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Random amplification of polymorphic DNA and inter-repeat polymerase chain reaction (IR-PCR) were compared with restriction fragment length polymorphism (RFLP) analysis as methods for DNA typing of Candida albicans. Forty-seven strains of Candida albicans isolated from the oral cavities of five AIDS patients undergoing fluconazole therapy were analyzed. There was an excellent correspondence between the DNA types obtained by both PCR-based techniques and by RFLP. With the exception of one patient who was infected with three DNA types of Candida albicans during a five-year observation period, the patients each harboured only one major strain, which became progressively less susceptible to fluconazole. Each DNA type was unique to a patient. The data suggest that these typing methods are suitable for investigating the epidemiology of oropharyngeal candidiasis in this patient group.

Objectives: The aim of the present study was to evaluate the effects of amphotericin B (AMB) on clinical isolates of Aspergillus flavus. Methods: MICs of both standard AMB and liposomal AMB (L-AMB) were determined using a broth dilution method for seven isolates ofA.flavus. AMB MICs were also determined using the Etest. The activity of the polyene was then investigated in a murine model of systemic aspergillosis in which animals were infected intravenously, treated intravenously with several doses of the polyene (1 –10 mg/kg/day) and observed for survival. Results: Broth dilution AMB, broth dilution L-AMB and Etest AMB MICs ranged from 0.5 to 2.0 mg/L, 0.06 to .16 mg/L and 1.0 to .32 mg/L, respectively. There were two isolates for which all doses were effective at prolonging the survival. Their AMB MICs were ≤1.0 mg/L, regardless of the method/drug formulation utilized for testing. There were four isolates for which no regimen was effective. Their broth dilution AMB, broth dilution L-AMB and Etest AMB MICs ranged from 1.0 to 2.0 mg/L, 0.06 to .16 mg/L and 2.0 to .32 mg/L, respectively. There was one isolate for which only L-AMB given at 10 mg/kg/day was effective; broth dilution MICs of AMB and L-AMB were 0.5 mg/L, while the Etest MIC of AMB was 2.0 mg/L. Conclusions: Our data indicate that not all isolates of A. flavus should be considered resistant to AMB. The Etest represented the in vitro method that best correlated with the experimental infection. Finally, a clinical isolate showing an MIC ≥2.0 mg/L may be reasonably considered resistantinvivo to any dose/formulation of the polyene.

The occurrence of resistance or side effects in patients receiving antifungal agents leads to failure in the treatment of mycosis. The aim of this experimental study was to investigate the in vitro effects of IB-367 alone and in combination with three standard antifungal drugs, fluconazole (FLU), itraconazole (ITRA) and terbinafine (TERB), against 20 clinical isolates of dermatophytes belonging to three species. Minimum inhibitory concentrations (MICs), minimal fungicidal concentrations (MFCs), synergy test, time-kill curves, fungal biomass (FB) and hyphal damage using 2,3-bis-(2-methoxy-4-nitro-5-sulfenylamino carbonil)-2H-tetrazolium hydroxide assay (XTT) were performed to study the efficacy of IB-367. In this study, we observed that TERB and ITRA had MICs lower values for all the strains compared to IB-367 and FLU. Synergy was found in 35%, 30% and 25% of IB-367/FLU, IB-367/ITRA and IB-367/TERB interactions respectively. IB-367 exerted a fungicidal activity against Trichophyton mentagrophytes, T. rubrum and Microsporum canis at concentrations starting from 1x MIC. At a concentration of 5x MIC, IB-367 showed the highest rates of hyphae damage for M. canis 53% and T. mentagrophytes 50%; against the same isolates it caused a reduction of 1 log of the total viable count cell hyphae damage. We propose IB-367 as a promising candidate for the future design of antifungal drugs.

Intestinal parasites are a serious problem in developing countries, but should not be underestimated in industrialised countries either. Between January 2006 and December 2011, stool specimens and the scotch tests of 5323 Italian and non Italian patients (adults and children) attending the laboratory of our Infectious Diseases Clinic in a teaching Hospital at Ancona were analyzed specifically for intestinal parasites. The present study shows that, over a six-year period, of a total of 5323 patients 305 harboured at least one species of parasite (5.7%). Among the pathogenic protozoa Giardia lamblia was the most common, the overall prevalence of giardiasis being 1.8 % (99/5323). Helminths were found in 0.9% of the patients (48/5323). In particular, Hymenolepis nana, Strongyloides stercoralis and Trichuris trichiura were most commonly recovered in non-Italian children, suggesting that certain intestinal parasites are restricted to endemic areas in the tropics. Eighteen of the 305 infected patients had more than one parasite in their stools. Our study demonstrates that intestinal parasites must be considered even in industrialised areas and stool examination should be supported by epidemiological data and clinical features.

Purpose We compared the risk factors, the diagnostic tools and the outcome of filamentous fungal infections (FFIs) in hematological patients (HAEs) and non-hematological patients (non-HAEs). Methods Prospective surveillance (2009–2011) of proven and probable FFIs was implemented in 23 Italian hospitals. Results Out of 232 FFIs, 113 occurred in HAEs and 119 in non-HAEs. The most frequent infection was invasive aspergillosis (76.1 % for HAEs, 56.3 % for non-HAEs), and the localization was principally pulmonary (83.2 % for HAEs, 74.8 % for non-HAEs). Neutropenia was a risk factor for 89.4 % HAEs; the main underlying condition was corticosteroid treatment (52.9 %) for non-HAEs. The distribution of proven and probable FFIs was different in the two groups: proven FFIs occurred more frequently in non-HAEs, whereas probable FFIs were correlated with the HAEs. The sensitivity of the galactomannan assay was higher for HAEs than for non-HAEs (95.3 vs. 48.1 %). The overall mortality rate was 44.2 % among the HAEs and 35.3 % among the non-HAEs. The etiology influenced the patient outcomes: mucormycosis was associated with a high mortality rate (57.1 % for HAEs, 77.8 % for non-HAEs). Conclusions The epidemiological and clinical data for FFIs were not identical in the HAEs and non-HAEs. The differences should be considered to improve the management of FFIs according to the patients’ setting.

Electrophoretic karyotype and restriction endonuclease analysis of genomic DNA were used for the typing of nine isolates of Candida albicans from the oral cavities of two patients with AIDS--a husband and wife--whose infections became resistant to treatment with fluconazole (400 mg/d). The in vitro susceptibilities of sequential isolates to fluconazole and two other triazoles, itraconazole and the investigational drug D0870, were also evaluated. DNA analysis showed that the isolates responsible for fluconazole-resistant episodes of oropharyngeal candidiasis in the two patients were genetically related. In vitro susceptibility to fluconazole correlated well with clinical outcome. Although the minimal inhibitory concentrations of itraconazole and D0870 for fluconazole-resistant isolates were higher than those for fluconazole-susceptible isolates, both of the former triazoles exhibited good in vitro activity against the isolates tested.

Sixty-two Candida albicans isolates from the oral cavities of 28 patients with AIDS who were receiving fluconazole therapy were typed by restriction endonuclease analysis followed by pulsed-field gel electrophoresis; these isolates were then tested for fluconazole susceptibility by a standard broth dilution method. Sequential isolates (range, 2-4) were evaluated for 22 patients; only one isolate was evaluated for six patients. DNA subtyping revealed a total of 37 different DNA subtypes. Twelve (54.5%) of 22 patients with multiple episodes of oropharyngeal candidiasis were infected with a single DNA subtype throughout the observation period. Ten (45.5%) of 22 patients with multiple episodes of oropharyngeal candidiasis were infected with two or three DNA subtypes during the observation period. In vitro susceptibility tests revealed that MICs of fluconazole ranged fromor = 0.125 microgram/mL to 64 micrograms/mL, with an MIC50 of 0.5 microgram/mL and an MIC90 of 4 micrograms/mL. A significant increase in the MICs (fourfold or greater) of fluconazole for sequential C. albicans isolates was found for 66.6% of the patients infected with a single DNA subtype and for 50% of the patients infected with multiple DNA subtypes. Despite a limited number of patients and isolates, our data suggest that C. albicans isolates that are susceptible to fluconazole at MICs ofor = 8 micrograms/mL in vitro will be less susceptible in vivo to standard doses (100-200 mg/d) of this drug.

The E test strip (AB Biodisk, Solna, Sweden) as an antimicrobial susceptibility testing method is a new and promising tool with broad application in microbiology. This method is less labour intensive than broth dilution methods and may be useful for testing individual clinical isolates. In contrast to several publications comparing E test antibacterial strips with NCCLS reference methods, there is little information about the performance of E test antifungal strips. This study compared fluconazole and itraconazole MICs obtained by E test with a microbroth dilution method performed to NCCLS guidelines. Fluconazole and itraconazole E test results exhibited good correlation with the broth microdilution (82% and 81% agreement within +/- 1 two-fold dilution, respectively). The E test is easy to perform and generates MICs for Candida species in 24 h.

OBJECTIVES The aim of the present study was to compare, in vitro and in vivo, the effects of caspofungin, micafungin and anidulafungin against Candida parapsilosis complex isolates. METHODS In vitro activities of all three echinocandins were assessed against C. parapsilosis sensu stricto (n = 4), Candida orthopsilosis (n = 4) and Candida metapsilosis (n = 3) using broth microdilution susceptibility testing, minimum fungicidal concentration determination and a killing-curve assay, in the absence and in the presence of 50% human serum. Then, the activities of all drugs were investigated in an immunocompromised murine model of systemic candidiasis. Animals were infected with six isolates (two for each species) and treated with the echinocandins administered at 0.25, 1, 5 and 10 mg/kg/day for six consecutive days. Fungal burdens were assessed in kidney tissues on day 7 post-infection. RESULTS Geometric mean MICs of caspofungin, micafungin and anidulafungin for C. parapsilosis sensu lato were, respectively, 0.09, 0.14 and 0.20 mg/L without serum, and 0.70, 3.92 and 5.84 mg/L with serum. The fungicidal activity of all three echinocandins was variable; however, the addition of serum reduced the fungicidal effects against these species. In vivo studies showed that caspofungin at 5 and 10 mg/kg/day significantly decreased the kidney burdens with respect to the controls for all isolates, while micafungin was active at 5 and/or 10 mg/kg/day only against C. metapsilosis. CONCLUSIONS Our susceptibility testing showed that caspofungin was the most active echinocandin against all three species. Also, caspofungin resulted in significant therapeutic effects for treatments of experimental systemic infections due to the three species, while micafungin was effective only against C. metapsilosis.

We investigated the in vitro activity of a new antifungal triazole, D0870, against 100 Candida albicans isolates from the oral cavities of patients infected with human immunodeficiency virus by using a broth macrodilution method following the recommendations provided by the National Committee for Clinical Laboratory Standards (document M27-P). All of the isolates were chosen from C. albicans isolates already tested for fluconazole susceptibility by the procedure of the National Committee for Clinical Laboratory Standards. Fifty isolates were considered fluconazole susceptible (MICs, < or = 4 micrograms/ml), and 50 isolates were considered fluconazole resistant (MICs, > or = 8 micrograms/ml). The in vitro data demonstrated that D0870 had good activity against isolates tested; for 90% of all strains of C. albicans, MICs were 0.5 micrograms/ml. However, the D0870 MICs for the fluconazole-susceptible isolates were lower than those for the fluconazole-resistant isolates; MICs for 50 and 90% of the isolates tested were < or = 0.0078 and 0.06 micrograms/ml, respectively, for fluconazole-susceptible isolates and 0.25 and 2 micrograms/ml, respectively, for fluconazole-resistant isolates (P < 0.001). Our data suggest that this new triazole could represent a valid alternative in the treatment of oral candidiasis in human immunodeficiency virus-infected patients.

SummaryWe compared the in vitro activity of amphotericin B, flucytosine, itraconazole, fluconazole, ketoconazole and miconazole against 18 strains of Cryptococcus neoformans by using two methods: microbroth dilution and semisolid agar dilution.By both of the methods minimum inhibitory concentrations (MICs) showed a wide range for all antifungal agents but not for amphotericin B. Statistically significant differences between the two methods were observed only with amphotericin B and flucytosine, p = 0.048 and p = 0.045 respectively.Our study suggests that azole susceptibility testing for C. neoformans may be performed by the broth microdilution as well as the semisolid agar test. The choice of the method when testing amphotericin B and flucytosine is more problematic.

The drug concentration which inhibited 50% of growth (IC50), the lowest drug concentration at which growth was less than 30% of that in a positive control well (IC30), the visual minimal inhibitory concentration (MIC), and the minimum fungicidal concentration (MFC), were applied to study the effects of fluconazole, itraconazole, ketoconazole, miconazole, flucytosine, and amphotericin B against 36 isolates ofCandida spp. andCryptococcus neoformans by a broth microdilution technique. When the recommendations established by the NCCLS Subcommittee on Antifungal Susceptibility Tests were applied for the visual reading of the microplates, the results were comparable with those obtained by the turbidimetric methods. Differences between MICs and IC30s were observed with miconazole against strains ofC. glabrata (p=0.014) and with flucytosine against strains ofC. neoformans (p=0.041). Differences between MICs and IC50s were observed with fluconazole against strains ofC. albicans (p=0.027),C. tropicalis (p=0.046), andC. neoformans (p=0.041); with miconazole against strains ofC. glabrata (p=0.014); and with amphotericin B against strains ofC. parapsilosis (p=0.025). Ten additional isolates ofC. albicans from AIDS patients suffering from recurrent episodes of oral candidiasis and clinically resistant to fluconazole also were included in this study. The MICs of fluconazole of these strains were significantly higher than those of the control group (p=0.003). When the turbidimetric parameters were applied for testing the in vitro activity of fluconazole against the above isolates, both IC30 and IC50 were capable of discriminating the strains of the two groups (p=0.002,p=0.001, respectively). Only amphotericin B demonstrated fungicidal activity against all the tested yeasts.

Objectives: We analysed the in vitro and in vivo effects of posaconazole and amphotericin B against three clinical isolates of zygomycetes: Lichtheimia corymbifera, F1; and Rhizopus oryzae, F5 and F6. Methods: In vitro activities of both drugs were assessed by determining MICs, minimum fungicidal concentrations (MFCs) and fungal damage measured by the XTT assay against either the spores or the hyphal forms. Additionally, the survival curves of neutropenic mice systemically infected with the zygomycete isolates were used as the marker of antifungal response to amphotericin B (1 mg/kg/day) or posaconazole (2.5, 10 and 50 mg/kg/day). Results: In terms of MICs, posaconazole proved to be active against the three isolates (MICs ranging from 0.125 to 1.0 mg/L). The median posaconazole MFCs were 0.25, 0.5 and .16 mg/L for F1, F5 and F6, respectively. The XTT assay showed that posaconazole was active against spores of all three isolates, but only partially effective against the hyphae. The survival studies showed that amphotericin B at 1 mg/kg/day and posaconazole at 10 mg/kg/day prolonged the survival of the animals infected with L. corymbifera F1. In mice infected with R. oryzae F5, only posaconazole at 50 mg/kg/day significantly prolonged survival, whereas amphotericin B at 1 mg/kg/day was the only regimen active against R. oryzae F6. Conclusions: Our findings showed that posaconazole could be useful in the treatment of zygomycosis. Also, we report that an isolate of R. oryzae with low MFC responded to posaconazole, while another isolate with high MFC did not.

Candida albicans is known to be the organism most often associated with serious fungal infection, but other Candida spp. are emerging as clinical pathogens associated with opportunistic infections. Among antimycotic treatments, increasing attention is currently given to anti-infective drugs based upon naturally occurring peptides, such as the short lipopeptide palmitoyl PAL-Lys-Lys-NH2 (PAL). The aim of this study is to evaluate the activity of this peptide compared to the traditional antifungal agents Fluconazole (FLU), amphotericin B (AMB) and caspofungin (CAS) on Candida spp. 24 clinical isolates of Candida spp. were tested against PAL, FLU, AMB and CAS using in vitro susceptibility tests, time killing and checkerboard assay. All of the drugs studied showed good activity against clinical isolates of candida; in particular CAS and AMB which have MICs value lower than PAL and FLU. Moreover we observed synergistic interactions for PAL/FLU (81.25%), PAL/AMB (75%) and particularly for PAL/CAS (87.5). We think that our results are interesting since synergy between PAL and CAS might be useful in clinic trails to treat invasive fungal infections.

SummaryIn out study 77 AIDS patients suffering from oral and/or esophageal candidiasis were evaluated: 38 received fluconazole, 39 ketoconazole. We analyzed the rates of clinical and mycological responses, relapses and toxicities. In vitro susceptibility tests for both antifungal drugs were performed by evaluating their Minimal Inhibitory Concentrations (MICs). The azole drugs investigated show a good activity in the treatment of oropharyngeal and esophageal candidiasis also in advanced stages of HIV infection. Clinical cure or improvement were achieved in 29 (76.3%) and 31 (79.4%) of the patients treated with fluconazole or ketoconazole respectively. Clinical or laboratory adverse experiences related to fluconazole were seen in 7 (21.2%) patients while ketoconazole provoked adverse experiences in 9 (26.4%) patients. In vitro susceptibility tests, if repeated more than once, both in primary infection and relapses, could be important to demonstrate a probable sensitivity change or resistance of the tested ...

The in vitro susceptibilities of 90 clinical isolates of gram-positive and gram-negative aerobic bacteria to six cationic peptides, buforin II, cecropin P1, indolicidin, magainin II, nisin, and ranalexin, were evaluated by two broth microdilution methods. The first method was performed according to the procedures outlined by the National Committee for Clinical Laboratory Standards for bacteria that grow aerobically, while the second was performed according to the procedures recently proposed by the R. E. W. Hancock laboratory for testing antimicrobial peptides. Overall, the first method produced MICs two- and fourfold higher than the second method.

The in vitro anticryptosporidial activities of ranalexin, lasalocid and azithromycin alone and in combination were investigated against four clinical isolates of Cryptosporidium parvum. Susceptibility was tested by inoculating the isolates on to cell monolayers and determining the parasite count after 48 h incubation at 37 degrees C. The culture medium was supplemented with Dulbecco's modified Eagle's medium containing serial dilutions of the above-mentioned compounds. Ranalexin showed moderate anticryptosporidial activity: at a concentration of 64 mg/L it reduced parasite counts by 33.8%. Azithromycin at a concentration of 8 mg/L gave inhibition comparable to that observed with the highest concentration of ranalexin. Lasalocid showed the highest activity, with a 70.3% reduction in parasite counts at 2 mg/L. The combination of ranalexin 64 mg/L and lasalocid 2 mg/L completely suppressed parasite growth without harming the monolayer.

We investigated the effects of anidulafungin alone and in combination with amphotericin B against Aspergillus fumigatus . Indifference was the only type of interaction observed in vitro. Anidulafungin at 1 and 5 mg/kg of body weight/day, amphotericin B at 1 mg/kg/day, and combination therapy prolonged the survival of mice with invasive aspergillosis. Anidulafungin at 5 mg/kg/day, alone and in combination with amphotericin B, reduced the kidney fungal burden. Overall, the combination was not superior to the most active single drug.

Aim of our study was to investigate the in vitro effects of Tachyplesin III (TP), a potent disulfide-linked peptide, in dermatophytes infections, with respect to or in combination with terbinafine (TERB), against 20 clinical isolates of dermatophytes belonging to four species. A broth microdilution method following the CLSI recommendations (M38-A) was used for testing drugs alone and in combination. TERB MICs were significantly lower than those observed for TP (p < 0.001). Testing for antifungal agents in combination was performed for TERB with TP for all the 20 isolates. TERB activity in combination with TP showed indifferent activity for 14 of the 20 isolates (70%); synergic activity for 6 of the 20 isolates (30%); no antagonistic activity was observed. Further experiments were conducted with Microsporum canis 133, Trichophyton rubrum 62 and Trichophyton mentagrophytes 91 for fungal biomass. TP and TERB did not show a significant growth reduction compared to the control against T. mentagrophytes and T. rubrum. A significant difference of growth reduction both for TP and TERB compared to controls was observed for M. canis (p < 0.01). In conclusion our study demonstrated that Tachyplesin III has potential activity against dermatophytes. In addition, we observed that the in vitro activity of Tachyplesin III can be enhanced upon combination with terbinafine. Synergy could permit lower doses of the individual antifungal agents to be used more effectively and/or safely.

We compared in-vitro activity of fluconazole, itraconazole and ketoconazole by evaluating their Minimal Inhibitory Concentrations (MICs) for 100 fungal strains isolated from different biological specimens of ARC/AIDS patients. A semisolid agar medium was used: this method is suitable for testing molds and yeasts, and can be applied to all azole antifungal drugs. Fluconazole had higher MICs than two other tested drugs, especially for Candida krusei strains; however it never had a MIC higher than 40 mg/l. Itraconazole and ketoconazole had MICs higher than 40mg/l for one Cryptococcus neoformans strain. There were no significant differences for itraconazole and ketoconazole among the tested strains.

A broth macrodilution method, performed as recommended by the National Committee for Clinical Laboratory Standards, was used for comparative testing of the new echinocandin antifungal agent MK-0991 and fluconazole against 50 yeast isolates belonging to 12 species of Candida. MK-0991 was shown to be highly effective against both fluconazole-susceptible and -resistant Candida spp., yielding minimum inhibitory concentrations ranging fromor = 0.19 to 0.78 microg/ml. Fungicidal activity was exerted ator = 1.5 microg/ml for 73% of the isolates tested. This study suggests that MK-0991 has significant potential for clinical development.

The minimum inhibitory concentrations (MICs), the minimal fungicidal concentrations (MFCs), the fungal biomass (FB) and hyphal viability employing the dye 3-4,5 dimethyl- 2-thiazolyl- 2,5- diphenyl- 2H tetrazolium bromide (MTT) were used to compare the in vitro effects of fluconazole (FLU) with those of the N-terminal palmitoyl-lipidated peptide, Pal-Lys-Lys-NH(2) (PAL), and a tea tree oil component, gamma-Terpinene (TER), against several clinical isolates of Microsporum canis and Trichophyton rubrum. In general, FLU and PAL MICs were significantly lower than those observed with TER, while no differences in the three drugs were found in the MFCs. However, they were from two to 16-times higher than their respective MICs. FB of M. canis treated with either FLU or PAL, but not with TER, was significantly reduced over untreated controls. Only PAL and TER, in a medium-dependent fashion, but not FLU, reduced the FB of T. rubrum. Finally, PAL was found to be significantly more active than FLU at reducing the hyphal viability against both genera of dermatophytes. This study shows that PAL exerts an in vitro activity against dermatophytes at least similar to that observed with FLU and suggests that this compound might be a promising candidate in the treatment of infections due to dermatophytes.

The in vitro activity of the peptide IB-367, alone or combined with either fluconazole (FLU) or amphotericin B (AMB), was investigated against 25 Candida isolates belonging to five species. IB-367 minimum inhibitory concentrations (MICs) ranged from 2.0 to 32 μg/ml and it was active against both FLU-susceptible and -resistant isolates. A rapid fungicidal activity was observed. Synergism was documented in 41.6% and 44% of IB-367/FLU and IB-367/AMB interactions, respectively. Antagonism was never observed. The broad antifungal activity and the positive interactions with AMB and FLU suggest that IB-367 represents a promising candidate against infections due to Candida spp.

Yeast-like fungi are the most common cause of fungal infections in humans. Actually, in the age of opportunistic infections and increasing resistance, development of modern antifungal agents becomes a very important challenge. This paper describes synthesis and antimicrobial assay of four naturally occurring peptide antibiotics (aurein 1.2, citropin 1.1, temporin A, uperin 3.6) and three chemically engineered analogues actually passing clinical trials (iseganan, pexiganan, omiganan) against Candida strains isolated from patients with infections of the oral cavity or respiratory tract. The peptides were synthesized using solid-phase method and purified by high-performance liquid chromatography. Biological tests were performed using the broth microdilution method. The antifungal activity of the peptide antibiotics was compared to that of nystatin and amphotericin B. We found synthetic peptides to be generally less potent than amphotericin B or nystatin. However, some of the naturally occurring peptides still retained reasonable antifungal activities which were higher than these of iseganan, pexiganan or omiganan. We think that the naturally occurring peptide antibiotics included in our study can be a good matrix for development of novel antifungal compounds.

The in vitro activity of fluconazole was investigated against 476 yeast isolates collected during a 9-year period (1997-2005) from patients hospitalised in a teaching hospital of Ancona. They included 373 isolates of Candida albicans, 53 of Candida glabrata and 50 of Candida parapsilosis. Minimum inhibitory concentrations (MICs) determined in accordance with the Clinical Laboratory Standards Institute methodology showed that 96% of the isolates were susceptible (MICor =8.0 microg/ml). The uncommon, resistant isolates (MICor =64 microg/ml) were randomly distributed over time. Our data show that resistance to fluconazole in this geographical area is a rare event and suggest that this triazole can still represent first-line therapy in our institution.

In vitro activity of voriconazole against fluconazole-resistant Candida albicans clinical isolates with identified molecular basis of multidrug resistance (MDR) and recombinant Saccharomyces cerevisiae expressing C. albicans genes coding for major multidrug transporters, CaCdr1p, CaCdr2p or CaMdr1p, was compared with that of fluconazole, ketoconazole and clotrimazole. It was found that overexpression of the MDR genes made the yeast cells less susceptible to voriconazole. The voriconazole resistance indexes, defined as a ratio of minimum inhibitory concentrations (MICs) determined for MDR and sensitive cells, were comparable with those determined for fluconazole. Voriconazole effectively competed with rhodamine 6G for the active efflux mediated by CaCdr1p and CaCdr2p.

A broth macro dilution method following the recommendations established by the National Committee for Clinical Laboratory Standards was employed for testing terbinafine against 20 clinical isolates of dermatophytes belonging to seven species. The minimal inhibitory concentrations resulting in either 80% or 100% inhibition of growth, compared to growth in drug-free control tubes, ranged from ≤0·03 to 0·25 g ml-1 and from

Organ transplant recipients are at increased risk for severe invasive aspergillosis, and amphotericin deoxycholate has been the standard treatment for many years. Currently, however, lipid formulations are preferred due to their few side effects. Also, a number of new antifungal drugs have been developed including new azoles and echinocandins. Caspofungin is the first of the echinocandin derivatives patented to treat patients with invasive aspergillosis who are refractory or intolerant to other therapies. A renal transplant patient on immunosuppressive treatment with chronic hepatitis B virus infection was admitted with fever, hemophthisis and lung consolidation, diagnosed to be probably caused by Aspergillus flavus. The patient developed cholestatic hepatitis most likely related to itraconazole. Clinical failure and in vitro itraconazole resistance of the isolate was also documented while the patient was receiving itraconazole at a reduced dosage. Caspofungin was administered once a day as ambulatory treatment and was well tolerated. Clinical improvement was observed after 6 weeks of treatment and no hepatic toxicity was documented. Caspofungin seems to be a potentially useful antifungal agent in renal transplant patients with invasive aspergillosis. Further evaluation of the efficacy of caspofungin is needed.

Three isolates of zygomycetes belonging to two different genera ( Rhizopus oryzae and Absidia corymbifera ) were used to produce a systemic infection in neutropenic mice. On days −2 and −1 and at 2 h prior to infection, the mice received either posaconazole (POS) at doses ranging from 20 to 80 mg/kg of body weight/day or amphotericin B (AMB) at 1 mg/kg/day. Antifungal drug efficacy was assessed by determination of the prolongation of survival, determination of the percentage of infected organs (brain, lung, spleen, and kidney), and histological examination for the number of infection foci and their sizes in brain and kidney tissues. AMB significantly prolonged the survival of mice infected with all isolates. POS significantly prolonged the survival of mice infected with zygomycetes. Cultured organs from mice infected with R. oryzae were all positive, while treated mice challenged with A. corymbifera generally showed lower percentages of infected organs compared with the percentages for the controls. Zygomycete isolates established an active infection (the presence of hyphae) in the brains and the kidneys of all controls. In mice challenged with R. oryzae , both antifungal drugs were effective at reducing the number and the size of infection foci in the kidneys. Only AMB reduced the numbers, but not the sizes, of infection foci in the brain. Finally, both drugs significantly reduced the numbers and the sizes of infection foci in both tissues of mice infected with A. corymbifera . Our data suggest that prophylaxis with POS has some potential to prevent zygomycosis.

The epidemiological and antifungal susceptibility data for 94 episodes of candidaemia in an Italian tertiary-care hospital between January 2000 and August 2003 were evaluated by prospective laboratory-based surveillance. The incidence of fungaemia was 0.90 episodes/10 000 patient-days, and the most common species isolated were Candida albicans (40.4%), Candida parapsilosis (22.3%), Candida tropicalis (16.0%) and Candida glabrata (12.8%). Among 24 patients who received antifungal prophylaxis, non-albicans Candida spp. were more prevalent than C. albicans (p 0.012). The 30-day mortality rate was high (38.2%), particularly for haematological (71.4%) and solid-organ transplant patients (50.0%), and in individuals with C. tropicalis and C. glabrata bloodstream infections (60.0% and 50.0%, respectively). In-vitro susceptibility tests demonstrated that 95% of the isolates were susceptible to amphotericin B (MIC < 2 mg/L), 98.1% to posaconazole (MIC < 1 mg/L), 95.8% to flucytosine (MIC < 32 mg/L) and fluconazole (MIC < 64 mg/L), and 94.7% to itraconazole (MIC < 1 mg/L). Posaconazole was active (MIC 0.5 mg/L) against all three isolates of Candida krusei, which had reduced susceptibility to both fluconazole and itraconazole. Overall, non-albicans Candida spp. accounted for 60% of the episodes of candidaemia, which could be related to the use of antifungal prophylaxis. Resistance is still uncommon in Candida spp. recovered from blood cultures. The in-vitro activity of posaconazole is encouraging, and this agent could play an important role in the management of invasive candidiasis, including episodes caused by inherently less susceptible species such as C. krusei.

A checkerboard microdilution method was applied to study the in vitro interaction of terbinafine with either fluconazole and itraconazole against 30 strains of Candida albicans. Synergy was observed in 40% of the terbinafine-fluconazole interactions and in 43% of the terbinafine-itraconazole interactions, while antagonism was not observed. Even when only additivity was achieved, the combinations still showed beneficial effects since at least twofold reductions in the MICs of both drugs were found in 100% of the terbinafine-fluconazole interactions and in 76% of the terbinafine-itraconazole interactions.

We investigated the in vitro and in vivo activities of caspofungin against Aspergillus terreus . The drug increased survival and reduced tissue fungal burden in neutropenic mice. Therefore, our data support the role of caspofungin in treating systemic infections due to this emerging pathogen.

A multicenter (six-center) study evaluated the performance (interlaboratory reproducibility, compatibility with reference methods, and categorical agreement) of 24-h visual and 48-h spectrophotometric MICs. MICs of fluconazole, itraconazole, voriconazole, and posaconazole were compared to reference 48-h microdilution broth visual MICs (CLSI [formerly NCCLS] M27-A2 document) for 71 isolates of Candida spp. that included 10 fluconazole-resistant strains. Twenty readings (5%) were reported as showing no growth at 24 h, mostly for Candida dubliniensis and from a single center. The overall interlaboratory agreement of 24-h visual readings and 48-h spectrophotometric MICs, as well their compatibility with reference values, were excellent with the four triazoles for most of the species (93 to 99%, within 3 dilutions). The categorical agreement between the investigational reading conditions and reference values was good with fluconazole and voriconazole (93 to 97%) but lower with itraconazole (86 to 88%), due primarily to minor errors. There were only 0 to 3% very major errors with these three triazoles; the number of substantial errors (more than three dilutions) was also low (

A broth microdilution method was used for testing amphotericin B against 33 clinical isolates of Candida tropicalis. All isolates were in vitro susceptible to the polyene (MIC [minimal inhibitory concentration] < or = 1.0 microg/mL). However, when the isolates were cultured in a medium containing amphotericin B at a concentration of 1.5 microg/mL, a wide interstrain variation of growth rate was observed. Five isolates (15%) proved to be highly tolerant to the drug and grew at a frequency ranging from 1 x 10(-1) to 2 x 10(-2). Twenty-three isolates (70%) grew at a frequency ranging from 1 x 10(-5) to 1 x 10(-8). The remaining five isolates (15%) failed to grow in drug-containing medium. In general, this growth variation was not associated with amphotericin B MICs displayed by the single isolates. In addition, the strains grown in drug-containing medium did not represent amphotericin B-resistant mutants, as shown by the maintenance of MICs similar to those of their respective parent isolates. Killing experiments conducted in selected isolates confirmed a variation of fungicidal activity of amphotericin B. To see whether this phenomenon was associated with a variation of amphotericin B response in vivo, we established an experimental model of systemic murine candidiasis in CD1 mice by intravenous injection of cells belonging to Candida tropicalis 3147 (growth rate at a frequency of 1 x 10(-1) in amphotericin B medium) and Candida tropicalis 4055 (no growth). Low (0.3 mg/kg/day) and high (1 mg/kg/day) doses of amphotericin B were both effective at reducing the fungal burdens in the kidneys of mice infected with either strain (p, 0.01 to 0.02). However, whereas the burden of mice infected with isolate 3147 and treated with the polyene at 0.3 mg/kg/day was reduced by 1.2 +/- 0.25 (mean +/- standard deviation) log10 cfu/g compared to untreated mice, the same dosing regimen yielded a burden reduction of 2.6 +/- 0.07 log10 cfu/g in mice infected with isolate 4055 (p < 0.001). Similarly, amphotericin B at 1 mg/kg/day yielded a burden reduction of 1.8 +/- 0.20 vs. 2.5 +/- 0.30 log10 cfu/g in mice infected with isolates 3147 and 4055, respectively (p < 0.001). Our data revealed a variable pattern of tolerance to amphotericin B among isolates of Candida tropicalis and showed that this phenomenon might influence the rate of organ clearance during therapy.

A sequential therapy of caspofungin (CAS) and fluconazole (FLC) administration for treatment of Candida albicans infection was investigated. Treatment with CAS followed by FLC was as effective as CAS treatment given alone for the same duration. Our data suggest that switching from CAS to FLC is a potentially explorable therapeutic option for treatment of systemic candidiasis.

To investigate the effects of posaconazole (POS) and amphotericin B (AMB) combination therapy in cryptococcal infection, we established an experimental model of systemic cryptococcosis in CD1 mice by intravenous injection of three distinct clinical isolates of Cryptococcus neoformans . Therapy was started 24 h after the infection and continued for 10 consecutive days. POS was given at 3 and 10 mg/kg of body weight/day, while AMB was given at 0.3 mg/kg/day. Combination therapy consisted of POS given at a low (combo 3) or at a high (combo 10) dose plus AMB. Survival studies showed that combo 3 was significantly more effective than POS at 3 mg/kg for two isolates tested ( P value, ≤0.001), while combo 10 was significantly more effective than POS at 10 mg/kg for all three isolates ( P values ranging from P values ranging from 0.001 to 0.015) but not in the lungs. This study demonstrates that the major impact of POS and AMB combination therapy is on brain fungal burden rather than on survival.

We report an outbreak of infection due to genotypically identical Candida parapsilosis isolates among patients hospitalized in a pediatric oncology unit. Control cultures showed genetic relatedness between strains isolated from the patients and those isolated from the hands of a health care worker. Our data underline the importance of an effective surveillance program for preventing nosocomial fungal infections.

Item does not contain fulltext OBJECTIVE: To evaluate the intra- and inter-laboratory reproducibility of a new standard for susceptibility testing of fermentative yeasts. This standard is based on the M27-A procedure of the National Committee for Clinical Laboratory Standards (NCCLS), but incorporates several modifications, including spectrophotometric growth-dependent endpoint reading. METHODS: Nine laboratories participated in the study. Common material lots were used to test six Candida species (one each of C. albicans, C. tropicalis, C. parapsilosis, C. glabrata, C. krusei, and C. lusitaniae), and two quality control strains (C. krusei ATCC6258 and C. parapsilosis ATCC22019). Triplicate testing on three separate days was performed in microtiter format with RPMI-2% glucose, pH 7.0. Flucytosine, fluconazole and itraconazole were tested. In total, 3888 MIC values were included in the analyses. Reproducibility was calculated by means of agreement (percentage of MICs within one two-fold dilution of the mode) and intraclass correlation coefficient (ICC, maximum value of 1). RESULTS: The average intra-laboratory agreements were 99% and 96% after 24 h and 48 h of incubation, respectively, with ICCs of 0.98 and 0.97 (P < 0.05). Two strains exhibiting a trailing effect showed intra-laboratory agreement of 92% and ICCs of < 0.91 at 48 h. The inter-laboratory agreement was 94% and 88% after 24 h and 48 h, respectively, with ICCs of 0.93 and 0.91 (P < 0.05). Lower values of agreement and ICCs were obtained for strains exhibiting trailing after 48 h of incubation. Itraconazole yielded the lowest values of reproducibility. CONCLUSION: The new procedure of EUCAST for antifungal susceptibility testing is a reproducible method within and between laboratories and offers several advantages over the NCCLS approved method.

A broth microdilution method following the NCCLS recommendations was used for testing fluconazole, itraconazole, posaconazole, 5-fluorocytosine and amphotericin B against 83 yeast isolates causing bloodstream infections in 59 patients hospitalized between January 1998 and June 2001 at the University Hospital of Bari, Italy. Isolates belonged to four species of Candida and three other yeast genera. Of the isolates, 97%, 95% and 100% were susceptible to fluconazole, itraconazole and posaconazole, respectively. Similarly, 97% and 100% of the isolates were susceptible to 5-fluorocytosine and amphotericin B, respectively.

We investigated the activity of a pyrazolo-isothiazole derivative (G8) against Cryptococcus neoformans. A first screening test showed that G8 at 10 mg/L inhibited the growth of 14 of 15 clinical isolates tested. Killing experiments showed that fungicidal activity was achieved after 8 h of treatment with G8 at concentrations > or =10 mg/L. In a murine model of systemic cryptococcosis, G8 was effective at prolonging survival compared with the controls. Our data indicate that this new derivative has a potential therapeutic role in infections caused by C. neoformans.

We conducted a prospective study to address the prevalence and microbiological characteristics of yeast isolates colonizing the oral cavities of HIV-infected patients undergoing highly active antiretroviral therapy. Sixty-eight patients (67%) from a total of 102 were found to be colonized with yeasts. Sixty-five patients carried a single species (60 Candida albicans, three Candida glabrata and two Candida krusei) and three patients had mixed colonization of C. albicans and C. krusei. The status of yeast carrier was not associated with the number of CD4 cells or the viral load. Similarly, the type of antiretroviral regimen was not associated with the carriage of Candida spp. The only predictor of Candida colonization was a previous history of oropharyngeal candidiasis (P = 0.009). Although many patients in this series had already been treated with repeated courses of fluconazole therapy for previous episodes of oropharyngeal candidiasis, fluconazole susceptibility patterns showed that 93% of yeasts were susceptible to this triazole in vitro (MIC < or = 8.0 mg/L).

The viability of Cryptosporidium parvum after exposure to peptide antibiotics was studied by two different methods, a cell culture system and a double fluorogenic staining. The peptides KFFKFFKFF and IKFLKFLKFL exerted high cytotoxic effects on sporozoites, as demonstrated by cell cultures (complete inhibition after 60 min at 100 microg/ml) and flow cytometry (30% after 20 min at 100 microg/ml), but did not affect consistently the oocysts. Clarithromycin and rifabutin demonstrated less activity against sporozoites but higher activity against oocysts (30% after 180 min at 10 microg/ml). The combination between peptides and azithromycin or rifabutin exerted the highest activities.

Subgingival colonization by Candida albicans has been described in human immunodeficiency virus (HIV)-infected individuals, but subgingival isolates have scarcely been characterized, particularly with respect to genotype and antifungal susceptibility. A series of 29 subgingival strains of C. albicans isolated from nine HIV-infected individuals was typed by electrophoretic karyotyping and tested for susceptibility to fluconazole, itraconazole, the new investigational triazole posaconazole and amphotericin B. DNA typing showed genetic heterogeneity within subgingival isolates, as almost every individual harbored his/her own specific isolate. Genetic identity was usually demonstrated within oral and subgingival isolates simultaneously collected from the same individual, but a number of DNA types were found to be unique to subgingival strains. These findings suggest that colonization is not just the result of Candida spreading from oral surfaces, and that subgingivally adapted strains could be involved. All isolates were susceptible to all the triazole drugs tested and amphotericin B. Additional studies on subgingival Candida colonization and further characterization of subgingival isolates are now required to clarify the role of Candida as opportunistic periodontal pathogen.

E. Gabrielli, A. W. Fothergill, L. Brescini, D. A. Sutton, E. Marchionni, E. Orsetti , S. Staffolani, P. Castelli , R. Gesuita and F. Barchiesi 1) Clinica Malattie Infettive, Universit a Politecnica delle Marche, Ancona, 2) Fungus Testing Laboratory, University of Texas Health Science Center, San Antonio, Texas and 3) Centro di Epidemiologia, Biostatistica e Informatica Medica, Universit a Politecnica delle Marche, Ancona, Italy

A broth microdilution method and an agar dilution method were used for testing fluconazole, itraconazole, ketoconazole, flucytosine and amphotericin B against 98 clinical isolates belonging to seven species of Candida. The approximate rank order of fluconazole MICs was Candida lusitaniae approximately Candida kefyr < Candida famata approximately Candida guilliermondii < Candida pelliculosa approximately C. lipolytica approximately Candida inconspicua. Candida lypolitica and C. pelliculosa were the species least susceptible to itraconazole and ketoconazole. Flucytosine MICs revealed the highest prevalence of resistant strains among C. lipolytica and C. lusitaniae. All isolates were susceptible to amphotericin B.

The in vitro anti-cryptosporidial activity of buforin II alone and in combination with azithromycin and minocycline was investigated. Buforin II showed moderate activity, which increased with increasing concentration to 55.7% suppression of growth at 20 microM. Moreover, its activity was enhanced when it was combined with either azithromycin or minocycline with >90% parasite reduction at the highest concentration tested. Buforin II may be active in inhibiting Cryptosporidium parvum growth in vitro upon combination with either azithromycin or minocycline.

A cell culture system and double fluorogenic staining were used to study the susceptibility of Cryptosporidium parvum to membrane-active antibiotics. Buforin II and magainin II exerted a cytotoxic effect on sporozoites but did not consistently affect oocyst viability. Lasalocid and nigericin demonstrated less activity against sporozoites but reduced the infectivity of oocysts.

The in vitro activity of buforin II, cecropin P1, indolicidin, magainin II and ranalexin, alone and in combination with eight clinically used antimicrobial agents was investigated against 12 multidrug-resistant strains of Acinetobacter baumannii isolated from immunocompromised patients. Antimicrobial activities were measured by MIC, MBC and viable count. The peptides had a varied range of inhibitory values: overall, the organisms were more susceptible to buforin II (MIC range 0.25-16 mg/L), cecropin P1 (0.50-32 mg/L) and magainin II (0.50-16 mg/L). Synergy occurred when magainin II was combined with beta-lactam antibiotics.

The interaction of amphotericin B (AmB) and azole antifungal agents in the treatment of fungal infections is still a controversial issue. A checkerboard titration broth microdilution-based method that adhered to the recommendations of the National Committee for Clinical Laboratory Standards was applied to study the in vitro interactions of AmB with fluconazole (FLC), itraconazole (ITC), and the new investigational triazole SCH 56592 (SCH) against 15 clinical isolates of Cryptococcus neoformans . Synergy, defined as a fractional inhibitory concentration (FIC) index of ≤0.50, was observed for 7% of the isolates in studies of the interactions of both FLC-AmB and ITC-AmB and for 33% of the isolates in studies of the SCH-AmB interactions; additivism (FICs, >0.50 to 1.0) was observed for 67, 73, and 53% of the isolates in studies of the FLC-AmB, ITC-AmB, and SCH-AmB interactions, respectively; indifference (FICs, >1.0 to ≤2.0) was observed for 26, 20, and 14% of the isolates in studies of the FLC-AmB, ITC-AmB, and SCH-AmB interactions, respectively. Antagonism (FIC >2.0) was not observed. When synergy was not achieved, there was still a decrease, although not as dramatic, in the MIC of one or both drugs when they were used in combination. To investigate the effects of FLC-AmB combination therapy in vivo, we established an experimental model of systemic cryptococcosis in BALB/c mice by intravenous injection of cells of C. neoformans 2337, a clinical isolate belonging to serotype D against which the combination of FLC and AmB yielded an additive interaction in vitro. Both survival and tissue burden studies showed that combination therapy was more effective than FLC alone and that combination therapy was at least as effective as AmB given as a single drug. On the other hand, when cells of C. neoformans 2337 were grown in FLC-containing medium, a pronounced increase in resistance to subsequent exposures to AmB was observed. In particular, killing experiments conducted with nonreplicating cells showed that preexposure to FLC abolished the fungicidal activity of the polyene. However, this apparent antagonism was not observed in vivo. Rather, when the two drugs were used sequentially for the treatment of systemic murine cryptococcosis, a reciprocal potentiation was often observed. Our study shows that (i) the combination of triazoles and AmB is significantly more active than either drug alone against C. neoformans in vitro and (ii) the concomitant or sequential use of FLC and AmB for the treatment of systemic murine cryptococcosis results in a positive interaction.

Candida tropicalis is less commonly isolated from clinical specimens than Candida albicans . Unlike C. albicans , which can be occasionally found as a commensal, C. tropicalis is almost always associated with the development of fungal infections. In addition, C. tropicalis has been reported to be resistant to fluconazole (FLC). To analyze the development of FLC resistance in C. tropicalis , an FLC-susceptible strain (ATCC 750) (MIC = 1.0 μg/ml) was cultured in liquid medium containing increasing FLC concentrations from 8.0 to 128 μg/ml. The strain developed variable degrees of FLC resistance which paralleled the concentrations of FLC used in the medium. The highest MICs of FLC were 16, 256, and 512 μg/ml for strains grown in medium with 8.0, 32, and 128 μg of FLC per ml, respectively. Development of resistance was rapid and could be observed already after a single subculture in azole-containing medium. The resistant strains were cross-resistant to itraconazole (MIC > 1.0 μg/ml) and terbinafine (MIC > 512 μg/ml) but not to amphotericin B. Isolates grown in FLC at concentrations of 8.0 and 32 μg/ml reverted to low MICs (1.0 μg/ml) after 12 and 11 passages in FLC-free medium, respectively. The MIC for one isolate grown in FLC (128 μg/ml) (128 R) reverted to 16 μg/ml but remained stable over 60 passages in FLC-free medium. Azole-resistant isolates revealed upregulation of two different multidrug efflux transporter genes: the major facilitators gene MDR1 and the ATP-binding cassette transporter CDR1 . The development of FLC resistance in vitro correlated well with the results obtained in an experimental model of disseminated candidiasis. While FLC given at 10 mg/kg of body weight/day was effective in reducing the fungal burden of mice infected with the parent strain, the same dosing regimen was ineffective in mice infected with strain 128 R. Finally, the acquisition of in vitro FLC resistance in strain 128 R was related to a loss of virulence. The results of our study elucidate important characteristics and potential mechanisms of FLC resistance in C. tropicalis .