In vitro activity of terbinafine and itraconazole against Aspergillus species isolated from otomycosis

The minimum inhibitory concentrations (MIC, microg ml-1) of itraconazole and terbinafine against overall 34 Aspergillus isolates from the external ear canals with otomycosis have been determined with M38-P microdilution method suggested by National Committee for Clinical Laboratory Standards (NCCLS). MIC intervals in 48 h determined by taking MIC-2 value of itraconazole (the lowest drug concentration causing 50% inhibition of visible fungal growth) and MIC-0 value of terbinafine (the lowest drug concentration causing 100% inhibition of visible fungal growth) as a basis have been found as follows: 0.125-1 and 0.06-0.5 microg ml-1 for A. niger (22 strains), 0.06-0.25 and 0.06-0.125 microg ml-1 for A. flavus (10 strains), 0.125 and 0.125-0.5 microg ml-1 for A. terreus (two strains). It has been observed that both of the antifungal agents showed an in vitro activity against all Aspergillus species tested.     

In-vitro activity of the synthetic protegrin IB-367 alone and in combination with antifungal agents against clinical isolates of Candida spp

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 microg/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.     

Kinetic study of antifungal activity of amphotericin B, 5-fluorocytosine and ketoconazole against clinical yeast isolates using liquid-phase turbidimetry

The fungal growth of clinical yeast isolates and of VW32 clone of Candida albicans were measured in vitro using a liquid-phase turbidimetric system (Bioscreen from Labsystems, France) in defined conditions. Cultures were performed in Shadomy's liquid medium and the fungal growth automatically evaluated every 10 minutes for 24 hours using various concentrations of drugs. The system made it possible to test 200 culture samples in one experiment. Yeast sensitivity to drugs was also measured by using our routine semi-automatic turbidimetric system. We observed that kinetic patterns of activity of each antifungal agent were typical. The in vitro tests showed that of 927 clinical yeast isolates 99.2% were sensitive to amphotericin B, 94.4% to 5-fluorocytosine and 69.7% to ketoconazole.     

Antifungal therapies in murine infections by Candida kefyr

Candida kefyr is an emerging pathogen able to cause disseminated infection, especially in immunocompromised patients. Although guidelines for the treatment of invasive candidiasis have been published, no specific recommendations against C. kefyr are available. We determine the in vitro killing activity of amphotericin B (AMB), fluconazole (FLC) and caspofungin (CFG) as well as their efficacy in a murine model of systemic infection by two C. kefyr strains. Time‐kill curves of AMB, FLC and CFG were determined in final volumes of 10 ml containing the assayed drugs ranged from 0.03 to 32 μg ml^?1 at different time points and efficacy of the drugs was evaluated in a systemic model of candidiasis, conducted in immunosuppressed mice, through survival, (1→3)‐β‐D‐glucan levels in serum and fungal load in kidneys. AMB and CFG showed fungicidal and FLC fungistatic activity against both isolates. The three drugs were able to reduce fungal burden in kidneys and (1→3)‐β‐D‐glucan concentration in serum of infected mice, with CFG showing the highest efficacy, followed by FLC. In conclusion, CFG showed efficacy over AMB and FLC against the systemic candidiasis by C. kefyr. The established epidemiological cut‐off for anidulafungin seems the best indicator of outcome for echinocandins.     

Activity and post antifungal effect of chlorpromazine and trifluopherazine against Aspergillus, Scedosporium and zygomycetes

The phenothiazine compounds chlorpromazine and trifluopherazine are antipsychotic agents that exhibit antimicrobial activity against bacteria, some protozoa and yeasts. Data of activity against filamentous fungi are lacking. The in vitro activity and postantifungal effect (PAFE) of chlorpromazine and trifluopherazine was determined against Aspergillus species, zygomycetes and Scedosporium species. In vitro susceptibility testing was performed with CLSI M38A and the PAFE was determined with previously established methods. Both drugs inhibited the growth of all fungi tested at concentrations of 16 to 64 microg ml(-1). For Aspergillus species the mean PAFE was 3.7 and 4.7 h; for zygomycetes, 3.1 and 3.4 h; for Scedosporium, 4.3 and 5.3 h for chlorpromazine and trifluoroperazine respectively. These are the first drugs shown to induce PAFE against Scedosporium. We show that phenothiazine compounds have in vitro antifungal activity and exhibit PAFE against a broad range of filamentous fungal pathogens. Although the exact mechanism of action is unknown, further studies are needed to explore the clinical usefulness of phenothiazine compounds.     

Activity of terbinafine against serious fungal pathogens

Although primarily indicated for dermatophyte infections, the allylamine terbinafine is active in vitro against a broad spectrum of filamentous and dimorphic fungi, in most cases with a primary fungicidal action. Using the standard NCCLS M27-A assay, recent studies confirmed the high activity of terbinafine against dematiaceous fungi and other medically important moulds such as Aspergillus and Penicillium marneffei. Terbinafine displayed a geometric mean MIC of 1.4 micrograms/ml against Candida albicans (n = 259) and has significant in vitro activity against other species of Candida, Cryptococcus, Trichosporon and Blastoschizomyces. As an approach to treatment of refractory infections, interactions of terbinafine with azoles and other agents are being investigated. Terbinafine was synergistic with azoles (and in some cases amphotericin B) against Candida species, Trichosporon beigelii, Aspergillus species, Pseudallescheria boydii and Scopulariopsis brevicaulis, some of which were unresponsive to any drug used singly. Terbinafine combined with fluconazole showed potent synergy against fluconazole- and multidrug-resistant C. albicans isolates. In conclusion, recent in vitro data suggest that terbinafine, either alone or in combination with other antifungal drugs, has potential in the therapy of a range of more severe fungal infections, in addition to its current widespread use against dermatomycoses.     

A head-on comparison of the in vitro antifungal activity of conventional and lipid-based amphotericin B: a multicenter study

A comparative study of conventional amphotericin B, Abelcet and AmBisome was performed using a microdilution format of the NCCLS M27-A methodology for susceptibility testing against 300 fungal isolates (152 yeasts, 148 filamentous fungi) in both RPMI-1640 and antibiotic medium #3 (AB3). The clinical isolates included Candida albicans (n=54), Candida glabrata (n=25), Candida parapsilosis (n=23), Candida krusei (n=19), Candida lusitaniae (n=14), Cryptococcus neoformans (n=5), Candida tropicalis (n=12), Aspergillus flavus (n=34), Aspergillus fumigatus (n=46) and 68 other filamentous fungi encompassing 22 different genera. The minimal inhibitory concentrations (MIC) for all drugs were defined as the lowest concentrations in which there was no visible growth. MICs were determined after 48 h for yeasts and 72 h for filamentous fungi. The mean MICs +/- standard error (microg/ml) for yeasts and filamentous fungi, respectively, were: Abelcet, 0.51+/-0.21, 4.34+/-0.61; AmBisome, 1.28+/-0.24, 5.68+/-0.57; amphotericin B, 0.29+/-0.11, 1.12+/-0.19, respectively. Overall, against both yeasts and filamentous fungi Abelcet proved to have more potent antifungal activity than AmBisome. Using AB3 as opposed to RPMI-1640 generally produced lower MIC values but did not have any effect on the order of relative activity with all of the antifungal agents tested. In conclusion, our data shows that Abelcet is more active than AmBisome against pathogenic yeast and filamentous fungi when assayed in AB3 in vitro. Comparison of the activities of these antifungals in experimental animal models is necessary to determine whether these in vitro findings are correlated with in vivo efficacy.     

Combined antifungal therapy against systemic murine infections by rare Cryptococcus species

Cryptococcus albidus and Cryptococcus laurentii are uncommon species of this genus that in recent decades have increasingly caused opportunistic infections in humans, mainly in immunocompromised patients; the best therapy for such infection being unknown. Using a murine model of systemic infection by these fungi, we have evaluated the efficacy of amphotericin B (AMB) at 0.8 mg/kg, administered intravenously, fluconazole (FLC) or voriconazole (VRC), both administered orally, at 25 mg/kg and the combination of AMB plus VRC against three C. albidus and two C. laurentii strains. All the treatments significantly reduced the fungal burden in all the organs studied. The combination showed a synergistic effect in the reduction in fungal load, working better than both monotherapies. The histopathological study confirmed the efficacy of the treatments.     

In vitro activity (MIC and MFC) of voriconazole, amphotericin B, and itraconazole against 192 filamentous fungi: the GISIA-2 study

We evaluated the in vitro activity of voriconazole, amphotericin B, and itraconazole against 192 clinical mould isolates recovered in twenty Italian microbiology laboratories. The vast majority of isolates belonged to the genus Aspergillus (94.2%) with A. fumigatus (58.3%) being the most frequently isolated species. Antifungal susceptibility testing was performed using the broth microdilution method defined by the CLSI M38-A standard, and results were compared to those obtained with Sensititre panels. Aspergillus flavus ATCC 204304 was employed as reference strain and results were within all expected ranges. Voriconazole's activity against the 192 mould isolates was comparable to that of amphotericin B and itraconazole: voriconazole MIC90 (CLSI 1 microg/ml, Sensititre 1 microg/ml), itraconazole MIC90 (CLSI 0.5 microg/ml, Sensititre 0.5 microg/ml), amphotericin B MIC90 (CLSI 1 microg/ml, Sensititre 1 microg/ml). In conclusion, these in vitro data highlight voriconazole's broad spectrum activity against filamentous fungi and support its use as a first line agent for the treatment of fungal diseases.     

Evaluation of the inhibitory effect of amphotericin B on the apical growth of F. solani using the BioCell-Tracer system

The BioCell-Tracer (BCT) system is an automatic microscopic method used for measuring the growth rate of a single fungal hyphae, which has not yet been applied to study Fusarium spp. Considering the large resistance of Fusarium species to the available chemotherapy and that hyphae is the morphological fungal form most often seen in vivo, in this work, Amphotericin B MIC and MFC values for a Fusarium solani strain were obtained by the conventional assay method testing conidia and also by the BCT monitoring system. Both MIC and MFC values of AMB against F. solani determined by broth dilution method resulted in 4.0 microg ml(-1). By the BCT system, their values were 1.0 microg ml(-1), with an inhibition rate of 99.5% (Exp-GR) and 100.0% (Post-GR), showing that when testing hyphae directly, MIC and MFC were determined at two lower dilutions than the MIC and MFC values obtained with conidia. Using the BCT system, 4.0, 2.0 and 1.0 microg ml(-1) of AMB concentrations inhibited hyphae growth in 50 min whereas 0.5 microg ml(-1) of AMB needed 100 min to start hyphae growth inhibition. These findings lead us to conclude that antifungal susceptibility varies between conidia and hyphae. For this strain of Fusarium solani, hyphae were more susceptible to AMB than conidia.     

In vitro activity of the protegrin IB‐367 alone and in combination compared with conventional antifungal agents against dermatophytes

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.     

Evaluation of antifungal combination against Cryptococcus spp.

The second cause of death among systemic mycoses, cryptococcosis treatment represents a challenge since that 5‐flucytosine is not currently available in Brazil. Looking for alternatives, this study evaluated antifungal agents, alone and combined, correlating susceptibility to genotypes. Eighty Cryptococcus clinical isolates were genotyped by URA5 gene restriction fragment length polymorphism. Antifungal susceptibility was assessed following CLSI‐M27A3 for amphotericin (AMB), 5‐flucytosine (5FC), fluconazole (FCZ), voriconazole (VRZ), itraconazole (ITZ) and terbinafine (TRB). Drug interaction chequerboard assay evaluated: AMB + 5FC, AMB + FCZ, AMB + TRB and FCZ + TRB. Molecular typing divided isolates into 14 C. deuterogattii (VGII) and C. neoformans isolates were found to belong to genotype VNI (n = 62) and VNII (n = 4). C. neoformans VNII was significantly less susceptible than VNI (P = 0.0407) to AMB; C. deuterogattii was significantly less susceptible than VNI and VNII to VRZ (P < 0.0001). C. deuterogattii was less susceptible than C. neoformans VNI for FCZ (P = 0.0170), ITZ (P < 0.0001) and TRB (P = 0.0090). The combination FCZ + TRB showed 95.16% of synergistic effect against C. neoformans genotype VNI isolates and all combinations showed 100% of synergism against genotype VNII isolates, suggesting the relevance of cryptococcal genotyping as it is widely known that the various genotypes (now species) have significant impact in antifungal susceptibilities and clinical outcome. In difficult‐to‐treat cryptococcosis, terbinafine and different antifungal combinations might be alternatives to 5FC.     

Study on the effect of neem (Azadirachta indica A. juss) leaf extract on the growth of Aspergillus parasiticus and production of aflatoxin by it at different incubation times

Aflatoxins are secondary metabolites that are produced by toxigenic strains of some Aspergillus species on foods. Neem plant is a known inhibitor of aflatoxin production. We studied the effects of different concentrations of aqueous neem leaf extract on fungal growth and aflatoxin production by Aspergillus parasiticus (NRRL 2999) at different incubation times. The toxigenic fungus was cultured on sucrose low salts medium in the presence of various concentrations of extracts (0.2, 0.8, 3.12, 12.5 and 50% v/v). After shaking incubation of cultures for 2, 4, 6, 8, 10 and 12 days at 28 degrees C, the fungal mycelia was collected and processed for determination of dry weight. Mycelia and culture media were assayed by TLC method to detect aflatoxin B(1) (AFB(1)). The extracts did not have any obvious effect on fungal growth. AFB(1) production in the control samples increased to the maximum level on the 8th day. The inhibition of aflatoxin synthesis by plant extracts was found to be time- and dose-dependent. The maximum inhibitory effect was 80-90% in the presence of 50% concentration that when compared with control samples was significant (P < 0.05). AFB(1) secretion/production ratio in all of control and treated samples, other than 2nd day, approximately stayed and neem had no effect on it.     

Voriconazole - applications and perspectives

Voriconazole (Vfend) is a new broadspectrum antifungal agent belonging to the group of triazole drugs. In vitro and in vivo efficacy was demonstrated against a large variety of yeasts with excellent activity against all Candida species but as well against Cryptococcus neoformans. Furthermore, voriconazole has shown excellent activity against many moulds in particular against Aspergillus species, but endemic fungi such as Histoplasma capsulatum are in the spectrum as well. Clinical efficacy was demonstrated in several large phase II/III studies in diseases such as oral and oesophageal candidosis, acute invasive aspergillosis or chronic invasive aspergillosis. New adverse events such as visual disturbancies has been described together with the use of voriconazole, but the majority of adverse events are similar to other triazole drugs and in particular not life-threatening. With the introduction of voriconazole a great progress in the therapy of invasive fungal infections was achieved. In the therapy of invasive aspergillosis, voriconazole is significantly more effective compared to amphotericin B desoxycholate.     

Antifungal susceptibilities of Cryptococcus neoformans cerebrospinal fluid isolates from AIDS patients in Kenya

Poor susceptibility of Cryptococcus neoformans to fluconazole (FLC) is a matter of concern among clinicians in Africa. The emergence of resistance to FLC was recently reported in Kenya, but it is not known whether it is widespread. Thus, there is need for more antifungal drug susceptibility studies in Kenya. The aim of this study was to measure the in vitro antifungal drug susceptibilities of incident C. neoformans isolates from acquired immunodeficiency syndrome patients in Kenya. Antifungal susceptibility testing was performed in 67 C. neoformans isolates by broth microdilution method as outlined in the Clinical and Laboratory Standards Institute document M27-A3 using FLC, amphotericin B (AMB), voriconazole (VOR), ravuconazole (RAV) and flucytosine (5-FC). Isolates were grown on l-canavanine glycine bromothymol blue medium for serotype identification. Six per cent of the isolates were identified as C. neoformans var. gattii serotype B or C and 94% as C. neoformans var. neoformans. All isolates tested were susceptible to AMB, VOR and RAV (100%), and high susceptibilities were seen to FLC (97%), and 5-FC (90%). Only 3% and 10% of the isolates' susceptibility to FLC and 5-FC, respectively, was dose-dependent or intermediate. These results demonstrate high susceptibilities of incident C. neoformans isolates to FLC and AMB, antifungals used for treatment of cryptococcal meningitis in Kenya.     

Clinicopathological and mycological spectrum of allergic fungal sinusitis in South India

In the present study, we describe characteristic clinicopathological and radiological features as well as fungal culture results in a series of 24 patients with allergic fungal sinusitis (AFS). Nasal obstruction and discharge with nasal polyposis was the commonest (95.8%) clinical presentation. Allergic mucin was uniformly present in all patients. Aspergillus species were the commonest fungal isolates (95.8%). One case of mixed Aspergillus and Curvularia sinusitis as well as one case of Drechslera sinusitis were also identified. Typical computerized tomography scan features of hyperdense areas interspersed with soft tissue densities in the affected sinuses were seen in all patients. Application of appropriate diagnostic criteria is essential to establish the diagnosis of AFS and distinguish it from invasive fungal sinus infections.     

Molecular detection and identification of Aspergillus spp. from clinical samples using real-time PCR

The definite and rapid diagnosis of invasive aspergillosis is necessary because of the high mortality caused. The objective of this study was to evaluate a real-time PCR assay to detect Aspergillus spp. in clinical samples, based on the Light Cycler technology. Specificity was assessed by using DNA extracted from pathogenic and non-pathogenic bacteria/fungi from Spanish Collection including: two Aspergillus flavus , four Aspergillus fumigatus , two Aspergillus nidulans , two Aspergillus niger and two Aspergillus terreus isolates. The analytical sensitivity was evaluated with different inocula (10¹–10⁵ conidia ml⁻¹), and serially diluted DNA of A. fumigatus. To assess clinical applicability, samples from patients at risk were analysed. Species identification was determined by analysing the melting curves. Reactions using genomic DNA from other species of different genera than Aspergillus were negative (specificity: 100%). Analytical sensitivity was 60 fg using DNA and 5–20 conidia using conidial suspensions. The linear range was from 60 to 6 × 10⁷ fg. The Tm ranged from 67.34 to 70.7 °C for the different Aspergillus spp. studied. Nine hundred and forty-eight consecutive blood samples from 127 patients were processed. In total, 10 (1%) of 948 samples from blood samples were PCR-positive. The real-time PCR assay provides a high sensitivity and specificity for detection of fungal DNA and rapidly identifies most of clinically relevant Aspergillus species.     

Itraconazole added to a lipid formulation of amphotericin B does not improve outcome of primary treatment of invasive aspergillosis

BACKGROUND: Invasive aspergillosis (IA) is associated with poor outcome in patients with hematologic malignancy treated with amphotericin B (AMB)-based therapy. Itraconazole (ITC), a triazole with activity against Aspergillus, has been used in combination with AMB or lipid formulations of AMB (LipoAMB) in the treatment of IA, although the efficacy of this strategy is uncertain. METHODS: To determine whether the addition of ITC to LipoAMB improves outcome of IA, the authors retrospectively studied 179 consecutive patients with hematologic malignancies and definite or probable IA who received primary antifungal therapy with either LipoAMB (n = 146), or lipoAMB plus ITC (n = 33) between June 1993 and June 2003. In view of the erratic absorption of ITC tablets, only patients who received either intravenous or liquid ITC were analyzed. Patients who received < 1 week of treatment were excluded. RESULTS: Evaluable patients in both groups (LipoAMB: n =101; ITC and LipoAMB: n = 11) had comparable distribution of risk factors of poor outcome such as neutropenia at onset of IA, persistent neutropenia, systemic steroids, previous antifungal prophylaxis, admission to the intensive care unit, disseminated IA, previous bone marrow transplant, and IA due to infection by a non-fumigatus Aspergillus species. Response to primary antifungal therapy was equally poor in both groups (LipoAMB group: 10%; ITC and LipoAMB group: 0%; P = not significant). CONCLUSIONS: In the authors' 10-year study of patients with hematologic malignancy and IA, the response rate to LipoAMB given as primary therapy was very poor. In a comparable group of patients, the addition of ITC did not result in a therapeutic benefit.     

Retention of amphotericin-B therapeutic efficacy at half doses by synergistic activation of phagocytes.

Amphotericin B (AMB) is a mainstay in the treatment of serious systemic fungal infections, such as those occurring prevalently in immuno-compromised patients treated with immunosuppressive agents or affected by Acquired Immunodeficiency Syndrome (AIDS). However AMB is an extremely toxic agent whose therapeutical utilization is often accompanied by acute side effects and chronic impairment of renal function. It is here reported that the preactivation of polymorphonucleated cells (PMN) in vivo, by a new immunomodulatory agent (PCF 39:N alpha-5[1,6,dihydro-(6-oxo-9 purinyl) pentoxycarbonyl]-L-Arginine) allows marked reduction of the AMB doses with full retention of therapeutic efficacy. This was observed in an experimental fungal infection induced in mice by intravenous inoculation of Candida albicans.     

In vitro activities of polyene and imidazole antifungal agents against unusual opportunistic fungal pathogens

Guidelines for the treatment of infections caused by unusual opportunistic fungi are limited and available in vitro data are scanty. In vitro susceptibility tests, employing an agar dilution procedure, were performed with amphotericin B (AMB), natamycin (NTC), itraconazole (ICZ), and ketoconazole (KTZ). Two media were used: Kimmig's agar (KA) and Yeast Morphology Agar (YMA). Fungi tested included isolates (n) of Acremonium spp. (10), Cunninghamella spp. (6), Fusarium spp. (18), Pseudallescheria boydii (14), and Trichosporon beigelii (5). All Acremonium and Cunninghamella isolates were susceptible to NTC (MIC less than or equal to 4 micrograms/ml) but many appeared to be resistant to AMB, (MIC greater than or equal to 32 micrograms/ml), KTZ and ICZ (MIC greater than or equal to 128 micrograms/ml). Most isolates of Fusarium spp. were susceptible to both AMB and NTC (MIC90 = 4 micrograms/ml); one isolate was cross-resistant to both polyenes (MIC greater than 32 micrograms/ml). Only two of 18 Fusarium isolates appeared susceptible to the imidazoles (MIC less than or equal to 4 micrograms/ml); the remaining isolates exhibited high MICs (greater than or equal to 64 micrograms/ml). All 14 isolates of P. boydii were susceptible to NTC (MIC less than or equal to 4 micrograms/ml) but four appeared to be resistant to AMB (MIC greater than or equal to 32 micrograms/ml). Most isolates of P. boydii were susceptible to both KTZ (MIC less than or equal to 4 micrograms/ml) and ICZ (MIC less than or equal to 16 micrograms/ml) but two isolates appeared to be resistant (MIC greater than or equal to 16 micrograms/ml).(ABSTRACT TRUNCATED AT 250 WORDS)