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Background: The standard of care for disinfecting needleless connectors (NCs) of central venous catheters includes alcohol-containing caps or up to a 15-second scrub with alcohol or chlorhexidine. Due to the clinical impact and high cost of treating Central line-associated bloodstream infections (CLABSI), reducing the incidence of CLABSI is a priority for public health and of the Centers for Disease Control. Alcohol-containing caps have been demonstrated to disinfect external NC surfaces, but not the internal surface. Ultraviolet light (UV-C) is a strategy for disinfection of NC internal and external surfaces., Methods: Four clinically relevant bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa) and Candida albicans were inoculated on NCs. Disinfection efficacy was measured after exposure to one second of 285 nm UV-C light at 48 mW/cm 2 in a proprietary handheld device and UV-C transparent NC or standard of care. Disinfection of internal and external surfaces of NC inoculated with S aureus using alcohol caps, and UV-C was also compared., Results: A 4-log reduction in colony forming units (CFUs) on the interior and exterior surfaces of the UV-transparent NC of clinically relevant pathogens was observed with UV-C light at this power for 1 second., Discussion: We demonstrated the efficacy of UV-C for the disinfection of NCs in one second using the UV-C device in benchtop studies., Conclusions: This device holds promise for reducing CLABSI, and clinical studies are planned., (Published by Elsevier Inc.)

A novel hybrid UV-C/microfiltration process for water disinfection is presented, and its application in continuous mode operation to the removal of different pathogen germs (Escherichia coli, Enterococcus faecalis, and Candida albicans) present in urban wastewater. The membrane photoreactor is based on porous stainless steel membranes coated with a TiO 2 layer and illuminated by a UV-C lamp (254 nm). A valve actuator in the outlet of the UV-C stream allows operation of the system under conditions of constant transmembrane pressure (TMP) keeping the UV-C contact time in few seconds, significantly lower than the typical irradiation time employed in TiO 2 photocatalytic processes. An E. coli removal of up to 4-log in the permeate stream and up to 2-log in the UV-C outlet was achieved with a 0.2 μm membrane operating with a TMP of 0.5 bar and a UV-C contact time as low as 8 s. The microbial balance data from the cells recovered from the membrane confirmed that 96-98% of the removed microorganisms died due to the UV-C action over the membrane surface. Modification of the membrane with a TiO 2 layer has been also shown to be a suitable way to improve both the UV-C inactivation and the filtration efficiency. The results reported in this work constitute a proof of concept of the synergy between UV-C and filtration that can be achieved in a hybrid UV-C/microfiltration system, being a good example of process intensification where two products of different quality can be simultaneously obtained.

Background: Ultraviolet irradiation is effectively used as a disinfection method for inactivating microorganisms. Methods: We investigated the bactericidal effects by irradiation with a deep-ultraviolet light-emitting diode (DUV-LED) on the causative microorganisms of catheter related blood stream infection contaminating the solution for intravenous infusion. For irradiation, prototype modules for water disinfection with a DUV-LED were used. Experiments were conducted on five kinds of microorganisms. We examined the dependence of bactericidal action on eleven solutions. Administration sets were carried out three types. Results: When the administration set JY-PB343L containing the infusion tube made of polybutadiene was used, the bactericidal action of the DUV-LED against all tested microorganisms in the physiological saline solutions was considered to be effective. We confirmed that the number of viable bacteria decreased in 5% glucose solution and electrolyte infusions with DUV-LED irradiation. Conclusions: These results indicate that the DUV-LED irradiation has bactericidal effects in glucose infusion and electrolyte infusions by irradiating via a plasticizer-free polybutadiene administration set. We consider DUV-LED irradiation to be clinically applicable., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Objective: The aim of this study was to quantitatively investigate the efficiency of the ultraviolet (UV) and visible light in eradication of Candida albicans in vitro; in particular, to determine, for selected wavelengths, the specific eradication coefficients and thresholds in terms of energy density levels required to effect 3.0log10 and 4.0log10 reduction., Background Data: Oral candidosis is the most common infection of the oral cavity and is caused by Candida species. The widespread use of topical and systemic antifungal agents as conventional treatment for oral candidosis has resulted in the development of resistance in C. albicans. Therefore, it has become necessary to develop alternative therapies for the treatment of oral candidosis., Methods: C. albicans ATCC(®) 90028(™) was irradiated with 254 nm, 365 nm, 406 nm, 420 nm, and broadband Xe spectrum. For each wavelength, a fit of experimental data (survival fraction vs. applied energy density) with an exponential decay function enabled estimation of the specific eradication coefficients and thresholds., Results: Based on estimated specific efficiencies (Δ) and eradication thresholds (ET) of the investigated wavelengths, the ranking in eradication efficiency of C. albicans (most to least effective) is: 254 nm (Δ=6.1 mJ/cm(-2), ET99.99=56 mJ/cm(-2)), broadband Xe spectrum (Δ=27.7 mJ/cm(-2), ET99.99=255 mJ/cm(-2)), 365 nm (Δ=4.3 J/cm(-2), ET99.99=39 J/cm(-2)), 420 nm (Δ=0.65 J/cm(-2), ET99.99=6 J/cm(-2)), and 406 nm (Δ=11.4 J/cm(-2), ET99.99=104 J/cm(-2))., Conclusions: The results provide insight into the wavelength-dependent dynamics of eradication of C. albicans. For each investigated wavelength, the eradication coefficient and corresponding eradication threshold were estimated. The observed different eradication efficiencies are consequence of different spectrally dependent inactivation mechanisms. The established methodology enables unambiguous quantitative comparison of eradication efficiencies of optical radiation and selection of most effective wavelengths for clinical and therapeutic use.

Background: Corneal collagen cross-linking (CXL), a technique that combines riboflavin administration with long-wave ultraviolet light irradiation, was primarily developed to increase the biomechanical strength of collagen fibrils of the cornea to avoid the progression of keratoconus. Recently, this method has been proposed to treat selected cases of infectious keratitis., Methods: To test the protocol used for progressive keratoconus in infectious keratitis, Candida albicans, and Fusarium solani, strains were exposed to irradiation using a wavelength of 365 nm at a power density of 3 mW/cm(2) for 30 min in the presence of riboflavin photosensitizer. All experiments were performed in triplicate. Qualitative and quantitative measurements of fungal viability used plate cultures and an automated trypan blue dye exclusion method respectively. Fungal cell diameter was also assessed in all groups. Statistical analyses were performed using the triplicate values of each experimental condition., Results: Experimental findings of photodynamic therapy applied to the cell inactivation of both yeasts and filamentous fungi were compared with control groups. Qualitative results were corroborated with quantitative findings which showed no statistical significance between challenged samples (experimental groups) and the control group (p-value = 1). In comparison with a control group of live cells, statistical significance was observed when riboflavin solution alone had an effect on the morphologic size of filamentous fungi, while ultraviolet light irradiation alone showed a slight decrease in the cell structure of C. albicans., Conclusions: The impact of long-wave ultraviolet combined with riboflavin photosensitizer showed no antifungal effect on C. albicans and F. solani. The significant decrease in cell morphology of both filamentous fungi and yeasts submitted to photosensitizing riboflavin and exposure to ultraviolet light, respectively, may be promising in the development and standardization of alternatives for fungal cell inactivation, because of their minimal cytotoxic effects on the corneal surface. The methodological improvement in the preparation and application of individual chemical compounds, such as riboflavin, or physical systems, such as a long-wave light source, as antifungal agents may also assist in establishing promising therapeutic procedures for keratomycosis.

Background: Candida albicans is known as part of human's skin normal flora; but simultaneously, is the most common opportunistic fungal pathogen of human which can cause a variety of infections including cutaneous candidiasis. Because of the importance of superficial infections like cutaneous candidiasis, we tried to use Ultraviolet B light as a method of phototherapy for inducing apoptosis in irradiated colonies of Candida albicans., Materials and Methods: The Ultraviolet B with the wavelength of 302 nanometer was used for irradiating the colonies of Candida albicans. For detecting the eventual apoptotic reactions, the DNA of irradiated colonies as well as control colonies were extracted and then were run in 1% agarose gel electrophoresis containing ethidium bromide to observe luminescent DNA bands., Results: Despite irradiating of Ultraviolet B to yeast cells, no abnormalities including DNA laddering bands or smears were detected in bands formed by total genomic DNA., Discussion: The applied irradiation protocol in this investigation was not successful to induce apoptotic reactions in Ultraviolet-exposed colonies. Maybe, Heat shock proteins as the important part of fungal protein pool, inhibit the inducing pathway of apoptosis in irradiated colonies of Candida albicans.

This constitutes the first study to report on the relationship between pulsed UV light (PL) irradiation and the simultaneous occurrence of molecular and cellular damage in clinical strains of Candida albicans. Microbial protein leakage and propidium iodide (PI) uptake assays demonstrated significant increases in cell membrane permeability in PL-treated yeast that depended on the amount of UV pulses applied. This finding correlated well with the measurement of increased levels of lipid hydroperoxidation in the cell membrane of PL-treated yeast. PL-treated yeast cells also displayed a specific pattern of intracellular reactive oxygen species (ROS) generation, where ROS were initially localised in the mitochondria after low levels of pulsing (UV dose 0.82 μJ/cm(2)) before more wide-spread cytosolic ROS production occurred with enhanced pulsing. Intracellular ROS levels were measured using the specific mitochondrial peroxide stain dihydrorhodamine 123 and the cytosolic oxidation stain dichloroflurescin diacetate. Use of the dihydroethidium stain also revealed increased levels of intracellular superoxide as a consequence of augmented pulsing. The ROS bursts observed during the initial phases of PL treatment was consistent with the occurrence of apoptotic cells as confirmed by detection of specific apoptotic markers, abnormal chromatin condensation and externalisation of cell membrane lipid phosphatidylserine. Increased amount of PL-irradiation (ca. UV does 1.24-1.65 μJ/cm(2)) also resulted in the occurrence of late apoptotic and necrotic yeast phenotypes, which coincided with the transition from mitochondrial to cytosolic localisation of ROS and with irreversible cell membrane leakage. Use of the comet assay also revealed significant nuclear damage in similarly treated PL samples. Although some level of cellular repair was observed in all test strains during sub-lethal exposure to PL-treatments (≤20 pulses or UV dose 0.55 μJ/cm(2)), this was absent in similar samples exposed to increased amounts of pulsing. This study showed that PL-irradiation inactivates C. albicans test strains through a multi-targeted process with no evidence of microbial ability to support cell growth after ≤20 pulses. Implications of our findings in terms of application of PL for contact-surface disinfection are discussed., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Aims: The major objective of the study was to evaluate the enhanced germicidal effects of low-frequency pulsed ultraviolet A (UVA)-light-emitting diode (LED) on biofilms., Methods and Results: The germicidal effects of UVA-LED irradiation (365 nm, 0·28 mW cm(-2) , in pulsed or continuous mode) on Candida albicans or Escherichia coli biofilms were evaluated by determining colony-forming units. The morphological change of microbial cells in biofilms was observed using scanning electron microscopy. After 5-min irradiation, over 90% of viable micro-organisms in biofilms had been killed, and pulsed irradiation (1-1000 Hz) had significantly greater germicidal ability than continuous irradiation. Pulsed irradiation (100 Hz, 60 min) almost completely killed micro-organisms in biofilm (>99·9%), and 20-min irradiation greatly damaged both microbial species. Interestingly, few hyphae were found in irradiated Candida biofilms. Moreover, mannitol treatment, a scavenger of hydroxyl radicals (OH(•) ), significantly protected viable micro-organisms in biofilms from UVA-LED irradiation., Conclusions: The study demonstrated that pulsed UVA-LED irradiation has a strong germicidal effect (maximum at 100 Hz, over 5-min irradiation) and causes the disappearance of hyphal forms of Candida., Significance and Impact of the Study: This study can assist in developing a low-frequency pulsed UVA-LED system to be applied to pathogenic biofilms for disinfection., (© 2010 The Authors. Journal of Applied Microbiology © 2010 The Society for Applied Microbiology.)

Purpose: To demonstrate the antimicrobial properties of riboflavin/UVA (365 nm) against common pathogens., Methods: One group of bacteria (Pseudomonas aeruginosa [PA], Staphylococcus aureus [SA], and Staphylococcus epidermidis [SE]) was tested by using Kirby-Bauer discs with (1) empty disc (Control - C); (2) riboflavin 0.1% (B2); (3) riboflavin 0.1% previously activated by UVA (B2'); (4) UVA alone (UVA); (5) group 2+additional UVA exposure (UVA+B2); and (6) group 3+additional UVA exposure (UVA+B2'). In addition, another group of microbes was tested with the same approach: methicillin-resistant S. aureus (MRSA), multidrug-resistant P. aeruginosa (MDRPA), drug-resistant Streptococcus pneumoniae (DRSP), and Candida albicans (CA). The mean growth inhibition zone (GIZ) in square millimeters was measured around the discs. The mean standard deviation (MSD) was calculated to be 3.65 when alpha = 0.01. A mean deviation (MD) > MSD indicates a significant difference., Results: In the first group, the GIZ was significantly greater after UVA (MD = 14.30), UVA+B2 (MD = 39.61), and UVA+B2' (MD = 40.45) when compared with C, B2, and B2'. UVA alone was less effective than UVA+B2 (MD = 25.31) and UVA+B2' (MD = 26.15). The second group demonstrated increased GIZ in UVA (MD = 6.98), UVA+B2 (MD = 17.80), and UVA+B2' (MD = 21.15) when compared with C, B2, and B2'. UVA alone was less effective against the second group of bacteria than was UVA+B2 (MD = 10.82) and UVA+B2' (MD = 14.17). CA did not show any GIZ after treatment., Conclusions: Riboflavin/UVA was effective against SA, SE, PA, MRSA, MDRPA, and DRSP, but was ineffective on CA and has the potential for use in treatment of microbial keratitis in the future.

The inactivation efficiencies of microorganisms were found to be enhanced by using silver solution together with ultraviolet light (UV-A, 395 nm) irradiation. The inactivation efficiencies were improved remarkably especially in eukaryotic microorganism. To make clear the inactivation mechanism of microorganisms by the combination effect of silver and ultraviolet light irradiation, the resultant solution was characterized by ESR (Electron spin resonance, ESR). Scanning electron microscopy (SEM) and the methnd for measuring enzyme activity of mitochondria for eukarvotic cells were used to conjecture the mechanism, by analysis of the morphological and physiologic changes in eukaryotic cells. It is proposed that silver oxide (Ag20) can be activated by ultraviolet light irradiation and react with water molecules to produce hydroxyl radical (.OH). Hydroxyl radical could damage cell wall of eukaryotic microorganisms, and inactivate the enzyme activity of mitochondria of eukaryotic microorganism cells. Accordingly, eukaryotic microorganism cells would die. In the experiment, Staphylococcus aureus was employed as the representative of prokaryotic microorganisms, and Candida albicans and Trichophyton mentagrophytes as the representative of eukaryotic microorganisms, respectively. Moreover, the results of the technology applied to washing machine were presented and discussed.

Candida albicans is a human fungal pathogen and has been extensively studied because of its clinical importance. Comprehensive gene analyses have, however, made little progress. This is because of the diploid and asexual characteristics of the fungus that hamper gene disruptions. In this study, we found that ultraviolet (UV) irradiation, as well as mutagen treatment, strongly stimulated loss of heterozygosity (LOH) in strains harboring artificially constructed heterozygosity. UV-induced LOH occurred more frequently in cells within the logarithmic phase of growth compared to those within the stationary phase of growth. This was observed at all loci tested on chromosome 7, except for a locus neighboring the centromere. C. albicans RAD52, whose orthologue in Saccharomyces cerevisiae was reported to be involved in DNA repair by homologous recombination, was shown to be required for UV-induced LOH. These results suggest that high efficiency LOH caused by UV irradiation could be a prominent tool for gene analyses in C. albicans.

In Malassezia furfur, tryptophan as the main nitrogen source induces production of the potent ultraviolet-absorbing indole compound pityriacitrin. An in vitro study about the effects of pityriacitrin on other human skin microorganisms is presented, with special focus on Candida albicans and staphylococci in which its toxicity and UV-protective capacity were investigated. Candida albicans was irradiated with UVB light either in the presence or in the absence of pityriacitrin (11 mmol) and the growth rate was determined. A UVB dose of 1 J cm(-2) caused death of the fungi without pityriacitrin, whereas those in the presence of pityriacitrin showed almost unaffected growth. A diffusion test in staphylococci revealed no antibiotic effects of pityriacitrin. For testing of an ultraviolet-protective effect, staphylococci were either inoculated and irradiated in a plate model for visual assessment of growth or inoculated and irradiated in square quartz cylinders for quantitative measurement of cell density, each time in the absence or presence of pityriacitrin. Cell density of the bacterial suspensions exhibited nearly no influence of pityriacitrin on growth rates, while again a UV-protective effect was observed. In summary, pityriacitrin has an ultraviolet-protective effect on Candida albicans and staphylococci with no toxicity in the range tested.

The effects of UV intensity and turbidity on selected microbial indicator inactivation were investigated. Results showed that UV disinfection was effective in killing all the selected microbial indicators, the resistance order of the microorganisms was as follows: MS-2 coliphage > Bacillus subtilis > E. coli > Staphylococcus aureus and Candida albicans. UV intensity had influence on the inactivation of all the microorganisms, high UV disinfection efficency was obtained with higher UV intensity. Turbidity had impact on the bacteria inactivation rate, but there was no evidence that turbidity had any negative contribution to MS-2 coliphage. Under the same UV dosage, higher UV intensity could overcome the negative influence of turbidity on UV performance, enhanced microorganism inactivation effect in turbidity water.

Ultraviolet B light (UVB) can have negative phototropic effects on fungi. Candida albicans is often found on human skin exposed to UVB. Therefore, it is of medical interest to know whether a negative phototropic response to UVB irradiation can support an invasive growth of this potentially dangerous agent. In our study we investigated how repeated irradiation with low doses of UVB can influence the hyphal growth of C. albicans. Six randomly chosen strains of C. albicans were tested. Formation of hyphae was induced and maintained within transparent agar plates. The fungi were exposed to UVB three times daily for 7 days from either the observe or the reverse side during incubation. The wavelength spectrum was in the range of 310-315 nm, single doses were between 0.0018 and 0.432 J cm(-2). After 7 days the morphology and growth direction of C. albicans cells were determined microscopically. All six strains showed a common and dose-dependent response to UVB irradiation: the progression of hyphal growth was inhibited, no phototropic effects were seen and as a new finding an increased formation of blastospores was observed. We conclude that an irradiation of human skin colonized by C. albicans with doses of UVB that can occur under natural or artificial conditions is unlikely to trigger skin invasion by C. albicans.

Aims: To evaluate the antimicrobial efficacy of a novel u.v. beaker, powered in a domestic microwave oven., Methods and Results: Three beakers were compared, with most rapid killing obtained in the Neutra Plasma 50. Ultraviolet light generated within the beakers efficiently killed planktonic and surface-associated Streptococcus mutans, Pseudomonas aeruginosa, vegetative Bacillus stearothermophilus, herpes simplex and polio viruses. Candida albicans and Mycobacterium phleii were less rapidly killed, and only 70% inactivation of B. stearothermophilus endospores was achieved. Irradiation for 45 s reduced viable bacterial counts in saliva by > 99%., Conclusions: The u.v.-generating beakers efficiently reduced viable counts of bacteria, yeast and viruses. Kinetics of killing varied, reflecting the fact that lethal mechanisms are complex, and probably depend on interplay between u.v. and heat., Significance and Impact of the Study: This novel method of generating u.v., using a cheap and widely available power source, provides a rapid, inexpensive and non-toxic method of disinfection with a wide range of applications in hospitals, clinics and the home.

Managing wounds infected with a mixture of several types of microorganisms such as bacteria (procaryotes) and fungi (eucaryotes) is a challenging clinical situation. The purpose of this study was to determine the effectiveness of ultraviolet light (UVC) in eradicating select procaryotic and eucaryotic organisms, in both pure culture and mixed cultures in vitro. Five replications of each organism or mixture of organisms (10(6) organisms/mL singly or 10(15) organisms/mL mixed culture) were plated. The cultures were treated with a UVC light 1 inch from the surface. Irradiation times were 0, 2, 3, 4, 5, 15, 30, 45, 60, 90, 120, and 180 seconds. Bacterial cultures were incubated and colony counts performed. Upon exposure to UVC, a 99.9% kill rate was obtained at 3 to 5 seconds for the procaryotic organisms (Pseudomonas aeruginosa and Mycobacterium abscessus) tested. However, 15 to 30 seconds of UVC treatment was required to obtain 99.9% kill of the eucaryotic organisms (Candida albicans, Aspergillus fumigatus) tested. This study demonstrates a decreasing sensitivity of evolutionarily more complex organisms to UVC. This study also provides further evidence that short exposure times to UVC are detrimental to procaryotic and simple unicellular eucaryotic organisms while sparing more complex multicellular organisms.

The antimicrobial efficiency of a disinfection box of radiology cassettes, by means of a device which emits ultraviolet rays, was studied according to a method defined by the laboratory. Germ-carriers contaminated with Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans were irradiated and compared to non irradiated reference ones. The bactericidal and fungicidal efficiency was better than 99.9% in 5 minutes of irradiation. This procedure represents an interesting alternative to the chemical treatment of radiology cassettes.

Exposure of mice to UV radiation inhibits the induction and elicitation of the delayed-type hypersensitivity (DTH) response to Candida albicans. To determine whether UV irradiation also affects the pathogenesis of systemic C. albicans infection, C3H mice were exposed to a single dose of 48 kJ/m2 UV-B radiation from FS40 sunlamps 5 days before or 5 days after sensitization with formalin-fixed C. albicans and challenged intravenously (i.v.) with a lethal dose of viable fungi 6 days after sensitization (11 or 1 days after UV irradiation). Exposing unsensitized mice to UV radiation 11 days before lethal challenge had no effect on survival, but the survival time of mice exposed to UV radiation 1 day before challenge was reduced by more than 50%. In the latter group, decreased survival time correlated with persistence of C. albicans in the brain and progressive growth of C. albicans in the kidneys. Sensitization of unirradiated mice with formalin-fixed C. albicans extended their survival time following lethal i.v. challenge with viable C. albicans. Exposing the mice to UV radiation 5 days before sensitization did not abrogate this beneficial effect of sensitization on survival, even though it significantly reduced the DTH response. Thus, immunity to systemic infection did not depend on the ability of the mice to exhibit a DTH response to C. albicans. The beneficial effect of sensitization on survival after lethal infection was abrogated, however, in mice exposed to UV radiation 1 day before lethal challenge with C. albicans. Furthermore, these mice were unable to contain the progressive growth of C. albicans in the kidneys, in contrast to sensitized, unirradiated mice.

Using three UV-plants of different technical designs for water disinfection, we studied the conformity between experimental germ reduction using standard test organisms and calculated UV-doses under various water flow conditions. Taking into consideration the style of construction of the UV-plants, the irradiation area and the layer thickness were used as constant parameters for dose calculations. This was also employed for the irradiation intensity, since the experiments were performed for a relatively short period compared of the life span of the UV-irradiators. Both exposure time and water transmission were employed as variable parameters in the dose calculations and experimental procedures respectively. The calculated UV-dose and experimentally obtained germ reduction values were comparatively the same for two of the three UV-plants studied. However, no correlation was observed between the reduction of E. coli and the corresponding calculated UV-dose values. Therefore, the calculated UV-dose values for any given UV-plant should be considered to be relative and by no means absolute values. We are of the opinion that within a certain range of water flow rate and transmission, antimicrobial effectiveness of different UV-plants should be demonstrated independent of dose values, technical and other construction characteristics. The applicability of the UV-plants studied is discussed.

Anecdotal reports suggest that the dihydropyridine calcium antagonist, nifedipine (NIF), may be phototoxic in human skin. We have studied NIF phototoxicity in vitro using UVA fluorescent tubes (Sylvania PUVA). NIF was phototoxic to Candida albicans and induced photohaemolysis both with NIF present during irradiation and with pre-irradiated drug. In V79 hamster fibroblasts, NIF (10 micrograms ml-1) was phototoxic MTT assay) 24 h after irradiation (0-112 kJ m-2); at 7.5 kJ m-2, about 70% of cells were damaged whilst at 37.5 kJ m-2, only about 45% of cells were damaged. A similar pattern was seen with pre-irradiated NIF. Absorption spectroscopy showed that the NIF absorption maximum (Amax approximately 340 nm) blue-shifted to 314 nm at low UVA doses (7.5 kJ m-2 or less) and red-shifted to 345 nm at higher doses (isosbestic point, 325 nm). Thin layer chromatography of irradiated NIF showed a single photoproduct (PP1; Amax approximately 314 nm) formed at 7.5 kJ m-2 or less which disappeared at higher UVA doses to give further photoproducts. PP1 was highly dark toxic to V79 cells (50% damage at about 5 micrograms ml-1) but PP1 pre-irradiated with UVA was non-toxic. Preliminary gas chromatography-mass spectroscopy studies suggest that PP1 is the nitroso derivative of NIF. These results indicate that NIF phototoxicity in vitro is partially mediated by initial formation of a toxic photoproduct (PP1) but, paradoxically, subsequent UVA irradiation may reduce phototoxicity. The NIF concentrations required to induce in vitro phototoxicity are much greater than therapeutic plasma levels. Unless there is skin accumulation of NIF or PP1, our in vitro results suggest that NIF may not be an important skin-photosensitizing agent in vivo.

Mercury vapor sourced ultraviolet light has been used for several years to disinfect connections that are integral to peritoneal dialysis solution delivery systems. This in vitro study evaluated the suitability of xenon flash sourced ultraviolet light as an effective alternative disinfecting agent. Candida albicans was determined to be at least four times more resistant than Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus mitis, and Pseudomonas aeruginosa to ultraviolet radiation generated by both mercury vapor and xenon flash devices. Both ultraviolet devices effectively disinfected transfer set spikes that were inoculated intraluminally with each of these five test microorganisms suspended in a pool of patient dialysis effluent. However, ultraviolet (254 nm) absorbance values of dialysis effluent was found to vary greatly between patients, with the most ultraviolet-opaque effluent significantly reducing the microbicidal effectiveness of the mercury vapor, but not the xenon flash system.

The efficiency of UV-irradiation for water disinfection was tested using an UV-plant with constant flow between 0.7 and 11.5 m3/h. Within the range of water transmission (254 nm) between 40% and 98% (1 cm), which naturally was found in tap water, water from the Neckar river and biologically treated sewage water in an amount of 96%, 78% and ca. 60%, simulation was performed by artificial addition of fluorescein to demineralized water. Data obtained show similar results for the reduction of E. faecium and C. albicans, which were used as test strains. The investigation show no major discrepancies in the range of transmission from 80-96%, which is relevant for tap water disinfection. Water transmission at 254 nm was confirmed as important factor for UV-irradiation. Also found suitable was fluorescein for simulation of the natural water transmission without affecting the test strains. Thus UV-sensitivity of test strains could be detected by combination of flow rate and simulated water transmission using fluorescein. P. aeruginosa and E. coli were found to be more UV-sensitive than S. enteritidis and S. marcescens. UV-resistance increased on the scale from E. faecium, S. aureus, M. luteus up to C. albicans. The efficiency of an UV-plant can be experimentally defined by a specific combination of flow rate (up to a certain amount) and water transmission. Thus the question can be answered whether and UV-plant is able to fulfill the specific requirements in use. These results, regarding the artificial variation of transmission values, are to be considered important for the specification of an additional measure of safety for minimal irradiation exposure times.

Two sources of ultraviolet (UV) radiation with peak wavelengths in the UV-C or UV-B ranges were compared for their ability to sterilize contact lenses infected with Pseudomonas aeruginosa, Streptococcus pneumoniae, Acanthamoeba castellani, Candida albicans, and Aspergillus niger. Also examined was the effect of prolonged UV light exposure on soft and rigid gas permeable (RGP) contact lenses. The UV-C lamp (253.7 nm, 250 mW/cm2 at 1 cm) was germicidal for all organisms within 20 minutes but caused destruction of the soft lens polymers within 6 hours of cumulative exposure. UV-C caused damage to RGP lenses in less than 100 hours. The UV-B lamp (290-310 nm, 500 mW/cm2 at 1 cm) was germicidal for all organisms tested (except Aspergillus) with a 180-minute exposure and caused less severe changes in the soft lens polymers than did the UV-C lamp, although cumulative exposure of 300 hours did substantially weaken the soft lens material. RGP materials were minimally affected by exposure to 300 hours of UV-B. Ultraviolet light is an effective germicidal agent but is injurious to soft lens polymers; its possible utility in the sterilization of RGP lenses and lens cases deserves further study.

Clinical photoreactions have been reported for quinine and quinidine after systemic and topical administration. We have investigated the phototoxic properties of these two quinoline methanol isomers in vitro using the Candida albicans inhibition test and photohemolysis, and in vivo with the mouse tail phototoxicity test. Both isomers were phototoxic in the hemolysis model, quinine being the more potent compound. In the Candida test only quinidine was phototoxically active. In the mouse tail model, measuring edema, the phototoxic activity of quinidine was comparatively low, causing a 7.3% wet weight increase of tail tissue at a dose of 150 mg/kg intraperitoneally of drug and 54 J/cm2 of UVA. In spite of its structural similarity to quinidine, quinine was not phototoxic in the mouse. These studies support the assumption, based on clinical data, that quinine photoreactions probably have a non-phototoxic mechanism. For quinidine, however, light-induced reactions based on phototoxicity can not be ruled out, since low-grade phototoxic properties were demonstrated in vivo.

Micro-organisms causing peritonitis during peritoneal dialysis or CAPD can reach the peritoneal cavity either along the Tenckhoff-catheter or via contaminated dialysis fluid. Laboratory experiments with UV irradiation of artificially contaminated CAPD fluid were made to demonstrate whether decontamination could be achieved during the flow through a UV-penetrable (lambda 2537 A) section of the CAPD catheter. Five CAPD bags were contaminated with a fluid culture of the following micro-organisms which frequently cause peritonitis (S. epidermidis, S. aureus, P. aeruginosa, E. coli, and Candida albicans). After UV irradiation positive cultures could not be demonstrated. UV irradiation of the peritoneal dialysis fluid before entering the peritoneal cavity is proposed as an additional preventive measure.

Pityrosporum orbiculare, Candida albicans, Staphylococcus epidermidis and S. aureus were irradiated with UVA and UVB light in vitro. UVB inhibited growth much more effectively than UVA. P. orbiculare was the most sensitive and S. aureus the least sensitive organism. With a dose of 900 mJ cm-2 of UVB a 50 times reduction in number of colony forming units was seen for S. aureus and for the other organisms a total inhibition of growth was seen. When P. orbiculare was irradiated with monochromatic light at 300, 330 and 360 nm the highest antimicrobial activity was seen at 300 nm. If these in vitro observations correlates with the effect of UV-light treatment of various skin disorders is still unclear.

The heat shock protocol described in this paper causes mitotic instability in log phase Candida albicans cells. Such instability is induced in diploid, aneuploid and tetraploid strains. The strains analysed are multiple heterozygotes which facilitates the detection of mitotic instability as manifested by the formation of homozygotes. Strains previously shown to be carrying cis linked mutant alleles show coincident segregation of the linked alleles. Conversely, strains which carry unlinked mutant alleles display no such coincident segregation. This segregation of complete linkage groups suggests that heat shock is inducing chromosome loss in C. albicans. The application of this protocol to the genetics of the imperfect fungus C. albicans has produced evidence of at least three chromosomes.

A method for introducing specific mutations into the diploid Candida albicans by one-step gene disruption and subsequent UV-induced recombination was developed. The cloned C. albicans URA3 gene was disrupted with the C. albicans ADE2 gene, and the linearized DNA was used for transformation of two ade2 mutants, SGY-129 and A81-Pu. Both an insertional inactivation of the URA3 gene and a disruption which results in a 4.0-kilobase deletion were made. Southern hybridization analyses demonstrated that the URA3 gene was disrupted on one of the chromosomal homologs in 15 of the 18 transformants analyzed. These analyses also revealed restriction site dimorphism of EcoRI at the URA3 locus which provides a unique marker to distinguish between chromosomal homologs. This enabled us to show that either homolog could be disrupted and that disrupted transformants of SGY-129 contained more than two copies of the URA3 locus. The A81-Pu transformants heterozygous for the ura3 mutations were rendered homozygous and Ura- by UV-induced recombination. The homozygosity of a deletion mutant and an insertion mutant was confirmed by Southern hybridization. Both mutants were transformed to Ura+ with plasmids containing the URA3 gene and in addition, were resistant to 5-fluoro-orotic acid, a characteristic of Saccharomyces cerevisiae ura3 mutants as well as of orotidine-5'-phosphate decarboxylase mutants of other organisms.

A 253.7-nm ultraviolet light with an intensity of 1,100 microW/cm2 was tested for its germicidal activity against contact lenses and storage solutions contaminated with various corneal pathogens. The exposure time necessary to reduce a concentration of organisms from 10(6)/ml to less than 10/ml was 30 seconds for Staphylococcus aureus, 60 seconds for Pseudomonas aeruginosa, and 84 seconds for Candida albicans. The time necessary to sterilize a suspension of 10(4)/ml Acanthamoeba polyphaga was less than three minutes with this technique. Four brands of soft contact lenses were exposed to ultraviolet light for over eight hours without changing their appearance, comfort, or refraction.

Candida (C) albicans cells were exposed to 8-Methoxypsoralen (8-MOP) concentrations of 1.0 microgram/ml and 10.0 microgram/ml medium and irradiated with 365 nm light. The amount of energy emitted was 4.8 J/cm2. Two divergent types of cell damage occured concerning yeast cell cytoplasm and cell wall. Two hours after exposure cytoplasmic changes involving mitochondria, which showed irregularities in shape, blurred appearance or loss of mitochondrial cristae and outer membrane were seen. The number of vacuoles was increased. The cytoplasm showed large electron transparent areas, the cytoplasmic membrane disappeared in some areas completely. Nucleus and nuclear envelope usually remained intact in early stages. 24h after exposure conspicuous cell wall alterations were observed in addition to cytoplasmic changes. Newly produced cell wall material formed ball-like protrusions or was adherent sickle-shaped to the cell wall. The investigations strongly suggest that the results found after 8-MOP-UVA treatment of C. albicans cells can not be interpreted in the sense of a general cytotoxic effect. Apparently it takes the form of a combination of events involving regressive and progressive alterations.

The red-shift of furocoumarin action spectra, compared with their absorption spectra, has been investigated. An action spectrum for 8-methoxypsoralen (8-MOP) monoadduct formation in the yeast Candida albicans has been determined. The yeast cells were initially exposed to sublethal doses of monochromatic UVA at different wavelengths. Monoadduct formation was monitored by growth inhibition induced, after washing out any unbound 8-MOP, by re-irradiation with a constant second (non-lethal) dose of 330 nm radiation. A comparison between this action spectrum and the absorption spectrum of the dark complex of 8-MOP and DNA was made. In addition, the action spectra of monoadduct formation of five monofunctional compounds including a coumarin derivative have been determined. These action spectra were compared with their respective DNA dark complex absorption spectra. In general, the peaks of the furocoumarin DNA dark complexes show a red-shift when compared with the free furocoumarin molecule and the action spectra show peaks which correspond with the peaks of the dark complexes. Such data indicate that the DNA dark complex is the chromophore for growth inhibition in yeast rather than the free furocoumarin. The similarity of the 8-MOP monoadduct formation action spectrum and 8-MOP action spectra suggests that spectral dependence for the photobiological effects (including the red-shift) is dependent on monoadduct formation rather than, as previously suggested by several authors, crosslink formation. The action spectrum for the coumarin derivative 4-methyl N-ethylpyrrolo (3,2-g) coumarin (PCNEt) correlated well with the free molecule absorption spectrum rather than DNA dark complex indicating that the free molecule is the chromophore.(ABSTRACT TRUNCATED AT 250 WORDS)

With the aim of finding new photoactive compounds that may reduce the side effects of 8-methoxypsoralen photochemotherapy we report on some photophysical, photochemical and photobiological properties of recently synthesized pyrrolocoumarins, in particular 4-methyl-N-ethyl-pyrrolo[3,2-g]coumarin (PCNEt) which has an absorption maximum in the UV-A (320-400 nm). Laser (347 nm) flash photolysis studies showed triplet transients that were quenched by O2 and by ground state PCNEt. Singlet minus triplet spectra were broad (350-550 nm) and, at 700 nm, indicated solvated electron and radical production. PCNEt complexes with DNA in the dark and photobinds to thymine but does not form DNA cross-links. PCNEt was phototoxic in yeast with an action spectrum similar to its absorption spectrum. PCNEt showed photohaemolytic activity but was not phototoxic on guinea pig skin. These data suggest that PCNEt may photosensitize via several mechanisms: direct DNA photobinding, photodynamic action, or photoproduction of radicals.

Candida (C.) albicans cells were exposed to 8-Methoxypsoralen (8-MOP) concentrations of 1.0 microgram/ml and 10.0 micrograms/ml medium and irradiated with 365 nm light. The amount of energy emitted was 4.8 J/cm2. Two divergent types of cell damage occured concerning yeast cell cytoplasm and cell wall. Two hours after exposure cytoplasmic changes involving mitochondria, which showed irregularities in shape, blurred appearance or loss of mitochondrial cristae and outer membrane were seen. The number of vacuoles was increased. The cytoplasm showed large electron transparent areas, the cytoplasmic membrane disappeared in some areas completely. Nucleus and nuclear envelope usually remained intact in early stages. 24 h after exposure conspicuous cell wall alterations were observed in addition to cytoplasmic changes. Newly produced cell wall material formed ball-like protrusions or was adherent sickle-shaped to the cell wall. The investigations strongly suggest that the results found after 8-MOP-UVA treatment of C. albicans cells can not be interpreted in the sense of a general cytotoxic effect. Apparently it takes the form of a combination of events involving regressive and progressive alterations.

The effect of PUVA therapy upon 12 dermatophyte strains, 3 of Candida albicans, and 4 of mould fungi was evaluated by the agar dilution method on Sabouraud agar. 8-MOP, 30 microgram/ml medium plus UVA, 4 Joule/cm2 proved to be fungicidal dose to all the strains studied, with the exception of three of the mould fungi. For dematophytes and Candida albicans the MIC was between 1 and 3 microgram, 8-MOP plus UVA, 4 J; and 3 microgram when the UVA dose was reduced to 1--2 J. For mould fungi it was higher. The MFC varied within the range 2 to 30 microgram, when the UVA dose was fixed at 4 J. It reached 16 J when the dose of 8-MOP was kept at 4 microgram. The wide range in the MFC of the dermatophytes could be due to resistant colonies. 8-MOP alone had a distinctly inhibitory effect upon dermatophytes, but not upon Candida albicans or mould fungi.

Auxotrophic variants were obtained following UV-irradiation of Candida albicans hybrids which were heterozygous (+/+/-/-/) for various genetic markers (met, ade, his, lys). Some variants contained less DNA (per cell) than did the hybrids from which they originated; such variants were considered to arise in a process which resulted in generalized reduction in ploidy. These results provide the basis for a cyclic parasexual system (2n X 2n----4n----2n) for genetic analysis in this amictic diploid species.

Prototrophic aneuploid and euploid derivatives of wild type Candida albicans strains 207 were produced by fusing protoplasts of complementing auxotrophs obtained from strain 207. Comparisons of cell survivals and incidences of mitotic recombinants occurring after ultraviolet irradiation (UV) of these strains indicate that (i) aneuploids are categorically less efficient than euploids for repair of pyrimidine dimers induced in DNA by UV and that (ii) such repair is enhanced by growing irradiated cells at 25 degrees C, on minimal medium or in the presence of ergosterol rather than at 37 degrees C, on amino acid enriched medium or medium unsupplemented with ergosterol. In addition, the comparisons establish than one cannot discriminate between strains of C. albicans which differ in cellular DNA contents or genomic constitutions on the basis of their UV survival curves.

Ultraviolet radiation is more effective than either ethyl methanesulfonate or nitrous acid in inducing reverse mutation from auxotrophy to prototrophy in C. albicans. The killing effect of each of the mutagens is greater for cells grown at 37 C than at 25 C after treatment; mutation frequencies are unaffected by post-treatment growth temperatures. Though caffeine depresses survival of mutagen treated cells at both 25 C or 37 C, its effect is more pronounced at 37 C. Caffeine has no effect on mutagenesis by nitrous acid or ethyl methanesulfonate; it depresses UV mutagenesis, but only at 37 C and at high UV dosages. These findings indicate that UV mutagenesis in C. albicans is mediated by a caffeine-sensitive, recombinational system for DNA repair analogous to those known to occur in other species of yeasts. The repair system of C. albicans is unique in being susceptible to caffeine only at high temperature and when the number of DNA lesions to be repaired is large. The caffeine-sensitive steps in repair critical to UV mutagenesis are not involved in fixing mutations induced by the chemical mutagens tested.

The resistances of 10 strains of Pseudomonas aeruginosa and other microorganisms to an ultraviolet (UV) intensity of 100 muW/cm2 were determined. Organisms were exposed in 2- or 15-ml saline suspensions contained in uncapped polyethylene bottles for increasing periods of time, and the surviving fractions were enumerated. Decimal reduction times were calculated by regression analysis, using the least-squares method. The 10 strains of P. aeruginosa, compared with Micrococcus radiodurans and Candida albicans, were very susceptible to low-intensity UV radiation. Results from this study showed that a UV intensity of 100 muW/cm2 penetrated saline suspensions up to 40 mm deep sufficiently to kill high levels of microbial cells, especially P. aeruginosa cells. These results allowed us to design a system for determining and monitoring the sterilization capability of low-intensity UV radiation. In our particular case, UV proved to be an efficient mode for sterilizing saline suspensions of P. aeruginosa in polyethylene bottles. The significance and application of these findings with regard to supporting UV as a sterilant are discussed.