Curso- Taller Internacional “Espiroquetas Habana 2016”. Rev Cubana Med Trop. 2017; 69.