R. Hervé

Current limitations about the cleaning of luminal endoscopes

BACKGROUND The presence and potential build-up of patient material such as proteins in endoscope lumens can have significant implications, including toxic reactions, device damage, inadequate disinfection/sterilization, increased risk of biofilm development and potential transmission of pathogens. AIM To evaluate potential protein deposition and removal in the channels of flexible luminal endoscopes during a simple contamination/cleaning cycle. METHODS The level of contamination present on disposable endoscopy forceps which come into contact with the lumen of biopsy channels was evaluated. Following observations in endoscopy units, factors influencing protein adsorption inside luminal endoscope channels and the action of current initial cleaning techniques were evaluated using a proteinaceous test soil and very sensitive fluorescence epimicroscopy. FINDINGS Disposable endoscope accessories appear to be likely to contribute to the contamination of lumens, and were useful indicators of the amount of proteinaceous soil transiting through the channels of luminal endoscopes. Enzymatic cleaning according to the manufacturers recommendations and brushing of the channels were ineffective at removing all proteinaceous residues from new endoscope channels after a single contamination. Rinsing immediately after contamination only led to a slight improvement in decontamination outcome. CONCLUSION Limited action of current decontamination procedures and the lack of applicable quality control methods to assess the cleanliness of channels between patients contribute to increasing the risk of cross-infection of potentially harmful micro-organisms and molecules during endoscopy procedures.

Doped diamond-like carbon coatings for surgical instruments reduce protein and prion-amyloid biofouling and improve subsequent cleaning.

Doped diamond-like carbon (DLC) coatings offer potential antifouling surfaces against microbial and protein attachment. In particular, stainless steel surgical instruments are subject to tissue protein and resilient prion protein attachment, making decontamination methods used in sterile service departments ineffective, potentially increasing the risk of iatrogenic Creutzfeldt-Jakob disease during surgical procedures. This study examined the adsorption of proteins and prion-associated amyloid to doped DLC surfaces and the efficacy of commercial cleaning chemistries applied to these spiked surfaces, compared to titanium nitride coating and stainless steel. Surfaces inoculated with ME7-infected brain homogenate were visualised using SYPRO Ruby/Thioflavin T staining and modified epi-fluorescence microscopy before and after cleaning. Reduced protein and prion amyloid contamination was observed on the modified surfaces and subsequent decontamination efficacy improved. This highlights the potential for a new generation of coatings for surgical instruments to reduce the risk of iatrogenic CJD infection.

A rapid dual staining procedure for the quantitative discrimination of prion amyloid from tissues reveals how interactions between amyloid and lipids in tissue homogenates may hinder the detection of prions

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases with no cure to this day, and are often associated with the accumulation of amyloid plaques in the brain and other tissues in affected individuals. The emergence of new variant Creutzfeldt-Jakob disease, an acquired TSE with a relatively long asymptomatic incubation period and unknown prevalence or incidence, which could potentially be iatrogenically transmitted, has prompted the need for sensitive and rapid methods of detection of the pathology indicator, the protease-resistant prion protein (PrP(Sc)), in tissues and on surgical instruments. To discriminate between common tissue proteins and amyloid-rich aggregates such as those formed by abnormal prion, we developed a quantitative thioflavin T/SYPRO Ruby dual staining procedure, used in combination with episcopic differential interference contrast/epifluorescence (EDIC/EF) microscopy for rapid scanning of samples. The detection limit of this direct observation technique applied to brain homogenates was greatly enhanced by the addition of Tween 20, as demonstrated in double-blind studies using various proportions of ME7-infected brain mixed with normal brain homogenate. The characteristic thioflavin T signal correlated with the relative amount of prion amyloid and proved at least 2-log more sensitive than the classic Western blot using the same prepared samples. This new sensitive microscopy procedure, which can be easily applied in instrument decontamination surveys, is likely to be more sensitive that Western blot in practice since it does not rely on the elution of resilient PrP(Sc) bound to the instrument surfaces. Our study also demonstrates how interactions between prion and lipid-rich tissue homogenates may reduce the sensitivity of such detection assays.

Persistent residual contamination in endoscope channels; a fluorescence epimicroscopy study

BACKGROUND AND STUDY AIMS The increasing demand for endoscopic procedures poses new contamination challenges, given developing antimicrobial resistance worldwide and potential viral or prion diseases in populations at risk. We examined working channels from reusable luminal endoscopes used in recent years. METHODS Very sensitive fluorescence epimicroscopy was used to examine working channels from 6 decommissioned and 6 factory-new channels, as received, or following spiking and washing in the laboratory. RESULTS After a single contamination and wash test cycle, new channels retained approximately 75 pg/mm(2) of proteins; through 7 subsequent cycles residual proteins fluctuated between 25 and 75 pg/mm(2). Decommissioned channels harbored 1 - 4 µg of proteins each, except in one gastroscope (33 µg), including up to 2 % amyloid proteins except in one gastroscope and one sigmoidoscope (with over 80 %); lumens showed wearing with established abraded biofilms in 3 cases. After spiking with scrapie-infected blood components and washing, residual protein levels in new channels varied following standard (17.23 pg/mm(2)), duplicated (2.39 pg/mm(2)) or extended (11.3 pg/mm(2)) washing; no changes were measured among the long-established contamination in old channels. CONCLUSIONS Our observations suggest that wear effects in endoscope lumens may contribute to the adsorption of proteins, thus facilitating retention and survival of bacteria. As demonstrated by recent outbreaks worldwide despite recommended reprocessing, the development of antimicrobial-resistant bacterial strains, and the estimated prevalence of variant Creutzfeldt-Jakob disease (vCJD) in the UK particularly, combined with increasing demand for endoscopic procedures, call for sustained precautions and improved methods for the reprocessing of nonautoclavable, reusable surgical instruments.

Down-regulation of DNA mismatch repair enhances initiation and growth of neuroblastoma and brain tumour multicellular spheroids

Multicellular tumour spheroid (MCTS) cultures are excellent model systems for simulating the development and microenvironmental conditions of in vivo tumour growth. Many documented cell lines can generate differentiated MCTS when cultured in suspension or in a non-adhesive environment. While physiological and biochemical properties of MCTS have been extensively characterized, insight into the events and conditions responsible for initiation of these structures is lacking. MCTS are formed by only a small subpopulation of cells during surface-associated growth but the processes responsible for this differentiation are poorly understood and have not been previously studied experimentally. Analysis of gene expression within spheroids has provided clues but to date it is not known if the observed differences are a cause or consequence of MCTS growth. One mechanism linked to tumourigenesis in a number of cancers is genetic instability arising from impaired DNA mismatch repair (MMR). This study aimed to determine the role of MMR in MCTS initiation and development. Using surface-associated N2a and CHLA-02-ATRT culture systems we have investigated the impact of impaired MMR on MCTS growth. Analysis of the DNA MMR genes MLH1 and PMS2 revealed both to be significantly down-regulated at the mRNA level compared with non-spheroid-forming cells. By using small interfering RNA (siRNA) against these genes we show that silencing of MLH1 and PMS2 enhances both MCTS initiation and subsequent expansion. This effect was prolonged over several passages following siRNA transfection. Down-regulation of DNA MMR can contribute to tumour initiation and progression in N2a and CHLA-02-ATRT MCTS models. Studies of surface-associated MCTS differentiation may have broader applications in studying events in the initiation of cancer foci.

Efficacy of humidity retention bags for the reduced adsorption and improved cleaning of tissue proteins including prion-associated amyloid to surgical stainless steel surfaces

Increasing drying time adversely affects attachment of tissue proteins and prion-associated amyloid to surgical stainless steel, and reduces the efficacy of commercial cleaning chemistries. This study tested the efficacy of commercial humidity retention bags to reduce biofouling on surgical stainless steel and to improve subsequent cleaning. Surgical stainless steel surfaces were contaminated with ME7-infected brain homogenates and left to dry for 15 to 1,440 min either in air, in dry polythene bags or within humidity retention bags. Residual contamination pre/post cleaning was analysed using Thioflavin T/SYPRO Ruby dual staining and microscope analysis. An increase in biofouling was observed with increased drying time in air or in sealed dry bags. Humidity retention bags kept both protein and prion-associated amyloid minimal across the drying times both pre- and post-cleaning. Therefore, humidity bags demonstrate a cheap, easy to implement solution to improve surgical instrument reprocessing and to potentially reduce associated hospital acquired infections.