Dynamized Aloysia Polystachya (Griseb.) Essential Oil: A Promising Antimicrobial Product.

BACKGROUND: Compounds from vegetal matter have therapeutic potential to control highly prevalent microorganisms that are resistant to commonly used antimicrobial drugs. Dynamization of compounds can either maintain or improve their therapeutic effects, and make their use safer, especially those compounds whose therapeutic dose is close to the toxic limit. Aloysia polystachya (Griseb.) stands out among aromatic plants with antimicrobial potential. OBJECTIVE: The aim of this study was to evaluate the antimicrobial activity of dynamized and crude forms of A. polystachya essential oil against Candida albicans, Escherichia coli and Staphylococcus aureus. METHODS: Essential oil was extracted from A. polystachya dry leaves, solubilized, and dynamized at 1 cH potency as recommended by the Brazilian Homeopathic Pharmacopoeia. Antimicrobial activity against C. albicans, E. coli and S. aureus of the samples was assayed using the plate microdilution method. RESULTS: Dynamized A. polystachya essential oil at the concentration of 1 µg/mL inhibited the growth of all the microbial species analyzed. The minimum inhibitory concentration of dynamized essential oil was smaller than crude essential oil for S. aureus, E. coli and C. albicans. CONCLUSION: It is reported for the first time that A. polystachya dynamized essential oil can effectively suppress microbial growth, and it is a promising adjuvant to treat infections with pathogenic S. aureus, E. coli and C. albicans.

Evaluation of the in vitrofungicidal activity of the dynamized essential oil of Aloysia polystachyabefore and after freezing

The method of preserving substances of natural origin should not only maintain the microbiological safety of the product but also the integrity of its therapeutic potential. Essential oils obtained from plants are complex mixtures of substances and it issuggested to keep them under refrigeration for better conservation. On the other hand, homeopathic mother tincture prepared from plant is kept at room temperature. Aim: This work aimed to evaluate if the freezing process changes the in vitro antifungal activity potential of the homeopathic preparation Aloysia polystachya1CH against Candida albicans. Methodology:The inoculum of C. albicansATCC 10231 was cultivated in culture médium Sabouroud (Himedia®), standardized on a spectrometer and distributed in a 96-well plate. Then, A. polystachya1CH was added to the wells, prepared accordingtothe Brazilian Homeopathic Pharmacopoeia (FHB, 3rd edition) from A. polystachya essencial oil. An aliquot of this homeopathic preparation was frozen and after 40 days it was submitted to the same methodology for evaluation of the antifungal activity. After incubation, the plates were read with triphenyltetrazolic (TTC) (Vetec®). Results and discussion: The results of the in vitroevaluation showed that the freezing process retained the antifungal activity of the dynamized essential oil of A. polystachya1CH against C. albicans. Conclusion: Under the conditions evaluated in this study, the freezing method presented as a viable method of conservation of dynamized plant material.

Therapeutic Antibodies Against Shiga Toxins: Trends and Perspectives.

Shiga toxins (Stx) are AB5-type toxins, composed of five B subunits which bind to Gb3 host cell receptors and an active A subunit, whose action on the ribosome leads to protein synthesis suppression. The two Stx types (Stx1 and Stx2) and their subtypes can be produced by Shiga toxin-producing Escherichia coli strains and some Shigella spp. These bacteria colonize the colon and induce diarrhea that may progress to hemorrhagic colitis and in the most severe cases, to hemolytic uremic syndrome, which could lead to death. Since the use of antibiotics in these infections is a topic of great controversy, the treatment remains supportive and there are no specific therapies to ameliorate the course. Therefore, there is an open window for Stx neutralization employing antibodies, which are versatile molecules. Indeed, polyclonal, monoclonal, and recombinant antibodies have been raised and tested in vitro and in vivo assays, showing differences in their neutralizing ability against deleterious effects of Stx. These molecules are in different phases of development for which we decide to present herein an updated report of these antibody molecules, their source, advantages, and disadvantages of the promising ones, as well as the challenges faced until reaching their applicability.

Modelos in vitro e in vivo e anticorpos recombinantes para o estudo da toxina termolábil produzida por Escherichia coli enterotoxigênica

Enterotoxigenic Escherichia coli (ETEC) is one of the main bacterial pathotypes in- volved in diarrhea, mainly affecting children under 5 years old and travelers to areas where this pathogen is endemic. Heat-labile type I toxin (LT-I), one of the main virulence factors of this pathotype, is associated with diarrhea in humans and is described as a potent mucosal and systemic immune adjuvant. Early diagnosis, therapeutic approaches, and in vitro and in vivo models are the main concerns in diarrhea caused by ETEC. Different in vitro and in vivo models have already been described, however, its use does not always allow screening of biomolecules and more systemic studies with the LT toxin. In addition, the generation of specific and high-affinity recombinant antibodies for diagnosis and therapy are of great importance. Thus, the present work aimed to establish strategies for the generation of recombinant antibodies such as scFv and Fab and evaluate and validate Caco-2 cells and zebrafish as models for studies with LT-I. Caco-2 cells were seeded in the 62nd passage and at different cells density per microplate well. The internalization of LT-I conjugated to FITC was visualized in Caco-2 cells at a density of 3x104 cells per well, both associated with the cell membrane and with the nucleus, thus demonstrating its retrograde transport and validating the use of this model. In a zebrafish, the fish embryo test revealed the sensitivity of embryos to different concentrations of LT-I, as well as evident malformation phenotypes, mainly in the pericardial and yolk region. Systemically, migration of the FITC labeled toxin was observed in the cardiac region, yolk, and intestine, suggesting the observed phenotypes of cardiac edema (100%), absence of swim bladder (100%), yolk edema (80%), in addition to growth delay in larvae, were caused by the toxin. None of these phenotypes were observed in the control group of animals. There was also a decrease in heart rate during the larvae' survival kinetics, showing the cardiotoxic effect of LT-I. In ELISA assays the scFv-LT obtained in the present study was reactive against the purified antigen as well to LT-I producing strains supernatant, but not to LT-I-non-producing strains. For the Fab-LT, a synthetic library was constructed, but no LT-I-reactive clones were generated. Therefore, herein we established in vitro and in vivo models that allowed us to validate and demonstrate unknown characteristics of the LT-I concerning its relationship with the host. Moreover, the generation of scFv-LT will allows us to employ recombinant antibodies for ETEC diagnosis avoiding animals immunization.

Escherichia coli enterotoxigênica (ETEC), é um dos principais patotipos bacterianos causadores de diarreia, afetando principalmente crianças menores de cinco anos e viajantes em áreas onde esse patógeno é endêmico. A toxina termolábil do tipo I (LT-I) é um dos principais fatores de virulência deste patotipo e está associada à diarreia em humanos; é também descrita como potente adjuvante de mucosa e sistêmico. O diagnóstico precoce, a abordagem terapêutica e os modelos experimentais são importantes aspectos que merecem atenção em relação à diarreia causada por ETEC. Diferentes modelos in vitro e in vivo já foram descritos, no entanto, seu uso nem sem- pre permite a triagem de biomoléculas e estudos mais sistêmicos com a toxina LT. Além disso, a geração de anticorpos específicos e de alta afinidade para uso no diagnóstico e terapia são de grande importância. Assim, o presente trabalho propôs estabelecer estratégias para geração de fragmentos de anticorpos do tipo scFv e Fab; bem como avaliar e validar as células Caco-2 e o zebrafish como modelos para estudos da LT-I. As células Caco-2 foram utilizadas na 62a passagem e em diferentes densidades de célula por poço da microplaca de cultivo. A internalização da LT-I conjugada ao FITC foi visualizada com 3x104 células Caco-2, tanto associada à membrana celular, quanto ao núcleo, evidenciando seu transporte retrógrado e validando o uso deste modelo. Em zebrafish, pelo teste de sensibilidade em embriões, foi observada sensibilidade dos embriões frente a diferentes concentrações de LT-I e fenótipos de malformações, principalmente na região pericárdica e no vitelo. Por via sistêmica, a migração da LT-I foi observada na região cardíaca, vitelo e intestino, sugerindo que os fenótipos observados de edema cardíaco (100%), ausência de bexiga natatória (100%), edema de vitelo (80%), além de retardo no crescimento nas larvas, tenham sido ocasionados pela toxina. Nenhum desses efeitos foi encontrado no grupo controle de animais. Houve também diminuição dos batimentos cardíacos durante a cinética de sobrevivência, mostrando o efeito cardiotóxico da LT-I. Em testes por técnica de ELISA o scFv-LT obtido no presente estudo mostrou-se reativo contra a toxina LT purificada, reconheceu as cepas produtoras de LT-I e não reconheceu cepas não produtoras da toxina. Para o Fab-LT foi estabelecida a biblioteca sintética, mas não foram gerados clones reativos contra a toxina LT-I. Assim, podemos afirmar que os modelos in vitro e in vivo utilizados permitiram-nos validar e demonstrar características até então não exploradas da LT-I na sua relação com o hospedeiro. A geração do scFv-LT nos permitirá utilizar anticorpos recombinantes no diagnóstico de ETEC sem depender da imunização de animais.

Therapeutic antibodies against shiga toxins: trends and perspectives

Shiga toxins (Stx) are AB5-type toxins, composed of five B subunits which bind to Gb3 host cell receptors and an active A subunit, whose action on the ribosome leads to protein synthesis suppression. The two Stx types (Stx1 and Stx2) and their subtypes can be produced by Shiga toxin-producing Escherichia coli strains and some Shigella spp. These bacteria colonize the colon and induce diarrhea that may progress to hemorrhagic colitis and in the most severe cases, to hemolytic uremic syndrome, which could lead to death. Since the use of antibiotics in these infections is a topic of great controversy, the treatment remains supportive and there are no specific therapies to ameliorate the course. Therefore, there is an open window for Stx neutralization employing antibodies, which are versatile molecules. Indeed, polyclonal, monoclonal, and recombinant antibodies have been raised and tested in vitro and in vivo assays, showing differences in their neutralizing ability against deleterious effects of Stx. These molecules are in different phases of development for which we decide to present herein an updated report of these antibody molecules, their source, advantages, and disadvantages of the promising ones, as well as the challenges faced until reaching their applicability.

Heat-Labile Toxin from Enterotoxigenic Escherichia coli Causes Systemic Impairment in Zebrafish Model.

Heat-labile toxin I (LT-I), produced by strains of enterotoxigenic Escherichia coli (ETEC), causes profuse watery diarrhea in humans. Different in vitro and in vivo models have already elucidated the mechanism of action of this toxin; however, their use does not always allow for more specific studies on how the LT-I toxin acts in systemic tracts and intestinal cell lines. In the present work, zebrafish (Danio rerio) and human intestinal cells (Caco-2) were used as models to study the toxin LT-I. Caco-2 cells were used, in the 62nd passage, at different cell concentrations. LT-I was conjugated to FITC to visualize its transport in cells, as well as microinjected into the caudal vein of zebrafish larvae, in order to investigate its effects on survival, systemic traffic, and morphological formation. The internalization of LT-I was visualized in 3 × 104 Caco-2 cells, being associated with the cell membrane and nucleus. The systemic traffic of LT-I in zebrafish larvae showed its presence in the cardiac cavity, yolk, and regions of the intestine, as demonstrated by cardiac edema (100%), the absence of a swimming bladder (100%), and yolk edema (80%), in addition to growth limitation in the larvae, compared to the control group. There was a reduction in heart rate during the assessment of larval survival kinetics, demonstrating the cardiotoxic effect of LT-I. Thus, in this study, we provide essential new depictions of the features of LT-I.

Heat-Labile toxin from enterotoxigenic Escherichia coli causes systemic impairment in Zebrafish model

Heat-labile toxin I (LT-I), produced by strains of enterotoxigenic Escherichia coli (ETEC), causes profuse watery diarrhea in humans. Different in vitro and in vivo models have already elucidated the mechanism of action of this toxin; however, their use does not always allow for more specific studies on how the LT-I toxin acts in systemic tracts and intestinal cell lines. In the present work, zebrafish (Danio rerio) and human intestinal cells (Caco-2) were used as models to study the toxin LT-I. Caco-2 cells were used, in the 62nd passage, at different cell concentrations. LT-I was conjugated to FITC to visualize its transport in cells, as well as microinjected into the caudal vein of zebrafish larvae, in order to investigate its effects on survival, systemic traffic, and morphological formation. The internalization of LT-I was visualized in 3 × 104 Caco-2 cells, being associated with the cell membrane and nucleus. The systemic traffic of LT-I in zebrafish larvae showed its presence in the cardiac cavity, yolk, and regions of the intestine, as demonstrated by cardiac edema (100%), the absence of a swimming bladder (100%), and yolk edema (80%), in addition to growth limitation in the larvae, compared to the control group. There was a reduction in heart rate during the assessment of larval survival kinetics, demonstrating the cardiotoxic effect of LT-I. Thus, in this study, we provide essential new depictions of the features of LT-I.

Zebrafish embryo sensitivity test as in vivo platform to anti-Shiga toxin compound screening.

Shiga toxin-producing Escherichia coli (STEC) pathotype secretes two types of AB5 cytotoxins (Stx1 and Stx2), responsible for complications such as hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) in infected patients, which could lead to sequels and death. Currently, there is no effective treatment against the cytotoxic effect of these toxins. However, in order to approve any therapy molecule, an animal experiment is required in order to evaluate the efficacy and safety of therapeutic approaches. The use of alternative small host models is growing among human infectious disease studies, particularly the vertebrate zebrafish model, since relevant results have been described for pathogen-host interaction. In this sense, the present work aimed to analyze the toxic effect of Shiga toxins in zebrafish embryo model in order to standardize this method in the future to be used as a fast, simple, and efficient methodology for the screening of therapeutic molecules. Herein, we demonstrated that the embryos were sensitive in a dose-dependent manner to both Stx toxins, with LD50 of 22 µg/mL for Stx1 and 33 µg/mL for Stx2, and the use of anti-Stx polyclonal antibody abolished the toxic effect. Therefore, this methodology can be a rapid alternative method for selecting promising compounds against Stx toxins, such as recombinant antibodies.

Large-scale evaluation of a rapid diagnostic test for diarrhea caused by enterotoxigenic Escherichia coli targeting the heat-labile toxin.

We standardized an immunochromatographic test (IC) for heat-labile toxin I (LT-I) detection using LT-I antibodies and a specific platform containing the apparatus for application, assembly and cutting. IC detected as little as 62.5ng/mL of purified LT-I toxin and presented 91% sensitivity, 99.5% specificity and 96.0% accuracy, thereby proving to be an excellent point-of-care test for the diagnosis of enterotoxigenic E. coli infection in low-income countries.

Large-scale evaluation of a rapid diagnostic test for diarrhea caused by enterotoxigenic Escherichia coli targeting the heat-labile toxin

We standardized an immunochromatographic test (IC) for heat-labile toxin I (LT-I) detection using LT-I antibodies and a specific platform containing the apparatus for application, assembly and cutting. IC detected as little as 62.5 ng/mL of purified LT-I toxin and presented 91% sensitivity, 99.5% specificity and 96.0% accuracy, thereby proving to be an excellent point-of-care test for the diagnosis of enterotoxigenic E. coli infection in low-income countries.

Fatores de risco no desenvolvimento da linguagem de crianças menores de dois anos atendidas em serviço especializado / Risk factors in language development of children under two years of age at a specialized service

Estudo exploratório, transversal que objetivou identificar os fatores de risco para o desenvolvimento associados às alterações de linguagem em crianças aos 18 meses acompanhadas em um serviço especializado de um município paulista. Participaram crianças atendidas no Serviço de Estimulação Precoce entre 01 de janeiro de 2013 e 31 de dezembro de 2015. Os dados foram obtidos através dos prontuários do serviço. As variáveis estudadas foram: peso ao nascer, idade gestacional, índice de Apgar, ocorrências clínicas (pré-natal, parto e pós-parto) e anomalias congênitas. Na fase descritiva, caracterizou-se os participantes, a partir das variáveis de interesse, sendo calculadas frequências e porcentagens das variáveis. Na fase analítica, buscou-se associações entre a variável dependente (atraso no desenvolvimento) e as demais variáveis, utilizou-se ainda modelos de regressão logística multinomial para estimar a chance de ocorrência das variáveis categóricas. O projeto de pesquisa foi aprovado pelo Comitê de Ética em Pesquisa da Escola de Enfermagem de Ribeirão Preto da Universidade de São Paulo (CAAE 61587916.0.0000.5393; Ofício CEP-EERP-USP nº373/2016), após anuência da Secretaria Municipal de Saúde. Nos três anos elencados para o estudo observou-se que 78 prontuários (56,9%) eram de crianças que deram entrada no serviço no ano de 2013, 49 (35,8%) em 2014, e 10 (7,3%) no ano de 2015. A maioria das crianças era do sexo masculino, com nascimentos distribuídos em sua maioria, entre os hospitais 1 e 2 e grande parte foi identificada com mais de um risco ao nascimento. A média da idade materna foi de 28 anos, com registros que indicavam, em sua maioria, mais de uma intercorrência durante a gestação, parto e pós-parto. Após a caracterização inicial das variáveis, as crianças foram divididas em três grupos: G1 - detectadas com atrasos no desenvolvimento da linguagem, encaminhadas para terapia fonoaudiológica aos 18 meses; G2 - crianças que passaram pelo retorno aos 18 meses e foram reagendadas para retorno aos dois anos, devido a dúvida quanto ao atraso ou não nas habilidades esperadas, recebendo alta neste; G3 - encaminhadas antes dos 18 meses para terapia, devido atraso significativo no desenvolvimento global. A partir dessa distribuição, buscou-se verificar associação entre os grupos e as variáveis mencionadas. O teste exato de Fisher indicou associação significante (p<0,001) entre os grupos em relação à variável anomalias congênitas. A análise de regressão logística indicou que crianças sem registros de intercorrências durante a gestação e no parto e nascimento, tiveram menores chances de evoluir com atrasos no desenvolvimento da linguagem. Aquelas nascidas prematuras apresentaram chance 8,51 vezes maior de terem, como desfecho, atraso na linguagem do que as crianças nascidas a termo e pós-termo. Crianças com valores de Apgar inferior a 4 no primeiro minuto e 7 no 5º minuto tiveram maior chance de apresentar atrasos; o baixo peso ao nascer também aumentou as chances deste desfecho. Os resultados deste estudo apontam para a necessidade da pronta detecção e encaminhamento precoce dessas crianças para serviços especializados, visando minimizar e/ou até evitar agravos futuros

This exploratory, cross-sectional study aimed at identifying the developmental risk factors associated with language changes in children at 18 months followed at a specialized service in a city of São Paulo. Participants were children attending the Early Stimulation Service between January 1, 2013 and December 31, 2015. Data were obtained from the service charts. The variables studied were birth weight, gestational age, Apgar score, clinical occurrences (prenatal, delivery and postpartum) and congenital anomalies. In the descriptive phase, the participants were characterized by the variables of interest, and the frequencies and percentages were calculated. In the analytical phase, we sought associations between the dependent variable (developmental delay) and the other variables, and multinomial logistic regression models were used to estimate the chance of occurrence of the categorical variables. The study was approved by the Research Ethics Committee of the University of São Paulo at Ribeirão Preto College of Nursing (CAAE 61587916.0.0000.5393; CEP-EERP-USP no. 373/2016), after consent of the Municipal Health Department. In the three years listed for the study, it was observed that 78 medical records (56.9%) belonged to the children who entered the service in 2013, 49 (35.8%) in 2014, and 10 (7.3%), in the year 2015. Most of the children were male, with births mostly distributed between hospitals 1 and 2, and most of them were identified as having more than one birth risk. The mean of maternal age was 28 years, with records indicating, for the most part, more than one intercurrence during pregnancy, delivery and postpartum. After the initial characterization of the variables, children were divided into three groups: G1 - children who were detected with delays in language development, referred for speech therapy at 18 months. G2 was related to children who returned for appointment at 18 months of age, and were rescheduled for return with two years, due to doubts related to delay or due they had no expected abilities; those children received discharge at two years old; and G3 was for children who was referred before 18 months for therapy, due to significant delay in overall development. From this distribution, we sought to verify association between the groups and the mentioned variables. The Fisher exact test indicated a significant association (p<0.001) between the groups in relation to the variable congenital anomalies. Logistic regression analysis indicated that children with no records of intercurrences during gestation, delivery and childbirth were less likely to evolve with delays in language development. Those children born premature presented an 8.51 times greater chance of having, as an outcome, delay in language than children born at term and post-term. Children with Apgar values less than 4 in the first minute and 7 in the fifth minute had a greater chance of delays; low birth weight also increased the odds of this outcome. The results of this study point to the need for prompt detection and early referral of these children to specialized services, in order to minimize and / or avoid future disorders

Fatores de risco no desenvolvimento da linguagem de crianças menores de dois anos atendidas em serviço especializado / Risk factors in language development of children under two years of age at a specialized service

Estudo exploratório, transversal que objetivou identificar os fatores de risco para o desenvolvimento associados às alterações de linguagem em crianças aos 18 meses acompanhadas em um serviço especializado de um município paulista. Participaram crianças atendidas no Serviço de Estimulação Precoce entre 01 de janeiro de 2013 e 31 de dezembro de 2015. Os dados foram obtidos através dos prontuários do serviço. As variáveis estudadas foram: peso ao nascer, idade gestacional, índice de Apgar, ocorrências clínicas (pré-natal, parto e pós-parto) e anomalias congênitas. Na fase descritiva, caracterizou-se os participantes, a partir das variáveis de interesse, sendo calculadas frequências e porcentagens das variáveis. Na fase analítica, buscou-se associações entre a variável dependente (atraso no desenvolvimento) e as demais variáveis, utilizou-se ainda modelos de regressão logística multinomial para estimar a chance de ocorrência das variáveis categóricas. O projeto de pesquisa foi aprovado pelo Comitê de Ética em Pesquisa da Escola de Enfermagem de Ribeirão Preto da Universidade de São Paulo (CAAE 61587916.0.0000.5393; Ofício CEP-EERP-USP nº373/2016), após anuência da Secretaria Municipal de Saúde. Nos três anos elencados para o estudo observou-se que 78 prontuários (56,9%) eram de crianças que deram entrada no serviço no ano de 2013, 49 (35,8%) em 2014, e 10 (7,3%) no ano de 2015. A maioria das crianças era do sexo masculino, com nascimentos distribuídos em sua maioria, entre os hospitais 1 e 2 e grande parte foi identificada com mais de um risco ao nascimento. A média da idade materna foi de 28 anos, com registros que indicavam, em sua maioria, mais de uma intercorrência durante a gestação, parto e pós-parto. Após a caracterização inicial das variáveis, as crianças foram divididas em três grupos: G1 - detectadas com atrasos no desenvolvimento da linguagem, encaminhadas para terapia fonoaudiológica aos 18 meses; G2 - crianças que passaram pelo retorno aos 18 meses e foram reagendadas para retorno aos dois anos, devido a dúvida quanto ao atraso ou não nas habilidades esperadas, recebendo alta neste; G3 - encaminhadas antes dos 18 meses para terapia, devido atraso significativo no desenvolvimento global. A partir dessa distribuição, buscou-se verificar associação entre os grupos e as variáveis mencionadas. O teste exato de Fisher indicou associação significante (p<0,001) entre os grupos em relação à variável anomalias congênitas. A análise de regressão logística indicou que crianças sem registros de intercorrências durante a gestação e no parto e nascimento, tiveram menores chances de evoluir com atrasos no desenvolvimento da linguagem. Aquelas nascidas prematuras apresentaram chance 8,51 vezes maior de terem, como desfecho, atraso na linguagem do que as crianças nascidas a termo e pós-termo. Crianças com valores de Apgar inferior a 4 no primeiro minuto e 7 no 5º minuto tiveram maior chance de apresentar atrasos; o baixo peso ao nascer também aumentou as chances deste desfecho. Os resultados deste estudo apontam para a necessidade da pronta detecção e encaminhamento precoce dessas crianças para serviços especializados, visando minimizar e/ou até evitar agravos futuros

This exploratory, cross-sectional study aimed at identifying the developmental risk factors associated with language changes in children at 18 months followed at a specialized service in a city of São Paulo. Participants were children attending the Early Stimulation Service between January 1, 2013 and December 31, 2015. Data were obtained from the service charts. The variables studied were birth weight, gestational age, Apgar score, clinical occurrences (prenatal, delivery and postpartum) and congenital anomalies. In the descriptive phase, the participants were characterized by the variables of interest, and the frequencies and percentages were calculated. In the analytical phase, we sought associations between the dependent variable (developmental delay) and the other variables, and multinomial logistic regression models were used to estimate the chance of occurrence of the categorical variables. The study was approved by the Research Ethics Committee of the University of São Paulo at Ribeirão Preto College of Nursing (CAAE 61587916.0.0000.5393; CEP-EERP-USP no. 373/2016), after consent of the Municipal Health Department. In the three years listed for the study, it was observed that 78 medical records (56.9%) belonged to the children who entered the service in 2013, 49 (35.8%) in 2014, and 10 (7.3%), in the year 2015. Most of the children were male, with births mostly distributed between hospitals 1 and 2, and most of them were identified as having more than one birth risk. The mean of maternal age was 28 years, with records indicating, for the most part, more than one intercurrence during pregnancy, delivery and postpartum. After the initial characterization of the variables, children were divided into three groups: G1 - children who were detected with delays in language development, referred for speech therapy at 18 months. G2 was related to children who returned for appointment at 18 months of age, and were rescheduled for return with two years, due to doubts related to delay or due they had no expected abilities; those children received discharge at two years old; and G3 was for children who was referred before 18 months for therapy, due to significant delay in overall development. From this distribution, we sought to verify association between the groups and the mentioned variables. The Fisher exact test indicated a significant association (p<0.001) between the groups in relation to the variable congenital anomalies. Logistic regression analysis indicated that children with no records of intercurrences during gestation, delivery and childbirth were less likely to evolve with delays in language development. Those children born premature presented an 8.51 times greater chance of having, as an outcome, delay in language than children born at term and post-term. Children with Apgar values less than 4 in the first minute and 7 in the fifth minute had a greater chance of delays; low birth weight also increased the odds of this outcome. The results of this study point to the need for prompt detection and early referral of these children to specialized services, in order to minimize and / or avoid future disorders