Jacques Bille

Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters.

Azole antifungal agents, and especially fluconazole, have been used widely to treat oropharyngeal candidiasis in patients with AIDS. An increasing number of cases of clinical resistance against fluconazole, often correlating with in vitro resistance, have been reported. To investigate the mechanisms of resistance toward azole antifungal agents at the molecular level in clinical C. albicans isolates, we focused on resistance mechanisms related to the cellular target of azoles, i.e., cytochrome P450(14DM) (14DM) and those regulating the transport or accumulation of fluconazole. The analysis of sequential isogenic C. albicans isolates with increasing levels of resistance to fluconazole from five AIDS patients showed that overexpression of the gene encoding 14DM either by gene amplification or by gene deregulation was not the major cause of resistance among these clinical isolates. We found, however, that fluconazole-resistant C. albicans isolates failed to accumulate 3H-labelled fluconazole. This phenomenon was reversed in resistant cells by inhibiting the cellular energy supply with azide, suggesting that resistance could be mediated by energy-requiring efflux pumps such as those described as ATP-binding cassette (ABC) multidrug transporters. In fact, some but not all fluconazole-resistant clinical C. albicans isolates exhibited up to a 10-fold relative increase in mRNA levels for a recently cloned ABC transporter gene called CDR1. In an azole-resistant C. albicans isolate not overexpressing CDR1, the gene for another efflux pump named BENr was massively overexpressed. This gene was cloned from C. albicans for conferring benomyl resistance in Saccharomyces cerevisiae. Therefore, at least the overexpression or the deregulation of these two genes potentially mediates resistance to azoles in C. albicans clinical isolates from AIDS patients with oropharyngeal candidiasis. Involvement of ABC transporters in azole resistance was further evidenced with S. cerevisiae mutants lacking specific multidrug transporters which were rendered hypersusceptible to azole derivatives including fluconazole, itraconazole, and ketoconazole.

Voriconazole Therapeutic Drug Monitoring in Patients with Invasive Mycoses Improves Efficacy and Safety Outcomes

BACKGROUND Voriconazole is the therapy of choice for aspergillosis and a new treatment option for candidiasis. Liver disease, age, genetic polymorphism of the cytochrome CYP2C19, and comedications influence voriconazole metabolism. Large variations in voriconazole pharmacokinetics may be associated with decreased efficacy or with toxicity. METHODS This study was conducted to assess the utility of measuring voriconazole blood levels with individualized dose adjustments. RESULTS A total of 181 measurements with high-pressure liquid chromatography were performed during 2388 treatment days in 52 patients. A large variability in voriconazole trough blood levels was observed, ranging from <or=1 mg/L (the minimum inhibitory concentration at which, for most fungal pathogens, 90% of isolates are susceptible) in 25% of cases to >5.5 mg/L (a level possibly associated with toxicity) in 31% of cases. Lack of response to therapy was more frequent in patients with voriconazole levels <or=1 mg/L (6 [46%] of 13 patients, including 5 patients with aspergillosis, 4 of whom were treated orally with a median dosage of 6 mg/kg per day) than in those with voriconazole levels >1 mg/L (15 [12%] of 39 patients; P=.02). Blood levels >1 mg/L were reached after increasing the voriconazole dosage, with complete resolution of infection in all 6 cases. Among 16 patients with voriconazole trough blood levels >5.5 mg/L, 5 patients (31%) presented with an encephalopathy, including 4 patients who were treated intravenously with a median voriconazole dosage of 8 mg/kg per day, whereas none of the patients with levels <or=5.5 mg/L presented with neurological toxicity (P=.002). Comedication with omeprazole possibly contributed to voriconazole accumulation in 4 patients. In all cases, discontinuation of therapy resulted in prompt and complete neurological recovery. CONCLUSIONS Voriconazole therapeutic drug monitoring improves the efficacy and safety of therapy in severely ill patients with invasive mycoses.

ESCMID* guideline for the diagnosis and management of Candida diseases 2012: non-neutropenic adult patients

This part of the EFISG guidelines focuses on non-neutropenic adult patients. Only a few of the numerous recommendations can be summarized in the abstract. Prophylactic usage of fluconazole is supported in patients with recent abdominal surgery and recurrent gastrointestinal perforations or anastomotic leakages. Candida isolation from respiratory secretions alone should never prompt treatment. For the targeted initial treatment of candidaemia, echinocandins are strongly recommended while liposomal amphotericin B and voriconazole are supported with moderate, and fluconazole with marginal strength. Treatment duration for candidaemia should be a minimum of 14 days after the end of candidaemia, which can be determined by one blood culture per day until negativity. Switching to oral treatment after 10 days of intravenous therapy has been safe in stable patients with susceptible Candida species. In candidaemia, removal of indwelling catheters is strongly recommended. If catheters cannot be removed, lipid-based amphotericin B or echinocandins should be preferred over azoles. Transoesophageal echocardiography and fundoscopy should be performed to detect organ involvement. Native valve endocarditis requires surgery within a week, while in prosthetic valve endocarditis, earlier surgery may be beneficial. The antifungal regimen of choice is liposomal amphotericin B +/- flucytosine. In ocular candidiasis, liposomal amphotericin B +/- flucytosine is recommended when the susceptibility of the isolate is unknown, and in susceptible isolates, fluconazole and voriconazole are alternatives. Amphotericin B deoxycholate is not recommended for any indication due to severe side effects.

Cloning of Candida albicans genes conferring resistance to azole antifungal agents: characterization of CDR2, a new multidrug ABC transporter gene

Resistance to azole antifungal agents in Candida albicans can be mediated by multidrug efflux transporters. In a previous study, we identified at least two such transporters, Cdr1p and Benp, which belong to the class of ATP-binding cassette (ABC) transporters and of major facilitators, respectively. To isolate additional factors potentially responsible for resistance to azole antifungal agents in C. albicans, the hypersusceptibility of a Saccharomyces cerevisiae multidrug transporter mutant, delta pdr5, to these agents was complemented with a C. albicans genomic library. Several new genes were isolated, one of which was a new ABC transporter gene called CDR2 (Candida drug resistance). The protein Cdr2p encoded by this gene exhibited 84% identity with Cdr1p and could confer resistance to azole antifungal agents, to other antifungals (terbinafine, amorolfine) and to a variety of metabolic inhibitors. The disruption of CDR2 in the C. albicans strain CAF4-2 did not render cells more susceptible to these substances. When the disruption of CDR2 was performed in the background of a mutant in which CDR1 was deleted, the resulting double delta cdr1 delta cdr2 mutant was more susceptible to these agents than the single delta cdr1 mutant. The absence of hypersusceptibility of the single delta cdr2 mutant could be explained by the absence of CDR2 mRNA in azole-susceptible C. albicans strains. CDR2 was overexpressed, however, in clinical C. albicans isolates resistant to azole antifungal agents as described previously for CDR1, but to levels exceeding or equal to those reached by CDR1. Interestingly, CDR2 expression was restored in delta cdr1 mutants reverting spontaneously to wild-type levels of susceptibility to azole antifungal agents. These data demonstrate that CDR2 plays an important role in mediating the resistance of C. albicans to azole antifungal agents.

Epidemiology of Candidemia in Swiss Tertiary Care Hospitals: Secular Trends, 1991–2000

Candida species are among the most common bloodstream pathogens in the United States, where the emergence of azole-resistant Candida glabrata and Candida krusei are major concerns. Recent comprehensive longitudinal data from Europe are lacking. We conducted a nationwide survey of candidemia during 1991-2000 in 17 university and university-affiliated hospitals representing 79% of all tertiary care hospital beds in Switzerland. The number of transplantations and bloodstream infections increased significantly (P<.001). A total of 1137 episodes of candidemia were observed: Candida species ranked seventh among etiologic agents (2.9% of all bloodstream isolates). The incidence of candidemia was stable over a 10-year period. C. albicans remained the predominant Candida species recovered (66%), followed by C. glabrata (15%). Candida tropicalis emerged (9%), the incidence of Candida parapsilosis decreased (1%), and recovery of C. krusei remained rare (2%). Fluconazole consumption increased significantly (P<.001). Despite increasing high-risk activities, the incidence of candidemia remained unchanged, and no shift to resistant species occurred.

Fluconazole prophylaxis prevents intra-abdominal candidiasis in high-risk surgical patients

OBJECTIVE To evaluate the efficacy and safety of intravenous fluconazole for the prevention of intra-abdominal Candida infections in high-risk surgical patients. DESIGN Randomized, prospective, double-blind, placebo-controlled study. SETTING Two university-affiliated hospitals in Switzerland. PATIENTS Forty-nine surgical patients with recurrent gastrointestinal perforations or anastomotic leakages. INTERVENTIONS Prophylaxis with intravenous fluconazole (400 mg per day) or placebo continued until resolution of the underlying surgical condition. MEASUREMENTS AND MAIN RESULTS Patients were evaluated daily, and specimens for culture were obtained three times per week during prophylaxis. The primary study end points were the frequency of and the time to intra-abdominal Candida infections. Secondary end points were the frequency of candidiasis (intra-abdominal and extra-abdominal) and the emergence or persistence of Candida colonization. Among patients who were not colonized at study entry, Candida was isolated from surveillance cultures during prophylaxis in 15% of the patients in the fluconazole group and in 62% of the patients in the placebo group (relative risk, 0.25; 95% confidence interval, 0.07 to 0.96; p = .04). Candida peritonitis occurred in one of 23 patients (4%) who received fluconazole and in seven of 20 patients (35%) who received placebo (relative risk, 0.12; 95% confidence interval, 0.02 to 0.93; p = .02). In addition, one catheter-related Candida albicans sepsis occurred in a fluconazole-treated patient. Thus, overall, candidiasis developed in two fluconazole patients and seven placebo patients (relative risk, 0.25; 95% confidence interval, 0.06 to 1.06; p = .06). C. albicans accounted for 87% of the Candida species isolated before or during prophylaxis, and all C. albicans strains were susceptible to fluconazole. Fluconazole was well tolerated, and adverse events occurred at similar frequencies in both treatment groups. CONCLUSIONS Fluconazole prophylaxis prevents colonization and invasive intra-abdominal Candida infections in high-risk surgical patients.

Performance of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of Bacterial Strains Routinely Isolated in a Clinical Microbiology Laboratory

ABSTRACT Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been introduced in diagnostic microbiology laboratories for the identification of bacterial and yeast strains isolated from clinical samples. In the present study, we prospectively compared MALDI-TOF MS to the conventional phenotypic method for the identification of routine isolates. Colonies were analyzed by MALDI-TOF MS either by direct deposition on the target plate or after a formic acid-acetonitrile extraction step if no valid result was initially obtained. Among 1,371 isolates identified by conventional methods, 1,278 (93.2%) were putatively identified to the species level by MALDI-TOF MS and 73 (5.3%) were identified to the genus level, but no reliable identification was obtained for 20 (1.5%). Among the 1,278 isolates identified to the species level by MALDI-TOF MS, 63 (4.9%) discordant results were initially identified. Most discordant results (42/63) were due to systematic database-related taxonomical differences, 14 were explained by poor discrimination of the MALDI-TOF MS spectra obtained, and 7 were due to errors in the initial conventional identification. An extraction step was required to obtain a valid MALDI-TOF MS identification for 25.6% of the 1,278 valid isolates. In conclusion, our results show that MALDI-TOF MS is a fast and reliable technique which has the potential to replace conventional phenotypic identification for most bacterial strains routinely isolated in clinical microbiology laboratories.

Calcineurin A of Candida albicans: involvement in antifungal tolerance, cell morphogenesis and virulence

The azole antifungal fluconazole possesses only fungistatic activity in Candida albicans and, therefore, this human pathogen is tolerant to this agent. However, tolerance to fluconazole can be inhibited when C. albicans is exposed to fluconazole combined with the immunosuppressive drug cyclosporin A, which is known to inhibit calcineurin activity in yeast. A mutant lacking both alleles of a gene encoding the calcineurin A subunit (CNA) lost viability in the presence of fluconazole, thus making calcineurin essential for fluconazole tolerance. Consistent with this observation, tolerance to fluconazole was modulated by calcium ions or by the expression of a calcineurin A derivative autoactivated by the removal of its C‐terminal inhibitory domain. Interestingly, CNA was also essential for tolerance to other antifungal agents (voriconazole, itraconazole, terbinafine, amorolfine) and to several other metabolic inhibitors (caffeine, brefeldin A, mycophenolic acid, fluphenazine) or cell wall‐perturbing agents (SDS, calcofluor white, Congo red), thus indicating that the calcineurin pathway plays an important role in the survival of C. albicans in the presence of external growth inhibitors. Several genes, including PMC1, a vacuolar calcium P‐type ATPase, were regulated in a calcineurin‐ and fluconazole‐dependent manner. However, PMC1 did not play a direct role in the survival of C. albicans when exposed to fluconazole. In addition to these different properties, calcineurin was found to affect colony morphology in several media known to modulate the C. albicans dimorphic switch. In particular, calcineurin was found to be essential for C. albicans viability in serum‐containing media. Finally, calcineurin was found to be necessary for the virulence of C. albicans in a mice model of infection, thus making calcineurin an important element for adequate adaptation to the conditions of the host environment.

Defining responses to therapy and study outcomes in clinical trials of invasive fungal diseases: Mycoses Study Group and European Organization for Research and Treatment of Cancer consensus criteria.

Invasive fungal diseases (IFDs) have become major causes of morbidity and mortality among highly immunocompromised patients. Authoritative consensus criteria to diagnose IFD have been useful in establishing eligibility criteria for antifungal trials. There is an important need for generation of consensus definitions of outcomes of IFD that will form a standard for evaluating treatment success and failure in clinical trials. Therefore, an expert international panel consisting of the Mycoses Study Group and the European Organization for Research and Treatment of Cancer was convened to propose guidelines for assessing treatment responses in clinical trials of IFDs and for defining study outcomes. Major fungal diseases that are discussed include invasive disease due to Candida species, Aspergillus species and other molds, Cryptococcus neoformans, Histoplasma capsulatum, and Coccidioides immitis. We also discuss potential pitfalls in assessing outcome, such as conflicting clinical, radiological, and/or mycological data and gaps in knowledge.

Detection of four Plasmodium species in blood from humans by 18S rRNA gene subunit-based and species-specific real-time PCR assays

ABSTRACT There have been reports of increasing numbers of cases of malaria among migrants and travelers. Although microscopic examination of blood smears remains the “gold standard” in diagnosis, this method suffers from insufficient sensitivity and requires considerable expertise. To improve diagnosis, a multiplex real-time PCR was developed. One set of generic primers targeting a highly conserved region of the 18S rRNA gene of the genus Plasmodium was designed; the primer set was polymorphic enough internally to design four species-specific probes for P. falciparum, P. vivax, P. malarie, and P. ovale. Real-time PCR with species-specific probes detected one plasmid copy of P. falciparum, P. vivax, P. malariae, and P. ovale specifically. The same sensitivity was achieved for all species with real-time PCR with the 18S screening probe. Ninety-seven blood samples were investigated. For 66 of them (60 patients), microscopy and real-time PCR results were compared and had a crude agreement of 86% for the detection of plasmodia. Discordant results were reevaluated with clinical, molecular, and sequencing data to resolve them. All nine discordances between 18S screening PCR and microscopy were resolved in favor of the molecular method, as were eight of nine discordances at the species level for the species-specific PCR among the 31 samples positive by both methods. The other 31 blood samples were tested to monitor the antimalaria treatment in seven patients. The number of parasites measured by real-time PCR fell rapidly for six out of seven patients in parallel to parasitemia determined microscopically. This suggests a role of quantitative PCR for the monitoring of patients receiving antimalaria therapy.