A importância do diagnóstico precoce no período neonatal para Estreptococo do grupo B

Streptococcus agalactiae or Group B Streptococcus (GBS) correspond to Gram positive diplococcus, usually residing in the microbiota of the gastrointestinal and genitourinary tract of pregnant women. The transmission of GBS occurs mainly through vertical ascent, especially in the intrapartum period, being one of the main bacteria responsible for the development of sepsis in the neonatal period. Early diagnosis associated with adequate antibiotic prophylaxis predicts lower risks of neonatal infection, in addition to a lower rate of complications and infant mortality. To carry out the research, we used high-impact articles taken from MEDLINE, SciELO, Pubmed, CNPq and LILACS platforms (2015–2022) and compiled a recent theoretical reference, with the exception of the historical reference, under which there was no time limitation. In reference to the levels of preventive medicine it can be observed: The primary prevention shows that the possibility of developing vaccines is viable, but so far in phase II in international studies and with a lack of epidemiological studies regarding the specific capsular serotypes of each Brazilian regions, hindering the development of the measure. Regarding the microbiological screening of GBS indicated by the Ministry of Health in 2012, it should be performed between the 35th and 37th week of pregnancy, in contrast to more recent international literature. In secondary prevention, there are new ways of diagnosing GBS infection, for example, the Xpert GBS, a rapid test indicated to test women at risk of preterm birth or in labor who did not undergo the test during prenatal care. The secondary still involves the existing treatment, which would be through the use of first-choice antimicrobials, such as crystalline penicillin or ampicillin, however, in some cases they already have resistance, requiring microbiological evaluation with antibiogram. In conclusion, the topic is extremely important for maternal and child health. GBS infection is a preventable disease with a simple diagnosis, however, there is still a huge literature divergence and lack of Brazilian protocols emphasizing the relevance of screening for GBS, which should be performed extensively in pregnant women, with antibiotic prophylaxis only in specific cases and, if necessary, with a previous sensitivity analysis to antimicrobials, in order to obtain better results.Streptococcus agalactiae ou Estreptococo do grupo B (EGB) correspondem às bactérias Gram positivas com formato de diplococo, geralmente residentes da microbiota do trato gastrointestinal e geniturinário de gestantes. A transmissão do EGB ocorre principalmente através da ascensão vertical, sobretudo no período intraparto, sendo uma das principais bactérias responsável pelo desenvolvimento da sepse no período neonatal. O diagnóstico precoce associado à adequada profilaxia antibiótica prevê menores riscos de infecção neonatal, além de menor taxa de complicações e mortalidade infantil. Para realização da pesquisa, utilizamos periódicos de alto impacto retirados de bases das plataformas MEDLINE, SciELO, Pubmed, CNPq e LILACS (2015-2022) compilou-se a utilização de um referencial teórico recente, com exceção do referencial histórico, sob o qual não houve limitação temporal. A partir dos resultados obtidos pode-se observar na medicina preventiva: A respeito da primária, a possibilidade de desenvolvimento de vacinas é viável, mas até o momento em fase II em estudos internacionais e com carência de estudos epidemiológicos a respeito dos sorotipos capsulares específicos das regiões brasileiras, dificultando o desenvolvimento da medida. Em relação ao rastreio microbiológico do EGB indicado pelo Ministério da Saúde em 2012 deve ser realizado entre a 35º e 37º semana de gestação, contrapondo literaturas internacionais mais recentes. Na prevenção secundária há novas formas de diagnóstico para a infecção por EGB, a exemplo, o Xpert GBS, teste rápido indicado para testar mulheres com risco de parto prematuro ou em trabalho de parto que não fizeram o exame durante o pré-natal. A secundária ainda envolve o tratamento existente, o qual seria através da utilização de antimicrobianos de primeira escolha, como a penicilina cristalina ou a ampicilina, entretanto, em alguns casos já apresentam resistência, necessitando de avaliação microbiológico com antibiograma. Entende-se, portanto, que o tema é de fundamental importância para a saúde materna e infantil. A infecção por EGB é uma doença prevenível e de simples diagnóstico, entretanto, ainda há uma grande divergência literária e falta de protocolos brasileiros enfatizando a relevância do rastreio para EGB, o qual deve ser realizado de forma ampla nas gestantes, com profilaxia antibiótica somente em casos específicos e se necessário com análise de sensibilidade aos antimicrobianos prévia, com intuito de melhores resultados

Análise imunológica comparada da eficiência e especificidade da hialuronidase recombinante de Polybia paulista (Hymenoptera, Vespidae) expressa em bactéria e levedura

A vespa social Polybia paulista tem sido muito estudada nos campos da bioquímica, proteômica e imunologia pelas suas características de habitat, composição de seu veneno e pelas conseqüencias dos acidentes decorrentes de suas ferroadas. O cDNA de hialuronidase (GI: 302201582), um dos principais alérgenos desse veneno, foi clonado em vetores de expressão - pET-28a e pPICZA - em bactéria Escherichia coli DE3 (BL21) e em levedura Pichia pastoris, respectivamente. A proteína Hyal recombinante (Pp-Hyal-rec) de bactéria foi expressa em corpúsculos de inclusão, enquanto que a de levedura na forma solúvel. Ambas foram purificadas por cromatografia de afinidade em resina Ni2+ (Ni-NTA-Agarose). A proteína nativa de Hyal (Pp-Hyal-nat) também foi obtida e purificada até a homogeneidade por meio de cromatografia de troca catiônica em coluna Hiprep FF CM, acoplado a um sistema de Akta FPLC, e as análises realizadas por espectrometria de massa em MALDI ToF/ToF-MS. Anticorpos policlonais foram produzidos em camundongos BALB/c contra a Pp-Hyal-nat e a Pp-Hyal-rec de bactéria, demonstrando alta especificidade nos testes de imunoblotting realizados. Estes alérgenos foram avaliados quanto ao reconhecimento de imunoglobulina E (IgE) Pp-Hyal-específico no soro de pacientes sabidamente alérgicos ao veneno desta vespa. Os soros imunes foram capazes de reconhecer especificamente, em maior intensidade as bandas correspondentes à proteína Pp-Hyal-rec de bactéria (43 kDa) e a Pp-Hyal-rec de levedura (37 kDa) em relação ao alérgeno Pp-Hyal-nat (39 kDa). No extrato de veneno bruto de P. paulista, os soros reconheceram outras proteínas provavelmente correspondentes aos demais alérgenos do veneno, tais como Fosfolipase (37 kDa), Antígeno 5 (25 kDa), e proteases. Os dados aqui obtidos com ambos...The social wasp Polybia paulista has been well studied in fields of biochemistry, proteomics and immunology due to its habitat characteristics, venom composition and the consequences of accidents arising from their stings. The hyaluronidase cDNA (GI: 302201582), one of the major venom allergens, was cloned into expression vectors - pET-28a and pPICZA - in Escherichia coli DE3 (BL21) and yeast Pichia pastoris, respectively. The recombinant protein Hyal (Pp-Hyal-rec) of bacteria was expressed in inclusion corpuscles, whereas the yeast in soluble form. Both were purified by affinity chromatography on Ni2+ (Ni-NTA-Agarose) resin. The native protein Hyal (Pp-Hyal-nat) was also obtained and purified to homogeneity by cation exchange chromatography on Hiprep CM FF column, to an Akta FPLC coupled system, and the analysis performed by mass spectrometry MALDI ToF / ToF-MS. Polyclonal antibodies were produced in BALB/c mice against Pp-Hyal-nat and Pp-Hyal-rec from bacteria, demonstrating an high specificity in the immunoblotting tests performed. These allergens were evaluated for recognition of immunoglobulin E (IgE) Pp-Hyal-specific in serum from patients known to be allergic to the venom of this wasp. The immune sera were able to recognize specifically in a higher intensity the bands corresponding to protein Pp-Hyal-rec of bacteria (43 kDa) and Pp-Hyal-rec of yeast (37 kDa) in relation to the allergen Pp-Hyal-nat (39 kDa). In the P. paulista crude venom extract, all sera recognized other proteins probably corresponding to other venom allergens, such as phospholipase (37 kD), antigen 5 (25 kDa), and protease. The data obtained here with both recombinant allergens strongly suggest the possibility of using... (Complete abstract click electronic access below)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Facing Hymenoptera venom allergy: from natural to recombinant allergens

Along with food and drug allergic reactions, a Hymenoptera insect Sting (Apoidea, Vespidae, Formicidae) is one of the most common causes of anaphylaxis worldwide. Diagnoses of Hymenoptera venom allergy (HVA) and specific immunotherapy (SIT) have been based on the use of crude venom extracts. However, the incidence of cross-reactivity and low levels of sensibility during diagnosis, as well as the occurrence of nonspecific sensitization and undesired side effects during SIT, encourage the search for novel allergenic materials. Recombinant allergens are an interesting approach to improve allergy diagnosis and SIT because they circumvent major problems associated with the use of crude venom. Production of recombinant allergens depends on the profound molecular characterization of the natural counterpart by combining some omics approaches with high-throughput screening techniques and the selection of an appropriate system for heterologous expression. To date, several clinically relevant allergens and novel venom toxins have been identified, cloned and characterized, enabling a better understanding of the whole allergenic and envenoming processes. Here, we review recent findings on identification, molecular characterization and recombinant expression of Hymenoptera venom allergens and on the evaluation of these heterologous proteins as valuable tools for tackling remaining pitfalls on HVA diagnosis and immunotherapy.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenadação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Heterologous Expression, Purification and Immunoreactivity of the Antigen 5 from Polybia paulista Wasp Venom

Polybia paulista (Hymenoptera: Vespidae) is responsible for a high number of sting accidents and anaphylaxis events in Southeast Brazil, Argentina and Paraguay. The specific detection of allergy to the venom of this wasp is often hampered by the lack of recombinant allergens currently available for molecular diagnosis. Antigen 5 (~23 kDa) from P. paulista venom (Poly p 5) is a highly abundant and glycosylated allergenic protein that could be used for development of component-resolved diagnosis (CRD). Here, we describe the cloning and heterologous expression of the antigen 5 (rPoly p 5) from P. paulista venom using the eukaryotic system Pichia pastoris. The expression as a secreted protein yielded high levels of soluble rPoly p 5. The recombinant allergen was further purified to homogeneity (99%) using a two-step chromatographic procedure. Simultaneously, the native form of the allergen (nPoly p 5) was purified from the wasp venom by Ion exchange chromatography. The rPoly p 5 and nPoly p 5 were then submitted to a comparative analysis of IgE-mediated immunodetection using sera from patients previously diagnosed with sensitization to wasp venoms. Both rPoly p 5 and nPoly p 5 were recognized by specific IgE (sIgE) in the sera of the allergic individuals. The high levels of identity found between nPoly p 5 and rPoly p 5 by the alignment of its primary sequences as well as by 3-D models support the results obtained in the immunoblot. Overall, we showed that P. pastoris is a suitable system for production of soluble rPoly p 5 and that the recombinant allergen represents a potential candidate for molecular diagnosis of P.paulista venom allergy

Hyaluronidase from the venom of the social wasp Polybia paulista (Hymenoptera, Vespidae): Cloning, structural modeling, purification, and immunological analysis

In this study, we describe the cDNA cloning, sequencing, and 3-D structure of the allergen hyaluronidase from Polybia paulista venom (Pp-Hyal). Using a proteomic approach, the native form of Pp-Hyal was purified to homogeneity and used to produce a Pp-specific polyclonal antibody. The results revealed that Pp-Hyal can be classified as a glycosyl hydrolase and that the full-length Pp-Hyal cDNA (1315 bp; GI: 302201582) is similar (80-90%) to hyaluronidase from the venoms of endemic Northern wasp species. The isolated mature protein is comprised of 338 amino acids, with a theoretical pI of 8.77 and a molecular mass of 39,648.8 Da versus a pI of 8.13 and 43,277.0 Da indicated by MS. The Pp-Hyal 3D-structural model revealed a central core (α/β)7 barrel, two sulfide bonds (Cys 19-308 and Cys 185-197), and three putative glycosylation sites (Asn79, Asn187, and Asn325), two of which are also found in the rVes v 2 protein. Based on the model, residues Ser299, Asp107, and Glu109 interact with the substrate and potential epitopes (five conformational and seven linear) located at surface-exposed regions of the structure. Purified native Pp-Hyal showed high similarity (97%) with hyaluronidase from Polistes annularis venom (Q9U6V9). Immunoblotting analysis confirmed the specificity of the Pp-Hyal-specific antibody as it recognized the Pp-Hyal protein in both the purified fraction and P. paulista crude venom. No reaction was observed with the venoms of Apis mellifera, Solenopsis invicta, Agelaia pallipes pallipes, and Polistes lanio lanio, with the exception of immune cross-reactivity with venoms of the genus Polybia (sericea and ignobilis). Our results demonstrate cross-reactivity only between wasp venoms from the genus Polybia. The absence of cross-reactivity between the venoms of wasps and bees observed here is important because it allows identification of the insect responsible for sensitization, or at least of the phylogenetically closest insect, in order to facilitate effective immunotherapy in allergic patients. © 2013 Elsevier Ltd