Mediterranean Journal of Hematology and Infectious Diseases

Despite availability of many antifungal agents, antifungal clinical resistance occurs, perhaps as a consequence of an infecting organism found to be resistant in vitro to one or more antifungals tested. From what derives the important current role of the in vitro antifungal susceptibility testing (AFST), that is to determine which agents are like to be scarcely effective for a given infection. Thus, AFST results, if timely generated by the clinical microbiology laboratory and communicated to clinicians, can aid them in the therapeutic decision making, especially for difficult-to-treat invasive candidiasis and aspergillosis. Although recently refined AFST methods are commercially available for allowing a close antifungal resistance surveillance in many clinical setting, novel assays such as flow cytometry or MALDI-TOF mass spectrometry are upcoming tools for AFST. Based on short-time antifungal drug exposure of fungal isolates, these assays could provide a reliable means for quicker and sensitive assessment of AFST. Introduction. Although several factors are key determinants of antifungal clinical resistance, which is referred to as the persistence or progression of a fungal infection despite the administration of appropriate antifungal therapy, there is a general consensus that clinical outcomes are better when treatments are started early. Almost all the classes of systemically active antifungal agents available to date, such as polyenes (i.e., amphotericin B), azoles, flucytosine, and the newest echinocandins contribute to improve the management of invasive fungal infections (IFIs). Nevertheless, the rate of antifungal failures is high, and the emergence of resistant fungal strains is a growing concern, particularly for strains capable of exhibiting resistance to commonly prescribed antifungal drugs. Eighteen (11.1%) of 162 fluconazole-resistant bloodstream isolates of Candida glabrata collected during two large surveillance programs were found to be cross-resistant to one or more of the echinocandins. Likewise, patients with chronic pulmonary Aspergillus infection who receive prolonged (tri)azole therapy are at risk of resistant aspergillosis, with an evolving Mediterr J Hematol Infect Dis 2014; 6; Open Journal System spectrum of resistance owing to the emergence of noncyp51A-mediated mechanisms, as well as are at risk azole-naïve patients due to the presence of resistant TR/L98H strains (i.e., carrying a substitution at codon 98 in the cyp51A gene in combination with a 34 basepair tandem repeat in the gene promoter) in the environment. Thus, while two-thirds of surveyed Dutch patients with azole-resistant Aspergillus disease had not history of previous azole exposure (with all A. fumigatus isolates from patients with invasive aspergillosis harboring the TR/L98H mutation), recent epidemiological data show that this resistance mechanism, first emerged in the Netherlands, is expanding not only in European countries but also in China, Iran, and India. Antifungal Susceptibility Testing to Aid the Management of IFI Patients. The primary utility of antifungal susceptibility testing (AFST) arises from the concept that susceptibility (or resistance) to an antifungal agent selected for the therapy would allow some prediction about the impact that administration of the agent tested in vitro has on the clinical outcome of infection caused by the treated organism. Therefore, clinical microbiologists are currently engaged to determine the growth of fungi under different drug concentrations so as to yield the minimum inhibitory concentration (MIC) for a specific infecting isolate, that is an in vitro measure of susceptibility (expressed as growth inhibition) which helps to predict the therapeutic efficacy. Thus, it is important that MIC results are timely communicated to physicians to guide them in the therapeutic decision making, in the same way that antibacterial testing aids in the clinical guidance of bacterial infections. As attested by several studies evaluating the role of “real-time” AFST in managing patients with invasive Candida infections, physicians frequently (and appropriately) adjust the therapy on the basis of MIC results, although a clearly defined association between the timely receipt of antifungal therapy and poor outcome after Candida bloodstream infection due to a resistant isolate is lacking to date. Indeed, Collins et al. reported that the susceptibility testing (especially when done in-house) of C. glabrata isolates may facilitate quicker interventions (i.e., de-escalation of therapy from an expensive echinocandin to fluconazole) for patients with documented C. glabrata fungemia, thereby resulting in lower overall treatment costs. Likewise, Grim et al. found that receipt of appropriate early antifungal therapy (i.e., administered within 72 h of a positive culture being drawn) was associated with a significant (P = 0.047) survival benefit for patients who were effectively treated for ≥24 h, and their results were supported by the inclusion of routine AFST to optimally assess the adequateness of therapy. Unlike Candida infection, there is only a limited number of reported Aspergillus infection cases that could elucidate the clinical impact of azole resistance on the patient’s outcome, and this situation has hindered the wide application of in vitro AFST of Aspergillus species. However, in an attempt to establish clinically derived breakpoints for Aspergilli that would help physicians to interpret the MIC values as produced from the clinical microbiology laboratory, a pragmatic (and not formal) approach was followed by Verweij et al. Thus, taking MIC distribution, pharmacokinetic/pharmacodynamic parameters of antifungal azoles, in vivo experimental correlation between cyp51A point mutations and failure, and clinical experience into account, interpretive breakpoints were proposed, that is MICs >2 μg/ml for itraconazole and voriconazole and >0.5 μg/ml for posaconazole. These breakpoints were able to discriminate between wild-type (that refers to isolates without mutational or acquired mechanisms of resistance) and non-wild-type (that refers to isolates with mutational or acquired mechanisms of resistance) MIC distributions for itraconazole and voriconazole among 325 consecutive clinical A. fumigatus isolates from the Nijmegen fungus culture collection. Based on these findings, a 4-well azole-agar dilution (4D) plate (i.e., 3 wells were each containing one of azoles: itraconazole 4 μg/ml, voriconazole 1 μg/ml, or posaconazole 0.5 μg/ml; and the fourth azole-free well served as control growth) was developed as a screening test for identifying potentially resistant A. fumigatus isolates. In parallel, Pfaller et al. used a collection of 637 geographically diverse, clinical isolates of A. fumigatus tested against itraconazole, posaconazole, and voriconazole, to assess the wild-type MIC distribution and epidemiological cutoff values (ECVs), that is MIC threshold values for differentiating wildtype isolates from non-wild-type isolates, for A. fumigatus and the mold-active triazoles. By contrast, due to scarce (and less frequent than for azoles) tendency to carrying out AFST for Aspergillus isolates, perhaps as a result of technical difficulties and suboptimal reproducibility of the methods employed, echinocandin resistance in Aspergillus species is much less known. Although the caspofungin is recommended as a second line treatment choice for invasive aspergillosis, and often administered in combination with amphotericin B, however, breakthrough infections (though sporadic) have been reported in patients under caspofungin therapy, and they involved A. fumigatus isolates, with elevated minimum effective concentrations (MECs) to caspofungin. The MEC endpoint, defined as Mediterr J Hematol Infect Dis 2014; 6: Open Journal System the lowest drug concentration that leads to the growth of small, rounded, compact hyphal forms as compared to the hyphal growth seen in the growth control well, was suggested for testing antifungal susceptibility of Aspergilli to echinocandins, rather than the MIC; nonetheless, MEC remains technically difficult to