Effects of a comprehensive brain computed tomography deep learning model on radiologist detection accuracy.

OBJECTIVES: Non-contrast computed tomography of the brain (NCCTB) is commonly used to detect intracranial pathology but is subject to interpretation errors. Machine learning can augment clinical decision-making and improve NCCTB scan interpretation. This retrospective detection accuracy study assessed the performance of radiologists assisted by a deep learning model and compared the standalone performance of the model with that of unassisted radiologists. METHODS: A deep learning model was trained on 212,484 NCCTB scans drawn from a private radiology group in Australia. Scans from inpatient, outpatient, and emergency settings were included. Scan inclusion criteria were age ≥ 18 years and series slice thickness ≤ 1.5 mm. Thirty-two radiologists reviewed 2848 scans with and without the assistance of the deep learning system and rated their confidence in the presence of each finding using a 7-point scale. Differences in AUC and Matthews correlation coefficient (MCC) were calculated using a ground-truth gold standard. RESULTS: The model demonstrated an average area under the receiver operating characteristic curve (AUC) of 0.93 across 144 NCCTB findings and significantly improved radiologist interpretation performance. Assisted and unassisted radiologists demonstrated an average AUC of 0.79 and 0.73 across 22 grouped parent findings and 0.72 and 0.68 across 189 child findings, respectively. When assisted by the model, radiologist AUC was significantly improved for 91 findings (158 findings were non-inferior), and reading time was significantly reduced. CONCLUSIONS: The assistance of a comprehensive deep learning model significantly improved radiologist detection accuracy across a wide range of clinical findings and demonstrated the potential to improve NCCTB interpretation. CLINICAL RELEVANCE STATEMENT: This study evaluated a comprehensive CT brain deep learning model, which performed strongly, improved the performance of radiologists, and reduced interpretation time. The model may reduce errors, improve efficiency, facilitate triage, and better enable the delivery of timely patient care. KEY POINTS: • This study demonstrated that the use of a comprehensive deep learning system assisted radiologists in the detection of a wide range of abnormalities on non-contrast brain computed tomography scans. • The deep learning model demonstrated an average area under the receiver operating characteristic curve of 0.93 across 144 findings and significantly improved radiologist interpretation performance. • The assistance of the comprehensive deep learning model significantly reduced the time required for radiologists to interpret computed tomography scans of the brain.

Droplet Volume Variability and Impact on Digital PCR Copy Number Concentration Measurements.

Digital polymerase chain reaction (dPCR) is increasingly being adopted by reference material producers and metrology institutes for value assignment, and for homogeneity and stability studies of nucleic acid reference materials. A reference method procedure should fulfill several requirements, and the uncertainty and biases should be completely understood. A bias in target concentration when inaccurate droplet volume is used in the droplet dPCR measurement equation has previously been documented. In this study, we characterize both intrawell and interwell droplet volume variability using optical microscopy and determine the impact of these two sources of variability on target concentration estimates. A small optical distortion across the image was measured which, without correction, biased droplet volume measurements. Longitudinal monitoring of interwell droplet volume over 39 weeks using several lots of Mastermix demonstrated a mean droplet volume of 0.786 nL and intermediate precision of 1.7%. The frequency distribution of intrawell droplet volumes varied. Some wells displayed a skewed distribution which resulted in a small bias in estimated target concentration for a simulated dPCR with target concentrations of between 62 and 8000 copies µL-1. The size and direction of this bias was influenced by the distribution pattern of the droplet volumes within the well. The proportion of Mastermix in dPCR mix affected droplet volume. A pipetting error of 10% during mixing of the premix and Mastermix resulted in a 2.6% change in droplet volume and, consequently, a bias in concentration measurements highlighting the advantages of gravimetric preparation of dPCR mixes for high accuracy measurements.

Interlaboratory Reproducibility of Droplet Digital Polymerase Chain Reaction Using a New DNA Reference Material Format.

Use of droplet digital PCR technology (ddPCR) is expanding rapidly in the diversity of applications and number of users around the world. Access to relatively simple and affordable commercial ddPCR technology has attracted wide interest in use of this technology as a molecular diagnostic tool. For ddPCR to effectively transition to a molecular diagnostic setting requires processes for method validation and verification and demonstration of reproducible instrument performance. In this study, we describe the development and characterization of a DNA reference material (NMI NA008 High GC reference material) comprising a challenging methylated GC-rich DNA template under a novel 96-well microplate format. A scalable process using high precision acoustic dispensing technology was validated to produce the DNA reference material with a certified reference value expressed in amount of DNA molecules per well. An interlaboratory study, conducted using blinded NA008 High GC reference material to assess reproducibility among seven independent laboratories demonstrated less than 4.5% reproducibility relative standard deviation. With the exclusion of one laboratory, laboratories had appropriate technical competency, fully functional instrumentation, and suitable reagents to perform accurate ddPCR based DNA quantification measurements at the time of the study. The study results confirmed that NA008 High GC reference material is fit for the purpose of being used for quality control of ddPCR systems, consumables, instrumentation, and workflow.

DNA copy number concentration measured by digital and droplet digital quantitative PCR using certified reference materials.

The value assignment for properties of six certified reference materials (ERM-AD623a-f), each containing a plasmid DNA solution ranging from 1 million to 10 copies per µL, by using digital PCR (dPCR) with the BioMark™ HD System (Fluidigm) has been verified by applying droplet digital PCR (ddPCR) using the QX100 system (Bio-Rad). One of the critical factors in the measurement of copy number concentrations by digital PCR is the partition volume. Therefore, we determined the average droplet volume by optical microscopy, revealing an average droplet volume that is 8 % smaller than the droplet volume used as the defined parameter in the QuantaSoft software version 1.3.2.0 (Bio-Rad) to calculate the copy number concentration. This observation explains why copy number concentrations estimated with ddPCR and using an average droplet volume predefined in the QuantaSoft software were systematically lower than those measured by dPCR, creating a significant bias between the values obtained by these two techniques. The difference was not significant anymore when the measured droplet volume of 0.834 nL was used to estimate copy number concentrations. A new version of QuantaSoft software (version 1.6.6.0320), which has since been released with Bio-Rad's new QX200 systems and QX100 upgrades, uses a droplet volume of 0.85 nL as a defined parameter to calculate copy number concentration.

Digital polymerase chain reaction measured pUC19 marker as calibrant for HPLC measurement of DNA quantity.

Digital polymerase chain reaction (dPCR) is potentially a primary method for quantifying target DNA regions in a background of nontarget material and is independent of external calibrators. Accurate dPCR measurements require single-molecule detection by conventional PCR assays that may be subject to bias due to inhibition, interference, or sequence-derived PCR inefficiency. Elimination or control of such biases is essential for validation of PCR assays, but this may require a substantial investment in resources. Here we present a mechanism for DNA quantification that does not require PCR assay validation in situations where target DNA quantity is high enough to be measured by physical techniques such as quantitative high-performance liquid chromatography (HPLC) or electrophoresis. A commercially available DNA marker derived from pUC19 was quantified by dPCR and was then used to calibrate an HPLC measuring system for quantifying a DNA amplicon that had a high content of guanidine and cytidine. The dPCR-calibrated HPLC measurement was verified by independent measurement using isotope dilution mass spectrometry (IDMS). HPLC quantification, calibrated with dPCR or IDMS measured DNA markers, provides an effective method for certifying the quantity of genetic reference materials that may be difficult to analyze by PCR. These secondary reference materials may then be used to validate and calibrate quantitative PCR measurements and thus could expand the breadth of applications for which traceability to the International System of Units is possible.

Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification.

Droplet digital polymerase chain reaction (ddPCR) is a new technology that was recently commercialized to enable the precise quantification of target nucleic acids in a sample. ddPCR measures absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined, water-in-oil droplet partitions. This novel ddPCR format offers a simple workflow capable of generating highly stable partitioning of DNA molecules. In this study, we assessed key performance parameters of the ddPCR system. A linear ddPCR response to DNA concentration was obtained from 0.16% through to 99.6% saturation in a 20,000 droplet assay corresponding to more than 4 orders of magnitude of target DNA copy number per ddPCR. Analysis of simplex and duplex assays targeting two distinct loci in the Lambda DNA genome using the ddPCR platform agreed, within their expanded uncertainties, with values obtained using a lower density microfluidic chamber based digital PCR (cdPCR). A relative expanded uncertainty under 5% was achieved for copy number concentration using ddPCR. This level of uncertainty is much lower than values typically observed for quantification of specific DNA target sequences using currently commercially available real-time and digital cdPCR technologies.

Between Laboratory Reproducibility of DNA Extraction from Human Blood and Fresh Frozen Tissue.

Standardization of molecular diagnostics is fundamental for effective application of genetic analyses in personalized medicine. The amount of DNA extracted from a specimen can have a significant impact on diagnostic accuracy, especially in cases where the diagnostic variant has a low concentration such as cancer. Blood and tissue samples were supplied to genetic laboratories to assess the reproducibility of extraction methodologies; DNA was extracted using participants' routine procedures and returned to the external quality assessment provider. The amount of DNA was measured by two independent analytical techniques, fluorescence intensity of intercalating dye and digital PCR; DNA quality was evaluated by DNA integrity number scores. The amount of DNA extracted varied widely between and within participants and for different blood volumes, indicating that consistent diagnostic quality is challenging even within a single test center. The median digital PCR-measured amount of DNA was on average six times higher than the intercalating dye measurements obtained in this study, indicating the possibility that the latter quantitative method may significantly underestimate the amount of DNA, thus making it not fit for purpose. Standardization of genetic diagnostic tests will require a significant improvement in the reproducibility of DNA extraction; this could be achieved if suppliers and users of DNA extraction kits validate their extraction methodology using reliable quantitative measurements or reference materials.

Fishing plastics: A high occurrence of marine litter in surf-zone trammel nets of Southern Brazil.

Plastic is one of the most commonly found residues in the marine environment, which can cause several impacts. This study evaluated the interaction of marine litter with surf-zone trammel nets in Southern Brazil. Fifty fishing operations were sampled, where 4213 items were captured: 1500 discarded fish, 1384 fragments of marine litter, and 1329 utilized fishes. Plastics were the most abundant items (n = 1363), representing 98.4% of the registered marine litter, especially plastic bags (n = 1191). 94.5% of registered marine litter is considered as single-use waste. The interaction with marine litter can cause negative impacts to small-scale fisheries (e.g. economic and damage to fishing nets). We highlight the urgency in implementing actions for urban solid waste management and public policies to reduce single-use plastics and educational campaigns on the environmental theme.

Calibre e formato de agulhas e sua relação com a incidência de cefaleia pós-punção dural: uma revisão sistemática / Needles gauge and shape and its relation to post-dural puncture headache incidence: a systematic review

Introdução: A cefaleia pós punção dural (CPPD) é uma complicação da punção lombar, um procedimento que, apesar de bem tolerado, está sujeito a adversidades, ocorrendo devido a um vazamento persistente do líquido cefalorraquidiano (LCR) do local da punção dural. A incidência de CPPD pode estar relacionada às características dos pacientes e dos procedimentos. Notou-se que em mulheres jovens até 30 anos, o risco de CPPD é maior quando comparado aos homens, não apresentando diferença a partir da quinta década de vida. Objetivo: investigar os diferentes sintomas e efeitos gerados pelos diferentes tipos de agulha, como calibre e modo de inserção, que visem reduzir a CPPD. Métodos: Trata-se de uma revisão sistemática de literatura realizada no período de 2 de agosto a 20 de novembro de 2023 por meio de pesquisas no PubMed. Foram utilizados os descritores: "Post-Dural Puncture Headache" e suas variações do MeSH, sendo submetidos aos critérios de inclusão: estudos em humanos, nos últimos 10 anos, ensaios clínicos e ensaios clínicos controlados e randomizados. Para garantir a qualidade da revisão sistemática foi aplicada a lista de verificação PRISMA de 2020. Resultados: Após investigação estatística, observou-se que as agulhas 25W e 25S demandaram maior tempo médio para a coleta de LCR (15 e 7 min, respectivamente). Ao se comparar 25W com 20Q (3 min), 22S (5 min) e 25S quanto à esta variável, observouse diferença significativa em todas as comparações. Conclusão: As agulhas do tipo atraumática foram associadas com redução do risco de desenvolvimento de CPPD quando comparadas às convencionais. Foi constatado que, dentre as agulhas convencionais, a traumática de 25G é melhor para a prevenção de CPPD que a de 22G.

Introduction: Post-Dural Puncture Headache (PDPH) is a complication of lumbar puncture, a procedure that, despite being well-tolerated, is subject to adversities, occurring due to a persistent leakage of cerebrospinal fluid (CSF) from the site of dural puncture. The incidence of PDPH may be related to patient and procedural characteristics. It has been noted that in young women up to 30 years old, the risk of CPPD is higher compared to men, with no difference between sexes from the fifth decade of life onward. Objective: To investigate the different symptoms and effects generated by different types of needles, such as gauge and insertion method, aiming to reduce CPPD. Methods: Is a systematic literature review conducted from August to October 2023 through searches on PubMed. The descriptors "Post-Dural Puncture Headache" and its MeSH variations were used. A total of 1,839 articles were found, which were then subjected to inclusion criteria: studies conducted in the last 10 years, controlled trials, and randomized clinical trials. Results: After statistical investigation, it was observed that the 25W and 25S needles required a longer average time for cerebrospinal fluid collection (15 and 7 minutes, respectively). When comparing 25W with 20Q (3 minutes), 22S (5 minutes), and 25S regarding this variable, a significant difference was observed in all comparisons. Conclusion: Atraumatic needles were associated with a reduction in the risk of developing CPPD compared to conventional needles. It was found that among conventional needles, the traumatic 25G needle is better for preventing CPPD than the 22G needle.

Basic Concepts and Validation of Digital PCR Measurements.

Use of digital polymerase chain reaction (dPCR) technology is rapidly growing and diversifying into a range of areas in life science. The release of dPCR commercial systems has facilitated access, leading to recognition of the potential advantages compared to previous quantitative PCR technologies, and the scope for novel applications. The capability of dPCR to deliver unprecedented levels of precision, accuracy, and resolution in quantification of nucleic acids has triggered a strong interest by academia and the life sciences industry in use of this technology as a molecular diagnostic tool. However, the performance of dPCR, as for a "classical" PCR assay, essentially still relies on enzyme-based amplification of nucleic acid using specific reagents and instrumentation. This chapter describes basic concepts, key properties, and important factors to consider for the verification and validation of dPCR measurements.

Changes in cellular proliferation and plasma products are associated with liver failure.

AIM: To study the differences in immune response and cytokine profile between acute liver failure and self-limited acute hepatitis. METHODS: Forty-six patients with self-limited acute hepatitis (AH), sixteen patients with acute liver failure (ALF), and twenty-two healthy subjects were involved in this study. The inflammatory and anti-inflammatory products in plasma samples were quantified using commercial enzyme-linked immunoassays and quantitative real-time PCR. The cellular immune responses were measured by proliferation assay using flow cytometry. The groups were divided into viral- and non-viral-induced self-limited AH and ALF. Thus, we worked with five groups: Hepatitis A virus (HAV)-induced self-limited acute hepatitis (HAV-AH), HAV-induced ALF (HAV-ALF), non-viral-induced self-limited acute hepatitis (non-viral AH), non-viral-induced acute liver failure (non-viral ALF), and healthy subjects (HC). Comparisons among HAV and non-viral-induced AH and ALF were performed. RESULTS: The levels of mitochondrial DNA (mtDNA) and the cytokines investigated [interleukin (IL)-6, IL-8, IL-10, interferon gamma, and tumor necrosis factor] were significantly increased in ALF patients, independently of etiology (P < 0.05). High plasma mtDNA and IL-10 were the best markers associated with ALF [mtDNA: OR = 320.5 (95%CI: 14.42-7123.33), P < 0.0001; and IL-10: OR = 18.8 (95%CI: 1.38-257.94), P = 0.028] and death [mtDNA: OR = 12.1 (95%CI: 2.57-57.07), P = 0.002; and IL-10: OR = 8.01 (95%CI: 1.26-50.97), P = 0.027]. In the cellular proliferation assay, NKbright, NKT and regulatory T cells (TReg) predominated in virus-specific stimulation in HAV-induced ALF patients with an anergic behavior in the cellular response to mitotic stimulation. Therefore, in non-viral-induced ALF, anergic behavior of activated T cells was not observed after mitotic stimulation, as expected and as described by the literature. CONCLUSION: mtDNA and IL-10 may be predictors of ALF and death. TReg cells are involved in immunological disturbance in HAV-induced ALF.

Measurement of absolute copy number variation reveals association with essential hypertension.

BACKGROUND: The role of copy number variation (CNV) has been poorly explored in essential hypertension in part due to technical difficulties in accurately assessing absolute numbers of DNA copies. Droplet digital PCR (ddPCR) provides a powerful new approach to CNV quantitation. The aim of our study was to investigate whether CNVs located in regions previously associated with blood pressure (BP) variation in genome-wide association studies (GWAS) were associated with essential hypertension by the use of ddPCR. METHODS: Using a "power of extreme" approach, we quantified nucleic acids using ddPCR in white subjects from the Victorian Family Heart Study with extremely high (n = 96) and low (n = 92) SBP, providing power equivalent to 1714 subjects selected at random. RESULTS: A deletion of the CNVs esv27061 and esv2757747 on chromosome 1p13.2 was significantly more prevalent in extreme high BP subjects after adjustment for age, body mass index and sex (12.6% vs. 2.2%; P = 0.013). CONCLUSIONS: Our data suggests that CNVs within regions identified in previous GWAS may play a role in human essential hypertension.

Método de cadastro de pacientes assistidos por uma unidade básica de saúde no Distrito Federal

INTRODUÇÃO No Brasil a Atenção Primária à Saúde (APS) está em um processo de transição. A opção pela reorganização desse modelo foi baseada na Estratégia Saúde da Família (ESF), com a implantação de equipes multiprofissionais em Unidades Básicas de Saúde (UBS), atuando com população e território definidos. Visando esse modelo, a atuação do agente comunitário de saúde (ACS) é essencial para a identificação de população assistida. Entretanto no Distrito Federal, pela carência desse profissional, há uma escassez de dados consolidados disponíveis a cada equipe para a devida programação de ações locais de saúde. OBJETIVOS 1- Avaliar método de cadastramento de pacientes acompanhados ESF. 2- Criação de banco de dados de pacientes atendidos por ESF. MÉTODO Utilizada ferramenta de computação em nuvem com serviço de resposta de questionário integrado com planilha pela plataforma do Google Apps e acesso universal tanto por computadores como smartphones. Esse serviço é disponibilizado online e gratuitamente possibilitando o preenchimento automático e ordenado de tabelas com respostas de questionário. Foi criado questionário com dados cadastrais: nome do paciente, data de nascimento, gênero, endereço, telefone, número do prontuário no trakcare/SESDF, número do cartão do SUS, se é tabagista, se é etilista, se é sabidamente hipertenso, se é sabidamente diabético e se possui diagnóstico prévio de outras doenças. Esse questionário poderia ser acessado por todos membros da ESF via email ou diretamente pelo site do Google Apps. Durante período de 01/2016 a 07/2016, todos pacientes que fossem marcar consulta na ESF n°36 de Samambaia-DF deveriam ser cadastrados pelo novo método. Os pacientes poderiam ser cadastrados em casa durante visita domiciliar por ACS, no acolhimento da UBS por técnico de enfermagem, ACS ou enfermeiro. Ou mesmo durante a consulta com próprio médico na UBS. Seriam contabilizados número de pacientes atendidos na consulta médica que não foram cadastrados previamente pelo novo método. RESULTADOS Todos pacientes atendidos pelo médico no período de avaliação foram cadastrados pelo novo método. Nas primeiras duas semanas de cadastramento, mais de dois terços dos pacientes (169 pacientes do total de 249 pacientes) foram cadastrados previamente à consulta médica na USB. Nas 2 semanas seguintes, houve aumento para 90% de pacientes previamente cadastrados. Durante os meses subsequentes, o cadastramento prévio à consulta médica foi superior a 99%. Totalizando no final de julho o cadastro de 1671 pacientes diferentes atendidos nesta UBS. Adesão ao novo método foi tão positiva que vários pacientes foram cadastros mesmo sem necessidade de agendamento de consultas e no último mês de acompanhamento taxa de novos cadastros já havia caído para 28% (113 novos cadastros em 390 atendimentos). CONCLUSÃO O novo método se mostrou prático e promissor, com adesão de toda equipe ESF n°36 de Samambaia-DF. Adotando-se a estimativa máxima de 3500 pacientes que deverão ser acompanhados por equipe, houve um cadastramento de quase 50% dos potenciais pacientes em 7 meses. Além da criação de um banco de dados atualizado que pode ser usado em diversas outras intervenções locais para saúde pública.

Avaliação do grau de implantação da estratégia de saúde da família na Regional de Saúde de Samambaia DF

Introdução A Atenção Primária à Saúde (APS) se constitui de cuidados essenciais baseados em métodos, teorias e práticas, bem aceitos socialmente, e representa a porta de entrada ao serviço de saúde. A Saúde da Família foi implantada no Brasil inicialmente na década de 90 e passou a ser visto pelo Ministério da Saúde como principal estratégia de expansão, qualificação e solidificação da atenção básica, buscando a reorientação do processo de trabalho para ter maior consolidação e qualificação do SUS. A Regional de Saúde de Samambaia é de 254.439 habitantes e 41 Equipes de Saúde da Família cadastradas no MS, Samambaia alcança 55% de cobertura por eSF (cerca de 140 mil pessoas). Será realizado uma Avaliação do grau de implantação da Estratégia Saúde da Família, intervenção de estudo, na Regional de Saúde de Samambaia, "cidade satélite" situada no Distrito Federal, estudo será realizado no segundo semestre de 2016. Algumas equipes de SF não contam com o número adequado de ACS e há 03 Unidades Básicas de Saúde que apresentam espaços insuficientes para acomodar o número de equipes existentes. Objetivos Descrever aspectos da Implantação da Estratégia Saúde da Família, relacionado a infraestrutura e recursos humanos, analisar fatores limitantes e facilitadores do contexto, que influenciam no processo de implantação, averiguar a consonância entre a organização do trabalho preconizado na Política Nacional de Atenção Básica e o realizado pela ESF, aerificar o grau de implantação da ESF de acordo com cada sub dimensão que será estudada. Metodologia Optou-se por Avaliação Normativa, com base nos critérios da conformidade ­ apreciação da estrutura e apreciação de processos. Objetiva comparar a maneira como se desenvolve a intervenção com aquilo que foi planejado, ou seja, analisar a estrutura e os processos de acordo com a norma. Será realizado um estudo de caso único, com níveis de análise imbricados. O componente 'estrutura' será abordado na sub dimensão disponibilidade. O componente 'atividade' será analisado acerca da sub dimensão adequação. Serão definidas 02 matrizes de análise e julgamento abordando cada sub dimensão do estudo, disponibilidade e adequação. Optou-se por realizar o pareamento do Modelo Lógico da intervenção (ESF). Os resultados serão obtidos através das entrevistas e check-list. Será calculado média de pontuação alcançada em cada critério, em seguida, se obterá a pontuação máxima atingida por sub dimensão. O Grau de implantação será classificado, em cada sub dimensão. Os sujeitos da pesquisa são os Enfermeiros, Médicos, Técnicos de Enfermagem, ACS, coordenadores das eSF e UBS selecionadas. Resultado Avaliação Normativa, se faz necessária para propor estratégias, para os Gestores da APS, que fortaleçam a intervenção e favoreçam a execução das ações normatizadas, identificando fatores influenciam, positivamente e/ou negativamente, o grau de implantação da intervenção, contribuindo na tomada de decisão. O Estudo ainda será concluído. Conclusão Conclui-se que estudos avaliativos auxiliam os gestores que atuam na APS, com recomendações que contribuem para a tomada de decisão após o término do processo avaliativo.