Alterations of T-cell surface markers in older women with persistent human papillomavirus infection.

We previously reported decreased lymphocyte proliferative responses among older women with persistent human papillomavirus (HPV) infection. To characterize the phenotype of peripheral lymphocytes associated with persistent HPV infection, we evaluated the expression of different cell surface markers in peripheral blood mononuclear cells (PBMCs) from a case-control study within a 10,049 woman population-based cohort study in Guanacaste, Costa Rica. Women in the cohort aged 46-74 and with HPV results at their 5th year anniversary visit were considered, and all women (n = 87) with persistent HPV infections, all women (n = 196) with transient HPV infections and a random sample of HPV DNA-negative women (n = 261) frequency-matched to cases on age were selected for this study. A median of 3 years after the case-control matching visit, cervical cells were collected for liquid-based cytology and repeat HPV DNA genotyping. Blood was obtained from which PBMCs were extracted and cryopreserved for immunological phenotyping via flow cytometry. Significant increases in risk of HPV persistence were observed for 3 marker subsets indicative of immune cell activation/differentiation. Relative risk estimates were 5.4 (95% CI = 2.2-13.3) for CD69(+)CD4(+), 2.6 (95% CI = 1.2-5.9) for HLADR(+)CD3(+)CD4(+) and 2.3 (95% CI = 1.1-4.7) for CD45RO(+)CD27(-)CD8(+). A significant decrease in HPV persistence was observed for a subset marker indicative of an immature, undifferentiated memory state CD45RO(+)CD27(+)CD4(+) (OR = 0.36; 95% CI = 0.17-0.76). Adjustment for these markers only partially explained the previously reported association between decreased lymphoproliferative responses and persistent HPV infection. Whether phenotypic alterations observed predispose to HPV persistence or result from it should be the focus of future studies.

Bacterial macroscopic rope-like fibers with cytopathic and adhesive properties.

We present a body of ultrastructural, biochemical, and genetic evidence that demonstrates the oligomerization of virulence-associated autotransporter proteins EspC or EspP produced by deadly human pathogens enterohemorrhagic and enteropathogenic Escherichia coli into novel macroscopic rope-like structures (>1 cm long). The rope-like structures showed high aggregation and insolubility, stability to anionic detergents and high temperature, and binding to Congo Red and thioflavin T dyes. These are properties also exhibited by human amyloidogenic proteins. These macroscopic ropes were not observed in cultures of nonpathogenic Escherichia coli or isogenic espP or espC deletion mutants of enterohemorrhagic or enteropathogenic Escherichia coli but were produced by an Escherichia coli K-12 strain carrying a plasmid expressing espP. Purified recombinant EspP monomers were able to self-assemble into macroscopic ropes upon incubation, suggesting that no other protein was required for assembly. The ropes bound to and showed cytopathic effects on cultured epithelial cells, served as a substratum for bacterial adherence and biofilm formation, and protected bacteria from antimicrobial compounds. We hypothesize that these ropes play a biologically significant role in the survival and pathogenic scheme of these organisms.

Elevated systemic levels of inflammatory cytokines in older women with persistent cervical human papillomavirus infection.

BACKGROUND: Defects in lymphoproliferative responses to mitogens/antigens in women >45 years old with a persistent type-specific human papillomavirus (HPV) infection have been reported. METHODS: To determine whether these defects were associated with altered cytokine profiles, plasma and peripheral blood mononuclear cell (PBMC) culture supernatants from 50 cases (oversampled for their reduced lymphoproliferative ability) and 50 uninfected controls (oversampled for their robust lymphoproliferative ability) were examined for 24 cytokines using multiplexed bead-based immunoassays and ELISA. RESULTS: The following plasma cytokines were significantly increased in cases relative to controls (cases versus controls; median pg/mL): interleukin (IL)-6, 393.1 versus 14.5; IL-8, 1,128.5 versus 43.9; tumor necrosis factor-alpha (TNF-alpha), 164.1 versus 9.2; macrophage inflammatory protein-1alpha (MIP-1alpha), 1,368.9 versus 25.5; granulocyte macrophage colony-stimulating factor (GM-CSF), 13.8 versus 7.3; IL-1beta, 8.3 versus 1.6 (all P < 0.0001); and IL-1alpha, 218.2 versus 169.5 (P = 0.02). We focused our analysis on the cytokines IL-6, IL-8, TNF-alpha, and MIP-1alpha due to their high fold change (>10) and highly statistically significant difference between cases and controls. Length of persistence or type of infection (high risk and low risk) did not affect these differences. IL-6, TNF-alpha, and MIP-1alpha levels were also increased in unstimulated PBMC culture supernatants from cases compared with controls (P < 0.05), however, the cytokine levels from phytohemagglutinin-stimulated PBMC culture supernatants were significantly lower in the cases (P < 0.0001). CONCLUSIONS: Persistent HPV infection in older women with evidence of immune deficit is associated with an increase in systemic inflammatory cytokines. IMPACT: Future studies are needed to determine whether the inflammatory profile is age dependent and to examine the role that inflammatory cytokines play in HPV-induced progression from infection to cervical cancer.

Interaction of Escherichia coli O157:H7 with leafy green produce.

Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen responsible for human diarrheal disease. EHEC lives in the intestinal tract of cattle and other farm and wild animals, which may be the source of environmental contamination particularly of agricultural fields. Human infections are associated with consumption of tainted animal products and fresh produce. How the bacteria interact with the plant phyllosphere and withstand industrial decontamination remain to be elucidated. The goals of the present study were to investigate the environmental conditions and surface structures that influence the interaction of EHEC O157:H7 with baby spinach and lettuce leaves in vitro. Independently of the production of Shiga toxin, EHEC O157:H7 colonizes the leaf surface via flagella and the type 3 secretion system (T3SS). Ultrastructural analysis of EHEC-infected leafy greens revealed the presence of flagellated bacteria, and mutation of the fliC flagellin gene in EHEC EDL933 rendered the bacteria significantly less adherent, suggesting the involvement of flagella in the bacteria-leaf interaction. EDL933 mutated in the escN (ATPase) gene associated with the function of the T3SS but not in the eae (intimin adhesin) gene required for adherence to host intestinal cells had significantly reduced adherence compared with that of the parental strain. The data suggest a compelling role of flagella and the T3SS in colonization of leafy green produce. Colonization of salad leaves by EHEC strains may be a strategy that ensures survival of these bacteria in the environment and allows transmission to the human host.

Comparison of two PCR-based human papillomavirus genotyping methods.

We compared two consensus primer PCR human papillomavirus (HPV) genotyping methods for the detection of individual HPV genotypes and carcinogenic HPV genotypes as a group, using a stratified sample of enrollment cervical specimens from sexually active women participating in the NCI/Costa Rica HPV16/18 Vaccine Efficacy Trial. For the SPF(10) method, DNA was extracted from 0.1% of the cervical specimen by using a MagNA Pure LC instrument, a 65-bp region of the HPV L1 gene was targeted for PCR amplification by using SPF(10) primers, and 25 genotypes were detected by reverse-line blot hybridization of the amplicons. For the Linear Array (LA) method, DNA was extracted from 0.5% of the cervical specimen by using an MDx robot, a 450-bp region of the HPV L1 gene was targeted for PCR amplification by using PGMY09/11 L1 primers, and 37 genotypes were detected by reverse-line blot hybridization of the amplicons. Specimens (n = 1,427) for testing by the LA method were randomly selected from strata defined on the basis of enrollment test results from the SPF(10) method, cytology, and Hybrid Capture 2. LA results were extrapolated to the trial cohort (n = 5,659). The LA and SPF(10) methods detected 21 genotypes in common; HPV16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, -68, and -73 were considered the carcinogenic HPV genotypes. There was no difference in the overall results for grouped detection of carcinogenic HPV by the SPF(10) and LA methods (35.3% versus 35.9%, respectively; P = 0.5), with a 91.8% overall agreement and a kappa value of 0.82. In comparisons of individual HPV genotypes, the LA method detected significantly more HPV16, HPV18, HPV39, HPV58, HPV59, HPV66, and HPV68/73 and less HPV31 and HPV52 than the SPF(10) method; inclusion of genotype-specific testing for HPV16 and HPV18 for those specimens testing positive for HPV by the SPF(10) method but for which no individual HPV genotype was detected abrogated any differences between the LA and SPF(10) methods. The LA method detected more carcinogenic-HPV-genotype infections per specimen than the SPF(10) method (P < 0.001). In conclusion, the LA method and the SPF(10) method with HPV16 and HPV18 genotype-specific detection among ungenotyped HPV-positive specimens were comparable for detection of HPV16 and HPV18, the two HPV genotypes targeted by current prophylactic HPV vaccines. Both approaches are suitable for monitoring the impact of HPV16/18 vaccines in clinical trials.

Rationale and design of a community-based double-blind randomized clinical trial of an HPV 16 and 18 vaccine in Guanacaste, Costa Rica.

We report the rationale, design, methods and details of participation of a community-based, double-blind, randomized clinical trial of an HPV 16 and 18 vaccine conducted in two provinces of Costa Rica to investigate the efficacy and population impact of the vaccine in the prevention of cervical cancer precursors. More than 24,000 women between 18 and 25 years of age were invited to participate and pre-screened for eligibility, with recruitment of 7466 women (30% of those pre-screened, 59% of those eligible) who were randomized to receive 3 doses of the HPV vaccine or hepatitis A vaccine as control. A complex protocol of data and specimen collection was applied, including an interview, pelvic exam for sexually active women, blood for serology and cell-mediated immunity, cervical secretions for local immunity and cells for HPV, Chlamydia trachomatis and gonorrhea testing. Eighty percent of the women received three doses, 12.4% two doses and 7.4% one dose. At visits, compliance with data and specimen collection was close to 100%. Baseline characteristics and age-specific prevalence of HPV and cervical neoplasia are reported. Overall prevalence of HPV was high (50%), with 8.3% of women having HPV 16 and 3.2% HPV 18. LSIL was detected in 12.7% of women at baseline and HSIL in 1.9%. Prevalence of Chlamydia was 14.2%. There was very good agreement in HPV detection between clinician-collected and self- collected specimens (89.4% agreement for all types, kappa 0.59). Follow up will continue with yearly or more frequent examinations for at least 4 years for each participant.

Epidemiology of genital Chlamydia trachomatis infection among young women in Costa Rica.

OBJECTIVE: To investigate Chlamydia trachomatis (Ct) epidemiology among 5829 women 18 to 25 years old, in Costa Rica. METHODS: Data are from a community-based human papillomavirus 16/18 vaccine trial. Before randomization, eligible women who reported previous sexual activity were interviewed and tested for Ct DNA by Hybrid Capture 2 and polymerase chain reaction-based genotyping. Multivariate models were developed. RESULTS: Overall prevalence was 14.2% (95% confidence interval, 13.3-15.1). Among Ct genotypes, serovar E was the most common (4.3%), followed by serovar F (3.0%), serovar D/Da (2.9%), and serovar I/Ia (2.1%).Ct increased with lifetime sexual partners of the women, and among women with 1 lifetime partner, with sexual partners of the partner. Current intrauterine device users had an increase in Ct detection [odds ratio (OR) 1.6, 1.1-2.5] but hormonal contraceptives or condom users did not. Miscarriages were associated with a reduction in Ct detection (OR 0.7, 0.5-1.0) while current regular smoking increased it (OR 1.7, 1.2-2.5). Vaginal discharge, reactive changes, ASCUS or LSIL and moderate to severe inflammation in the cytology were significantly more common among Ct positive women (P <0.001). Gonorrhea prevalence was 0.8%, and it was, as other STIs, highly correlated with Ct detection. CONCLUSIONS: This is a high-prevalence population where we confirmed the strong link between Ct and sexual behavior of women and their partners. The establishment of a screening program in the age group included in this study should be considered. More studies are needed in developing countries to further investigate the role of intrauterine devices and the lack of protection by condoms, in addition to the interplay between Ct and other STIs, ectopy, inflammation, and epithelial abnormalities.

Evaluation of a novel PCR-based assay for detection and identification of Chlamydia trachomatis serovars in cervical specimens.

The aims of this study were to compare a novel PCR-based Chlamydia trachomatis detection and genotyping (Ct-DT) assay with the FDA-approved, commercially available C. trachomatis detection Hybrid Capture 2 (HC2) assay and to investigate the C. trachomatis serovar distribution among young women in a rural Costa Rican study population. A total of 5,828 sexually active women participating in a community-based trial in Costa Rica were tested for C. trachomatis by HC2. A sample of 1,229 specimens consisting of 100% HC2 C. trachomatis-positive specimens (n = 827) and a random sample of 8% HC2 C. trachomatis-negative specimens (n = 402) were tested with the Ct-DT assay. Agreement between the two assays was determined by the unweighted kappa statistic. Discrepant specimens were tested with a second commercially available test (COBAS TaqMan). The Ct-DT-positive specimens were further analyzed with the Ct-DT genotyping step to investigate the distribution of 14 different C. trachomatis serovars (A, B/Ba, C, D/Da, E, F, G/Ga, H, I/Ia, J, K, L1, L2/L2a, and L3). After accounting for the sampling fraction selected for Ct-DT testing, crude agreement with the HC2 assay was 98% and the kappa was 0.92 (95% confidence interval [CI], 0.89 to 0.97). The 33 discordant samples that were further analyzed with the COBAS TaqMan test showed better agreement with the Ct-DT assay (31/33, P < 0.001). Among the 806 Ct-DT-positive samples, serovar E was the most common serovar (31%), followed by serovars F and D (both 21%) and serovar I (15%). In conclusion, the novel Ct-DT assay permits reliable detection and identification of C. trachomatis serovars.

Comparison of the SPF10-LiPA system to the Hybrid Capture 2 Assay for detection of carcinogenic human papillomavirus genotypes among 5,683 young women in Guanacaste, Costa Rica.

The objective of this analysis was to compare the performance characteristics of two human papillomavirus (HPV) DNA detections assays, the Hybrid Capture 2 assay (HC2) and the SPF(10) assay, for the detection of carcinogenic HPV. Data are from the enrollment visits of women who participated in the randomized, double-blind, placebo-controlled phase III HPV16/18 Vaccine Trial in Guanacaste, Costa Rica. We compared the results of HC2 and SPF(10) testing of cervical specimens. Since the line probe assay (LiPA) detection system does not distinguish between HPV type 68 (HPV68; which is targeted by HC2) and HPV73 (which is not targeted by HC2), for SPF(10)-LiPA, we defined the carcinogenic HPV types as the 12 HC2-targeted types (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59), HPV68/73, and the HC2-cross-reactive, carcinogenic type HPV66. The kappa values and the performance characteristics for the detection of cervical abnormalities were ascertained. Paired observations were available for 5,683 sexually active, young women (median age, 21 years). The prevalence of carcinogenic HPV types was 35% (n=1,962) by HC2 and 35% (n=2,003) by SPF(10)-LiPA. There were no differences in the prevalence of carcinogenic HPV types by HC2 and SPF(10)-LiPA among women with normal, atypical squamous cells of undetermined significance and high-grade squamous intraepithelial lesion cytology. Among women with low-grade squamous intraepithelial lesion cytology, HC2 was more likely to test positive than SPF(10)-LiPA for the carcinogenic HPV types (87% and 79%, respectively; P=0.001) as a result of HC2 cross-reactivity with HPV types 40, 43, 44, 53, 54, 60, 70, and 74. The crude agreement between the two assays was 88%, with a kappa value of 0.75 (95% confidence limits, 0.73 to 0.76). We observed very good agreement between HC2 and SPF(10)-LiPA for carcinogenic HPV type detection.

Persistent human papillomavirus infection is associated with a generalized decrease in immune responsiveness in older women.

The development of cervical cancer and its precursors are linked to persistent infection with oncogenic types of human papillomavirus (HPV). Host immune responses seem to be determinants of risk for this disease. However, little is known about the immunologic determinants of HPV persistence. Here, we examined the association between lymphoproliferative responses to antigens/mitogens and persistent HPV infection in women older than 45 years. Women included in this study were participants in a 10,000-woman population-based cohort study of cervical neoplasia in Costa Rica. Women older than 45 years and HPV DNA positive at a screening visit were selected as cases (n = 283). We selected a comparably sized control group of HPV DNA-negative women, matched to cases on age and time since enrollment (n = 261). At an additional clinical visit, women were cytologically and virologically rescreened, and cervical and blood specimens were collected. Proliferative responses to phytohemagglutinin (PHA), influenza virus (Flu), and HPV16 virus-like particle (VLP) were lower among women with persistent HPV infection [median counts per minute (cpm): 72,849 for PHA, 1,241 for Flu, and 727 for VLP] than for the control group (median cpm: 107,049 for PHA, 2,111 for Flu, and 2,068 for VLP). The decreases were most profound in women with long-term persistence and were only observed for the oldest age group (>/=65 years). Our results indicate that an impairment in host immunologic responses is associated to persistent HPV infection. The fact that effects were evident for all studied stimuli is suggestive of a generalized effect.

Presencia de los dinoflagelados Ceratium dens, C. fusus y C. furca (Gonyaulacales: Ceratiaceae) en el Golfo de Nicoya, Costa Rica / Presence of the dinoflagellates Ceratium dens, C. fusus and C. furca (Gonyaulacales: Ceratiaceae) in Golfo de Nicoya, Costa Rica

Las proliferaciones algales nocivas (PAN) son un fenómeno frecuente en el Golfo de Nicoya. La morfología y fisiología de estas microalgas son muy importantes debido al hecho de que las especies involucradas en estos eventos presentan características adaptativas. PAN producidas por dinoflagelados del género Ceratium son comunes en algunas partes del mundo, sin embargo, a pesar del hecho de que la producción de altas concentraciones de toxinas paralíticas sólo se ha documentado a nivel experimental, éste género ha sido asociado con la mortalidad de organismos acuáticos y con la disminución en la calidad del agua, así como el responsable de la mortalidad de ostras y larvas de gambas. Algunas especies del género Ceratium han sido asociados con eventos de mortalidad en peces. Durante un reciente fenómeno PAN que ocurrió cerca de la Isla Tortuga en el Golfo de Nicoya, los pescadores en el área informaron muertes masivas de peces enjaulados. Se tomaron muestras de una PAN que producía una discoloración anaranjada en el agua y tenía un olor fétido. Estas muestras fueron procesadas para observar el detalle ultraestructural usando el microscopio electrónico. En muestras tomadas en superficie, los dinoflagelados observados fueron Ceratium furca, C. fusus y C. dens. El conteo celular reveló cuatro millones de células de este género por litro. Los cambios morfológicos de estas especies son muy variables; por consiguiente la microscopía electrónica es una herramienta muy útil en el estudio ultraestructural de estos organismos. Esta es la primera vez que tres especies del género Ceratium se conocen como productoras concurrentes de PAN en Costa Rica

Harmful Algae Blooms (HAB) are a frequent phenomenon in the Gulf of Nicoya, Costa Rica, as in other parts of the world. The morphology and physiology of these microalgae are important because HAB species have adaptive characteristics. The production of high concentrations of paralytic toxins by Ceratium dinoflagellates has only been documented at the experimental level. However, this genus has been associated with the mortality of aquatic organisms, including oyster and shrimp larva, and fish, and with decreased water quality. Recently, fishermen reported massive mortality of encaged fish near Tortuga Island (Gulf of Nicoya). Samples were taken from an algal bloom that had produced an orange coloration and had a strong foul-smelling odor. Ultrastructural details were examined with scanning electron microscopy. The dinoflagellates Ceratium dens, C. furca and C. fusus were found in samples taken at the surface. The cell count revealed four million cells of this genus per liter. The morphological variability of these species is high; therefore electron microscopy is an useful tool in the ultrastructural study of these organisms. This is the first time that three Ceratium species are reported concurrently producing harmful blooms in Costa Rica

Proliferaciones algales nocivas de cianobacterias (Oscillatoriaceae) y dinoflagelados (Gymnodiniaceae) en el Golfo de Nicoya, Costa Rica / Harmful blooms of cyanobacteria (Oscillatoriaceae) and dinoflagellates (Gymnodiniaceae) in the Golfo de Nicoya, Costa Rica

Recently, the Pacific coast of Costa Rica has experienced an increase in both magnitude and frequency of harmful algae blooms (HAB). The lack of data regarding the dynamics of these events in the area, and the species of microalgae that produce them, are themes of great interest. The blooms have produced negative impacts on fishery resources and on human health in Costa Rica. In May 2002 a HAB left a large number of dead fish along the central Pacific coast. Water samples were collected using a phytoplankton net and fixed for subsequent processing by electron microscopy. In addition, a one liter sample of surface water was taken for later cell count. In the observed HAB, the dominating organisms found were the cyanobacteria Trichodesmiun erythraeum surrounded by high concentrations of Gram - bacteria and the dinoflagellate Cochlodinium cf. polykrikoides. T. erythraeum, is one of the most important N 2 fixing cyanobacteria in marine waters that has been associated with HAB events in diverse parts of the world as well as with symptoms that produce contact dermatitis and other discomforts. C. cf. polykrikoides is a dinoflagellete associated with fish kills; although the type of associated toxins are unknown. In a national newspaper 17 cases of intoxication in humans were reported during this same period, which presented respiratory disorders and burning of the eyes. This is the first report in Costa Rica where a cyanobacteria and a dinoflagellate were observed together producing HAB

Recientemente, la costa del Pacífico de Costa Rica ha experimentado un aumento en magnitud y frecuencia de proliferaciones algales nocivas (PAN). La falta de datos con respecto a la dinámica de estos eventos en el área, y las especies de microalgas que los producen, son temas de mucho interés. Las PAN han producido impactos negativos en los recursos pesqueros y en la salud humana en Costa Rica. En mayo de 2002, una PAN se observó frente a las playas a lo largo del Pacífico central; además, se observó un gran número y diversidad de peces muertos. Las muestras de agua fueron recolectadas usando una red de fitoplancton y se fijaron para ser procesadas posteriormente con microscopía electrónica. Asimismo, se tomó un litro de muestra de la superficie para su posterior conteo celular. En la PAN observada, los organismos dominantes fueron la cianobacteria Trichodesmiun erythraeum, rodeada por altas concentraciones de bacterias Gram -, y el dinoflagelado Cochlodinium cf. polykrikoides. T. erythraeum, es una de las principales cianobacterias fijadoras de N2 atmosférico en aguas marinas, y se ha asociado con los eventos PAN en diversas partes del mundo, produciendo síntomas como dermatitis por contacto con la brisa marina y otras afecciones. C. cf. polykrikoides es un dinoflagelado asociado con mortalidad de peces; aunque el tipo de toxinas asociadas aún es desconocido. En un periódico nacional se reportaron 17 casos de intoxicación en humanos durante este mismo periodo de PAN, los afectados presentaron desórdenes respiratorios e irritación en los ojos. Éste es el primer informe en Costa Rica dónde se observaron cianobacterias y dinoflagelados juntos produciendo PAN. Este tipo de fenómenos debe analizarse y debe documentarse, para habilitar una comprensión en la dinámica y los factores asociados con el aumento de estos eventos en el Pacífico costarricense

Proliferaciones algales de la diatomea toxigénica Pseudo-Nitzschia (Bacillariophyceae) en el Golfo de Nicoya, Costa Rica / Algal blooms of the toxigenic diatom Pseudo-Nitzschia (Bacillariophyceae) in the Golfo de Nicoya, Costa Rica

En el mes de noviembre de 2001, se aisló de una marea roja cerca de la Isla San Lucas, las diatomeas Pseudo-Nitzschia pungens f. pungens seguida por Skeletonema costatum, Chaetoceros lorenzianus y en menor concentración Thalassiosira spp. las cuales fueron caracterizadas por microscopía electrónica de rastreo y transmisión, siendo esta la primera vez que se describe la presencia de estas especies produciendo proliferaciones en Costa Rica. Actualmente existe un aumento en el reporte de mareas rojas donde predominan las diatomeas, y los conteos celulares indican el aumento en el número de las especies conocidas como Pseudo-nitzschia pungens f. multiseries. A estas se les atribuye la producción del ácido domóico, un aminoácido de bajo peso molecular. Las intoxicaciones amnésicas por mariscos (IAM) que afectan a los humanos, se producen por la ingesta de mariscos contaminados con este ácido. En Costa Rica, hasta la fecha, solo se han reportado casos de intoxicación paralítica por mariscos contaminados por los dinoflagelados Pyrodinium bahamense var. compressum y Gymnodinium catenatum, sin embargo existe la posibilidad de producirse intoxicaciones humanas por la ingesta de mariscos y peces contaminados con toxinas amnésicas las cuales son hidrosolubles y termoestables. Debe entonces considerarse el peligro potencial de aparición de casos de intoxicaciones amnésicas y se sugiere la inclusión de estas especies en los programas de monitoreo permanente para tomar las medidas preventivas de salud pública

Water samples were collected during a red tide event in November 2001, near San Lucas Island (Gulf of Nicoya, Costa Rica). Superficial temperature was 27ºC and water was turbid, with no fetid smell. One sample was treated with negative staining and observed using a transmission electron microscope (TEM); another sample was observed using a scanning electron microscope (SEM). Samples had high concentrations of the diatom Pseudo-Nitzschia pungens f. pungens (characterized by two rows of poroids in the external channel), and lower concentrations of Skeletonema costatum (chains joined by external microtubules) and Chaetoceros lorenzianus (oval apertures and long chains, having setae with distinctive transverse rows and spines). This is the first time that the first species was described producing red tides in Costa Rica. However, reports about red tides with high concentration of species like P. pungens (variety multiseries) are increasing. These species have been related to the production of domoic acid, a low molecular weight amino acid which in humans can cause amnesic intoxications with seafood. Previously, Costa Rican reports of toxic accidents only referred to seafood contaminated with Pyrodinium bahamense var. compressum and Gymnodinium catenatum dinoflagellates. The increase in the number of Pseudo-Nitzschia causing harmful algae blooms is of interest for scientists around the world and must be documented. Similarly, some Chaetoceros species have been reported to be harmful to fish. We strongly recommend the establishment of a permanent surveillance program monitoring the presence of these species new at Costa Rican Pacific coast. Since the amnesic toxin is soluble in water and heat-resistant, we want to stress the possibility of having human cases of amnesic intoxication

Adherence of Brucella to human epithelial cells and macrophages is mediated by sialic acid residues.

The basis for the interaction of Brucella species with the surface of epithelial cells before migration in the host within polymorphonuclear leucocytes is largely unknown. Here, we studied the ability of Brucella abortus and Brucella melitensis to adhere to cultured epithelial (HeLa and HEp-2) cells and THP-1-derived macrophages, and to bind extracellular matrix proteins (ECM). The brucellae adhered to epithelial cells forming localized bacterial microcolonies on the cell surface, and this process was inhibited significantly by pretreatment of epithelial cells with neuraminidase and sodium periodate and by preincubation of the bacteria with heparan sulphate and N-acetylneuraminic acid. Trypsinization of epithelial cells yielded increased adherence, suggesting unmasking of target sites on host cells. Notably, the brucellae also adhered to cultured THP-1 cells, and this event was greatly reduced upon removal of sialic acid residues from these cells with neuraminidase. B. abortus bound in a dose-dependent manner to immobilized fibronectin and vitronectin and, to a lesser extent, to chondroitin sulphate, collagen and laminin. In sum, our data strongly suggest that the adherence mechanism of brucellae to epithelial cells and macrophages is mediated by cellular receptors containing sialic acid and sulphated residues. The recognition of ECM (fibronectin and vitronectin) by the brucellae may represent a mechanism for spread within the host tissues. These are novel findings that offer new insights into understanding the interplay between Brucella and host cells.

[Presence of the dinoflagellates Ceratium dens, C. fusus and C. furca (Gonyaulacales: Ceratiaceae) in Golfo de Nicoya, Costa Rica]. / Presencia de los dinoflagelados Ceratium dens, C. fusus y C. furca (Gonyaulacales: Ceratiaceae) en el Golfo de Nicoya, Costa Rica.

Harmful Algae Blooms (HAB) are a frequent phenomenon in the Gulf of Nicoya, Costa Rica, as in other parts of the world. The morphology and physiology of these microalgae are important because HAB species have adaptive characteristics. The production of high concentrations of paralytic toxins by Ceratium dinoflagellates has only been documented at the experimental level. However, this genus has been associated with the mortality of aquatic organisms, including oyster and shrimp larva, and fish, and with decreased water quality. Recently, fishermen reported massive mortality of encaged fish near Tortuga Island (Gulf of Nicoya). Samples were taken from an algal bloom that had produced an orange coloration and had a strong foul-smelling odor. Ultrastructural details were examined with scanning electron microscopy. The dinoflagellates Ceratium dens, C. furca and C. fusus were found in samples taken at the surface. The cell count revealed four million cells of this genus per liter. The morphological variability of these species is high; therefore electron microscopy is an useful tool in the ultrastructural study of these organisms. This is the first time that three Ceratium species are reported concurrently producing harmful blooms in Costa Rica.

[Harmful blooms of cyanobacteria (Oscillatoriaceae) and dinoflagellates (Gymnodiniaceae) in the Golfo de Nicoya, Costa Rica]. / Proliferaciones algales nocivas de cianobacterias (Oscillatoriaceae) y dinoflagelados (Gymnodiniaceae) en el Golfo de Nicoya, Costa Rica.

Recently, the Pacific coast of Costa Rica has experienced an increase in both magnitude and frequency of harmful algae blooms (HAB). The lack of data regarding the dynamics of these events in the area, and the species of microalgae that produce them, are themes of great interest. The blooms have produced negative impacts on fishery resources and on human health in Costa Rica. In May 2002 a HAB left a large number of dead fish along the central Pacific coast. Water samples were collected using a phytoplankton net and fixed for subsequent processing by electron microscopy. In addition, a one liter sample of surface water was taken for later cell count. In the observed HAB, the dominating organisms found were the cyanobacteria Trichodesmiun erythraeum surrounded by high concentrations of Gram-bacteria and the dinoflagellate Cochlodinium cf. polykrikoides. T. erythraeum, is one of the most important N2 fixing cyanobacteria in marine waters that has been associated with HAB events in diverse parts of the world as well as with symptoms that produce contact dermatitis and other discomforts. C. cf. polykrikoides is a dinoflagellete associated with fish kills; although the type of associated toxins are unknown. In a national newspaper 17 cases of intoxication in humans were reported during this same period, which presented respiratory disorders and burning of the eyes. This is the first report in Costa Rica where a cyanobacteria and a dinoflagellate were observed together producing HAB.

[Algal blooms of the toxigenic diatom Pseudo-Nitzschia (Bacillariophyceae) in the Golfo de Nicoya, Costa Rica]. / Proliferaciones algales de la diatomea toxigénica Pseudo-Nitzschia (Bacillariophyceae) en el Golfo de Nicoya, Costa Rica.

Water samples were collected during a red tide event in November 2001, near San Lucas Island (Gulf of Nicoya, Costa Rica). Superficial temperature was 27 degrees C and water was turbid, with no fetid smell. One sample was treated with negative staining and observed using a transmission electron microscope (TEM); another sample was observed using a scanning electron microscope (SEM). Samples had high concentrations of the diatom Pseudo-Nitzschia pungensf pungens (characterized by two rows of poroids in the external channel), and lower concentrations of Skeletonema costatum (chains joined by external microtubules) and Chaetoceros lorenzianus (oval apertures and long chains, having setae with distinctive transverse rows and spines). This is the first time that the first species was described producing red tides in Costa Rica. However, reports about red tides with high concentration of species like P. pungens (variety multiseries) are increasing. These species have been related to the production of domoic acid, a low molecular weight amino acid which in humans can cause amnesic intoxications with seafood. Previously, Costa Rican reports of toxic accidents only referred to seafood contaminated with Pyrodinium bahamense var. compressum and Gymnodinium catenatum dinoflagellates. The increase in the number of Pseudo-Nitzschia causing harmful algae blooms is of interest for scientists around the world and must be documented. Similarly, some Chaetoceros species have been reported to be harmful to fish. We strongly recommend the establishment of a permanent surveillance program monitoring the presence of these species new at Costa Rican Pacific coast. Since the amnesic toxin is soluble in water and heat-resistant, we want to stress the possibility of having human cases of amnesic intoxication.

[Ultrastructural description of Euglena pailasensis (Euglenozoa) from Rincón de la Vieja volcano, Guanacaste, Costa Rica]. / Descripción ultraestructural de Euglena pailasensis (Euglenozoa) del Volcán Rincón de la Vieja, Guanacaste, Costa Rica.

The euglenoids are unicellular eukaryotic flagellates living in a diversity of soils and aquatic environments and ecosystems. This study describes the ultrastructure of an euglenoid isolated from the surface of a boiling mud pool with temperatures ranging from 38 to 98 degrees C and pH 2 - 4. The hot mud pool is located in Area de Pailas de Barro, Las Pailas, Rincón de la Vieja Volcano, Guanacaste, Costa Rica. The morphological characterization of the Euglena pailasensis was performed by SEM and TEM. It was determined that, although the euglenoid was obtained from an extreme volcanic environment, the general morphology corresponds to that of a typical member of Euglena of 30-45 microm long and 8-10 microm wide, with membrane, pellicle, chloroplasts, mitochondria, nucleus, pigments and other cytoplasmic organelles. E. pailasensis is delimited by a membrane and by 40 to 90 pellicle strips. It was observed up to 5 elongated chloroplasts per cell. The chloroplast contains several osmiophilic globules and a pyrenoid penetrated by few thylakoid pairs. The nutritious material is reserved in numerous small paramylon grains located at the center of the cell, mitocondria are characterized by the presence of crests in radial disposition toward the interior of the lumen. It was also observed around the external surface "pili" like filaments originating from the pellicle strips. There is no evidence for the presence of flagella in the ampulla (reservoir/canal area), a fact confirmed by negative staining, and a difference regarding other species of Euglena. The observed ultrastructural characteristics are not sufficient to explain the adaptation of this species to acid and hot environments.

Floraciones algales nocivas en la costa pacífica de Costa Rica: toxicología y sus efectos en el ecosistema y salud pública / Harmful Algae Blooms in Costa Rica

Justificación y objetivos: Los primeros casos de intoxicación por floraciones algales nocivas (FAN) se reportaron en el país en 1990. Sin embargo desde finales de 1999 se ha observado un aumento drástico en el número de personas intoxicadas, especialmente en la costa pacífica del país. En Costa Rica existe gran desconocimiento acerca del problema. Debido al impacto que producen los fenómenos FAN en la salud pública, el turismo local y el ecosistema, es necesario describir las especies tóxicas y toxinas para tomar las medidas adecuadas de control y prevención. Métodos: Se realizaron muestreos de agua en varios sectores de la costa pacífica de nuestro país, con el objetivo de detectar las especies de microalgas potencialmente tóxicas y nocivas, y se interrogó a los pescadores locales. La identificacón de las especies se realizó morfológicamente mediante microscopía de luz y electrónica de rastreo. Los hallazgos se compararon con la bibliografía y se propuso el potencial tóxico de estas microalgas, así como los métodos de análisis toxicológicos, y la sintomatología de los afectados. Resultados: Observamos en Costa Rica un aumento en número de especies de microalgas productoras de FAN. Se reportaron las especies tóxicas productoras de FAN descritas por varios autores y las encontradas en este estudio. En algunas muestras de floraciones algales hemos encontrado bacterias grandes intracelulares, de forma bacilar, principalmente dentro del dinoflagelado Prorocentrum micans. Esta relación podría estar asociada a la toxigenicidad del alga. Hemos descrito la presencia de 26 dinoflagelados, 8 diatomeas y 1 cianobacteria tóxica en las FAN de las costas del Océano Pacifíco costarricense. Conclusión: Se piensa que el aumento en la contaminación de las zonas costeras y el transporte de especies por medio de navíos pueden ser la causa de la aparición de estas microalgas. Estas son capaces de producir: intoxicación paralizante, amnésica, diarréica, neurotóxica, ciguatera y fiebre de Tamandré. Es importante que el personal de salud tenga un buen conocimiento de las FAN y se necesita con urgenica un adecuado programa de monitoreo y control en Costa Rica, para proteger a la población. Descriptores: mareas rojas, floraciones algales nocivas, dinoflagelados, toxicología.