Isavuconazole MIC distribution of 29 yeast species responsible for invasive infections (2015-2017).

OBJECTIVES: Isavuconazole is a recent extended-spectrum triazole with activity against yeasts. However, few data are available about the in vitro activity of rare yeast species. We report the MIC distribution of isavuconazole compared with fluconazole for a large collection of common or rare yeasts. METHODS: Isavuconazole and fluconazole MICs were determined using the EUCAST method for 1457 clinical isolates, mainly recovered from invasive infections, belonging to 29 species. They were sent to the National Reference Centre for Invasive Mycoses & Antifungals between January 2015 and October 2017 and species identification was performed using a polyphasic approach (matrix-assisted laser desorption/ionization time of flight analysis and a molecular method). RESULTS: Isavuconazole had effective in vitro activity against Cryptococcus neoformans (MIC90 < 0.25 mg/L), the five most common Candida spp. (MIC90 ≤ 0.5 mg/L for Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, and Candida krusei) and also against the majority of rare species, including Candida kefyr and Candida lusitaniae. A few isolates of C. albicans (0.7%, 3/404), C. glabrata (2.7%, 5/184), C. tropicalis (1.0%, 1/96) and C. parapsilosis (0.8%, 1/127) exhibited MIC ≥4 mg/L. All were also resistant to fluconazole according to the EUCAST breakpoints. Some isolates with isavuconazole MIC ≥4 mg/L were also observed among rarer species: Meyerozyma guilliermondii (8.7%, 2/23), Wickerhamomyces anomalus (10.0%, 1/10). Other rare species Saprochaete clavata, Magnusiomyces capitatus, and Rhodotorula mucilaginosa had high MIC50 (≥1 mg/L) and MIC90 (≥4 mg/L) and could be considered as resistant to isavuconazole. CONCLUSIONS: We confirmed the good in vitro activity of isavuconazole against common Candida, Cryptococcus species and the majority of the rare yeast species studied.

Estimating the burden of mucormycosis infections in France (2005-2007) through a capture-recapture method on laboratory and administrative data.

BACKGROUND: Mucormycoses are rare but severe fungal infections whose incidence is increasing, particularly in immunosuppressed and diabetic patients. Following a retrospective study on the characteristics and outcomes of cases who were identified through two sources of information, we carried out a capture-recapture method to estimate the actual burden of the disease in France, 2005-2007. METHODS: An administrative dataset from the national hospital discharge system and a laboratory dataset from the National Reference Centre for Mycoses and Antifungals were combined to identify patients from 2005 to 2007. We applied capture-recapture equations to estimate the number of cases missed by both sources and to assess the advantages of each dataset, especially in terms of sensitivity. RESULTS: There were 94 mucormycosis cases included in the study: 30 and 31 in each respective source and 33 common to both. Capture-recapture showed that 28 cases were missed (expected total: 122 cases, CI95: 102-142). Each dataset had a sensitivity value below 53%. The merged set yielded a 77% sensitivity (66%-92%). CONCLUSION: This study highlights the importance of combining available sources when analysing rare infectious diseases. The proportion of 23% missed cases might seem acceptable given the scarcity of the disease, for which further knowledge is needed. However this proportion could decrease in the future, through the sensitization of clinicians, pathologists and mycologists together with the improving quality of discharge datasets.

A global analysis of mucormycosis in France: the RetroZygo Study (2005-2007).

BACKGROUND: Mucormycosis is a deadly invasive fungal infection whose characteristics are only partially understood. METHODS: Data on mucormycosis obtained in France between 2005 and 2007 from 2 notification systems were merged. The 2008 European Organisation for Research and Treatment of Cancer/Mycoses Study Group definition criteria were applied and risk factors for death were analyzed by hazard ratios (HRs) calculated from the Cox proportional hazards regression model. RESULTS: A total of 101 cases (60 proven, 41 probable), mostly in men (58%) >50 years (mean age, 50.7 ± 19.9) were recorded. Hematological malignancies represented 50% (median time for occurrence, 8.8 months after disease onset), diabetes 23%, and trauma 18% of cases. Sites of infection were lungs (28%; 79% in hematology patients), rhinocerebral (25%; 64% in diabetic patients), skin (20%), and disseminated (18%). Median time between first symptoms and diagnosis was 2 weeks. The main fungal species were Rhizopus oryzae (32%) and Lichtheimia species (29%). In cases where the causative species was identified, R. oryzae was present in 85% of rhinocerebral forms compared with only 17% of nonrhinocerebral forms (P < .001). Treatment consisted of surgery in 59% and antifungals in 87% of cases (liposomal amphotericin B in 61%). Ninety-day survival was 56%; it was reduced in cases of dissemination compared with rhinocerebral (HR, 5.38 [2.0-14.1]; P < .001), pulmonary (HR, 2.2 [1.0-4.7]; P = .04), or skin localization (HR, 5.73 [1.9-17.5]; P = .002); survival was reduced in cases of hematological malignancies compared with diabetes mellitus (HR, 2.3 [1.0-5.2]; P < .05) or trauma (HR, 6.9 [1.6-28.6], P = .008) and if ≥2 underlying conditions (HR, 5.9 [1.8-19.0]; P = .004). Mucormycosis localization remained the only independent factor associated with survival. CONCLUSIONS: This 3-year study performed in one country shows the diverse clinical presentation of mucormycosis with a high prevalence of primary skin infection following trauma and a prognosis significantly influenced by localization.

Epidemiological trends in invasive aspergillosis in France: the SAIF network (2005-2007).

A prospective (2005-2007) hospital-based multicentre surveillance of EORTC/MSG-proven or probable invasive aspergillosis (IA) cases whatever the underlying diseases was implemented in 12 French academic hospitals. Admissions per hospital and transplantation procedures were obtained. Cox regression models were used to determine risk factors associated with the 12-week overall mortality. With 424 case-patients included, the median incidence/hospital was 0.271/10(3) admissions (range 0.072-0.910) without significant alteration of incidence and seasonality over time. Among the 393 adults (62% men, 56 years (16-84 years)), 15% had proven IA, 78% haematological conditions, and 92.9% had lung involvement. Acute leukaemia (34.6%) and allogeneic stem cell transplantation (21.4%) were major host factors, together with chronic lymphoproliferative disorders (21.6%), which emerged as a new high-risk group. The other risk host factors consisted of solid organ transplantation (8.7%), solid tumours (4.3%), systemic inflammatory diseases (4.6%) and chronic respiratory diseases (2.3%). Serum galactomannan tests were more often positive (≥69%) for acute leukaemia and allogeneic stem cell transplantation than for the others (<42%; p <10(-3)). When positive (n = 245), cultures mainly yielded Aspergillus fumigatus (79.7%). First-line antifungal therapy consisted of voriconazole, caspofungin, lipid formulations of amphotericin, or any combination therapy (52%, 14%, 8% and 19.9%, respectively). Twelve-week overall mortality was 44.8% (95% CI, 39.8-50.0); it was 41% when first-line therapy included voriconazole and 60% otherwise (p <0.001). Independent factors for 12-week mortality were older age, positivity for both culture and galactomannan and central nervous system or pleural involvement, while any strategy containing voriconazole was protective.

Molecular phylogeny and proposal of two new species of the emerging pathogenic fungus Saksenaea.

Saksenaea is a monotypic genus belonging to the order Mucorales and capable of producing severe human infections. Through a polyphasic study based on analysis of the sequences of the internal transcribed spacer (ITS) region, domains D1 and D2 of the 28S rRNA gene, and the elongation factor 1α (EF-1α) gene, as well as by evaluation of relevant morphological and physiological characteristics of a set of clinical and environmental strains, we have demonstrated that Saksenaea vasiformis is a complex of species. We propose as new species Saksenaea oblongispora, characterized by oblong sporangiospores and unable to grow at 42°C, and Saksenaea erythrospora, characterized by large sporangiophores and sporangia and by ellipsoid sporangiospores, biconcave in the lateral view. Itraconazole, posaconazole, and terbinafine were active against all isolates included in the study, while amphotericin B, voriconazole, and the echinocandins showed low activity.

Comparison of antifungal MICs for yeasts obtained using the EUCAST method in a reference laboratory and the Etest in nine different hospital laboratories.

In routine laboratory practice, the determination of MICs of antifungals for yeasts often relies on the Etest, because of a good correlation with reference methods. However, this correlation was established through predesigned studies, rather than prospective testing. The surveillance programme of fungaemia (YEASTS programme), implemented since 2003, facilitated our comparison of the Etest and the EUCAST results, obtained on a routine basis in nine different hospitals and in a reference laboratory, respectively. The analysis included 690 isolates recovered from blood culture (362 Candida albicans, 113 Candida glabrata, 69 Candida parapsilosis, 55 Candida tropicalis, 31 Cryptococcus neoformans, and 60 other yeast species) that were tested for their susceptibility to amphotericin B (n = 655), fluconazole (n = 669), itraconazole (n = 198), voriconazole (n = 588), flucytosine (n = 314), and caspofungin (n = 244). Agreement between the Etest and EUCAST datasets was calculated and categorized on the basis of previously published breakpoints. The level of agreement at ±2 dilutions was 75% for amphotericin B and 90% for flucytosine; for the azoles, it ranged from 71% for itraconazole to 87% for voriconazole. No significant difference was observed among the yeast species, except for Cryptococcus neoformans and flucytosine, with an agreement <40%. Categorical agreement ranged from 60% for itraconazole to 90% for flucytosine. Major and very major discrepancies occurred in <12% and 6%, respectively. The Etest, even when performed on a routine basis, shows a ≥71% agreement with the EUCAST reference method.

Comparative in vitro activities of caspofungin and micafungin, determined using the method of the European Committee on Antimicrobial Susceptibility Testing, against yeast isolates obtained in France in 2005-2006.

The in vitro activities of caspofungin and micafungin against 1,038 yeast isolates have been determined. The caspofungin and micafungin MICs were lower for Candida albicans, Candida glabrata, and Candida tropicalis than for Candida parapsilosis, Candida guilliermondii, and Candida krusei. A clear correlation was seen between the MICs for the two drugs.

Platelia Aspergillus assay for diagnosis of disseminated histoplasmosis.

The Platelia Aspergillus assay tested positive in 8 of 11 patients with disseminated histoplasmosis. While other available methods for diagnosis have several drawbacks, this cross-reactivity is particularly valuable in the perspective of practitioners outside the USA who cannot use the test detecting antigen of Histoplasma capsulatum.

Peritonitis due to Blastobotrys proliferans in a patient undergoing continuous ambulatory peritoneal dialysis.

Blastobotrys proliferans is an ascomycetous yeast never previously reported as a human pathogen. Here we report a case of peritonitis due to Blastobotrys proliferans in a 46-year-old man undergoing peritoneal dialysis.

Results obtained with various antifungal susceptibility testing methods do not predict early clinical outcome in patients with cryptococcosis.

The in vitro susceptibilities of Cryptococcus neoformans isolates from consecutive human immunodeficiency virus-positive and -negative patients to the antifungal agents fluconazole, amphotericin B, and flucytosine were determined by different techniques, including the CLSI method, Etest, and broth microdilution in yeast nitrogen base (YNB) medium, during a multicenter prospective study in France. The relationship between the in vitro data and the clinical outcome 2 weeks after the initiation of antifungal therapy was assessed. In addition, the correlation between the strain serotype and the in vitro activities of the antifungals was determined, and the susceptibility results obtained with the different techniques were also compared. Thirty-seven patients received a combination of amphotericin B with flucytosine as first-line therapy, 22 were treated with amphotericin B alone, and 15 received fluconazole alone. Whatever the antifungal tested, there was no trend toward higher MICs for strains isolated from patients who failed to respond to a given therapy compared to those from patients who did not with either the CLSI method, Etest, or broth microdilution in YNB medium. The MICs obtained by the CLSI or Etest method were significantly lower for serotype D strains than for serotype A strains for both fluconazole and amphotericin B, while flucytosine MICs were not different according to serotype. These findings suggest that the in vitro antifungal susceptibility of C. neoformans, as determined with the techniques used, is not able to predict the early clinical outcome in patients with cryptococcosis.

Fluconazole for the management of invasive candidiasis: where do we stand after 15 years?

Candida spp. are responsible for most of the fungal infections in humans. Available since 1990, fluconazole is well established as a leading drug in the setting of prevention and treatment of mucosal and invasive candidiasis. Fluconazole displays predictable pharmacokinetics and an excellent tolerance profile in all groups, including the elderly and children. Fluconazole is a fungistatic drug against yeasts and lacks activity against moulds. Candida krusei is intrinsically resistant to fluconazole, and other species, notably Candida glabrata, often manifest reduced susceptibility. Emergence of azole-resistant strains as well as discovery of new antifungal drugs (new triazoles and echinocandins) have raised important questions about its use as a first line drug. The aim of this review is to summarize the main available data on the position of fluconazole in the prophylaxis or curative treatment of invasive Candida spp. infections. Fluconazole is still a major drug for antifungal prophylaxis in the setting of transplantation (solid organ and bone marrow), intensive care unit, and in neutropenic patients. Prophylactic fluconazole still has a place in HIV-positive patients in viro-immunological failure with recurrent mucosal candidiasis. Fluconazole can be used in adult neutropenic patients with systemic candidiasis, as long as the species identified is a priori susceptible. Among non-neutropenic patients with candidaemia fluconazole is one of the first line drugs for susceptible species. Cases reports and uncontrolled studies have also reported its efficacy in the setting of osteoarthritis, endophthalmitis, meningitis, endocarditis and peritonitis caused by Candida spp. among immunocompetent adults. In paediatrics, fluconazole is a well tolerated and major prophylactic drug for high-risk neonates, as well as an alternative treatment for neonatal candidiasis. Importantly 15 years after its introduction in the antifungal armamentarium, fluconazole is still a first line treatment option in several cases of invasive candidiasis. Its prophylactic use should however be limited to selected high-risk patients to limit the risk of emergence of azole-resistant strains.

Evidence for human immunodeficiency virus and Cryptococcus neoformans interactions in the pro-inflammatory and anti-inflammatory responses in blood during AIDS-associated cryptococcosis.

Tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-6, -8 and-10 and soluble TNF receptor II (sTNFR II) levels were measured at baseline, and after antifungal therapy for 2 weeks and 3 months, in plasma from 75 human immunodeficiency virus (HIV)-positive and 14 HIV-negative patients with cryptococcosis, and in plasma from 14 HIV-positive controls. At baseline, TNF-alpha, IL-6 and sTNFR II levels, and cryptococcal antigen titres, were increased in patients with fungaemia compared to controls (p < 0.02). The mediator levels were not influenced by the severity of the disease or subsequent death, but sTNFR II and IL-10 levels were reduced, together with virus load, in patients receiving anti-retroviral agents (p < 0.01). During antifungal therapy, sTNFR II levels decreased (p 0.003) in parallel with the virus load and with an increase in CD4 T-cell numbers.

Multicenter evaluation of the reproducibility of the proposed antifungal susceptibility testing method for fermentative yeasts of the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antimicrobial Susceptibility Testing (AFST-EUCAST).

OBJECTIVE: To evaluate the intra- and inter-laboratory reproducibility of a new standard for susceptibility testing of fermentative yeasts. This standard is based on the M27-A procedure of the National Committee for Clinical Laboratory Standards (NCCLS), but incorporates several modifications, including spectrophotometric growth-dependent endpoint reading. METHODS: Nine laboratories participated in the study. Common material lots were used to test six Candida species (one each of C. albicans, C. tropicalis, C. parapsilosis, C. glabrata, C. krusei, and C. lusitaniae), and two quality control strains (C. krusei ATCC6258 and C. parapsilosis ATCC22019). Triplicate testing on three separate days was performed in microtiter format with RPMI-2% glucose, pH 7.0. Flucytosine, fluconazole and itraconazole were tested. In total, 3888 MIC values were included in the analyses. Reproducibility was calculated by means of agreement (percentage of MICs within one two-fold dilution of the mode) and intraclass correlation coefficient (ICC, maximum value of 1). RESULTS: The average intra-laboratory agreements were 99% and 96% after 24 h and 48 h of incubation, respectively, with ICCs of 0.98 and 0.97 (P < 0.05). Two strains exhibiting a trailing effect showed intra-laboratory agreement of 92% and ICCs of < 0.91 at 48 h. The inter-laboratory agreement was 94% and 88% after 24 h and 48 h, respectively, with ICCs of 0.93 and 0.91 (P < 0.05). Lower values of agreement and ICCs were obtained for strains exhibiting trailing after 48 h of incubation. Itraconazole yielded the lowest values of reproducibility. CONCLUSION: The new procedure of EUCAST for antifungal susceptibility testing is a reproducible method within and between laboratories and offers several advantages over the NCCLS approved method.

An outbreak of acute pulmonary histoplasmosis in members of a trekking trip in Martinique, French West Indies.

BACKGROUND: Thirteen clustered cases of American histoplasmosis, a deep mycosis caused by Histoplasma capsulatum and acquired through inhalation of airborne spores was reported. Twenty-five persons traveled in Martinique, French West Indies. Thirteen underwent trekking and passed through a mountain tunnel full of bats (tunnel group). The 12 others performed canyoning and did not go through the tunnel (control group). Fifteen days after exposure, 1 patient of the tunnel group developed fever, chills, and cough. METHODS: The index case was diagnosed in the hospital, but 12 cases where initially diagnosed as prolonged influenza. All individuals were contacted and submitted to a phone questionnaire. They were asked about eventual occurrence of influenzalike symptoms, about activities practiced, and the notion of contact with bats. All were invited to have clinical examinations, chest x-ray films, and blood samplings. Serologic testing for histoplasmosis was performed by immunodiffusion. Clinical evidence of infection with H. capsulatum was obtained in all the remaining patients of the tunnel group and in none in the control group. Symptoms occurred with an acute onset in 11 to 23 days: fever and chills, severe asthenia, headaches, digestive tract involvement, and then cough, dyspnea, hepatic involvement. Pulmonary micro- or macronodules and mediastinal adenopathies were seen on radiograph and/or computed tomography scan. RESULTS: H. capsulatum serologic tests were positive in all 13 cases with presence of specific M and or H precipitins, 5 to 13 weeks after exposure, and were negative in control group. All patients were treated with itraconazole 200 mg per day during at least 2 months. Treatment was well tolerated; patients progressively recovered. Clinical and serologic follow-up was obtained for some patients at 1 and 4 years. The present study reports the first large outbreak of histoplasmosis acquired in Martinique. CONCLUSION: Histoplasmosis still occurs and is potentially serious. In patients returning from endemic areas, presenting prolonged influenzalike symptoms, clinicians should look for previous possible exposure to Histoplasma.

Influence of gender and age on course of infection and cytokine responses in mice with disseminated Cryptococcus neoformans infection.

OBJECTIVE: To study the influence of gender and age on the course of infection and the cytokine response in a murine model of disseminated cryptococcosis. METHODS: The course of the infection (survival and fungal load in blood and tissues) as well as pro-inflammatory and anti-inflammatory cytokine responses in plasma and organs were compared according to gender and age in outbred mice previously infected with Cryptococcus neoformans NIH52D. RESULTS: Although survival and fungal load were similar in male and female mice, the expression of all cytokines in plasma and of tumour necrosis factor-alpha and interferon-gamma in spleen was significantly increased in female mice compared to male mice in two independent experiments. Young male mice had a significantly shortened survival, were significantly more infected and had predominant tumour necrosis factor-alpha and interferon-gamma responses in comparison with older male mice. CONCLUSION: Host factors should be taken into account when studying the immune response to experimental C. neoformans infection. Our data support epidemiological and clinical data showing differences in susceptibility to cryptococcosis according to gender and age.

Cryptococcal meningitis caused by Cryptococcus neoformans var. gattii, serotype C, in AIDS patients in Soweto, South Africa.

We present four patients from South Africa with meningitis caused by Cryptococcus neoformans var. gattii, serotype C. These are the first patients with human immunodeficiency virus (HIV) infection to be reported with serotype C meningitis.

Cas1p is a membrane protein necessary for the O-acetylation of the Cryptococcus neoformans capsular polysaccharide.

The capsule is certainly the most obvious virulence factor for Cryptococcus neoformans. The main capsule constituents are glucuronoxylomannans (GXM). Several studies have focused on the structure and chemistry of the GXM component of the capsule, yet little is known about the genetic basis of the capsule construction. Using a monoclonal antibody specific to a sugar epitope, we isolated a capsule-structure mutant strain and cloned by complementation a gene named CAS1 that codes for a putative membrane protein. Although no sequence homology was found with any known protein in the different databases, protein analysis using the PROPSEARCH software classified Cas1p as a putative glycosyltransferase. Cas1p is a well-conserved evolutionary protein, as we identified one orthologue in the human genome, one in the drosophila genome and four in the plant Arabidopsis thaliana genome. Analysis of the capsule structure after CAS1 deletion showed that it is required for GXM O-acetylation.

[Cryptococcosis]. / Cryptococcose.

Cryptococcus neoformans, an encapsulated yeast is responsible for life-threatening infection in patients with cellular immune defect especially those with AIDS. The most frequent presentation of cryptococcosis is a disseminated meningoencephalitis without acute onset. The diagnosis is based on direct examination with India ink staining, detection of soluble capsular polysaccharide in body fluids, culture of the micro-organism or histology. Looking for other localisations and for factors of poor prognosis is mandatory before selecting the right treatment regimen. In case of meningitis, one should prefer a combination of amphotericin B and 5 fluorocytosin, followed by a triazole, usually fluconazole until sterilisation of all infected sites is achieved or even for life-time maintenance therapy in case of sustained immune defect.

Are two Cryptococcus neoformans strains epidemiologically linked?

The aim of this study was to standardize a method to determine whether two strains of Cryptococcus neoformans could be considered epidemiologically linked. We hypothesized that strains isolated from the same patient were epidemiologically linked and that those isolated from different patients were unrelated. We used 17 environmental isolates and 97 clinical isolates from 31 patients diagnosed with cryptococcosis (1 to 14 isolates per patient). Using the plasmid pCnTel-1-labeled probe CENTEL, we were able to differentiate some unrelated strains that yielded the same hybridization profile with the C. neoformans middle-repetitive-element CNRE-1 probe. The genetic distances separating the strains isolated from the same patient and those separating the strains isolated from different patients were estimated, and the results obtained with the two probes were compared. Analysis of the results enabled the calculation of two Dice coefficient limits defining the zones containing the pairs of linked strains and the pairs of unrelated strains, as well as an intermediate uncertainty zone for which it was not possible to establish whether the pairs of strains were linked.