The lack of robust rapid diagnostic tests, safe and effective drugs, and preventative vaccines hampers treatment of patients with invasive fungal infections.

SUMMARY: In the past decade, the frequency of diagnosed fungal infections has risen sharply due to several factors, including the increase in the number of immunosuppressed patients resulting from the AIDS epidemic and treatments during and after organ and bone marrow transplants. Linked with the increase in fungal infections is a recent increase in the frequency with which these infections are recalcitrant to standard antifungal therapy. This review summarizes the factors that contribute to antifungal drug resistance on three levels: (i) clinical factors that result in the inability to successfully treat refractory disease; (ii) cellular factors associated with a resistant fungal strain; and (iii) molecular factors that are ultimately responsible for the resistance phenotype in the cell. Many of the clinical factors that contribute to resistance are associated with the immune status of the patient, with the pharmacology of the drugs, or with the degree or type of fungal infection present. At a cellular level, antifungal drug resistance can be the result of replacement of a susceptible strain with a more resistant strain or species or the alteration of an endogenous strain (by mutation or gene expression) to a resistant phenotype. The molecular mechanisms of resistance that have been identified to date in Candida albicans include overexpression of two types of efflux pumps, overexpression or mutation of the target enzyme, and alteration of other enzymes in the same biosynthetic pathway as the target enzyme. Since the study of antifungal drug resistance is relatively new, other factors that may also contribute to resistance are discussed.

Resistance to antifungal drugs, specifically azoles such as fluconazole, in the opportunistic yeast Candida albicans has become an increasing problem in human immunodeficiency virus (HIV)-infected individuals. The molecular mechanisms responsible for this resistance have only recently become apparent and can include alterations in the target enzyme of the azole drugs (lanosterol 14alpha demethylase [14DM]), or in various efflux pumps from both the ABC transporter and major facilitator gene families. To determine which of these possible mechanisms was associated with the development of drug resistance in a particular case, mRNA levels have been studied in a series of 17 clinical isolates taken from a single HIV-infected patient over 2 years, during which time the levels of fluconazole resistance of the strain increased over 200-fold. Using Northern blot analysis of steady-state levels of total RNA from these isolates, we observed increased mRNA levels of ERG16 (the 14DM-encoding gene), CDR1 (an ABC transporter), and MDR1 (a major facilitator) in this series. The timing of the increase in mRNA levels of each of these genes correlated with increases in fluconazole resistance of the isolates. Increased mRNA levels were not observed for three other ABC transporters, two other genes in the ergosterol biosynthetic pathway, or the NADPH-cytochrome P-450 oxidoreductase gene that transfers electrons from NADPH to 14DM. Increases in mRNA levels of ERG16 and CDR1 correlated with increased cross-resistance to ketoconazole and itraconazole but not to amphotericin B. A compilation of the genetic alterations identified in this series suggests that resistance develops gradually and is the sum of several different changes, all of which contribute to the final resistant phenotype.

Resistance to azole antifungals continues to be a significant problem in the common fungal pathogen Candida albicans . Many of the molecular mechanisms of resistance have been defined with matched sets of susceptible and resistant clinical isolates from the same strain. Mechanisms that have been identified include alterations in the gene encoding the target enzyme ERG11 or overexpression of efflux pump genes including CDR1 , CDR2 , and MDR1 . In the present study, a collection of unmatched clinical isolates of C. albicans was analyzed for the known molecular mechanisms of resistance by standard methods. The collection was assembled so that approximately half of the isolates were resistant to azole drugs. Extensive cross-resistance was observed for fluconazole, clotrimazole, itraconazole, and ketoconazole. Northern blotting analyses indicated that overexpression of CDR1 and CDR2 correlates with resistance, suggesting that the two genes may be coregulated. MDR1 overexpression was observed infrequently in some resistant isolates. Overexpression of FLU1 , an efflux pump gene related to MDR1 , did not correlate with resistance, nor did overexpression of ERG11 . Limited analysis of the ERG11 gene sequence identified several point mutations in resistant isolates; these mutations have been described previously. Two of the most common point mutations in ERG11 associated with resistance, D116E and E266D, were tested by restriction fragment length polymorphism analysis of the isolates from this collection. The results indicated that the two mutations occur frequently in different isolates of C. albicans and are not reliably associated with resistance. These analyses emphasize the diversity of mechanisms that result in a phenotype of azole resistance. They suggest that the resistance mechanisms identified in matched sets of susceptible and resistant isolates are not sufficient to explain resistance in a collection of unmatched clinical isolates and that additional mechanisms have yet to be discovered.

The prophylactic use of fluconazole is common in blood and marrow transplant (BMT) recipients. To evaluate how fluconazole has influenced the develop

Candida albicans is both a member of the healthy human microbiome and a major pathogen in immunocompromised individuals. Infections are typically treated with azole inhibitors of ergosterol biosynthesis often leading to drug resistance. Studies in clinical isolates have implicated multiple mechanisms in resistance, but have focused on large-scale aberrations or candidate genes, and do not comprehensively chart the genetic basis of adaptation. Here, we leveraged next-generation sequencing to analyze 43 isolates from 11 oral candidiasis patients. We detected newly selected mutations, including single-nucleotide polymorphisms (SNPs), copy-number variations and loss-of-heterozygosity (LOH) events. LOH events were commonly associated with acquired resistance, and SNPs in 240 genes may be related to host adaptation. Conversely, most aneuploidies were transient and did not correlate with drug resistance. Our analysis also shows that isolates also varied in adherence, filamentation, and virulence. Our work reveals new molecular mechanisms underlying the evolution of drug resistance and host adaptation.