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1.  A Phase 2, Randomized, Double-Blind, Multicenter Trial To Evaluate the Safety and Efficacy of Three Dosing Regimens of Isavuconazole Compared with Fluconazole in Patients with Uncomplicated Esophageal Candidiasis 
Esophageal candidiasis is a frequent cause of morbidity in immunocompromised patients. Isavuconazole is a novel, broad-spectrum antifungal developed for the treatment of opportunistic fungal infections. This phase 2 trial compared the efficacy and safety of three oral dosing regimens of isavuconazole with an oral fluconazole regimen in the primary treatment of uncomplicated esophageal candidiasis. The isavuconazole regimens were as follows: 200 mg on day 1 and then 50 mg once daily (arm A), 400 mg on day 1 and then 400 mg once-weekly (arm B), and 400 mg on day 1 and then 100 mg once daily (arm C). Patients in arm D received fluconazole at 200 mg on day 1 and then 100 mg once daily. The minimum treatment duration was 14 days. The primary endpoint was the rate of endoscopically confirmed clinical response at end of therapy. Safety and tolerability were also assessed. Efficacy was evaluated in 153 of 160 enrolled patients. Overall, 146 (95.4%) achieved endoscopically confirmed clinical success. Each of the isavuconazole regimens was shown to be not inferior to fluconazole, i.e., arm A versus D, −0.5% (95% confidence interval [CI] −10.0 to 9.4), arm B versus D, 3.5% (95% CI, −5.6 to 12.7), and arm C versus D, −0.2% (95% CI, −9.8 to 9.4). The frequency of adverse events was similar in arm A (n = 22; 55%), arm B (n = 18; 45%), and arm D (n = 22; 58%), but higher in arm C (n = 29; 71%). In summary, efficacy and safety of once-daily and once-weekly isavuconazole were comparable with once-daily fluconazole in the primary treatment of uncomplicated esophageal candidiasis.
PMCID: PMC4325824  PMID: 25561337
2.  Role of oral candidiasis in TB and HIV co-infection: AIDS Clinical Trial Group Protocol A5253 
To evaluate the association between oral candidiasis and tuberculosis (TB) in human immunodeficiency virus (HIV) infected individuals in sub-Saharan Africa, and to investigate oral candidiasis as a potential tool for TB case finding.
Protocol A5253 was a cross-sectional study designed to improve the diagnosis of pulmonary TB in HIV-infected adults in high TB prevalence countries. Participants received an oral examination to detect oral candidiasis. We estimated the association between TB disease and oral candidiasis using logistic regression, and sensitivity, specificity and predictive values.
Of 454 participants with TB culture results enrolled in African sites, the median age was 33 years, 71% were female and the median CD4 count was 257 cells/mm3. Fifty-four (12%) had TB disease; the prevalence of oral candidiasis was significantly higher among TB cases (35%) than among non-TB cases (16%, P < 0.001). The odds of having TB was 2.4 times higher among those with oral candidiasis when controlling for CD4 count and antifungals (95%CI 1.2–4.7, P = 0.01). The sensitivity of oral candidiasis as a predictor of TB was 35% (95%CI 22–48) and the specificity 85% (95%CI 81–88).
We found a strong association between oral candidiasis and TB disease, independent of CD4 count, suggesting that in resource-limited settings, oral candidiasis may provide clinical evidence for increased risk of TB and contribute to TB case finding.
PMCID: PMC4157598  PMID: 24903939
oral candidiasis; tuberculosis; HIV; acquired immunedeficiency syndrome
3.  In Vitro Antifungal Activity of Naftifine Hydrochloride against Dermatophytes 
The incidence of superficial dermatophytoses is high in developed countries, and there remains a need for effective topical antifungals. In this study, we evaluated the in vitro antifungal activity of naftifine hydrochloride, the active ingredient in naftifine hydrochloride cream and gel 1% and 2%, against dermatophytes. The MICs and minimum fungicidal concentrations (MFCs) of naftifine hydrochloride against 350 clinical strains, including Trichophyton rubrum, T. mentagrophytes, T. tonsurans, Epidermophyton floccosum, and Microsporum canis, were determined using the CLSI methodology. Subsets from this test panel were subsequently tested in a time-kill assay at 0.125×, 0.25×, 0.5×, and 1× the MFC for each isolate. CFU counts were performed over a period of 48 h of incubation. Additionally, in order to determine the potential for resistance development, six strains were subjected to 15 serial passages in concentrations higher than the MIC for each strain. MICs were determined following each passage. The MIC range against the dermatophyte isolates tested was 0.015 to 1.0 μg/ml, with naftifine hydrochloride being fungicidal against 85% of the Trichophyton species. The time-kill assay showed dose-dependent activity, with the greatest reduction in the numbers of CFU corresponding to the highest drug concentration. There was no increase in MIC for any strains following repeated exposure to naftifine hydrochloride. Naftifine hydrochloride demonstrated potent activity against all dermatophytes tested, and none of the isolates within this test panel demonstrated the potential for the development of resistance. Thus, future clinical studies of naftifine hydrochloride against dermatophytes may be warranted for the treatment of superficial dermatophytoses.
PMCID: PMC3754295  PMID: 23817365
4.  Nonspecific Effect of Mycograb on Amphotericin B MIC 
Mycograb C28Y is a recombinant human antibody fragment thought to target HSP-90 and potentiate amphotericin B (AMB). Absence of in vivo efficacy led us to reevaluate its in vitro activity. Interactions between AMB and Mycograb were investigated using a checkerboard design. Addition of Mycograb or various unrelated proteins, including human serum, resulted in similar decreases in the MIC of AMB. Potentiation of AMB by Mycograb appears to be a nonspecific protein effect.
PMCID: PMC3393390  PMID: 22508314
5.  Wild-Type MIC Distributions and Epidemiological Cutoff Values for Amphotericin B, Flucytosine, and Itraconazole and Candida spp. as Determined by CLSI Broth Microdilution 
Journal of Clinical Microbiology  2012;50(6):2040-2046.
Clinical breakpoints (CBPs) and epidemiological cutoff values (ECVs) have been established for several Candida spp. and the newer triazoles and echinocandins but are not yet available for older antifungal agents, such as amphotericin B, flucytosine, or itraconazole. We determined species-specific ECVs for amphotericin B (AMB), flucytosine (FC) and itraconazole (ITR) for eight Candida spp. (30,221 strains) using isolates from 16 different laboratories in Brazil, Canada, Europe, and the United States, all tested by the CLSI reference microdilution method. The calculated 24- and 48-h ECVs expressed in μg/ml (and the percentages of isolates that had MICs less than or equal to the ECV) for AMB, FC, and ITR, respectively, were 2 (99.8)/2 (99.2), 0.5 (94.2)/1 (91.4), and 0.12 (95.0)/0.12 (92.9) for C. albicans; 2 (99.6)/2 (98.7), 0.5 (98.0)/0.5 (97.5), and 2 (95.2)/4 (93.5) for C. glabrata; 2 (99.7)/2 (97.3), 0.5 (98.7)/0.5 (97.8), and 05. (99.7)/0.5 (98.5) for C. parapsilosis; 2 (99.8)/2 (99.2), 0.5 (93.0)/1 (90.5), and 0.5 (97.8)/0.5 (93.9) for C. tropicalis; 2 (99.3)/4 (100.0), 32 (99.4)/32 (99.3), and 1 (99.0)/2 (100.0) for C. krusei; 2 (100.0)/4 (100.0), 0.5 (95.3)/1 (92.9), and 0.5 (95.8)/0.5 (98.1) for C. lusitaniae; −/2 (100.0), 0.5 (98.8)/0.5 (97.7), and 0.25 (97.6)/0.25 (96.9) for C. dubliniensis; and 2 (100.0)/2 (100.0), 1 (92.7)/−, and 1 (100.0)/2 (100.0) for C. guilliermondii. In the absence of species-specific CBP values, these wild-type (WT) MIC distributions and ECVs will be useful for monitoring the emergence of reduced susceptibility to these well-established antifungal agents.
PMCID: PMC3372147  PMID: 22461672
6.  Evaluation of the Morphological Effects of TDT 067 (Terbinafine in Transfersome) and Conventional Terbinafine on Dermatophyte Hyphae In Vitro and In Vivo 
TDT 067 is a novel, carrier-based dosage form of terbinafine in Transfersome (1.5%) formulated for topical delivery of terbinafine to the nail, nail bed, and surrounding tissue. We examined the effects of TDT 067 and conventional terbinafine on the morphology of dermatophytes. Trichophyton rubrum hyphae were exposed to TDT 067 or terbinafine (15 mg/ml) and examined under white light, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Subungual debris from patients treated with TDT 067 in a clinical trial was also examined. Exposure of T. rubrum hyphae to TDT 067 led to rapid and extensive ultrastructural changes. Hyphal distortion was evident as early as 4 h after exposure to TDT 067. After 24 h, there was complete disruption of hyphal structure with few intact hyphae remaining. Exposure to terbinafine resulted in morphological alterations similar to those seen with TDT 067; however, the effects of TDT 067 were more extensive, whereas a portion of hyphae remained intact after 24 h of exposure to terbinafine. Lipid droplets were observed under TEM following 30 min of exposure to TDT 067, which after 24 h had filled the intracellular space. These effects were confirmed in vivo in subungual debris from patients with onychomycosis who received topical treatment with TDT 067. The Transfersome in TDT 067 may potentiate the action of terbinafine by delivering terbinafine more effectively to its site of action inside the fungus. Our in vivo data confirm that TDT 067 can enter fungus in the nail bed of patients with onychomycosis and exert its antifungal effects.
PMCID: PMC3346586  PMID: 22354309
7.  Identification of gentian violet concentration that does not stain oral mucosa, possesses anti-candidal activity and is well tolerated 
Gentian violet (GV) is recommended for initial treatment of oral candidiasis in HIV-infected patients in resource-limited settings. Currently GV is not used because of its staining effects. In this study, we investigated the staining capacity of three different concentrations of GV to determine a concentration that does not cause staining. The selected concentration that did not cause staining was evaluated for its physical stability and antifungal activity. Fifteen healthy participants were randomized to rinse twice daily for 14 days with one of three GV concentrations: 0.1%, 0.0085%, or 0.00165%. Oral examination and intra-oral photographs were performed at baseline and at the end of therapy. Participants responded to a questionnaire to assess adverse events. Antifungal activity was evaluated using the Clinical and Laboratory Standard Institute methodology. GV at a concentration of 0.00165% did not stain the oral mucosa and was well tolerated. GV at a concentration of 0.00165% was stable and possessed antifungal activity when stored at certain temperatures for different time periods. Gentian violet solution at the concentration of 0.00165% does not stain the oral mucosa, is stable and possesses potent antifungal activity.
PMCID: PMC3076549  PMID: 21210170
8.  Wild-Type MIC Distributions and Epidemiological Cutoff Values for Amphotericin B and Aspergillus spp. for the CLSI Broth Microdilution Method (M38-A2 Document)▿ 
Antimicrobial Agents and Chemotherapy  2011;55(11):5150-5154.
Although clinical breakpoints have not been established for mold testing, epidemiological cutoff values (ECVs) are available for Aspergillus spp. versus the triazoles and caspofungin. Wild-type (WT) MIC distributions (organisms in a species-drug combination with no acquired resistance mechanisms) were defined in order to establish ECVs for six Aspergillus spp. and amphotericin B. Two sets (CLSI/EUCAST broth microdilution) of available MICs were evaluated: those for A. fumigatus (3,988/833), A. flavus (793/194), A. nidulans (184/69), A. niger (673/140), A. terreus (545/266), and A. versicolor (135/22). Three sets of data were analyzed: (i) CLSI data gathered in eight independent laboratories in Canada, Europe, and the United States; (ii) EUCAST data from a single laboratory; and (iii) the combined CLSI and EUCAST data. ECVs, expressed in μg/ml, that captured 95%, 97.5%, and 99% of the modeled wild-type population (CLSI and combined data) were as follows: for A. fumigatus, 2, 2, and 4; for A. flavus, 2, 4, and 4; for A. nidulans, 4, 4, and 4; for A. niger, 2, 2, and 2; for A. terreus, 4, 4, and 8; and for A. versicolor, 2, 2, and 2. Similar to the case for the triazoles and caspofungin, amphotericin B ECVs may aid in the detection of strains with acquired mechanisms of resistance to this agent.
PMCID: PMC3195003  PMID: 21876047
9.  Overview of the Oral HIV/AIDS Research Alliance Program 
Advances in Dental Research  2011;23(1):28-33.
The Oral HIV/AIDS Research Alliance is part of the AIDS Clinical Trials Group, the largest HIV clinical trial organization in the world, and it is funded by the National Institute of Dental and Craniofacial Research, in collaboration with the National Institute of Allergy and Infectious Diseases. The alliance’s main objective is to investigate the oral complications associated with HIV/AIDS as the epidemic is evolving—in particular, the effects of potent antiretrovirals on the development of oral mucosal lesions and associated fungal and viral pathogens. Furthermore, oral fluids are being explored for their potential monitoring and diagnostic role with respect to HIV disease and coinfections. This article presents an overview of the alliance, its scientific agenda, and an outline of the novel interventional and noninterventional clinical studies ongoing and developing within the AIDS Clinical Trials Group infrastructure in the United States and internationally.
PMCID: PMC3144044  PMID: 21441477
HIV/AIDS; OHARA; infectious diseases; AIDS Clinical Trials Group; oral cavity
10.  Antimicrobial Activity of B-Lock against Bacterial and Candida spp. Causing Catheter-Related Bloodstream Infections▿ 
The triple combination trimethoprim, EDTA, and ethanol (B-Lock), is an antimicrobial lock solution for use in indwelling intravascular catheters to prevent and treat catheter-associated infections. B-Lock demonstrated MICs of ≤0.05% (percentage of solution) against Candida spp. (n = 125) and 0.003% to 25% against bacterial strains (n = 175). B-Lock was also fungicidal against the majority of the Candida strains at 6% to 25%. B-Lock demonstrates potential value for the prevention and treatment of catheter-associated infections.
PMCID: PMC3165356  PMID: 21730123
11.  Quality Control Guidelines for Amphotericin B, Itraconazole, Posaconazole, and Voriconazole Disk Diffusion Susceptibility Tests with Nonsupplemented Mueller-Hinton Agar (CLSI M51-A Document) for Nondermatophyte Filamentous Fungi▿ 
Journal of Clinical Microbiology  2011;49(7):2568-2571.
Although Clinical and Laboratory Standards Institute (CLSI) disk diffusion assay standard conditions are available for susceptibility testing of filamentous fungi (molds) to antifungal agents, quality control (QC) disk diffusion zone diameter ranges have not been established. This multicenter study documented the reproducibility of tests for one isolate each of five molds (Paecilomyces variotii ATCC MYA-3630, Aspergillus fumigatus ATCC MYA-3626, A. flavus ATCC MYA-3631, A. terreus ATCC MYA-3633, and Fusarium verticillioides [moniliforme] ATCC MYA-3629) and Candida krusei ATCC 6258 by the CLSI disk diffusion method (M51-A document). The zone diameter ranges for selected QC isolates were as follows: P. variotii ATCC MYA-3630, amphotericin B (15 to 24 mm), itraconazole (20 to 31 mm), and posaconazole (33 to 43 mm); A. fumigatus ATCC MYA-3626, amphotericin B (18 to 25 mm), itraconazole (11 to 21 mm), posaconazole (28 to 35 mm), and voriconazole (25 to 33 mm); and C. krusei, amphotericin B (18 to 27 mm), itraconazole (18 to 26 mm), posaconazole (28 to 38 mm), and voriconazole (29 to 39 mm). Due to low testing reproducibility, zone diameter ranges were not proposed for the other three molds.
PMCID: PMC3147863  PMID: 21543581
12.  Agar-Based Disk Diffusion Assay for Susceptibility Testing of Dermatophytes ▿  
Journal of Clinical Microbiology  2010;48(10):3750-3752.
Currently, no agar-based susceptibility testing method has been standardized for testing dermatophytes. We describe a newly developed agar-based method employing disk diffusion assay to test the susceptibility of 47 isolates of dermatophytes against 8 antifungals. Our results show that the method is reproducible, is simple, and could be used to determine the antifungal susceptibility of dermatophytes.
PMCID: PMC2953072  PMID: 20668120
13.  Wild-Type MIC Distribution and Epidemiological Cutoff Values for Aspergillus fumigatus and Three Triazoles as Determined by the Clinical and Laboratory Standards Institute Broth Microdilution Methods▿  
Journal of Clinical Microbiology  2009;47(10):3142-3146.
Antifungal susceptibility testing of Aspergillus species has been standardized by both the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Recent studies suggest the emergence of strains of Aspergillus fumigatus with acquired resistance to azoles. The mechanisms of resistance involve mutations in the cyp51A (sterol demethylase) gene, and patterns of azole cross-resistance have been linked to specific mutations. Studies using the EUCAST broth microdilution (BMD) method have defined wild-type (WT) MIC distributions, epidemiological cutoff values (ECVs), and cross-resistance among the azoles. We tested a collection of 637 clinical isolates of A. fumigatus for which itraconazole MICs were ≤2 μg/ml against posaconazole and voriconazole using the CLSI BMD method. An ECV of ≤1 μg/ml encompassed the WT population of A. fumigatus for itraconazole and voriconazole, whereas an ECV of ≤0.25 μg/ml was established for posaconazole. Our results demonstrate that the WT distribution and ECVs for A. fumigatus and the mold-active triazoles were the same when determined by the CLSI or the EUCAST BMD method. A collection of 43 isolates for which itraconazole MICs fell outside of the ECV were used to assess cross-resistance. Cross-resistance between itraconazole and posaconazole was seen for 53.5% of the isolates, whereas cross-resistance between itraconazole and voriconazole was apparent in only 7% of the isolates. The establishment of the WT MIC distribution and ECVs for the azoles and A. fumigatus will be useful in resistance surveillance and is an important step toward the development of clinical breakpoints.
PMCID: PMC2756953  PMID: 19692559
14.  Correlation of MIC with Outcome for Candida Species Tested against Caspofungin, Anidulafungin, and Micafungin: Analysis and Proposal for Interpretive MIC Breakpoints▿  
Journal of Clinical Microbiology  2008;46(8):2620-2629.
The CLSI Antifungal Subcommittee followed the M23-A2 “blueprint” to develop interpretive MIC breakpoints for anidulafungin, caspofungin, and micafungin against Candida species. MICs of ≤2 μg/ml for all three echinocandins encompass 98.8 to 100% of all clinical isolates of Candida spp. without bisecting any species group and represent a concentration that is easily maintained throughout the dosing period. Data from phase III clinical trials demonstrate that the standard dosing regimens for each of these agents may be used to treat infections due to Candida spp. for which MICs are as high as 2 μg/ml. An MIC predictive of resistance to these agents cannot be defined based on the data from clinical trials due to the paucity of isolates for which MICs exceed 2 μg/ml. The clinical data set included only three isolates from patients treated with an echinocandin (caspofungin) for which the MICs were >2 μg/ml (two C. parapsilosis isolates at 4 μg/ml and one C. rugosa isolate at 8 μg/ml). Based on these data, the CLSI subcommittee has decided to recommend a “susceptible only” breakpoint MIC of ≤2 μg/ml due to the lack of echinocandin resistance in the population of Candida isolates thus far. Isolates for which MICs exceed 2 μg/ml should be designated “nonsusceptible” (NS). For strains yielding results suggestive of an NS category, the organism identification and antimicrobial-susceptibility test results should be confirmed. Subsequently, the isolates should be submitted to a reference laboratory that will confirm the results by using a CLSI reference dilution method.
PMCID: PMC2519503  PMID: 18579718
15.  Clinical Evaluation of the Sensititre YeastOne Colorimetric Antifungal Panel for Antifungal Susceptibility Testing of the Echinocandins Anidulafungin, Caspofungin, and Micafungin▿  
Journal of Clinical Microbiology  2008;46(7):2155-2159.
A commercially prepared, dried colorimetric microdilution panel (Sensititre YeastOne Trek Diagnostic Systems, Cleveland, OH) was compared in three different laboratories with the Clinical and Laboratory Standards Institute (CLSI) reference microdilution method by testing 2 quality control strains, 25 reproducibility strains, and 404 isolates of Candida spp. against anidulafungin, caspofungin, and micafungin. Reference MIC endpoints and YeastOne colorimetric endpoints were read after 24 h of incubation. YeastOne endpoints were determined to be the lowest concentration at which the color in the well changed from red (positive, indicating growth) to blue (negative, indicating no growth). Excellent essential agreement (within 2 dilutions) between the reference and colorimetric MICs was observed. Overall agreement was 100% for all three agents. Categorical agreement ranged from 99.3% (anidulafungin) to 100% (caspofungin, micafungin) and interlaboratory reproducibility was 99%. The YeastOne colorimetric method appears to be comparable to the CLSI reference method for testing the susceptibility of Candida spp. to the echinocandins anidulafungin, caspofungin, and micafungin.
PMCID: PMC2446941  PMID: 18463213
16.  Quality Control and Reference Guidelines for CLSI Broth Microdilution Method (M38-A Document) for Susceptibility Testing of Anidulafungin against Molds▿  
Journal of Clinical Microbiology  2007;45(7):2180-2182.
The CLSI (formerly NCCLS) M38-A document for antifungal susceptibility testing of filamentous fungi does not describe guidelines for echinocandins. A multicenter study (eight centers) evaluated inter- and intralaboratory reproducibilities of two reading times (24 and 48 h or 48 and 72 h) and two end points (MICs and minimum effective concentrations [MECs]) for evaluating anidulafungin against molds. Anidulafungin MICs (≥50% inhibition) and MECs (morphological hyphal changes) were determined for seven Aspergillus isolates (four species) and one isolate each of Fusarium moniliforme, Fusarium solani, and Paecilomyces variotii and for two Scedosporium apiospermum isolates. The inter- and intralaboratory reproducibilities of 10 replicate tests performed in each laboratory on 10 different days for each isolate was 100% at 24 h (MECs, ≤0.015 μg/ml) for six Aspergillus and P. variotii isolates. The reproducibility was 94 to 96.7% at 72 h (MECs, 1 to 8 μg/ml) for S. apiospermum and 96.7 to 97.5% at 48 h (MICs, ≥32 μg/ml) for both Fusarium isolates. Introduction of these identified optimum testing conditions for anidulafungin into future versions of the M38 document is warranted.
PMCID: PMC1932988  PMID: 17475760
17.  Interlaboratory Study of Quality Control Isolates for a Broth Microdilution Method (Modified CLSI M38-A) for Testing Susceptibilities of Dermatophytes to Antifungals▿  
Journal of Clinical Microbiology  2006;44(12):4353-4356.
The Clinical and Laboratory Standards Institute (CLSI; formerly National Committee for Clinical Laboratory Standards, or NCCLS) M38-A standard for the susceptibility testing of filamentous fungi does not specifically address the testing of dermatophytes. In 2003, a multicenter study investigated the reproducibility of the microdilution method developed at the Center for Medical Mycology, Cleveland, Ohio, for testing the susceptibility of dermatophytes. Data from that study supported the introduction of this method for testing dermatophytes in the future version of the CLSI M38-A standard. In order for the method to be accepted by CLSI, appropriate quality control isolates needed to be identified. To that end, an interlaboratory study, involving the original six laboratories plus two additional sites, was conducted to evaluate potential candidates for quality control isolates. These candidate strains included five Trichophyton rubrum strains known to have elevated MICs to terbinafine and five Trichophyton mentagrophytes strains. Antifungal agents tested included ciclopirox, fluconazole, griseofulvin, itraconazole, posaconazole, terbinafine, and voriconazole. Based on the data generated, two quality control isolates, one T. rubrum isolate and one T. mentagrophytes isolate, were identified and submitted to the American Type Culture Collection (ATCC) for inclusion as reference strains. Ranges encompassing 95.2 to 97.9% of all data points for all seven drugs were established.
PMCID: PMC1698433  PMID: 17050812
18.  Determination of MICs of Aminocandin for Candida spp. and Filamentous Fungi▿  
Journal of Clinical Microbiology  2006;44(12):4342-4344.
Candida and Aspergillus spp., as well as other filamentous molds, have increasingly been reported as the causes of severe invasive fungal infections. We evaluated the new echinocandin aminocandin (AMN) for its antifungal activities against a range of fungal pathogens by determination of the MICs for the organisms. The MICs of the comparator drugs amphotericin B, caspofungin, micafungin, and voriconazole were also determined. The MICs of AMN for 25 strains each of non-Candida albicans Candida spp. (including Candida parapsilosis, Candida krusei, Candida guilliermondii, and Candida tropicalis), Aspergillus fumigatus, Scedosporium spp., Fusarium spp., and zygomycetes (including Absidia, Mucor, and Rhizopus spp.) were determined by using the Clinical and Laboratory Standards Institute M27-A2 and M38-A methodologies for yeasts and filamentous molds, respectively. The MIC ranges of AMN for all yeasts were similar (0.03 to 4.0 μg/ml), while the MIC ranges of AMN for filamentous fungi were species specific. AMN demonstrated potent antifungal activity against A. fumigatus, limited activity against Scedosporium spp., and no activity against zygomycetes or Fusarium spp. Our data showed that AMN demonstrated potent antifungal activities against all of the yeasts and Aspergillus isolates tested, suggesting that AMN could be an important addition to our arsenal of antifungals for the treatment of invasive fungal disease.
PMCID: PMC1698398  PMID: 17021057
19.  Voriconazole Susceptibilities of Dermatophyte Isolates Obtained from a Worldwide Tinea Capitis Clinical Trial 
Journal of Clinical Microbiology  2006;44(7):2579-2580.
In this study, the voriconazole susceptibilities of dermatophyte isolates obtained from a worldwide tinea capitis trial were compared to their susceptibilities to fluconazole and griseofulvin. The MIC ranges of voriconazole, fluconazole, and griseofulvin, were 0.002 to 0.06 μg/ml, 0.25 to 32 μg/ml, and 0.125 to 2.0 μg/ml, respectively.
PMCID: PMC1489484  PMID: 16825385
20.  Correlation of MIC with Outcome for Candida Species Tested against Voriconazole: Analysis and Proposal for Interpretive Breakpoints 
Journal of Clinical Microbiology  2006;44(3):819-826.
Developing interpretive breakpoints for any given organism-drug combination requires integration of the MIC distribution, pharmacokinetic and pharmacodynamic parameters, and the relationship between the in vitro activity and outcome from both in vivo and clinical studies. Using data generated by standardized broth microdilution and disk diffusion test methods, the Antifungal Susceptibility Subcommittee of the Clinical and Laboratory Standards Institute has now proposed interpretive breakpoints for voriconazole and Candida species. The MIC distribution for voriconazole was determined using a collection of 8,702 clinical isolates. The overall MIC90 was 0.25 μg/ml and 99% of the isolates were inhibited at ≤1 μg/ml of voriconazole. Similar results were obtained for 1,681 Candida isolates (16 species) from the phase III clinical trials. Analysis of the available data for 249 patients from six phase III voriconazole clinical trials demonstrated a statistically significant correlation (P = 0.021) between MIC and investigator end-of-treatment assessment of outcome. Consistent with parallel pharmacodynamic analyses, these data support the following MIC breakpoints for voriconazole and Candida species: susceptible (S), ≤1 μg/ml; susceptible dose dependent (SDD), 2 μg/ml; and resistant (R), ≥4 μg/ml. The corresponding disk test breakpoints are as follows: S, ≥17 mm; SDD, 14 to 16 mm; and R, ≤13 mm.
PMCID: PMC1393146  PMID: 16517860
21.  Quality Control and Reference Guidelines for CLSI Broth Microdilution Susceptibility Method (M38-A Document) for Amphotericin B, Itraconazole, Posaconazole, and Voriconazole 
Journal of Clinical Microbiology  2005;43(10):5243-5246.
Although standard conditions are available for testing the susceptibilities of filamentous fungi to antifungal agents by the Clinical and Laboratory Standards Institute (CLSI; formerly National Committee for Clinical Laboratory Standards) broth microdilution assay, quality control (QC) MIC limits have not been established for any mold-agent combination. This multicenter (eight-center) study documented the reproducibility of tests for one isolate of Paecilomyces variotii ATCC MYA-3630 and 11 other mold isolates (three isolates of Aspergillus fumigatus; two isolates of A. terreus; one isolate each of A. flavus, A. nidulans, Fusarium moniliforme, and F. solani; and two isolates of Scedosporium apiospermum) by the CLSI reference broth microdilution method (M38-A document). Control limits (amphotericin B, 1 to 4 μg/ml; itraconazole, 0.06 to 0.5 μg/ml; posaconazole, 0.03 to 0.25 μg/ml; voriconazole, 0.015 to 0.12 μg/ml) for the selected QC P. variotii ATCC MYA-3630 were established by the analysis of replicate MIC results. Reference isolates and corresponding MIC ranges were also established for 6 of the 12 molds evaluated. MIC limits were not proposed for the other five molds tested due to low testing reproducibility for these isolates.
PMCID: PMC1248457  PMID: 16207990
22.  Intra- and Interlaboratory Study of a Method for Testing the Antifungal Susceptibilities of Dermatophytes 
Journal of Clinical Microbiology  2004;42(7):2977-2979.
The National Committee for Clinical Laboratory Standards (NCCLS) M38-A standard for the susceptibility testing of conidium-forming filamentous fungi does not explicitly address the testing of dermatophytes. This multicenter study, involving six laboratories, investigated the MIC reproducibility of seven antifungal agents tested against 25 dermatophyte isolates (5 blinded pairs of five dermatophyte species per site for a total of 300 tests), using the method of dermatophyte testing developed at the Center for Medical Mycology, Cleveland, Ohio. The dermatophytes tested included Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton tonsurans, Epidermophyton floccosum, and Microsporum canis. Seven antifungals with activity against dermatophytes were tested, including ciclopirox, fluconazole, griseofulvin, itraconazole, posaconazole, terbinafine, and voriconazole. Interlaboratory MICs for all isolates were in 92 to 100% agreement at a visual endpoint reading of 50% inhibition as compared to the growth control and 88 to 99% agreement at a visual endpoint reading of 80% inhibition as compared to the growth control. Intralaboratory MICs between blinded pairs were in 97% agreement at a visual endpoint reading of 50% inhibition as compared to the growth control and 96% agreement at a visual endpoint reading of 80% inhibition as compared to the growth control. Data from this study support consideration of this method as an amendment to the NCCLS M38-A standard for the testing of dermatophytes.
PMCID: PMC446243  PMID: 15243047
23.  Indoor Mold, Toxigenic Fungi, and Stachybotrys chartarum: Infectious Disease Perspective 
Clinical Microbiology Reviews  2003;16(1):144-172.
Damp buildings often have a moldy smell or obvious mold growth; some molds are human pathogens. This has caused concern regarding health effects of moldy indoor environments and has resulted in many studies of moisture- and mold-damaged buildings. Recently, there have been reports of severe illness as a result of indoor mold exposure, particularly due to Stachybotrys chartarum. While many authors describe a direct relationship between fungal contamination and illness, close examination of the literature reveals a confusing picture. Here, we review the evidence regarding indoor mold exposure and mycotoxicosis, with an emphasis on S. chartarum. We also examine possible end-organ effects, including pulmonary, immunologic, neurologic, and oncologic disorders. We discuss the Cleveland infant idiopathic pulmonary hemorrhage reports in detail, since they provided important impetus for concerns about Stachybotrys. Some valid concerns exist regarding the relationship between indoor mold exposure and human disease. Review of the literature reveals certain fungus-disease associations in humans, including ergotism (Claviceps species), alimentary toxic aleukia (Fusarium), and liver disease (Aspergillys). While many papers suggest a similar relationship between Stachybotrys and human disease, the studies nearly uniformly suffer from significant methodological flaws, making their findings inconclusive. As a result, we have not found well-substantiated supportive evidence of serious illness due to Stachybotrys exposure in the contemporary environment. To address issues of indoor mold-related illness, there is an urgent need for studies using objective markers of illness, relevant animal models, proper epidemiologic techniques, and examination of confounding factors.
PMCID: PMC145304  PMID: 12525430
24.  Uses and Limitations of the XTT Assay in Studies of Candida Growth and Metabolism 
Journal of Clinical Microbiology  2003;41(1):506-508.
Colorimetric tetrazolium assays are used increasingly in studies of fungi, often in the absence of standardization or correlation with other methods. We examined species- and strain-related tetrazolium metabolism in Candida albicans and Candida parapsilosis by using XTT {2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide} and WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphonyl)-2H-tetrazolium] and found marked variations. Also, significant signal was often missed in the absence of dimethyl sulfoxide extraction.
PMCID: PMC149594  PMID: 12517908
25.  Antifungal Susceptibility of Candida Biofilms: Unique Efficacy of Amphotericin B Lipid Formulations and Echinocandins 
Biofilms, likely the predominant mode of device-related microbial infection, exhibit resistance to antimicrobial agents. Evidence suggests that Candida biofilms have dramatically reduced susceptibility to antifungal drugs. We examined antifungal susceptibilities of Candida albicans and Candida parapsilosis biofilms grown on a bioprosthetic model. In addition to conventional agents, we determined if new antifungal agents (triazoles, amphotericin B lipid formulations, and echinocandins) have activities against Candida biofilms. We also explored effects of preincubation of C. albicans cells with subinhibitory concentrations (sub-MICs) of drugs to see if they could modify subsequent biofilm formation. Finally, we used confocal scanning laser microscopy (CSLM) to image planktonic- and biofilm-exposed blastospores to examine drug effects on cell structure. Candida biofilms were formed on silicone elastomer and quantified by tetrazolium and dry weight (DW) assays. Susceptibility testing of fluconazole, nystatin, chlorhexidine, terbenafine, amphotericin B (AMB), and the triazoles voriconazole (VRC) and ravuconazole revealed resistance in all Candida isolates examined when grown as biofilms, compared to planktonic forms. In contrast, lipid formulations of AMB (liposomal AMB and AMB lipid complex [ABLC]) and echinocandins (caspofungin [Casp] and micafungin) showed activity against Candida biofilms. Preincubation of C. albicans cells with sub-MIC levels of antifungals decreased the ability of cells to subsequently form biofilm (measured by DW; P < 0.0005). CSLM analysis of planktonic and biofilm-associated blastospores showed treatment with VRC, Casp, and ABLC resulted in morphological alterations, which differed with each agent. In conclusion, our data show that Candida biofilms show unique susceptibilities to echinocandins and AMB lipid formulations.
PMCID: PMC127206  PMID: 12019089

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