Synthesis of the Antimicrobial Peptide Murepavadin Using Novel Coupling Agents.

The problem of antimicrobial resistance is becoming a daunting challenge for human society and healthcare systems around the world. Hence, there is a constant need to develop new antibiotics to fight resistant bacteria, among other important social and economic measures. In this regard, murepavadin is a cyclic antibacterial peptide in development. The synthesis of murepavadin was undertaken in order to optimize the preparative protocol and scale-up, in particular, the use of new activation reagents. In our hands, classical approaches using carbodiimide/hydroxybenzotriazole rendered low yields. The use of novel carbodiimide and reagents based on OxymaPure® and Oxy-B is discussed together with the proper use of chromatographic conditions for the adequate characterization of peptide crudes. Higher yields and purities were obtained. Finally, the antimicrobial activity of different synthetic batches was tested in three Pseudomonas aeruginosa strains, including highly resistant ones. All murepavadin batches yielded the same highly active MIC values and proved that the chiral integrity of the molecule was preserved throughout the whole synthetic procedure.

SARS-CoV-2 uses CD4 to infect T helper lymphocytes.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent of a major global outbreak of respiratory tract disease known as Coronavirus Disease 2019 (COVID-19). SARS-CoV-2 infects mainly lungs and may cause several immune-related complications, such as lymphocytopenia and cytokine storm, which are associated with the severity of the disease and predict mortality. The mechanism by which SARS-CoV-2 infection may result in immune system dysfunction is still not fully understood. Here, we show that SARS-CoV-2 infects human CD4+ T helper cells, but not CD8+ T cells, and is present in blood and bronchoalveolar lavage T helper cells of severe COVID-19 patients. We demonstrated that SARS-CoV-2 spike glycoprotein (S) directly binds to the CD4 molecule, which in turn mediates the entry of SARS- CoV-2 in T helper cells. This leads to impaired CD4 T cell function and may cause cell death. SARS-CoV-2-infected T helper cells express higher levels of IL-10, which is associated with viral persistence and disease severity. Thus, CD4-mediated SARS-CoV-2 infection of T helper cells may contribute to a poor immune response in COVID-19 patients.

Polyethersulfone (PES) Filters Improve the Recovery of Legionella spp. and Enhance Selectivity against Interfering Microorganisms in Water Samples.

In the analysis of water samples, the type of filtration membrane material can influence the recovery of Legionella species, although this issue has been poorly investigated. Filtration membranes (0.45 µm) from different materials and manufacturers (numbered as 1, 2, 3, 4, and 5) were compared: mixed cellulose esters (MCEs), nitrocellulose (NC), and polyethersulfone (PES). After membrane filtration of samples, filters were placed directly onto GVPC agar and incubated at 36 ± 2 °C. The highest mean counts of colony-forming units and colony sizes for Legionella pneumophila and Legionella anisa were obtained with PES filters (p < 0.001). All membranes placed on GVPC agar totally inhibited Escherichia coli and Enterococcus faecalis ATCC 19443 and ATCC 29212, whereas only the PES filter from manufacturer 3 (3-PES) totally inhibited Pseudomonas aeruginosa. PES membrane performance also differed according to the manufacturer, with 3-PES providing the best productivity and selectivity. In real water samples, 3-PES also produced a higher Legionella recovery and better inhibition of interfering microorganisms. These results support the use of PES membranes in methods where the filter is placed directly on the culture media and not only in procedures where membrane filtration is followed by a washing step (according to ISO 11731:2017).

Use of Blood Powder (Ground and Irradiated) for the Manufacture of Chocolate Agar.

Chocolate agar (CA) is an enriched medium for the isolation and identification of fastidious bacteria. Defibrinated blood is used to manufacture CA, but this expensive product is not always affordable for companies in developing countries. Blood powder (BP) is potentially a cheaper alternative, although its pre-treatment using autoclaving can impair the quality of the media. Therefore, optimization of BP as a substitute for defibrinated blood for CA manufacture deserves further research. CA was manufactured with irradiated BP (dehydrated bovine blood powder) and its physical and microbiological characteristics were compared with those of conventional CA and CA prepared with autoclaved BP. Each medium was seeded with 20-200 CFU of target bacteria using the spiral pouring method. Finally, another medium was prepared using BP pre-treated by grinding and gamma irradiation and its performance assessed. Compared to conventional CA, the medium containing ground and irradiated BP provided a similar CFU count for both fastidious (Neisseria, Haemophilus, Campylobacter, and Streptococcus) and non-fastidious (Moraxella, Staphylococcus, Enterococcus, Klebsiella, and Pseudomonas) species, unlike the medium prepared with BP subjected only to irradiation, which provided a lower growth of fastidious species. Morphology and characteristics of all bacterial colonies were very similar in conventional CA and the new medium, the number of Pseudomonas CFU being higher in the latter. The medium prepared with ground plus irradiated vs. irradiated BP more closely resembled conventional CA, having a browner background. The new CA medium prepared with ground and gamma irradiation-sterilized BP has comparable productivity properties to conventional CA. Therefore, it could be a more practical and economical methodology to facilitate large-scale CA manufacture.

Obesity increases blood-brain barrier permeability and aggravates the mouse model of multiple sclerosis.

Obesity-induced insulin resistance (OIR) has been associated with an increased prevalence of neurodegenerative disorders such as multiple sclerosis. Obesity results in increased blood-brain barrier (BBB) permeability, specifically in the hypothalamic regions associated with the control of caloric intake. In obesity, the chronic state of low-grade inflammation has been implicated in several chronic autoimmune inflammatory disorders. However, the mechanisms that connect the inflammatory profile of obesity with the severity of experimental autoimmune encephalomyelitis (EAE) are poorly defined. In this study, we show that obese mice are more susceptible to EAE, presenting a worse clinical score with more severe pathological changes in the spinal cord when compared with control mice. Analysis of immune infiltrates at the peak of the disease shows that high-fat diet (HFD)- and control (chow)-fed groups do not present any difference in innate or adaptive immune cell compartments, indicating the increased severity occurs prior to disease onset. In the setting of worsening EAE in HFD-fed mice, we observed spinal cord lesions in myelinated regions and (blood brain barrier) BBB disruption. We also found higher levels of pro-inflammatory monocytes, macrophages, and IFN-γ+CD4+ T cells in the HFD-fed group compared to chow-fed animals. Altogether, our results indicate that OIR promotes BBB disruption, allowing the infiltration of monocytes/macrophages and activation of resident microglia, ultimately promoting CNS inflammation and exacerbation of EAE.

Magnitude of tuberculosis cases notified in a municipality: epidemiological profile and risk factors, a temporal description / Magnitude dos casos de tuberculose notificados em um município: perfil epidemiológico e fatores de risco, uma descrição temporal

Background: Tuberculosis remains among the most prevalent infectious diseases worldwide, Risk factors include sociodemographic factors and comorbidities such as alcohol consumption, diabetes mellitus, the use of legal and illegal drugs and HIV. This research aimed to estimate the incidence of tuberculosis cases and describe the epi-demiological profile of all tuberculosis cases reported to the National System of Notifiable Diseases in the city of Campo Grande/MS, correlating with national and international literature. Methods: This is a cross-sectional survey of secondary data that analyzed all cases reported in the city from January 2014 to December 2019. Results: There was a predominance of males (80.3%). The most affected age group corresponded to individuals of working age (20 to 59 years), with 84% of the total data. As for comorbidities, 70.5% had at least one. Smoking was the most prevalent health issue (27%), followed by the use of psychoactive substances, alcohol consumption and HIV/Aids co-infection (13.8%). Diabetes was the lowest rated disease, with 5,4%. Conclusions: The data obtained points to the need to assess the directly observed treatment (DOT) in the capital, given the alarming rate of adherence od 3.8%. About one third of tuberculosis cases were diagnosed in hospitals, supporting the hypothesis of delayed diagnosis, which leverages the need for health actions on the subject (AU)

ntrodução: A tuberculose permanece entre as doenças infecciosas mais prevalentes em todo o mundo, os fatores de risco incluem fatores sociodemográficos e comorbidades como consumo de álcool, diabetes mellitus, uso de dro-gas lícitas e ilícitas e HIV. Esta pesquisa teve como objetivo estimar a incidência de casos de tuberculose e descrever o perfil epidemiológico de todos os casos notificados ao Sistema Nacional de Agravos de Notificação do município de Campo Grande/MS, correlacionando com a literatura nacional e internacional. Métodos: Trata-se de um levanta-mento transversal de dados que analisou todos os casos notificados em um período de 6 anos. Resultados: Houve predomínio do sexo masculino (80,3%). A faixa etária mais acometida correspondeu aos indivíduos em idade ativa (20 a 59 anos), com 84% do total de dados. O tabagismo foi o problema de saúde mais prevalente (27%), seguido pelo uso de substâncias psicoativas, alcoolismo e coinfecção HIV/Aids. Conclusões: Os dados obtidos apontam para a necessidade de avaliar o tratamento diretamente observado (TDO) na capital, dada a alarmante taxa de adesão. Cerca de 33% dos casos de tuberculose foram diagnosticados em hospitais, corroborando a hipótese de diagnóstico tardio, a qual alavanca a necessidade de ações de saúde sobre a temática (AU)

GVPC Medium Manufactured without Oxygen Improves the Growth of Legionella spp. and Exhibits Enhanced Selectivity Properties.

Glycine-vancomycin-polymyxin-cycloheximide agar (GVPC) is a recommended medium for the detection of Legionella spp. in water samples. However, its quality could be improved in terms of recovery of Legionella spp. and selectivity properties. Modifications were introduced in GVPC manufacture: autoclaving conditions (115°C, 15 min) and atmosphere during component-stirring (removal of oxygen and N2 injection). The use of softer autoclaving conditions (115°C, 15 min) improved the growth of Legionella anisa by the spiral method and Legionella pneumophila after membrane filtration. The medium manufactured with O2 removal and autoclaving for 15 min at 115°C allowed a faster growth of L. pneumophila (colonies visible at day 2) and a notable increase of L. anisa growth (colonies appearing at day 3, and statistically significant numbers of CFU at day 5). After 3 to 5 days of incubation, the improved media showed higher selectivity properties, particularly for Enterococcus faecalis ATCC 29212 and Pseudomonas aeruginosa ATCC 9027. A further improvement was achieved by the addition of N2 during ingredient stirring, leading to a statistically significant faster growth of L. pneumophila at days 2 and 3 and L. anisa at day 3. Selectivity properties were also enhanced, resulting in the complete inhibition of both E. faecalis strains and Escherichia coli and complete-partial inhibition of P. aeruginosa. Oxygen removal during GVPC manufacture using a vacuum pump system promotes the growth of L. pneumophila and L. anisa, and markedly inhibits the growth of E. coli, P. aeruginosa, and E. faecalis. IMPORTANCE Currently, GVPC is a recommended medium for the detection of Legionella spp. in water samples. However, recovery of Legionella spp. and selectivity properties can be improved. GVPC medium manufactured without oxygen improved the growth of Legionella pneumophila and Legionella anisa. Oxygen removal during GVPC manufacture also improved selectivity properties. A further improvement was achieved by the addition of N2 during ingredient stirring, leading to a faster growth of L. pneumophila at days 2 and 3 and L. anisa at day 3 and enhancement of selectivity properties. The introduction of the modified GVPC medium in routine practice can allow a better detection of Legionella spp. in water samples.

Unveiling the Membrane and Cell Wall Action of Antimicrobial Cyclic Lipopeptides: Modulation of the Spectrum of Activity.

Antibiotic resistance is a major public health challenge, and Gram-negative multidrug-resistant bacteria are particularly dangerous. The threat of running out of active molecules is accelerated by the extensive use of antibiotics in the context of the COVID-19 pandemic, and new antibiotics are urgently needed. Colistin and polymyxin B are natural antibiotics considered as last resort drugs for multi-resistant infections, but their use is limited because of neuro- and nephrotoxicity. We previously reported a series of synthetic analogues inspired in natural polymyxins with a flexible scaffold that allows multiple modifications to improve activity and reduce toxicity. In this work, we focus on modifications in the hydrophobic domains, describing analogues that broaden or narrow the spectrum of activity including both Gram-positive and Gram-negative bacteria, with MICs in the low µM range and low hemolytic activity. Using biophysical methods, we explore the interaction of the new molecules with model membranes that mimic the bacterial inner and outer membranes, finding a selective effect on anionic membranes and a mechanism of action based on the alteration of membrane function. Transmission electron microscopy observation confirms that polymyxin analogues kill microbial cells primarily by damaging membrane integrity. Redistribution of the hydrophobicity within the polymyxin molecule seems a plausible approach for the design and development of safer and more selective antibiotics.

Surface-Modified Multifunctional Thymol-Loaded Biodegradable Nanoparticles for Topical Acne Treatment.

The present work is focused on the development of novel surface-functionalized poly(lactic-co-glycolic acid) nanoparticles loaded with thymol (TH-NPs) for topical administration enhancing thymol anti-inflammatory, antioxidant and wound healing activities against acne. TH-NPs were prepared by solvent evaporation method using different surface functionalization strategies and obtaining suitable physicochemical parameters and a good short-term stability at 4 °C. Moreover, TH-NPs skin penetration and antioxidant activity were assessed in ex vivo pig skin models. Skin penetration of TH-NPs followed the follicular route, independently of the surface charge and they were able to enhance antioxidant capacity. Furthermore, antimicrobial activity against Cutibacterium acnes was evaluated in vitro by the suspension test showing improved antibacterial performance. Using human keratinocyte cells (HaCat), cytotoxicity, cellular uptake, antioxidant, anti-inflammatory and wound healing activities were studied. TH-NPs were non-toxic and efficiently internalized inside the cells. In addition, TH-NPs displayed significant anti-inflammatory, antioxidant and wound healing activities, which were highly influenced by TH-NPs surface modifications. Moreover, a synergic activity between TH-NPs and their surface functionalization was demonstrated. To conclude, surface-modified TH-NPs had proven to be suitable to be used as anti-inflammatory, antioxidant and wound healing agents, constituting a promising therapy for treating acne infection and associated inflammation.

Lipopeptides production by a newly Halomonas venusta strain: Characterization and biotechnological properties.

A halotolerant marine strain PHKT of Halomonas venusta was isolated from contaminated seawater as an efficient biosurfactant producer candidate, on low-value substrate (glycerol). The produced biosurfactants (Bios-PHKT) were characterized as lipopeptides molecules, belonging to surfactin and pumilacidin families, by using Thin Layer Chromatography (TLC), Fourier Transform Infrared Spectroscopy (FT-IR) and Tandem Mass Spectrometry (MALDI-TOF/MS-MS). Bios-PHKT has a critical micelle concentration (CMC) of 125 mg/L, and showed a high steadiness against a wide spectrum of salinity (0-120 g/L NaCl), temperature (4-121 °C) and pH (2-12), supporting its powerful tensioactive properties under various environmental conditions. Likewise, the cytotoxic test revealed that the biosurfactant Bios-PHKT, at concentrations lower than 125 µg/mL, was not cytotoxic for human HEK-293 cells since the cell survival is over than 80%. Furthermore, Bios-PHKT lipopeptides showed excellent anti-adhesive and anti-biofilm activities, being able to avoid and disrupt the biofilm formation by certain pathogenic microorganisms. In addition, the biosurfactant Bios-PHKT showed a remarkable anti-proliferative activity towards tumor B16 melanoma cell line. Besides, Bios-PHKT exhibited an excellent in vitro and in vivo wound healing process. In light of these promising findings, Bios-PHKT could be successfully used in different biotechnological applications.

Rationalisation of the caesarean section rate in a tertiary referral hospital using the Robson classification.

The aim of this study was to implement the Robson Ten Groups Classification System (RTGCS) to identify the main contributors to the caesarean section (CS) rate and to evaluate whether the introduction of a plan of obstetrical interventions reduced this rate. An observational retrospective cross-sectional study was conducted during two time periods at Alicante University General Hospital. In the pre-implementation period (2009-2012), RTGCS was applied to identify the main groups contributing to the overall CS rate. In the post-implementation period (2013-2017), RTGCS was applied again to identify changing trends in CS rates. In all, 11,034 deliveries during the pre-intervention period and 11,453 during the post-intervention period were analysed. The overall CS rate was 23.9% and 20.9%, respectively. There were no changes in perinatal outcomes. In the post-intervention period, there was a significant decrease of the CS rate in the groups of targeted interventions 1, 2, 3, 4, 5, and 8B.Impact statementWhat is already known on this subject? High CS rates are becoming a public health problem because of risks, costs, excessive medicalisation, and abuse of resources. RTGCS provides a framework for auditing and analysing CS rates.What do the results of this study add? RTGCS can identify the groups that have the greatest impact on the CS rate and monitor changes in it consequent to policy changes.What are the implications of these findings for clinical practice? The introduction of a strategic plan with evidence-based clinical interventions may have a greater effect on the CS rate than other features justifying the increase in the incidence of CS.

Isolation and characterization of a newly naphthalene-degrading Halomonas pacifica, strain Cnaph3: biodegradation and biosurfactant production studies.

A newly marine Halomonas pacifica strain Cnaph3 was isolated, as a naphthalene degrader and biosurfactant producer, from contaminated seawater collected in Ataya's fishing harbor, located in Kerkennah Islands, Tunisia. Chromatography flame ionization detector analysis revealed that 98.8% of naphthalene (200 mg/L) was degraded after 7 days of incubation, at 30 g/L NaCl and 37 °C. Strain Cnaph3 showed also a noticeable capacity to grow on a wide range of aliphatic, aromatic, and complex hydrocarbons. Interestingly, strain Cnaph3 showed a significant potential to produce biosurfactants in the presence of all tested substrates, particularly on glycerol (1%, v/v). Electrospray ionization analysis of the biosurfactant, designated Bios-Cnaph3, suggested a lipopeptide composition. The critical micelle concentration of Bios-Cnaph3 was about 500 mg/L. At this concentration, the surface tension of the water was reduced to 27.6 mN/m. Furthermore, Bios-Cnaph3 displayed interesting stabilities over a wide range of temperatures (4-105 °C), salinities (0-100 g/L NaCl), and pH (2.2-12.5). In addition, it showed promising capacities to remove used motor oil from contaminated soils. The biodegradation and biosurfactant-production potential of the Halomonas sp. strain Cnaph3 would present this strain as a favorite agent for bioremediation of hydrocarbon-contaminated sites under saline conditions.

Frecuencia de episiotomías en un hospital de tercer nivel / Frequency of episiotomy in a third-level hospital

OBJETIVO: Determinar la frecuencia y correlación de las episiotomías y desgarros en pacientes con parto eutócico. MATERIALES Y MÉTODOS: Estudio observacional y retrospectivo efectuado en el Hospital General Universitario de Alicante en dos periodos: 1) 2008-2012 y 2) 2013-2018. Se incluyen los partos eutócicos de embarazos únicos, a término, en presentación cefálica. Variables de estudio: episiotomía, desgarro, edad, semanas de embarazo, paridad, inducción del parto, cesárea anterior, analgesia regional, peso y sexo del recién nacido. Los resultados se expresan en porcentaje, coeficiente de correlación y las variables implicadas con razón de momios (RM). RESULTADOS: La tasa de episiotomía entre el periodo 1 vs el 2 disminuyó de manera muy importante: de 42.3 a 32.8%; [p < 0.001; RM: 0.81 (0.78-0.84)]. En cambio, el porcentaje de desgarros aumentó: de 42.7 a 50.8%; (p < 0.001; RM: 1.16[1.13-1.20]). El coeficiente de correlación fue de -0.91. Uno de los factores de riesgo asociados con la episiotomía fue la edad menor de 35 años, con RM de 1.25; IC95%: 1.16-1.35; p < 0.001, pero fue un factor protector de desgarros con RM de 0.76; IC95%: 0.71-0.81; p < 0.001. CONCLUSIONES: La tendencia de la episiotomía es decreciente, mientras que la de desgarro es inversamente proporcional. No se encontraron diferencias en la tasa de desgarro de III y IV grado.

Abstract OBJECTIVE: To determine the frequency and correlation of episiotomies and tears of patients with eutocic deliveries. MATERIALS AND METHODS: Observational and retrospective study carried out at the Hospital General de Alicante, Spain, in two periods: 1) 2008-2012 and 2) 2013-2018. Eutocic deliveries of single pregnancies, at term, in cephalic presentation are included. Study variables: episiotomies, tear, age, weeks of pregnancy, parity, labor induction, previous caesarean section, regional analgesia, weight and sex of the newborn. The results are expressed as a percentage, correlation coefficient and the variables involved with odds ratio. RESULTS: The episiotomy rate between period 1 vs. 2 decreased very significantly: 42.3% vs 32.8%; [p <0.001; OR: 0.81 (0.78-0.84)]. In contrast, the percentage of index tears: 42.7% vs 50.8%; (p <0.001; OR: 1.16 [1.13-1.20]). The correlation coefficient was -0.91. Regarding risk factors associated with episiotomy, one of them was the age under 35 years with an OR (95% CI): 1.25 (1.16-1.35; p <0.001), but it was a OR protective factor (95% CI): 0.76 (0.71-0.81; p <0.001) for the appearance of tears. CONCLUSIONS: The tendency in the realization of episiotomies is decreasing, while the tendency of the appearance of tears is inversely proportional. No differences were found in the tear rate of III and IV grade.

Hamartoma perineal congénito con malformación genital y anorrectal, asociado a alteración genética: duplicidad del gen TGIF2LX / Congenital perineal hamartoma with genital and anorectal malformation associated with genetic alteraction: duplicity of the TGIF2LX gene

Se presenta el caso de una gestante de 29 años, en la que en la ecografía de la semana 20 se evidencia un varón con genitales externos ambiguos, sospecha de hipospadias y una masa perineal, con el resto de hallazgos normales. La amniocentesis informa de cariotipo 46 XY normal y microarray CGH con duplicidad del gen TGIF2LX, cuya expresión es testicular y su función parece relacionada con la espermatogénesis, y también duplicidad de su gen homólogo TGIF2LY, cuya significación es desconocida. El seguimiento prenatal es normal, con persistencia de la alteración en genitales externos. Al nacimiento se detecta hipospadias con meato en linea media, escroto bífido y masa de consistencia blanda de 4 cm con apéndice cutáneo, asociado a fístula rectoperineal. Las pruebas de imagen indican que se trata de una masa perineal exófitica de 2,2 x 4 cm, de apariencia grasa con nódulo sólido de 1,4 cm en su interior, que podría corresponder a tumor de estirpe grasa de aspecto benigno. Intervenido para resección de la masa a los 11 días de vida, anorrectoplastia posterior y localización de neoano mediante neuroestimulación. El diagnóstico anatomopatológico es hamartoma perineal congénito, con músculo liso, cartílago maduro y tejido adiposo, sin evidencia de malignidad. El postoperatorio precisa de dilataciones anales hasta conseguir defecación espontánea, pero en general es normoevolutivo. El hamartoma perineal congénito es un tumor raro y casi siempre aparece asociado a alguna malformación genital o anorrectal, pero no hay publicaciones que describan correlación con alteraciones genéticas, como podría ser nuestro caso

We present the case of a 29 years old patient, in which the ultrasound of week 20 shows a male with ambiguous external genitals, suspected hypospadias and a perineal mass, with the rest of normal findings. Amniocentesis reports normal 46 XY karyotype and CGH microarray with duplicity of the TGIF2LX gene, whose expression is testicular and its function seems related to spermatogenesis, and also duplicity of its homologous gene TGIF2LY, whose significance is unknown. The prenatal follow-up is normal, with persistence of the alteration in external genitals. At birth, hypospadias was detected with a midline meatus, bifid scrotum, and a soft consistency mass of 4 cm with cutaneous appendage, associated with rectoperineal fistula. The imaging tests indicate an exophytic perineal mass of 2.2 x 4 cm, with a greasy appearance and a solid nodule measuring 1.4 cm inside, which could correspond to a benign-looking fat line tumor. Intervened for resection of the mass at 11 days of life, posterior anoreplasty and neoano localization by neurostimulation. The anatomopathological diagnosis is congenital perineal hamartoma, with smooth muscle, mature cartilage and adipose tissue, without evidence of malignancy. The postoperative period requires anal dilations until spontaneous defecation, but in general it is normal. The congenital perineal hamartoma is a rare tumor and almost always appears associated with some genital or anorectal malformation, but there are not publications that describe correlation with genetic alterations, how could our case be

Estimating the age of the p.Cys433Arg variant in the MYOC gene in patients with primary open-angle glaucoma.

The aim of this study was to estimate the age of the Cys433Arg (c.1297T>C, p.Cys433Arg) variant by comparing the genotypes of individuals affected and not affected by primary open angle glaucoma juvenile onset (JOAG). Our sample consisted of 35 JOAG-affected individuals from three families, 16 unrelated patients with the MYOC p.Cys433Arg variant and 16 unaffected individuals. Genomic DNA was amplified by PCR; nine short tandem repeats were genotyped through automated electrophoresis and three single nucleotide polymorphisms through Sanger sequencing. The determination of haplotypes was performed using Arlequin software and age estimation was performed using DMLE+ 2.3 and BDMC21 softwares. Four markers constituted the haplotypes associated with the p.Cys433Arg variant. The software DMLE+2.3 predicted an age of 43 generations for this variant with a 95% confidence interval ranging from 28 to 76 generations (560-1520 years) and BDMC21 predicted an age of 59 generations (1180 years) (95% CI: 40 to 100).

Atenção em rede às pessoas com amputação: a ação da Enfermagem sob o olhar da Bioética / Atención en red para las personas con amputaciones: la acción de la Enfermería bajo la mirada de la Bioética / The healthcare network for people with amputation: Nursing action in the view of Bioethics

RESUMO Objetivo: analisar o processo de referência e contrarreferência na assistência à saúde das pessoas com amputação, na perspectiva dos enfermeiros, sob o olhar da bioética. Método: estudo descritivo, exploratório-analítico com abordagem qualitativa, realizado por entrevista semiestruturada com 21 enfermeiros que prestam assistência à pessoa com amputação alocada na rede de atenção à saúde de Florianópolis. Os dados foram organizados com auxílio do software Atlas ti 7.5.6 e analisados com base na análise de conteúdo temática. Resultados: emergiram duas categorias temáticas: O fazer do enfermeiro no processo de referência e contrarreferência e Avanços e desafios de um processo em construção. Evidenciou-se que sem o respaldo legal, a maioria dos encaminhamentos são realizados com base nas relações interpessoais e na informalidade. Logo, a responsabilização compartilhada e a troca de saberes possibilitam ações fundamentadas nas necessidades e complexidades intrínsecas na assistência à saúde dessa população. Conclusão: a análise sob o olhar da bioética permitiu concluir que, não só a pessoa com amputação como também o enfermeiro, ocupam posição de vulnerabilidade. Estes mostraram o empenho para oferecer um atendimento integral. O comprometimento ético, a criatividade profissional, o matriciamento e a interconsulta foram pontuadas como potencialidades. As fragilidades foram a inexistência de protocolos, a comunicação ineficaz entre os níveis de atenção, o acesso restrito à informação e a lentidão nos referenciamentos.

RESUMEN Objetivo: analizar el proceso de referencia y contra referencia en la asistencia para la salud de las personas con amputaciones, en la perspectiva de los enfermeros y bajo la mirada de la bioética. Método: estudio descriptivo, exploratorio-analítico y con abordaje cualitativo realizado por entrevistas semiestructuradas con 21 enfermeros que dan asistencia a personas con amputaciones asignadas por la red de atención a la salud de Florianópolis. Los datos fueron organizados con el auxilio del software Atlas ti 7.5.6 y analizados en base al análisis del contenido temático. Resultados: aparecieron dos categorías temáticas: El hacer del enfermero en el proceso de referencia y contra referencia y Los avances y desafíos de un proceso en construcción. Quedó evidente que sin el respaldo legal, la mayoría de los encaminamientos son realizados en base a las relaciones interpersonales y la informalidad. Así, la responsabilidad compartida y el intercambio de saberes posibilitan acciones fundamentadas en las necesidades y complejidades intrínsecas en la asistencia de la salud de esa población. Conclusión: el análisis bajo la mirada de la bioética permitió concluir que no solo la persona con amputación sino también el enfermero ocupan una posición de vulnerabilidad. Los mismos demostraron empeño para ofrecer un atendimiento integral. El comprometimiento ético, la creatividad profesional, el apoyo matricial y la interconsulta fueron considerados como potencialidades. Las fragilidades fueron la inexistencia de protocolos, la comunicación ineficaz entre los niveles de atención, el acceso restringido a la información y la lentitud en la referenciación.

ABSTRACT Objective: to analyze the referral and counter-referral process in the health care of people with amputations, in the perspective of nurses, in the view of bioethics. Method: a descriptive, exploratory-analytical study with a qualitative approach, performed with semi-structured interviews with 21 nurses who provide care to people with amputations in the health care network of Florianópolis (Santa Catarina/ Brazil). The data were organized using Atlas ti 7.5.6 software and analyzed based on thematic content analysis. Results: two thematic categories emerged: The nurse's role in the referral and counter-referral process and Advances and challenges of a process under construction. It was evidenced that without legal backing, most referrals are made based on interpersonal relationships and informality. Therefore, shared responsibility and the exchange of knowledge allows actions based on the intrinsic needs and complexities in the health care of this population. Conclusion: the analysis in the view of bioethics allowed to conclude that not only the person with the amputation but also the nurse occupies a vulnerable position. They have shown the commitment to offer a comprehensive service. The ethical commitment, professional creativity, specialist orientation and consultation-liaison were highlighted as potentialities. The weaknesses were the lack of protocols, ineffective communication between levels of care, restricted access to information and slow referral time.

Atuação do enfermeiro no referenciamento e contrarreferenciamento de pessoas com amputação / Nursing actions in reference and counter-reference in health care for persons with amputation* / Actuación del enfermero en el sistema de referencia y contrarreferencia de personas con amputación

O objetivo foi identificar referenciamentos e contrarreferenciamentos realizados pelos enfermeiros da Rede de Atenção à Saúde, descrevendo sua atuação na assistência à pessoa com amputação. Estudo qualitativo, com coleta de dados realizada por entrevista semiestruturada com 21 enfermeiros dos três níveis de atenção à saúde de Florianópolis. A coleta ocorreu nos meses de abril e maio de 2015. Os dados foram organizados com auxílio do software ATLAS.ti 7.5.6. e analisados com base na técnica da Análise Temática. Emergiram duas categorias: Portas de entrada da pessoa com amputação; O enfermeiro na assistência e as implicações de sua atuação no referenciamento e contrarreferenciamento. Identificou-se como portas de entrada mais procuradas Unidades Básicas de Saúde, serviço de Emergência e o Centro Catarinense de Reabilitação. O foco inicial dos enfermeiros é oferecer melhor assistência à pessoa com amputação realizando referência e contrarreferência quando necessário (AU).

The present study aimed to identify reference and counter-reference activities performed by nurses of the Health Care Network, by describing their actions in the delivery of care to persons with amputation. Qualitative study, with data collection performed by semi-structured interviews with 21 nurses of primary, secondary and tertiary levels of health care in Florianópolis. Data was collected in April and May 2015 and was organized with the aid of ATLAS.ti 7.5.6 software and analyzed with the use of Thematic Analysis technique. Two categories emerged: Entry points to the healthcare network (RAS) for persons with amputation, and Nurses role in care and the implications of the actions of these professionals in the reference and counter-reference system. Basic Health Units, Emergency services and the Rehabilitation Center of Santa Catarina (CCR) were identified as the most frequently accessed entry points. The primary focus of nursing professionals is to deliver higher quality care to persons with amputation through reference and counter-reference in health care when necessary (AU).

El objetivo del estudio fue identificar los sistemas de referencia y contrarreferencia que describen la actuación de enfermeros de la Red de Atención a la Salud en la asistencia a la persona con amputación. Es un estudio cualitativo, cuyos datos fueron obtenidos por medio de entrevista semi estructurada con 21 enfermeros de los tres niveles de atención a la salud de Florianópolis en los meses de abril y maio de 2015. Las informaciones fueron organizadas con por medio del software ATLAS.ti 7.5.6. y analizadas con base en la técnica del Análisis Temático. Resultaron de eso dos categorías: Puertas de entrada de la persona con amputación; El enfermero en la asistencia y las implicaciones de su actuación en sistema de referencia y contrarreferencia. Se identificaron como puertas de entrada más buscadas Unidades Básicas de Salud, servicio de Emergencia y el Centro Catarinense de Rehabilitación. El infoque inicial de los enfermeros es ofrecer mejor asistencia a la persona con amputación, realizando referencia y contrarreferencia cuando sea necesario (AU).

[Antituberculosis-drug resistance in the border of Brazil with Paraguay and Bolivia]. / Resistência às drogas antituberculose na fronteira do Brasil com Paraguai e Bolívia.

OBJECTIVE: To estimate the rate of drug resistance among pulmonary tuberculosis (PTB) cases in the state of Mato Grosso do Sul, Brazil, and specifically in the border areas with Paraguay and Bolivia, as well as to identify associated risk factors. METHOD: The present cross-sectional, epidemiological study focused on PTB cases recorded between January 2007 and December 2010 in the State Reportable Disease Information System with results of susceptibility tests to rifampicin, isoniazid, ethambutol, and streptomycin. Dependent variables were development of resistance to a single drug or any combination of drugs. Independent variables were being a new or treated case, living in border areas, presence/absence of diabetes, and history of alcoholism. RESULTS: There were 789 TBP cases with susceptibility testing. The following characteristics were associated with resistance: treated case (P = 0.0001), border region (P = 0.0142), alcoholism (P = 0.0451), and diabetes (P = 0.0708). The rates of combined, primary, and acquired resistance for the state were 16.3%, 10.6%, and 39.0%, vs. 22.3%, 19.2%, and 37.5% for the border region. The rates of combined, primary, and acquired multidrug resistance for the state were 1.8%, 0.6%, and 6.3%, vs. 3.1%, 1.2%, and 12.5% for the border region. CONCLUSIONS: In the border region, the state should investigate drug resistance in all patients with respiratory symptoms, determine the pattern of resistance in confirmed cases, adopt directly observed treatment for cases of PTB, and develop health actions together with neighboring countries. Across the state, the levels of acquired resistance should be monitored, with investigation of resistance in all treated cases and implementation of directly observed treatment especially among patients with diabetes or alcoholism.

Resistência às drogas antituberculose na fronteira do Brasil com Paraguai e Bolívia / Antituberculosis-drug resistance in the border of Brazil with Paraguay and Bolivia

Objetivo. Estimar as taxas de resistência às drogas entre casos de tuberculose pulmonar (TBP) para o estado de Mato Grosso do Sul, Brasil, e especificamente para a região da fronteira com Paraguai e Bolívia, além de identificar fatores de risco associados. Métodos. O presente estudo epidemiológico, transversal, enfocou os casos de TBP registrados de janeiro de 2007 a dezembro de 2010 no Sistema de Informação de Agravos de Notificação da Secretaria de Estado de Saúde com resultados do teste de suscetibilidade a rifampicina, isoniazida, etambutol e estreptomicina. Definiram-se como variáveis dependentes o desenvolvimento de resistência a uma única droga e a qualquer combinação de drogas. As variáveis independentes foram ser caso novo ou tratado, residência em região de fronteira ou outra região, presença ou ausência de diabetes e história de alcoolismo. Resultados. Foram identificados 789 casos de TBP com teste de suscetibilidade. As características associadas à resistência foram: caso tratado (P=0,0001), região de fronteira (P=0,0142), alcoolismo (P=0,0451) e diabetes (P=0,0708). As taxas de resistência combinada, primária e adquirida no estado foram de 16,3%, 10,6% e 39,0%, e na fronteira, de 22,3%, 19,2% e 37,5%. As taxas de resistência a múltiplas drogas combinada, primária e adquirida no estado foram de 1,8%, 0,6% e 6,3%, e na fronteira, de 3,1%, 1,2% e 12,5%. Conclusões. O estado deve, na região de fronteira, realizar cultura em todos os sintomáticos respiratórios, investigar o padrão de resistência nos casos confirmados, adotar o tratamento diretamente observado nos casos de TBP e desencadear ações de saúde conjuntas com os países fronteiriços. Em todo o estado, é necessário monitorar os níveis de resistência adquirida, ampliar a investigação de resistência para todos os casos tratados e adotar o tratamento diretamente observado prioritariamente entre pacientes com alcoolismo e diabetes.

Objective. To estimate the rate of drug resistance among pulmonary tuberculosis (PTB) cases in the state of Mato Grosso do Sul, Brazil, and specifically in the border areas with Paraguay and Bolivia, as well as to identify associated risk factors. Method. The present cross-sectional, epidemiological study focused on PTB cases recorded between January 2007 and December 2010 in the State Reportable Disease Information System with results of susceptibility tests to rifampicin, isoniazid, ethambutol, and streptomycin. Dependent variables were development of resistance to a single drug or any combination of drugs. Independent variables were being a new or treated case, living in border areas, presence/absence of diabetes, and history of alcoholism. Results. There were 789 TBP cases with susceptibility testing. The following characteristics were associated with resistance: treated case (P=0.0001), border region (P=0.0142), alcoholism (P=0.0451), and diabetes (P=0.0708). The rates of combined, primary, and acquired resistance for the state were 16.3%, 10.6%, and 39.0%, vs. 22.3%, 19.2%, and 37.5% for the border region. The rates of combined, primary, and acquired multidrug resistance for the state were 1.8%, 0.6%, and 6.3%, vs. 3.1%, 1.2%, and 12.5% for the border region. Conclusions. In the border region, the state should investigate drug resistance in all patients with respiratory symptoms, determine the pattern of resistance in confirmed cases, adopt directly observed treatment for cases of PTB, and develop health actions together with neighboring countries. Across the state, the levels of acquired resistance should be monitored, with investigation of resistance in all treated cases and implementation of directly observed treatment especially among patients with diabetes or alcoholism.

Resistência às drogas antituberculose na fronteira do Brasil com Paraguai e Bolívia / Antituberculosis-drug resistance in the border of Brazil with Paraguay and Bolivia

RESUMO Objetivo Estimar as taxas de resistência às drogas entre casos de tuberculose pulmonar (TBP) para o estado de Mato Grosso do Sul, Brasil, e especificamente para a região da fronteira com Paraguai e Bolívia, além de identificar fatores de risco associados. Métodos O presente estudo epidemiológico, transversal, enfocou os casos de TBP registrados de janeiro de 2007 a dezembro de 2010 no Sistema de Informação de Agravos de Notificação da Secretaria de Estado de Saúde com resultados do teste de suscetibilidade a rifampicina, isoniazida, etambutol e estreptomicina. Definiram-se como variáveis dependentes o desenvolvimento de resistência a uma única droga e a qualquer combinação de drogas. As variáveis independentes foram ser caso novo ou tratado, residência em região de fronteira ou outra região, presença ou ausência de diabetes e história de alcoolismo. Resultados Foram identificados 789 casos de TBP com teste de suscetibilidade. As características associadas à resistência foram: caso tratado (P=0,0001), região de fronteira (P=0,0142), alcoolismo (P=0,0451) e diabetes (P=0,0708). As taxas de resistência combinada, primária e adquirida no estado foram de 16,3%, 10,6% e 39,0%, e na fronteira, de 22,3%, 19,2% e 37,5%. As taxas de resistência a múltiplas drogas combinada, primária e adquirida no estado foram de 1,8%, 0,6% e 6,3%, e na fronteira, de 3,1%, 1,2% e 12,5%. Conclusões O estado deve, na região de fronteira, realizar cultura em todos os sintomáticos respiratórios, investigar o padrão de resistência nos casos confirmados, adotar o tratamento diretamente observado nos casos de TBP e desencadear ações de saúde conjuntas com os países fronteiriços. Em todo o estado, é necessário monitorar os níveis de resistência adquirida, ampliar a investigação de resistência para todos os casos tratados e adotar o tratamento diretamente observado prioritariamente entre pacientes com alcoolismo e diabetes.

ABSTRACT Objective To estimate the rate of drug resistance among pulmonary tuberculosis (PTB) cases in the state of Mato Grosso do Sul, Brazil, and specifically in the border areas with Paraguay and Bolivia, as well as to identify associated risk factors. Method The present cross-sectional, epidemiological study focused on PTB cases recorded between January 2007 and December 2010 in the State Reportable Disease Information System with results of susceptibility tests to rifampicin, isoniazid, ethambutol, and streptomycin. Dependent variables were development of resistance to a single drug or any combination of drugs. Independent variables were being a new or treated case, living in border areas, presence/absence of diabetes, and history of alcoholism. Results There were 789 TBP cases with susceptibility testing. The following characteristics were associated with resistance: treated case (P = 0.0001), border region (P = 0.0142), alcoholism (P = 0.0451), and diabetes (P = 0.0708). The rates of combined, primary, and acquired resistance for the state were 16.3%, 10.6%, and 39.0%, vs. 22.3%, 19.2%, and 37.5% for the border region. The rates of combined, primary, and acquired multidrug resistance for the state were 1.8%, 0.6%, and 6.3%, vs. 3.1%, 1.2%, and 12.5% for the border region. Conclusions In the border region, the state should investigate drug resistance in all patients with respiratory symptoms, determine the pattern of resistance in confirmed cases, adopt directly observed treatment for cases of PTB, and develop health actions together with neighboring countries. Across the state, the levels of acquired resistance should be monitored, with investigation of resistance in all treated cases and implementation of directly observed treatment especially among patients with diabetes or alcoholism.