Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Proteômica de Paracoccidioides brasiliensis : uma análise quantitativa das fases miceliana e leveduriforme e da transição dimórfica

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Programa de Pós-Graduação em Biologia Molecular, 2011.Dimorfismo é uma característica importante para sobrevivência fúngica em diferentes ambientes e tem sido relacionado com a virulência. O fungo ascomiceto, Paracoccidioides brasiliensis, agente causador da paracoccidioidomicose, pode crescer nas fases de micélio ou de levedura. A patogenicidade do P. brasiliensis é frequentemente associada com a transição dimórfica, de saprófita a patogênico, mas os mecanismos que regulam esse processo permanecem obscuros. Uma das maneiras de estudar esse fenômeno seria isolar e caracterizar proteínas que são especificamente expressas em um dos estágios do ciclo de vida e/ou durante a diferenciação. Eletroforese bidimensional (2-DE) foi utilizada para comparar o proteoma de micélio e de levedura do isolado Pb01 e após 22 h de transição de micélio para levedura. Os géis corados pela prata de três replicatas biológicas independentes foram digitalizados e as imagens foram analisadas utilizando-se o software Image Master 2D Platinum 6.0 software (GE Healthcare). A detecção dos spots e o pareamento foram realizados. A intensidade dos spots foi normalizada e as análises estatísticas avaliada por one-way ANOVA. Os spots de interesse obtidos dos géis 2-D foram retirados, digeridos com tripsina e os peptídeos foram então analisados por MS e/ou MS/MS. Um total de 100 proteínas/isoformas foi identificado; 81 foram diferencialmente expressas nas três fases do fungo, enquanto que 19 proteínas/isoformas foram constitutivamente expressas. A expressão de proteínas como superóxido dismutase e peroxiredoxina mitocondrial foi mais abundante na fase miceliana. Nos estágios iniciais da transição (22 h) algumas enzimas envolvidas na glicólise, como enolase e fosfoglicomutase, estão aumentadas. Proteínas de choque térmico e ATP sintase também estão significantemente aumentadas durante o evento de transição. Proteínas preferencialmente expressas na fase leveduriforme foram identificadas. Muitas das enzimas da via glicolítica e algumas enzimas do ciclo do glioxalato e metabolismo de lipídeos foram mais abundantes em levedura. Para validar os resultados dos géis 2-D foi realizado western blotting de seis proteínas diferentes, e os resultados foram confirmados. Os resultados foram validados por RT-PCR em tempo real nas três condições estudadas, incluindo conídio e transição de conídio para levedura. Os resultados demonstram uma mudança no metabolismo durante a transição de micélio para levedura; o mesmo padrão foi evidenciado na transição de conídio para levedura. ______________________________________________________________________________ ABSTRACTFungal dimorphism is important for survival in different environments and has been related to virulence. The ascomycete Paracoccidioides brasiliensis, the causative agent of paracoccidioidomycosis, can grow as mycelia or yeast. The pathogenicity of P. brasiliensis is frequently associated with the dimorphic shift, from a saprobe to a pathogenic lifestyle, but the mechanisms that regulate the process is still poorly understood. One way to study this phenomenon is to isolate and characterize proteins that are specifically expressed in one of the stages of the life cycle and/or during differentiation. Two-dimensional gel electrophoresis we used to investigate the proteins expressed differentially during transition from mycelia to yeast forms of isolate Pb01 after 22 h of temperature shift. Silver-stained gels of three independent biological replicates were digitalized and the images were analyzed using the ImageMaster 2D Platinum 6.0 software (GE Healthcare). Spot detection and matching was performed. Spot intensities were normalized and the statistics analyses were estimated by one-way ANOVA. The spots of interest were excised, in-gel digested with trypsin, and the peptides were then analyzed by MS and/or MS/MS. A total of 100 proteins/isoforms were identified; 81 were differentially expressed in the three phases of the fungus, whereas 19 proteins/isoforms were constitutively expressed. Enzymes such as superoxide dismutase and mitochondrial peroxiredoxin have been detected as abundant in the mycelium phase. After the early stage of transition (22 h) some enzymes involved in glycolysis, such as enolase and phosphoglutomutase, were increased. Heat shock proteins and ATP synthase were also significantly increased in the transition event. Proteins preferentially expressed in the yeast phase were identified. Most of the enzymes of glycolysis, and some of the glyoxylate cycle and lipid metabolism were more abundant in yeast cells. To validate the 2-D gels results we performed western blotting of six different proteins, and the results were confirmed. The results were also validated by real-time RT-PCR in the three studied conditions, including conidia and conidia-yeast transition. The results demonstrated a shift in the metabolism during transition from mycelia to yeast cell; the same patterns were evidenced in the conidia to yeast transition

Identificação de um novo Antígeno de Paracoccidioides brasiliensis (Lumazina Sintase) através da Técnica de IVIAT

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Medicina, 2006.Paracoccidioides brasiliensis é um fungo termodimórfico agente causador da paracoccidioidomicosse (PCM), uma micose que afeta 10 milhões de indivíduos na América Latina. A infecção é adquirida pela inalação de propágulos aéreos produzidos pela forma miceliana do fungo, os quais se convertem à morfologia leveduriforme na temperatura do hospedeiro. P brasiliensis expressa in-vivo importantes genes que podem contribuir para a patogênese do fungo. Nós utilizamos a tecnologia do antígeno induzido in-vivo (IVIAT) para identificar novos genes de P. brasiliensis que podem ser expressos durante o processo de infecção. A técnica IVIAT é uma modificação do rastreamento imunológico que evita o uso de modelo animal e permite a identificação de antígenos expressos em vários estágios da infecção. Nós utilizamos a estratégia de IVIAT para identificar genes possivelmente induzidos in-vivo. Usando esta técnica nós selecionamos proteínas imunogênicas que poderiam ser expressas especificamente durante a infecção e não durante o crescimento sob condições padrões do laboratório. O soro de onze pacientes com PCM obtidos em Goiânia, foram combinados e, em seguida, adsorvidos com extratos de células totais e lisados de células leveduriformes cultivadas in-vitro. Esses soros foram utilizados para rastrear proteínas de uma biblioteca de cDNA da fase leveduriforme de P. brasiliensis construída no vetor ZAPII. Clones foram obtidos e caracterizados. O cDNA que codifica para uma proteína de 174 resíduos de aminoácidos foi caracterizado como lumazina sintase (PbLS) de P.brasiliensis (GenBank: DQ081183). Análises comparativas entre a PbLS e outros organismos foram feitas. Essa proteína catalisa o penúltimo passo na síntese de riboflavina em plantas, fungos e bactérias. Para produzir anticorpos contra a PbLS recombinante, uma construção no pGEX-4T-3-LS foi realizada e introduzida dentro das células de Escherichia coli e a expressão e purificação da proteína recombinante foi obtida. A análise da reatividade imunológica da proteína recombinante mostrou que esta é reconhecida por soros de pacientes com PCM e não é reativa com soros de indivíduos controle. Para analisarmos a expressão do gene Pbls nas duas formas de P.brasiliensis utilizamos a técnica RT-PCR semiquantitativo. Os transcritos para LS foram preferencialmente expressos na forma leveduriforme do fungo. Pneumócitos humanos infectados com P.brasiliensis foram utilizados para investigar a expressão dos transcritos num modelo de infecção. O gene Pbls foi detectado em células de pneumócitos humanos infectados com células leveduriformes. A LS representa um alvo atrativo para o desenvolvimento de drogas contra patógenos, visto que, bactérias, fungos e plantas são dependentes da síntese endógena da vitamina B2. Estudos mostram que a LS recombinante são fortemente imunogênicos e elucidam resposta immune celular e humoral, conferindo proteção em camundongos. Esses dados são muito interessantes e despertam o interesse em se estudar essa proteína do fungo P. brasiliensis. _________________________________________________________________________________ ABSTRACTParacoccidioides brasiliensis is a thermally dimorphic fungus causing paracoccidioidomycosis (PCM), a mycosis that affects 10 million individuals in Latin America. The infection is acquired by inhaling airborne propagules produced by the fungal mycelium which transforms into the pathogenic yeast form, when at the body temperature. P brasiliensis expresses invivo many important virulence genes that may contribute to the overall fungus pathogenesis. We utilized in vivo-induced antigen technology (IVIAT) to identify new P. brasiliensis antigens that could be expressed during the infection process. IVIAT is a modified immunoscreening that circumvents the need for animal models and permits identification of antigens expressed at various stages of infection. We used the IVIAT strategy to identify P. brasiliensis genes putatively induced in vivo. Using this technique we selected immunogenic proteins which should be expressed specifically during human infection and not during growth under standard laboratory conditions. Sera from eleven patients with PCM infection obtained in Goiânia were pooled and after that were adsorbed with whole cells and lysates of the in vitro cultured yeast phase. These sera were probed to induced proteins from a cDNA expression library of the yeast phase of P. brasiliensis constructed in ZAPII. Clones were obtained and characterized. Of special note is a cDNA (Pbls) encoding a 174 amino acid residues protein characterized as lumazine synthase (PbLS) homologue of P.brasiliensis (GenBank: DQ081183).This protein catalyzes the penultimate step in the synthesis of riboflavin in plants, fungi, and microrganisms. In order to produce antibodies against the recombinant PbLS the expression construct pGEX-4T-3-LS was introduced into Escherichia coli cells and the expression and purification of the recombinant protein was obtained. The analysis of the immunological reactivity of the recombinant protein showed that this is recognized by sera of patients with PCM and is not reactive with sera of control individuals. To analyze the expression of the Pbls gene in the two forms of P.brasiliensis we use semiquantitative RT-PCR. The transcripts for LS had been preferentially expressed in the yeast form of fungus. Human pneumocytes infected with P.brasiliensis had been used to investigate the expression of transcripts in an infection model. The Pbls gene was detected in yeast cells infecting human pneumocytes. The lumazine synthase represents an attractive targets for the development of drugs against pathogens, since bacteria, fungus and plants are dependent of the endogenous synthesis of the B2 vitamin. Studies shown that recombinant lumazine synthase is strongly immunogenic and elicits both humoral and cellular immune responses confering protection in mice. Those data make very interesting the study of this protein in P. brasiliensis

Identification and characterization of antigenic proteins potentially expressed during the infectious process of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis causes paracoccidioidomycosis (PCM), a systemic mycosis presenting clinical manifestations ranging from mild to severe forms. A P. brasiliensis cDNA expression library was produced and screened with pooled sera from PCM patients adsorbed against antigens derived from in vitro-grown P. brasiliensis yeast cells. Sequencing DNA inserts from clones reactive with PCM patients sera indicated 35 open reading frames presenting homology to genes involved in metabolic pathways, transport, among other predicted functions. The complete cDNAs encoding aromatic-L-amino-acid decarboxylase (Pbddc), lumazine synthase (Pbls) and a homologue of the high affinity copper transporter (Pbctr3) were obtained. Recombinant proteins PbDDC and PbLS were obtained; a peptide was synthesized for PbCTR3. The proteins and the synthetic peptide were recognized by sera of patients with confirmed PCM and not by sera of healthy patients. Using the in vivo-induced antigen technology (IVIAT), we identified immunogenic proteins expressed at high levels during infection. Quantitative real time RTPCR demonstrated high transcript levels of Pbddc, Pbls and Pbctr3 in yeast cells infecting macrophages. Transcripts in yeast cells derived from spleen and liver of infected mice were also measured by qRT-PCR. Our results suggest a putative role for the immunogenic proteins in the infectious process of P. brasiliensis. (C) 2009 Elsevier Masson SAS. All rights reserved.Financiadora de Estudos e Projetos (FINEP)FINEPConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)CNPqSECTEC-GO, FAPEGSECTEC-GO, FAPE

Genomic Epidemiology of SARS-CoV-2 in Tocantins State and the Diffusion of P.1.7 and AY.99.2 Lineages in Brazil

Tocantins is a state in the cross-section between the Central-West, North and Northeast regions of Brazilian territory; it is a gathering point for travelers and transportation from the whole country. In this study, 9493 genome sequences, including 241 local SARS-CoV-2 samples (collected from 21 December 2020, to 16 December 2021, and sequenced in the MinION platform) were analyzed with the following aims: (i) identify the relative prevalence of SARS-CoV-2 lineages in the state of Tocantins; (ii) analyze them phylogenetically against global SARS-CoV-2 sequences; and (iii) hypothesize the viral dispersal routes of the two most abundant lineages found in our study using phylogenetic and phylogeographic approaches. The performed analysis demonstrated that the majority of the strains sequenced during the period belong to the Gamma P.1.7 (32.4%) lineage, followed by Delta AY.99.2 (27.8%), with the first detection of VOC Omicron. As expected, there was mainly a dispersion of P.1.7 from the state of S&atilde;o Paulo to Tocantins, with evidence of secondary spreads from Tocantins to Goi&aacute;s, Mato Grosso, Amap&aacute;, and Par&aacute;. Rio de Janeiro was found to be the source of AY.99.2 and from then, multiple cluster transmission was observed across Brazilian states, especially S&atilde;o Paulo, Paraiba, Federal District, and Tocantins. These data show the importance of trade routes as pathways for the transportation of the virus from Southeast to Northern Brazil

Genomic Epidemiology of SARS-CoV-2 in Tocantins State and the Diffusion of P.1.7 and AY.99.2 Lineages in Brazil

Tocantins is a state in the cross-section between the Central-West, North and Northeast regions of Brazilian territory; it is a gathering point for travelers and transportation from the whole country. In this study, 9493 genome sequences, including 241 local SARS-CoV-2 samples (collected from 21 December 2020, to 16 December 2021, and sequenced in the MinION platform) were analyzed with the following aims: (i) identify the relative prevalence of SARS-CoV-2 lineages in the state of Tocantins; (ii) analyze them phylogenetically against global SARS-CoV-2 sequences; and (iii) hypothesize the viral dispersal routes of the two most abundant lineages found in our study using phylogenetic and phylogeographic approaches. The performed analysis demonstrated that the majority of the strains sequenced during the period belong to the Gamma P.1.7 (32.4%) lineage, followed by Delta AY.99.2 (27.8%), with the first detection of VOC Omicron. As expected, there was mainly a dispersion of P.1.7 from the state of São Paulo to Tocantins, with evidence of secondary spreads from Tocantins to Goiás, Mato Grosso, Amapá, and Pará. Rio de Janeiro was found to be the source of AY.99.2 and from then, multiple cluster transmission was observed across Brazilian states, especially São Paulo, Paraiba, Federal District, and Tocantins. These data show the importance of trade routes as pathways for the transportation of the virus from Southeast to Northern Brazil

NEOTROPICAL CARNIVORES: a data set on carnivore distribution in the Neotropics

Mammalian carnivores are considered a key group in maintaining ecological health and can indicate potential ecological integrity in landscapes where they occur. Carnivores also hold high conservation value and their habitat requirements can guide management and conservation plans. The order Carnivora has 84 species from 8 families in the Neotropical region: Canidae; Felidae; Mephitidae; Mustelidae; Otariidae; Phocidae; Procyonidae; and Ursidae. Herein, we include published and unpublished data on native terrestrial Neotropical carnivores (Canidae; Felidae; Mephitidae; Mustelidae; Procyonidae; and Ursidae). NEOTROPICAL CARNIVORES is a publicly available data set that includes 99,605 data entries from 35,511 unique georeferenced coordinates. Detection/non-detection and quantitative data were obtained from 1818 to 2018 by researchers, governmental agencies, non-governmental organizations, and private consultants. Data were collected using several methods including camera trapping, museum collections, roadkill, line transect, and opportunistic records. Literature (peer-reviewed and grey literature) from Portuguese, Spanish and English were incorporated in this compilation. Most of the data set consists of detection data entries (n = 79,343; 79.7%) but also includes non-detection data (n = 20,262; 20.3%). Of those, 43.3% also include count data (n = 43,151). The information available in NEOTROPICAL CARNIVORES will contribute to macroecological, ecological, and conservation questions in multiple spatio-temporal perspectives. As carnivores play key roles in trophic interactions, a better understanding of their distribution and habitat requirements are essential to establish conservation management plans and safeguard the future ecological health of Neotropical ecosystems. Our data paper, combined with other large-scale data sets, has great potential to clarify species distribution and related ecological processes within the Neotropics. There are no copyright restrictions and no restriction for using data from this data paper, as long as the data paper is cited as the source of the information used. We also request that users inform us of how they intend to use the data