Comparison between visual analysis and microscope assessment of surgical instrument cleanliness from sterile service departments.

Modern hospital sterile service departments (SSDs) routinely inspect instruments visually to assess the degree of contamination present after the washer/disinfector cycle. We aimed to test the effectiveness and reliability of this method. Surgical instrument sets were obtained from nine anonymous National Health Service (NHS) Primary Care Trust SSDs to investigate the efficacy of 'in-place' cleaning procedures. The instruments were first inspected visually, followed by a novel technique called episcopic differential interference contrast microscopy. This was combined with a sensitive fluorescent reagent, SYPRO Ruby, to rapidly visualise and assess contamination levels on the medical devices. The application of a Contamination Index (0-4) for both proteinaceous and non-proteinaceous deposits on the surface allowed quantitative assessment. Close correlation was seen for simple instruments between visual assessment and microscopic analysis. For more complex instruments, however, there was a marked difference between the two assessment techniques and the microscopy procedure showed areas of proteinaceous and non-proteinaceous crystalline soiling that was difficult or impossible to see by eye. Visual assessment of cleaning is fraught with possible error. This survey shows how large amounts of contamination could pass undetected using such antiquated methods. The new methodology applied in the assessment of surface contamination is rapid and generally applicable and could be used more widely for routine monitoring of instrument cleanliness.

Comparative study of surgical instruments from sterile-service departments for presence of residual gram-negative endotoxin and proteinaceous deposits.

The ineffective cleaning of surgical instruments may be a vector for the transmission of hospital-acquired infections. The aim of this research was to investigate whether further decontamination procedures need to be instigated in sterile-service departments (SSDs) to reduce the risk of nosocomial illnesses, such as endotoxemia, sepsis, or iatrogenic Creutzfeldt-Jakob disease (to date, 1,147 cases of confirmed Creutzfeldt-Jakob disease deaths in the United Kingdom since 1990 have been reported). Instrument sets were obtained from nine anonymous United Kingdom National Health Service (NHS) primary care trust SSDs. The investigation implemented an advanced light microscopy technique, episcopic differential interference contrast microscopy with the sensitive fluorescent reagents SYPRO Ruby and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI), to detect proteinaceous and microbial contamination levels. Gram-negative lipopolysaccharide (LPS) endotoxin was monitored using a dansylated polymyxin B fluorochrome agent. None of the 260 instruments examined displayed signs of microbial colonization or LPS endotoxin contamination. However, over 60 percent of the instruments showed a high degree of protein soiling (0.4 to 4.2 mug protein/mm(2)). Some instruments appeared soiled with crystalline deposits that may consist of a potentially hazardous material contributing to inflammation and/or surgical shock. It is clear that the overall standard for cleaning must be raised in order to fulfill the imminent introduction of new European standards and to reduce the risk of cross-patient contamination and iatrogenic transmission.

Rapid method for the sensitive detection of protein contamination on surgical instruments.

Hospital sterile service departments (SSDs) currently rely on simple visual confirmation of cleanliness as an assessment of the efficacy of cleaning surgical instruments. The inherent inability to monitor low levels of infectious or proteinaceous contamination on surgical instruments creates the possibility that highly dangerous and robust biological agents may remain infectious and undetected even after standard cleaning and sterilization procedures have been employed. This paper describes the development of a novel microscopy technique, episcopic differential interference contrast microscope, combined with the fluorescent reagent, SYPRO Ruby, to rapidly detect brain tissue protein to below 400 pg/mm(2) on an instrument surface. This technique has displayed a minimum level of detection observed by 50% of volunteers of 85 pg/mm(2) (95% confidence intervals 67-112 pg/mm(2)). Quantitative assessment of instruments supplied from various SSDs enabled the establishment of a 'contamination index' of both proteinaceous and non-proteinaceous deposits on the surface. This new methodology for the assessment of surface contamination is generally applicable and should facilitate future quantitative surveys of instrument contamination in hospitals and other healthcare environments.

Determining calmodulin binding to metabotropic glutamate receptors with distinct protein-interaction methods.

mGluRs (metabotropic glutamate receptors) are G-protein-coupled receptors that modulate synaptic transmission. The eight mammalian mGluRs form three groups based on sequence and functional similarities: group I (1 and 5), group II (2 and 3) and group III (4, 6-8) mGluRs. In the present study, we used a Y2H (yeast two hybrid) screen to identify proteins that interact with the C-terminal intracellular tail of mGluR3. Prominent among the candidate receptor interacting proteins was calmodulin, a Ca(2+) sensor known to bind identifiable sequences in group I and III mGluRs. The Y2H method was used to investigate calmodulin binding to mGluRs but failed to confirm the documented interaction with group III mGluRs. Furthermore, subsequent biochemical analysis showed that calmodulin does not interact with group II mGluRs. This illustrates that certain Ca(2+)-dependent interactions are not recapitulated in yeast. Moreover, it highlights the necessity for supporting biochemical data to substantiate interactions identified with Y2H methods.