Margarita Ramírez

The vaccine candidate Vibrio cholerae 638 is protective against cholera in healthy volunteers.

ABSTRACT Vibrio cholerae 638 is a living candidate cholera vaccine strain attenuated by deletion of the CTXΦ prophage from C7258 (O1, El Tor Ogawa) and by insertion of the Clostridium thermocellum endoglucanase A gene into the hemagglutinin/protease coding sequence. This vaccine candidate was previously found to be well tolerated and immunogenic in volunteers. This article reports a randomized, double-blind, placebo-controlled trial conducted to test short-term protection conferred by 638 against subsequent V. cholerae infection and disease in volunteers in Cuba. A total of 45 subjects were enrolled and assigned to receive vaccine or placebo. The vaccine contained 109 CFU of freshly harvested 638 buffered with 1.3% NaHCO3, while the placebo was buffer alone. After vaccine but not after placebo intake, 96% of volunteers had at least a fourfold increase in vibriocidal antibody titers, and 50% showed a doubling of at least the lipopolysaccharide-specific immunoglobulin A titers in serum. At 1 month after vaccination, five volunteers from the vaccine group and five from the placebo group underwent an exploratory challenge study with 109 CFU of ΔCTXΦ attenuated mutant strain V. cholerae 81. Only two volunteers from the vaccine group shed strain 81 in their feces, but none of them experienced diarrhea; in the placebo group, all volunteers excreted the challenge strain, and three had reactogenic diarrhea. An additional 12 vaccinees and 9 placebo recipients underwent challenge with 7 × 105 CFU of virulent strain V. cholerae 3008 freshly harvested from a brain heart infusion agar plate and buffered with 1.3% NaHCO3. Three volunteers (25%) from the vaccine group and all from the placebo group shed the challenge agent in their feces. None of the 12 vaccinees but 7 volunteers from the placebo group had diarrhea, and 2 of the latter exhibited severe cholera (>5,000 g of diarrheal stool). These results indicate that at 1 month after ingestion of a single oral dose (109 CFU) of strain 638, volunteers remained protected against cholera infection and disease provoked by the wild-type challenge agent V. cholerae 3008. We recommend that additional vaccine lots of 638 be prepared under good manufacturing practices for further evaluation.

Randomized, double-blind, placebo-controlled trial to evaluate the safety and immunogenicity of live oral cholera vaccine 638 in Cuban adults

A randomized, double-blind, placebo-controlled clinical trial was conducted to evaluate the safety, reactogenicity and the immunogenicity of a 2 x 10(9)CFU dose of the 638 lyophilized live attenuated cholera vaccine for oral administration, formulated and produced at Finlay Institute, City of Havana, Cuba. Thirty-six healthy female and male adult volunteers from 18 to 40 years old were involved, clinically examined and laboratory tested after the informed consent signature. Adverse events were monitored and seroconversion rates and geometrical mean titer (GMT) of vibriocidal antibodies were tested in volunteers sera samples. Neither serious adverse events nor other damages to the volunteers due to vaccine or placebo feeding were reported during the clinical follow-up period of this study; none of the adverse events registered within the first 72 h after inoculation were life-threatening for volunteers. Neither severe nor moderate adverse events were reported. Sixty-one percent of subjects showed mild expected adverse events in an interval lower than 24h up to the first 72 h, 75% of these in the vaccinated group and 18% in the placebo group. Fourteen days after inoculation the GMT of vibriocidal antibodies in the vaccine group significantly increased in comparison to the placebo group. All subjects in the vaccine group (24) seroconverted (100%). Results show that this vaccine is safe, well tolerated and immunogenic in healthy female and male volunteers.

Acute Diarrhea Associated with Heat-stable Enteroxin Producing Strains of Vibrio cholerae Non-O1: First Report from Cuba

A five month old male infant was referred withwatery diarrhea, vomiting and fever, to the paedi-atric ward of the provincial Pediatric Hospital ofLas Tunas, Cuba. Abdominal examination revealedtenderness; liver and spleen were normal.Chest, cardiovascular and other systems werenormal. The patient was rehydratated with oralrehydratation with solution according to the WorldHealth Organization formula and breast-feedingwas continued. The clinical picture improvedgradually and the patient was discharged after sixdays without diarrhea and in good condition. Onadmission stool sample was taken and cultured onSalmonella-Shigella and MacConkey agar for iso-lation of Salmonella, Shigella spp., enterotoxigenicand enteropathogenic Escherichia coli, Vibrio spp .,Aeromonas spp ., and Plesiomonas shigelloides (JFMcFaddin 1980, p. 19-34. Biochemical Test forIdentification of Medical Bacteria, 2nd ed., TheWilliams and Wilkins Co, Baltimore).Blood agar suplemented with Butzler’s antimi-crobials was employed for the isolation ofCampylobacter spp. The presence of Rotavirus wasruled out by latex agglutination assay. Three dif-ferent samples of feaces were analyzed by conven-tional microscopic examination for the presenceof ova, cyst or vegetative forms of parasites(Organizacion Mundial de la Salud 1992, p. 10-20, Metodos Basicos de Laboratorio enParasitologia Medica, OMS, Geneva).The stool specimen plated on MacConkey agaryielded a pure culture of lactose non-fermentingGram-negative rods, moved by polar flagella, iden-tified at genus level as Vibrio. The national strat-egy for cholera control in Cuba shows that thecountry is still free from cholera. A slide aggluti-nation with polyvalent O1 group antiserum for pre-sumptive identification of V. cholerae O1 strain wasperformed; the test was negative.The coproparasitological examination wasnegative and no Rotavirus or other bacterialenterophatogen were found or isolated from stoolsamples. The Vibrio strain isolated was sent to theNational Reference Laboratory for Diarrhoeal Dis-eases and Cholera Control at the “Pedro Kouri”Institute of Tropical Medicine, Havana City, andconfirmed to be a non-O1 and non-O139 V.cholerae by standard methods (JJ Farmer & FWHickman Brenner 1992, p. 2552-3011. In ABallows, HG Truper, M Dworkin, W Harder, KHSchleifer (eds), The Prokaryotes: a Handbook onthe Biology of Bacteria, Vol. 3, Springer, Verlag,New York). The strain was susceptible to 10- and150 µg disks of the vibriostatic agent O/129; theantimicrobial susceptibility test, performed with theKirby-Bauer method, showed that the strain wassusceptible to tetracycline, ampicillin, cloram-phenicol, trimethoprim, sulphametoxazol, nalid-ixic acid, ciprofloxacin, streptomycin, erytromycin,gentamicin, cefuroxime and furazolidone (CWStratton & RC Cooksey 1991, p. 1153-1165. In ABalows, WJ Hausler Jr, KL Herrmann, HDIsenberg, HJ Shadomy (eds), Manual of ClinicalMicrobiology, 5th ed., ASM, Washington, DC). Thestrain was analyzed for the presence of genomicsequences related to the V. cholerae O1 ctx ge-netic element, that includes the cep, orFU, ace, zot,and ctxAB genes, by using a colony-blot hybrid-ization procedure (T Karasawa et al. 1993 FEMSMicrobiol Letters 106: 143-146). The hybridiza-tion probe used was a 4.4 kbp cloned DNA frag-ment containing the entire ctx element of V.cholerae, and was labeled with (a 32P) dCTP bythe random priming technique.The presence of aheat-stable enterotoxin gene was investigated byPCR amplification as previously described (PGuglielmetti et al. 1994 Mol Cell Probes 8: 39-44). The biological activity of the strain was testedby the conventional suckling mouse assay (YTakeda et al. 1979 Infec Immun 25 : 978-985). Theresults of the molecular characterization of the

Two cases of laboratory-confirmed leptospirosis in travellers returning to Spain from Thailand, September 2013

In September 2013, leptospirosis was diagnosed in two Spanish travellers returning from Thailand. The first case walked in floodwater in the Phi Phi Islands in pouring rain: 20 days later he presented with fever and acute hepatitis. The second presented with fever and renal failure 17 days after visiting the islands. These cases remind clinicians to consider leptospirosis in febrile patients with a history of contact with flood or fresh water while travelling to tropical countries.

Fatal Plesiomonas shigelloides in a Newborn

A case of fatal Plesiomonas shigelloides men-ingitis is reported in a newborn. In spite of a courseof intravenous ampicillin and gentamicin he diedin cardiac arrest. The patient in this case, is thefirst to be documented in Cuba and the Caribbeanregion.P. shigelloides is a member of the familyVibrionaceae. The organism is a facultative,anaerobic, polarly flagellated, Gram-negative bac-terium (RA Brendan et al. 1988 Rev Infect Dis 10:303-316). Its primary natural reservoirs are sur-face water and soil, as well as fish and other ma-rine animals (T Arai et al. 1980 J Hyg 84: 203-211, LR Van Dame & J Vandepitte 1980 ApplEnviron Microbiol 39: 475-479). It is not consid-ered to be part of the normal flora of the humanintestinal tract (SD Holmberg & JJ Farmer 1984Rev Infect Dis 6: 633-639). In humans infectionswith P. shigelloides can cause a usually mild gas-troenteritis, septicemia, cellulitis, arthritis, chole-cystitis and osteomyelitis. Some cases in whichneonates developed P. shigelloides sepsis and men-ingitis have been described (A Pathak et al. 1981Pediatrics 71: 389-391, C Terpeluk et al. 1992 EurJ Pediatr: 499-501).Case report - A ten day-old newborn infant wasadmitted at the Carlos Manuel de CespedesPedriatric Hospital in Granma, Cuba, with fever(39°C), irritability and anorexia. Physical exami-nation revealed a bulging, tense anterior fontanel;respiratory rate 32 per min with intermittent pausesno reales; heart rate of 140 per min, no murmurs;normal abdominal palpation; nervous central sys-tem signs: hypotonia and bilateral mydriasis.Laboratory tests revealed hemoglobin 13.2 g/l,WBC 6,75.10

Chalicogloea cavernicola gen. nov., sp. nov. (Chroococcales, Cyanobacteria), from low-light aerophytic environments: combined molecular, phenotypic and ecological criteria.

This work characterizes a unicellular cyanobacterium with nearly spherical cells and thin-outlined sheaths that divide irregularly, forming small packets immersed in a diffluent mucilaginous layer. It was isolated growing on calcite speleothems and walls in a show cave in Collbató (Barcelona, Spain). Spectral confocal laser and transmission electron microscopy were used to describe the morphology, fine structure and thylakoid arrangement. The pigments identified were phycoerythrin, phycocyanin, allophycocyanin and chlorophyll a. Three-dimensional reconstructions, generated from natural fluorescence z-stacks, revealed a large surface area of nearly flat, arm-like thylakoidal membranes connected to each other and forming a unified structure in a way that, to our knowledge, has never been described before. Phylogenetic analyses using the 16S rRNA gene sequence showed 95% similarity to strain Chroococcus sp. JJCM (GenBank accession no. AM710384). The diacritical phenotypic features do not correspond to any species currently described, and the genetic traits support the strain being classified as the first member of an independent genus in the order Chroococcales and the family Chroococcaceae. Hence, we propose the name Chalicogloea cavernicola gen. nov., sp. nov. under the provisions of the International Code of Nomenclature for Algae, Fungi and Plants. The type strain of Chalicogloea cavernicola is COLL 3(T) (=CCALA 975(T) =CCAP 1424/1(T)).

Caracterización fenotípica y factores de virulencia en cepas de Aeromonas aisladas de pacientes con enfermedad diarreica aguda en Cuba

RESUMEN O BJETIVO : se realizo un estudio en cepas de Aeromonas aisladas de pacientes con enfermedad diarreica aguda en Cuba, paraconocer la expresion fenotipica de la citotoxina y la enterotoxina como factores de virulencia. M ETODOS : se investigaron46 cepas ( A. hydrophila, A. veronii biovar sobria, A. caviae, A. veronii biovar veronii y Aeromonas spp.) , aisladas de heces depacientes con enfermedad diarreica aguda, en el periodo comprendido entre 2005 y 2006. Todas las cepas tenian identificado supatron de susceptibilidad antimicrobiana. Se comprobo la expresion fenotipica de la citotoxina y la enterotoxina en la lineacelular Vero. R ESULTADOS : el estudio demostro que 91,31 % de las cepas mostraron actividad citotoxica y 43,48 % actividadenterotoxica. De las cepas multirresistentes, 93,75 % presento al menos un factor de virulencia estudiado. C ONCLUSIONES : losresultados demostraron que los 2 factores de virulencia investigados estuvieron presentes en las cepas estudiadas, contribuyendoasi a los multiples esfuerzos que se realizan para conocer los mecanismos de enteropatogenicidad de este genero bacteriano.

Preoperative levosimendan. A new way for organoprotection.

Preoperative use of levosimendan in cardiac surgery patients is one of the most attractive therapeutic alternatives in subjects with left or right ventricular dysfunction. Our review explores the pharmacological bases and clinical evidence for the use of levosimendan, with the intention of making a series of recommendations regarding its use in preoperative optimization prior to cardiac surgery. We carried out a survey of 102 Spanish anesthesiologists in order to shed light on the grey areas regarding the use of this drug prior to surgery. Our findings suggest that levosimendan is recommended for patients with severe left or right ventricular dysfunction, moderate left ventricular dysfunction in which Intra-aortic Balloon Counterpulsation (IABC) is necessary and severe pulmonary hypertension. Administration of levosimendan prior to surgical cardiac intervention without an initial bolus reduces the likelihood of complications.

O5.105-ALA FLUORESCENCE GUIDED RESECTION OF HEMANGIOBLASTOMAS - IS IT USEFUL WITH CONVENTIONAL SURGICAL TOOLS?

BACKGROUND: The use of 5-aminolevulinic acid (5-ALA) for fluorescent guided malignant glioma surgery has proved to be useful to ensure a complete resection and thus, a best survival result. One report has described its usefulness for the detection of hemangioblastoma fragments at the cyst wall after apparently complete tumor resection. Von Hippel-Lindau disease patients suffer from many CNS hemangioblastomas, requiring multiple surgical procedures for resection of tumors, many times obscured because distorted structures and dense gliotic scars. MATERIAL AND METHODS: In order to test the eventual utility of 5-ALA fluorescent guided surgery in multiple hemangioblastomas, we have performed surgical procedures in two VHL patients for resection of multiple hemangioblastomas. After patient consent and approval by the Ethical Committee, 5-ALA was administered as oral dose 20 mg/kg 3 hours before anesthesia induction. A surgical microscope modified for Protoporphyrin IX (PpIX)fluorescence guidance, and frameless neuronavigation were used for the surgical procedure. After neuronavigated location and microsurgical dissection of tumors, some involved in dense scars, surgical field visualization was performed alternating standard light illumination and violet-blue light mode, to excite PpIX and collect fluorescence by visualization in surgical oculars and registration on a color camera. RESULTS: Both VHL patients had been previously subjected to surgical procedures for resection of previous posterior fossa tumors. In one case, resection of 7 cerebellar and brainstem tumors was planned on neuronavegator after MRI images, and performed; postsurgical MRI showed a one residual tumor, unnoticed during the surgical procedure because dense scarring tissue. In the second patient, 4 tumors were planned for resection, 3 in cerebellum and 1 in brainstem, and all of them were found and resected. In no case red remitted fluorescence was observed during the location, dissection or resection of tumors. Histopathology analysis proved that all 11 tumors were typical hemangioblastomas. One small area with pink remitted fluorescence in the surgical bed of a tumor proved to be just gliosis without tumor cells. CONCLUSIONS: In the present cases, 5-ALA fluorescent guided surgery for multiple hemangioblastoma resections has not been of utility with the regular surgical armamentarium, instead of the above mentioned experience from one author. The different system for fluorescence evaluation (adapted surgical microscope visualization versus spectroscopy adapted probe system and digitized evaluation) could explain the different results. Other studies are necessary to confirm the results with regular surgical armamentarium, and to explore ancillary tools for detection of localization and extension of CNS tumors under fluorescent conditions during surgical procedures.