Cécile Garnaud

Resistance of Candida spp. to antifungal drugs in the ICU: where are we now?

Current increases in antifungal drug resistance in Candida spp. and clinical treatment failures are of concern, as invasive candidiasis is a significant cause of mortality in intensive care units (ICUs). This trend reflects the large and expanding use of newer broad-spectrum antifungal agents, such as triazoles and echinocandins. In this review, we firstly present an overview of the mechanisms of action of the drugs and of resistance in pathogenic yeasts, subsequently focusing on recent changes in the epidemiology of antifungal resistance in ICU. Then, we emphasize the clinical impacts of these current trends. The emergence of clinical treatment failures due to resistant isolates is described. We also consider the clinical usefulness of recent advances in the interpretation of antifungal susceptibility testing and in molecular detection of the mutations underlying acquired resistance. We pay particular attention to practical issues relating to ICU patient management, taking into account the growing threat of antifungal drug resistance.

Performance of an antigen-antibody combined assay for hepatitis C virus testing without venipuncture.

BACKGROUND Hepatitis C virus (HCV) is underdiagnosed and therefore increasing the opportunities for HCV testing without venipuncture may be useful. OBJECTIVES We evaluated the analytical performance of a modified, commercially available, combined HCV antigen-antibody assay (cEIA) (Monolisa(®) HCV-Ag-Ab-ULTRA) and a commercially available point-of-care (POC) device (OraQuick(®) HCV) on fingerstick blood (FSB) and oral mucosal transudate (OMT). STUDY DESIGN FSB, OMT and serum samples were collected from 113 cases of HCV-antibody-positive patients and 88 HCV-antibody-negative controls. The HCV-antibody-positive group included 63 patients with quantifiable HCV-RNA (56%) and 17 HIV/HCV co-infected patients (15%). FSB and OMT specimens were collected as dried blood spots (DBSs) or with the OraSure collection system, before testing with cEIA. RESULTS With FSB specimens, the cEIA and the POC device exhibited 100% specificity and 98.2% and 97.4% sensitivity, respectively. The specificity of the cEIA in FSB sharply decreased if stored 3days at room temperature. With OMT specimens, the cEIA sensitivity (71.7%) and specificity (94.3%) were significantly lower than the performance of OraQuick(®) HCV (sensitivity, 94.6%; specificity, 100%). The optical densities obtained with the cEIA in FSB and OMT were lower in HIV/HCV co-infected patients compared with HCV monoinfected patients. CONCLUSION The cEIA using FSB specimens collected on DBSs preserved in appropriate storage conditions was a reliable alternative, equivalent to the POC assay, for HCV testing without venipuncture. The cEIA was not adapted for HCV testing on OMT.

Next-generation sequencing offers new insights into the resistance of Candida spp. to echinocandins and azoles

OBJECTIVES MDR Candida strains are emerging. Next-generation sequencing (NGS), which enables extensive and deep genome analysis, was used to investigate echinocandin and azole resistance in clinical Candida isolates. METHODS Six genes commonly involved in antifungal resistance (ERG11, ERG3, TAC1, CgPDR1, FKS1 and FKS2) were analysed using NGS in 40 Candida isolates (18 Candida albicans, 15 Candida glabrata and 7 Candida parapsilosis). The strategy was validated using strains with known sequences. Then, 8 clinical strains displaying antifungal resistance and 23 sequential isolates collected from 10 patients receiving antifungal therapy were analysed. RESULTS A total of 391 SNPs were detected, among which 6 coding SNPs were reported for the first time. Novel genetic alterations were detected in both azole and echinocandin resistance genes. A C. glabrata strain, which was resistant to echinocandins but highly susceptible to azoles, harboured an FKS2 S663P mutation plus a novel presumed loss-of-function CgPDR1 mutation. This isolate was from a patient with deep-seated and urinary candidiasis. Another C. glabrata isolate, with an MDR phenotype, carried a new FKS2 S663A mutation and a new putative gain-of-function CgPDR1 mutation (T370I); this isolate showed mutated (80%) and WT (20%) populations and was collected after 75 days of exposure to caspofungin from a patient who underwent complicated abdominal surgery. CONCLUSIONS This study shows that NGS can be used for extensive assessment of genetic mutations involved in antifungal resistance. This type of wide genome approach will become very valuable for detecting mechanisms of resistance in clinical strains subjected to multidrug pressure.

Seven-year surveillance of nosocomial invasive aspergillosis in a French University Hospital

OBJECTIVES This study aims at describing the evolution of the epidemiology of invasive aspergillosis (IA) in a French University Hospital focussing on nosocomial cases, in order to assess the efficiency of the environmental preventive measures which were implemented. METHODS From 2003 to 2009, IA cases were reviewed monthly and classified according to the EORTC/MSG criteria and the origin of contamination. RESULTS Five proven and 65 probable IA cases were diagnosed. Most of the cases (74.3%) occurred in patients with haematological malignancies. Incidences of IA and nosocomial IA (NIA) were 0.106 and 0.032 cases per 1000 admissions, respectively. All the 21 NIA cases occurred in the absence of air treatment (laminar air flow facilities or Plasmair decontamination units) and/or during construction works. The 3-month and 1-year overall survival rates were 50.6% [38.2-61.7] and 31.1% [20-42.9] respectively, and did not differ according to the origin of contamination. CONCLUSION Nosocomial IA still accounted for a third of all IA cases diagnosed from 2003 to 2009 and mainly occurred in the absence of environmental protective measures, which were confirmed to be effective when applied. Our results show that extension and/or reinforcement of these measures is needed, especially in the haematology unit and during construction works.

Direct Molecular Diagnosis of Aspergillosis and CYP51A Profiling from Respiratory Samples of French Patients

Background: Microbiological diagnosis of aspergillosis and triazole resistance is limited by poor culture yield. To better estimate this shortcoming, we compared culture and molecular detection of A. fumigatus in respiratory samples from French patients at risk for aspergillosis. Methods: A total of 97 respiratory samples including bronchoalveolar lavages (BAL), bronchial aspirates (BA), tracheal aspirates, sputa, pleural fluids, and lung biopsy were collected from 33 patients having invasive aspergillosis (n = 12), chronic pulmonary aspergillosis (n = 3), allergic bronchopulmonary aspergillosis (n = 7), or colonization (n = 11) and 28 controls. Each specimen was evaluated by culture, pan-Aspergillus qPCR, and CYP51A PCR and sequencing. Results: One A. flavus and 19 A. fumigatus with one multiazole resistant strain (5.3%) were cultured from 20 samples. Culture positivity was 62.5, 75, 42.9, and 15.8% in ABPA, CPA, IA, and colonized patients, respectively. Aspergillus detection rate was significantly higher by pan-Aspergillus qPCR than by culture in IA (90.5 vs. 42.9%; P < 0.05) and colonization group (73.7 vs. 15.8%; P < 0.05). The CYP51A PCR found one TR34/L98H along with 5 novel cyp51A mutations (4 non-synonymous and 1 promoter mutations), yet no association can be established currently between these novel mutations and azole resistance. The analysis of 11 matched pairs of BA and BAL samples found that 9/11 BA carried greater fungal load than BAL and CYP51A detection was more sensitive in BA than in BAL. Conclusion: Direct molecular detection of Aspergillus spp. and azole resistance markers are useful adjunct tools for comprehensive aspergillosis diagnosis. The observed superior diagnostic value of BAs to BAL fluids warrants more in-depth study.

Histone Deacetylases and Their Inhibition in Candida Species

Fungi are generally benign members of the human mucosal flora or live as saprophytes in the environment. However, they can become pathogenic, leading to invasive and life threatening infections in vulnerable patients. These invasive fungal infections are regarded as a major public health problem on a similar scale to tuberculosis or malaria. Current treatment for these infections is based on only four available drug classes. This limited therapeutic arsenal and the emergence of drug-resistant strains are a matter of concern due to the growing number of patients to be treated, and new therapeutic strategies are urgently needed. Adaptation of fungi to drug pressure involves transcriptional regulation, in which chromatin dynamics and histone modifications play a major role. Histone deacetylases (HDACs) remove acetyl groups from histones and actively participate in controlling stress responses. HDAC inhibition has been shown to limit fungal development, virulence, biofilm formation, and dissemination in the infected host, while also improving the efficacy of existing antifungal drugs toward Candida spp. In this article, we review the functional roles of HDACs and the biological effects of HDAC inhibitors on Candida spp., highlighting the correlations between their pathogenic effects in vitro and in vivo. We focus on how HDAC inhibitors could be used to treat invasive candidiasis while also reviewing recent developments in their clinical evaluation.

Added Value of Next-Generation Sequencing for Multilocus Sequence Typing Analysis of a Pneumocystis jirovecii Pneumonia Outbreak1

Pneumocystis jirovecii is a major threat for immunocompromised patients, and clusters of pneumocystis pneumonia (PCP) have been increasingly described in transplant units during the past decade. Exploring an outbreak transmission network requires complementary spatiotemporal and strain-typing approaches. We analyzed a PCP outbreak and demonstrated the added value of next-generation sequencing (NGS) for the multilocus sequence typing (MLST) study of P. jirovecii strains. Thirty-two PCP patients were included. Among the 12 solid organ transplant patients, 5 shared a major and unique genotype that was also found as a minor strain in a sixth patient. A transmission map analysis strengthened the suspicion of nosocomial acquisition of this strain for the 6 patients. NGS-MLST enables accurate determination of subpopulation, which allowed excluding other patients from the transmission network. NGS-MLST genotyping approach was essential to deciphering this outbreak. This innovative approach brings new insights for future epidemiologic studies on this uncultivable opportunistic fungus.

Profile of GenMark’s ePlex® blood culture identification fungal pathogen panel

ABSTRACT Introduction: Fungemia presents high morbi-mortality and thus rapid microbiological diagnosis may contribute to appropriate patient management. In the last decade, kits based on molecular technologies have become available and health care institutes are increasingly facing critical investment choices. Although all these tools aim to achieve rapid fungal detection and species identification, they display different inherent characteristics. Areas covered: Considering technologies allowing detection and identification of fungal species in a sepsis context, the market proposes either tests on positive blood culture or tests on patient’s whole blood. In this review, the authors describe and compare the ePlex® Blood Culture Identification Fungal Pathogen (BCID-FP) test, a fully automated one-step single-use cartridge assay that has been designed to detect identify frequent or rare but emerging, fungal species, from positive blood culture. A comparison with the competing kits is provided. Expert commentaries: The ePlex BCID-FP test provides a diversified and rather relevant panel. Its easy-to-use cartridges allow flexible use around the clock. Nevertheless, prospective clinical studies assessing the time-to-result benefit on antifungal stewardship and on hospital length of stay are not available yet. New tools aim to benefit clinicians and patients, but they should be accompanied by supervision of result interpretation and adaptation of antifungal stewardship.

Superiority of SDS lysis over saponin lysis for direct bacterial identification from positive blood culture bottle by MALDI-TOF MS.

Fast species diagnosis has an important health care impact, as rapid and specific antibacterial therapy is of clear benefit for patients outcome. Here, a new protocol for species identification directly from positive blood cultures is proposed.

Selective BET bromodomain inhibition as an antifungal therapeutic strategy.

Invasive fungal infections cause significant morbidity and mortality among immunocompromised individuals, posing an urgent need for new antifungal therapeutic strategies. Here we investigate a chromatin-interacting module, the bromodomain (BD) from the BET family of proteins, as a potential antifungal target in Candida albicans, a major human fungal pathogen. We show that the BET protein Bdf1 is essential in C. albicans and that mutations inactivating its two BDs result in a loss of viability in vitro and decreased virulence in mice. We report small-molecule compounds that inhibit C. albicans Bdf1 with high selectivity over human BDs. Crystal structures of the Bdf1 BDs reveal binding modes for these inhibitors that are sterically incompatible with the human BET-binding pockets. Furthermore, we report a dibenzothiazepinone compound that phenocopies the effects of a Bdf1 BD-inactivating mutation on C. albicans viability. These findings establish BET inhibition as a promising antifungal therapeutic strategy and identify Bdf1 as an antifungal drug target that can be selectively inhibited without antagonizing human BET function.