Improving drinking water quality through proficiency testing-the impact of testing method and accreditation status on Escherichia coli detection by Canadian environmental testing laboratories.

Water quality testing is crucial for protecting public health, especially considering the number of boil water advisories annually issued across Canada that impact daily life for residents in affected areas. To overcome these challenges, the development of drinking water safety plans and accessibility to regular testing using simple, rapid, and accurate materials are necessary. However, the significance of monitoring the accuracy of environmental microbiology testing laboratories cannot be overlooked. Participation in external quality assessment programs, such as those that include proficiency testing (PT), is a necessary risk management resource that ensures the effectiveness of these testing processes. Proficiency Testing Canada (PTC), in collaboration with the Canadian Microbiological Proficiency Testing (CMPT) program based at the University of British Columbia, have implemented a drinking-water microbiology PT program since 1996. Both PTC and CMPT are ISO/IEC 17043:2010-accredited EQA providers. The drinking water program provided PT challenges to subscribing testing laboratories twice per year. Each challenge consisted of four samples containing unknown concentrations of Escherichia coli (E. coli) and Enterobacter spp. Results from participants were assessed for accuracy based on the method of testing. This cross-sectional study evaluated 150 rural and metropolitan testing sites across Canada between 2016 and 2022. Multivariable logistic regression analysis was conducted to examine the impact of different testing methods and laboratory accreditation status on the proficiency scores. This approach enabled us to assess the association between multiple independent variables and the likelihood of achieving specific proficiency scores, providing insights into how testing methods and accreditation status affect overall performance. After adjusting for rural residence, testing time, and survey year, the membrane filtration method was positively associated with the likelihood of scoring satisfactory results compared to the enzyme-substrate method (OR: 1.75; CI: 1.37-2.24), as well as accreditation status (OR: 1.47; CI: 1.16-1.85). The potential for improvement in environmental laboratory testing performance through the implementation of regulated PT in drinking water safety plans is proposed, along with the need for reliable testing methods applicable to rapid drinking water microbiology testing.

Ensuring diagnostic testing accuracy for patient care and public health- COVID-19 testing scale-up from an EQA provider's perspective.

In response to the coronavirus pandemic (COVID-19) and scale up of diagnostic testing, the Canadian Microbiology Proficiency Testing program created a new proficiency testing (PT) program for the molecular and antigen detection of SARS-CoV-2. The program was geared to point of care testing (POCT) sites located in each of the eight provincial Health Authorities across British Columbia, Canada, with the intention to monitor testing quality. The PT program consisted of 6 shipments in a year, each containing a set of 4 samples either positive for SARS-CoV-2 virus or negative. The program began with initial 23 sites enrolling in March 2021, expanding to >100 participants by December 2021. After the first two surveys, it was observed that testing performance (accuracy) was consistently acceptable for sites using nucleic acid technology (NAT), however performance by sites using rapid antigen detection (RAD) methods was poor, especially when testing the weakly positive samples. A root cause investigation of poor testing performance revealed gaps in the execution of testing methods and also in results interpretation. These quality issues were most commonly associated with new testers who lacked experience with diagnostic testing. Tester training and mentoring was reinforced as was retraining of personnel; sample processing instructions were modified, and a training video was also created for testing sites. As a result of these interventions, sites improved their testing accuracy and the performance of POCT sites using RAD methods came to more closely match the performance of sites utilizing NAT. Overall, the PT program was highly successfully and improved quality of testing in the province. This work demonstrates the critical value of an external quality assessment (EQA) partner towards improving patient and public health and safety, especially when testing is conducted outside of an accredited medical laboratory setting.

Laboratory error reporting rates can change significantly with year-over-year examination.

BACKGROUND: Incident reporting systems are useful tools to raise awareness of patient safety issues associated with healthcare error, including errors associated with the medical laboratory. METHODS: Previously, we presented the analysis of data compiled by the British Columbia Patient Safety & Learning System over a 3-year period. A second comparable set was collected and analyzed to determine if reported error rates would tend to remain stable or change. RESULTS: Compared to the original set, the second set presented changes that were both materially and statistically significant. Overall, the total number of reports increased by 297% with substantial changes between the pre-examination, examination and post-examination phases (χ2: 993.925, DF=20; p<0.00001). While the rate of change for pre-examination (clerical and collection) errors were not significantly different than the total year results, the rate of change for reporting examination errors rose by 998%. While the exact reason for dramatic change is not clear, possible explanations are provided. CONCLUSIONS: Longitudinal error rate tracking is a useful approach to monitor for laboratory quality improvement.

Structural and functional insights into asymmetric enzymatic dehydration of alkenols.

The asymmetric dehydration of alcohols is an important process for the direct synthesis of alkenes. We report the structure and substrate specificity of the bifunctional linalool dehydratase isomerase (LinD) from the bacterium Castellaniella defragrans that catalyzes in nature the hydration of ß-myrcene to linalool and the subsequent isomerization to geraniol. Enzymatic kinetic resolutions of truncated and elongated aromatic and aliphatic tertiary alcohols (C5-C15) that contain a specific signature motif demonstrate the broad substrate specificity of LinD. The three-dimensional structure of LinD from Castellaniella defragrans revealed a pentamer with active sites at the protomer interfaces. Furthermore, the structure of LinD in complex with the product geraniol provides initial mechanistic insights into this bifunctional enzyme. Site-directed mutagenesis confirmed active site amino acid residues essential for its dehydration and isomerization activity. These structural and mechanistic insights facilitate the development of hydrating catalysts, enriching the toolbox for novel bond-forming biocatalysis.

Medical laboratory associated errors: the 33-month experience of an on-line volunteer Canadian province wide error reporting system.

BACKGROUND: This article reports on the findings of 12,278 laboratory related safety events that were reported through the British Columbia Patient Safety & Learning System Incident Reporting System. METHODS: The reports were collected from 75 hospital-based laboratories over a 33-month period and represent approximately 4.9% of all incidents reported. RESULTS: Consistent with previous studies 76% of reported incidents occurred during the pre-analytic phase of the laboratory cycle, with twice as many associated with collection problems as with clerical problems. Eighteen percent of incidents occurred during the post-analytic reporting phase. The remaining 6% of reported incidents occurred during the actual analytic phase. Examination of the results suggests substantial under-reporting in both the post-analytic and analytic phases. Of the reported events, 95.9% were reported as being associated with little or no harm, but 0.44% (55 events) were reported as having severe consequences. CONCLUSIONS: It is concluded that jurisdictional reporting systems can provide valuable information, but more work needs to be done to encourage more complete reporting of events.

Analysis of the interactions of cytochrome b5 with flavocytochrome P450 BM3 and its domains.

Interactions between a soluble form of microsomal cytochrome b(5) (b(5)) from Musca domestica (housefly) and Bacillus megaterium flavocytochrome P450 BM3 and its component reductase (CPR), heme (P450) and FAD/NADPH-binding (FAD) domains were analyzed by a combination of steady-state and stopped-flow kinetics methods, and optical spectroscopy techniques. The high affinity binding of b(5) to P450 BM3 induced a low-spin to high-spin transition in the P450 heme iron (K(d) for b(5) binding = 0.44 microM and 0.72 microM for the heme domain and intact flavocytochrome, respectively). The b(5) had modest inhibitory effects on steady-state turnover of P450 BM3 with fatty acids, and the ferrous-carbon monoxy P450 complex was substantially stabilized on binding b(5). Single turnover reduction of b(5) by BM3 using stopped-flow absorption spectroscopy (k(lim) = 116 s(-1)) was substantially faster than steady-state reduction of b(5) by P450 BM3 (or its CPR and FAD domains), indicating rate-limiting step(s) other than BM3 flavin-to-b(5) heme electron transfer in the steady-state reaction. Steady-state b(5) reduction by P450 BM3 was considerably accelerated at high ionic strength. Pre-reduction of P450 BM3 by NADPH decreased the k(lim) for b(5) reduction approximately 10-fold, and also resulted in a lag phase in steady-state b(5) reduction that was likely due to BM3 conformational perturbations sensitive to the reduction state of the flavocytochrome. Ferrous b(5) could not reduce the ferric P450 BM3 heme domain under anaerobic conditions, consistent with heme iron reduction potentials of the two proteins. However, rapid oxidation of both hemoproteins occurred on aeration of the ferrous protein mixture (and despite the much slower autoxidation rate of b(5) in isolation), consistent with electron transfer occurring from b(5) to the oxyferrous P450 BM3 in the complex. The results demonstrate that strong interactions occur between a eukaryotic b(5) and a model prokaryotic P450. Binding of b(5) perturbs BM3 heme iron spin-state equilibrium, as is seen in many physiologically relevant b(5) interactions with eukaryotic P450s. These results are consistent with the conservation of structure of P450s (particularly at the heme proximal face) between prokaryotes and eukaryotes, and may point to as yet undiscovered roles for b(5)-like proteins in the control of activities of certain prokaryotic P450s.

Does external evaluation of laboratories improve patient safety?

Laboratory accreditation and External Quality Assessment (also called proficiency testing) are mainstays of laboratory quality assessment and performance. Both practices are associated with examples of improved laboratory performance. The relationship between laboratory performance and improved patient safety is more difficult to assess because of the many variables that are involved with patient outcome. Despite this difficulty, the argument to continue external evaluation of laboratories is too compelling to consider the alternative.

Coordinated response to SARS, Vancouver, Canada.

Two Canadian urban areas received travelers with severe acute respiratory syndrome (SARS) before the World Health Organization issued its alert. By July 2003, Vancouver had identified 5 cases (4 imported); Toronto reported 247 cases (3 imported) and 43 deaths. Baseline preparedness for pandemic threats may account for the absence of sustained transmission and fewer cases of SARS in Vancouver.

Interaction of nitric oxide with cytochrome P450 BM3.

The interaction of nitric oxide with cytochrome P450 BM3 from Bacillus megaterium has been analyzed by spectroscopic techniques and enzyme assays. Nitric oxide ligates tightly to the ferric heme iron, inducing large changes in each of the main visible bands of the heme and inhibiting the fatty acid hydroxylase function of the protein. However, the ferrous adduct is unstable under aerobic conditions, and activity recovers rapidly after addition of NADPH to the flavocytochrome due to reduction of the heme via the reductase domain and displacement of the ligand. The visible spectral properties revert to that of the oxidized resting form. Aerobic reduction of the nitrosyl complex of the BM3 holoenzyme or heme domain by sodium dithionite also displaces the ligand. A single electron reduction destabilizes the ferric-nitrosyl complex such that nitric oxide is released directly, as shown by the trapping of released nitric oxide. Aerobically and in the absence of exogenous reductant, nitric oxide dissociates completely from the P450 over periods of several minutes. However, recovery of the nativelike visible spectrum is accompanied by alterations in the catalytic activity of the enzyme and changes in the resonance Raman spectrum. Specifically, resonance Raman spectroscopy identifies the presence of internally located nitrated tyrosine residue(s) following treatment with nitric oxide. Analysis of a Y51F mutant indicates that this is the major nitration target under these conditions. While wild-type P450 BM3 does not form an aerobically stable ferrous-nitrosyl complex, a site-directed mutant of P450 BM3 (F393H) does form an isolatable ferrous-nitrosyl complex, providing strong evidence for the role of this residue in controlling the electronic properties of the heme iron. We report here the spectroscopic characterization of the ferric- and ferrous-nitrosyl complexes of P450 BM3 and describe the use of resonance Raman spectroscopy to identify nitrated tyrosine residue(s) in the enzyme. Nitration of tyrosine in P450 BM3 may exemplify a typical mechanism by which the ubiquitous messenger molecule nitric oxide exerts a regulatory function over the cytochromes P450.

Expression, purification, and characterization of Bacillus subtilis cytochromes P450 CYP102A2 and CYP102A3: flavocytochrome homologues of P450 BM3 from Bacillus megaterium.

The cyp102A2 and cyp102A3 genes encoding the two Bacillus subtilis homologues (CYP102A2 and CYP102A3) of flavocytochrome P450 BM3 (CYP102A1) from Bacillus megaterium have been cloned, expressed in Escherichia coli, purified, and characterized spectroscopically and enzymologically. Both enzymes contain heme, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) cofactors and bind a variety of fatty acid molecules, as demonstrated by conversion of the low-spin resting form of the heme iron to the high-spin form induced by substrate-binding. CYP102A2 and CYP102A3 catalyze the fatty acid-dependent oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and reduction of artificial electron acceptors at high rates. Binding of carbon monoxide to the reduced forms of both enzymes results in the shift of the heme Soret band to 450 nm, confirming the P450 nature of the enzymes. Reverse-phase high-performance liquid chromatography (HPLC) of products from the reaction of the enzymes with myristic acid demonstrates that both catalyze the subterminal hydroxylation of this substrate, though with different regioselectivity and catalytic rate. Both P450s 102A2 and 102A3 show kinetic and binding preferences for long-chain unsaturated and branched-chain fatty acids over saturated fatty acids, indicating that the former two molecule types may be the true substrates. P450s 102A2 and 102A3 exhibit differing substrate selectivity profiles from each other and from P450 BM3, indicating that they may fulfill subtly different cellular roles. Titration curves for binding and turnover kinetics of several fatty acid substrates with P450s 102A2 and 102A3 are better described by sigmoidal (rather than hyperbolic) functions, suggesting binding of more than one molecule of substrate to the P450s, or possibly cooperativity in substrate binding. Comparison of the amino acid sequences of the three flavocytochromes shows that several important amino acids in P450 BM3 are not conserved in the B. subtilis homologues, pointing to differences in the binding modes for the substrates that may explain the unusual sigmoidal kinetic and titration properties.

ISO 15190:2003 Medical Laboratories - Requirements for Safety.

ISO 15190:2003 is a new standard developed by the International Organization for Standardization to address the safety aspects of medical laboratories. It is directly linked to the ISO 15189:2003, the standard for quality and competence. It provides a framework for a safety program based upon the principles of quality management, including designation of responsibilities and authorities, regular audits, and continuous improvement.

Variation in the pH-dependent pre-steady-state and steady-state kinetic characteristics of cysteine-proteinase mechanism: evidence for electrostatic modulation of catalytic-site function by the neighbouring carboxylate anion.

The acylation and deacylation stages of the hydrolysis of N -acetyl-Phe-Gly methyl thionoester catalysed by papain and actinidin were investigated by stopped-flow spectral analysis. Differences in the forms of pH-dependence of the steady-state and pre-steady-state kinetic parameters support the hypothesis that, whereas for papain, in accord with the traditional view, the rate-determining step is the base-catalysed reaction of the acyl-enzyme intermediate with water, for actinidin it is a post-acylation conformational change required to permit release of the alcohol product and its replacement in the catalytic site by the key water molecule. Possible assignments of the kinetically influential p K (a) values, guided by the results of modelling, including electrostatic-potential calculations, and of the mechanistic roles of the ionizing groups, are discussed. It is concluded that Asp(161) is the source of a key electrostatic modulator (p K (a) 5.0+/-0.1) in actinidin, analogous to Asp(158) in papain, whose influence is not detected kinetically; it is always in the 'on' state because of its low p K (a) value (2.8+/-0.06).

Catalytically functional flavocytochrome chimeras of P450 BM3 and nitric oxide synthase.

P450 BM3 and the nitric oxide synthases are related classes of flavocytochrome mono-oxygenase enzymes, containing NADPH-dependent FAD- and FMN-containing oxidoreductase modules fused to heme b-containing oxygenase domains. Domain-swap hybrids of these two multi-domain enzymes were created by genetic engineering of different segments of reductase and heme domains from neuronal nitric oxide synthase and P450 BM3, as a means of investigating the catalytic competence and substrate-binding properties of the fusions and the influence of tetrahydrpbiopterin and calmodulin binding regions on the electron transfer kinetics of the chimeras. Despite marked differences in hybrid stability and solubility, four catalytically functional chimeras were created that retained good reductase activity and which could be expressed successfully in Escherichia coli and purified. All of the BM3 reductase domain chimeras (chimeras I-III) exhibited inefficient flavin-to-heme inter-domain electron transfer, diminishing their oxygenase activity. However, the chimera containing the neuronal nitric oxide synthase reductase domain (chimera IV) showed good oxygenase domain activity, indicating that the flavin-to-heme electron transfer reaction is relatively efficient in this case. The data reinforce the importance of the nature of inter-domain linker constitution in multi-domain enzymes, and the difficulties posed in attempts to create chimeric enzymes with enhanced catalytic properties.