Implementation and evaluation of a SARI surveillance system in a tertiary hospital in Scotland in 2021/2022.

OBJECTIVE: To set up and evaluate a new surveillance system for severe acute respiratory infection (SARI) in Scotland. STUDY DESIGN: Cross-sectional study and evaluation of surveillance system. METHODS: The SARI case definition comprised patients aged 16 years or over with an acute respiratory illness presentation requiring testing for influenza and SARS-CoV-2 and hospital admission. Data were collected from SARI cases by research nurses in one tertiary teaching hospital using a bespoke data collection tool from November 2021 to May 2022. Descriptive analyses of SARI cases were carried out. The following attributes of the surveillance system were evaluated according to Centers for Disease Control and Prevention (CDC) guidelines: stability, data quality, timeliness, positive predictive value, representativeness, simplicity, acceptability and flexibility. RESULTS: The final surveillance dataset comprised 1163 records, with cases peaking in ISO week 50 (week ending 19/12/2021). The system produced a stable stream of surveillance data, with the proportion of SARI records with sufficient information for effective surveillance increasing from 65.4% during the first month to 87.0% over time. Similarly, the proportion where data collection was completed promptly was low initially, but increased to 50%-65% during later periods. CONCLUSION: SARI surveillance was successfully established in one hospital, but for a national system, additional sentinel hospital sites across Scotland, with flexibility to ensure consistently high data completeness and timeliness are needed. Data collection should be automated where possible, and demands on clinicians minimised. SARI surveillance should be embedded and resourced as part of a national respiratory surveillance strategy.

Vibrational spectroscopic analysis of blood for diagnosis of infections and sepsis: a review of requirements for a rapid diagnostic test.

Infections and sepsis represent a growing global burden. There is a widespread clinical need for a rapid, high-throughput and sensitive technique for the diagnosis of infections and detection of invading pathogens and the presence of sepsis. Current diagnostic methods primarily consist of laboratory-based haematology, biochemistry and microbiology that are time consuming, labour- and resource-intensive, and prone to both false positive and false negative results. Current methods are insufficient for the increasing demands on healthcare systems, causing delays in diagnosis and initiation of treatment, due to the intrinsic time delay in sample preparation, measurement, and analysis. Vibrational spectroscopic techniques can overcome these limitations by providing a rapid, label-free and low-cost method for blood analysis, with limited sample preparation required, potentially revolutionising clinical diagnostics by producing actionable results that enable early diagnosis, leading to improved patient outcomes. This review will discuss the challenges associated with the diagnosis of infections and sepsis, primarily within the UK healthcare system. We will consider the clinical potential of spectroscopic point-of-care technologies to enable blood analysis in the primary-care setting.

Electron-electron double resonance measurements on xanthine oxidase.

Electron-electron double resonance measurements were carried out on milk xanthine oxidase (xanthine:oxygen oxidoreductase EC 1.2.3.2) and the spectra obtained supported a previous model, based on EPR data, proposing a spin-spin interaction between unpaired electrons associated with Fe-S and Mo. The technique demonstrated that the additional apparently isotropic, splitting in the Mo EPR spectra observed at low temperature is produced by a single site giving two spectra interconverting at a rate consistent with the Fe-S spin lattice relaxation time. Other data concerning the model and the relaxation behaviour of the species are discussed.

A precursor in the photolytic cleavage of the Co (III)-C bond of coenzyme B-12 unobservable by electron paramagnetic resonance.

Photolysis of a frozen (80--200 K) anaerobic solution of 5'-deoxyadenosyl-cobalamin in aqueous propan-1,2-diol produces only a small Co(II) signal detectable by electron paramagnetic resonance (EPR). Upon warming to room temperature and refreezing without further irradiation the Co(II) signal increases many-fold. The interpretation is that at low temperature there is an EPR-undetectable "incipient" homolysis of the Co-C bond of the coenzyme which is revealed at higher temperature. The possible implications of this observation for the coenzyme B-12-dependent enzymes are noted.

Oxo-vanadium as a spin probe for the investigation of the metal coordination environment of imidazole glycerol phosphate dehydratase.

Imidazole glycerol phosphate dehydratase (IGPD) catalyses the dehydration of imidazole glycerol phosphate to imidazole acetol phosphate, an important late step in the biosynthesis of histidine. IGPD, isolated as a low molecular weight and inactive apo-form, assembles with specific divalent metal cations to form a catalytically active high molecular weight metalloenzyme. Oxo-vanadium ions also assemble the protein into, apparently, the same high molecular weight form but, uniquely, yield a protein without catalytic activity. The VO2+ derivative of IGPD has been investigated by electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) spectroscopy. The spin Hamiltonian parameters indicate the presence of multiple 14N nuclei in the inner coordination sphere of VO2+ which is corroborated by ENDOR and ESEEM spectra showing resonances attributable to interactions with 14N nuclei. The isotropic superhyperfine coupling component of about 7 MHz determined by ENDOR is consistent with a nitrogen of coordinated histidine imidazole(s). The ESEEM Fourier-transform spectra further support the notion that the VO2+ substituted enzyme contains inner-sphere nitrogen ligands. The isotropic and anisotropic 14N superhyperfine coupling components are similar to those reported for other equatorially coordinated enzymatic histidine imidazole systems. ESEEM resonances from axial 14N ligands are discussed.

Development of a medical supply set for corpsmen in the field.

Fleet Marine Force corpsmen are the first medical responders to treat casualties in the field. They carry an outdated bag of supplies called the surgical instrument and supply set. The purpose of this investigation is to develop an updated supply set for field corpsmen by linking each supply item to specific medical tasks conducted in the field, which then creates an audit trail. The review of medical supplies generated an updated list of supplies to be carried by corpsmen in a new medical module and a list of items that corpsmen can pull from the battalion aid station authorized medical allowance lists as needed. Items without a clinical requirement were not included. This improved set of supplies for corpsmen will greatly enhance treatment capability in the field. As technology and needs change, replacements, additions, and deletions of the items can easily be made.

The microbial flora of in-use blood pressure cuffs.

The capacity of blood pressure cuffs to act as vehicles of hospital infection has been recognised. We describe the microbial flora of in-use DINAMAP blood pressure cuffs used in the operating theatres and one recovery room in a teaching hospital. Our results show significant microbial contamination of in-use blood pressure cuffs.

Electron paramagnetic resonance in biochemistry. Computer simulation of spectra from frozen aqueous samples.

Two computer programs are described; they can be used to simulate e.p.r. spectra of effective spin-1/2 systems in frozen aqueous samples. One program is written in BASIC and the other in FORTRAN IV. Both programs are deposited as a Supplementary Publication (SUP 50082; 27 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7QB, U.K. References are given to the applications of these programs in biochemical work.

Simulation of the electron-paramagnetic-resonance spectrum of the iron-protein of nitrogenase. A prediction of the existence of a second paramagnetic centre.

The e.p.r. spectra of the Fe-proteins of nitrogenase from all sources studied have unusual features in that they have very anisotropic linewidths and low integrated intensities. These characteristics can be explained by assuming that one of the two electrons accepted by these proteins is located at a rapidly relaxing paramagnetic centre that is unobservable by e.p.r., but causes anisotropic broadening of the e.p.r. signal of the other electron. Complex-formation between Fe-proteins and MgATP is described in terms of a 50-60 degrees rotation of the e.p.r.-observable centre.

Nitrogenase of Klebsiella pneumoniae. Kinetics of the dissociation of oxidized iron protein from molybdenum-iron protein: identification of the rate-limiting step for substrate reduction.

Stopped-flow spectrophotometry and e.p.r. spectroscopy were used to study the kinetics of reduction by dithionite of the oxidized Fe protein of nitrogenase from Klebsiella pneumoniae (Kp2ox.) in the presence of MgADP at 23 degrees C at pH 7.4. The active reductant, SO2.-, produced by the predissociation of S2O4(2-) in equilibrium 2SO2.-, reacts with Kp2ox. (MgADP)2, with k4 = 3.0 X 10(6) +/- 0.4 X 10(6) M-1 X s-1. The inhibition of this reaction by the Mo-Fe protein (Kp1) has enabled the rate of dissociation of Kp2ox. (MgADP)2 from Kp1+ (the Kp2-binding site on Kp1) to be measured (k-3 = 6.4 +/- 0.8 s-1). Comparison with the steady-state rate of substrate reduction shows that the dissociation (k-3) of the complex Kp2ox. (MgADP)2-Kp1+, which is formed after MgATP-induced electron transfer from Kp2 to Kp1+, is the rate-limiting step in the catalytic cycle for substrate reduction.

The mechanism of Klebsiella pneumoniae nitrogenase action. Pre-steady-state kinetics of H2 formation.

A comprehensive model for the mechanism of nitrogenase action is used to simulate pre-steady-state kinetic data for H2 evolution in the presence and in the absence of N2, obtained by using a rapid-quench technique with nitrogenase from Klebsiella pneumoniae. These simulations use independently determined rate constants that define the model in terms of the following partial reactions: component protein association and dissociation, electron transfer from Fe protein to MoFe protein coupled to the hydrolysis of MgATP, reduction of oxidized Fe protein by Na2S2O4, reversible N2 binding by H2 displacement and H2 evolution. Two rate-limiting dissociations of oxidized Fe protein from reduced MoFe protein precede H2 evolution, which occurs from the free MoFe protein. Thus Fe protein suppresses H2 evolution by binding to the MoFe protein. This is a necessary condition for efficient N2 binding to reduced MoFe protein.

The mechanism of Klebsiella pneumoniae nitrogenase action. Pre-steady-state kinetics of an enzyme-bound intermediate in N2 reduction and of NH3 formation.

The reduction of N2 to 2NH3 by Klebsiella pneumoniae nitrogenase was studied by a rapid-quench technique. The pre-steady-state time course for N2H4, formed on quenching by the acid-induced hydrolysis of an enzyme-bound intermediate in N2 reduction, showed a 230 ms lag followed by a damped oscillatory approach to a constant concentration in the steady state. The pre-steady-state time course for NH3 formation exhibited a lag of 500 ms and a burst phase that was essentially complete at 1.5s, before a steady-state rate was achieved. These time courses have been simulated by using a previously described kinetic model for the mechanism of nitrogenase action [Lowe & Thorneley (1984) Biochem. J. 224, 877-886]. A hydrazido(2-) structure (=N-NH2) is favoured for the intermediate that yields N2H4 on quenching. The NH3-formation data indicate enzyme-bound metallo-nitrido (identical to N) or -imido (=NH) intermediates formed after N-N bond cleavage to produce the first molecule of NH3 and which subsequently give the second molecule of NH3 by hydrolysis on quenching. The simulations require stoichiometric reduction of one N2 molecule at each Mo and the displacement of one H2 when N2 binds to the MoFe protein. Inhibition by H2 of N2-reduction activity occurs before the formation of the proposed hydrazido(2-) species, and is explained by H2 displacement of N2 at the active site.

The mechanism of Klebsiella pneumoniae nitrogenase action. The determination of rate constants required for the simulation of the kinetics of N2 reduction and H2 evolution.

Kinetic data for Klebsiella pneumoniae nitrogenase were used to determine the values of nine of the 17 rate constants that define the scheme for nitrogenase action described by Lowe & Thorneley [(1984) Biochem. J. 224, 877-886]. Stopped-flow spectrophotometric monitoring of the MgATP-induced oxidation of the Fe protein (Kp2) by the MoFe protein (Kp1) was used to determine the rates of association (k+1) and dissociation (k-1) of reduced Kp2(MgATP)2 with Kp1. The dependences of the apparent KNm2 on Fe protein/MoFe protein ratio and H2 partial pressure were used to determine the mutual displacement rates of N2 and H2 (k+10, k-10, k+11 and k-11). These data also allowed the rate constants for H2 evolution from progressively more reduced forms of Kp1 to be determined (k+7, k+8 and k+9). A mechanism for N2-dependent catalysis of 1H2H formation from 2H2 that requires H2 to be a competitive inhibitor of N2 reduction is also presented.

The mechanism of Klebsiella pneumoniae nitrogenase action. Simulation of the dependences of H2-evolution rate on component-protein concentration and ratio and sodium dithionite concentration.

The rate constants from Table 1 and Scheme 2 of Lowe & Thorneley [(1984) Biochem. J. 224, 877-886] were used to simulate the rate of H2 evolution, under various conditions, from nitrogenase isolated from Klebsiella pneumoniae. These rates depend on both the ratio and concentrations of the MoFe protein and Fe protein that comprise nitrogenase. The simulations explain the shapes of 'protein titration' and 'dilution effect' curves. The concept of an apparent Km for the reductant Na2S2O4 is shown to be invalid, since the dependence of H2-evolution rate on the square root of S2O4(2-) concentration is not hyperbolic and depends on the ratio and absolute concentrations of the MoFe protein and Fe protein.

Magnetic coupling of the molybdenum and iron-sulphur centres in xanthine oxidase and xanthine dehydrogenases.

Magnetic interaction between molybdenum and one of the iron-sulphur centres in milk xanthine oxidase [Lowe, Lynden-Bell & Bray (1972) Biochem. J. 130, 239-249] was studied further, with particular reference to the newly discovered Mo(V) e.p.r.(electron-paramagnetic-resonance) signal, Resting II [Lowe, Barber, Pawlik & Bray (1976) Biochem. J. 155, 81-85]. E.p.r. measurements at 35GHz near to 4.2K showed that the interaction has the same sign at all molybdenum orientations and is ferromagnetic. The predicted splitting of the e.p.r. signal from the reduced iron-sulphur centre, Fe/S I, was observed, Providing positive identification of this as the other interacting species. Chemical modification of the molybdenum environment in xanthine oxidase can change the size of the interaction severalfold, but interaction always remains approximately isotropic. The interaction in turkey liver xanthine dehydrogenase is indistinguishable from that in the oxidase. However, a bacterial xanthine dehydrogenase with different iron-sulphur centres shows rather larger interaction. Guanidinium chloride disturbs the iron-sulphur centres of the oxidase, and when this occurs there is a parallel and relatively small change in the interaction. Removal of flavin from the molecule, or raising the pH to 12.0, changes the interaction slightly without affecting the chromophores themselves. It is concluded that the Fe/S I centre and the Mo are at least 1.0nm and probably nearer 2.5nm apart, and that the conformation of the protein between them is relatively stable up to pH 12.

Purification and characterization of the dissimilatory nitrite reductase from Alcaligenes xylosoxidans subsp. xylosoxidans (N.C.I.M.B. 11015): evidence for the presence of both type 1 and type 2 copper centres.

Dissimilatory nitrite reductase was isolated from extracts of Alcaligenes xylosoxidans subsp. xylosoxidans (N.C.I.M.B. 11015), after activation of crude extracts by the addition of copper(II) sulphate. The enzyme was purified by a combination of (NH4)2SO4 fractionation and cationic-exchange chromatography to 93% homogeneity as judged by SDS/PAGE. SDS/PAGE and spray m.s. showed that the enzyme had a subunit M(r) of 36.5 kDa. The copper content was 3.5 +/- 0.8 Cu atoms/trimer of M(r) 109,500. E.p.r. spectroscopy of nitrite reductase as isolated showed that both type 1 (g parallel = 2.208, A parallel = 6.3 mT) and type 2 (g parallel = 2.298, A parallel = 14.2 mT) Cu centres were present, in contrast with published data [Masuko, Iwasaki, Sakurai, Suzuki and Nakahara (1984) J. Biochem. (Tokyo) 96, 447-454], where only type 1 copper centres were reported. Our preparations had a specific activity of 150-300 mumol of NO2- reduced/min per mg of protein, 6-12-fold higher than reported previously. As isolated, the oxidized form of our preparations of the enzyme showed absorption maxima in the visible region at 460, 593 and 770 nm. The ratio of the absorption bands at 460 nm and 593 nm resulted in this protein having a strong blue colour, in contrast with the green colour of other purified copper-containing nitrite reductases. We conclude that, in contrast with previous reports, this 'blue' nitrite reductase requires both type 1 and type 2 copper centres for optimal activity.