Detection of Lactobacillus and Gardnerella species in vaginal samples by PNA-FISH

Bacterial vaginosis (BV) is a common vaginal infection of women in reproductive age. This infection is initially asymptomatic and late diagnosis can increase the health costs and hamper treatment. Therefore, an easy and quick method to accurately detect the transition between normal microflora to the predecessors of infection is of upmost importance. Despite the fact that BV etiology remains unknown, it is commonly accepted that the microbial switch from normal to BV state is characterized by a decrease in vaginal colonization by Lactobacillus species together with an increase in the number of Gardnerella vaginalis and others anaerobes. G. vaginalis is found in normal vaginal epithelium but recent findings suggested that G. vaginalis biofilm can be responsible for the establishment of BV, allowing other anaerobes to grow. In this work, our goal was to develop two novel Peptid Nucleic Acid (PNA) probes for Fluorescence in situ Hybridization (FISH) multiplex analysis, aimed to detect Lactobacillus spp. and G. vaginalis in vaginal samples. We designed, evaluated and validated both probes by using 36 representative Lactobacillus strains, 22 representative Gardnerella vaginalis strains and 27 others taxonomically related or pathogenic bacteria strains usually found in vaginal samples. The probes were also tested in 17 vaginal samples, collected from women with normal vaginal microflora or women with BV. These probes proved to be a powerful tool for the simultaneous detection and visualization of Lactobacillus spp. and G. vaginalis in bacterial strain collections and vaginal samples, with higher specificity and sensitivity than similar reported probes . More precisely, theoretical specificity and sensitivity from PNA probes were 99.65% and 91.50%, respectively, for Lactobacillus spp. and 99.9999% and 95.00%, respectively, for G. vaginalis

Presence of Gardnerella vaginalis in healthy portuguese women: a pilot study

Bacterial vaginosis (BV) has an important position worldwide, as the leading vaginal disorder in women, and affects 30-50% of African women and 10-20% of White women of reproductive age. This condition although not mortal causes great discomfort and may lead to other complications such as pre-term labour or increase susceptibility for HIV infection. During BV occur a decrease of Lactobacillus spp. present in the vaginal epithelium and an increase in the number of anaerobic microorganisms like Gardnerella vaginalis, Pretovella spp., Mobilincus spp.; Mycoplasma hominis and Atopobium vaginae. Gardnerella vaginalis is also responsible for the formation of a biofilm in the vaginal epithelium in sick women’s. However, the direct correlation between the pathology and the causing agent (or agents) has not been clearly established. Currently there is only one article in PubMed (Guerreiro et. al, 1998) referring to the prevalence of bacterial vaginosis in Portugal and we aim to extend the research in this field specifically to the portuguese population. As part of this effort one of our aims it to characterise the bacterial population of portuguese women both healthy and diagnosed with BV. As such we collected swab samples of vaginal fluids from protuguese women with the help of health professionals and using self collection. The swabs were collected and treated within 24 hours at the University of Minho for the characterization of the bacterial population present, by using conventional microbiological growth techniques, PNA-FISH microscopy and 16S PCR. It was found that about 20% of the samples tested possessed G. vaginalis and all possessed Lactobacillus spp. using all 3 identification techniques described. This result is consistent with previous reports of G. vaginalis prevalence althought slightly lower, and shows that traditional microbiological techniques, microscopy and molecular methods were consistent in terms of results

Cost of Bordetella pertussis illness in tertiary hospitals in Argentina

La Comisión Nacional de Inmunizaciones y el ProNaCEI (Programa Nacional de Control de Enfermedades Inmunoprevenibles) actualizaron la política de vacunación por Bordetella pertussis (BP) a partir del año 2009 con el objetivo de optimizar el control de esta enfermedad, de acuerdo con las recomendaciones internacionales. Para evaluar el impacto económico de esta nueva política de vacunación resulta necesario conocer inicialmente el costo que implica para el sistema de salud un niño internado o ambulatorio con infección por BP. El objetivo de este estudio fue describir el perfl de costos en niños internados o tratados ambulatoriamente, con infección confrmada por laboratorio de BP en tres hospitales de la Argentina. Estudio prospectivo de costo de la enfermedad durante el período diciembre de 2010 a marzo de 2012. Resultados. El costo total para toda la cohorte fue de 1 170 663,32 pesos (236 497,64 dólares); los costos médicos directos, de 1 124 052,31 pesos (227 081,27 dólares); los costos indirectos y gastos de bolsillo, de 46611 pesos (9 416,36 dólares), lo que permite inferir un costo total promedio por paciente de 10 546,52 pesos (IC 95% 9009 a 13 840) (2130,60 dólares, IC 95% 1820 a 2795), costos médicos directos por paciente de 10 126,6 pesos (IC 95% 8607 a 13 171) (2045,77 dólares, IC 95%1738 a 2660) y costos indirectos más de bolsillo (viajes y extras) de 419,92 pesos (IC 95% 344,7 a 565,3), (84 dólares, IC 95% 69 a 115). Conclusión. El costo de un caso confrmado hospitalizado por BP es 10 546,52 pesos (IC 95% 9009 a 13 840) (2130,60 dólares, IC 95% 1820 a 2795). Los costos directos no médicos y costos indirectos constituyen el 4% del total, lo que corresponde a 419,91 pesos por familia (84 dólares, IC 95% 69 a 115), un 8% del salario promedio.Fil: Gentile, Angela. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "ricardo Gutierrez"; ArgentinaFil: Salgueiro, Ana L.. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "ricardo Gutierrez"; ArgentinaFil: Garcia Bournissen, Facundo. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "r. Gutierrez". Servicio de Parasitología y Chagas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Romanin, Viviana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "ricardo Gutierrez"; ArgentinaFil: Bulgheroni, Sonia. Hospital Materno Infantil de San Isidro "Dr. Gianantonio"; ArgentinaFil: Gaiano, Alejandra. Hospital Materno Infantil de San Isidro "Dr. Gianantonio"; ArgentinaFil: Benegas, Liliana. Hospital de Niños "Víctor J. Vilela"; ArgentinaFil: Uboldi, Andrea. Hospital de Niños "Víctor J. Vilela"; ArgentinaFil: Giglio, Norberto. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "ricardo Gutierrez"; Argentin

Comparison between classical and molecular (FISH and PCR) methods for Lactobacillus spp. detection in clinical samples

Lactobacillus species constitute the main beneficial bacteria in our body by inhibiting the growth from pathogenic microorganisms. Fluorescence In Situ Hybridization (FISH) is an ideal method for cultivation-independent detection of microorganisms in microbial communities or clinical samples. Therefore, the current aims of this research are to identify and discriminate Lactobacillus spp. contained in clinical samples by the use of PNA-FISH methodology. In spite this method is proved to be useful to visualize target cells in natural habitats, it wasn't possible to find a Lactobacillus spp. 16S conservative region that allowed an unique and efficient identification in clinical samples. To overcome this problem, we used morphological visualization to differentiate Lactobacillus genus from another relative genera of the same Lactobacillaceae order. In addition, we also needed to overcome some methodological limitations, such as minimizing probe penetration problems and increasing hybridization efficiencies. As a result, we investigated the effect of different pre-treatment procedures of the exopolymer cell walls prior to the hybridization step, such as, several types of fixation compounds (paraformaldehyde and ethanol percentages), buffer steps and enzymatic (lysozyme and protease) pre-treatment. Furthermore, we modified PNA FISH protocol in several steps, for instance, hybridization and washing steps. In resume, the use of PNA probe specific for Lactobacillus spp. in situ hybridization by fluorescence microscopy could be perfectly used to study the complex and spatial organization of vaginal microbial samples. To conclude, we validate Lactobacillus spp. PNA probe by FISH to quantify and characterize in mixed microbiologic populations present in clinical samples

Fluorescence in situ hybridization method using peptide nucleic acid probes for rapid detection of Lactobacillus and Gardnerella spp

Background: Bacterial vaginosis (BV) is a common vaginal infection occurring in women of reproductive age. It is widely accepted that the microbial switch from normal microflora to BV is characterized by a decrease in vaginal colonization by Lactobacillus species together with an increase of Gardnerella vaginalis and other anaerobes. Our goal was to develop and optimize a novel Peptide Nucleic Acid (PNA) Fluorescence in situ Hybridization assay (PNA FISH) for the detection of Lactobacillus spp. and G. vaginalis in mixed samples. Results: Therefore, we evaluated and validated two specific PNA probes by using 36 representative Lactobacillus strains, 22 representative G. vaginalis strains and 27 other taxonomically related or pathogenic bacterial strains commonly found in vaginal samples. The probes were also tested at different concentrations of G. vaginalis and Lactobacillus species in vitro, in the presence of a HeLa cell line. Specificity and sensitivity of the PNA probes were found to be 98.0% (95% confidence interval (CI), from 87.8 to 99.9%) and 100% (95% CI, from 88.0 to 100.0%), for Lactobacillus spp.; and 100% (95% CI, from 92.8 to 100%) and 100% (95% CI, from 81.5 to 100.0%) for G. vaginalis. Moreover, the probes were evaluated in mixed samples mimicking women with BV or normal vaginal microflora, demonstrating efficiency and applicability of our PNA FISH. Conclusions: This quick method accurately detects Lactobacillus spp. and G. vaginalis species in mixed samples, thus enabling efficient evaluation of the two bacterial groups, most frequently encountered in the vagina.This work was supported by European Union funds (FEDER/COMPETE) and by national funds (FCT) under the project with reference FCOMP-01-0124-FEDER-008991 (PTDC/BIA-MIC/098228/2008). AM acknowledges the FCT individual fellowship - SFRH/BD/62375/2009)

Costs of Bordetella pertussis in tertiary hospitals of Argentina

The National Immunization Commission and the National Program for the Control of Vaccine-Preventable Diseases (Programa Nacional de Control de Enfermedades Inmunoprevenibles, ProNaCEI) updated the immunization policy in relation to Bordetella pertussis (BP) in 2009 in order to improve the control of this disease in accordance with international recommendations. To evaluate the financial impact of this new immunization policy, we must first know the cost on the health system of having a hospitalized or outpatient child infected with BP. The objective of this study was to describe the profile of costs of hospitalized or outpatient children with laboratory-confirmed BP infection in three hospitals of Argentina. This was a prospective study of the cost of BP in the period between December 2010 and March 2012. Results. The total cost for the entire cohort was 1 170 663.32 ARS (236 497.64 USD); direct medical costs were 1 124 052.31 ARS (227 081.27 USD); indirect costs and out-of-pocket expenses were 46 611 ARS (9416.6 USD). From this data, it is possible to conclude that the total average cost per patient was 10 546.52 ARS (95% CI: 9009-13 840) (2130.60 USD, 95% CI: 1820-2795), the direct medical cost per patient was 10 126.6 ARS (95% CI: 8607-13 171) (2045.77 USD, 95% CI: 1738-2660), and the indirect plus out-of-pocket costs (transportation and extras) were 419.92 ARS (95% CI: 344.7-565.3) (84 USD, 95% CI: 69-115). Conclusion. The cost of a hospitalized child with confirmed BP is 10 546.52 ARS (95% CI: 9009-13 840) (2130.60 USD, 95% CI: 1820-2795). Direct non-medical costs and overhead costs account for 4% of the total cost, amounting to 419.91 ARS per family (84 USD, 95% CI: 69-115), approximately an 8% of an average salary. © 2013 Sociedad Argentina de Pediatría

Cripto-independent Nodal signaling promotes positioning of the A-P axis in the early mouse embryo

During early mouse development, the TGF beta-related protein Nodal specifies the organizing centers that control the formation of the anterior-posterior (A-P) axis. EGF-CFC proteins are important components of the Nodal signaling pathway, most likely by acting as Nodal coreceptors. However, the extent to which Nodal activity depends on EGF-CFC proteins is still debated. Cripto is the earliest EGF-CFC gene expressed during mouse embryogenesis and is involved in both A-P axis orientation and mesoderm formation. To investigate the relation between Cripto and Nodal in the early mouse embryo, we removed the Nodal antagonist Cerberus 1 (Cer1) and simultaneously Cripto, by generating Cer1;Cripto double mouse mutants. We observed that two thirds of the Cer1,Cripto double mutants are rescued in processes that are severely compromised in Cripto(-/-) embryos, namely A-P axis orientation, anterior mesendoderm and posterior neuroectoderin formation. The observed rescue is strongly reduced in Cer1;Cripto;Nodal triple mutants, suggesting that Nodal can signal extensively in the absence of Cripto, if Cer1 is also inhibited. This signaling activity drives A-P axis positioning. Our results provide evidence for the existence. of Cripto-independent signaling mechanisms, by which Nodal controls axis specification in the early mouse embryo. (C) 2007 Elsevier Inc. All rights reserved

Nursing Students' Perceptions on Healthcare-Associated Infection Control and Prevention Teaching and Learning Experience: Development and Validation of a Scale in Four European Countries

Healthcare-associated infections are one of the major concerns worldwide. This study presents the development and the validation process of the InovSafeCare scale and aimed at identifying and measuring the ecosystem variables related to healthcare-associated infection (HCAI) prevention and control practices in European nurse students. Qualitative and quantitative approaches were used to (1) elaborate an item pool related to the educational environment, the healthcare setting environment, and the attitudes, beliefs, and performance of the nursing students regarding HCAI prevention and control and (2) analyze psychometric properties of the scale using factor analysis. The validated InovSafeCare scale was applied to undergraduate nursing students of five European Higher Education Institutions. The partial least square structural equation modeling (PLS-SEM) method with SMART-PLS3 software was used. The study sample consists of 657 nursing students, who responded a self-report inventory. From the analyzed data were identified 14 factors. The InovSafeCare scale reveals good validity and reliability of the dimensions in different European countries.info:eu-repo/semantics/publishedVersio

Nursing Students’ Perceptions on Healthcare-Associated Infection Control and Prevention Teaching and Learning Experience in Portugal

Healthcare-associated infections (HAI) are one of the major concerns worldwide, posing significant challenges to healthcare professionals’ education and training. This study intended to measure nursing students’ perceptions regarding their learning experiences on HAI prevention and control. In the first phase of the study, a cross-sectional and descriptive study with a convenience sample composed of undergraduate nursing students from Portugal, Spain, Poland, and Finland was conducted to develop the InovSafeCare questionnaire. In the second phase, we applied the InovSafeCare scale in a sample of nursing students from two Portuguese higher education institutions to explore which factors impact nursing students’ adherence to HAI prevention and control measures in clinical settings. In phase one, the InovSafeCare questionnaire was applied to 1326 students internationally, with the instrument presenting adequate psychometric qualities with reliability results in 14 dimensions. During phase two, the findings supported that Portuguese nursing students’ adherence to HAI prevention and control measures is influenced not only by the curricular offerings and resources available in academic settings, but also by the standards conveyed by nursing tutors during clinical placements. Our findings support the need for a dedicated curricular focus on HAI prevention and control learning, not only through specific classroom modules, innovative resources, and pedagogical approaches, but also through a complementary and coordinated liaison between teachers and tutors in academic and clinical settings.info:eu-repo/semantics/publishedVersio