Quantification of Dermatophyte Viability for Evaluation of Antifungal Effect by Quantitative PCR

Dermatophytosis is a common disease caused by dermatophyte fungi such as Trichophyton rubrum and Trichophyton mentagrophytes. A method of quantifying fungal viability in the lesions of dermatophytosis is indispensable for understanding the therapeutic process and outcome; however, no such method has yet been developed. The aim of this study was to develop a method for quantifying dermatophyte viability by quantitative polymerase chain reaction (qPCR). The internal transcribed spacer (ITS) and D1/D2 regions, including each of rRNA and rDNA, were chosen as the targets, and dermatophyte-specific primer pairs were designed corresponding to ITS and D1/D2 regions. The amounts of target RNA and DNA after heat or antifungal treatment were measured by qPCR and compared with colony-forming unit (CFU) counts. RNA and DNA could extract from dermatophytes by mechanical pulverization of conidia using a Multi-Beads Shocker cell disruptor. Our method was sufficiently sensitive to detect 10 copies by qPCR using both ITS and D1/D2 primer pairs. The most sensitive target was ITS-cDNA after heat or antifungal treatment, and essentially consistent with CFU counts. On the other hands, ITS-DNA and D1/D2-DNA were not decreased soon after heat or antifungal treatment, but those were decreased significantly and reflected the CFU counts after 48 h of antifungal treatment. We conclude that ITS-cDNA is useful mainly for quantifying dermatophyte viability at early responses, but ITS-DNA and D1/D2-DNA are also available for evaluation, which does not need an early response.     

Rotenone Enhances the Antifungal Properties of Staurosporine

We studied staurosporine-induced cell death in the filamentous fungus Neurospora crassa. The generation of reactive oxygen species during the process appears to be an important signaling event, since addition of the antioxidant glutathione prevents the effects of staurosporine on fungal growth. Selected mutants with mutations in respiratory chain complex I are extremely sensitive to the drug, stressing the involvement of complex I in programmed cell death. Following this finding, we determined that the complex I-specific inhibitor rotenone used in combination with staurosporine results in a synergistic and specific antifungal activity, likely through a concerted action on intracellular glutathione depletion. Paradoxically, the synergistic antifungal activity of rotenone and staurosporine is observed in N. crassa complex I mutants and in Saccharomyces cerevisiae, which lacks complex I. In addition, it is not observed when other complex I inhibitors are used instead of rotenone. These results indicate that the rotenone effect is independent of complex I inhibition. The combination of rotenone and staurosporine is effective against N. crassa as well as against the common pathogens Aspergillus fumigatus and Candida albicans, pointing to its usefulness as an antifungal agent.Programmed cell death (PCD) refers to a genetically controlled process of cellular suicide initiated by endogenous or extrinsic signals. Many of the genes involved are widely conserved from unicellular to multicellular organisms (46). Apoptosis and autophagy, with its particular characteristics, have been recognized as the main categories of PCD (27). The process of PCD is crucial for the development and homeostasis of metazoan organisms and has been implicated in a number of human disorders, including cancer and neurodegenerative and infectious diseases (3, 10, 25, 55).The participation in PCD of mitochondria, the cellular organelles responsible for the production of most cellular ATP in eukaryotes (30), has been well established. Particularly, these organelles have a central role in the intrinsic (mitochondrion-dependent) pathway of apoptosis, which includes production of reactive oxygen species (ROS), membrane depolarization, ultrastructural changes, and the release of cytochrome c and other proteins (18, 50, 55). Drugs like staurosporine (STS), an inhibitor of protein kinases, have been used to induce the mitochondrion-dependent pathway of apoptosis (24, 35). Staurosporine (48) and derivatives have been used in clinical trials for cancer therapy (63). The complex I inhibitor rotenone too has been widely used to induce PCD and also extensively applied as a pesticide (11, 39, 56). Thus, these types of drugs can be employed for the acquisition of fundamental knowledge and for more practical applications, like modulation of the progression of PCD.Modulation of PCD by targeting metabolic pathways involved in the process can be exploited to the benefit of human health in several very significant situations, from cancer therapy (4, 57) to the treatment of fungal infections (3, 52). However, the molecular basis of PCD involves complex metabolic networks (32, 38, 59), and further work is required for their identification. Neurospora crassa has many advantages for biochemical and genetic experiments (14, 17, 23) and is thus a good model organism for the study of mechanisms of PCD. We are interested in identifying the cell molecular pathways associated with PCD and using this knowledge to devise strategies to modulate the process. In this work, we analyze the effects of STS on the N. crassa wild type and mitochondrial complex I mutants and describe the synergistic effect of combining STS and rotenone on this organism and other human-pathogenic fungi.     

Antifungal Properties of Cranberry Juice

Cranberry juice exerts a significant in vitro antifungal effect on eight representative species of dermatophytes, whereas it has no apparent effect on Candida albicans. The antifungal effect is fungistatic. Benzoic acid or other small molecular weight components, or both, were responsible for the fungistatic action. Studies with C. albicans on the effect of pH alone and the effect of pH on the ionization of benzoic acid indicate that cranberry juice would exert an even more significant antifungal action if the pH were left at its native value of 2.8; not adjusted to 5.6. This would probably be due to pH and a larger amount of free benzoic acid. Further investigation suggested that benzoic acid loses some of its antifungal properties in cranberry juice at pH 5.6. This investigation suggests that the dermatophytes may have a higher sensitivity to benzoic acid or other small molecular weight components of cranberry juice, or to both.     

A Sunflower Lectin with Antifungal Properties and Putative Medical Mycology Applications

Using a new culture method for unculturable soil bacteria, we discovered a novel species, NHI-38^T, from the forest soil of Kyonggi University campus, South Korea. It was a Gram-positive, rod-shaped, and endospore-forming bacterial strain. It grew over a wide pH range (6.5–9.5), with an optimum range of pH 7–9, and in a wide range of temperatures (15–60 °C), with an optimum range of 35–45 °C. Growth was possible at 0–2 % NaCl concentration, and the optimal range was between 0.5 and 1.5 % NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that this new species clustered within the genus Bacillus; it was closely related to “Bacillus abyssalis” SCSIO 15042^T (98.86 %), B. methanolicus NCIMB 13113^T (95.97 %), B. vietnamensis 15-1^T (95.8 %), B. seohaeanensis BH724^T (95.5 %), B. timonensis MM10403188^T (95.33 %), and B. subtilis subsp. subtilis NCIB 3610^T (94.87 %). The main fatty acid components of this bacterium were iso-C_15:0 (35.92 %), summed feature 3 (C_16:1ω7c/C_16:1ω6c; 16.92 %), and anteiso-C_15:0 (14.19 %). The predominant quinone in this bacterial strain was MK-7. The polar lipid profile primarily comprised phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. The genomic DNA G+C composition of the isolate was 40.7 mol%. The DNA–DNA hybridization results indicated that this strain was distinct from other Bacillus species, the degree of similarity being 50 % with “B. abyssalis”, 56 % with B. methanolicus, 47 % with B. vietnamensis, 43 % with B. seohaeanensis, 46 % with B. timonensis, and 32 % with B. subtilis. Based on our results, we regard strain NHI-38^T as a novel member of the Bacillus genus, and we propose the name Bacillus thaonhiensis (=KACC 17216^T = KEMB 9005-019^T = JCM 18863^T).     

Glutaraldehyde: nature of the reagent

Aqueous solutions of glutaraldehyde used for the chemical modification and stabilization of proteins have been found to consist of free glutaraldehyde (I), the cyclic hemiacetal of its hydrate (II) and oligomers of this (III) in equilibrium with each other. Ultraviolet absorption spectra of these solutions at wavelengths greater than 200 nm should have only a single maximum at 280 nm. Absorption at 235 nm is due to an impurity which can be removed by various means. Reactions of the reagent with proteins involve principally the lysinyl (and hydroxylysinyl) residues in the relative amounts of four moles of glutaraldehyde to one of lysine. Three unstable products can be partially isolated from acid hydrolyzates of glutaraldehyde-treated proteins or from the reaction mixtures of glutaraldehyde and model compounds; two of these products have strong ultraviolet absorption near 265 nm.     

Investigation of the Physiological,Biochemical and Antifungal Susceptibility Properties of Candida auris

Background

Candida auris is an emerging pathogen associated with outbreaks in clinical settings. Isolates of the pathogen have been geographically clustered into four clades with high intra-clade clonality. Pathogenicity varies among the clades, highlighting the importance of understanding these differences.

Objectives

To examine the physiological and biochemical properties of each clade of C. auris to improve our understanding of the fungus.

Methods

Optimal growth temperatures of four strains from three clades, East Asia, South Asia and South Africa, were explored. Moreover, assimilation and antifungal susceptibility properties of 22 C. auris strains from the three clades were studied.

Results

The optimal growth temperatures of all strains were 35–37 °C. Assimilation testing demonstrated that the commercial API ID 32 C system can be used to reliably identify C. auris based on the biochemical properties of the yeast. Notably, C. auris can be uniquely differentiated from commonly clinical fungi by its ability to assimilate raffinose and inability to utilize D-xylose, suggesting a useful simple screening tool. The antifungal susceptibility results revealed that all strains are resistant against fluconazole (minimal inhibitory concentration (MIC) 4 to?>?64 µg/mL) and miconazole (MIC 8 to?>?16 µg/mL), with strains from the Japanese lineage showing relatively lower MIC values (1–4 µg/mL). Conversely, itraconazole, voriconazole, amphotericin B, micafungin and caspofungin were active against most of the tested strains. On the clade level, East Asian strains generally showed lower MICs against azoles comparing to the other clades, while they displayed MICs against flucytosine higher than those of strains from South Africa and South Asia clades.

Conclusion

Our data suggest a simple identification approach of C. auris based on its physiological and biochemical properties and highlight aspects of C. auris population from various clades.

     

Glutaraldehyde pretreatment blocks phospholipase A2 modulation of adrenergic receptors

Treatment of rat cerebral cortical membranes with phospholipase A2 affects, in a parallel fashion, beta-, alpha 1- and alpha 2-adrenergic receptor binding, but not the affinity of these receptors for their respective ligands. Pretreatment of membranes with 0.1 percent glutaraldehyde blocks the effects of phospholipase A2 on adrenergic receptor binding. The results support the hypothesis that desensitization or "masking" of adrenergic receptors may involve changes in membrane lipid composition. Furthermore, glutaraldehyde may prove a useful tool in the investigation of the dynamic roles of lipids in receptor function and more specifically, their regulation and coupling to physiological events.     

A Case for Antifungal Stewardship

Purpose of Review

The expanding utilization of limited available antifungal agents has led to a pressing need to implement interventions to ensure appropriate usage. The global emergence of resistant, difficult-to-treat invasive fungal infections among the most vulnerable patient populations is a call to action to develop a multifaceted antifungal stewardship approach.

Recent Findings

Candida species demonstrating multi-drug resistance, including highly resistant Candida auris, are emerging threats. Azole-resistant Aspergillus fumigatus, likely initially originating in the environment, likewise presents a treatment challenge. Routine empiric and prophylactic antifungal use, though effective, further complicates this issue, with the emergence of breakthrough mold infections. Early evidence supports success with antifungal stewardship programs.

Summary

Broad antifungal stewardship approaches that optimize antifungal drug usage, facilitate provider education, and monitor fungal epidemiology are crucial steps to preserve the antifungal armamentarium. Future development of novel diagnostic and treatment strategies will further facilitate management of invasive fungal infections.

     

Inactivation of Enterovirus by Glutaraldehyde

A study on the rate of inactivation by glutaraldehyde of coxsackievirus was conducted using different concentrations, temperatures, and pH values. It was found, that 2 percent glutaraldehyde at pH 7.4 and 25 C, as recommended for a sporicide, reduced the titer of infectious virus by 2 log10U in 1 min or less. The reduction was not negatively affected by high concentrations of organic matter (serum and animal spillings) in the reaction mixtures.     

A Quantitative Health Evaluation of an Eco-Economy in the Semi-Arid Loess Plateau of China

Understanding the factors that affect the health of a semi-arid region's eco-economy is necessary for its sustainable development. The health evaluation, or diagnoses, of an eco-economy at the small watershed scale requires the integrated analysis of ecological, economic, and social factors, yet few studies have achieved this. The health of an eco-economy comprises three components: vigor, organization, and resilience. We use an analytic hierarchy process to develop a health evaluation index system that evaluates the health of an eco-economy system. We then use this diagnostic method to explore the factors affecting the health status of a semi-arid loess watershed in China in 2007 and 2009. The results show that between 2007 and 2009 the health status of the eco-economy improved from the “better” stage to the “benign circle” stage. The primary productivity of grassland, land productivity, rural per capita net income, number of livestock per household, input–output ratio, commodity rate of farm produce, and labor productivity were the main factors influencing the health of this eco-economy. Furthermore, this study shows that the eco-economy depends on material input from regions outside the watershed.     

Antifungal and Root Surface Colonization Properties of GFP-Tagged Paenibacillus brasilensis PB177

Summary This study evaluates the potential of Paenibacillus brasilensis strain PB177 to inhibit phytopathogenic fungi commonly causing maize diseases and to colonize maize plants. In vitro assays demonstrated antagonistic activity against the fungal pathogens, Fusarium moniliforme and Diplodia macrospora. The PB177 strain was tagged with the gfp gene, encoding the green fluorescent protein (GFP) and GFP-tagged bacteria were detected attached to maize roots by stereo- and confocal microscopy. The GFP-tagged bacteria were also used to treat maize seeds before challenging the seeds with two phytopathogenic fungi. The results demonstrated that the bacterial cells are mobilized to the maize roots in the presence of the fungal pathogens. The ability of P. brasilensis PB177 to inhibit fungal growth in vitro and its capability of colonization of maize roots in vivo suggest a potential application of this strain as a biological control agent. This is the first report on the successful introduction of the GFP marker gene into a P. brasilensis strain, enabling the direct observation of these promising plant growth promoting bacteria on maize roots in situ.     

Microemulsion Formulation of Carbendazim and Its In Vitro Antifungal Activities Evaluation

The fungus Rhizoctonia solani Kuhn is a widespread and destructive plant pathogen with a very broad host range. Although various pathogens, including R. solani, have been traditionally controlled using chemical pesticides, their use faces drawbacks such as environmental pollution, development of pesticide resistance, and other negative effects. Carbendazim is a well-known antifungal agent capable of controlling a broad range of plant diseases, but its use is hampered by its poor aqueous solubility. In this study, we describe an environmentally friendly pharmaceutical microemulsion system using carbendazim as the active ingredient, chloroform and acetic acid as solvents, and the surfactants HSH and 0204 as emulsifiers. This system increased the solubility of carbendazim to 30 g/L. The optimal microemulsion formulation was determined based on a pseudo-ternary phase diagram; its physicochemical characteristics were also tested. The cloud point was greater than 90°C and it was resistant to freezing down to −18°C, both of which are improvements over the temperature range in which pure carbendazim can be used. This microemulsion meets the standard for pesticide microemulsions and demonstrated better activity against R. solani AG1-IA, relative to an aqueous solution of pure carbendazim (0.2 g/L). The mechanism of activity was reflected in the inhibition of against R. solani AG1-IA including mycelium growth, and sclerotia formation and germination were significantly better than that of 0.2 g/L carbendazim water solution according to the results of t-test done by SPSS 19.     

Fornix-Based versus Limbus-Based Conjunctival Flap in Trabeculectomy: A Quantitative Evaluation of the Evidence

Objective

To evaluate the efficacy and tolerability of limbus-based (LBCF) compared with fornix-based conjunctival flaps (FBCF) for trabeculectomy in the treatment of patients with uncontrolled glaucoma.

Methods

A comprehensive literature meta-analysis was performed according to the Cochrane Collaboration methodology to identify controlled clinical trials comparing LBCF with FBCF in trabeculectomy. The efficacy measures were the weighted mean differences (WMDs) for intraocular pressure reduction (IOPR), the reduction in glaucoma medications, and the relative risks (RRs) for success rates. Tolerability estimates were measured by RR for adverse events. The pooled effects were calculated using the random effects model.

Results

Sixteen controlled clinical trials meeting the predefined criteria were included in the meta-analysis. A total of 1,825 eyes from 1,392 patients with medically uncontrolled glaucoma were included. The WMD of the IOPR from baseline was 1.12 (95% CI: −0.88 to 3.12) when comparing LBCF with FBCF. LBCF was associated with numerically greater but non-significant IOP lowering efficacy than FBCF (P = 0.270). LBCF was comparable with FBCF in the reduction of glaucoma medication, with a WMD of 0.15 (−0.05 to 0.36) at the follow-up endpoint (P = 0.141). The pooled RR comparing LBCF with FBCF were 1.08 (0.94, 1.23) for the complete success rate and 1.01 (0.92, 1.10) for the qualified success rate. Rates of adverse events did not differ significantly between LBCF and FBCF.

Conclusions

There is no significant difference in IOP lowering, number of glaucoma medications, or proportion of patients who reached the IOP target between LBCF and FBCF trabeculectomy. Both incision techniques may contribute equally to adverse events.     

Glutaraldehyde Inactivation of Virus in Tissue

High concentrations of influenza virus and T3 coliphage were inoculated into mouse tissue blocks. Exposure of the inoculated tissue blocks to 5% alkaline glutaraldehyde resulted in rapid inactivation of both viral agents.     

Studies on the Mechanism of the Sporicidal Action of Glutaraldehyde

S ummary . Low concentrations (0.025–0.125%) of glutaraldehyde inhibited or prevented colony formation by Escherichia coli, Bacillus subtilis and B. pumilis in agar, and inhibited germination of spores of the Bacillus spp. in L-alanine plus D-glucose. Higher concentrations (2%) of glutaraldehyde at pH 8.5 were sporicidal. Pre-treatment of spores with glutaraldehyde lessened release of dipicolinic acid when the spores were subsequently heated at 100°, but not at 121°. Spores treated with glutaraldehyde and then with 0.5 M thioglycollic acid in 6 M urea at 70° were less sensitive to lysis by hydrogen peroxide than spores which had not been exposed to glutaraldehyde. Glutaraldehyde was less effective in preventing peroxide induced lysis if added to spores which had been previously exposed to thioglycollic acid plus urea at 70°. The mechanism of the sporicidal activity of glutaraldehyde is discussed in relation to these findings.     

Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks performed by the RPE are phagocytosis and processing of outer photoreceptor segments through lysosome-derived organelles. These degradation products, stored and referred to as lipofuscin granules, are composed partially of bisretinoids, which have broad fluorescence absorption and emission spectra that can be detected clinically as fundus autofluorescence with confocal scanning laser ophthalmoscopy (cSLO). Lipofuscin accumulation is associated with increasing age, but is also found in various patterns in both acquired and inherited degenerative diseases of the retina. Thus, studying its pattern of accumulation and correlating such patterns with changes in the overlying sensory retina are essential to understanding the pathophysiology and progression of retinal disease. Here, we describe a technique employed by our lab and others that uses cSLO in order to quantify the level of RPE lipofuscin in both healthy and diseased eyes.