Etonogestrel implant as a contraceptive choice; patient acceptability and adverse effect profile in a general practice setting.

The aim of this study was to evaluate the experience of a cohort of patients utilizing the etonogestrel implant (Implanon) in an Irish General Practice setting. This study involved a survey administered as a telephone questionnaire, to a cohort of women (n=75) who opted to use the etonogestrel implant. 53% reported problems with the implant post insertion, the commonest problem being irregular bleeding in 22% cases. Early removals were documented in 28% cases, of which, 29%, were caused by irregular bleeding. Based on this study, it is evident that women have high expectations of the implant, and counselling about what they can expect during use is important in order to avoid unreasonable expectations. This study also demonstrates that the use of the etonogestrel implant is a valuable contraceptive option, which can be successfully delivered in a GP setting, for both patients of the practice as well as patients referred by colleagues locally.

Microbial contamination of dental unit water systems.

INTRODUCTION: Dental unit water systems (DUWS) may serve as a reservoir for biofilms that contribute to high numbers of bacteria in the water used during dental treatment. These microbes are predominantly harmless but potentially pathogenic organisms can also be present in the biofilm. This may pose a potential health risk for patients and dental personnel. AIM: to determine the microbial levels of DUWS in dental practices. MATERIALS AND METHOD: A cross-sectional study of water and tubing samples from 30 general dental practices (15 health board and 15 private surgeries) was undertaken as part of a pan-European investigation of the microbial qualitative and quantitative aspects of DUWS. RESULTS: Microbial loads ranged from 100 to 104 cfu ml-1 and exceeded the European guidelines for drinking water in many cases. The available evidence suggested the presence of isolates most likely belonging to families of aquatic and soil bacteria. It was not possible to draw distinct conclusions correlating microbial loads with dental unit parameters, including age of the unit, water source and chemistry and presence or absence of anti-retraction devices. Opportunistic or true pathogens were not detected. Yeasts were observed in samples from three units although further analysis confirmed that these were not Candida albicans. A decontamination strategy applied to one of the units eliminated the yeasts completely. CONCLUSIONS: Dental practitioners must be knowledgeable regarding microbial contamination and biofilm formation in dental unit waterlines. There is a need for development of European evidence-based guidelines and reliable control regimes for microbial contamination of DUWS.

Microbiological evaluation of dental unit water systems in general dental practice in Europe.

A range of opportunistic pathogens have been associated with dental unit water systems (DUWS), particularly in the biofilms that can line the tubing. This study therefore aimed to assess the microbiology of DUWS and biofilms in general dental practices across seven European countries, including the United Kingdom (UK), Ireland (IRL), Greece (GR), Spain (ES), Germany (D), Denmark (DK) and the Netherlands (NL). Water supplied by 51% of 237 dental unit water lines exceeded current American Dental Association recommendations of < or = 200 colony-forming units (CFU) ml(-1). Microbiological loading of the source waters was between 0 (Denmark, the Netherlands and Spain) and 4.67 (IRL) log CFU ml(-1); water line samples from the DUWS ranged from 1.52 (ES) to 2.79 (GR) log CFU ml(-1); and biofilm counts ranged from 1.49 (GR) to 3.22 (DK) log CFU.cm(-2). Opportunistic pathogens such as legionellae (DK and ES), including Legionella pneumophila SG1 (DK and GR), and Mycobacterium spp. (DK, NL, GR, D and ES) were recovered occasionally. Presumptive oral streptococci (ES and NL), oral anaerobes (GR), Candida spp. (UK, NL and ES) and blood (GR and IRL) were detected at relatively low frequencies, but their presence indicated a failure of the 3-in-1 antiretraction valve, leading to back siphonage of oral fluids into the water and biofilm phase. These findings confirm that a substantial proportion of DUWS have high levels of microbial contamination, irrespective of country, type of equipment and source water. The study emphasizes the need for effective mechanisms to reduce the microbial burden within DUWS, and highlights the risk of occupational exposure and cross-infection in general dental practice.

Characterization of a novel EGFP reporter mouse to monitor Cre recombination as demonstrated by a Tie2 Cre mouse line.

The use of the Cre/loxP system has greatly empowered the field of gene targeting. Here we describe the successful establishment of a novel knock-in EGFP reporter mouse line to monitor Cre-induced recombination in the vast majority of cell types. The value of this reporter mouse line is demonstrated by the use of a novel Tie2Cre transgenic mouse line that facilitates gene targeting in endothelial and hematopoietic cells. High efficiency of recombination was found in all endothelial cells and in the majority of hematopoietic cells but was absent in other tissues. Furthermore, in the second generation, the Tie2Cre mouse can be used to get 100% recombination of one allele, whilst allowing tissue specific in the second, therefore offering excellent efficiency.

A cytotoxicity assay for the detection and differentiation of two families of shellfish toxins.

There is an urgent need for an alternative to the mouse bioassay for the detection of algal toxins in shellfish on both analytical and animal welfare grounds. Several alternative methodologies have been described, but have not gained widespread acceptance to date, because each assay measures only one or a small number of related phycotoxins out of the increasing range that needs to be detected. A simple cytotoxicity assay using either the HepG2 or ECV-304 cell lines is described with two end-point measurements, which can detect and distinguish between two unrelated classes of phycotoxins. Morphological examination following 3h exposure to the sample enables the detection of the diarrhetic shellfish poisons, including okadaic acid and related toxins. Viability testing using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide), following 24h exposure of the same cells to the sample, reveals a second class of toxin, which is most probably the newly-described toxin, azaspiracid. This assay should play an important role in shellfish monitoring in the future.

Biotechnological approaches to the understanding and improvement of mature cheese flavour.

There have been important milestones in biotechnological practice that have led to the determination and production of superior cheese flavours. Within the past year, the use of gas chromatographic techniques and sensory methodologies has been optimised by several groups in efforts to evaluate the organoleptic properties of a number of mature cheeses. The hydrolysis of milk caseins, small peptides, free amino acids and fatty acids, and the generation of sulfur-containing compounds are uniformly assumed to result in the formation of specific cheese aromas. Giant strides have been taken in molecular technology to aid the dissection and exploitation of the metabolic pathways that lead to the formation of these flavour constituents. Specific advances in molecular technology have included metabolic engineering of lactic acid bacteria for enhanced flavour development.

Bacteriophage defence systems in lactic acid bacteria.

The study of the interactions between lactic acid bacteria and their bacteriophages has been a vibrant and rewarding research activity for a considerable number of years. In the more recent past, the application of molecular genetics for the analysis of phage-host relationships has contributed enormously to the unravelling of specific events which dictate insensitivity to bacteriophage infection and has revealed that while they are complex and intricate in nature, they are also extremely effective. In addition, the strategy has laid solid foundations for the construction of phage resistant strains for use in commercial applications and has provided a sound basis for continued investigations into existing, naturally-derived and novel, genetically-engineered defence systems. Of course, it has also become clear that phage particles are highly dynamic in their response to those defence systems which they do encounter and that they can readily adapt to them as a consequence of their genetic flexibility and plasticity. This paper reviews the exciting developments that have been described in the literature regarding the study of phage-host interactions in lactic acid bacteria and the innovative approaches that can be taken to exploit this basic information for curtailing phage infection.

Identification of four phage resistance plasmids from Lactococcus lactis subsp. cremoris HO2.

The bacteriophage-host sensitivity patterns of 16 strains of Lactococcus lactis originally isolated from a mixed strain Cheddar cheese starter culture were determined. Using phages obtained from cheese factory whey, four of the strains were found to be highly phage resistant. One of these isolates, Lactococcus lactis subsp. cremoris HO2, was studied in detail to determine the mechanisms responsible for the phage insensitivity phenotypes. Conjugal transfer of plasmid DNA from strain HO2 allowed a function to be assigned to four of its six plasmids. A 46-kb molecule, designated pCI646, was found to harbor the lactose utilization genes, while this and plasmids of 58 kb (pCI658), 42 kb (pCI642), and 4.5 kb (pCI605) were shown to be responsible for the phage resistance phenotypes observed against the small isometric-headed phage phi712 (936 phage species) and the prolate-headed phage phic2 (c2 species). pCI658 was found to mediate an adsorption-blocking mechanism and was also responsible for the fluffy pellet phenotype of cells containing the molecule. pCI642 and pCI605 were both shown to be required for the operation of a restriction-modification system.

Anti-phagocyte antibodies and infection.

Autoantibodies specific to the cytoplasmic components of neutrophils and monocytes are associated with vasculitis and other idiopathic inflammatory disorders. In this study, using enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assays, sera from patients with acute and chronic infection were examined for the presence of anti-neutrophil and anti-monocyte antibodies: cystic fibrosis (n = 23), acute appendicitis (n = 22), tuberculosis (n = 26), acute gastroenteritis (n = 38), bronchiectasis (n = 9) and chronic granulomatous disease (n = 6). Sera from patients with Wegener's granulomatosis (n = 14), rheumatoid factor positive (n = 15) and healthy volunteers (n = 20) were used as positive and negative controls. In patients with chronic infection, using an ELISA assay, antibodies reactive with neutrophil or monocyte components (% reacting with monocyte components in parenthesis) were found in: 70% (39%) of patients with cystic fibrosis, 4% (38%) of patients with tuberculosis, 0% (33%) of patients with bronchiectasis and 0% (17%) of patients with chronic granulomatous disease. When these sera were examined using an immunofluorescence assay, all of the positive samples were found to react with the cytoplasmic component of neutrophils or monocytes. In patients with acute infection no antibodies (either IgG or IgM) were detected against neutrophils or monocytes. These findings imply that antibodies directed against neutrophil cytoplasmic components are predominantly associated with chronic pyogenic infection and antibodies specific to monocyte cytoplasmic components are predominantly associated with chronic granulomatous infection. This mirrors the findings in idiopathic inflammatory disease where anti-monocyte antibodies are associated with granulomatous disorders such as sarcoidosis, and anti-neutrophil antibodies are associated with neutrophilic disorders such as ulcerative colitis. These results suggest that chronic stimulation of phagocytes by infectious agents may result in the generation of a humoral response against phagocyte cytoplasmic components. This furthers our understanding of humoral immune responses against phagocytic cell components during infection.

Characterisation of anti-neutrophil cytoplasmic antibody target antigens using electrophoresis and western blotting techniques.

Anti-neutrophil cytoplasmic antibodies (ANCA) are a family of autoantibodies which react with components of phagocytic cells, and are associated with vasculitis and other idiopathic inflammatory disorders. However, the antigenic targets of many of these autoantibodies have not been defined yet. In this study, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) were evaluated for characterising the antigenic specificity of unidentified ANCA. The uncharacterised sera included those from patients with ulcerative colitis (n = 21), Crohn's disease (n = 5), cystic fibrosis (n = 16) and sarcoidosis (n = 2). In addition, sera from patients with antibodies to the phagocytic enzymes proteinase 3 (PR3) (n = 11) and myeloperoxidase (MPO) (n = 5) were also included. The sub-cellular localisation of antigens was determined by testing sera against crude neutrophil extract and sub-cellular fractions consisting of azurophilic granules, specific granules and cytosolic, fractions using enzyme-linked immunosorbent assays (ELISAs). All sera reacted with the crude and azurophilic granule extracts. The native system of IEF followed by capillary immunoblotting successfully detected anti-PR3 and anti-MPO in azurophilic granule extracts. In contrast, SDS-PAGE Western blotting failed to detect any reactivity, either to PR3 or MPO, in the crude extract or azurophilic granule extract. However, the antibody specificity of patient sera with uncharacterised autoantibodies could not be detected by IEF/capillary immunoblotting or SDS-PAGE. This study showed that the sub-cellular azurophilic granules are the antigenic target of a variety of uncharacterised ANCA. It also showed that IEF characterised both anti-PR3 and anti-MPO but failed to detect other forms of ANCA. In contrast, the majority of common ANCA were not detected by SDS-PAGE.

Anti-monocyte cytoplasmic antibodies in granulomatous disease.

Antibodies reactive with cytoplasmic components of neutrophils (ANCA) have received much attention because of their association with vasculitis and other inflammatory disorders. In this study, ELISA and immunofluorescence assays were employed to detect antimonocyte-specific antibodies and similar assays were used in parallel to detect anti-neutrophil cytoplasmic antibodies in a range of patient sera: Wegener's granulomatosis (14), Crohn's disease (46), ulcerative colitis (43), sarcoidosis (40), rheumatoid factor positive (15), and normal controls (22). Using an ELISA, anti-monocyte antibodies were detected in (figures in parentheses represent anti-neutrophil results): Wegener's granulomatosis, 79% (93%); sarcoidosis, 30% (5%); Crohn's disease, 20% (11%); ulcerative colitis, 0% (49%); rheumatoid factor-positive sera, 0% (0%). Excellent concordance (>98%) was found between the ELISA and immunofluorescence assays and all of the anti-monocyte antibodies gave a cytoplasmic pattern of staining. This is the first report demonstrating the presence of antibodies specific to the cytoplasmic components of monocytes in patients with granulomatous disease and these findings enhance our understanding of auto-antibody responses against phagocytic cell components.

Comparison of five cardiac markers in the detection of reperfusion after thrombolysis in acute myocardial infarction.

OBJECTIVE: To investigate and compare the clinical usefulness of serial measurements of five cardiac marker proteins, namely creatine kinase (CK), CK-MB mass, myoglobin, troponin T, and myosin light chain 1, in the early detection of reperfusion after thrombolytic treatment. METHOD: Serial blood samples were taken from 26 patients presenting with acute myocardial infarction. Concentrations of the five markers were assayed in each sample. Thrombolytic treatment was given to the patients who were divided into those who reperfused (n = 17, group A) and those who failed to reperfuse (n = 9, group B) on the basis of clinical signs and angiography within 24 h. RESULTS: The release profiles of CK, CK-MB mass, myoglobin, and troponin T for patients in group A differed from those of patients in group B. No difference was observed in the release profile of myosin light chain 1 between the two groups. The time to peak concentration of CK, CK-MB mass, myoglobin, and troponin T occurred significantly earlier in patients of group A than in those of group B, with myoglobin peaking earlier than the other markers. An index, defined as the ratio of the concentration of each marker immediately before and 2 h after the start of thrombolytic treatment, was calculated for each marker in groups A and B. The 2 h myoglobin and troponin T indices were significantly different between groups A and B. The diagnostic efficiency of the myoglobin index, however, was best at 85%. CONCLUSIONS: These studies suggest that myoglobin has greater potential than the other markers examined in the detection of reperfusion after thrombolytic treatment.

In situ detection of PCR-amplified HIV-1 nucleic acids and tumor necrosis factor cDNA in cervical tissues.

This study determined the histological distribution of polymerase chain reaction-amplified human immunodeficiency virus 1 (HIV-1) DNA and RNA in cervical tissues. Amplified HIV-1 DNA and complementary DNA were detected in each of 21 cervical biopsies from women with acquired immunodeficiency syndrome. The viral nucleic acids were most abundant in the endocervical aspect of the transformation zone at the interface of the glandular epithelium and the submucosa and in the deep submucosa around microvessels. Many virally infected cells colabeled with leukocyte common antigen, Mac387, and polymerase chain reaction-amplified tumor necrosis factor complementary DNA, demonstrating that they were activated macrophages. Virally amplified nucleic acids were not detected in 10 controls and in only one of eight cervical tissues from children less than 3 years of age who died due to immunodeficiency syndrome acquired in utero. Determining whether the HIV-1-infected macrophages consistently present in the cervix of adult seropositive women may represent primary infection and, if so, whether they can transport the virus to regional lymph nodes and thus initiate systemic infection requires further study.

In situ analysis of Paget's disease of bone for measles-specific PCR-amplified cDNA.

Paget's disease of bone is a disease of unknown etiology. The demonstration of viral-like particles on ultrastructural examination and the putative detection of viral antibodies and nucleic acids in the tissues suggest a possible viral association. The purpose of this study was to search for nucleic acid sequences homologous to measles virus using the recently described reverse transcriptase (RT) polymerase chain reaction (PCR) in situ hybridization (ISH) technique. After performing RT PCR ISH utilizing primers specific for the nucleocapsid region of the measles virus, an intense signal was evident in most measles-infected HeLa cells compared with a weak signal in few of these cells using standard cDNA-RNA ISH analysis. Amplified measles nucleic acid was detected in tissue from a patient who died of measles infection and was not detected in any of the 11 cases of Paget's disease of bone studied or in a giant cell tumor of bone that had tubuloreticular inclusions on electron microscopy. Therefore, these data suggest that infection by the measles virus is not associated with Paget's disease of bone.

Detection of human papillomavirus DNA in formalin-fixed tissues by in situ hybridization after amplification by polymerase chain reaction.

The authors describe the detection of human papillomavirus (HPV) 16 DNA in paraffin-embedded, formalin-fixed tissues of cervical squamous intraepithelial lesions (SILs) by in situ hybridization after amplification by the polymerase chain reaction (PCR). Using conventional in situ hybridization and a biotin-labeled probe, variable numbers of superficial cells and none of the basal cells in the SILs showed detectable HPV 16 DNA. When the in situ assay was done after amplification, increased numbers of superficial cells had detectable HPV DNA, and the hybridization signal was much more intense. HPV DNA was also detected in basal and parabasal cells at the site of the lesion whereas not detectable in directly adjacent, normal squamous epithelium. Amplified HPV DNA was demonstrated in formalin-fixed SiHa cells using a biotin-labeled probe, demonstrating the ability to detect one copy of HPV 16 DNA. This technique should allow for direct visualization in cells of other DNA sequences of low copy number from achival specimens otherwise undetectable by conventional in situ hybridization analysis.

Value of intracranial pressure monitoring of asphyxiated newborn infants.

Twenty-three infants suffering the effects of moderate or severe hypoxic-ischaemic encephalopathy were continuously monitored for intracranial pressure (ICP) by means of a subarachnoid catheter for a total of 1083 hours. Cerebral perfusion pressure (CPP) was also continuously monitored for 21 of the infants. The median age at the start of ICP monitoring was 17 hours, and the opening pressure correlated poorly with maximum sustained pressures. Maximum sustained ICP allowed the infants to be divided into three groups: (1) those with no elevation of ICP (nine), of whom two died and five had a normal outcome; (2) those with sustained rises in ICP which were resistent to treatment (nine), of whom seven died and two survivors are severely handicapped; and (3) those in whom the pressure was elevated but could be controlled medically (five), of whom two survived to be quite normal. No infant with a sustained elevation of ICP of 15mmHg or more survived to be normal, nor any who had had a CPP below 20mmHg for one hour or more. Hypotension was the cause of low CPP in most cases. There was a highly significant correlation between sustained elevation of ICP above 10mmHg and poor outcome, but no correlation between outcome and minimum CPP. It was not possible to predict clinically which infants would develop intracranial hypertension, and some infants with very severe perinatal asphyxia did not develop intracranial hypertension, and some infants with very severe perinatal asphyxia did not develop raised intracranial pressure at any time.