Comparison of the culture method with multiplex PCR for the confirmation of Legionella spp. and Legionella pneumophila.

AIMS: The detection and enumeration of Legionella spp. in water samples are typically performed via a cultural technique standardized in ISO 11731. This method is time-consuming (up to 15 days), and the specificity of the confirmation step is questionable. This study proposes the use of multiplex polymerase chain reaction (PCR) to confirm presumptive Legionella colonies directly from the culture plate; this shortens the response time by 2-5 days while still reporting results in colony forming units (CFU). METHODS AND RESULTS: Two laboratories analysed a total of 290 colonies to compare the confirmation step of Legionella spp. and Legionella pneumophila in accordance with ISO 11731 by culture growth and agglutination vs multiplex PCR. Discordant results were resolved by the swiss national reference laboratory. The data were evaluated following ISO 16140 and showed that the PCR-technique had higher specificity. CONCLUSIONS: The confirmation of Legionella spp., L. pneumophila and L. pneumophila serogroup 1 by multiplex PCR allows detection of positive colonies more rapidly and with higher specificity. SIGNIFICANCE AND IMPACT OF THE STUDY: The study highlights a possibility to shorten the response time significantly during the enumeration of Legionella spp. and achieving a higher specificity while adhering to the legally recognized reporting in CFU.

Evaluation of critical congenital heart defects screening using pulse oximetry in the neonatal intensive care unit.

OBJECTIVE: To evaluate the implementation of early screening for critical congenital heart defects (CCHDs) in the neonatal intensive care unit (NICU) and potential exclusion of sub-populations from universal screening. STUDY DESIGN: Prospective evaluation of CCHD screening at multiple time intervals was conducted in 21 NICUs across five states (n=4556 infants). RESULTS: Of the 4120 infants with complete screens, 92% did not have prenatal CHD diagnosis or echocardiography before screening, 72% were not receiving oxygen at 24 to 48 h and 56% were born ⩾2500 g. Thirty-seven infants failed screening (0.9%); none with an unsuspected CCHD. False positive rates were low for infants not receiving oxygen (0.5%) and those screened after weaning (0.6%), yet higher among infants born at <28 weeks (3.8%). Unnecessary echocardiograms were minimal (0.2%). CONCLUSION: Given the majority of NICU infants were ⩾2500 g, not on oxygen and not preidentified for CCHD, systematic screening at 24 to 48 h may be of benefit for early detection of CCHD with minimal burden.

Photoplethysmographic waveform characteristics of newborns with coarctation of the aorta.

OBJECTIVE: Fetal echocardiography, physical examination and pulse oximetry detect only half of coarctation of aorta (CoA) cases. We aimed to quantify delayed arrival and diminished amplitude of lower extremity photoplethysmographic (PPG) pulses relative to the right hand in affected patients. STUDY DESIGN: We studied 8 CoA infants and 32 healthy controls. The pulse arrival time difference between foot and hand (f-hTD) and pulse amplitude ratio (F/H) were measured on PPG signal waveforms by digitally-determining maxima and minima of systolic decrease of light transmission. Mann-Whitney test was used for group comparisons. RESULTS: In comparison to healthy newborns, CoA infants' PPG waveforms demonstrated prolonged f-hTD (mean±s.d. of 73.2±26.6 versus 35.2±8.3 ms, P<0.001) and lower F/H (0.57±0.26 versus 0.99±0.58, P=0.014). CONCLUSIONS: F-hTD and F/H are quantifiable from hand- and foot-derived PPG waveforms and are significantly different in CoA versus healthy newborns. Larger studies are needed to validate PPG for improved critical congenital heart disease screening.

Unintended Consequences: New Problems, New Solutions. Contributions From 2015.

OBJECTIVE: To select the best of the 2015 published papers on unintended consequences of healthcare information technology (HIT). METHOD: Literature searches in several areas of scholarship, including IT, human factors, evaluation studies, medical errors, medical informatics, and implementation science. Also, because the specific terms "unintended consequences" were not often included in abstracts and titles, a more nuanced search algorithm was developed. RESULTS: We identified 754 papers that had some empirical research on unintended consequences of HIT. An initial screen of titles and abstracts reduced this to 171 papers of potential interest. We then further filtered out papers that did not meet the following criteria: 1) the paper had to report an original empirical investigation, and 2) the impact reported had to be not negligible, i.e., in quantitative studies, the results related to unintended consequences were statistically significant; and in qualitative studies the relevant themes emerged were prominent. This resulted in 33 papers of which 15 were selected as best paper candidates. Each of these 15 papers was then separately evaluated by four reviewers. The final selection of four papers was made jointly by the external reviewers and the two section editors. CONCLUSIONS: There is a growing awareness of the importance of HIT's unintended consequences-be they generated by the HIT vendors, the implementation process, the consultants, the users, or most probably, some combination of the above. There has also been greater creativity in use of data sources, including secondary data (e.g., medical malpractice cases and surveys) and a wider acceptance of mixed methods to identify unintended consequences. Unfortunately, the complexity of causes mitigates the value of recommendations to avoid unwanted outcomes. Suggestions are often contentious rather than obvious, setting-specific, and not universally applicable. "Lessons learned" often take on generalized-and perhaps platitudinous-forms, such as: "plan extra time," "involve all of the stakeholders," "recognize the different needs of different units or disciplines." The greater awareness of these problems, and the increased desire to identify and eliminate them is clearly reflected in the area's growing literature. We are hopeful the topic will receive additional attention and the discipline will improve its ability to identify and address these unexpected and usually adverse outcomes.

Computerised physician order entry-related medication errors: analysis of reported errors and vulnerability testing of current systems.

IMPORTANCE: Medication computerised provider order entry (CPOE) has been shown to decrease errors and is being widely adopted. However, CPOE also has potential for introducing or contributing to errors. OBJECTIVES: The objectives of this study are to (a) analyse medication error reports where CPOE was reported as a 'contributing cause' and (b) develop 'use cases' based on these reports to test vulnerability of current CPOE systems to these errors. METHODS: A review of medication errors reported to United States Pharmacopeia MEDMARX reporting system was made, and a taxonomy was developed for CPOE-related errors. For each error we evaluated what went wrong and why and identified potential prevention strategies and recurring error scenarios. These scenarios were then used to test vulnerability of leading CPOE systems, asking typical users to enter these erroneous orders to assess the degree to which these problematic orders could be entered. RESULTS: Between 2003 and 2010, 1.04 million medication errors were reported to MEDMARX, of which 63 040 were reported as CPOE related. A review of 10 060 CPOE-related cases was used to derive 101 codes describing what went wrong, 67 codes describing reasons why errors occurred, 73 codes describing potential prevention strategies and 21 codes describing recurring error scenarios. Ability to enter these erroneous order scenarios was tested on 13 CPOE systems at 16 sites. Overall, 298 (79.5%) of the erroneous orders were able to be entered including 100 (28.0%) being 'easily' placed, another 101 (28.3%) with only minor workarounds and no warnings. CONCLUSIONS AND RELEVANCE: Medication error reports provide valuable information for understanding CPOE-related errors. Reports were useful for developing taxonomy and identifying recurring errors to which current CPOE systems are vulnerable. Enhanced monitoring, reporting and testing of CPOE systems are important to improve CPOE safety.

Discussion of "Biomedical informatics: we are what we publish".

This article is part of a For-Discussion-Section of Methods of Information in Medicine about the paper "Biomedical Informatics: We Are What We Publish", written by Peter L. Elkin, Steven H. Brown, and Graham Wright. It is introduced by an editorial. This article contains the combined commentaries invited to independently comment on the Elkin et al. paper. In subsequent issues the discussion can continue through letters to the editor.

Is healthcare information technology based on evidence?

Is healthcare information technology (HIT) based on evidence of efficacy? Are the trillions of dollars already devoted and in the pipeline for HIT implementations based on systematic evaluations? If evaluated, would those evaluations focus on patient safety, return on investment, clinical efficiency, improved clinician satisfaction, and/or workflow integration? Do we have reliable evidence of usable interfaces, of successful implementations, of data standards allowing interoperability, of continuous improvement, of responsiveness to clinician feedback? While measurement of HIT's efficacy is extraordinarily difficult-complicated by a myriad of other factors involved in providing healthcare and in organizational dynamics-it is not impossible. But is such evidence required before most implementations? Any implementation? Or are the goals of patient safety and efficiency so self-evident, profoundly desired, and laudable that HIT's beneficence is accepted without rigorous data? Note that lack of systematic evidence does not mean HIT is ineffective. HIT may provide untold benefits even if there is no hard proof of those benefits. We find that HIT is seldom objectively measured, and that evidence of its efficacy is at best spotty, and often influenced by self-promotion. Most measures, especially those associated with cost-benefit analyses, are aspirational or hubris transubstantiated into numbers.

Determination of microbial transglutaminase in meat and meat products.

Transglutaminase is an enzyme that can be used to cross-link pieces of meat, fish or meat products. The resulting product gives the optical impression of an intact chunk of meat. The usage of transglutaminase as a food additive is permitted in some countries. However, its utilisation has to be declared to ensure transparency for consumers. This paper describes two orthogonal analytical methods suited for the detection of technological relevant transglutaminase concentrations (around 25 mg pure enzyme in 1 kg of product) in meat and meat products. The mass spectrometry-based approach relies on a previous digestion with Achromobacter lyticus protease and LC-MS/MS separation and detection. Sufficient selectivity was obtained by monitoring four different peptides. The orthogonal (complementary and independent), ELISA-based approach relies on two commercially available bacterial transglutaminase-specific antibodies, combined to a sandwich ELISA. The two methods were tested by analysing some 60 samples obtained from the market.

IgM detection via selective recognition by mannose-binding protein.

Ca(2+)-dependent mannose-binding proteins (MBPs) belong to the family of animal lectins isolated from the liver and serum of rabbits, humans and rodents. They perform in vivo as defense molecules that act as opsonins by enhancing the clearance of mannose-rich pathogens and have been used in vitro for the purification of immunoglobulin M (IgM). In this study, we used MBPs as a sensitive and specific reagent for the detection of IgM due to their high specificity for mannose found only in IgM carbohydrate regions. MBP performed as a sensitive alternative to the usually used anti-IgM, where very low concentrations of IgM should be detected. IgM plays a central role in the initial response of the immune system to the invasion of foreign pathogens, as the early detection of the appearance of pathogenic IgM in biological fluids is of great significance in the diagnosis and treatment of many acute pathological cases. The development of a highly sensitive and reliable assay for the detection of low concentrations of IgM based on covalent binding on epoxy film-coated surfaces and selective recognition of IgM by MBP may be of clinical importance.

Immunostaining of calmodulin and aluminium in Alzheimer's disease-affected brains.

Previous in vitro studies have shown that Al(3+) binds to calmodulin, inducing alterations in its capability to interact with target proteins, accompanied by loss of immunological recognition by its conformational specific monoclonal antibody CAM1. In spite of the wealth of data of calmodulin action in vitro, little information is available on the possible involvement of this protein in the pathology typical of Alzheimer's disease. In the present study, we investigated calmodulin immunoreactivity in post-mortem human brains affected by Alzheimer's disease, compared with age-matched control brains. Conformational monoclonal antibodies raised against Ca(2+)-calmodulin, namely CAM1 and CAM4, were used in this study for the characterization of calmodulin. Calmodulin immunorecognition by monoclonal antibody CAM1 was found to be lost in cortical tissue sample from brains affected by Alzheimer's disease. This finding leads to the hypothesis of a new, possibly inactive, conformation of the molecule during the disease. On the other hand, CAM4 immunoreactivity was decreased in neurons of brains affected by Alzheimer's disease. Anti-Al(3+) monoclonal antibodies revealed instead more marked aluminium immunoreactivity in the affected brains compared to normal ones. The loss of CAM1 immunoreactivity and the occurrence of large amounts of aluminium suggest an alteration of the active conformation of calmodulin in disease-affected brains. These alterations could be involved in the development of Alzheimer's disease pathology.

Cutaneous reactions to chemotherapeutic agents.

The task of evaluating a cutaneous eruption in the patient receiving chemotherapy can be quite formidable. Most of the time, these patients are receiving a multitude of agents and have profound immunosuppression. These factors may alter the more common manifestations of cutaneous eruptions. This article presents some of the more common cutaneous eruptions that may occur in an oncology patient receiving chemotherapy. It is hoped we may recognize clinical patterns seen with chemotherapeutic agents in the immunosuppressed population and, by recognizing these cutaneous eruptions, we may avoid the pitfalls of discontinuing medicines that may certainly be needed or altering the treatment course in a patient.

The efficacy of EMLA versus ELA-Max for pain relief in medium-depth chemical peeling: a clinical and histopathologic evaluation.

BACKGROUND: Medium-depth chemical peels are an effective and popular treatment for actinic damage, fine wrinkles, and pigmentary dyschromias. However, they are also uncomfortable. A previous attempt to study the effectiveness of a topical anesthetic gel in 35% trichloroacetic acid (TCA) peeling found a reduction in discomfort but an increased depth of penetration and delayed healing. OBJECTIVE: To evaluate both the efficacy of two topical anesthetic agents in medium-depth combination peeling as well as the histologic result from chemical peeling combined with topical anesthesia. METHOD: Seventy percent glycolic acid (GA) was applied to the entire face of 10 patients and diluted with water after 2 minutes. This was followed by the sequential application of EMLA cream (lidocaine 2.5% and prilocaine 2.5%), ELA-Max cream (lidocaine 4%), and placebo to selected areas on the face for 30 minutes without occlusion. These agents were then removed and 35% TCA was applied to the entire face. The level of discomfort felt by the patients during the TCA peel was recorded, clinical photographs were taken, and bilateral preauricular biopsies were performed at baseline, 48 hours, and 90 days postoperatively. RESULTS: Clinically there was a statistically significant decrease in pain felt during the 70% GA-35% TCA peel with topical anesthesia when compared to the control. There was no statistically significant difference in efficacy between EMLA and ELA-Max. There was also no difference in either the clinical or the histopathologic appearance between the medium-depth peel combined with topical anesthesia and the medium-depth peel with control. CONCLUSION: Both EMLA and ELA-Max decrease the discomfort felt during medium-depth combination chemical peeling without influencing either the clinical or the histopathologic result.

Construction and characterization of a stably transformed HeLa cell line in which the expression of bovine herpesvirus 1 ICP0 (BICP0) is induced by tetracycline.

To explore the effects BICP0 (a principal transactivator of BHV-1 gene expression) on viral promoter elements, we established a cell line in which the expression of BICP0 is regulated by tetracycline. A hybrid promoter containing reiterated copies of the tet-operator (tet-O) and a minimal herpesviral alpha gene transinducing factor (alpha TIF) responsive element (minimal human cytomegalovirus immediate early promoter) was fused to the BICP0 gene and used to transform a HeLa cell line which expressed a fusion protein consisting of the repressor of the tet-O and the transactivating domain of alpha TIF. Simultaneously, the hygromycin resistance gene was transfected to select cells in media containing either hygromycin alone or both hygromycin and tetracycline. Immunofluorescent assays indicated that BICP0 was synthesised in the transformed cell lines solely upon induction of the gene by tetracycline removal. Only cells which had been kept constantly in medium containing tetracycline were able to synthesise BICP0 upon induction. Induced cell lines transactivated the native BICP0 promoter as well as the herpes simplex virus thymidine kinase promoter and the long terminal repeat sequences of human immunodeficiency virus in a dose dependent manner. These cell lines may help to further explore the functions of BICP0 as well as to investigate the molecular basis of interactions between herpes- and retroviruses.

alpha1-Proteinase inhibitor therapy for the prevention of chronic lung disease of prematurity: a randomized, controlled trial.

BACKGROUND: An imbalance between increased neutrophil elastase and a decreased antiprotease shield has been suggested as a factor contributing to the development of chronic lung disease (CLD). We hypothesized that administration of alpha1-proteinase inhibitor (A1PI), also known as alpha1-antitrypsin, to premature neonates would prevent CLD. DESIGN: A randomized, placebo-controlled, prospective study of A1PI supplementation was performed. Neonates <24 hours of age with birth weights 600-1000 g on respiratory support, and 1001-1250 g with respiratory distress syndrome (RDS) were eligible. Intravenous A1PI (60 mg/kg) or placebo was infused on days 0, 4, 7, and 14. Primary outcome was CLD in survivors, defined as the need for supplemental oxygen on day 28. RESULTS: A total of 106 patients were recruited. There were no significant differences between groups in birth weight or incidence of RDS. The incidence of CLD in survivors was lower in the treated group, but the difference did not reach statistical significance (relative risk [RR], 0.79; confidence interval [CI], 0.60-1.02). This beneficial trend persisted at 36 weeks corrected gestational age (RR, 0.48; CI, 0.23-1.00). The incidence of pulmonary hemorrhage was lower in the treated group (RR, 0.22; CI, 0.05-0.98). Other complications were not significantly different between groups. CONCLUSIONS: In this, the first trial of a protease inhibitor for the prevention of CLD in premature infants, the infusions were well-tolerated. A1PI therapy may impede the development of CLD and appears to reduce the incidence of pulmonary hemorrhage in some neonates born prematurely.

Disaggregation of Alzheimer beta-amyloid by site-directed mAb.

In Alzheimer disease, beta-amyloid peptide accumulates in the brain as insoluble amyloid plaques. Amyloid filaments, similar to those found in amyloid plaques, can be assembled in vitro from chemically synthesized beta-peptides. In this study, we report that antibodies raised against the N-terminal region (1-28) of the beta-amyloid peptide bind to the in vitro-formed beta-amyloid assemblies, leading to disaggregation of the fibrils and partial restoration of the peptide's solubility. The concomitant addition of fibrillar beta-amyloid with these antibodies to PC 12 cells leads to the inhibition of the neurotoxic effects of beta-amyloid. Some of the mAbs raised against soluble beta-peptide (1-28) have been found to prevent in vitro fibrillar aggregation of beta-amyloid peptide. These experimental data suggest that site-directed mAbs interfere with the aggregation of beta-amyloid and trigger reversal to its nontoxic, normal components. The above findings give hints on how to convert in vivo senile plaques into nontoxic, diffuse components and may have therapeutic interest for those studying Alzheimer disease and other human diseases related to amyloidogenic properties of physiological peptides and proteins.

Immediate-early protein BICP22 of bovine herpesvirus 1 trans-represses viral promoters of different kinetic classes and is itself regulated by BICP0 at transcriptional and posttranscriptional levels.

Bovine herpesvirus 1 (BHV-1) encodes four immediate-early (IE) proteins. The transactivators BICP0 and BICP4 are key regulatory elements in viral replication, and circ is a myristylated virion component, whereas BICP22--originating from a spliced 1.7-kb transcript synthesized with dual IE and late kinetics--has not yet been characterized as a protein. In this study, Western blot and immunofluorescence analysis using antisera against a C-terminal oligopeptide revealed major 50-kDa and minor 35-kDa species of BICP22, predominantly located in the nuclei of BHV-1 infected cells. In transient expression assays, BICP22 acted as transrepressor protein on viral promoters of different kinetic classes, e.g. the IE promoter of the BICP4/BICP0 gene, early promoter of the BICP0 gene, and late promoter of the gC gene. The BICP22 gene promoter itself was not repressed by BICP22; it could be dissected into a proximal region stimulated by BICP0 and a distal region stimulated by BHV-1 alpha-transinducing factor. Replacement of the BICP22 promoter by cytomegalovirus IE promoter revealed an additional posttranscriptional level of regulation whereby more BICP22 accumulated in cells when functional BICP0 was present. Interplay of BICP22 and BICP0 might involve the recently described nuclear domains (ND10) and ubiquitin-dependent pathway.

Recombinant bovine herpesvirus-1 (BHV-1) lacking transactivator protein BICPO entails lack of glycoprotein C and severely reduced infectivity.

The immediate-early transactivator protein BICPO is a key regulatory element of bovine herpesvirus 1 (BHV-1) replication based on transient expression assays. To examine BICPO function in the context of the viral genome, we created recombinant BHV-1 expressing beta-galactosidase instead of BICPO. To complement the defect, a neomycin resistant MDBK cell line (M164) expressing BICPO was established, permitting selection of a blue-staining BHV-1 recombinant (A2G2). Southern blot and PCR analysis confirmed that the BICPO gene was interrupted by the beta-galactosidase gene and that wt progeny was absent. Compared with wt BHV-1, A2G2 reached lower titers in M164 cells but replicated with similar kinetics. Once isolated, A2G2 also grew in MDBK cells although the titer was reduced a further 10-fold and the virus remained strongly cell-associated. Thus, BICPO is not absolutely required for replication in cell culture. Gene expression of A2G2 was investigated by Western blots and immunofluorescence. Surprisingly, not only was BICPO absent, but glycoprotein C (gC) was also missing. Other viral genes were expressed normally. Semiquantitative PCR showed that A2G2 produced similar amounts of viral DNA as wt but a much smaller number of infectious particles. Cotransfection of A2G2 DNA and a plasmid containing the BICPO gene yielded revertant virus with fully restored wt properties. We conclude that BICPO is required for gC expression, and that the missing gC partly accounts for the reduced A2G2 infectivity.

Congenital vocal cord paralysis with possible autosomal recessive inheritance: case report and review of the literature.

We describe an infant with congenital vocal cord paralysis born to consanguineous parents. While autosomal dominant and X-linked inheritance have been previously reported in this condition, we conclude that the degree of parental consanguinity in this case strongly suggests autosomal recessive inheritance. Although we cannot exclude X-linked inheritance, evidence from animal studies demonstrates autosomal recessive inheritance and provides a possible molecular basis for congenital vocal cord paralysis.

Monoclonal antibodies inhibit in vitro fibrillar aggregation of the Alzheimer beta-amyloid peptide.

The beta-amyloid peptide, the hallmark of Alzheimer disease, forms fibrillar toxic aggregates in brain tissue that can be dissolved only by strong denaturing agents. To study beta-amyloid formation and its inhibition, we prepared immune complexes with two monoclonal antibodies (mAbs), AMY-33 and 6F/3D, raised against beta-amyloid fragments spanning amino acid residues 1-28 and 8-17 of the beta-amyloid peptide chain, respectively. In vitro aggregation of beta-amyloid peptide was induced by incubation for 3 h at 37 degrees C and monitored by ELISA, negative staining electron microscopy, and fluorimetric studies. We found that the mAs prevent the aggregation of beta-amyloid peptide and that the inhibitory effect appears to be related to the localization of the antibody-binding sites and the nature of the aggregating agents. Preparation of mAbs against "aggregating epitopes," defined as sequences related to the sites where protein aggregation is initiated, may lead to the understanding and prevention of protein aggregation. The results of this study may provide a foundation for using mAbs in vivo to prevent the beta-amyloid peptide aggregation that is associated with Alzheimer disease.