Antifungal therapy of dermatophytosis in guinea pigs and congenitally athymic rats

Guinea pigs and athymic nude (RNU/RNU) rats were used to assess the efficacy of three orally administered antifungal agents — Tolciclate, Tolnaftate, and Ketoconazole — against Trichophyton mentagrophytes dermatophytosis. All three antifungal agents inhibited the test strain of T. mentagrophytes in vitro. Antifungal agents were tested in intervention (oral therapy started 5 days after challenge) or prophylaxis (oral therapy started 5 days before challenge) protocols. Oral treatment of dermatophytosis on guinea pig skin demonstrated that Tolciclate and Tolnaftate alleviated clinical symptoms and shortened the duration of the dermatophytosis, in comparison to nontreated controls. Assessment of antifungal efficacy in the guinea pig model was time consuming (30–35 days) and variability in the duration and severity of clinical symptoms on guinea pig skin was common.Oral therapy of chronically infected athymic rats demonstrated that Tolciclate, Tolnaftate, and Ketoconazole were effective antifungal agents in vivo. Obvious improvement in clinical symptoms of dermatophytosis (i.e. less erythema and fewer lesions) was evident with all three antifungal agents within 10 days of starting oral therapy. By day 20, athymic rats that were treated with either Tolciclate or Ketoconazole showed marked clinical improvement of the chronic dermatophytosis.Chronically infected athymic rats, which lack thymus matured T-cells, are a promising new model to evaluate the efficacy of antifungal agents by culture, histology, and visual observations of clinical symptoms.     

Sensitivity of some strains of the genus Epidermophyton to different antifungal agents

The inhibitory activity of some antifungal agents against 17 strains of genus Epidermophyton have been studied. The behaviour of Epidermophyton stockdaleae against antifungal agents tested is clearly different from that observed in the strains belonging to Epidermophyton floccosum, since all of the latter were sensitive to the antifungal agents used and the geophilic species showed resistance to griseofulvin, isoconazole, natamycin and nystatin, and intermediate sensitivity to ketoconazole and miconazole. The low sensitivity of E. stockdaleae (and other geophilic dermatophytes) to antifungal agents could be a problem in the current therapy if further studies or case reports demonstrate their pathogenic role.     

Treatment of Invasive Candida Infections in the Neonatal Intensive Care Unit

Invasive Candida infections are a leading cause of morbidity and mortality in the neonatal intensive care unit (NICU). Extremely preterm and very low birth weight infants are at the highest risk of infection. There are currently no antifungal agents that have FDA-labeling for the treatment of invasive candidiasis in the neonatal population. Based on the current IDSA guidelines, amphotericin and fluconazole are considered first-line options for neonatal candidiasis. The newer antifungal agents (i.e., echinocandins and voriconazole) are currently considered second-line or salvage therapy; however, evidence supporting their use is emerging. This review focuses on the supporting evidence for the selection of antifungal agents for treatment of invasive Candida infections in the NICU.     

In Vitro Antifungal Activity of Dihydroxyacetone Against Causative Agents of Dermatomycosis

Dihydroxyacetone (DHA), a three-carbon sugar, is the browning ingredient in commercial sunless tanning formulations. DHA preparations have been used for more than 50 years and are currently highly popular for producing temporary pigmentation resembling an ultraviolet-induced tan. In this work, the in vitro antifungal activity of dihydroxyacetone was tested against causative agents of dermatomycosis, more specifically against dermatophytes and Candida spp. The antifungal activity was determined by the broth microdilution method according to the Clinical and Laboratory Standards Institute guidelines for yeasts and filamentous fungi. The data obtained show that the fungicidal activity varied from 1.6 to 50 mg ml⁻¹. DHA seems to be a promising substance for the treatment of dermatomycosis because it has antifungal properties at the same concentration used in artificial suntan lotions. Therefore, it is a potential low-toxicity antifungal agent that may be used topically because of its penetration into the corneal layers of the skin.     

Characterization and <Emphasis Type="Italic">In Vitro</Emphasis> Evaluation of the Complexes of Posaconazole with β- and 2,6-di-<Emphasis Type="Italic">O</Emphasis>-methyl-β-cyclodextrin

Posaconazole is a triazole antifungal drug that with extremely poor aqueous solubility. Up to now, this drug can be administered via intravenous injection and oral suspension. However, its oral bioavailability is greatly limited by the dissolution rate of the drug. This study aimed to improve water solubility and dissolution of posaconazole through characterizing the inclusion complexes of posaconazole with β-cyclodextrin (β-CD) and 2,6-di-O-methyl-β-cyclodextrin (DM-β-CD). Phase solubility studies were performed to calculate the stability constants in solution. The results of FT-IR, PXRD, ¹H and ROESY 2D NMR, and DSC all verified the formation of the complexes in solid state. The complexes showed remarkably improved water solubility and dissolution rate than pure posaconazole. Especially, the aqueous solubility of the DM-β-CD complex is nine times higher than that of the β-CD complex. Preliminary in vitro antifungal susceptibility tests showed that the two inclusion complexes maintained high antifungal activities. These results indicated that the DM-β-CD complexes have great potential for application in the delivery of poorly water-soluble antifungal agents, such as posaconazole.     

Antifungal Therapeutic Drug Monitoring

A growing body of evidence suggests that patient-to-patient variability in the pharmacokinetics of some antifungals, particularly the mold-active triazoles (itraconazole, voriconazole, and posaconazole) may contribute to therapeutic failure or unexpected toxicity. As a result, many clinicians have recognized a need for therapeutic drug monitoring (TDM) to individualize drug dosing in select patients with suspected or documented invasive fungal infections. However, approaches for performing and interpreting plasma concentrations are not well standardized, and logistical issues such as the turnaround time of test results can limit the clinical usefulness of testing in acutely ill patients. This article summarizes the pharmacologic rationale for TDM of antifungal agents, with a particular focus on recently published data for the newer triazoles, voriconazole and posaconazole. Practical recommendations for TDM-guided dosing are also provided, based on a critical evaluation of literature published over the past 5 years.     

CLSI M38-A2肉汤稀释法对蝙蝠蛾拟青霉(Paecilomyces hepiali)的药敏检测

[目的]对保健食品生产菌株蝙蝠蛾拟青霉(Paecilomyces hepiali)进行6种常见抗真菌药物:伊曲康唑、酮康唑、伏立康唑、环吡酮胺、5-氟胞嘧啶、氟康唑的药敏检测.[方法]参考美国国家临床实验室标准化委员会CLSI M38-A2《肉汤稀释法抗真菌药敏试验参考方案》.[结果]质控菌株MIC值均在参考范围之内,蝙蝠蛾拟青霉(P.hepiali)对以上6种药物的MIC值分别为1 mg/L、0.5 mg/L、0.25 mg/L、≥128 mg/L、64 mg/L、2 mg/L.[结论]M38-A2方案适合用来测定蝙蝠蛾拟青霉(P.hepiali)对抗真菌药物的敏感性,但获得菌株耐药敏感性的判别标准,仍需进一步的试验数据.     

Comparative evaluation of E-test and CLSI methods for Itraconazole,Fluconazole and Ketoconazole susceptibilities of Microsporum canis strains

The incidence of resistance to antifungal agents for dermatophytes is increasing, but most of the methods currently available to test the antifungal susceptibility of Microsporum canis still require standardization. The aims of this study were: (i) to evaluate the antifungal susceptibility of M. canis strains recovered from animals to ketoconazole (KTZ), fluconazole (FLZ) and itraconazole (ITZ) using a modified CLSI broth microdilution (CLSI M38-A2-BMD) and the E-test® protocols and (ii) to estimate the agreement between the methods. Tentative azole epidemiological cutoff values (ECVs) were also proposed in order to interpret the results of in vitro susceptibility tests and to establish the agreement between the E-test and CLSI BMD methods. A total of forty clinical M. canis strains from animals with skin lesions were tested, and the essential (EA) and categorical agreement (CA) between the two methods were determined. KTZ displayed the lowest MIC values, while ITZ and FLZ the highest. The ECV for KTZ and ITZ were 4 μg/ml, while those of FLZ was 64 μg/ml. Based on ECVs, about 88% of M. canis strains were susceptible to all azoles being a cross-resistance with ITZ-FLZ registered for one strain. A total of five M. canis strains showed MIC?>?ECV for FLZ using CLSI, while one strain showed MIC?>?ECV for ITZ using both tests. KTZ, ITZ and FLZ showed EA ranging from 92.5 to 95%, for all azoles and CA?>?97% except for FLZ (87.5%). The good CA between the E-test and the CLSI BMD provides evidence of the reliability of the former method to test the antifungal susceptibility of M. canis for ITZ and KTZ and not for FLZ.

     

Synthesis and evaluation of novel azoles as potent antifungal agents

Using a rational approach to the design of antifungal agents, a series of azole agents with 1,3,4-oxadiazole side chains were designed and synthesized. The results of preliminary in vitro antifungal tests with eight human pathogenic compounds showed that all of the title compounds exhibited excellent activities against all of the tested fungi except Aspergillus fumigatus. Compounds 11e and 11f were found to be the most effective, with a minimum inhibitory concentration of 0.0039 μg/mL, followed by voriconazole, which has a MIC of 0.0625 μg/mL. The 1,3,4-oxadiazole side chain is not the major contributor but plays a role in eliciting the observed antifungal activity.     

Silver nanoparticles,a promising treatment against clinically important fluconazole-resistant Candida glabrata

Resistance to azole antifungal agents is a challenging limitation in Candida glabrata treatment. It is associated with decreased intracellular concentrations of antifungal agents as a result of overexpression of efflux pumps on the cellular plasma membranes. This work evaluates the potential of silver nanoparticles (AgNPs) to reverse the resistance of fungal cells to fluconazole. Silver nanoparticles were prepared using wet chemical method and characterised by UV-Vis spectrophotometry, dynamic light scattering, and zeta potential. Broth microdilution and pour plates methods were used to study the anticandidal activity using two C. glabrata fluconazole-resistant strains (DSY565 and CBS138) known to overexpress active efflux pumps, and a standard fluconazole sensitive strain ATCC 22553. Silver nanoparticles–fluconazole combinations decreased concentrations of fluconazole substantially without compromising the activity. These findings suggest that AgNPs enhance the efficacy of fluconazole and offer a promising application in therapy of C. glabrata infections.     

Identification and Characterization of a Dual-Acting Antinematodal Agent against the Pinewood Nematode,Bursaphelenchus xylophilus

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is a mycophagous and phytophagous pathogen responsible for the current widespread epidemic of the pine wilt disease, which has become a major threat to pine forests throughout the world. Despite the availability of several preventive trunk-injection agents, no therapeutic trunk-injection agent for eradication of PWN currently exists. In the characterization of basic physiological properties of B. xylophilus YB-1 isolates, we established a high-throughput screening (HTS) method that identifies potential hits within approximately 7 h. Using this HTS method, we screened 206 compounds with known activities, mostly antifungal, for antinematodal activities and identified HWY-4213 (1-n-undecyl-2-[2-fluorphenyl] methyl-3,4-dihydro-6,7-dimethoxy-isoquinolinium chloride), a highly water-soluble protoberberine derivative, as a potent nematicidal and antifungal agent. When tested on 4 year-old pinewood seedlings that were infected with YB-1 isolates, HWY-4213 exhibited a potent therapeutic nematicidal activity. Further tests of screening 39 Caenorhabditis elegans mutants deficient in channel proteins and B. xylophilus sensitivity to Ca²⁺ channel blockers suggested that HWY-4213 targets the calcium channel proteins. Our study marks a technical breakthrough by developing a novel HTS method that leads to the discovery HWY-4213 as a dual-acting antinematodal and antifungal compound.     

Calcium Dependence of Eugenol Tolerance and Toxicity in Saccharomyces cerevisiae

Eugenol is a plant-derived phenolic compound which has recognised therapeutical potential as an antifungal agent. However little is known of either its fungicidal activity or the mechanisms employed by fungi to tolerate eugenol toxicity. A better exploitation of eugenol as a therapeutic agent will therefore depend on addressing this knowledge gap. Eugenol initiates increases in cytosolic Ca²⁺ in Saccharomyces cerevisiae which is partly dependent on the plasma membrane calcium channel, Cch1p. However, it is unclear whether a toxic cytosolic Ca²⁺elevation mediates the fungicidal activity of eugenol. In the present study, no significant difference in yeast survival was observed following transient eugenol treatment in the presence or absence of extracellular Ca²⁺. Furthermore, using yeast expressing apoaequorin to report cytosolic Ca²⁺ and a range of eugenol derivatives, antifungal activity did not appear to be coupled to Ca²⁺ influx or cytosolic Ca²⁺ elevation. Taken together, these results suggest that eugenol toxicity is not dependent on a toxic influx of Ca²⁺. In contrast, careful control of extracellular Ca²⁺ (using EGTA or BAPTA) revealed that tolerance of yeast to eugenol depended on Ca²⁺ influx via Cch1p. These findings expose significant differences between the antifungal activity of eugenol and that of azoles, amiodarone and carvacrol. This study highlights the potential to use eugenol in combination with other antifungal agents that exhibit differing modes of action as antifungal agents to combat drug resistant infections.     

Glucose Directly Promotes Antifungal Resistance in the Fungal Pathogen,Candida spp.

Effects of glucose on the susceptibility of antifungal agents were investigated against Candida spp. Increasing the concentration of glucose decreased the activity of antifungal agents; voriconazole was the most affected drugs followed by amphotericin B. No significant change has been observed for anidulafungin. Biophysical interactions between antifungal agents with glucose molecules were investigated using isothermal titration calorimetry, Fourier transform infrared, and ¹H NMR. Glucose has a higher affinity to bind with voriconazole by hydrogen bonding and decrease the susceptibility of antifungal agents during chemotherapy. In addition to confirming the results observed in vitro, theoretical docking studies demonstrated that voriconazole presented three important hydrogen bonds and amphotericin B presented two hydrogen bonds that stabilized the glucose. In vivo results also suggest that the physiologically relevant higher glucose level in the bloodstream of diabetes mellitus mice might interact with the available selective agents during antifungal therapy, thus decreasing glucose activity by complex formation. Thus, proper selection of drugs for diabetes mellitus patients is important to control infectious diseases.     

Usefulness of tilt testing in children with syncope: a survey of pediatric electrophysiologists

The role of tilt table testing as a diagnostic modality in children with unexplained syncope is unclear. We sent a questionnaire to members of the Pediatric and Congenital Electrophysiology Society to assess the current practice pattern. Of the 186 members, 97 (52%) replied. Twenty four percent of the pediatric electrophysiologists have completely stopped doing tilt table tests and of those performing the tests, a majority (76%) did < 10 tests/yr (median=3 tilts/yr, range 0-100/yr). Of those performing the test, 95% rarely or never accepted direct referrals from the general practioners and 62% felt that the frequency of tilt table tests being performed had decreased since they had started practicing. The median usefulness of the test was rated at 3 (range 1-9) on a scale of 1 to 10 with 10 being very useful. A majority (68%) felt they rarely or never altered treatment based on the results of the tilt test. Wide variability was noted in the test protocol including the tilt angle, tilt duration, use of pharmacologic agents and the duration of fasting prior to the test. We therefore conclude that there is significant lack of standardization in tilt table tests performed in children. Tilt table testing, as perceived by pediatric electrophysiologists, is of limited utility and progressively less used in children with syncope.     

A comparison of methods for evaluating seed quality in carrots (Daucus carota)

Comparisons of embryo length, ISTA (International Seed Testing Association), cold (10 ^oC), controlled-deterioration, and slope tests for estimating seedling size variability and percentage seedling emergence were made using eighteen stocks of carrot seeds. The coefficient of variation (C.V.) of seedling weight was closely correlated with the C.V. of embryo length (r = 0–87 D.F., 16) and also with the C.V. of root length from the slope test (r = 0–63 D.F., 16). Poorer estimates of percentage seedling emergence were obtained from the cold test in all three sowings (correlation coefficients were between 0–65 and 0–70 D.F., 16) compared with the ISTA, controlled deterioration, and slope tests. For these three tests the correlation coefficients varied between 0–80 and 0–87 (D.F., 16) for all sowings. The embryo test is a rapid (1 man hour per seed lot) and accurate method of assessing potential variability between seed lots. The slope test could provide a less labour demanding method of assessing potential variability than the embryo length test though the results would not be available immediately. However, the slope test could also provide as good an estimate of seedling emergence in the field as the ISTA or controlled-deterioration tests.     

Design,synthesis, and biological evaluation of novel 1-(1H-1,2,4-triazole-1-yl)-2-(2,4-difluorophenyl)-3-substituted benzylamino-2-propanols

Based on the results of computational docking to the active site of the cytochrome P450 14α-demethylase (CYP51), a series of 1-(1H-1,2,4-triazole-1-yl)-2-(2,4-difluorophenyl)-3-substituted benzylamino-2-propanols as analogs of fluconazole were designed, synthesized, and evaluated as antifungal agents. Results of preliminary antifungal tests against eight human pathogenic fungi in vitro showed that all the title compounds exhibited excellent activities with broad spectrum.     

Standardization of hyphal growth inhibition rate as a means of evaluating Microsporum spp. in vitro susceptibility to terbinafine, griseofulvin, and ciclopiroxolamine

Reference methods for antifungal susceptibility tests recommend the use of conidia as inoculum. However, some isolates produce few conidia, while the invasive form of filamentous fungi in general is hyphae making susceptibility tests infeaseble. These facts suggest that other than conidia broth dilution method is required for susceptibility tests. The aim of this study was to clarify if the hyphal growth inhibition rate could be used as a method of determining the antifungal susceptibility of genus Microsporum. For this reason, a method which traces hyphal tips automatically and measures their growth rate was standardized for Microsporum spp. Control growth curves and test growth curves obtained by real-time observation of the hyphae groups responses to different concentrations of terbinafine, griseofulvin, and ciclopiroxolamine were used to compare with minimum inhibitory concentrations (MICs) obtained by conidia broth microdilution method. A visible reduction in the growth inhibition rate was observed when hyphal activity was evaluated using the third or fourth serial two-fold dilution below the MIC determined by broth microdilution for terbinafine and ciclopiroxolamine. For griseofulvin, this reduction occurred after the fifth dilution below the MIC. This study highlights the importance of the inoculum type used to determine the in vitro susceptibility of Microsporum strains. We conclude that measurement of hyphal growth inhibition, despite being time consuming, could be a suitable method for evaluating antifungal susceptibility, particularly for fungi as Microsporum spp. that produce a small (or not at all) number of conidia.     

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals¹. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents². Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip³. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 °C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)⁴. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation⁵), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.     

Purification and characterization of a potential antifungal protein from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> E1R-J against <Emphasis Type="Italic">Valsa mali</Emphasis>

In order to identify the antagonistic substances produced by Bacillus subtilis E1R-J as candidate of biocontrol agents for controlling Apple Valsa Canker, hydrochloric acid precipitation, reverse phase chromatography, gel filtration, and ion exchange chromatography were used. The purified fraction EP-2 showed a single band in native-polyacrylamide gel electrophoresis (native-PAGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Fraction EP-2 was eluted from native-PAGE and showed a clear inhibition zone against V. mali 03-8. These results prove that EP-2 is one of the most important antifungal substances produced by B. subtilis E1R-J in fermentation broth. SDS-PAGE and Nano-LC–ESI–MS/MS analysis results demonstrated that EP-2 was likely an antifungal peptide (trA0A086WXP9), with a relative molecular mass of 12.44 kDa and isoelectric point of 9.94. The examination of antagonistic mechanism under SEM and TEM showed that EP-2 appeared to inhibit Valsa mali 03-8 by causing hyphal swelling, distortion, abnormality and protoplasts extravasation. Inhibition spectrum results showed that antifungal protein EP-2 had significantly inhibition on sixteen kinds of plant pathogenic fungi. The stability test results showed that protein EP-2 was stable with antifungal activity at temperatures as high as 100 °C for 30 min and in pH values ranging from 1.0 to 8.0, or incubated with each 5 mM Cu²⁺, Zn²⁺, Mg²⁺, or K⁺. However, the antifungal activity was negatively affected by Proteinase K treatment.     

Systemic Candida infection in University Hospital 1997–1999: the distribution of Candida biotypes and antifungal susceptibility patterns

A total of 102 Candida species were isolated from blood cultures from January 1997 to October 1999. Using assimilation of carbohydrate test, 52 (51.0%) of the Candida sp. were identified as C. parapsilosis, 25.5% (26) were C. tropicalis. C. albicans made up 11.8% (12), 6.9% (7) were C. rugosa, 3.8% (4) C. glabrata and 1% (1) C. guilliermondii. No C. dubliniensis was found in the study. In vitro antifungal susceptibility tests showed that all Candida species were sensitive to nystatin, amphotericin B and ketoconazole. Although all isolates remained sensitive to fluconazole, intermediate susceptibility was found in 3 C. rugosa isolates. Antifungal agents with high frequency of resistance were econazole, clotrimazole, miconazole and 5-fluorocytosine. Candida species found to have resistance to these antifungal agents were non-C. albicans. This revised version was published online in June 2006 with corrections to the Cover Date.