Management and cost of surgical site infection in patients undergoing surgery for spinal metastasis.

BACKGROUND: Surgical site infection (SSI) is a serious potential complication of spinal surgery. SSI may impact significantly on inpatient hospitalization and the costs associated with extra care. AIM: To investigate the management of patients experiencing SSI following surgery for spinal metastatic tumours, and to estimate the costs associated with SSI in this context. METHODS: Patients experiencing SSI following spinal tumour surgery at a large spinal surgery centre between January 2009 and December 2012 were identified. Existing case notes were reviewed and patient and procedural data, details of the infection, and treatment interventions were recorded. A bottom-up approach to calculating costs associated with infection was used for patients experiencing SSI and compared with a quasi-random sample of similar patients without SSI. FINDINGS: The mean cost of treating patients with SSI was significantly greater than costs associated with those without SSI (P=0.019). Mean cost of inpatient hospital stay was 60% higher in patients with SSI compared to those without SSI (P=0.004). Inpatient hospital stay alone accounted for 59% of total costs. Return to theatre was the second most costly intervention overall, accounting for 38% of costs, and was the most expensive single intervention involved in the treatment of SSI. CONCLUSION: SSI significantly increases healthcare costs for patients undergoing surgery for spinal metastasis, with prolonged inpatient hospitalization and return to theatre for wound management being major contributors. The actual total cost to society derived from SSI in this patient group is likely to be far beyond just the direct costs to healthcare providers.

An assessment of key risk factors for surgical site infection in patients undergoing surgery for spinal metastases.

OBJECTIVE: This study aimed to determine the rate of surgical site infection (SSI) in patients undergoing surgery for spinal metastases, and identify key risk factors for SSI among this patient group. METHOD: A retrospective case note review was undertaken in adult patients being treated at a single specialist centre for spinal surgery. RESULTS: There were 152 patients identified for inclusion. Overall SSI rate was 11.2 per 100 patients (9.7 per 100 procedures). An increase in the risk of SSI was observed when surgery involved a greater number of vertebral levels (odds ratio 1.26, p=0.019) when controlling for primary spinal region. Controlling for the number of spinal levels, the odds of SSI increased by a factor of 5.6 (p=0.103) when the primary surgical region was thoracic, as opposed to cervical or lumbar. CONCLUSION: In conclusion, surgery associated with multiple vertebral levels for treatment of spinal metastases, particularly of the thoracic spine, is associated with increased risk of SSI.

Survival of patients undergoing surgery for metastatic spinal tumours and the impact of surgical site infection.

BACKGROUND: Patients with metastatic spinal tumours have a limited prognosis. Surgical complications that may result in prolonged hospitalization or readmission are highly undesirable. Surgical site infection (SSI) is one such complication, which can, in extreme cases, lead to death. AIM: To assess the impact of SSI on patient survival after surgery for spinal metastases. METHODS: Demographic, operative, and survival data were collected on 152 patients undergoing surgery for spinal metastasis at a large UK tertiary referral centre. American Society of Anesthesiologists (ASA) grade and the Revised Tokuhashi Score (RTS) were determined as measures of health status and prognosis, respectively, at baseline. A semi-parametric Cox proportional hazards survival analysis was used to assess the relationships between covariates and survival. FINDINGS: Seventeen patients (11.2%) experienced SSI. Overall, median survival time from operation was 262 days (95% confidence interval: 190-334 days) and 12-month survival was 42.1%. RTS (hazard ratio: 0.82; 95% confidence interval: 0.76-0.89; P < 0.001) and ASA grade (1.37; 1.03-1.82; P=0.028) were significantly associated with survival, with better survival found in patients with higher RTS and lower ASA scores. Infection status was of substantive importance, with better survival in those without SSI (P=0.075). CONCLUSION: Twelve-month survival in patients undergoing surgery for spinal metastasis is ∼42%. RTS and ASA scores may be used as indicators of patient survival either in combination or individually. Whereas SSI has some negative impact on survival, a larger study sample would be needed to confirm whether this is statistically significant.

Qualitative grading of disc degeneration by magnetic resonance in the lumbar and cervical spine: lack of correlation with histology in surgical cases.

BACKGROUND: Clinically, magnetic resonance (MR) imaging is the most effective non-invasive tool for assessing IVD degeneration. Histological examination of the IVD provides a more detailed assessment of the pathological changes at a tissue level. However, very few reports have studied the relationship between these techniques. Identifying a relationship may allow more detailed staging of IVD degeneration, of importance in targeting future regenerative therapies. OBJECTIVES: To investigate the relationship between MR and histological grading of IVD degeneration in the cervical and lumbar spine in patients undergoing discectomy. METHODS: Lumbar (N = 99) and cervical (N = 106) IVD samples were obtained from adult patients undergoing discectomy surgery for symptomatic IVD herniation and graded to ascertain a histological grade of degeneration. The pre-operative MR images from these patients were graded for the degree of IVD (MR grade) and vertebral end-plate degeneration (Modic Changes, MC). The relationship between histological and MR grades of degeneration were studied. RESULTS: In lumbar and cervical IVD the majority of samples (93%) exhibited moderate levels of degeneration (ie MR grades 3-4) on pre-operative MR scans. Histologically, most specimens displayed moderate to severe grades of degeneration in lumbar (99%) and cervical spine (93%). MR grade was weakly correlated with patient age in lumbar and cervical study groups. MR and histological grades of IVD degeneration did not correlate in lumbar or cervical study groups. MC were more common in the lumbar than cervical spine (e.g. 39 versus 20% grade 2 changes; p < 0.05), but failed to correlate with MR or histological grades for degeneration. CONCLUSIONS: In this surgical series, the resected IVD tissue displayed moderate to severe degeneration, but there is no correlation between MR and histological grades using a qualitative classification system. There remains a need for a quantitative, non-invasive, pre-clinical measure of IVD degeneration that correlates with histological changes seen in the IVD.

Silver-impregnated external-ventricular-drain-related cerebrospinal fluid infections: a meta-analysis.

BACKGROUND: Cerebrospinal fluid (CSF) infection is the primary complication associated with placement of an external ventricular drain (EVD). The use of silver-impregnated EVD catheters has become commonplace in many neurosurgical centres. AIM: To assess the effect of silver-impregnated EVD catheter usage on catheter-related CSF infections. METHODS: A meta-analysis was performed by systematically searching Medline, Embase and the Cochrane Library. All randomized controlled trials (RCTs) and non-RCTs comparing silver-impregnated and plain EVD catheters were identified and analysed. FINDINGS: Six non-RCTs were included. The crude infection rate was 10.8% for plain catheters and 8.9% for silver-impregnated catheters [pooled odds ratio (OR) 0.71, 95% confidence interval (CI) 0.46-1.08; P = 0.11]. In a microbiological spectrum analysis, silver-impregnated catheters demonstrated a significantly lower rate of CSF infections caused by Gram-positive organisms (2.0% vs 6.7% in the silver-impregnated and plain catheter groups, respectively; pooled OR 0.27, 95% CI 0.11-0.63; P = 0.002). CONCLUSION: The antimicrobial effects of silver-impregnated EVD catheters may be selective, and may need to be evaluated further in a prospective, controlled manner.

The structure-activity relationship of ferric pyoverdine bound to its outer membrane transporter: implications for the mechanism of iron uptake.

Under iron limitation, Pseudomonas aeruginosa ATCC 15692 secretes a major siderophore, pyoverdine I (PvdI). This molecule chelates iron in the extracellular medium and shuttles it into the cells via a specific outer membrane transporter, FpvAI. PvdI consists of a fluorescent chromophore derived from 2,3-diamino-6,7-dihydroxyquinoline and containing one of the bidentate groups involved in iron chelation, linked to a peptide moiety containing the two other bidentate groups required for binding to Fe(3+). Kinetic studies, based on the fluorescence properties of this siderophore, showed that pH 8.0 was optimal for the binding of PvdI and PvdI-Fe to FpvAI. We investigated the mechanism of interaction of PvdI and PvdI-Fe with FpvAI, by synthesizing various analogues of this siderophore, determining their affinity for FpvAI in vitro and in vivo and their ability to transport iron, and interpreting the results obtained in light of the structure of FpvAI-PvdI. Our findings demonstrate that the succinyl moiety linked to the chromophore of PvdI and the first amino acid of the peptide moiety can be sterically hindered with no effect on binding or the iron uptake properties of PvdI-Fe. Moreover, the sequence and the structure of the peptide moiety of PvdI seems to be more important for the iron uptake step than for the binding of the siderophore to FpvAI. Finally, the efficiency of iron uptake and of recycling of the various PvdI analogues after iron release suggests that iron dissociates from PvdI on FpvAI or in the periplasm. All these data have serious implications for the specificity and mechanism of PvdI-mediated iron transport in P. aeruginosa.

A protective and agonistic function of IL-12p40 in mycobacterial infection.

IL-12p35(-/-)p40(-/-) mice are highly susceptible to Mycobacterium bovis bacillus Calmette-Guérin (BCG) or Mycobacterium tuberculosis infection. In this study IL-12p35(-/-) mice, which are able to produce endogenous IL-12p40, cleared M. bovis BCG and showed reduced susceptibility to pulmonary M. tuberculosis infection, which was in striking contrast to the outcome of mycobacterial infection in IL-12p35(-/-)p40(-/-) mice. Resistance in wild-type and IL-12p35(-/-) mice was accompanied by protective granuloma formation and Ag-specific delayed-type hypersensitivity responses, which were impaired in susceptible IL-12p35(-/- )p40(-/-) mice. Furthermore, IL-12p35(-/-) mice, but not IL-12p35(-/-)p40(-/-) mice, mounted Ag-specific Th1 and cytotoxic T cell responses. In vivo therapy with rIL-12p40 homodimer restored the impaired delayed-type hypersensitivity responses in M. bovis BCG-infected IL-12p35(-/-)p40(-/-) mice and reverted them to a more resistant phenotype. Together, these results show evidence for a protective and agonistic role of endogenous and exogenous IL-12p40 in mycobacterial infection, which is independent of IL-12p70.

Ca2+-independent binding of an EF-hand domain to a novel motif in the alpha-actinin-titin complex.

The interaction between alpha-actinin and titin, two modular muscle proteins, is essential for sarcomere assembly. We have solved the solution structure of a complex between the calcium-insensitive C-terminal EF-hand domain of alpha-actinin-2 and the seventh Z-repeat of titin. The structure of the complex is in a semi-open conformation and closely resembles that of myosin light chains in their complexes with heavy chain IQ motifs. However, no IQ motif is present in the Z-repeat, suggesting that the semi-open conformation is a general structural solution for calcium-independent recognition of EF-hand domains.

A structural characterization of the interactions between titin Z-repeats and the alpha-actinin C-terminal domain.

Titin and alpha-actinin, two modular muscle proteins, are with actin the major components of the Z-band in vertebrate striated muscles where they serve to organize the antiparallel actin filament arrays in adjacent sarcomeres and to transmit tension between sarcomeres during activation. Interactions between titin and alpha-actinin have been mainly localized in a 45-amino acid multiple motif (Z-repeat) in the N-terminal region of titin and the C-terminal region of alpha-actinin. In this study, we provide the first quantitative characterization of alpha-actinin-Z-repeat recognition and dissect the interaction to its minimal units. Different complementary techniques, such as circular dichroism, calorimetry, and nuclear magnetic spectroscopy, were used. Two overlapping alpha-actinin constructs (Act-EF34 and Act-EF1234) containing two and four EF-hand motifs, respectively, were produced, and their folding properties were examined. Complex formation of Act-EF34 and Act-EF1234 with single- and double-Z-repeat constructs was studied. Act-EF34 was shown quantitatively to be necessary and sufficient for binding to Z-repeats, excluding the presence of additional high-affinity binding sites in the remaining part of the domain. The binding affinities of the different Z-repeats for Act-EF34 range from micromolar to millimolar values. The strongest of these interactions are comparable to those observed in troponin C-troponin I complexes. The binding affinities for Act-EF34 are maximal for Zr1 and Zr7, the two highly homologous sequences present in all muscle isoforms. No cooperative or additional contributions to the interaction were observed for Z-repeat double constructs. These findings have direct relevance for evaluating current models of Z-disk assembly.

Isolation of Neospora caninum genes detected during a chronic murine infection.

In order to isolate genes coding for antigens of Neospora caninum which are recognised by the host immune system during a chronic murine infection, a cDNA library was immunoscreened with pooled sera from mice which survived three independent infections by N. caninum. Two new genes from N. caninum were isolated and expressed in Escherichia coli. The genes identified include one homologous to GRA1 of Toxoplasma gondii, plus another (NCP20) previously unknown in any taxon. Both genes encode small polypeptides which induced an IgG response in the mouse and were also recognised by IgG from a cow chronically infected with N. caninum. These results are consistent with the hypothesis that the polypeptides encoded by these genes are a target for the host immune system during chronic infections of N. caninum.

Seroprevalence of Neospora caninum infection following an abortion outbreak in a dairy cattle herd.

OBJECTIVE: To investigate the seroprevalence of Neospora caninum infection in a commercial dairy cattle herd, 15 months after detection of an abortion outbreak. PROCEDURE: Sera from the whole herd (n = 266) were examined for N caninum antibodies by indirect fluorescent antibody test (IFAT) and immunoblot analysis. Herd records were reviewed to collate serological results with abortion history, proximity to calving, and pedigree data. RESULTS: The seroprevalence of N caninum infection was 24% (63/266) for IFAT titre > or = 160, 29% (78/266) for immunoblot positive (+ve), and 31% (82/266) for IFAT > or = 160 and/or immunoblot +ve; 94% (59/63) of animals with IFAT > or = 160 were immunoblot +ve. The association between seropositivity (IFAT > or = 160 and/or immunoblot +ve) and history of abortion was highly significant (P < 0.001); the seroprevalence was 86% (18/21) in aborting cows, compared with 30% (50/164) in non-aborting animals. The abortion rate for seropositive cows was 26% (18/68) compared with 3% (3/117) for seronegative animals. IFAT titres of infected cows were higher within 2 months of calving than at other times (P < 0.001). The association between seropositivity in dams and daughters was highly significant (P = 0.009). CONCLUSIONS: The abortions were associated with N caninum infection and there was evidence of reactivation of latent infection close to calving and congenital transmission of infection. Immunodominant antigens identified by immunoblots may prove useful for improved diagnostic tests.

Binding of alpha-actinin to titin: implications for Z-disk assembly.

Titin is an exceptionally large protein (M.Wt. approximately 3 MDa) that spans half the sarcomere in muscle, from the Z-disk to the M-line. In the Z-disk, it interacts with alpha-actinin homodimers that are a principal component of the Z-filaments linking actin filaments. The interaction between titin and alpha-actinin involves repeating approximately 45 amino acid sequences (Z-repeats) near the N-terminus of titin and the C-lobe of the C-terminal calmodulin-like domain of alpha-actinin. The conformation of Z-repeat 7 (ZR7) of titin when complexed with the 73-amino acid C-terminal portion of alpha-actinin (EF34) was studied by heteronuclear NMR spectroscopy using (15)N-labeling of ZR7 and found to be helical over a stretch of 18 residues. Complex formation resulted in the protection of one site of preferential cleavage of EF34 at Phe14-Leu17, as determined by limited proteolysis experiments coupled to mass spectrometry measurements. Intermolecular NOEs show Val16 of ZR7 to be positioned close in space to the backbone of EF34 around Phe14. These observations suggest that the mode of binding of ZR7 to EF34 is similar to that of troponin I to troponin C and of peptide C20W to calmodulin. These complexes would appear to represent a general alternative binding mode of calmodulin-like domains to target peptides.

Structural and dynamic characterization of omega-conotoxin MVIIA: the binding loop exhibits slow conformational exchange.

omega-Conotoxin MVIIA is a 25-residue, disulfide-bridged polypeptide from the venom of the sea snail Conus magus that binds to neuronal N-type calcium channels. It forms a compact folded structure, presenting a loop between Cys8 and Cys15 that contains a set of residues critical for its binding. The loop does not have a unique defined structure, nor is it intrinsically flexible. Broadening of a subset of resonances in the NMR spectrum at low temperature, anomalous temperature dependence of the chemical shifts of some resonances, and exchange contributions to J(0) from (13)C relaxation measurements reveal that conformational exchange affects the residues in this loop. The effects of this exchange on the calculated structure of omega-conotoxin MVIIA are discussed. The exchange appears to be associated with a change in the conformation of the disulfide bridge Cys8-Cys20. The implications for the use of the omega-conotoxins as a scaffold for carrying other functions is discussed.

Reduced spectral density mapping for proteins: Validity for studies of 13C relaxation.

Spectral density mapping provides direct access to protein dynamics with no assumptions as to the nature of the molecule or its dynamic behaviour. Reduced spectral density mapping characterises a protein's motions at a lower experimental burden, assuming that the spectral density function J(ω) is flat around ωH. This introduces little error for 15N relaxation data but is less valid for 13C studies, perturbing J(ωC) considerably to an extent that depends on the nature of the molecule's motions. We propose the fitting of spectral density at high frequencies to a single Lorentzian and show that the true values of the spectral density lie between those determined by the two approximations.

Bacterial iron transport: 1H NMR determination of the three-dimensional structure of the gallium complex of pyoverdin G4R, the peptidic siderophore of Pseudomonas putida G4R.

Among the fluorescent Pseudomonas species, Pseudomonas putida is a rare case of a nitrogen-fixing bacterium that transforms nitrogen into ammonia. When grown under iron-deficient conditions, it produces two major pyoverdins: pyoverdin G4R and pyoverdin G4RA. Their primary structures have been established using FAB-MS and one- and two-dimensional 15N, 13C, and 1H NMR on both the unlabeled and 15N-labeled compounds [Salah El Din, A. L. M., et al. (1997) Tetrahedron 53, 12539-12552]. The two pyoverdins have a common chromophore derived from 2,3-diamino-6,7-dihydroxyquinoline. The chromophore is bound to the linear heptapeptide L-Asp-L-Orn-D-beta-threo-OHAsp-L-Dab-Gly-L-Ser-L-cyclo-OHOrn . Circular dichroism spectra suggest that the absolute configuration of the metal complex is Delta. The three-dimensional structure in solution of pyoverdin G4R-Ga(III) was determined after interpretation of two-dimensional 1H NMR spectra recorded at 283 and 303 K. The complex is tightly defined with a compact structure with a Delta absolute configuration. The site of complexation of the metal ion is found to be located on the surface of the molecule, showing that the ion can be released without large conformational changes, while the polar groups of the peptide chain, which may be responsible for the recognition of the receptor, are placed on the opposite side of the overall shape. The three-dimensional structure of pyoverdin G4R-Ga(III) is compared with those of other pyoverdins, and the role of the structure in iron uptake is discussed.

The assembly of immunoglobulin-like modules in titin: implications for muscle elasticity.

Titin, a giant muscle protein, forms filaments that span half of the sarcomere and cover, along their length, quite diversified functions. The region of titin located in the sarcomere I-band is believed to play a major rôle in extensibility and passive elasticity of muscle. In the I-band, the titin sequence contains tandem immunoglobulin-like (Ig) modules intercalated by a potentially non-globular region. By a combined approach making use of small angle X-ray scattering and nuclear magnetic resonance techniques, we have addressed the questions of what are the average mutual orientation of poly-Igs and the degree of flexibility around the domain interfaces. Various recombinant fragments containing one, two and four titin I-band tandem domains were analysed. The small-angle scattering data provide a picture of the domains in a mostly extended configuration with their long axes aligned head-to-tail. There is a small degree of bending and twisting of the modules with respect to each other that results in an overall shortening in their maximum linear dimension compared with that expected for the fully extended, linear configurations. This shortening is greatest for the four module construct ( approximately 15%). 15N NMR relaxation studies of one and two-domain constructs show that the motions around the interdomain connecting regions are restricted, suggesting that titin behaves as a row of beads connected by rigid hinges. The length of the residues in the interface seems to be the major determinant of the degree of flexibility. Possible implications of our results for the structure and function of titin in muscles are discussed.

Mapping the active site of factor Xa by selective inhibitors: an NMR and MD study.

The structure of two selective inhibitors, Ac-Tyr-Ile-Arg-Ile-Pro-NH2 and Ac-(4-Amino-Phe)-(Cyclohexyl-Gly)-Arg-NH2, in the active site of the blood clotting enzyme factor Xa was determined by using transferred nuclear Overhauser effect nuclear magnetic resonance (NMR) spectroscopy. They represent a family of peptidic inhibitors obtained by the screening of a vast combinatorial library. Each structure was first calculated by using standard computational procedures (distance geometry, simulated annealing, energy minimization) and then further refined by systematic search of the conformation of the inhibitor docked in the active site and repeating the simulated annealing and energy minimization. The final structure was optimized by molecular dynamics simulations of the inhibitor-complex in water. The NMR restraints were kept throughout the refinement. The inhibitors assume a compact, very well defined conformation, embedded into the substrate binding site not in the same way as a substrate, blocking thus the catalysis. The model allows to explain the mode of action, affinity, and specificity of the peptides and to map the active site.

7Li nuclear-magnetic-resonance study of lithium binding to myo-inositolmonophosphatase.

The interaction of Li+ with myo-inositol monophosphatase was studied by 7Li-NMR spectroscopy. Li+ binding to the enzyme induces a downfield shift and broadening of the 7Li-NMR signal. Changes of the chemical shift were used to follow the titration of the enzyme with lithium and to determine a dissociation constant, Kd = (1.0 +/- 0.1) mM. Only one major binding site/enzyme subunit was inferred. The complex forms independently of the presence of inorganic phosphate. Metals from the group IIa of the periodic table compete with Li+ binding with the affinity increasing in the order Mg2+ < Ca2+ < Be2+. In contrast to lithium, their binding is enhanced by phosphate.

Conformational studies of osteocalcin in solution.

1H-NMR and circular dichroism studies have been carried out on osteocalcin, a 49-residue, calcium-binding protein, the sequence of which contains a disulphide bridge, a proline-rich segment and three gamma-carboxyglutamic acid (Gla) residues. These latter residues have been proposed to lie on one face of an alpha helix and interact with the mineral phase, leading to incorporation of the protein into the bone matrix. Circular dichroism shows an increase in the alpha-helical structure on Ca2+ binding to bovine osteocalcin. This induced structure is lost on heating the protein, giving a spectrum close to that of the Ca(2+)-free protein. 1H-NMR studies of rabbit osteocalcin gave a set of resonance assignments and NOEs which could be interpreted in terms of distance constraints. These did not allow a single conformation to be defined for the protein in solution but reflect rather a flexible structure which may be essential for the function of the protein. The calculated structures contain a hydrophobic core (comprising Leu2, Leu32, Val36 and Tyr42, seen to be slowly flipping in the Ca(2+)-bound form) and have the gamma-carboxyglutamic acid side chains exposed on one face of the molecule.