Chromoblastomycosis is one of the most frequent infections caused by melanized fungi. It is a subcutaneous fungal infection, usually an occupational related disease, mainly affecting individuals in tropical and temperate regions. Although several species are etiologic agents, Fonsecaea pedrosoi and Cladophialophora carrionii are prevalent in the endemic areas. Chromoblastomycosis lesions are polymorphic and must be differentiated from those associated with many clinical conditions. Diagnosis is confirmed by the observation of muriform cells in tissue and the isolation and the identification of the causal agent in culture. Chromoblastomycosis still is a therapeutic challenge for clinicians due to the recalcitrant nature of the disease, especially in the severe clinical forms. There are three treatment modalities, i.e., physical treatment, chemotherapy and combination therapy but their success is related to the causative agent, the clinical form and severity of the chromoblastomycosis lesions. There is no treatment of choice for this neglected mycosis, but rather several treatment options. Most of the patients can be treated with itraconazole, terbinafine or a combination of both. It is also important to evaluate the patient's individual tolerance of the drugs and whether the antifungal will be provided for free or purchased, since antifungal therapy must be maintained in long-term regimens. In general, treatment should be guided according to clinical, mycological and histopathological criteria.

The in vitro susceptibilities of 66 molecularly identified strains of the Mucorales to eight antifungals (amphotericin B, terbinafine, itraconazole, posaconazole, voriconazole, caspofungin, micafungin, and 5-fluorocytosine) were tested. Molecular phylogeny was reconstructed based on the nuclear ribosomal large subunit to reveal taxon-specific susceptibility profiles. The impressive phylogenetic diversity of the Mucorales was reflected in susceptibilities differing at family, genus, and species levels. Amphotericin B was the most active drug, though somewhat less against Rhizopus and Cunninghamella species. Posaconazole was the second most effective antifungal agent but showed reduced activity in Mucor and Cunninghamella strains, while voriconazole lacked in vitro activity for most strains. Genera attributed to the Mucoraceae exhibited a wide range of MICs for posaconazole, itraconazole, and terbinafine and included resistant strains. Cunninghamella also comprised strains resistant to all azoles tested but was fully susceptible to terbinafine. In contrast, the Lichtheimiaceae completely lacked strains with reduced susceptibility for these antifungals. Syncephalastrum species exhibited susceptibility profiles similar to those of the Lichtheimiaceae. Mucor species were more resistant to azoles than Rhizopus species. Species-specific responses were obtained for terbinafine where only Rhizopus arrhizus and Mucor circinelloides were resistant. Complete or vast resistance was observed for 5-fluorocytosine, caspofungin, and micafungin. Intraspecific variability of in vitro susceptibility was found in all genera tested but was especially high in Mucor and Rhizopus for azoles and terbinafine. Accurate molecular identification of etiologic agents is compulsory to predict therapy outcome. For species of critical genera such as Mucor and Rhizopus, exhibiting high intraspecific variation, susceptibility testing before the onset of therapy is recommended.

The increase in fungal infections and the change in fungal epidemiology is caused by the extensive use of antifungal agents to treat fungal infections that are being diagnosed in severly immunocompromised hosts. In addition, opportunistic fungal infections resistant to antifungal drugs have become increasingly common, and the armamentarium for treatment remains limited. A possible approach to overcoming these problems is to combine antifungal drugs, especially if the mechanisms of action are different. The in vitro test is the first step to evaluate possible antifungal combinations. In this chapter, the three most frequently used metholodologies are described: checkerboard, E-test, and time-kill curves. The description of each technique and intrepretaion of the results are addressed in detail.

To develop new approaches for the treatment of invasive infections caused byInvasive aspergillosis causes approximately 30% of invasive fungal infections in patients treated for cancer (11). Until 1990 only one drug was available for the treatment of invasive Aspergillus disease, amphotericin B, which must be given intravenously and which has a number of serious toxicities. In 1990 itraconazole (ITZ) capsules became available and Aspergillus species were included in the spectrum of activity of the drug, although the drug was mainly used in the prophylactic setting due to poor bioavailability (11). Ten years later an intravenous formulation of ITZ became available and allowed the drug to be used for the empirical or preemptive treatment of high-risk patients. With the registration of voriconazole and caspofungin, the arsenal of available drugs has increased further. However, despite antifungal therapy, the rate of mortality in patients with invasive aspergillosis remains very high, and clearly, new therapeutic approaches are needed. Combination therapy is one approach that can be used to improve the efficacy of antimicrobial therapy for difficult-to-treat infections, such as human immunodeficiency virus and mycobacterial infections. By analogy, the combination of ITZ with other compounds could represent a possible approach for the treatment of patients with invasive aspergillosis or patients infected with strains with reduced susceptibilities to antifungal agents. Resistance to antifungal azoles has been studied in yeasts and molds, especially Aspergillus. Resistance mechanisms include changes in the cellular azole content (an altered uptake or efflux mechanism), mutations in sterol desaturation during ergosterol biosynthesis, and mutations in or elevated levels of 14␣-demethylase (12, 42). The recent discovery of drug effluxmediated resistance mechanisms in yeasts and Aspergillus opens new therapeutic concepts. It has been recognized that Candida albicans and Aspergillus nidulans express multidrug efflux transporter (MET) genes belonging to different classes, i.e., the ATP-binding cassette (ABC) transporters and the major facilitators (13,48). The expression of these genes and their targeted deletion determine the level of azole resistance.In this study we investigated the in vitro interactions between ITZ and different nonantimicrobial membrane-active compounds against clinical ITZ-resistant (ITZ-R) and ITZsusceptible (ITZ-S) strains using four different drug interaction models. MATERIALS AND METHODSStrains. Fourteen clinical isolates of Aspergillus fumigatus were tested. These included seven ITZ-S isolates (isolates V09-22, V09-23, AZN5161, AZN7820, AZN8248, AZN9339, and AZN9362) and seven ITZ-R isolates (isolates V09-18, V09-19, AZN5241, AZN5242, AZN7720, AZN7722, and AZG7). The strains numbered AZN and V09 were obtained from the private collection of the Department of Medical Microbiology, University Medical Center Nijmegen, and strain AZG7 was obtained from the University Hospital Groningen, Groningen, The ...