The impact of endoscopic activity on musculoskeletal disorders of high-volume endoscopists in Germany.

Physical stress is common in GI endoscopists, leading to musculoskeletal disorders. Considering the increasing complexity of interventional GI endoscopy with prolonged examination time, work-related musculoskeletal disorders have come into focus. However, data on work-related health stress in German endoscopists are elusive. The aim of this study was therefore to investigate the prevalence and consequences of work-related musculoskeletal disorders in German endoscopists. A 24-item questionnaire on endoscopy-associated musculoskeletal disorders and standardized pain assessment was developed by an interdisciplinary team of endoscopists and sports medics. The survey was distributed online by the leading German societies for gastroenterology and endoscopy. Overall, 151 German practicing endoscopists took part in the study. Regarding the average number of endoscopic procedures per week, the study collective consisted mainly of high-volume endoscopists. The survey showed that most participants suffered from general musculoskeletal disorders (82.8%) and from work-related musculoskeletal disorders (76.8%). The most affected body parts were the neck, low back, thumb, and shoulder. Temporary absence from work due to symptoms was reported by 9.9% of the respondents. Over 30% of participating endoscopists stated the need for analgesics or physiotherapy due to musculoskeletal disorders. Age, professional experience and work time were identified as relevant risk factors for musculoskeletal health issues. A high number of German endoscopists are affected by musculoskeletal disorders due to specific working postures and repetitive movements with a large impact on personal health. Further interventional studies are mandatory to improve the risk prevention of endoscopic activity.

Outcomes of normothermic machine perfusion of liver grafts in repeat liver transplantation (NAPLES initiative).

BACKGROUND: Retransplantation candidates are disadvantaged owing to lack of good-quality liver grafts. Strategies that can facilitate transplantation of suboptimal grafts into retransplant candidates require investigation. The aim was to determine whether late liver retransplantation can be performed safely with suboptimal grafts, following normothermic machine perfusion. METHODS: A prospectively enrolled group of patients who required liver retransplantation received a suboptimal graft preserved via normothermic machine perfusion. This group was compared with both historical and contemporaneous cohorts of patient who received grafts preserved by cold storage. The primary outcome was 6-month graft and patient survival. RESULTS: The normothermic machine perfusion group comprised 26 patients. The historical (cold storage 1) and contemporaneous (cold storage 2) groups comprised 31 and 25 patients respectively. The 6-month graft survival rate did not differ between groups (cold storage 1, 27 of 31, cold storage 2, 22 of 25; normothermic machine perfusion, 22 of 26; P = 0.934). This was despite the normothermic machine perfusion group having significantly more steatotic grafts (8 of 31, 7 of 25, and 14 of 26 respectively; P = 0.006) and grafts previously declined by at least one other transplant centre (5 of 31, 9 of 25, and 21 of 26; P < 0.001). CONCLUSION: In liver retransplantation, normothermic machine perfusion can safely expand graft options without compromising short-term outcomes.

Liver transplantation is a life-saving procedure for many different diseases. In the UK, one in 10 patients awaiting transplant have had a previous liver transplant. These retransplant operations are complex, and the general belief is that a good-quality donor liver graft is required for best outcomes. However, there is a significant shortage of good-quality organs for liver transplantation, so many patients awaiting retransplantation spend longer on the waiting list. This study investigated whether a new technology, called normothermic machine perfusion, could be used to preserve lower-quality donor livers and have successful outcomes for patients undergoing retransplantation. Traditionally, good-quality livers are preserved in an ice box and the study compared the outcomes of these two different approaches. The aim was to prove that normothermic machine perfusion improves access to transplantation for this group of patients, without compromising outcomes. A group of patients who underwent retransplantation and received a lesser-quality liver preserved with normothermic machine perfusion was compared with two groups of patients who had received a transplant with traditional ice-box preservation. The complications, graft, and patient survival of the former group was compared with those in the latter two groups who underwent liver retransplantation with better-quality liver grafts. The rate of survival and adverse surgical outcomes were comparable between the groups of patients who received a liver preserved via traditional ice-box preservation, and those who received a lesser-quality liver preserved via normothermic machine perfusion. Normothermic machine perfusion can potentially expand the number of suitable donor livers available for retransplant candidates.

Severe Sepsis Mimicking Primary Nonfunction Following Liver Transplantation: Normothermic Machine Perfusion Is a Potential Environment for Bacterial Overgrowth and Transmission From Donor to Recipient. A Case Report.

Primary nonfunction (PNF) in the early postoperative period following liver transplantation is fatal if not managed appropriately with early retransplantation. Severe early allograft dysfunction can mimic PNF. The identification of treatable causative factors such as sepsis, hepatic artery, or portal vein thrombosis is essential to distinguish it from PNF, and their early management may avoid the need for retransplantation. In this article, we describe a case of sepsis-induced severe liver dysfunction from a contaminated graft perfused with normothermic machine perfusion (NMP), which presented in a manner similar to PNF. The implications of graft contamination are poorly described. To our knowledge, this is the first report of bacterial contamination of a graft that underwent NMP and subsequently caused severe sepsis in the recipient. The conditions created with NMP may be optimal for certain micro-organisms to thrive. The role of the liver in the immune system is complex as it provides an essential barrier to enterically derived portal venous pathogens and produces numerous acute phase proteins that augment the systemic immune response. Additionally, the liver is also known to restrain harmful and excessive systemic immune responses such as those that occur with the sepsis syndrome. The relationship between bacterial graft contamination, sepsis, and graft dysfunction may be multidirectional.

Improvement in advanced pancreatic cancer survival with novel chemotherapeutic strategies - experience of a community based hospital.

Background: New chemotherapeutic strategies for locally advanced or metastatic pancreatic ductal adenocarcinoma (PDAC) have been shown to improve survival in randomized clinical trials. Little is known about the use of such chemotherapies and their benefit in community-based hospitals. This retrospective study analyzes the overall survival of these patients under "real life conditions" before and after the introduction of FOLFIRINOX in 2011. Methods: We retrospectively identified consecutive patients with PDAC who were treated at our hospital from 2011 to June 2014 (2011+ cohort) and 2004 to 2010 (historical cohort). Patients were included if PDAC was diagnosed in a locally advanced or metastatic state and at least 1 cycle of chemotherapy was given. Survival was assessed until April 2016. Patients with FOLFIRINOX were further analyzed regarding drug administration and side effects. Results: 128 patients met the inclusion criteria. Of the 74 patients in the historical cohort, 62 patients received Gemcitabine. Of the 54 patients diagnosed between 2011 and June 2014, 28 patients received FOLFIRINOX and 22 Gemcitabine as the first-line chemotherapy. Only 34 % of the patients in the historical cohort received a second-line chemotherapy in comparison to 69 % in the 2011+ cohort. Median overall survival (OS) showed a survival of 13.1 months (95 % CI; 11.6 - 14.5) for the 2011+ cohort compared to 9.6 months (95 % CI; 6.1 - 13.1) in the historical group. Conclusion: This study shows a marked improvement in survival of patients diagnosed with locally advanced or metastatic PDAC in a community-based hospital during the past 4 years. The most likely reasons are the use of new polychemotherapies like FOLFIRINOX and the use of second-line chemotherapy.

A randomised controlled trial of the effect of continuous electronic physiological monitoring on the adverse event rate in high risk medical and surgical patients.

We conducted a randomised controlled trial of mandated five-channel physiological monitoring vs standard care, in acute medical and surgical wards in a single UK teaching hospital. In all, 402 high-risk medical and surgical patients were studied. The primary outcome was the proportion of patients experiencing one or more major adverse events, including urgent staff calls, changes to higher care levels, cardiac arrests or death, in 96 h following randomisation. Secondary outcomes were the proportion of patients requiring acute treatment changes, and the 30-day and hospital mortality. In the 96 h following randomisation, 113 (56%) patients in the monitored arm and 116 (58%) in the control arm (OR 0.94, 95% CI 0.63-1.40, p = 0.76) had a major event. An acute change in treatment was necessary in 107 (53%) monitored patients and 101 (50%) control patients (OR 0.55, 95% CI 0.87-1.29). Thirty-four (17%) monitored patients and 35 (17%) control patients died within 30 days. Thirteen patients in the control group received full five-channel monitoring at the request of the ward staff. We conclude that mandated electronic vital signs monitoring in high risk medical and surgical patients has no effect on adverse events or mortality.

Integrated monitoring and analysis for early warning of patient deterioration.

Recently there has been an upsurge of interest in strategies for detecting at-risk patients in order to trigger the timely intervention of a Medical Emergency Team (MET), also known as a Rapid Response Team (RRT). We review a real-time automated system, BioSign, which tracks patient status by combining information from vital signs monitored non-invasively on the general ward. BioSign fuses the vital signs in order to produce a single-parameter representation of patient status, the Patient Status Index. The data fusion method adopted in BioSign is a probabilistic model of normality in five dimensions, previously learnt from the vital sign data acquired from a representative sample of patients. BioSign alerts occur either when a single vital sign deviates by close to +/-3 standard deviations from its normal value or when two or more vital signs depart from normality, but by a smaller amount. In a trial with high-risk elective/emergency surgery or medical patients, BioSign alerts were generated, on average, every 8 hours; 95% of these were classified as 'True' by clinical experts. Retrospective analysis has also shown that the data fusion algorithm in BioSign is capable of detecting critical events in advance of single-channel alerts.

Glycogen autophagy in the liver and heart of newborn rats. The effects of glucagon, adrenalin or rapamycin.

The effects of glucagon, adrenalin or rapamycin on glycogen autophagy in the liver and heart of newborn rats were studied using biochemical determinations and electron microscopy. Glucagon or adrenalin increased autophagic activity in the hepatocytes and myocardiocytes, glycogen-hydrolyzing acid glucosidase activity in the liver and heart and degradation of glycogen inside the autophagic vacuoles. Glucagon or adrenalin also increased the maltose-hydrolyzing acid glucosidase activity in the liver, but not in the heart. Similar effects were produced in the newborn heart by rapamycin. These observations support previous studies suggesting that the cellular machinery which controls glycogen autophagy in the liver and heart of newborn animals, is regulated by the cyclic AMP and the mTOR pathways.

Use of the 'ex vivo' test to study long-term bacterial survival on human skin and their sensitivity to antisepsis.

AIMS: To determine bacterial survival on human skin and their sensitivity to antisepsis. METHODS AND RESULTS: An 'ex vivo' protocol which uses human skin samples placed into diffusion cells, and electron microscopy (EM), were used to study the growth of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa inoculated onto skin samples over a 46-h incubation period at 32 degrees C. Concurrently variation in skin pH was evaluated at different time intervals during this period. In addition the antimicrobial activity of three antiseptics against the incubated micro-organisms was assessed quantitatively with the 'ex vivo' test, while their detrimental effects against bacteria were observed by EM. All three bacteria were still present in high number after 46 h inoculation on skin, although the concentration of E. coli and S. aureus were reduced by 2.74 and 1.58 log(10) reduction, respectively, over this period of time. Electron micrographs showed clear evidence of cell division and some bacteria appeared to be embedded into the skin layers. The antiseptics tested had some antibacterial activity against bacteria incubated on skin for 3 and 10 h, and EM evidence showed some morphological damages including cellular blebbing and the presence of fibrillar material around the cells. All micro-organisms had an acidifying effect on skin samples. CONCLUSIONS: Here, it was shown that bacterial pathogens can survive and grow when incubated on human skin. In addition, it is possible that they can penetrate the stratum corneum, which can provide some protection against antisepsis. SIGNIFICANCE AND IMPACT OF THE STUDY: The apparent low bactericidal activity of biocides attributed in part to bacterial protection from skin layers is particularly important to assess in order to ensure antisepsis efficacy.

Microfabricated silicon microneedles for nonviral cutaneous gene delivery.

BACKGROUND: The skin represents an accessible somatic tissue for therapeutic gene transfer. The superficial lipophilic layer of the skin, the stratum corneum, however, constitutes a major obstacle to the cutaneous delivery of charged macromolecules such as DNA. OBJECTIVES: To determine whether silicon-based microneedles, microfabricated via a novel isotropic etching/BOSCH reaction process, could generate microchannels in the skin of sufficient dimensions to facilitate access of lipid : polycation : pDNA (LPD) nonviral gene therapy vectors. METHODS: Scanning electron microscopy was used to visualize the microconduits created in heat-separated human epidermal sheets after application of the microneedles. Following confirmation of particle size and particle surface charge by photon correlation spectrocopy and microelectrophoresis, respectively, the diffusion of fluorescent polystyrene nanospheres and LPD complexes through heat-separated human epidermal sheets was determined in vitro using a Franz-type diffusion cell. In-vitro cell culture with quantification by flow cytometry was used to determine gene expression in human keratinocytes (HaCaT cells). RESULTS: The diffusion of 100 nm diameter fluorescent polystyrene nanospheres, used as a readily quantifiable predictive model for LPD complexes, through epidermal sheets was significantly enhanced following membrane treatment with microneedles. The delivery of LPD complexes either into or through the membrane microchannels was also demonstrated. In both cases considerable interaction between the particles and the epidermal sheet was observed. In-vitro cell culture was used to confirm that LPD complexes mediated efficient reporter gene expression in human keratinocytes in culture when formulated at the appropriate surface charge. CONCLUSIONS: These studies demonstrate the utility of silicon microneedles in cutaneous gene delivery. Further studies are required to elucidate fully the influence of the physicochemical characteristics of gene therapy vectors, e.g. particle diameter and surface charge, on their diffusion through microchannels and to quantify gene expression in vivo.

Electron microscopic and biochemical study of the effects of rapamycin on glycogen autophagy in the newborn rat liver.

The effects of rapamycin on glycogen autophagy in the newborn rat liver were studied using biochemical determinations, electron microscopy, and morphometric analysis. Rapamycin increased the fractional volume of hepatocytic autophagic vacuoles, the liver lysosomal glycogen-hydrolyzing activity of acid glucosidase, the degradation of glycogen inside the autophagic vacuoles, and decreased the activity of acid mannose 6-phosphatase. These findings suggest that rapamycin, a known inhibitor of the mammalian target of rapamycin (mTOR) signaling, induces glycogen autophagy in the newborn rat hepatocytes. mTOR may participate in the regulation of this process.

Histochemical localization of acid mannose 6-phosphatase activity in the newborn rat hepatocytes.

The localization of acid mannose 6-phosphatase activity in newborn rat hepatocytes was demonstrated at the electron microscopic level by using a histochemical method based on the work of Robinson and Karnovsky. Reaction product was virtually restricted to the lysosomes. Most of them exhibited various grades of reactivity. Some were devoid of activity. Our observations suggested that this histochemical method could be used to differentiate distinct subpopulations of lysosomes on the basis of their acid mannose 6-phosphatase activity.

Assessment of skin viability: is it necessary to use different methodologies?

BACKGROUND/AIM: Skin is complex and may display variable structural and metabolic change 'ex vivo'. The present study aimed to follow measures of skin viability and evaluate their usefulness as markers of viability. MATERIALS AND METHODS: We evaluated the viability of skin samples fresh or after being frozen and subsequently thawed. Assessments included histopathological appearance, lactate dehydrogenase (LDH) activity, oxygen consumption and skin pH. RESULTS: Morphological investigations of fresh and frozen skin samples using light and electron microscopy showed samples with relatively well-defined epidermis and dermis. Frozen samples showed some sign of stratum corneum fragmentation, although this was not obvious. LDH activity measured in fresh samples kept at 4 degrees C was low, but it was stable up to 7 days. Fresh samples kept at 32 degrees C had a comparable LDH activity to the ones kept in the fridge up to 4 days. Frozen samples, thawed and then kept at 4 degrees C showed a stable LDH activity after 24 h of incubation. However, frozen samples incubated at 32 degrees C demonstrated a high variability in results, with up to 800 U/L of LDH activity after 5 days of incubation. Freshly excised as well as freshly thawed samples showed the highest respiration rates. Fresh and thawed samples stored for a long period of time had a significantly lower (sometimes non-existent) oxygen consumption rate. Our results also showed an increase in the oxygen consumption rate of fresh samples being incubated at 32 degrees C for 24 h. The oxygen consumption rate for all samples reached a plateau within the 15-min measurement period and even the fresh samples did not deplete all the oxygen from the medium. Skin samples ex vivo showed a significantly higher pH than human skin in vivo, and when incubated for 46 h at 32 degrees C, fresh samples had a significantly lower pH than frozen samples. All protocols were reproducible and freshly excised and freshly thawed skin samples showed the highest rates of viability. CONCLUSION: ex vivo skin shows variation of several parameters over time. It is recommended to use two or three techniques for evaluation of skin viability including at least oxygen measurement and an enzyme assay.

An electron microscopic and biochemical study of the effects of propranolol on the glycogen autophagy in newborn rat hepatocytes.

The effects of propranolol on the glycogen autophagy in newborn rat hepatocytes were studied by using biochemical determinations, electron microscopy and morphometric analysis. Propranolol lowered the liver cyclic AMP and cyclic AMP-dependent protein kinase activity. It also decreased the formyl-methionyl-leucyl-phenylalanine (FMLP)-inhibitable Ca2+-ATPase activity including lysosomal calcium uptake pump. The normal postnatal increase in the volume of autophagic vacuoles and the activity of acid glycogen-hydrolyzing alpha glucosidase were inhibited. Also, the degradation of glycogen inside the autophagic vacuoles was apparently inhibited. The activity of acid mannose 6-phosphatase was increased. These findings indicate that propranolol influences several steps in the sequence of events leading to the breakdown of glycogen in the autophagic vacuoles of newborn rat hepatocytes. This supports our previous studies suggesting that cyclic AMP regulates glycogen autophagy.

Effects of ortho-phthalaldehyde, glutaraldehyde and chlorhexidine diacetate on Mycobacterium chelonae and Mycobacterium abscessus strains with modified permeability.

The mechanisms of the mycobactericidal action of ortho-phthalaldehyde (OPA), glutaraldehyde (GTA) and chlorhexidine diacetate (CHA) were investigated using mycobacterial spheroplasts of two reference strains, Mycobacterium chelonae NCTC 946, Mycobacterium abscessus NCTC 10882 and two GTA-resistant strains, M. chelonae Epping and M. chelonae Harefield. Transmission electron microscopy of the spheroplasts revealed an altered cell wall structure compared with the parent cells. Structural alterations resulting from the spheroplasting process were in part correlated to a loss of lipid content. Low concentrations of CHA induced protein coagulation in M. chelonae NCTC 946 spheroplasts, which also exhibited the highest loss of free non-polar lipids. Higher concentrations of CHA were required to produce similar results to the other spheroplasts investigated in which there was a less substantial decrease in lipid content. OPA (0.5% w/v) readily penetrated the residual cell wall and cytoplasmic membrane, producing significant protein coagulation in M. chelonae NCTC 946. GTA (0.5% v/v) induced a similar effect but to a lesser extent. Pre-treatment of the spheroplasts with OPA and GTA and their subsequent suspension in water demonstrated that GTA was a more potent cross-linking agent. This protective effect of GTA results from extensive cross-linking of amino and/or sulphydryl side-chain groups of proteins. The rapid mycobactericidal effect of OPA probably arises from its more efficient penetration across biological membranes. Mycobacterial spheroplasts represented a useful cellular model with an altered cell wall permeability. This study also showed the importance of the mycobacterial cell wall in conferring intrinsic resistance to CHA.

Apoptosis in the connective tissues of the periodontal ligament and gingivae of rat incisor and molar teeth at various stages of development.

Apoptosis in the periodontal connective tissues was studied using the TUNEL technique, supported by electron microscopy. For 16 rats (aged 3, 8, or 104 weeks), nuclear fragmentation was assessed using the TUNEL technique (for 4 of the animals aged 8 weeks, incisor eruption was experimentally increased by trimming of teeth to the gingival margin--unimpeded eruption). A further 8 rats (aged 8 and 104 weeks) were employed for electron microscopy. For the incisor, prior to aging, and regardless of eruptive behavior (i.e., for both impeded and unimpeded incisors), there was little evidence of apoptosis in the periodontal ligament or gingival connective tissues. For the molar, apoptosis was also not usually detected when the teeth were erupting or in the mature, erupted state. In the aged animals, however, there was a marked increase in apoptosis (as assessed by the TUNEL technique) within the periodontal ligament and gingivae of both the molars and incisors (where eruption rates also increased). Nevertheless, electron microscopy indicated that significant numbers of apoptotic cells were only in the incisor periodontium. These findings are not consistent with the view that the periodontal fibroblasts provide a component of the force(s) responsible for eruption.

Studies on glycogen autophagy: effects of phorbol myristate acetate, ionophore A23187, or phentolamine.

The effects of agents that could manipulate the lysosomal calcium such as phorbol myristate acetate, ionophore A23187, and phentolamine on the lysosomal glycogen degradation were studied by electron microscopy, morphometric analysis, and biochemical assays in newborn rat hepatocytes. Phorbol myristate acetate, which promotes the input of calcium to lysosomes, increased the total volume of autophagic vacuoles and the activity of lysosomal glycogen-hydrolyzing acid alpha 1,4 glucosidase and decreased the fractional volume of undigested glycogen inside the autophagic vacuoles and also decreased the activity of acid mannose 6-phosphatase. Ionophore A23187, which releases lysosomal calcium, produced opposite results in these enzyme activities. Phentolamine, an alpha-adrenergic blocking agent which interferes with the generation of phosphoinositides and may activate the lysosomal calcium uptake pump, increased the total volume of autophagic vacuoles and the activity of lysosomal glycogen-hydrolyzing acid glucosidase and decreased the fractional volume of undigested glycogen inside the autophagic vacuoles. The results of this study constitute evidence that changes in lysosomal calcium may influence certain aspects of autophagy, including the degradation of glycogen inside the autophagic vacuoles. They also support our previous postulate [Kalamidas and Kotoulas (2000a,b) Histol Histopathol 15:29-35, 1011-1018] that stimulation of autophagic mechanisms in newborn rat hepatocytes may be associated with acid mannose 6-phosphatase activity-deficient lysosomes.

The North-West Diabetes Foot Care Study: incidence of, and risk factors for, new diabetic foot ulceration in a community-based patient cohort.

AIMS: To determine the incidence of, and clinically relevant risk factors for, new foot ulceration in a large cohort of diabetic patients in the community healthcare setting. METHODS: Diabetic patients (n = 9710) underwent foot screening in six districts of North-west England in various healthcare settings. All were assessed at baseline for demographic information, medical and social history, neuropathy symptom score, neuropathy disability score, cutaneous pressure perception (insensitivity to the 10 g monofilament), foot deformities, and peripheral pulses. Two years later, patients were followed up via postal questionnaire to determine the incidence of new foot ulcers. Cox's proportional hazards regression analysis was used to determine the independent, relative risk of baseline variables for new foot ulceration. RESULTS: New foot ulcers occurred in 291/6613 patients who completed and returned their 2-year follow-up questionnaire (2.2% average annual incidence). The following factors were independently related to new foot ulcer risk: ulcer present at baseline (relative risk (95% confidence interval)) 5.32 (3.71-7.64), past history of ulcer 3.05 (2.16-4.31), abnormal neuropathy disability score (> or = 6/10) 2.32 (1.61-3.35), any previous podiatry attendance 2.19 (1.50-3.20), insensitivity to the 10 g monofilament 1.80 (1.36-2.39), reduced pulses 1.80 (1.40-2.32), foot deformities 1.57 (1.22-2.02), abnormal ankle reflexes 1.55 (1.01-2.36) and age 0.99 (0.98-1.00). CONCLUSIONS: More than 2% of community-based diabetic patients develop new foot ulcers each year. The neuropathy disability score, 10 g monofilament and palpation of foot pulses are recommended as screening tools in general practice.

Possible mechanisms for the relative efficacies of ortho-phthalaldehyde and glutaraldehyde against glutaraldehyde-resistant Mycobacterium chelonae.

AIMS: This investigation compared glutaraldehyde (GTA)-sensitive and -resistant strains of Mycobacterium chelonae and examined the effects of pretreatment of GTA-sensitive and -resistant strains of Myco. chelonae with chemical agents that interfere with cell wall synthesis. METHODS AND RESULTS: When exposed to 2% (v/v) GTA at 25 degrees C, GTA-resistant strains of Myco. chelonae dried on to glass carriers were not inactivated to any significant extent. By contrast, GTA-sensitive strains of Myco. chelonae and a strain of Myco. terrae suffered a > 6 log reduction in viability in 5 min. However, ortho-phthalaldehyde (OPA; 0.5% w/v) achieved a corresponding inactivation against two GTA-resistant strains within 5-10 and 10-20 min, respectively. Electron microscopy, using a non-aldehyde fixation process and also negative staining, failed to detect any extensive changes in GTA-sensitive and -resistant cultures exposed to GTA or OPA. Thin-layer chromatography was unsuccessful in detecting differences between GTA-resistant and -sensitive strains of Myco. chelonae. However, pretreatment of GTA-resistant cells with mycobacterial cell wall synthesis inhibitors increased their subsequent susceptibility further to OPA but not to GTA. CONCLUSION: Ortho-phthalaldehyde is an effective new biocidal agent that, at its in-use concentration, is rapidly bactericidal to non-sporulating bacteria, including GTA-sensitive and -resistant mycobacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Pretreatment of GTA-resistant cells with mycobacterial cell wall synthesis inhibitors increased their subsequent susceptibility to OPA but not to GTA.

Encystation in Acanthamoeba castellanii: development of biocide resistance.

Since the early 1960s, axenic culture and the development of procedures for the induction of encystation have made Acanthamoeba spp. superb experimental systems for studies of cell biology and differentiation. More recently, since their roles as human pathogens causing keratitis and encephalitis have become widely recognized, it has become urgent to understand the parameters that determine differentiation, as cysts are much more resistant to biocides than are the trophozoites. Viability of trophozoites of the soil amoeba Acanthamoeba castellanii (Neff), is conveniently measured by its ability to form plaques on a lawn of Escherichia coli. Use of confocal laser scanning microscopy with Calcofluor white, Congo Red or the anionic oxonol dye, DiBAC4(3) or flow cytometry with propidium iodide diacetate and fluorescein or oxonol provides more rapid assessment. For cysts, the plaque method is still the best, because dye exclusion does not necessarily indicate viability and therefore the plate count method has been used to study the sequence of development of biocide resistance during the differentiation process. After two hours, resistance to HCl was apparent. Polyhexamethylene biguanide, benzalkonium chloride, propamidine isethionate, pentamidine isethionate, dibromopropamine isethionate, and H2O2 and moist heat, all lost effectiveness at between 14 and 24 h after trophozoites were inoculated into encystation media. Chlorhexidine diacetate resistance was observed at between 24 and 36 h. The molecular biology and biochemistry of the modifications that underlie these changes are now being investigated.