Analysis of the impact of a diabetes education program on glycemic control and prevalence of chronic complications

ABSTRACT Objective: Diabetes mellitus (DM) is a chronic disease of increasing importance in public health, associated with chronic complications including retinopathy, neuropathy, and kidney, cardiovascular and cerebrovascular disease. This study assessed the impact of strategic DM education actions on glycemic control and prevalence of chronic complications in patients with DM. Subjects and methods: Retrospective, quantitative, cohort study at a diabetes patients association comprised of a multidisciplinary team. In all, 533 individuals with DM were included. Sociodemographic and clinical data were collected using questionnaire and medical records. Of these, 333 patients evaluated for 12 to 24 months, with type 2 DM (T2DM, n = 317) and other types of DM (n = 16), were selected to collect data on retinopathy and diabetes kidney disease (DKD). Results: There was a predominance of elderly individuals, low education level, women, high rate of overweight and obesity, physical inactivity, dietary errors, dyslipidemia, and T2DM. More patients with T2DM versus type 1 DM had optimal glycemic control (46.3% vs. 12.2%, respectively; p < 0.001). The impact of the educational processes was demonstrated by the analysis of the initial and final glycated hemoglobin (HBA1c) levels. There was an increased prevalence of individuals with well-controlled DM during follow-up (prevalence ratio [PR] 2.76, 95%, p = 0.001), along with a significant reduction in retinopathy (PR: 0.679, p = 0.001) and albuminuria (PR: 0.637, 95%, p = 0.002) when these variables were evaluated in well-controlled versus uncontrolled HbA1c groups. Conclusions: A multidisciplinary approach with integration and quality was associated with improvements in DM control and reduced occurrence of chronic DM complications.

Analysis of the impact of a diabetes education program on glycemic control and prevalence of chronic complications.

Objective: Diabetes mellitus (DM) is a chronic disease of increasing importance in public health, associated with chronic complications including retinopathy, neuropathy, and kidney, cardiovascular and cerebrovascular disease. This study assessed the impact of strategic DM education actions on glycemic control and prevalence of chronic complications in patients with DM. Subjects and methods: Retrospective, quantitative, cohort study at a diabetes patients association comprised of a multidisciplinary team. In all, 533 individuals with DM were included. Sociodemographic and clinical data were collected using questionnaire and medical records. Of these, 333 patients evaluated for 12 to 24 months, with type 2 DM (T2DM, n = 317) and other types of DM (n = 16), were selected to collect data on retinopathy and diabetes kidney disease (DKD). Results: There was a predominance of elderly individuals, low education level, women, high rate of overweight and obesity, physical inactivity, dietary errors, dyslipidemia, and T2DM. More patients with T2DM versus type 1 DM had optimal glycemic control (46.3% vs. 12.2%, respectively; p < 0.001). The impact of the educational processes was demonstrated by the analysis of the initial and final glycated hemoglobin (HBA1c) levels. There was an increased prevalence of individuals with well-controlled DM during follow-up (prevalence ratio [PR] 2.76, 95%, p = 0.001), along with a significant reduction in retinopathy (PR: 0.679, p = 0.001) and albuminuria (PR: 0.637, 95%, p = 0.002) when these variables were evaluated in well-controlled versus uncontrolled HbA1c groups. Conclusion: A multidisciplinary approach with integration and quality was associated with improvements in DM control and reduced occurrence of chronic DM complications.

Microsatellite instability detection in breast cancer using drop-off droplet digital PCR.

The use of conventional methods (immunohistochemistry, pentaplex PCR) for detecting microsatellite instability (MSI), a predictive biomarker of immunotherapy efficacy, is debated for cancers with low MSI prevalence, such as breast cancer (BC). We developed two multiplex drop-off droplet digital PCR (ddPCR) assays targeting four microsatellites, initially identified from public BC whole-genome sequencing dataset. Performances of the assays were investigated and 352 tumor DNA and 28 circulating cell-free DNA from BC patients, with unknown MSI status were blindly screened. Cross-validation of ddPCR MSI status with other MSI detection methods was performed. We then monitored circulating tumor DNA (ctDNA) dynamics before and during pembrolizumab immunotherapy in one patient with MSI-high (MSI-H) metastatic BC. The assays showed high analytical specificity and sensitivity (limit of detection = 0.16%). Among N = 380 samples, seven (1.8%) were found as MSI-H by ddPCR with six of them confirmed by next-generation sequencing (NGS). Specificity was 100% in N = 133 microsatellite stable BC submitted to NGS. In the patient with MSI-H metastatic BC, ctDNA monitoring revealed an early decrease of microsatellite mutant allelic frequencies during immunotherapy. These results demonstrated MSI detection by ddPCR, a non-invasive, fast and cost-effective approach, allowing for large pre-screening of BC patients who may benefit from immunotherapy.

A fast and low-cost approach for the discrimination of commercial aged cachaças using synchronous fluorescence spectroscopy and multivariate classification.

BACKGROUND: Cachaça is the distilled beverage typical of Brazil and can be subjected to the aging process in wooden barrels. In addition to oak barrels, cachaça is also aged in barrels of different Brazilian native woods, resulting in a wide variety of its sensory characteristics. In this work, 172 cachaças aged in bálsamo, jequitibá, oak, and umburana barrels were analyzed by synchronous fluorescence spectroscopy and by the classification methods of principal component analysis and partial least squares discriminant analysis. Spectra were preprocessed by the first derivative by Savitzky-Golay smoothing, using a filter width and polynomial order determined through face-centered central composite designs. Multivariate analysis was realized using the spectra recorded at different wavelength differences, and models were compared by the classification errors in the test sets. RESULTS: The principal component analysis applied to the synchronous fluorescence spectra presented a tendency of separation by the wood used in the aging process, and the partial least squares discriminant analysis model constructed using the fluorescence spectra recorded at a wavelength difference of 30 nm provided better performance parameters (efficiency 91-97%, sensitivity 81-100%, and specificity 91-100%). CONCLUSION: Synchronous fluorescence spectroscopy offers a promising approach for the classification of cachaças aged in bálsamo, oak, jequitibá, and umburana barrels, and the discriminant model can be used for routine analysis as a screening method. © 2022 Society of Chemical Industry.

Synchronous fluorescence spectroscopy and multivariate classification for the discrimination of cachaças and rums.

Although cachaça and rum are distilled beverages obtained from the same raw material, they present differences in their chemical compositions. In this study, synchronous fluorescence spectroscopy was used combined with supervised classification models based on the partial least squares discriminant analysis to develop a rapid and low-cost model for discriminating between 50 cachaça and 40 rum samples. Partial least squares discriminant analysis models were constructed using synchronous fluorescence spectra recorded at wavelength differences of 10-100 nm. Initially, spectra were preprocessed by the first derivative with the Savitzky-Golay smoothing, and filter width and polynomial order were selected through face-centered central composite designs. For the construction and validation models, the spectra data were split into two datasets: the training and the test sets containing 60 (C, n = 33; R, n = 27) and 30 (C, n = 17; R, n = 13) samples, respectively. The best discrimination was achieved using fluorescence spectra recorded at wavelength difference 10 nm, allowing the discrimination of cachaça and rum with a classification efficiency of 98%. These results indicate that synchronous fluorescence spectroscopy offers a promising approach for the authentication of cachaças and rums.

The importance of active surveillance of carbapenem-resistant Enterobacterales (CRE) in colonization rates in critically ill patients.

OBJECTIVE: This study aimed to demonstrate the importance of active carbapenem-resistant Enterobacterales (CRE) surveillance and evaluate the prevalence of invasive infections, risk factors, and mortality risk in CRE-colonized patients. METHODS: Retrospective cohort study analyzing 1,920 patients identified using an active CRE surveillance protocol, admitted to an adult intensive care unit in southeastern Brazil from January 2014 to December 2018. RESULTS: There were 297 (15.47%) CRE colonized patients, with one colonized for every six control patients. CRE-colonized patients demonstrated an increased chance of infection (odds ratio [OR] 7.967, p < 0.001). Overall, 20.54% of the colonized patients presented invasive infection (81.96% due to Klebsiella pneumoniae). The colonization and infection ratio demonstrated the important role of the active CRE surveillance protocol. There were identified multiple risk factors for CRE colonization, including long-term mechanical ventilation (OR 1.624, p = 0.019) and previous exposure to aminopenicillins (OR 5.204, p < 0.001), carbapenems (OR 3.703, p = 0.017), cephalosporins (OR 12.036, p < 0.001), and fluoroquinolones (OR 5.238, p = 0.012). The mortality risk was significantly higher among colonized (OR 2.356, p < 0.001) and colonized-infected (OR 2.000, p = 0.009) patients and in those with Enterobacter cloacae colonization (OR 5.173, p < 0,001) and previous aminopenicillins exposure (OR 3.452, p = 0.007). CONCLUSIONS: Early detection of CRE colonization through screening testing proved to be an important tool to control CRE spread. However, observation over the years has shown no effective control of colonization and infection. The prevalence rates of CRE colonization and colonization-infection were high, as were the mortality rates. In conclusion, an active CRE surveillance protocol is essential, but its impact depends on the effective implementation of preventive measures and feedback between team members.

Frequency distribution of HCV resistance-associated variants in infected patients treated with direct-acting antivirals.

BACKGROUND: Hepatitis C virus (HCV) is a global public health problem. Second-generation direct-acting antivirals targeting non-structural regions on the viral genome are the cornerstone for treatment of chronic infection. However, resistance-associated variants (RAVs) have been reported to be associated with therapeutic failure. The aim of this study was to assess the frequency of variants, including RAVs, in the NS3, NS5A and NS5B regions at baseline in Brazilian patients with chronic hepatitis C with HCV genotypes 1a, 1b and 3a. METHODS: Serum samples from 13 patients were used to obtain viral RNA. Massively parallel sequencing was performed using genotype-specific amplicons and a panel of Ampliseq technology for all genotypes. RESULTS: Several non-synonymous substitutions were detected at baseline for 11 responders and pre-/post-treatment for two non-responders. HCV genotype 3a was found to have significantly more non-synonymous substitutions than HCV genotype 1 in the NS3 and NS5A regions. Analyses were conducted using quantitative and qualitative inter- and intrapatient comparisons. Variants that confer resistance to the treatment used by the patients were found in both responders and non-responders. CONCLUSIONS: A wide frequency distribution of RAVs was found at baseline, and this did not interfere with the achievement of a sustained response. Evaluation of the presence of RAVs requires additional study in order to determine clinical relevance.

Polymorphism PLIN1 11482 G>A interacts with dietary intake to modulate anthropometric measures and lipid profile in adults with normal-weight obesity syndrome.

Evidence shows that genetic polymorphisms in perilipin 1 gene (PLIN1) are associated with excessive accumulation of body fat and disturbances in cardiometabolic markers. Therefore, the aim of this study was to verify whether the SNP PLIN1 11482 G>A (rs894160) interacts with nutrient intake, anthropometric, body composition and cardiometabolic markers in adults with normal-weight obesity (NWO) syndrome. A cross-sectional study was carried out with 116 individuals aged 20-59 years, with normal BMI and high percentage of body fat. Anthropometric and body composition measures, glycaemic control and serum lipid markers, SNP PLIN1 11482 G>A and nutrient intake were evaluated. Interactions between nutrient intake and the SNP were determined by regression models and adjusted for potential confounders. The SNP frequency was 56·0 % GG, 38·8 % GA and 5·2 % AA. Anthropometric measures and biochemical markers were not different according to genotype, except for total cholesterol (TC), LDL-cholesterol and non-HDL-cholesterol concentrations. However, important interactions between the SNP and dietary intake were observed. Carbohydrate intake interacted with the SNP PLIN1 11482 G>A to modulate waist circumference (WC) and the homeostatic model assessment of insulin resistance index. Interaction of lipid intake and the SNP modulated TC and LDL-cholesterol concentrations, and the interaction between protein intake and the SNP tended to modulate weight, WC and BMI. The SNP PLIN1 11482 G>A seems to modulate responses in anthropometric and lipid profile biomarkers of subjects with NWO depending on the dietary macronutrient composition, which may have long-term impact on cardiometabolic markers.

Avaliação do aporte proteico e de indicadores antropométricos no desfecho clínico de pacientes críticos / Evaluation of protein support and anthropometric indicators in the clinical outcome of critical patients

A terapia nutricional é uma das intervenções terapêuticas mais utilizadas em cuidados intensivos, quando aplicada de forma correta auxilia na diminuição de complicações metabólicas, no equilíbrio imunológico, além de amenizar a perda de massa corporal do paciente gravemente enfermo. Objetivo: avaliar a associação dos indicadores antropométricos e o consumo proteico com o desfecho clínico de pacientes críticos em uso de terapia nutricional enteral (TNE) exclusiva. Materiais e Métodos: Trata-se de um estudo prospectivo de caráter observacional analítico, com a utilização de dados de pacientes adultos e idosos em uso exclusivo de TNE desenvolvido em uma Unidade de Terapia Intensiva (UTI). Realizado por meio da coleta de dados secundários registrados nos prontuários de pacientes ≥ 18 anos, que receberam terapia nutricional enteral exclusiva por pelo menos 72 horas. Utilizou-se os indicadores antropométricos índice de massa corporal (IMC) e circunferência do braço. Para a estimativa das necessidades proteicas, considerou-se a recomendação da American Society for Parenteral and Enteral Nutrition. A adequação nutricional foi realizada por meio da coleta diária do volume da formula enteral prescrita e administrada. Nas análises utilizaram-se testes paramétricos e não paramétricos e foi considerado significante p < 0,05. Resultado: Participaram do estudo 52 pacientes que estiveram em uso de nutrição enteral exclusiva internados na UTI no hospital por pelo menos de 72 horas durante a etapa de coleta de dados. Desses pacientes a maioria era do sexo masculino (76,9%), com idade média de 45,7 anos (DP=15,0, mínimo 20 e máximo 71). O tempo de internação médio foi de 12 dias. Os diagnósticos de AIDS (síndrome da imunodeficiência adquirida) (46,1%) e COVID-19 (13,4%) foram os mais prevalentes. Foram identificados que quase 60% da amostra não tinham comorbidades e o desfecho clínico mais frequente foi a alta hospitalar. Conclusão: Neste estudo não foi verificado associações entre os desfechos clínicos avaliados (alta ou óbito) com o aporte proteico ofertado e os indicadores antropométricos, assim também como não tiveram entre as variáveis demográficas e clínicas investigadas

Nutritional therapy is one of the most used therapeutic interventions in intensive care, when correctly applied, it helps to reduce metabolic complications, immune balance, in addition to mitigating the loss of body mass in seriously ill patients. Objective: to evaluate the association of anthropometric indicators and protein consumption with the clinical outcome of critically ill patients using exclusive enteral nutritional therapy (EN). Materials and Methods: This is a prospective, observational, analytical study, using data from adult and elderly patients exclusively using EN developed in an Intensive Care Unit (ICU). Performed by collecting secondary data recorded in the medical records of patients ≥ 18 years old, who received exclusive enteral nutritional therapy for at least 72 hours. Anthropometric indicators body mass index (BMI) and arm circumference were used. For the estimation of protein requirements, the recommendation of the American Society for Parenteral and Enteral Nutrition was considered. Nutritional adequacy was performed through the daily collection of the volume of the prescribed and administered enteral formula. Parametric and non-parametric tests were used in the analyses, and p < 0.05 was considered significant. Result: The study included 52 patients who had been using exclusive enteral nutrition and were hospitalized in the ICU for at least 72 hours during the data collection stage. Of these patients, the majority were male (76.9%), with a mean age of 45.7 years (SD=15.0, minimum 20 and maximum 71). The mean hospital stay was 12 days. The diagnoses of AIDS (acquired immunodeficiency syndrome) (46.1%) and COVID-19 (13.4%) were the most prevalent. It was identified that almost 60% of the sample had no comorbidities and the most frequent clinical outcome was hospital discharge. Conclusion: In this study, there were no associations between the evaluated clinical outcomes (discharge or death) with the protein intake offered and the anthropometric indicators, as well as they did not have between the demographic and clinical variables investigated

Multimodal liquid biopsy for early monitoring and outcome prediction of chemotherapy in metastatic breast cancer.

Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) are two cancer-derived blood biomarkers that inform on patient prognosis and treatment efficacy in breast cancer. We prospectively evaluated the clinical validity of quantifying both CTCs (CellSearch) and ctDNA (targeted next-generation sequencing). Their combined value as prognostic and early monitoring markers was assessed in 198 HER2-negative metastatic breast cancer patients. All patients were included in the prospective multicenter UCBG study COMET (NCT01745757) and treated by first-line chemotherapy with weekly paclitaxel and bevacizumab. Blood samples were obtained at baseline and before the second cycle of chemotherapy. At baseline, CTCs and ctDNA were respectively detected in 72 and 74% of patients and were moderately correlated (Kendall's τ = 0.3). Only 26 (13%) patients had neither detectable ctDNA nor CTCs. Variants were most frequently observed in TP53 and PIK3CA genes. KMT2C/MLL3 variants detected in ctDNA were significantly associated with a lower CTC count, while the opposite trend was seen with GATA3 alterations. Both CTC and ctDNA levels at baseline and after four weeks of treatment were correlated with survival. For progression-free and overall survival, the best multivariate prognostic model included tumor subtype (triple negative vs other), grade (grade 3 vs other), ctDNA variant allele frequency (VAF) at baseline (per 10% increase), and CTC count at four weeks (≥5CTC/7.5 mL). Overall, this study demonstrates that CTCs and ctDNA have nonoverlapping detection profiles and complementary prognostic values in metastatic breast cancer patients. A comprehensive liquid-biopsy approach may involve simultaneous detection of ctDNA and CTCs.

Detection of Transposable Element Insertions in Arabidopsis Using Sequence Capture.

Transposable elements (TEs) are repetitive DNA sequences that have the ability to mobilize in the genome and create major effect mutations. Despite the importance of transposition as a source of genetic novelty, we still know little about the rate, landscape, and consequences of TE mobilization. This situation stems in large part from the repetitive nature of TEs, which complicates their analysis. Moreover, TE mobilization is typically rare and therefore new TE (i.e., non-reference) insertions tend to be missed in small-scale population studies. This chapter describes a TE-sequence capture approach designed to identify transposition events for most of the TE families that are potentially active in Arabidopsis thaliana. We show that our TE-sequence capture design provides an efficient means to detect with high sensitivity and specificity insertions that are present at a frequency as low as 1/1000 within a DNA sample.

Circulating tumor DNA as a dynamic biomarker of response to palbociclib and fulvestrant in metastatic breast cancer patients.

BACKGROUND: Following the PALOMA-3 study results, the combination of palbociclib, a CDK4/6 inhibitor, with fulvestrant, a selective estrogen receptor degrader, has become a standard therapy in women with estrogen receptor-positive (ER+) HER2-negative (HER2-) metastatic breast cancer (MBC). Palbociclib has been shown to increase the progression-free survival (PFS) overall but no predictive biomarker of palbociclib efficacy has been validated so far. We thus evaluated whether early changes of circulating tumor DNA (ctDNA) levels are associated with palbociclib plus fulvestrant efficiency. METHODS: ER+ HER2- MBC patients were included in a prospective observational cohort before treatment initiation. Tumor response was assessed by radiological evaluation (RECIST v1.1) every 3 months. Plasma samples were collected before treatment (baseline), at day 15 (D15), at day 30 (D30), and at disease progression. We searched for somatic mutations from archived tumor tissues by targeted deep sequencing. For patients with somatic mutations identified, circulating tumor DNA (ctDNA) was tracked using digital droplet PCR. Ratios of ctDNA levels ([D15/baseline] and [D30/baseline]) were then correlated with prospectively registered patient characteristics and outcomes. RESULTS: Twenty-five of the 61 patients enrolled had a somatic mutation testable in plasma (NPIK3CA = 21, NTP53 = 2, NAKT1 = 2). At baseline, 84% of patients had detectable ctDNA levels but ctDNA levels had no prognostic impact on PFS (p = 0.10). Among those patients, ctDNA was still detected in 82% at D15 and 68% at D30. ctDNA clearance observed at day 30 was associated with longer PFS (HR = 7.2, 95% CI = 1.5-32.6, p = 0.004). On the contrary, a [D30/baseline] ctDNA ratio > 1 was associated with a shorter PFS (HR = 5.1, 95% CI = 1.4-18.3, p = 0.02) and all 5 patients with increased ctDNA levels at D30 showed disease progression after 3 months under palbociclib-fulvestrant. Finally, at the time of radiological tumor progression, ctDNA was detected in all patients tested. CONCLUSION: Our study demonstrates that the efficiency of palbociclib and fulvestrant can be monitored by serial analyses of ctDNA before radiological evaluation and that early ctDNA variation is a prognostic factor of PFS.

Mild drought in the vegetative stage induces phenotypic, gene expression, and DNA methylation plasticity in Arabidopsis but no transgenerational effects.

There is renewed interest in whether environmentally induced changes in phenotypes can be heritable. In plants, heritable trait variation can occur without DNA sequence mutations through epigenetic mechanisms involving DNA methylation. However, it remains unknown whether this alternative system of inheritance responds to environmental changes and if it can provide a rapid way for plants to generate adaptive heritable phenotypic variation. To assess potential transgenerational effects induced by the environment, we subjected four natural accessions of Arabidopsis thaliana together with the reference accession Col-0 to mild drought in a multi-generational experiment. As expected, plastic responses to drought were observed in each accession, as well as a number of intergenerational effects of the parental environments. However, after an intervening generation without stress, except for a very few trait-based parental effects, descendants of stressed and non-stressed plants were phenotypically indistinguishable irrespective of whether they were grown in control conditions or under water deficit. In addition, genome-wide analysis of DNA methylation and gene expression in Col-0 demonstrated that, while mild drought induced changes in the DNA methylome of exposed plants, these variants were not inherited. We conclude that mild drought stress does not induce transgenerational epigenetic effects.

High-Accuracy Determination of Microsatellite Instability Compatible with Liquid Biopsies.

BACKGROUND: Microsatellite instability (MSI) has recently emerged as a predictive pan-tumor biomarker of immunotherapy efficacy, stimulating the development of diagnostic tools compatible with large-scale screening of patients. In this context, noninvasive detection of MSI from circulating tumor DNA stands as a promising diagnostic and posttreatment monitoring tool. METHODS: We developed drop-off droplet-digital PCR (ddPCR) assays targeting BAT-26, activin A receptor type 2A (ACVR2A), and defensin beta 105A/B (DEFB105A/B) microsatellite markers. Performances of the assays were measured on reconstitution experiments of various mutant allelic fractions, on 185 tumor samples with known MSI status, and on 72 blood samples collected from 42 patients with advanced colorectal or endometrial cancers before and/or during therapy. RESULTS: The 3 ddPCR assays reached analytical sensitivity <0.1% variant allelic frequency and could reliably detect and quantify MSI in both tumor and body fluid samples. High concordance between MSI status determination by the three-marker ddPCR test and the reference pentaplex method were observed (100% for colorectal tumors and 93% for other tumor types). Moreover, the 3 assays showed correlations with r ≥ 0.99 with other circulating tumor DNA markers and their dynamic during treatment correlated well with clinical response. CONCLUSIONS: This innovative approach for MSI detection provides a noninvasive, cost-effective, and fast diagnostic tool, well suited for large-scale screening of patients that may benefit from immunotherapy agents, as well as for monitoring treatment responses.

Transposition favors the generation of large effect mutations that may facilitate rapid adaption.

Transposable elements (TEs) are mobile parasitic sequences that have been repeatedly coopted during evolution to generate new functions and rewire gene regulatory networks. Yet, the contribution of active TEs to the creation of heritable mutations remains unknown. Using TE accumulation lines in Arabidopsis thaliana we show that once initiated, transposition produces an exponential spread of TE copies, which rapidly leads to high mutation rates. Most insertions occur near or within genes and targets differ between TE families. Furthermore, we uncover an essential role of the histone variant H2A.Z in the preferential integration of Ty1/copia retrotransposons within environmentally responsive genes and away from essential genes. We also show that epigenetic silencing of new Ty1/copia copies can affect their impact on major fitness-related traits, including flowering time. Our findings demonstrate that TEs are potent episodic (epi)mutagens that, thanks to marked chromatin tropisms, limit the mutation load and increase the potential for rapid adaptation.

Comparison of rRNA-based reverse transcription PCR and rDNA-based PCR for the detection of streptococci in root canal infections.

OBJECTIVE: The rDNA-based method is unable to distinguish between alive and dead cells. Alternatively, bacterial viability can be assessed by molecular methods based on ribosomal RNA (rRNA). Therefore, this study aimed to detect viable streptococci in root canal samples using rRNA-based reverse transcription polymerase chain reaction (RT-PCR), compared to an rDNA-based PCR assay. METHODOLOGY: Microbiological root canal samples were obtained from 32 teeth with primary endodontic infections before (S1) and after chemomechanical preparation (S2), and after removal of intracanal medication (S3). RNA and DNA were extracted, and complementary DNA (cDNA) was synthesized from RNA using RT reaction. cDNA and genomic DNA were subjected to PCR with primers complementary to the 16S rRNA sequences of Streptococcus spp. McNemar's test was used to compare the detection rate of both assays (P<0.05). RESULTS: Streptococci were detected in 28.12% (9/32) and 37.5% (12/32) of S1 samples using rRNA- and rDNA-based PCR assays, respectively. In contrast, they were detected in only 6.25% (2/32) of S2 samples using rRNA-based RT-PCR, compared to 15.62% (5/32) using rDNA-based PCR. Finally, in S3 samples, streptococci were not detected by rRNA, whereas rDNA-based PCR still detected the bacteria in 12.5% (4/32) of the samples. The total number of PCR-positive reactions in the rDNA-based PCR was higher than in the rRNA-based assay (P<0.05). CONCLUSIONS: The rRNA-based RT-PCR showed a lower detection rate of streptococci when compared to the rDNA-based PCR, suggesting that the latter may have detected dead cells of streptococci in root canal samples.

Patient safety culture in the intensive care unit: cross-study.

INTRODUCTION: Patient safety culture has been the reason for great concern for the scientific community due to the high number of failures resulting from the provision of health care. The objective of this study was to evaluate the perception regarding the patient safety culture and their differences between categories, in the professional teams of the adult intensive care unit (ICU). METHODOLOGY: This is a cross-sectional descriptive study, with a quantitative approach, to evaluate the patient safety culture developed in the unit adult ICU of a public university hospital. RESULTS: In this survey, 138 employees of the ICU participated, among them: physicians, psychologists, nutritionists, physiotherapists, nurses, nursing technicians, and secretaries. There was a predominance of nursing technicians (76.8%) and work experience time from 5 to ≥ 21 years (62.3%). The overall mean of the safety culture in the ICU was 57.80, and the domains with the best average were stress perception (73.84) and satisfaction at work (72.38) and with the worst mean was the perception of hospital management (42.69). The perception of safety attitudes in the professional category of physicians presented a general average of 61.63, being strengthened to job satisfaction (77,89) and with a higher perception in relation to nurses. CONCLUSIONS: The overall ICU average for the patient safety culture was less than 75, which demonstrates a team with weakened safety attitude and, in addition, low perceptions of safety attitudes based on the results of management domains, working conditions and communication failures.

Comparison of rRNA-based reverse transcription PCR and rDNA-based PCR for the detection of streptococci in root canal infections

Abstract Objective The rDNA-based method is unable to distinguish between alive and dead cells. Alternatively, bacterial viability can be assessed by molecular methods based on ribosomal RNA (rRNA). Therefore, this study aimed to detect viable streptococci in root canal samples using rRNA-based reverse transcription polymerase chain reaction (RT-PCR), compared to an rDNA-based PCR assay. Methodology Microbiological root canal samples were obtained from 32 teeth with primary endodontic infections before (S1) and after chemomechanical preparation (S2), and after removal of intracanal medication (S3). RNA and DNA were extracted, and complementary DNA (cDNA) was synthesized from RNA using RT reaction. cDNA and genomic DNA were subjected to PCR with primers complementary to the 16S rRNA sequences of Streptococcus spp. McNemar's test was used to compare the detection rate of both assays (P<0.05). Results Streptococci were detected in 28.12% (9/32) and 37.5% (12/32) of S1 samples using rRNA- and rDNA-based PCR assays, respectively. In contrast, they were detected in only 6.25% (2/32) of S2 samples using rRNA-based RT-PCR, compared to 15.62% (5/32) using rDNA-based PCR. Finally, in S3 samples, streptococci were not detected by rRNA, whereas rDNA-based PCR still detected the bacteria in 12.5% (4/32) of the samples. The total number of PCR-positive reactions in the rDNA-based PCR was higher than in the rRNA-based assay (P<0.05). Conclusions The rRNA-based RT-PCR showed a lower detection rate of streptococci when compared to the rDNA-based PCR, suggesting that the latter may have detected dead cells of streptococci in root canal samples.

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions.

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation into thousands of nano-sized water-in-oil individual reactions. Recently, ddPCR has become one of the most accurate and sensitive tools for circulating tumor DNA (ctDNA) detection. One of the major limitations of the standard ddPCR technique is the restricted number of mutations that can be screened per reaction, as specific hydrolysis probes recognizing each possible allelic version are required. An alternative methodology, the drop-off ddPCR, increases throughput, since it requires only a single pair of probes to detect and quantify potentially all genetic alterations in the targeted region. Drop-off ddPCR displays comparable sensitivity to conventional ddPCR assays with the advantage of detecting a greater number of mutations in a single reaction. It is cost-effective, conserves precious sample material, and can also be used as a discovery tool when mutations are not known a priori.

Multiresidue Determination of the Anabolic-Agent Residues Steroids, Stilbenes, and Resorcylic Acid Lactones in Bovine Urine by GC-MS/MS with Microwave-Assisted Derivatization.

In this work, a GC-MS/MS method was developed for the determination of anabolic-agent residues in bovine urine. The optimized sample preparation was as follows: enzymatic hydrolysis by ß-glucuronidase-sulfatase enzyme from Helix pomatia for 16 h at 37.5 °C, liquid-liquid extraction with diethyl ether, solid-phase extraction with HLB and aminopropylsilane cartridges, and microwave-assisted derivatization using 25 µL of MSTFA/NH4I/ethanethiol and full microwave power for 2 min. The method was validated according to Decision 657/2002/EC, Codex Alimentarius, and Manual da Garantia da Qualidade Analítica guidelines. The acceptability criteria for quantitative analysis were met for α-ethinylestradiol, α-nandrolone, ß-estradiol, ß-zearalanol, ß-zearalenol, drostanolone, ethisterone, dienestrol, diethylstilbestrol, hexestrol, megestrol, methyltestosterone, and zearalenone. The analytes α-zearalenol, α-zearalanol, and norethandrolone were validated for qualitative analysis.