Decentralization of the health system - experiences from Pakistan, Portugal and Brazil.

BACKGROUND: Decentralization of a health system is a complex and multidimensional phenomenon that demands thorough investigation of its process logistics, predisposing factors and implementation mechanisms, within the broader socio-political environment of each nation. Despite its wide adoption across both high-income countries (HICs) and low-and-middle-income countries (LMICs), empirical evidence of whether decentralization actually translates into improved health system performance remains inconclusive and controversial. This paper aims to provide a comprehensive description of the decentralization processes in three countries at different stages of their decentralization strategies - Pakistan, Brazil and Portugal. MAIN BODY: This study employed a systematic analysis of peer-reviewed academic journals, official government reports, policy documents and publications from international organizations related to health system decentralization. A comprehensive search was conducted using reputable databases such as PubMed, Google Scholar, the WHO repository and other relevant databases, covering the period up to the knowledge cutoff date in June 2023. Information was systematically extracted and organized into the determinants, process mechanics and challenges encountered during the planning, implementation and post-decentralization phases. Although decentralization reforms have achieved some success, challenges persist in their implementation. Comparing all three countries, it was evident that all three have prioritized health in their decentralization reforms and aimed to enhance local decision-making power. Brazil has made significant progress in implementing decentralization reforms, while Portugal and Pakistan are still in the process. Pakistan has faced significant implementation challenges, including capacity-building, resource allocation, resistance to change and inequity in access to care. Brazil and Portugal have also faced challenges, but to a lesser extent. The extent, progress and challenges in the decentralization processes vary among the three countries, each requiring ongoing evaluation and improvement to achieve the desired outcomes. CONCLUSION: Notable differences exist in the extent of decentralization, the challenges faced during implementation and inequality in access to care between the three countries. It is important for Portugal, Brazil and Pakistan to address these through reinforcing implementation strategies, tackling inequalities in access to care and enhancing monitoring and evaluation mechanism. Additionally, fostering knowledge sharing among these different countries will be instrumental in facilitating mutual learning.

Cashew By-Product as a Functional Substrate for the Development of Probiotic Fermented Milk.

Cashew (Anacardium occidentale) processing generates a by-product (CB) with potential for health benefits and that could be a favorable ingredient to be added to a probiotic food matrix. This study aimed to assess the functional attributes of CB in fermented milk with a probiotic and a starter culture using in vitro gastrointestinal conditions. Two formulations were tested, without CB (Control Formulation-CF) and with CB (Test Formulation-TF), and the two strains most adapted to CB, the probiotic Lacticaseibacillus paracasei subsp. paracasei F19® and the starter Streptococcus thermophilus ST-M6®, were chosen to be fermented in the CF and the TF. During a 28-day period of refrigeration (4 °C), both strains used in the CF and TF maintained a population above 8.0 log CFU/mL. Strains cultured in the TF had a significant increase in total phenolic compounds and greater antioxidant potential during their shelf life, along with improved survival of F19® after in vitro-simulated gastrointestinal conditions. Our study revealed the promising potential of CB in the probiotic beverage. The CB-containing formulation (TF) also exhibited higher phenolic content and antioxidant activity. Furthermore, it acted as a protector for bacteria during gastrointestinal simulation, highlighting its potential as a healthy and sustainable product.

Genetic Structure and Biodiversity in Wild Centropomus parallelus and in Wild and Recently Domesticated Centropomus undecimallis Populations.

Centropomus undecimalis (common snook) and Centropomus parallelus (fat snook) have a wide distribution from southern Florida to southern Brazil. Due to their value as a food source, these species have been heavily exploited through predatory fishing, posing a conservation challenge. To assess their genetic diversity and population structure, we used microsatellite markers. Our findings revealed genetic differences among populations of the same species, highlighting the need for targeted conservation efforts. The microsatellite markers proved effective in assessing genetic variability, providing valuable insights for management and conservation. The parameters Ho (observed heterozygosity) and He (expected heterozygosity) were reliable indicators of genetic diversity, and specific loci showed varying allele numbers across populations. Our study contributes to understanding population genetics in these snook species and supports their conservation. Despite not being classified as endangered, genetic differences among populations emphasize the importance of considering population-level characteristics in conservation strategies. This research lays the foundation for future studies and actions aimed at preserving these valuable fish species. In summary, our study demonstrates the significance of microsatellite markers in assessing genetic variability and population structure in common snook and fat snook, informing conservation efforts for these species.

The combination of omics strategies to evaluate starter and probiotic strains in the Catharina sour Brazilian-style beer.

Catharina sour, the first internationally recognized Brazilian beer, is characterized by fermentation with lactic acid bacteria (LAB), which may have probiotic potential, and the addition of fruit juice. This study aimed to evaluate the use of the starter Streptococcus thermophilus TH-4 (TH-4) and the probiotics Lacticaseibacillus paracasei F19 and 431, associated with Saccharomyces cerevisiae US-05, in the absence (control)/presence of passion fruit or peach juices. Evaluation proceeded during fermentation and storage by enumeration using pour-plate and qPCR; gene expressions of hop resistance; proteome by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS); and odor, flavor, and metabolome by Headspace Solid-Phase Microextraction (HS-SPME), coupled with the gas chromatography-mass spectrometry (GC-MS) analysis. We concluded that the strains studied are recommended for applications in sour beers, due to the presence of defense mechanisms like membrane adhesion and H + pump. Furthermore, HS-SPME/GC-MS indicated that the strains may contribute to the beer flavor and odor.

Brewer's Spent Grain Enhanced the Recovery of Potential Probiotic Strains in Fermented Milk After Exposure to In Vitro-Simulated Gastrointestinal Conditions.

Brewer's spent grain (BSG) is a beer industry by-product with interesting functional properties by its high fiber content and bioactive compounds, which may be possibly employed as a prebiotic ingredient. The fermentability of BSG by ten probiotics and two starter cultures was evaluated, and the co-culture of Lacticaseibacillus paracasei subsp. paracasei F-19® (probiotic) and Streptococcus thermophilus TH-4® (starter) was selected to produce a potentially probiotic fermented milk (FM). Four formulations of FM were studied: FM1 (control), FM2 (probiotic - /BSG +), FM3 (probiotic + /BSG -), and FM4 (probiotic + /BSG +). The viability of the microorganisms in the FM was monitored throughout 28 days of storage. The resistance of the microorganisms in the FM to in vitro-simulated gastrointestinal tract (GIT) conditions was also evaluated. Even though the BSG did not influence the fermentation kinetics or increase the populations of both microorganisms in the FM, a significant improvement on the survival of TH-4® against in vitro-simulated GIT stress was observed in the formulations containing BSG alone or in combination with F-19®. All formulations showed potential as probiotic FM, since total probiotic populations were kept above 1010 CFU in a daily portion of 200 mL, and a minimum of 1010 and 108 CFU equivalent of, respectively, TH-4® and F-19® was recovered after the GIT stress. Therefore, TH-4® has potential as a probiotic strain in addition to its starter feature, while BSG may be employed as a possible prebiotic ingredient in a synbiotic approach. Nonetheless, further studies to evaluate possible health benefits are needed.

Sour Beer with Lacticaseibacillus paracasei subsp. paracasei F19: Feasibility and Influence of Supplementation with Spondias mombin L. Juice and/or By-Product.

This study aimed to evaluate the probiotic strain Lacticaseibacillus (L.) paracasei subsp. paracasei F19 (F19) with the yeast Saccharomyces cerevisiae US-05 (US-05), using Spondias mombin L. ('taperebá' or 'cajá') juice and by-product, in four sour-type beer formulations: control, with bagasse, juice, and juice and bagasse. The viability of F19 was evaluated by pour-plating and PMA-qPCR. Fermentability, in addition to physicochemical and sensory parameters, and aroma and flavor, were evaluated during brewery by using Headspace Solid-Phase Microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS). F19 was successful in fermenting bagasse in a MRS medium (9.28 log CFU/mL in 24 h) but had a low viability in hopped wort, growing better in formulations without bagasse or juice. No difference between formulations was observed regarding sensory acceptability, and the HS-SPME/GC-MS revealed different flavors and aroma compounds. In conclusion, the production of a potential probiotic sour beer with F19 and US-05 is feasible regarding probiotic viability. However, S. mombin, as juice or bagasse, threatened probiotic survival. Different flavors and aroma compounds were detected, whereas no difference between formulations was found regarding sensory acceptability. The moderate alcohol content achieved is important for bacterial survival and for the development of a probiotic beer with health claims.

MicroRNA roles in regeneration: Multiple lessons from zebrafish.

MicroRNAs (miRNAs) are small noncoding RNAs with pivotal roles in the control of gene expression. By comparing the miRNA profiles of uninjured vs. regenerating tissues and structures, several studies have found that miRNAs are potentially involved in the regenerative process. By inducing miRNA overexpression or inhibition, elegant experiments have directed regenerative responses validating relevant miRNA-to-target interactions. The zebrafish (Danio rerio) has been the epicenter of regenerative research because of its exceptional capability to self-repair damaged tissues and body structures. In this review, we discuss recent discoveries that have improved our understanding of the impact of gene regulation mediated by miRNAs in the context of the regeneration of fins, heart, retina, and nervous tissue in zebrafish. We compiled what is known about the miRNA control of regeneration in these tissues and investigated the links among up-regulated and down-regulated miRNAs, their putative or validated targets, and the regenerative process. Finally, we briefly discuss the forthcoming prospects, highlighting directions and the potential for further development of this field.

MicroRNA roles in regeneration: multiple lessons from zebrafish

MicroRNAs (miRNAs) are small noncoding RNAs with pivotal roles in the control of gene expression. By comparing the miRNA profiles of uninjured vs. regenerating tissues and structures, several studies have found that miRNAs are potentially involved in the regenerative process. By inducing miRNA overexpression or inhibition, elegant experiments have directed regenerative responses validating relevant miRNA-to-target interactions. The zebrafish (Danio rerio) has been the epicenter of regenerative research because of its exceptional capability to self-repair damaged tissues and body structures. In this review, we discuss recent discoveries that have improved our understanding of the impact of gene regulation mediated by miRNAs in the context of the regeneration of fins, heart, retina, and nervous tissue in zebrafish. We compiled what is known about the miRNA control of regeneration in these tissues and investigated the links among up-regulated and down-regulated miRNAs, their putative or validated targets, and the regenerative process. Finally, we briefly discuss the forthcoming prospects, highlighting directions and the potential for further development of this field.

Vitamin D Modulates Intestinal Microbiota in Inflammatory Bowel Diseases.

Inflammatory bowel disease (IBD) is a chronic inflammation of the gastrointestinal tract (GIT), including Crohn's disease (CD) and ulcerative colitis (UC), which differ in the location and lesion extensions. Both diseases are associated with microbiota dysbiosis, with a reduced population of butyrate-producing species, abnormal inflammatory response, and micronutrient deficiency (e.g., vitamin D hypovitaminosis). Vitamin D (VitD) is involved in immune cell differentiation, gut microbiota modulation, gene transcription, and barrier integrity. Vitamin D receptor (VDR) regulates the biological actions of the active VitD (1α,25-dihydroxyvitamin D3), and is involved in the genetic, environmental, immune, and microbial aspects of IBD. VitD deficiency is correlated with disease activity and its administration targeting a concentration of 30 ng/mL may have the potential to reduce disease activity. Moreover, VDR regulates functions of T cells and Paneth cells and modulates release of antimicrobial peptides in gut microbiota-host interactions. Meanwhile, beneficial microbial metabolites, e.g., butyrate, upregulate the VDR signaling. In this review, we summarize the clinical progress and mechanism studies on VitD/VDR related to gut microbiota modulation in IBD. We also discuss epigenetics in IBD and the probiotic regulation of VDR. Furthermore, we discuss the existing challenges and future directions. There is a lack of well-designed clinical trials exploring the appropriate dose and the influence of gender, age, ethnicity, genetics, microbiome, and metabolic disorders in IBD subtypes. To move forward, we need well-designed therapeutic studies to examine whether enhanced vitamin D will restore functions of VDR and microbiome in inhibiting chronic inflammation.

Prevalence of Anaplasma marginale, Babesia bovis, and Babesia bigemina in cattle in the Campos de Lages region, Santa Catarina state, Brazil, estimated by multiplex-PCR.

This study aimed to determine the prevalence of three common hemoparasites (Anaplasma marginale, Babesia bovis and Babesia bigemina) in cattle from 16 counties in the Campos de Lages region, Santa Catarina state, Brazil, and the factors affecting disease occurrence. The study population consisted of 257 clinically healthy animals from 21 rural farms. Bovine blood samples were collected by jugular venipuncture. DNA was extracted from whole blood by the phenol/ chloroform method. Genomic DNA extracted from blood samples was subjected to Multiplex PCR for screening of B. bovis, B. bigemina, and A. marginale using specific primers. Prevalences of A. marginale, B. bigemina, and B. bovis were 27%, 16%, and 29%, respectively. Mixed infection was observed in 17.5% of samples. The most frequent was Babesia bovis and Babesia bigemina in 6.62% of samples. A. marginale infection rates were statistically correlated with age groups of cattle. The infections detected in the study population were considered to be subclinical, based on the presence pathogen DNA and absence of clinical symptoms. Seasonality of the pathogens resulted in various degrees of infection, related to the age of the animals and the season. The Campos de Lages region is characterized by enzootic instability for these pathogens because of its climatic and geographic features.

Fishing Into the MicroRNA Transcriptome.

In the last decade, several studies have been focused on revealing the microRNA (miRNA) repertoire and determining their functions in farm animals such as poultry, pigs, cattle, and fish. These small non-protein coding RNA molecules (18-25 nucleotides) are capable of controlling gene expression by binding to messenger RNA (mRNA) targets, thus interfering in the final protein output. MiRNAs have been recognized as the main regulators of biological features of economic interest, including body growth, muscle development, fat deposition, and immunology, among other highly valuable traits, in aquatic livestock. Currently, the miRNA repertoire of some farmed fish species has been identified and characterized, bringing insights about miRNA functions, and novel perspectives for improving health and productivity. In this review, we summarize the current advances in miRNA research by examining available data on Neotropical and other key species exploited by fisheries and in aquaculture worldwide and discuss how future studies on Neotropical fish could benefit from this knowledge. We also make a horizontal comparison of major results and discuss forefront strategies for miRNA manipulation in aquaculture focusing on forward-looking ideas for forthcoming research.

Combining Results from Distinct MicroRNA Target Prediction Tools Enhances the Performance of Analyses.

Target prediction is generally the first step toward recognition of bona fide microRNA (miRNA)-target interactions in living cells. Several target prediction tools are now available, which use distinct criteria and stringency to provide the best set of candidate targets for a single miRNA or a subset of miRNAs. However, there are many false-negative predictions, and consensus about the optimum strategy to select and use the output information provided by the target prediction tools is lacking. We compared the performance of four tools cited in literature-TargetScan (TS), miRanda-mirSVR (MR), Pita, and RNA22 (R22), and we determined the most effective approach for analyzing target prediction data (individual, union, or intersection). For this purpose, we calculated the sensitivity, specificity, precision, and correlation of these approaches using 10 miRNAs (miR-1-3p, miR-17-5p, miR-21-5p, miR-24-3p, miR-29a-3p, miR-34a-5p, miR-124-3p, miR-125b-5p, miR-145-5p, and miR-155-5p) and 1,400 genes (700 validated and 700 non-validated) as targets of these miRNAs. The four tools provided a subset of high-quality predictions and returned few false-positive predictions; however, they could not identify several known true targets. We demonstrate that union of TS/MR and TS/MR/R22 enhanced the quality of in silico prediction analysis of miRNA targets. We conclude that the union rather than the intersection of the aforementioned tools is the best strategy for maximizing performance while minimizing the loss of time and resources in subsequent in vivo and in vitro experiments for functional validation of miRNA-target interactions.

Prevalence of C282Y and H63D mutations in the HFEgene in patients from São Paulo and Southern Brazil / Prevalência das mutações C282Y e H63D no gene HFEem pacientes de São Paulo e do Sul do Brasil

ABSTRACT Hereditary hemochromatosis (HH) is an autosomal recessive disorder caused by mutations in the HFE gene; it is characterized by the risk of iron overload. C282Y and H63D are the most associated mutations in HH. This study aimed to determine the frequency of mutations in the south of Brazil and São Paulo. It used the real-time polymerase chain reaction (PCR) technique and the results collected from Genolab data. In 90 individuals, 46.67% had at least one of the mutations for HH. There is a high prevalence of these mutations in both populations, therefore searching for patients under clinical suspicion is recommended.

RESUMO A hemocromatose hereditária (HH) é uma desordem autossômica recessiva provocada por mutações no gene HFE e caracterizada pelo risco de uma sobrecarga de ferro. As mutações mais conhecidas associadas à HH são a C282Ye a H63D. Este estudo teve como objetivo determinar a frequência das mutações no sul do Brasil e em São Paulo. Ele utilizou a técnica de reação em cadeia da polimerase (PCR) em tempo real e o resultado coletado dos dados do Genolab. De 90 indivíduos, 46,67%possuíam pelo menos uma das mutações para HH. Existe alta prevalência dessas mutações nas duas populações, portanto recomenda-se a busca em pacientes sob suspeita clínica.

Genotypic characterization of ten microsatellite loci in two Brazilian free range (Caipira) chicken lines / Caracterização genotípica de dez loci de microssatélites em duas linhagens de galinhas caipiras brasileiras

This study aimed to investigate the genetic variability of two Brazilian free range (Caipira) chickens lines using microsatellites analysis of ten loci. It was collected a total of 99 blood samples, which 49 were from Paraíso Pedrês (PP) and 50 were from Rubro Negra (RN) lines. The amplification of the DNA fragments was performed by polymerase chain reaction (PCR) and the genotyping was conduct using ABI 3130 sequencer. The allele number variation was among 3 (LEI0254) to 32 (LEI0212) in the PP line, and 4 (LEI0254) to 31 (LEI0212) in the RN line. The allelic average per locus was 13.3 and 13.1 in the PP and RN lines, respectively. The average observed and the expected heterozygosity were 0.650 and 0.820 in the PP line, and 0.671 and 0.804 in the RN line. All of the analyzed loci were informative (PIC>0.5). These results indicate that these free-range animals have a high genetic variability, at least for the majority of the analyzed loci, and this genetic variation is higher than the commercial chickens and similar for the no-commercial birds.(AU)

Este trabalho teve como objetivo investigar a variabilidade genética de duas linhagens de galinhas caipiras brasileiras usando 10 locos de microssatélites. Noventa e nove amostras de sangue total foram coletadas, sendo 49 da linhagem Paraíso Pedrês (PP) e 50 da linhagem Rubro Negra (RN). As amplificações dos fragmentos do DNA foram realizadas pela técnica da reação em cadeia pela polimerase (PCR), e a genotipagem ocorreu em um sequenciador ABI 3130. O número de alelos variou de 3 (LEI0254) a 32 (LEI0212), na linhagem PP, e de 4 (LEI0254) a 31 (LEI0212), na linhagem RN. O número médio de alelos por loco foi de 13,3 e de 13,1 nas linhagens PP e RN, respectivamente. A heterozigosidade observada média e a heterozigosidade esperada média foram 0,650 e 0,820, na linhagem PP, e 0,671 e 0,804, na linhagem RN. Todos os locos analisados foram informativos (PIC>0,5). Estes resultados indicam que estes animais caipiras têm uma grande variabilidade genética, pelo menos para a maioria dos locos analisados, e que esta variação genética é maior do que a das galinhas comerciais e semelhante à de aves não comerciais.(AU)

Genotypic characterization of ten microsatellite loci in two Brazilian free range (Caipira) chicken lines / Caracterização genotípica de dez loci de microssatélites em duas linhagens de galinhas caipiras brasileiras

This study aimed to investigate the genetic variability of two Brazilian free range (Caipira) chickens lines using microsatellites analysis of ten loci. It was collected a total of 99 blood samples, which 49 were from Paraíso Pedrês (PP) and 50 were from Rubro Negra (RN) lines. The amplification of the DNA fragments was performed by polymerase chain reaction (PCR) and the genotyping was conduct using ABI 3130 sequencer. The allele number variation was among 3 (LEI0254) to 32 (LEI0212) in the PP line, and 4 (LEI0254) to 31 (LEI0212) in the RN line. The allelic average per locus was 13.3 and 13.1 in the PP and RN lines, respectively. The average observed and the expected heterozygosity were 0.650 and 0.820 in the PP line, and 0.671 and 0.804 in the RN line. All of the analyzed loci were informative (PIC>0.5). These results indicate that these free-range animals have a high genetic variability, at least for the majority of the analyzed loci, and this genetic variation is higher than the commercial chickens and similar for the no-commercial birds.

Este trabalho teve como objetivo investigar a variabilidade genética de duas linhagens de galinhas caipiras brasileiras usando 10 locos de microssatélites. Noventa e nove amostras de sangue total foram coletadas, sendo 49 da linhagem Paraíso Pedrês (PP) e 50 da linhagem Rubro Negra (RN). As amplificações dos fragmentos do DNA foram realizadas pela técnica da reação em cadeia pela polimerase (PCR), e a genotipagem ocorreu em um sequenciador ABI 3130. O número de alelos variou de 3 (LEI0254) a 32 (LEI0212), na linhagem PP, e de 4 (LEI0254) a 31 (LEI0212), na linhagem RN. O número médio de alelos por loco foi de 13,3 e de 13,1 nas linhagens PP e RN, respectivamente. A heterozigosidade observada média e a heterozigosidade esperada média foram 0,650 e 0,820, na linhagem PP, e 0,671 e 0,804, na linhagem RN. Todos os locos analisados foram informativos (PIC>0,5). Estes resultados indicam que estes animais caipiras têm uma grande variabilidade genética, pelo menos para a maioria dos locos analisados, e que esta variação genética é maior do que a das galinhas comerciais e semelhante à de aves não comerciais.

Efficiency of DNA extraction from different carp tissue using the sodium chloride (NaCl) protocol / Eficiência na extração de DNA de diferentes tecidos de carpas através do protocolo de cloreto de sódio (NaCl)

With advances of farming and research in the area of molecular genetics, studies that can help with obtaining good quality DNA are necessary. The objective was to evaluate the efficiency of DNA extraction in different types of tissues of three species of carp by using the protocol of DNA extraction with Sodium Chloride (NaCl). The samples were subjected to DNA extraction, and the integrity was visualized on 1.5% agarose gel. In a total of 72 samples used for DNA extraction, all of them were positive, confirmed by the presence of bands on agarose gel. The capacity of amplifying the extracted DNA was tested by amplification reactions using the RAPD (random amplification of polymorphic DNA) technique, confirming that the DNA was of good quality for use in subsequent studies with molecular markers.(AU)

Com o avanço da piscicultura e das pesquisas na área da genética molecular, existe a necessidade de estudos que possam aperfeiçoar a obtenção de DNA com boa qualidade. Diante disso, o objetivo desse trabalho foi avaliar a eficiência da extração de DNA em diferentes tipos de tecidos de três espécies de carpas, através do protocolo de extração de DNA com cloreto de sódio (NaCl). As amostras foram submetidas à extração de DNA e a integridade foi visualizada em gel de agarose 1,5%. Em um total de 72 amostras utilizadas na extração de DNA, todas foram positivas, confirmadas com a presença de banda no gel de agarose. A capacidade de amplificação do DNA extraído foi testada através de reações de amplificação utilizando a técnica RAPD (polimorfismos de DNA amplificados ao acaso), confirmando que o DNA apresenta boas condições para utilização em estudos posteriores com marcadores moleculares.(AU)

Efficiency of DNA extraction from different carp tissue using the sodium chloride (NaCl) protocol / Eficiência na extração de DNA de diferentes tecidos de carpas através do protocolo de cloreto de sódio (NaCl)

With advances of farming and research in the area of molecular genetics, studies that can help with obtaining good quality DNA are necessary. The objective was to evaluate the efficiency of DNA extraction in different types of tissues of three species of carp by using the protocol of DNA extraction with Sodium Chloride (NaCl). The samples were subjected to DNA extraction, and the integrity was visualized on 1.5% agarose gel. In a total of 72 samples used for DNA extraction, all of them were positive, confirmed by the presence of bands on agarose gel. The capacity of amplifying the extracted DNA was tested by amplification reactions using the RAPD (random amplification of polymorphic DNA) technique, confirming that the DNA was of good quality for use in subsequent studies with molecular markers.

Com o avanço da piscicultura e das pesquisas na área da genética molecular, existe a necessidade de estudos que possam aperfeiçoar a obtenção de DNA com boa qualidade. Diante disso, o objetivo desse trabalho foi avaliar a eficiência da extração de DNA em diferentes tipos de tecidos de três espécies de carpas, através do protocolo de extração de DNA com cloreto de sódio (NaCl). As amostras foram submetidas à extração de DNA e a integridade foi visualizada em gel de agarose 1,5%. Em um total de 72 amostras utilizadas na extração de DNA, todas foram positivas, confirmadas com a presença de banda no gel de agarose. A capacidade de amplificação do DNA extraído foi testada através de reações de amplificação utilizando a técnica RAPD (polimorfismos de DNA amplificados ao acaso), confirmando que o DNA apresenta boas condições para utilização em estudos posteriores com marcadores moleculares.

Analysis of factor V Leiden and prothrombin mutations in patients with suspected thrombophilia in São Paulo state-Brazil / Análise da mutação no fator V de Leiden e na protrombina em pacientes do estado de São Paulo, Brasil

INTRODUCTION: Prothrombin (factor II) is a thrombin precursor, which induces fibrin formation. A mutation in the prothrombin gene (G20210A) has been described, which is directly associated with high prothrombin levels, hence thrombophilia. G1691A mutation in the factor V Leiden (FVL) gene occurs on exon 10, one of the main cleavage sites for protein C activation, resulting in protein alteration. OBJECTIVE: To identify and estimate the genotype frequency of the three possible genotypes and the frequency of the two existing alleles in the FVL and prothrombin genes in patients with suspected thrombophilia in the state of Sao Paulo. This study may provide more literature and reference data on the incidence of prothrombin genotypes among individuals in Brazil. MATERIAL AND METHODS: Analysis of point mutation by real time polymerase chain reaction (RT-PCR). RESULTS: We obtained a total of 100 individuals, from which 94% had the homozygous G genotype. Only 6% had heterozygous genotype and there was no individual with the homozygous genotype A for FVL gene. As to the prothrombin gene, the frequency was 97% for homozygous G genotype and 3% for the heterozygous genotype. There was no patient with the homozygous A genotype. CONCLUSION: This study demonstrated that the genotype identification of these genes is advisable for patients with suspected thrombophilia in this region.

INTRODUÇÃO: A protrombina (fator II) é a precursora da trombina, que induz a formação de fibrina. Foi descrita uma mutação no gene da protrombina (G20210A), associado diretamente a altos níveis de protrombina no sangue e, consequentemente, a trombofilia. A mutação G1691A no gene do fator V de Leiden (FLV) localiza-se no éxon 10, resultando na alteração da proteína, um dos principais sítios de clivagem para ativação da proteína C. OBJETIVOS: Identificar e estimar a frequência genotípica dos três possíveis genótipos, assim como estimar a frequência dos dois alelos existentes no gene do FLV e na protrombina em pacientes com suspeita de trombofilia no estado de São Paulo. Este estudo poderá fornecer mais dados para a literatura e para consulta da incidência dos genótipos da protrombina em indivíduos no Brasil. MATERIAL E MÉTODOS: Análise de mutação pontual por reação em cadeia da polimerase em tempo real (RT-PCR). RESULTADO: Obtivemos o número de 100 indivíduos e, desse total, 94% possuíam o genótipo para homozigoto G; apenas 6%, genótipo heterozigoto; nenhum indivíduo foi encontrado com genótipo homozigoto A no gene do FLV. No gene da protrombina, a frequência foi de 97% para o genótipo homozigoto G e 3% para o genoma heterozigoto; não foi encontrado nenhum indivíduo com o genoma homozigoto A. CONCLUSÃO: Este estudo mostrou que é recomendável a identificação do genótipo para esses genes em pacientes com suspeita de trombofilia nessa região.

Prevalência de Chlamydia trachomathis em amostras endocervicais de mulheres em São Paulo e Santa Catarina pela PCR / Prevalence of Chlamydia trachomatis in endocervical samples by PCR in São Paulo and Santa Catarina

INTRODUÇÃO: Nenhuma outra doença sexualmente transmissível (DST) tem mostrado frequência tão elevada quanto a infecção por Chlamydia trachomatis (CT). É frequente a detecção de mulheres portadoras de danos tubários causados por esse agente, determinando infertilidade permanente e as intervenções cirúrgicas não têm demonstrado sucesso em reparar esses danos. A reação em cadeia da polimerase (PCR) se mostrou mais sensível do que a cultura para a identificação de CT, principalmente em cervicite clamidiana nas mulheres. A PCR promove a detecção de sequências específicas de nucleotídeos para a CT. OBJETIVO: Analisar a prevalência de infecções causadas pela CT em mulheres nos estados de São Paulo e Santa Catarina utilizando amostras endocervicais. MATERIAIS E MÉTODOS Utilizaram-se para o presente trabalho amostras enviadas pelos laboratórios conveniados ao Genolab, pertencentes aos estados de São Paulo e de Santa Catarina. Foram consultados os resultados dos laudos de exames para CT oriundos do banco de dados do Genolab no ano de 2010. Para a obtenção e o isolamento do ácido desoxirribonucleico (DNA), utilizou-se a técnica de fenol-clorofórmio e para a amplificação do material genético, a técnica de PCR. RESULTADOS: Obteve-se uma amostra de 287 indivíduos, e desse total 56,45% das mulheres eram positivas. A amostra que obteve o maior número de positivos foi o swab endocervical, com 75%. CONCLUSÃO: As amostras biológicas provenientes do endocérvix apresentaram detecção eficiente da CT na população feminina. A alta prevalência salienta a importância no emprego do diagnóstico molecular, principalmente por este trabalho apontar esse aspecto.

INTRODUCTION: No other sexually transmitted disease (STD) has been as frequent as Chlamydia trachomatis (CT) infection. Tubal damage caused by this agent has been frequently detected among women. This infection causes permanent infertility. Furthermore, surgical interventions have not demonstrated success in repairing tubal damage. The polymerase chain reaction (PCR) has proved to be more sensitive than culture to the identification of CT mainly in women with chlamydial cervicitis.PCR promotes the detection of specific nucleotide sequences in CT. OBJECTIVE: To analyze the prevalence of infections caused by CT in women in São Paulo and Santa Catarina states by use of endocervical samples. MATERIAL AND METHODS: In this study we used samples from laboratories in São Paulo and Santa Catarina states, which are associated with Genolab. CT examination result reports from 2010 obtained from Genolab database were analyzed. The phenol-chloroform protocol was used to obtain and isolate deoxyribonucleic acid (DNA) and the (PCR) method was used to amplify genetic material. RESULTS: We obtained a sample of 287 individuals, of which 56.45% were positive. Endocervical swab samples showed the highest positive results (75%). CONCLUSION: Endocervical samples constituted an accurate detection of CT. The high prevalence emphasizes the importance of molecular diagnosis, which is also corroborated by this study.

Análise da mutação G20210A no gene da protrombina (fator II) em pacientes com suspeita de trombofilia no sul do Brasil / Analysis of prothrombin G20210A mutation (factor II) in patients with suspected trombophilia in Southern Brazil

INTRODUÇÃO: A protrombina (fator II) é uma proteína sanguínea sintetizada no fígado com a presença de vitamina K. É a precursora da trombina, que induz a formação de fibrina. Foi descrita uma mutação no gene da protrombina G20210A, associada diretamente a altos níveis de protrombina no sangue e, consequentemente, à trombofilia. Essa variante alélica consiste em mutação pontual, também chamada de polimorfismo de nucleotídeo simples (SNP), ocasionando a troca de uma guanina por uma adenina no nucleotídeo 20210, localizado em um sítio de clivagem do precursor do ácido ribonucleico mensageiro (mRNA). Essa troca caracteriza o alelo A e a ausência da mutação do alelo G. OBJETIVO: Quantificar o número de indivíduos homozigotos para alelo G, homozigotos para alelo A e heterozigotos, cujas amostras foram enviadas para o laboratório Genolab Análises Genéticas, abrangendo os estados do Paraná e Santa Catarina, no período de 1º de janeiro de 2009 a 10 de outubro de 2010. MÉTODOS: Análise de mutação pontual por reação em cadeia da polimerase em tempo real (RT-PCR). RESULTADOS: Obtivemos o número de 243 indivíduos e desse total 51,03% eram oriundos do estado do Paraná, enquanto 48,97%, oriundos do estado de Santa Catarina. Do total analisado, 88,89% possuíam o genótipo para homozigoto G, e nenhum indivíduo foi encontrado com mutação para homozigoto A. Apenas 11,11% possuíam genótipo heterozigoto. O estado de Santa Catarina apresentou frequência superior para genótipo heterozigoto em relação ao Paraná. CONCLUSÃO: Este estudo mostrou que é recomendável a identificação do genótipo para esse gene em pacientes com suspeita de trombofilia nos dois estados.

INTRODUCTION: Prothrombin (factor II) is a blood protein synthesized in the liver in the presence of vitamin K. It is a thrombin precursor, which induces fibrin formation. Prothrombin G20210A mutation and high prothrombin levels have been closely associated with thrombophilia. This allelic variant is a single mutation, also denominated single nucleotide polymorphism (SNP), in which guanine is replaced with adenine in the messenger ribonucleic acid (mRNA) cleavage of nucleotide 20210. The replacement is characterized by the presence of allele A and the absence of mutation in allele G. OBJECTIVE: To quantify the number of individuals homozygous for allele G, allele A and heterozygotes. The samples were collected in Paraná and Santa Catarina from January 1st, 2009 to October 10th, 2010 and were sent to Genolab Análises Genéticas. METHODS: Analysis of single mutation by polymerase chain reaction in real time (RT-PCR). RESULTS: From 243 individuals, 51.03% were from Paraná and 48.97% were from Santa Catarina. 88.89% individuals were homozygous for G genotype, none of them were homozygous for A. Only 11.11% were heterozygotes. Santa Catarina presented a higher frequency in heterozygous genotype in comparison with Paraná. CONCLUSION: This study showed that patients with suspected thrombophilia should undergo genotype identification in both states.