Diagnosis of Human Leptospirosis in a Clinical Setting: Real-Time PCR High Resolution Melting Analysis for Detection of Leptospira at the Onset of Disease:

Currently, direct detection of Leptospira can be done in clinical laboratories by conventional and by real-time PCR (qRT-PCR). We tested a biobank of paired samples of serum and urine from the same patient (202 patients) presenting at the hospital in an area endemic for leptospirosis using qRT-PCR followed by high resolution melting (HRM) analysis. The results were compared with those obtained by conventional nested PCR and with the serologic gold standard microscopic agglutination test (MAT). Differences were resolved by sequencing. qRT-PCR-HRM was positive for 46 of the 202 patients (22.7%, accuracy 100%) which is consistent with known prevalence of leptospirosis in the Azores. MAT results were positive for 3 of the 46 patients (6.5%). Analysis of paired samples allowed us to identify the illness point at which patients presented at the hospital: onset, dissemination or excretion. The melting curve analysis of Leptospira species revealed that 60.9% (28/46) of patients were infected with L. interrogans and 39.1% (18/46) were infected with L. borgpetersenii, both endemic to the Azores. We validated the use of qRT-PCR-HRM for diagnosis of leptospirosis and for identification of the Leptospira species at the earliest onset of infection in a clinical setting, in less than 2 hours.publishersversionpublishe

Detection of Escherichia coli O157 by Peptide Nucleic Acid Fluorescence In Situ Hybridization (PNA-FISH) and Comparison to a Standard Culture Method

Despite the emergence of non-O157 shiga toxin-producing Escherichia coli (STEC) infections, E. coli O157 serotype is still the most commonly identified STEC in world. It causes high morbidity and mortality, and has been responsible for a number of outbreaks in many parts of the world. Various methods have been developed to detect this particular serotype, but standard bacteriological methods remain as the gold standard.In here, we propose a new peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method for the rapid detection of E. coli O157.Testing on 54 representative strains showed that the PNA probe is highly sensitive and specific to E. coli O157. Then, the method was optimized for the detection in food samples. Ground beef and unpasteurized milk samples were artificially contaminated with E. coli O157 concentrations ranging from 1×10-2 to 1×102 CFU per 25g or ml of food. Samples were then pre-enriched and analyzed by both the traditional bacteriological method (ISO 16654:2001) and PNA-FISH. The PNA-FISH method performed well in both types of food matrixes with a detection limit of 1 CFU/25 g or ml of food samples. Tests on 60 food samples have shown a specificity value of 100% (95% CI 82.83 - 100), a sensitivity of 97.22% (95% CI, 83.79 - 99.85) and an accuracy of 98,33% (CI 95%, 83.41 - 99.91). Results indicate that PNA-FISH performed as well as the traditional culture methods and can reduce the diagnosis time to 1 day.This work was supported by the Portuguese Institute Fundacao para a Ciencia e Tecnologia (FCT), project PIC/IC/82815/2007. C.A. acknowledges FCT for individual postdoctoral fellowship SFRH/BPD/74480/2010. We also acknowledge Biomode S.A. for providing some supplies for this project

Effect of chitosan-Aloe vera coating on postharvest quality of blueberry (Vaccinium corymbosum) fruit

The present study was carried out to evaluate the effect of chitosan-based edible coatings with Aloe vera extract on the postharvest blueberry fruit quality during storage at 5 °C. Firstly, A. vera fractions (pulp and liquid) were extracted from leaves and evaluated in terms of antifungal and antioxidant capacities. The choice of the most adequate chitosan and A. vera fraction concentrations to be incorporated in coating formulation was made based on the wettability of the corresponding coating solutions. Coatings with 0.5% (w/v) chitosan + 0.5% (w/v) glycerol + 0.1% (w/v) Tween 80 + 0.5% (v/v) A. vera liquid fraction presented the best characteristics to uniformly coat blueberry surface. Physico-chemical (i.e., titratable acidity, pH, weight loss) and microbiological analyses of coated blueberries (non-inoculated or artificially inoculated with Botrytis cinerea) were performed during 25 d. Microbiological growth and water loss levels were approximately reduced by 50% and 42%, respectively, in coated blueberries after 25 d compared to uncoated blueberries. After 15 d, weight loss values were 6.2% and 3.7% for uncoated and chitosanA. vera coated blueberries, respectively. Uncoated fruits presented mold contamination after 2 d of storage (2.0 ± 0.32 log CFU g1), whilst fruits with chitosan-based coatings with A. vera presented mold contamination only after 9 d of storage (1.3 ± 0.35 log CFU g1). Overall, coatings developed in this study extend blueberries shelf-life for about 5 d, demonstrating for the first time that the combination of chitosan and A. vera liquid fraction as edible coating materials has great potential in expanding the shelf-life of fruits.Joana T. Martins (SFRH/BPD/89992/2012) is the recipient of a fellowship from the Fundação para a Ciência e Tecnologia (FCT, Portugal). María L. Flores-López thanks Mexican Science and Technology Council(CONACYT, Mexico) for PhD fellowship support (CONACYT Grant number: 215499/310847). The authors thank the FCT the strategic funding of UID/BIO/04469/2013 unit and the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER- 027462)

Primiparous mothers' knowledge about child development: a study in Itajaí, SC

A presente pesquisa teve como objetivo avaliar conhecimentos que mães primíparas têm ao longo de primeiro ano de vida de seu filho sobre o processo de desenvolvimento infantil. Participaram da pesquisa 109 mães primíparas, maiores de 18 anos, com filho ou filha de até um ano de vida e que viviam na cidade de Itajaí (SC). Para sua realização foi utilizado o KIDI (Knowledge of Infant Development Inventory) e para identificar o nível socioeconômico dos pais foi utilizada a Escala de Avaliação de Status Socioeconômico de Hollingshead. Através de análise estatística, foi verificado que fatores sócio-demográficos, tais como: escolaridade e ocupação da mãe estão correlacionados significativamente com seu conhecimento sobre cuidados parentais. Foi constatado que o maior conhecimento das mães é sobre cuidados parentais (crenças, estratégias e comportamentos dos pais). Conclui-se que o status socioeconômico é um fator importante na determinação do conhecimento da mãe sobre sua criança.The central focus of this study was to evaluate primiparous mothers' knowledge about parenting and child development throughout the first year of life of their children. Participants were 109 primiparous mothers, aged 18 years or older, living in Itajaí (Santa Catarina), with a child aged 1-12 months. The KIDI (Knowledge of Infant Development Inventory) was employed to evaluate mothers' conceptions of infant development and the Hollingshead Four Factor Index of Socioeconomic Status was used to evaluate the socioeconomic level of mothers' and their families'. Through statistical analysis it was verified that mothers' educational attainment and occupation correlated significantly with their knowledge on parenting and child development. The study revealed that mothers had more knowledge about parenting (beliefs, strategies and practices of the parents) when compared with other types of knowledge. The study concluded that the mothers' socioeconomic level is an important factor related to their knowledge about the child development

Metal accumulation in female green sea turtles (Chelonia mydas) from Eastern Atlantic affects their egg quality with potential implications for embryonic development

Sea turtles, with their global distribution and complex life cycle, often accumulate pollutants such as metals and metalloids due to their extended lifespan and feeding habits. However, there are limited studies exploring the impact of metal pollution on the reproductive health of female sea turtles, specifically focusing on the quality of their eggs, which has significant implications for the future generations of these charismatic animals. São Tomé Island, a crucial nesting and feeding habitat for green sea turtles, underscores the urgent need for comprehensive research in this ecologically significant area. This study aimed to investigate whether metals and metalloids in the blood of nesting female green sea turtles induce genotoxic effects in their erythrocytes and affect their egg morphometric characteristics and the composition of related compartments. Additionally, this study aimed to evaluate whether the quality of energetic reserves for embryo development (fatty acids in yolk's polar and neutral lipids) is influenced by the contamination status of their predecessors. Results revealed correlations between Cu and Hg levels and increased "lobed" erythrocytes, while As and Cu negatively influenced shell thickness. In terms of energy reserves, both polar and neutral lipid fractions contained primarily saturated and monounsaturated fatty acids, with prevalent 18:1n-9, 18:0, 16:0, 14:0, and 12:0 fatty acids in yolk samples. The yolk polar fraction was more susceptible to contaminant levels in female sea turtles, showing consistent negative correlations between pollution load index and essential n3 fatty acids, including linolenic, eicosatrienoic, eicosapentaenoic, and docosapentaenoic acids, crucial for embryonic development. These metals accumulation, coupled with the reduced availability of these key fatty acids, may disrupt the eicosanoid and other important pathways, affecting reproductive development. This study reveals a negative correlation between metal contamination in female sea turtles' blood and egg lipid reserves, raising concerns about embryonic development and the species' future generations

Effect of chronic treatment with Rosiglitazone on Leydig cell steroidogenesis in rats: In vivo and ex vivo studies

<p>Abstract</p> <p>Background</p> <p>The present study was designed to examine the effect of chronic treatment with rosiglitazone - thiazolidinedione used in the treatment of type 2 diabetes mellitus for its insulin sensitizing effects - on the Leydig cell steroidogenic capacity and expression of the steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage enzyme (P450scc) in normal adult rats.</p> <p>Methods</p> <p>Twelve adult male Wistar rats were treated with rosiglitazone (5 mg/kg) administered by gavage for 15 days. Twelve control animals were treated with the vehicle. The ability of rosiglitazone to directly affect the production of testosterone by Leydig cells <it>ex vivo </it>was evaluated using isolated Leydig cells from rosiglitazone-treated rats. Testosterone production was induced either by activators of the cAMP/PKA pathway (hCG and dbcAMP) or substrates of steroidogenesis [22(R)-hydroxy-cholesterol (22(R)-OH-C), which is a substrate for the P450scc enzyme, and pregnenolone, which is the product of the P450scc-catalyzed step]. Testosterone in plasma and in incubation medium was measured by radioimmunoassay. The StAR and P450scc expression was detected by immunocytochemistry.</p> <p>Results</p> <p>The levels of total circulating testosterone were not altered by rosiglitazone treatment. A decrease in basal or induced testosterone production occurred in the Leydig cells of rosiglitazone-treated rats. The ultrastructural and immunocytochemical analysis of Leydig cells from rosiglitazone-treated rats revealed cells with characteristics of increased activity as well as increased StAR and P450scc expression, which are key proteins in androgen biosynthesis. However, a number of rosiglitazone-treated cells exhibited significant mitochondrial damage.</p> <p>Conclusion</p> <p>The results revealed that the Leydig cells from rosiglitazone-treated rats showed significant reduction in testosterone production under basal, hCG/dbcAMP- or 22 (R)-OH-C/pregnenolone-induced conditions, although increased labeling of StAR and P450scc was detected in these cells by immunocytochemistry. The ultrastructural study suggested that the lower levels of testosterone produced by these cells could be due to mitochondrial damage induced by rosiglitazone.</p