A low-cost tebuconazole-based screening test for azole-resistant aspergillus fumigatus.

The global emergence of azole resistance in Aspergillus fumigatus is resulting in health and food security concerns. Rapid diagnostics and environmental surveillance methods are key to understanding the distribution and prevalence of azole resistance. However, such methods are often associated with high costs and are not always applicable to laboratories based in the least-developed countries. Here, we present and validate a low-cost screening protocol that can be used to differentiate between azole-susceptible "wild-type" and azole-resistant A. fumigatus isolates. © 2020 The Authors. Basic Protocol 1: Preparation of Tebucheck multi-well plates Basic Protocol 2: Inoculation of Tebucheck multi-well plates

First hospital outbreak of the globally emerging Candida auris in a European hospital

Background: Candida auris is a globally emerging multidrug resistant fungal pathogen causing nosocomial transmission. We report an ongoing outbreak of C. auris in a London cardio-thoracic center between April 2015 and July 2016. This is the first report of C. auris in Europe and the largest outbreak so far. We describe the identification, investigation and implementation of control measures. Methods: Data on C. auris case demographics, environmental screening, implementation of infection prevention/control measures, and antifungal susceptibility of patient isolates were prospectively recorded then analysed retrospectively. Speciation of C. auris was performed by MALDI-TOF and typing of outbreak isolates performed by amplified fragment length polymorphism (AFLP). Results: This report describes an ongoing outbreak of 50 C. auris cases over the first 16 month (April 2015 to July 2016) within a single Hospital Trust in London. A total of 44 % (n = 22/50) patients developed possible or proven C. auris infection with a candidaemia rate of 18 % (n = 9/50). Environmental sampling showed persistent presence of the yeast around bed space areas. Implementation of strict infection and prevention control measures included: isolation of cases and their contacts, wearing of personal protective clothing by health care workers, screening of patients on affected wards, skin decontamination with chlorhexidine, environmental cleaning with chorine based reagents and hydrogen peroxide vapour. Genotyping with AFLP demonstrated that C. auris isolates from the same geographic region clustered. Conclusion: This ongoing outbreak with genotypically closely related C. auris highlights the importance of appropriate species identification and rapid detection of cases in order to contain hospital acquired transmission

The serum opsonin L-ficolin is detected in lungs of human transplant recipients following fungal infections and modulates inflammation and killing of Aspergillus fumigatus.

BACKGROUND: Invasive aspergillosis (IA) is a life-threatening systemic fungal infection in immunocompromised individuals that is caused by Aspergillus fumigatus. The human serum opsonin, L-ficolin, has been observed to recognize A. fumigatus and could participate in fungal defense. METHODS: Using lung epithelial cells, primary human monocyte-derived macrophages (MDMs), and neutrophils from healthy donors, we assessed phagocytosis and killing of L-ficolin-opsonized live A. fumigatus conidia by flow cytometry and microscopy. Additionally, cytokines were measured by cytometric bead array, and L-ficolin was measured in bronchoalveolar lavage (BAL) fluid from lung transplant recipients by enzyme-linked immunosorbent assay. RESULTS: L-ficolin opsonization increased conidial uptake and enhanced killing of A. fumigatus by MDMs and neutrophils. Opsonization was also shown to manifest an increase in interleukin 8 release from A549 lung epithelial cells but decreased interleukin 1β, interleukin 6, interleukin 8, interleukin 10, and tumor necrosis factor α release from MDMs and neutrophils 24 hours after infection. The concentration of L-ficolin in BAL fluid from patients with fungal infection was significantly higher than that for control subjects (P = .00087), and receiving operating characteristic curve analysis highlighted the diagnostic potential of L-ficolin for lung infection (area under the curve, 0.842; P < .0001). CONCLUSIONS: L-ficolin modulates the immune response to A. fumigatus. Additionally, for the first time, L-ficolin has been demonstrated to be present in human lungs

MARDy: Mycology Antifungal Resistance Database

This is the final version. Available from the publisher via the DOI in this record.Summary: The increase of antifungal drug resistance is a major global human health concern and threatens agriculture and food security; in order to tackle these concerns, it is important to understand the mechanisms that cause antifungal resistance. The curated Mycology Antifungal Resistance Database (MARDy) is a web-service of antifungal drug resistance mechanisms, including amino acid substitutions, tandem repeat sequences and genome ploidy. MARDy is implemented on a Linux, Apache, MySQL and PHP web development platform and includes a local installation of BLASTn of the database of curated genes.Antimicrobial Research Collaborative (ARC)Natural Environment Research Council (NERC

Microbial contamination of domiciliary nebulisers and clinical implications in chronic obstructive pulmonary disease.

BACKGROUND AND PURPOSE: Domiciliary nebulisers are widely used in chronic obstructive pulmonary disease (COPD) but nebuliser cleaning practice has not been assessed in patients with COPD who are often elderly and may have severe disease and multiple comorbidities. We aimed to evaluate microbial contamination of home nebulisers used by patients with COPD. METHODS: Random microbiological assessment of domiciliary nebulisers was undertaken together with an enquiry into cleaning practices. We also examined the effectiveness of the trust-wide cleaning instructions in eradicating isolated microorganisms in a laboratory setting. RESULTS: The mean age of patients in this study was 71 (range 40-93) years, and in 68% of patients a large number of significant comorbidities were present. Forty-four nebuliser sets were obtained and 73% were contaminated with microorganisms at >100 colony forming units/plate. Potentially pathogenic bacteria colonised 13 of the 44 nebulisers (30%) and organisms isolated included Pseudomonas aeroginosa, Staphylococcus aureus, multidrug resistant Serratia marcesans, Escherichia coli and multiresistant Klebsiella spp, Enterobacteriaceae and fungus Fusarium oxysporum. Washing of nebuliser masks, chambers and mouthpieces achieved complete eradication of Gram-positive bacterial and fungal flora. Gram-negative organisms were incompletely eradicated, which may be attributed to the presence of biofilms. We also found that in patients with pathogenic organisms cultured on the nebuliser sets, there was a higher probability of occurrence of a COPD exacerbation with a mean number of exacerbations of 3.3 (SD=1) per year in the group in whom pathogens were isolated compared with 1.7 (SD=1.2) exacerbations per year in those whose sets grew non-pathogenic flora (p=0.02). CONCLUSIONS: Nebulisers contaminated with microorganisms are potential reservoirs delivering serious pathogens to the lung. Relationships between nebuliser contamination, clinical infection and exacerbations require further examination, but is a potential concern in elderly patients with COPD with comorbidities who fail to effectively maintain reasonable standards of nebuliser cleanliness

Genomic Context of Azole Resistance Mutations in Aspergillus fumigatus Determined Using Whole-Genome Sequencing.

A rapid and global emergence of azole resistance has been observed in the pathogenic fungus Aspergillus fumigatus over the past decade. The dominant resistance mechanism appears to be of environmental origin and involves mutations in the cyp51A gene, which encodes a protein targeted by triazole antifungal drugs. Whole-genome sequencing (WGS) was performed for high-resolution single-nucleotide polymorphism (SNP) analysis of 24 A. fumigatus isolates, including azole-resistant and susceptible clinical and environmental strains obtained from India, the Netherlands, and the United Kingdom, in order to assess the utility of WGS for characterizing the alleles causing resistance. WGS analysis confirmed that TR34/L98H (a mutation comprising a tandem repeat [TR] of 34 bases in the promoter of the cyp51A gene and a leucine-to-histidine change at codon 98) is the sole mechanism of azole resistance among the isolates tested in this panel of isolates. We used population genomic analysis and showed that A. fumigatus was panmictic, with as much genetic diversity found within a country as is found between continents. A striking exception to this was shown in India, where isolates are highly related despite being isolated from both clinical and environmental sources across >1,000 km; this broad occurrence suggests a recent selective sweep of a highly fit genotype that is associated with the TR34/L98H allele. We found that these sequenced isolates are all recombining, showing that azole-resistant alleles are segregating into diverse genetic backgrounds. Our analysis delineates the fundamental population genetic parameters that are needed to enable the use of genome-wide association studies to identify the contribution of SNP diversity to the generation and spread of azole resistance in this medically important fungus. IMPORTANCE: Resistance to azoles in the ubiquitous ascomycete fungus A. fumigatus was first reported from clinical isolates collected in the United States during the late 1980s. Over the last decade, an increasing number of A. fumigatus isolates from the clinic and from nature have been found to show resistance to azoles, suggesting that resistance is emerging through selection by the widespread usage of agricultural azole antifungal compounds. Aspergillosis is an emerging clinical problem, with high rates of treatment failures necessitating the development of new techniques for surveillance and for determining the genome-wide basis of azole resistance in A. fumigatus

MARDy : Mycology Antifungal Resistance Database

J.R. was supported by an Antimicrobial Research Collaborative (ARC) early career research fellowship, Imperial College London (RSRO_54990). T.S. and J.M.G.S. were supported by a Natural Environment Research Council grant awarded to MCF (NE/P001165/1).Peer reviewedPublisher PD

Rapid detection of mobilized colistin resistance using a nucleic acid based lab-on-a-chip diagnostic system

The increasing prevalence of antimicrobial resistance is a serious threat to global public health. One of the most concerning trends is the rapid spread of Carbapenemase-Producing Organisms (CPO), where colistin has become the last-resort antibiotic treatment. The emergence of colistin resistance, including the spread of mobilized colistin resistance (mcr) genes, raises the possibility of untreatable bacterial infections and motivates the development of improved diagnostics for the detection of colistin-resistant organisms. This work demonstrates a rapid response for detecting the most recently reported mcr gene, mcr−9, using a portable and affordable lab-on-a-chip (LoC) platform, offering a promising alternative to conventional laboratory-based instruments such as real-time PCR (qPCR). The platform combines semiconductor technology, for non-optical real-time DNA sensing, with a smartphone application for data acquisition, visualization and cloud connectivity. This technology is enabled by using loop-mediated isothermal amplification (LAMP) as the chemistry for targeted DNA detection, by virtue of its high sensitivity, specificity, yield, and manageable temperature requirements. Here, we have developed the first LAMP assay for mcr−9 - showing high sensitivity (down to 100 genomic copies/reaction) and high specificity (no cross-reactivity with other mcr variants). This assay is demonstrated through supporting a hospital investigation where we analyzed nucleic acids extracted from 128 carbapenemase-producing bacteria isolated from clinical and screening samples and found that 41 carried mcr−9 (validated using whole genome sequencing). Average positive detection times were 6.58 ± 0.42 min when performing the experiments on a conventional qPCR instrument (n = 41). For validating the translation of the LAMP assay onto a LoC platform, a subset of the samples were tested (n = 20), showing average detection times of 6.83 ± 0.92 min for positive isolates (n = 14). All experiments detected mcr−9 in under 10 min, and both platforms showed no statistically significant difference (p-value > 0.05). When sample preparation and throughput capabilities are integrated within this LoC platform, the adoption of this technology for the rapid detection and surveillance of antimicrobial resistance genes will decrease the turnaround time for DNA detection and resistotyping, improving diagnostic capabilities, patient outcomes, and the management of infectious diseases

Confronting and mitigating the risk of COVID-19 associated pulmonary aspergillosis.

Cases of COVID-19 associated pulmonary aspergillosis (CAPA) are being increasingly reported and physicians treating patients with COVID-19-related lung disease need to actively consider these fungal co-infections. The SARS-CoV-2 (COVID-19) virus causes a wide spectrum of disease in healthy individuals as well as those with common comorbidities [1]. Severe COVID-19 is characterised acute respiratory distress syndrome (ARDS) secondary to viral pneumonitis, treatment of which may require mechanical ventilation or extracorporeal membrane oxygenation (ECMO) [2]. Clinicians are alert to the possibility of bacterial co-infection as a complication of lower respiratory tract viral infection; for example a recent review found that 72% of patients with COVID-19 received antimicrobial therapy [3]. However, the risk of fungal co-infection, in particular COVID-19 associated pulmonary aspergillosis (CAPA), remains underappreciated. Fungal disease consistent with invasive aspergillosis (IA) has been observed with other severe Coronaviruses such as Severe Acute Respiratory Syndrome (SARS-CoV-2003) [4, 5] and Middle East Respiratory Syndrome (MERS-CoV) [6]. From the outset of the COVID-19 pandemic, there were warning signs of secondary invasive fungal infection; Aspergillus flavus was isolated from the respiratory tract from one of 99 patients in the first COVID-19 cohort from Wuhan to be reported in any detail [2] and Aspergillus spp. were isolated from 2/52 (3.8%) of a subsequent cohort of critically unwell patients from this region [7]. More recently, retrospective case series from Belgium [8], France [9], The Netherlands [10] and Germany [11] have reported evidence of CAPA in an alarming 20–35% of mechanically ventilated patients