Association between the thrombophilic polymorphisms MTHFR C677T, Factor V Leiden, and prothrombin G20210A and recurrent miscarriage in Brazilian women.

Some cases of recurrent first trimester miscarriage have a thrombotic etiology. The aim of this study was to investigate the prevalence of the most common thrombophilic mutations - factor V (FV) Leiden G1691A (FVL), prothrombin (FII) G20210A, and methylenetetrahydrofolate reductase (MTHFR) C677T - in women with recurrent miscarriages. In this case-control study, we included 137 women with two or more consecutive first-trimester miscarriages (£12 weeks of gestation) and 100 healthy women with no history of pregnancy loss, and with at least one living child. DNA was extracted from the patient samples, and the relevant genes (FVL, FII, and MTHFR) were amplified by PCR, followed by restriction fragment length polymorphism, to assess the polymorphisms in these genes. The allelic frequencies of polymorphisms were not significantly different between the case and control groups. Polymorphisms in the MTHFR, FVL, and FII genes were not associated with recurrent miscarriage during the first trimester of pregnancy in Brazilian women (P = 0.479; P = 0.491 and P = 0.107, respectively). However, the etiologic identification of genetic factors is important for genetic counseling.

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 serotype O157 is still the most commonly identified STEC in the 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 the gold standard. 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. The method then was optimized for 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 × 10(2) CFU per 25 g or ml of food. Samples were then preenriched 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 matrices 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% confidence interval [CI], 82.83 to 100), a sensitivity of 97.22% (95% CI, 83.79 to 99.85%), and an accuracy of 98.33% (CI 95%, 83.41 to 99.91%). Results indicate that PNA-FISH performed as well as the traditional culture methods and can reduce the diagnosis time to 1 day.

Rapid detection of urinary tract infections caused by Proteus spp. using PNA-FISH.

We developed a fluorescence in situ hybridization (FISH) method for the rapid detection of Proteus spp. in urine, using a novel peptide nucleic acid (PNA) probe. Testing on 137 urine samples from patients with urinary tract infections has shown specificity and sensitivity values of 98 % (95 % CI, 93.2-99.7) and 100 % (95 % CI, 80,8-100), respectively, when compared with CHROMagar Orientation medium. Results indicate that PNA-FISH is a reliable alternative to traditional culture methods and can reduce the diagnosis time to approximately 2 h.

Application of flow cytometry for the identification of Staphylococcus epidermidis by peptide nucleic acid fluorescence in situ hybridization (PNA FISH) in blood samples.

Staphylococcus epidermidis is considered to be one of the most common causes of nosocomial bloodstream infections, particularly in immune-compromised individuals. Here, we report the development and application of a novel peptide nucleic acid probe for the specific detection of S. epidermidis by fluorescence in situ hybridization. The theoretical estimates of probe matching specificity and sensitivity were 89 and 87%, respectively. More importantly, the probe was shown not to hybridize with closely related species such as Staphylococcus aureus. The method was subsequently successfully adapted for the detection of S. epidermidis in mixed-species blood cultures both by microscopy and flow cytometry.

Effect of chlorine on incorporation of Helicobacter pylori into drinking water biofilms.

The use of a specific peptide nucleic acid (PNA) probe demonstrated that Helicobacter pylori persisted inside biofilms exposed to low concentrations of chlorine (0.2 and 1.2 mg liter(-1)) for at least 26 days, although no culturable cells were recovered. Coupled with data obtained using viability stains in pure culture, this result suggests that H. pylori can survive chlorination but remain undetectable by culture methods, which can be effectively replaced by PNA hybridization.

Fluorescence in situ hybridization method using a peptide nucleic acid probe for identification of Salmonella spp. in a broad spectrum of samples.

A fluorescence in situ hybridization (FISH) method for the rapid detection of Salmonella spp. using a novel peptide nucleic acid (PNA) probe was developed. The probe theoretical specificity and sensitivity were both 100%. The PNA-FISH method was optimized, and laboratory testing on representative strains from the Salmonella genus subspecies and several related bacterial species confirmed the predicted theoretical values of specificity and sensitivity. The PNA-FISH method has been successfully adapted to detect cells in suspension and is hence able to be employed for the detection of this bacterium in blood, feces, water, and powdered infant formula (PIF). The blood and PIF samples were artificially contaminated with decreasing pathogen concentrations. After the use of an enrichment step, the PNA-FISH method was able to detect 1 CFU per 10 ml of blood (5 x 10(9) +/- 5 x 10(8) CFU/ml after an overnight enrichment step) and also 1 CFU per 10 g of PIF (2 x 10(7) +/- 5 x 10(6) CFU/ml after an 8-h enrichment step). The feces and water samples were also enriched according to the corresponding International Organization for Standardization methods, and results showed that the PNA-FISH method was able to detect Salmonella immediately after the first enrichment step was conducted. Moreover, the probe was able to discriminate the bacterium in a mixed microbial population in feces and water by counter-staining with 4',6-diamidino-2-phenylindole (DAPI). This new method is applicable to a broad spectrum of samples and takes less than 20 h to obtain a diagnosis, except for PIF samples, where the analysis takes less than 12 h. This procedure may be used for food processing and municipal water control and also in clinical settings, representing an improved alternative to culture-based techniques and to the existing Salmonella PNA probe, Sal23S10, which presents a lower specificity.

Development and application of a novel peptide nucleic acid probe for the specific detection of Cronobacter genomospecies (Enterobacter sakazakii) in powdered infant formula.

Here, we report a fluorescence in situ hybridization (FISH) method for rapid detection of Cronobacter strains in powdered infant formula (PIF) using a novel peptide nucleic acid (PNA) probe. Laboratory tests with several Enterobacteriaceae species showed that the specificity and sensitivity of the method were 100%. FISH using PNA could detect as few as 1 CFU per 10 g of Cronobacter in PIF after an 8-h enrichment step, even in a mixed population containing bacterial contaminants.

Validation of SYTO 9/propidium iodide uptake for rapid detection of viable but noncultivable Legionella pneumophila.

Legionella pneumophila is an ubiquitous environmental microorganism that can cause Legionnaires' disease or Pontiac fever. As a waterborne pathogen, it has been found to be resistant to chlorine disinfection and survive in drinking water systems, leading to potential outbreaks of waterborne disease. In this work, the effect of different concentrations of free chlorine was studied (0.2, 0.7, and 1.2 mg l(-1)), the cultivability of cells assessed by standard culture techniques (buffered charcoal yeast extract agar plates) and viability using the SYTO 9/propidium iodide fluorochrome uptake assay (LIVE/DEAD BacLight). Results demonstrate that L. pneumophila loses cultivability after exposure for 30 min to 0.7 mg l(-1) of free chlorine and in 10 min when the concentration is increased to 1.2 mg l(-1). However, the viability of the cells was only slightly affected even after 30 min exposure to the highest concentration of chlorine; good correlation was obtained between the rapid SYTO 9/propidium iodide fluorochrome uptake assay and a longer cocultivation with Acanthamoeba polyphaga assay, confirming that these cells could still recover their cultivability. These results raise new concerns about the assessment of drinking water disinfection efficiency and indicate the necessity of further developing new validated rapid methods, such as the SYTO 9/propidium iodide uptake assay, to assess viable but noncultivable L. pneumophila cells in the environment.

Incorporation of natural uncultivable Legionella pneumophila into potable water biofilms provides a protective niche against chlorination stress.

Legionella pneumophila is a waterborne pathogen that has been isolated sporadically from drinking water distribution systems (DWDS). Resistance to disinfectants is mainly attributed to the association of cells with amoebae, but biofilms are also thought to provide some degree of protection. In the present work, a two-stage chemostat was used to form heterotrophic biofilms from drinking water to study the influence of chlorine on the presence of naturally occurring L. pneumophila. The pathogen was tracked in planktonic and sessile biofilm phases using standard culture recovery techniques for cultivable cells and a peptide nucleic acid fluorescence in situ hybridisation assay for total cells. The results showed that the total number of L. pneumophila cells in biofilms was not affected by the concentrations of chlorine tested, and the presence of L. pneumophila could not be detected by culturing. To restrict the outbreaks of disease caused by this bacterium, efforts need to be concentrated on preventing L. pneumophila from re-entering an infectious state by maintaining residual disinfectant levels through the entire DWDS network so that the resuscitation of cells via contact with amoebae is prevented.

Incorporation of natural uncultivable Legionella pneumophila into potable water biofilms provides a protective niche against chlorination stress.

Legionella pneumophila is a waterborne pathogen that has been isolated sporadically from drinking water distribution systems (DWDS). Resistance to disinfectants is mainly attributed to the association of cells with amoebae, but biofilms are also thought to provide some degree of protection. In the present work, a two-stage chemostat was used to form heterotrophic biofilms from drinking water to study the influence of chlorine on the presence of naturally occurring L. pneumophila. The pathogen was tracked in planktonic and sessile biofilm phases using standard culture recovery techniques for cultivable cells and a peptide nucleic acid fluorescence in situ hybridisation assay for total cells. The results showed that the total number of L. pneumophila cells in biofilms was not affected by the concentrations of chlorine tested, and the presence of L. pneumophila could not be detected by culturing. To restrict the outbreaks of disease caused by this bacterium, efforts need to be concentrated on preventing L. pneumophila from re-entering an infectious state by maintaining residual disinfectant levels through the entire DWDS network so that the resuscitation of cells via contact with amoebae is prevented.

Comparison between standard culture and peptide nucleic acid 16S rRNA hybridization quantification to study the influence of physico-chemical parameters on Legionella pneumophila survival in drinking water biofilms.

Legionella pneumophila is a waterborne pathogen that is mainly transmitted by the inhalation of contaminated aerosols. In this article, the influence of several physico-chemical parameters relating to the supply of potable water was studied using a L. pneumophila peptide nucleic acid (PNA) specific probe to quantify total L. pneumophila in addition to standard culture methods. A two-stage chemostat was used to form the heterotrophic biofilms, with biofilm generating vessels fed with naturally occurring L. pneumophila. The substratum was the commonly used potable water pipe material, uPVC. It proved impossible to recover cultivable L. pneumophila due to overgrowth by other microorganisms and/or the loss of cultivability of this pathogen. Nevertheless, results obtained for total L. pneumophila cells in biofilms using a specific PNA probe showed that for the two temperatures studied (15 and 20 degrees C), there were no significant differences when shear stress was increased. However, when a source of carbon was added there was a significant increase in numbers at 20 degrees C. A comparison of the two temperatures showed that at 15 degrees C, the total cell numbers for L. pneumophila were generally higher compared with the total microbial flora, suggesting that lower temperatures support the inclusion of L. pneumophila in drinking water biofilms. The work reported in this article suggests that standard culture methods are not accurate for the evaluation of water quality in terms of L. pneumophila. This raises public health concerns since culture methods are still considered to be the gold standard for assessing the presence of this opportunistic pathogen in water.

Persistence of Helicobacter pylori in heterotrophic drinking-water biofilms.

Although the route of transmission of Helicobacter pylori remains unknown, drinking water has been considered a possible transmission vector. It has been shown previously that, in water, biofilms are a protective niche for several pathogens, protecting them from stressful conditions, such as low carbon concentration, shear stress, and less-than-optimal temperatures. In this work, the influence of these three parameters on the persistence and cultivability of H. pylori in drinking-water biofilms was studied. Autochthonous biofilm consortia were formed in a two-stage chemostat system and then inoculated with the pathogen. Total numbers of H. pylori cells were determined by microscopy using a specific H. pylori 16S rRNA peptide nucleic acid probe, whereas cultivable cells were assessed by standard plating onto selective H. pylori medium. Cultivable H. pylori could not be detected at any time point, but the ability of H. pylori cells to incorporate, undergo morphological transformations, persist, and even agglomerate in biofilms for at least 31 days without a noticeable decrease in the total cell number (on average, the concentration was between 1.54 x 10(6) and 2.25 x 10(6) cells cm(-2)) or in the intracellular rRNA content may indicate that the loss of cultivability was due to entry into a viable but noncultivable state. Unlike previous results obtained for pure-culture H. pylori biofilms, shear stress did not negatively influence the numbers of H. pylori cells attached, suggesting that the autochthonous aquatic bacteria have an important role in retaining this pathogen in the sessile state, possibly by providing suitable microaerophilic environments or linking biomolecules to which the pathogen adheres. Therefore, biofilms appear to provide not only a safe haven for H. pylori but also a concentration mechanism so that subsequent sloughing releases a concentrated bolus of cells that might be infectious and that could escape routine grab sample microbiological analyses and be a cause of concern for public health.

Survival of gastric and enterohepatic Helicobacter spp. in water: implications for transmission.

Part of the reason for rejecting aquatic environments as possible vectors for the transmission of Helicobacter pylori has been the preference of this microorganism to inhabit the human stomach and hence use a direct oral-oral route for transmission. On the other hand, most enteric bacterial pathogens are well known for being able to use water as an environmental reservoir. In this work, we have exposed 13 strains of seven different Helicobacter spp. (both gastric and enterohepatic) to water and tracked their survival by standard plating methods and membrane integrity assessment. The influence of different plating media and temperatures and the presence of light on recovery was also assessed. There was good correlation between cultivability and membrane integrity results (Pearson's correlation coefficient = 0.916), confirming that the culture method could reliably estimate differences in survival among different Helicobacter spp. The species that survived the longest in water was H. pylori (>96 h in the dark at 25 degrees C), whereas H. felis appeared to be the most sensitive to water (<6 h). A hierarchical cluster analysis demonstrated that there was no relationship between the enterohepatic nature of Helicobacter spp. and an increased time of survival in water. This work assesses for the first time the survival of multiple Helicobacter spp., such has H. mustelae, H. muridarum, H. felis, H. canadensis, H. pullorum, and H. canis, in water under several conditions and concludes that the roles of water in transmission between hosts are likely to be similar for all these species, whether enterohepatic or not.

Real-time quantification of Pseudomonas fluorescens cell removal from glass surfaces due to bacteriophage varphiS1 application.

AIMS: To study the efficacy of the lytic phage varphiS1 in eliminating Pseudomonas fluorescens in the early stage of biofilm formation, using an in situ and real time methodology for cell quantification. METHODS AND RESULTS: Cell adhesion and phage infection studies were carried out in a parallel plate flow chamber under laminar conditions. Cells were allowed to adhere until reaching 1.7-1.8 x 10(6) cells cm(-2) and phage infection was performed with two different phage concentrations (2 x 10(9) PFU ml(-1) and 1 x 10(10) PFU ml(-1)). Phage concentration clearly affects the speed of infection. The less concentrated phage solution promoted a three times slower rate of cell removal but did not affect the overall percentage of cell removal. In fact, after a longer infection period the less concentrated phage solution reached the same 93% cell removal value. CONCLUSIONS: Phages are efficient in the eradication of bacterial cells at the early stage of biofilm formation and their presence at the surface did not allow bacterial recolonization of the surface. SIGNIFICANCE AND IMPACT OF THE STUDY: To date, no published studies have been made concerning in situ and real time quantification of cell removal from surfaces due to phage action.

The role of hydrodynamic stress on the phenotypic characteristics of single and binary biofilms of Pseudomonas fluorescens.

This study investigates the phenotype of turbulent (Re = 5,200) and laminar (Re = 2,000) flow-generated Pseudomonas fluorescens biofilms. Three P. fluorescens strains, the type strain ATCC 13525 and two strains isolated from an industrial processing plant, D3-348 and D3-350, were used throughout this study. The isolated strains were used to form single and binary biofilms. The biofilm physiology (metabolic activity, cellular density, mass, extracellular polymeric substances, structural characteristics and outer membrane proteins [OMP] expression) was compared. The results indicate that, for every situation, turbulent flow-generated biofilms were more active (p < 0.05), had more mass per cm(2) (p < 0.05), a higher cellular density (p < 0.05), distinct morphology, similar matrix proteins (p > 0.1) and identical (isolated strains -single and binary biofilms) and higher (type strain) matrix polysaccharides contents (p < 0.05) than laminar flow-generated biofilms. Flow-generated biofilms formed by the type strain revealed a considerably higher cellular density and amount of matrix polysaccharides than single and binary biofilms formed by the isolated strains (p < 0.05). Similar OMP expression was detected for the several single strains and for the binary situation, not dependent on the hydrodynamic conditions. Binary biofilms revealed an equal coexistence of the isolated strains with apparent neutral interactions. In summary, the biofilms formed by the type strain represent, apparently, the worst situation in a context of control. The results obtained clearly illustrate the importance of considering strain variation and hydrodynamics in biofilm development, and complement previous studies which have focused on physical aspects of structural and density differences.

Influence of biofilm composition on the resistance to detachment.

Bacillus cereus and Pseudomonas fluorescens were used to develop monoculture biofilms in a bioreactor rotating system using a stainless steel cylinder for biofilm formation. The biofilms were allowed to grow for 7 days, exposed continuously to a Reynolds number of agitation (ReA) of 2,400. Afterwards, the biofilms were characterised in terms of respiratory activity, amount of biomass, cellular density, cellular size and total and extracellular proteins and polysaccharides. The biofilm mechanical stability was assessed by sequential submission of the biofilms to increasing ReA, respectively, 4,000, 8,100, 12,100 and 16,100. The results showed that P. fluorescens biofilms were five times more active, had a higher amount of biomass, cellular density, a reduced cellular size and a four-fold higher amount of extracellular proteins and polysaccharides than B. cereus biofilms. The application of shear stress forces higher than the one under which the biofilm was formed (ReA = 2,400) caused biomass removal. The high percentage of removal occurred with the implementation of a ReA of 8,100 for both B. cereus and P. fluorescens biofilms. The total series of ReA did not give rise to total biofilm removal, as only about 76% of P. fluorescens biofilm mass and 53% of B. cereus biofilm mass were detached from the cylinders. This latter result evidences that B. cereus had a higher mechanical stability than P. fluorescens biofilms. The overall results demonstrate that P. fluorescens and B. cereus formed physiologically distinct biofilms, B. cereus biofilms mainly being constituted by cells and P. fluorescens biofilms largely constituted by extracellular proteins and polysaccharides. B. cereus biofilms had a substantially higher mechanical stability than P. fluorescens biofilms.

Use of fluorescent in situ hybridisation for the visualisation of Helicobacter pylori in real drinking water biofilms.

A fluorescently labelled peptide nucleic acid (PNA) probe has been applied for the in situ detection of Helicobacter pylori in drinking water biofilms. The method was originally applied to real pipe samples removed from a drinking water distribution system (DWDS) but the curvature and the heavy fouling of the pipes prevented an accurate detection of the bacterium by epifluorescence microscopy. Therefore, two semi-circular flow cells were placed in a bypass of the DWDS, and coupons with up to 72 days of exposure were regularly sampled and analysed for the presence of H. pylori. In the flat surfaces of the coupons, it was possible to sparsely detect cells exhibiting similar morphology to H. pylori that were emitting the PNA probe fluorescent signal. Coupons were also visualised under the microscope before the hybridisation procedure to serve as negative controls and ensure the validity of the method. This work corroborates the findings already published elsewhere that this bacterium might be present in DWDS biofilms. The method requires, however, highly trained personnel for an accurate detection of the pathogen and will need simplification before being routinely used in standard water analysis laboratories.

Genetic Analysis of Feline Panleukopenia Virus Full-length VP2 Gene in Domestic Cats Between 2006-2008 and 2012-2014, Portugal.

Feline panleukopenia virus (FPV) and canine parvovirus (CPV) are two closely related viruses, which cause acute gastroenteritis in carnivores, and cats may be infected by strains of both viruses. The viruses are found worldwide and may have changing host ranges and genetic variation that can be found around the world in some cases. Here, we screened a Portuguese population of cats by a conventional PCR assay for the presence of FPV/CPV viruses in faecal samples and tissues between 2006-2008 and 2012-2014. The sequence analysis of the complete VP2 gene showed that 18 of 31 animals tested were positive for FPV DNA, and no case of CPV infection was detected. The analysis of specific DNA detected three new non-synonymous substitutions in the VP2 gene that were found in single groups and were related to viruses reported elsewhere by phylogenetic analysis - some were related to Italian isolates, one was closely related to isolates from Vietnam and China, and two were related with older strains from the USA. The results of our study show that FPV circulated in the Portuguese cat population and as expected the found strains are slightly divergent from those reported previously.

Cytochemical detection of calcium in a case of calcinosis circumscripta in a dog.

A 9-month-old intact female Serra-da-Estrela dog (giant Portuguese breed) was presented with a 2-month history of lingual lesions. Two hard, nodular, nonpainful lesions were observed on the lateral border of the tongue. Fine-needle aspiration yielded a chalky white material. Hemacolor-stained smears showed an amorphous granular basophilic background with crystal-like structures and granulomatous inflammation consisting of foamy macrophages and giant cells. Cytochemical staining with von Kossa and Alizarin red S confirmed the calcareous nature of the material. The cytologic diagnosis was lingual calcinosis circumscripta; this was later confirmed by histopathologic examination. To our knowledge, this case is the first to document the use of von Kossa and Alizarin red S on cytologic smears to obtain a definitive diagnosis of lingual calcinosis circumscripta.

Preoperative assessment in cases of adult megacolon suffering from moderate malnutrition.

Acquired megacolon is a chronic disease associated with constipation and malnutrition. Surgical treatment may be required for the alleviation of the intestinal symptoms, emphasizing the interest of nutritional assessment in this population. In a prospective study of 33 patients suffering from acquired megacolon and requiring either anterior resection or pull-through operation, standard anthropometric and biochemical measurements as well as the PNI of Buzby and Mullen were preoperatively assessed. Mean age of the population was 49 +/- 13 y, with 17 males and 16 females. The combined nutritional score indicated 63.6% of the population were malnourished (21/33) of the patients, whereas the Prognostic nutritional index (PNI) revealed 3 high-risk cases (9.1%), 9 with moderate risk (27.3%), and reduced risk for all others. Indeed, 39.4% (13/33) of the patients displayed surgical or septic problems. Chi-square analysis confirmed that both studied criteria were significantly associated with complications (p < 0.05), as morbidity was restricted predominantly to cases with unfavorable Prognostic nutritional index (PNI) results or clear signs of nutritional deficit. Both PNI and conventinoal nutritional assessment are valuable tools for the screening of these surgical candidates. Artificial alimentation was not used in this experience, but deserves consideration in selected patients. Nutritional status improved in the late postoperative period, with normalization of bowel function.