Estimating influenza vaccine effectiveness in Spain using sentinel surveillance data.

We aimed to estimate influenza vaccine effectiveness (VE) against laboratory-confirmed influenza during three influenza seasons (2010/11 to 2012/2013) in Spain using surveillance data and to compare the results with data obtained by the cycEVA study, the Spanish component of the Influenza Monitoring Vaccine Effectiveness (I-MOVE) network. We used the test-negative case­control design, with data from the Spanish Influenza Sentinel Surveillance System (SISS) or from the cycEVA study. Cases were laboratory-confirmed influenza patients with the predominant influenza virus of each season, and controls were those testing negative for any influenza virus. We calculated the overall and age-specific adjusted VE. Although the number of patients recorded in the SISS was three times higher than that in the cycEVA study, the quality of information for important variables, i.e. vaccination status and laboratory results, was high in both studies. Overall, the SISS and cycEVA influenza VE estimates were largely similar during the study period. For elderly patients (> 59 years), the SISS estimates were slightly lower than those of cycEVA, and estimates for children (0­14 years) were higher using SISS in two of the three seasons studied. Enhancing the SISS by collecting the date of influenza vaccination and reducing the percentage of patients with incomplete information would optimise the system to provide reliable annual influenza VE estimates to guide influenza vaccination policies.

Trends in systemic lupus erythematosus mortality in Spain from 1981 to 2010.

BACKGROUND: Incidence and mortality of systemic lupus erythematosus (SLE) seem to be increasing in the last few decades, in contrast to the survival rate that has improved over time. The objective of this study was to examine the trends in the SLE mortality in Spain over a 30-year period (1981-2010). METHODS: Data on SLE deaths were drawn from the National Statistics Institute of Spain. Crude and overall age-standardized SLE mortality rates were calculated and joinpoint regression models were used to describe trend changes. Mean age of deaths by SLE each year was also assessed. RESULTS: The overall age-standardized SLE mortality rate was 1.82 per million in 1981 and 2.24 in 2010. It was higher in women, 1.39 vs 0.43 in 1981 and 1.96 vs 0.28 in 2010. There was a statistically significant change in 1999. The overall age-standardized mortality rate increased from 1981 to 1999 and stabilized from 2000 to 2010. Only male rates decreased from 2000 to 2010. The mean age at death increased with time, from 42 years in 1981 to 61 years in 2010. CONCLUSIONS: In conclusion, a slight decrease in SLE mortality has been observed in Spain over the last decade and future studies would be needed to explain the factors contributing to the improvement in the mortality rates.

Influenza vaccine effectiveness in Spain 2013/14: subtype-specific early estimates using the cycEVA study.

Adjusted early estimates of the 2013/14 influenza vaccine effectiveness (VE) in Spain for all age groups was 35% (95% CI: -9 to 62), 33% (95% CI: -33 to 67) and 28% (95% CI: -33 to 61) against any influenza virus type, A(H1N1)pdm09 and A(H3N2) viruses, respectively. For the population targeted for vaccination, the adjusted VE was 44% (95% CI: -11 to 72), 36% (95% CI: -64 to 75) and 42% (95% CI: -29 to 74), respectively. These preliminary results in Spain suggest a suboptimal protective effect of the vaccine against circulating influenza viruses.

Early estimates of the effectiveness of the 2011/12 influenza vaccine in the population targeted for vaccination in Spain, 25 December 2011 to 19 February 2012.

We present early estimates of influenza vaccine effectiveness (VE) in the population targeted for vaccination, during 25 December 2011 to 19 February 2012. The adjusted VE was 55% (95% CI: 3 to 79) against any type of influenza virus and 54% (95% CI: 1 to 79) against influenza A(H3N2) virus. This suggests a moderate protective effect of the vaccine in the targeted population in a late influenza epidemic with limited match between vaccine and circulating strains.

The Role of Knowledge and Attitudes About Nonsteroidal Anti-inflammatory Drugs in Determining Treatment Use.

OBJECTIVE: The objective of this study was to evaluate how patient knowledge and beliefs regarding nonsteroidal anti-inflammatory drugs (NSAIDs) may influence the use of NSAIDs for osteoarthritis (OA). METHODS: Surveys of 334 adults with knee and/or hip OA were analyzed in this cross-sectional study. Familiarity with and perceptions of benefits/risks of NSAID use were measured to assess associations with the use of prescription and nonprescription oral NSAIDs. Multinomial logistic regression models were adjusted for sociodemographic and clinical variables. RESULTS: In this sample, 35.9% and 35.6% reported use of oral prescription and nonprescription-only NSAIDs, respectively. Hispanic participants, compared with non-Hispanic White participants, had lower perceived benefit (P = 0.005) and risk (P = 0.001) of prescription NSAIDs. The following were associated with prescription NSAID use instead of no NSAID use: having family/friends who used prescription (relative risk ratio [RRR] 3.91; 95% confidence interval [CI] 2.05-7.47) and over-the-counter (OTC) (RRR 3.10; 95% CI 1.65-5.83) NSAIDs for OA, understanding the consequences of using both prescription (RRR 3.50; 95% CI 1.79-6.86) and OTC (RRR 2.80; 95% CI 1.39-5.65) NSAIDs, higher perceived benefit of both prescription (RRR 2.51; 95% CI 1.71-3.66) and OTC (RRR 1.44; 95% CI 1.01-2.06) NSAIDs, and lower perceived risk of both types of NSAIDs (prescription: RRR 0.63 [95% CI 0.46-0.87]; OTC: RRR 0.53 [95% CI 0.37-0.75]). Similar results were found when we assessed the relationship between these variables and OTC NSAID use versus no oral NSAID use. CONCLUSION: Adults with knee and/or hip OA were more likely to use NSAIDs if they were more familiar with, had an increased perceived benefit of, and had a decreased perceived risk of these drugs. Patients' perceptions and beliefs about NSAIDs should be evaluated when considering them for treatment.

An outbreak of Mycobacterium fortuitum cutaneous infection associated with mesotherapy.

OBJECTIVE: We describe an outbreak of Mycobacterium fortuitum cutaneous infections associated with mesotherapy in La Rioja, Spain. DESIGN: Descriptive epidemiology. SETTING: Private practice. PATIENTS OR OTHER PARTICIPANTS: Case subjects were customers of a single beauty salon who were treated with mesotherapy injections. INTERVENTION(S): Two skin biopsies were taken from each patient. RESULTS: Over the designated period, 138 women received mesotherapy. Of these women, 39, or 28.3%, developed lesions ultimately thought to be caused by Mycobacterium fortuitum infection. The number of lesions per patient varied from 3 to 20 in the most severe case. Most of the lesions were indurated, erythematous or violaceous papules, some progressing to become fluctuant boils with suppuration, fistulization and scarring. The individual lesions varied in diameter from 0.5 to 6 cm. Two patients (5.1%) developed inguinal or axillary adenopathy. Two others presented with fever. One reported muscular pain. In 12 of the 39 cases, M. fortuitum was isolated from the wound cultures. The patients were all successfully treated with clarithromycin and levofloxacin. CONCLUSIONS: We identified a large outbreak of rapidly growing mycobacterial lesions among women who received mesotherapy injections in a single beauty salon.

Scattering proton CT.

Proton computed tomography (CT) is an imaging modality investigated mainly in the context of proton therapy as a complement to x-ray CT. It uses protons with high enough energy to fully traverse the imaged object. Common prototype systems measure each proton's position and direction upstream and downstream of the object as well as the energy loss which can be converted into the water equivalent thickness. A reconstruction algorithm then produces a map of the relative stopping power in the object. As an alternative to energy-loss proton CT, it has been proposed to reconstruct a map of the object's scattering power based on the protons' angular dispersion which can be estimated from the measured directions. As in energy-loss proton CT, reconstruction should best be performed considering the non-linear shape of proton trajectories due to multiple Coulomb scattering (MCS), but no algorithm to achieve this is so far available in the literature. In this work, we propose a filtered backprojection algorithm with distance-driven binning to account for the protons' most likely path. Furthermore, we present a systematic study of scattering proton CT in terms of inherent noise and spatial resolution and study the artefacts which arise from the physics of MCS. Our analysis is partly based on analytical models and partly on Monte Carlo simulations. Our results show that the proposed algorithm performs well in reconstructing relative scattering power maps, i.e. scattering power relative to that of water. Spatial resolution is improved by almost a factor of three compared to straight line projection and is comparable to energy-loss proton CT. Image noise, on the other hand, is inherently much higher. For example, in a water cylinder of 20 cm diameter, representative of a human head, noise in the central image pixel is about 40 times higher in scattering proton CT than in energy-loss proton CT. Relative scattering power in dense regions such as bone inserts is systematically underestimated by a few percent, depending on beam energy and phantom geometry.

Low allelic heterogeneity in a sample of Mexican patients with classical galactosaemia.

Classical galactosaemia is an autosomal recessive disease of galactose metabolism caused by a deficiency of the enzyme galactose-1-phosphate uridyltransferase (GALT). Galactosaemia is not included in the neonatal screening programme in Mexico and it is necessary to implement methodologies for prompt diagnosis of these patients to establish treatment. To date, more than 190 mutations in the GALT gene have been reported, most in caucasian populations, but there have been no reports of mutations in Latin-American populations. We report here the mutational spectrum in 19 Mexican galactosaemic patients. The most frequent mutations were p.Q188R, p.N314D and IVS2-2A>G, which together represented 71% of detected mutations. The mutation IVS2-2A>G, which has been detected only in Hispanics, was thought to generate a null allele; we identified one patient with a homozygous IVS2-2A>G mutation who showed a mild deficiency of enzyme value in erythrocytes. One patient homozygous for Duarte 2 (p.N314D, IVS5+62G>A) is probably due to a partial uniparental disomy of chromosome 9. In addition, a novel mutation c.336T>C (p.S112R) was detected in one patient with severe enzymatic deficiency. Despite the small number of patients studied, our results suggest that classical galactosaemia shows low allelic heterogeneity in Mexican patients, in contrast what is observed in other Mendelian disorders such as cystinosis or autosomal dominant hypercholesterolaemia. This low allelic heterogeneity might be explained by a "population of origin" effect in the central region of Mexico, as has been described for phenylketonuria.

[Huntington's disease mortality in Spain in the period 1981-2004]. / Mortalidad por la enfermedad de Huntington en Espana en el periodo 1981-2004.

INTRODUCTION: Huntington's disease (HD) is an autosomic dominant neurodegenerative disease characterized by neuromuscular, cognitive and psychiatric symptoms. AIM: To analyze the mortality trend for HD from 1981-2004 in Spain. PATIENTS AND METHODS: Both crude and specific rates adjusted to the European population were used to show the evolution of mortality. Rates are showed by age and gender per million of inhabitants. Joinpoint regression model was used to analyze mortality trends. RESULTS: 866 deaths under HD codes were recorded in Spain during the study period (452 males and 414 females). Adjusted rates ranged from 0.64 in 1981 to 1.65 in 2004 in males and from 0.40 in 1981 to 1.16 in 2004 in females. The trend of the mortality rates in both genders followed a slight and steady increase during the whole period and dramatic changes were not detected. The average yearly percentage of this increase was 3.76% in males and 3.67% in females. CONCLUSIONS: The study has showed a yearly age adjusted mortality rates increase close to 4%. No differences have been seen between males and females. The follow up of this trend should be monitored to test if it stabilizes or it rises.

A measles outbreak in children under 15 months of age in La Rioja, Spain, 2005-2006.

This paper describes a measles outbreak in La Rioja, Spain, which began in December 2005 and mainly affected children under 15 months of age who were not yet immunised with MMR vaccine. The measles cases were detected by the mandatory reporting system, under which laboratories must report every confirmed measles case. Cases were classified in accordance with the National Measles Elimination Plan: suspected and laboratory-confirmed. In the period 14 December 2005 to 19 February 2006, 29 suspected cases of measles were investigated, and 18 were confirmed. The mean incubation period was 13.8 days (range: 9 to 18). Of the 18 confirmed cases, only two were in adults. MMR vaccination was recommended for all household contacts, as well as for children aged 6 to 14 months who attended the daycare centres where the cases had appeared. At these centres, the second dose of MMR was administered ahead of schedule for children under three years of age. It was recommended that the first dose of MMR vaccine be administered ahead of schedule for all children aged 9 to 14 months. During an outbreak of measles, children aged 6 months or older, who have not previously been vaccinated against measles, mumps and rubella, should receive a first dose as soon as possible, and those who have had a first dose should receive a second dose as soon as possible, provided that a minimum of one month has elapsed between the two doses.

A measles outbreak in children under 15 months of age in La Rioja, Spain, 2005-2006.

This paper describes a measles outbreak in La Rioja, Spain, which began in December 2005 and mainly affected children under 15 months of age who were not yet immunised with MMR vaccine. The measles cases were detected by the mandatory reporting system, under which laboratories must report every confirmed measles case. Cases were classified in accordance with the National Measles Elimination Plan: suspected and laboratory-confirmed. In the period 14 December 2005 to 19 February 2006, 29 suspected cases of measles were investigated, and 18 were confirmed. The mean incubation period was 13.8 days (range: 9 to 18). Of the 18 confirmed cases, only two were in adults. MMR vaccination was recommended for all household contacts, as well as for children aged 6 to 14 months who attended the daycare centres where the cases had appeared. At these centres, the second dose of MMR was administered ahead of schedule for children under three years of age. It was recommended that the first dose of MMR vaccine be administered ahead of schedule for all children aged 9 to 14 months. During an outbreak of measles, children aged 6 months or older, who have not previously been vaccinated against measles, mumps and rubella, should receive a first dose as soon as possible, and those who have had a first dose should receive a second dose as soon as possible, provided that a minimum of one month has elapsed between the two doses.

Filtered back-projection reconstruction for attenuation proton CT along most likely paths.

This work investigates the attenuation of a proton beam to reconstruct the map of the linear attenuation coefficient of a material which is mainly caused by the inelastic interactions of protons with matter. Attenuation proton computed tomography (pCT) suffers from a poor spatial resolution due to multiple Coulomb scattering (MCS) of protons in matter, similarly to the conventional energy-loss pCT. We therefore adapted a recent filtered back-projection algorithm along the most likely path (MLP) of protons for energy-loss pCT (Rit et al 2013) to attenuation pCT assuming a pCT scanner that can track the position and the direction of protons before and after the scanned object. Monte Carlo simulations of pCT acquisitions of density and spatial resolution phantoms were performed to characterize the new algorithm using Geant4 (via Gate). Attenuation pCT assumes an energy-independent inelastic cross-section, and the impact of the energy dependence of the inelastic cross-section below 100 MeV showed a capping artifact when the residual energy was below 100 MeV behind the object. The statistical limitation has been determined analytically and it was found that the noise in attenuation pCT images is 411 times and 278 times higher than the noise in energy-loss pCT images for the same imaging dose at 200 MeV and 300 MeV, respectively. Comparison of the spatial resolution of attenuation pCT images with a conventional straight-line path binning showed that incorporating the MLP estimates during reconstruction improves the spatial resolution of attenuation pCT. Moreover, regardless of the significant noise in attenuation pCT images, the spatial resolution of attenuation pCT was better than that of conventional energy-loss pCT in some studied situations thanks to the interplay of MCS and attenuation known as the West-Sherwood effect.

[Prevalence of non-traumatic musculoskeletal pathology as main complaint and its impact in a emergency department]. / Prevalencia de la afección musculoesquelética no traumática como motivo de consulta y su impacto asistencial en un servicio de urgencias.

BACKGROUND: Non-traumatic musculoskeletal pathology (NTMP) generates a high healthcare demand in primary care, however, European studies designed to assess its real impact in Emergency Departments are scarce. The present study aims to determine the prevalence of NTMP and its impact in Emergency Department of a university hospital in Madrid. MATERIAL AND METHOD: Two thousand randomized medical registries were reviewed from 2008 to 2011. The epidemiological data collected were, main complaints, time consumed, image test requests, and need of further assessment within a month. RESULTS: Prevalence of NTMP was 13.8% (95% CI; 12.1%-15.4%) of all patients. The most frequent musculoskeletal complaint was lumbar pain. An imaging test was requested in 79.1% of all the NTMP cases assessed. Patients with NTMP consumed an average of 79 minutes, with 17% of them requesting a new urgent assessment within the first month. CONCLUSIONS: The results of this study show that NTMP is the leading cause for emergency department visits in our area, producing the highest consumption of time and the highest frequency of new queries for the same reason within a month. The overuse of the emergency services and the lack of medical training in the management of this type of pathology can cause this phenomenon. During the design of strategies to optimize patients care in emergency departments, the importance of this type of pathology should be taken into account.

Monte Carlo comparison of x-ray and proton CT for range calculations of proton therapy beams.

Proton computed tomography (CT) has been described as a solution for imaging the proton stopping power of patient tissues, therefore reducing the uncertainty of the conversion of x-ray CT images to relative stopping power (RSP) maps and its associated margins. This study aimed to investigate this assertion under the assumption of ideal detection systems. We have developed a Monte Carlo framework to assess proton CT performances for the main steps of a proton therapy treatment planning, i.e. proton or x-ray CT imaging, conversion to RSP maps based on the calibration of a tissue phantom, and proton dose simulations. Irradiations of a computational phantom with pencil beams were simulated on various anatomical sites and the proton range was assessed on the reference, the proton CT-based and the x-ray CT-based material maps. Errors on the tissue's RSP reconstructed from proton CT were found to be significantly smaller and less dependent on the tissue distribution. The imaging dose was also found to be much more uniform and conformal to the primary beam. The mean absolute deviation for range calculations based on x-ray CT varies from 0.18 to 2.01 mm depending on the localization, while it is smaller than 0.1 mm for proton CT. Under the assumption of a perfect detection system, proton range predictions based on proton CT are therefore both more accurate and more uniform than those based on x-ray CT.

CD40L is critical for protection from demyelinating disease and development of spontaneous remyelination in a mouse model of multiple sclerosis.

Theiler's murine encephalomyelitis virus (TMEV) induces acute neuronal disease followed by chronic demyelination in susceptible strains of mice. In this study we examined the role of a limited immune defect (deletion or blocking of CD40 ligand [CD40L]) on the extent of brain disease, susceptibility to demyelination, and the ability of demyelinated mice to spontaneously remyelinate following TMEV infection. We demonstrated that CD40L-dependent immune responses participate in pathogenesis in the cerebellum and the spinal cord white matter but protect the striatum of susceptible SJL/J mice. In mice on a background resistant to TMEV-induced demyelination (C57BL/6), the lack of CD40L resulted in increased striatal disease and meningeal inflammation. In addition, CD40L was required to maintain resistance to demyelination and clinical deficits in H-2b mice. CD40L-mediated interactions were also necessary for development of protective H-2b-restricted cytotoxic T cell responses directed against the VP2 region of TMEV as well as for spontaneous remyelination of the spinal cord white matter. The data presented here demonstrated the critical role of this molecule in both antibody- and cell-mediated protective immune responses in distinct phases of TMEV-mediated pathology.

Proliferative response of CD4+ and CD8+ T cell subsets from Hispanics with HIV+ and AIDS: the superantigen hypothesis.

It has been hypothesized that the progressive deletion of CD4+ T cells in the course of infection due to human immunodeficiency virus (HIV) may be mediated in part by interaction with a superantigen inherent in an HIV protein. Consequently, selective loss of CD4+ cells with a T cell receptor V beta-chain capable of interaction with superantigen would produce a CD4+ population less or totally unresponsive to superantigen such as staphylococcal enterotoxins B and A (SEB and SEA respectively), but not to other mitogens such as concanavalin A, anti-CD3 (OKT3), or pokeweed mitogen. We tested this hypothesis by comparing the proliferative response of SEB and SEA with the other mitogens for 25 controls, 20 HIV+, and 15 donors with acquired immune deficiency syndrome (AIDS). We found that peripheral blood mononuclear cells, as well as the CD4+ and CD8+ subsets from both HIV+ and AIDS sources, the degree of suppression of mitogenesis for SEB and SEA was approximately equal to or less than that of the other mitogens. Moreover, suppression of HIV+ CD4+ and CD8+ T cell responses to SEB and SEA was equal (26%). If HIV superantigens exist, our data suggest that they are not responsible for the selective depletion of the CD4+ T cell subset as evaluated by SEB and SEA specificity.

Failure of treatment with Linomide or oral myelin tolerization to ameliorate demyelination in a viral model of multiple sclerosis.

Both Linomide (quinoline-3-carboxamide) and tolerization with self-antigens have been demonstrated to successfully ameliorate demyelinating disease in experimental autoimmune encephalomyelitis (EAE). Based on the autoimmune hypothesis of multiple sclerosis (MS), both agents have been tested in clinical trials but have been found to be toxic or not efficacious. We investigated the efficacy of these immunomodulators in an alternative experimental model of MS, a virus-induced demyelinating disease. Oral administration of Linomide to Theiler's virus-infected mice beginning either at time of infection or at day 15 post-infection (p.i.) resulted in an increased percentage of spinal cord quadrants with demyelination. Administration of Linomide beginning at day 15 p.i. increased lesion size as compared to infected control-treated mice. Treatment with 80 mg kg(-1) day(-1) of Linomide beginning at the time of infection significantly increased the number of Theiler's murine encephalomyelitis virus (TMEV)-positive cells mm(-2) of spinal cord white matter. There were no differences in the amount of remyelination between mice treated with Linomide or water. However, chronically infected mice treated with Linomide had severely reduced spontaneous vertical activity as measured using a activity wheel. Oral tolerization of mice with mouse or bovine myelin had no effect on virus-induced demyelination or virus antigen expression. The contrasting results obtained between the TMEV model and the autoimmune model of demyelination do not support recent reports suggesting that the underlying mechanism of demyelination in the Theiler's model is autoimmune.

Comparative mitogenic response of old and young rhesus monkey T cells to lectin from Erythrina cristagalli.

The lectin (EC) from the coral tree, E. cristagalli, while less mitogenic on a molar or weight basis than PHA or ConA, strongly activates both Rhesus monkey and human T cells. The optimal mitogenic concentrations for both Rhesus and human T cells are 0.25, 2.5, and 25 micrograms/ml, respectively, for PHA, ConA, and EC. Aged Rhesus T cells were profoundly suppressed in mitogenic response to EC (approx. 80%) compared to young Rhesus cells. However, in the presence of supplemental interleukin 2 (20 U/ml), the age-related defect was reversed; the average mitogenic response of the old Rhesus T cells was increased sixfold.

Inhibition of T cell mitogenesis by nitrofurans.

A group of nitrofurans (5-nitro-2-furaldehyde, nifuroxime, nitrofurazone, nitrofurantoin, 5-nitro-2-furoic acid and 2-nitrofuran) were evaluated for inhibition of mitogenesis (DNA synthesis) in human peripheral blood T cells. T cells, either triggered by phorbol myristate acetate (PMA) or in the presence of accessory cells, were activated with a specified mitogen [phytohemagglutin (PHA), concanavalin A (ConA), or anti-CD3] and the amount of tritiated thymidine incorporated into DNA was determined. The results obtained indicate that nitrofurans inhibit mitogenesis irrespective of activator. 5-Nitro-2-furaldehyde was much more inhibitory than the other compounds, while 2-nitrofuran was less inhibitory. When the aldehyde group (5-nitro-2-furaldehyde) was replaced by a carboxyl group (5-nitro-2-furoic acid), the inhibitory activity was also reduced greatly. These results show that while the nitro group alone confers inhibitory activity to the furan ring, the group at the 2 position is crucial. In general, the mitogenic response of purified T cells (lacking accessory cells) triggered by PMA (phorbol ester) was inhibited less than that of the T cell-accessory cell system. With the latter, 50% inhibition of T cell mitogenesis was achieved by nifuroxime, nitrofurazone, and nitrofurantoin at 45-51 and 34-39 microM with PHA and ConA respectively. When purified T cells were used, the values were 71-85 and 55-60 microM respectively. For a given drug concentration, mitogenesis was more inhibited when induced by ConA or anti-CD3 than by PHA. The importance of using a single cell system (purified T cells) was emphasized by the interesting finding that only this system showed enhancement of mitogenesis, up to 35-40% at low drug levels. With the exception of the nitrofuraldehyde, the nitrofurans at strongly inhibitory levels were only moderately cytotoxic, exhibiting 62-85% cell survival after exposure to drug for 68 hr. Our results suggest that nitrofurans inhibit T cell mitogenesis by a relatively non-toxic mechanism; these results are comparable to those obtained for mammalian cells under aerobic conditions.

Chronic cavitary histoplasmosis. Failure of oral treatment with ketoconazole.

Ketoconazole appears to be a safe drug in the treatment of chronic cavitary histoplasmosis. Primary failure and relapse have been described, requiring amphotericin B, even after long therapy with ketoconazole. Four typical cases are presented. We caution about such potential failures and stress the importance of close observation of patients begun on therapy with ketoconazole for chronic cavitary histoplasmosis.