Talaromycosis in a renal transplant recipient returning from South China.

Talaromycosis is a fungal infection endemic in Southeast Asia. We report a case of a renal transplant recipient who developed infection after a trip to South China. She presented with constitutional symptoms and was found to have an FDG-avid lung mass. Histopathology demonstrated small yeast cells and culture grew Talaromyces marneffei. The patient was treated with 2 weeks of liposomal amphotericin B followed by itraconazole. The dose of tacrolimus was significantly reduced because of the interaction with itraconazole. Mycophenolate mofetil was discontinued. After 12 months of treatment, the mass had completely resolved. Talaromycosis has mainly been reported in patients with AIDS and is uncommon among solid organ transplant recipients. The immune response against T. marneffei infection is mediated predominantly by T cells and macrophages. The diagnosis may not be suspected outside of endemic areas. We propose a therapeutic approach in transplant patients by extrapolating the evidence from the HIV literature and following practices applied to other endemic mycoses.

Isavuconazole Therapeutic Drug Monitoring during Long-Term Treatment for Chronic Pulmonary Aspergillosis.

Isavuconazole is the newest triazole antifungal, and it displays a favorable pharmacokinetic and safety profile. Less is known about its long-term use in immunocompetent hosts. We performed a retrospective service evaluation of isavuconazole therapeutic drug monitoring in patients with chronic pulmonary aspergillosis. Adverse events (AEs) and dose adjustments made during routine clinical practice were recorded, and AEs were classified based on Common Terminology Criteria for Adverse Events v5.0. Forty-five patients (mean age, 64 years) had 285 isavuconazole blood drug levels measured (mean level, 4.1 mg/liter). A total of 117 measurements (41%) were performed on patients on a 100-mg daily dose instead of 200 mg, and all had blood levels of >1 mg/liter. Age (P = 0.012) and a daily dose of 200 mg versus 100 mg (P = 0.02) were independent predictors of levels of >6 mg/liter. AEs were recorded for 25 patients (56%). The mean drug level at the first measurement was 5.5 ± 2 mg/liter for patients reporting AEs, compared with 4.2 ± 1.7 mg/liter for those not reporting AEs (P = 0.032). The cutoff threshold best predictive of an AE was 4.6 mg/liter (area under the concentration-time curve, 0.710). Sixteen patients (36%) discontinued isavuconazole therapy due to AEs. Twenty-six patients (58%) continued on isavuconazole beyond 6 months. Asthma (P = 0.022) and a daily dose of 200 mg versus 100 mg (P = 0.048) were associated with AEs of grade 2 or higher. A reduced daily dose (100 mg versus 200 mg) of isavuconazole resulted in satisfactory drug levels in a substantial number of patients; it was better tolerated and enabled continuation of therapy for prolonged periods.

Deciphering Aspergillus fumigatus cyp51A-mediated triazole resistance by pyrosequencing of respiratory specimens.

BACKGROUND: Infections caused by triazole drug-resistant Aspergillus fumigatus are an increasing problem. The sensitivity of standard culture is poor, abrogating susceptibility testing. Early detection of resistance can improve patient outcomes, yet tools for this purpose are limited. OBJECTIVES: To develop and validate a pyrosequencing technique to detect resistance-conferring cyp51A polymorphisms from clinical respiratory specimens and A. fumigatus isolates. METHODS: Method validation was performed by Sanger sequencing and pyrosequencing of 50 A. fumigatus isolates with a spectrum of triazole susceptibility patterns. Then, 326 Aspergillus quantitative PCR (qPCR)-positive respiratory samples collected over a 27 month period (January 2017-March 2019) from 160 patients at the UK National Aspergillosis Centre were assessed by cyp51A pyrosequencing. The Sanger sequencing and pyrosequencing results were compared with those from high-volume culture and standard susceptibility testing. RESULTS: The cyp51A genotypes of the 50 isolates analysed by pyrosequencing and Sanger sequencing matched. Of the 326 Aspergillus qPCR-positive respiratory specimens, 71.2% were reported with no A. fumigatus growth. Of these, 56.9% (132/232) demonstrated a WT cyp51A genotype and 31.5% (73/232) a resistant genotype by pyrosequencing. Pyrosequencing identified the environmental TR34/L98H mutation in 18.7% (61/326) of the samples in contrast to 6.4% (21/326) pan-azole resistance detected by culture. Importantly, pyrosequencing detected resistance earlier than culture in 23.3% of specimens. CONCLUSIONS: The pyrosequencing assay described could detect a wide range of cyp51A polymorphisms associated with triazole resistance, including those not identified by commercial assays. This method allowed prompt recognition of resistance and the selection of appropriate antifungal treatment when culture was negative.

Positive Aspergillus PCR as a marker of azole resistance or sub-therapeutic antifungal therapy in patients with chronic pulmonary aspergillosis.

BACKGROUND: Chronic pulmonary aspergillosis (CPA) is a progressive respiratory disease, caused most commonly by A fumigatus, with significant morbidity and mortality. Azole resistance in A fumigatus is a growing concern worldwide, with resistance to itraconazole reported in up to 50% of patients. AIM: The aim of this study was to determine whether a positive Aspergillus PCR (polymerase chain reaction) is a marker of resistance in CPA patients on azole therapy. METHODS: Patients were selected via a consecutive database search for the first 50 CPA patients with a positive Aspergillus PCR from January to September 2016. Data were collected regarding concurrent and subsequent culture results, current therapy and serum antifungal levels. PCR-positive patients not on therapy were included as the control group. RESULTS: Twenty-three patients were on therapy (15 itraconazole, 4 voriconazole and 4 posaconazole). Cycle threshold (Ct) values ranged from 20.8 to 37.9; no significant difference was found between each treatment and the control group (P = .47). In treated patients, concurrent azole-resistant A fumigatus was found in 75% of A fumigatus-positive cultures (6/8). All of the resistant isolates in the itraconazole group showed therapy resistance. Twenty per cent of all itraconazole levels were sub-therapeutic. No significant difference was found in serum itraconazole levels for patients on itraconazole with a positive PCR versus negative PCR (P = .44). CONCLUSION: Positive sputum, Aspergillus-specific PCR can be associated with azole resistance in CPA patients on therapy.

Therapeutic drug monitoring and adverse events of delayed-release posaconazole tablets in patients with chronic pulmonary aspergillosis.

BACKGROUND: Posaconazole delayed-release tablets offer better bioavailability than the liquid suspension, but no post-marketing data are available in immunocompetent hosts such as those with chronic pulmonary aspergillosis (CPA). OBJECTIVES: To explore the pharmacokinetics and adverse event (AE) profile of posaconazole tablets in patients with CPA. METHODS: Patients started on posaconazole tablets at the National Aspergillosis Centre (NAC), Manchester, UK between February 2014 and October 2015 were identified from the NAC database and analysed retrospectively. The medical records were reviewed for factors that could affect posaconazole serum levels and the development of AEs. RESULTS: Seventy-two patients were included; 50 (69%) were male and the mean age was 48.5 ±âŸ12 years. Therapeutic levels (≥1 mg/L) were achieved in 90% of cases on 200 mg versus 90% of cases on 300 mg daily (P = not significant). Based on multivariate analysis, female sex (P = 0.041), a 100 mg daily dose (P < 0.001), asthma (P = 0.01) and bronchiectasis (P = 0.001) were associated with subtherapeutic levels. Forty-nine (68%) patients developed AEs, mainly fatigue (37%), dyspnoea (18%) and nausea (12%). AEs were present on 115/196 (59%) occasions on 300 mg/day and on 45/115 (39%) occasions on 200 mg/day (P < 0.01). The mean level was 1.81 ±âŸ0.96 mg/L for patients reporting no AEs and 1.90 ±âŸ1.11 mg/L for those reporting AEs (P = not significant). Factors associated with AEs of grade ≥2 were a daily dose of 300 versus 200 mg (P = 0.001) and asthma (P = 0.008). CONCLUSIONS: A lower-than-recommended posaconazole tablet dose achieved therapeutic levels in most patients and was better tolerated. Males were more likely to achieve a therapeutic level. Underlying conditions affected the degree and frequency of AEs.

Isavuconazole and voriconazole for the treatment of chronic pulmonary aspergillosis: A retrospective comparison of rates of adverse events.

BACKGROUND: Long-term oral triazole antifungal therapy is the cornerstone of management for patients with chronic pulmonary aspergillosis (CPA). Itraconazole is the first-line choice of treatment. Voriconazole, posaconazole or isavuconazole can be used as alternative treatments in case of resistance or intolerance. All of these can cause significant adverse drug reactions. OBJECTIVES: To evaluate how CPA patients tolerate voriconazole and isavuconazole after prior triazole therapy. METHODS: We performed a retrospective observational study at the UK National Aspergillosis Centre. Medical records for all consecutive CPA patients started on isavuconazole and voriconazole during an observation period of 12 and 6 months respectively were analysed. RESULTS: During this study period, 20 patients were started on isavuconazole and 21 patients on voriconazole. Adverse events were seen in 18 of 21 (86%) the patients in the voriconazole group and 12 of 20 (60%) in the isavuconazole group (P = 0.02). For those who developed adverse events to these agents, the rates of discontinuation of therapy were comparable (ie 10/18 [56%], voriconazole vs 8/12 [67%], isavuconazole; P = 0.54). Five (25%) patients in the isavuconazole group who were intolerant to other triazoles tolerated the standard dose of isavuconazole. CONCLUSIONS: Compared with isavuconazole, adverse events were significantly higher in CPA patients commenced on voriconazole. Isavuconazole may be an option for those patients who are intolerant to other triazoles.

A systematic review of fluconazole resistance in clinical isolates of Cryptococcus species.

Fluconazole is the most commonly used antifungal agent for both the treatment of cryptococcal meningitis, and for prophylaxis against the disease. However, its prolonged use has the potential to exert selection pressure in favour of fluconazole-resistant strains. We evaluated the prevalence of fluconazole resistance in Cryptococcus spp. clinical isolates in 29 studies from 1988 to May 2017 included in EMBASE and MEDLINE databases. A total of 4995 Cryptococcus isolates from 3210 patients constituted this study; 248 (5.0%) of the isolates from relapsed episodes of cryptococcosis were included in this analysis. Eleven (38%) of the studies used minimum inhibitory concentrations (MICs) breakpoints of ≥64 µg/mL to define fluconazole resistance, 6 (21%) used ≥32 µg/mL, 11 (38%) used ≥16 µg/mL and 1 (3%) used ≤20 µg/mL. Overall, mean prevalence of fluconazole resistance was 12.1% (95% confidence interval [CI]: 6.7-17.6) for all isolates (n = 4995). Mean fluconazole resistance was 10.6% (95% CI: 5.5-15.6) for the incident isolates (n = 4747) and 24.1% (95% CI: -3.1-51.2) for the relapse isolates (n = 248). Of the 4995 isolates, 936 (18.7%) had MICs above the ecological cut-off value. Fluconazole resistance appears to be an issue in Cryptococcus isolates from patients with relapses. It remains unclear whether relapses occur due to resistance or other factors. There is an urgent need to establish antifungal breakpoints for Cryptococcus spp.

Volume dependency for culture of fungi from respiratory secretions and increased sensitivity of Aspergillus quantitative PCR.

Diagnosis of aspergillosis is often difficult. We compared fungal yields from respiratory specimens using the Health Protection Agency standard culture method (BSOP57), a higher volume undiluted culture method Mycology Reference Centre Manchester (MRCM) and Aspergillus quantitative real time polymerase chain reaction (qPCR). Sputum, bronchial aspirate and bronchoalveolar lavage (BAL) samples (total 23) were collected from aspergillosis patients. One fraction of all samples was cultured using the MRCM method, one BSOP57 and one was used for qPCR. The recovery rate for fungi was significantly higher by MRCM (87%) than by BSOP57 (8.7%) from all 23 specimens. Sputum samples were 44% positive by MRCM compared to no fungi isolated (0%) by BSOP57. Bronchial aspirates were 75% positive by MRCM and 0% by BSOP57. BAL samples were positive in 20% by MRCM and 10% by BSOP57. qPCR was always more sensitive than culture (95.6%) from all samples. In general, over 100 mould colonies (81 Aspergillus fumigatus) were grown using the MRCM method compared with only one colony from BSOP57. This study provides a reference point for standardisation of respiratory sample processing in diagnostic laboratories. Culture from higher volume undiluted respiratory specimens has a much higher yield for Aspergillus than BSOP57. qPCR is much more sensitive than culture and the current UK method requires revision.

In vitro susceptibility of Aspergillus fumigatus to isavuconazole: correlation with itraconazole, voriconazole, and posaconazole.

Triazoles are first-line agents for treating aspergillosis. The prevalence of azole resistance in Aspergillus fumigatus is increasing, and cross-resistance is a growing concern. In this study, the susceptibilities of 40 A. fumigatus clinical isolates were tested by using the CLSI method with amphotericin B, itraconazole, voriconazole, posaconazole, and the new triazole isavuconazole. Isavuconazole MICs were higher in strains with reduced susceptibilities to other triazoles, mirroring changes in voriconazole susceptibility. Isavuconazole MICs differed depending on the Cyp51A substitution.

The cdr1B efflux transporter is associated with non-cyp51a-mediated itraconazole resistance in Aspergillus fumigatus.

OBJECTIVES: Recent increases in triazole resistance in Aspergillus fumigatus have been attributed primarily to target site (cyp51A) mutations. A recent survey of resistant isolates in Manchester showed that >50% of resistant isolates had no mutation in cyp51A or its promoter. We investigated the mechanisms of resistance in clinical azole-resistant isolates without cyp51A mutations. METHODS: Twelve azole-resistant isolates, 10 of which were itraconazole resistant, were studied. Bioinformatic comparisons between Candida albicans efflux genes and A. fumigatus genome data identified 20 putative azole transporter genes. Basal and azole-induced expression of these genes and cyp51A was quantified using RT-PCR with comparison with clinical azole-susceptible isolates. Function of high basal or itraconazole-induced expression transporters was tested by gene knockout in azole-susceptible and azole-resistant isolates. RESULTS: All susceptible strains showed minimal basal expression of cdr1B compared with 8 of 10 azole-resistant strains with high basal expression of this gene (>5-fold), 3 of which showed >30-fold increased expression. Knockout of this gene resulted in a 4-fold reduction in itraconazole, posaconazole and voriconazole MICs for a susceptible clinical isolate and a 4-fold reduction in itraconazole susceptibility in a clinical resistant isolate. One strain showed a >500-fold induction of cyp51A. No increase in basal expression or expression after induction was seen for the 18 remaining putative transporters. CONCLUSIONS: The reasons behind the shift away from target site mutation in azole-resistant isolates from Manchester are unknown. The modest change in expression of cdr1B in azole-susceptible strains implies that only study of resistant isolates will lead to further understanding of resistance mechanisms in A. fumigatus.

The ELSI Virtual Forum, 30 Years of the Genome: Integrating and Applying ELSI Research.

This paper reports our analysis of the ELSI Virtual Forum: 30 Years of the Genome: Integrating and Applying ELSI Research, an online meeting of scholars focused on the ethical, legal, and social implications (ELSI) of genetics and genomics.

Comparison of ß-1-3-D-Glucan and Candida Mannan Biomarker Assays with Serological Tests for the Diagnosis of Candidemia.

Invasive candidiasis, including bloodstream infection (candidemia), encompasses the most severe forms of Candida infection. Several species-specific and non-specific serological assays are commercially available to aid in diagnosis. This study compared the performance of five such biomarker assays. Serum samples from 14 patients with proven or probable invasive candidiasis, and from 10 control patients, were included in the analysis. A total of 50 serum samples were tested using C. albicans germ tube antibody (CAGTA) assay (Vircell), C. albicans IgM, C. albicans IgG and Candida mannan assays (Dynamiker Biotechnology). Among these samples, the ß-1-3-D-glucan (BDG) assay (Fungitell), a laboratory standard for the diagnosis of invasive candidiasis, was positive in 20 (40%), intermediate in five (10%) and negative in 25 (50%). In cases of proven or probable candidemia, the sensitivity and specificity of the BDG assay was 86% and 80%, respectively; the Candida mannan assay, 14% and 86%; the CAGTA test, 57% and 60%; the C. albicans IgM assay, 71% and 60%; and C. albicans IgG assay 29% and 90%. In 4/8 (50%) cases with multiple serum samples, C. albicans IgM was positive sooner than BDG. Thus, when used as a rule-out test for invasive candidiasis, our data suggest that the C. albicans IgM assay may assist antifungal stewardship (over serum BDG).

Risk factors associated with an outbreak of equine coronavirus at a large farm in North Carolina.

Background: Equine coronavirus (ECoV) leads to outbreaks with variable morbidity and mortality. Few previous reports of risk factors for infection are available in the literature. Objectives: To describe unique clinical findings and risk factors for infection and development of clinical disease. Animals: 135 horses on a farm affected by ECoV outbreak. Methods: Retrospective cohort study. Data obtained included age, breed, gender, activity level, housing, and feed at the onset of the outbreak. Factors were evaluated for assessment of risk of infection using simple logistic regression or Fisher's exact test. Significance was set at p ≤ 0.05. Results and findings: Forty-three of 54 (79.6%) horses tested on the farm were positive on fecal PCR for ECoV, and 17 horses (12.6%) developed clinical signs consistent with ECoV. Out of 17 horses in which the presence or absence of signs of colic was noted, 6 of 17 (35.3%) showed signs of colic. Three of these horses had small colon impactions, 2 of which required surgical intervention. Significant risk factors for having positive PCR results included being primarily stalled (OR 167.1, 95% CI 26.4-1719), housing next to a positive horse (OR 7.5, 95% CI 3.1-19.0), being in work (OR 26.9, 95% CI 4.6-281.9), being fed rationed hay vs. ad libitum (OR 1,558, 95% CI 130.8-15,593), and being fed alfalfa hay (OR 1,558, 95% CI 130.8-15,593). Conclusions and clinical importance: This report describes risk factors for ECoV infection many of which were associated with intensive management of show horses. Clinicians should be aware that clinical signs vary and can include severe colic.

COVID-19-Associated Pulmonary Aspergillosis Isolates Are Genomically Diverse but Similar to Each Other in Their Responses to Infection-Relevant Stresses.

Secondary infections caused by the pulmonary fungal pathogen Aspergillus fumigatus are a significant cause of mortality in patients with severe coronavirus disease 19 (COVID-19). Even though epithelial cell damage and aberrant cytokine responses have been linked to susceptibility to COVID-19-associated pulmonary aspergillosis (CAPA), little is known about the mechanisms underpinning copathogenicity. Here, we analyzed the genomes of 11 A. fumigatus isolates from patients with CAPA in three centers from different European countries. CAPA isolates did not cluster based on geographic origin in a genome-scale phylogeny of representative A. fumigatus isolates. Phenotypically, CAPA isolates were more similar to the A. fumigatus A1160 reference strain than to the Af293 strain when grown in infection-relevant stresses, except for interactions with human immune cells wherein macrophage responses were similar to those induced by the Af293 reference strain. Collectively, our data indicate that CAPA isolates are genomically diverse but are more similar to each other in their responses to infection-relevant stresses. A larger number of isolates from CAPA patients should be studied to better understand the molecular epidemiology of CAPA and to identify genetic drivers of copathogenicity and antifungal resistance in patients with COVID-19. IMPORTANCE Coronavirus disease 2019 (COVID-19)-associated pulmonary aspergillosis (CAPA) has been globally reported as a life-threatening complication in some patients with severe COVID-19. Most of these infections are caused by the environmental mold Aspergillus fumigatus, which ranks third in the fungal pathogen priority list of the WHO. However, little is known about the molecular epidemiology of Aspergillus fumigatus CAPA strains. Here, we analyzed the genomes of 11 A. fumigatus isolates from patients with CAPA in three centers from different European countries, and carried out phenotypic analyses with a view to understanding the pathophysiology of the disease. Our data indicate that A. fumigatus CAPA isolates are genomically diverse but are more similar to each other in their responses to infection-relevant stresses.

High-frequency triazole resistance found In nonculturable Aspergillus fumigatus from lungs of patients with chronic fungal disease.

BACKGROUND: Oral triazole therapy is well established for the treatment of invasive (IPA), allergic (ABPA), and chronic pulmonary (CPA) aspergillosis, and is often long-term. Triazole resistance rates are rising internationally. Microbiological diagnosis of aspergillosis is limited by poor culture yield, leading to uncertainty about the frequency of triazole resistance. METHODS: Using an ultrasensitive real-time polymerase chain reaction (PCR) assay for Aspergillus spp., we assessed respiratory fungal load in bronchoalveolar lavage (BAL) and sputum specimens. In a subset of PCR-positive, culture negative samples, we further amplified the CYP51A gene to detect key single-nucleotide polymorphisms (SNPs) associated with triazole resistance. RESULTS: Aspergillus DNA was detected in BAL from normal volunteers (4/11, 36.4%) and patients with culture or microscopy confirmed IPA (21/22, 95%). Aspergillus DNA was detected in sputum in 15 of 19 (78.9%) and 30 of 42 (71.4%) patients with ABPA and CPA, compared with 0% and 16.7% by culture, respectively. In culture-negative, PCR-positive samples, we detected triazole-resistance mutations (L98H with tandem repeat [TR] and M220) within the drug target CYP51A in 55.1% of samples. Six of 8 (75%) of those with ABPA and 12 of 24 (50%) with CPA had resistance markers present, some without prior triazole treatment, and in most despite adequate plasma drug concentrations around the time of sampling. CONCLUSIONS: The very low organism burdens of fungi causing infection have previously prevented direct culture and detection of antifungal resistance in clinical samples. These findings have major implications for the sustainability of triazoles for human antifungal therapy.

Azole antifungal resistance today: focus on Aspergillus.

Oral triazole therapy is well established for the treatment of invasive aspergillosis (IPA), allergic aspergillosis (ABPA), and chronic pulmonary aspergillosis (CPA), and is often long-term. Resistance to triazole azole antifungal drugs in Aspergillus fumigatus is now a major clinical problem in a number of European locations, in China, Canada and the USA with particularly high frequencies from the north-west of the UK, and The Netherlands. A number of centers are reporting the continuing increasing frequency and evolution of resistance mechanisms in A. fumigatus, in both azole-naïve and patients treated with azoles. The increasing rate of resistance is of concern. A number of resistance mechanisms have been found. The biofilm modality of Aspergillus growth may have a number of therapeutic implications for aspergillosis, including antifungal resistance. Microbiological diagnosis of aspergillosis is limited by poor culture yield, leading to uncertainty about the frequency of triazole resistance. Direct resistance testing in culture-negative clinical samples may add additional insights into the prevalence of azole resistance in A. fumigatus.

Efficacy and safety of posaconazole for chronic pulmonary aspergillosis.

BACKGROUND: Chronic pulmonary aspergillosis (CPA) is a severe, progressive respiratory infection characterized by multiple pulmonary cavities and increased levels of antibodies to Aspergillus species. We report the first use of posaconazole in patients with CPA. METHODS: A retrospective study was performed. A composite clinical and radiological evaluation was used to assess response to posaconazole therapy. The rates of clinical response and failure after 6 and 12 months of therapy were determined. Kaplan-Meier survival models were developed to describe the time to clinical response and failure. The underlying diagnosis, the type of therapy (primary or salvage), Aspergillus antibody titer, and posaconazole serum concentrations were assessed as covariates. Aspergillus species were identified and minimum inhibitory concentrations (MICs) of triazoles were determined using standard techniques. RESULTS: There were 79 patients that initially received posaconazole 400 mg twice per day. The median age of patients was 61 years, and 57% were male. Response to posaconazole was observed in 61% of patients at 6 months and in 46% at 12 months. Kaplan-Meier plots showed that the first response to posaconazole was observed in some patients only after approximately 1 year of therapy. Covariates were not significant. Adverse reactions were observed in 12 patients (15%) (nausea in 5, rash in 5, headache in 1, and lethargy in 1), leading to withdrawal of treatment for 9 patients. Aspergillus species were recovered from 22 patients. A posaconazole MIC of >8 mg/L was found in 4 isolates; in 1 of these isolates, this emerged during therapy. Treatment failed in all 4 patients from whom these 4 isolates had been recovered. CONCLUSION: Posaconazole is a safe and partially effective treatment for CPA. Prospective comparative studies are now required.

Azole antifungal resistance in Aspergillus fumigatus: 2008 and 2009.

OBJECTIVES: Resistance to azole antifungal drugs in Aspergillus fumigatus is now a major clinical problem in some locations. Here we update our previous experience with data from 2008-09. METHODS: We tested all A. fumigatus isolates submitted to the Mycology Reference Centre Manchester in 2008 and 2009 for susceptibility to itraconazole, voriconazole and posaconazole. We undertook CYP51A sequencing for most of the azole-resistant isolates. RESULTS: Of 230 isolates, 64 (28%) were azole resistant. In 2008 and 2009, 14% and 20% of patients had resistant isolates, respectively. During this period 62 of 64 (97%) were itraconazole resistant, 2 of 64 (3%) were only voriconazole resistant and 78% of cases were multi-azole resistant. Forty-three percent of isolates did not carry a cyp51A mutation (previously the most common azole resistance mechanism), indicating that other mechanisms must be responsible and are increasing in frequency. CONCLUSIONS: Azole resistance is evolving and growing in frequency. Established and novel mechanisms may be responsible.

Absence of Azole Antifungal Resistance in Aspergillus fumigatus Isolated from Root Vegetables Harvested from UK Arable and Horticultural Soils.

The emergence of azole-resistant Aspergillus fumigatus (ARAf) complicates the treatment of aspergillosis and can nearly double the mortality from invasive aspergillosis (IA). ARAf has been isolated from many different environmental sites and indoor environments and thus presents a significant risk for susceptible patients. Local surveillance of environmental ARAf can guide antifungal prescribing and improve patient outcomes. In this study, seventy-four soils samples collected from the surface of a variety of root vegetables from farm shops and private gardens covering a wide geographical area of the UK, were cultured to assess the presence of A. fumigatus, and the prevalence and nature of any resistance mechanisms. A high-throughput in-house antifungal susceptibility screening method was developed and validated using the EUCAST MIC reference method, E.DEF 9.3.1. A total of 146 isolates were recovered and analysed. Even though the study premise was that soil-covered root vegetables and other fresh produce could represent a conduit for ARAf exposure in vulnerable patients, no ARAf were found in the soil samples despite 55% of samples harbouring A. fumigatus. The sample type and screening method used could be suitable for more extensive monitoring of the soil to detect trends in the prevalence of ARAf.

Toxicodynamics of itraconazole: implications for therapeutic drug monitoring.

We explored concentration-toxicity relationships for itraconazole among 216 patients. Logistic regression revealed a progressive increase in the probability of toxicity with increasing concentrations of itraconazole. Classification and regression tree analysis suggested that 17.1 mg/L of itraconazole (measured using a bioassay) was the concentration level at which the population of patients was separated into 2 groups, each with a high and a low probability of toxicity.