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Yu L-S, Rodriguez-Manzano J, Malpartida-Cardenas K, et al., 2019, Rapid and sensitive detection of azole-resistant Aspergillus fumigatus by tandem-repeat loop-mediated isothermal amplification, Journal of Molecular Diagnostics, Vol: 21, Pages: 286-295, ISSN: 1525-1578
Invasive human fungal infections caused by multi-azole resistant Aspergillus fumigatus are associated with increasing rates of mortality in susceptible patients. Current methods of diagnosing infections caused by multi-azole resistant A. fumigatus are, however, not well suited for use in clinical point-of-care testing or in the field. Loop-mediated isothermal amplification (LAMP) is a widely used method of nucleic acid amplification with rapid and easy-to-use features, making it suitable for use in different resource settings. Here, we developed a LAMP assay to detect a 34 bp tandem repeat, named TR34-LAMP. TR34 is a high-prevalence allele that, in conjunction with the L98H single nucleotide polymorphism, is associated with the occurrence of multi-azole resistance in A. fumigatus in the environment and in patients. This process was validated with both synthetic double stranded DNA and genomic DNA prepared from azole-resistant isolates of A. fumigatus. Use of our assay resulted in rapid and specific identification of the TR34 allele with high sensitivity, detecting down to 10 genomic copies per reaction within 25 minutes. Fluorescent and colorimetric detections were used for the analysis of 11 clinical isolates as cross validation. These results show that the TR34-LAMP assay has the potential to accelerate the screening of clinical and environmental A. fumigatus to provide a rapid and accurate diagnosis of azole resistance, which current methods struggle to achieve.
Abdolrasouli A, Scourfield A, Rhodes J, et al., 2018, High prevalence of triazole resistance in clinical Aspergillus fumigatus isolates in a specialist cardiothoracic centre, International Journal of Antimicrobial Agents, Vol: 52, Pages: 637-642, ISSN: 0924-8579
OBJECTIVES: To evaluate the prevalence of triazole-resistant Aspergillus fumigatus and common molecular cyp51A polymorphisms amongst clinical isolates in a specialised cardiothoracic centre in London, UK. METHODS: All A. fumigatus isolates were prospectively analysed from April 2014 to March 2016. Isolates were screened with a four-well VIPcheck™ plate to assess triazole susceptibility. Resistance was confirmed with a standard microbroth dilution method according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. Triazole-resistant A. fumigatus isolates were subjected to a mixed-format real time polymerase chain reaction (RT-PCR) assay (AsperGenius®) to detect common cyp51A alterations. RESULTS: We identified 167 clinical A. fumigatus isolates from 135 patients. Resistance to at least one azole antifungal drug was confirmed in 22/167 (13.2%) of isolates from 18/135 (13.3%) patients, including 12/74 (16.2%) patients with cystic fibrosis (CF). The highest detection rate of azole-resistant A. fumigatus was among the 11- to 20-y age group. All triazole-resistant isolates (n = 22) were resistant to itraconazole, 18 showed cross-resistance to posaconazole and 10 displayed reduced susceptibility to voriconazole. No pan-azole-resistant A. fumigatus was identified. TR34/L98H was identified in 6/22 (27.3%) of azole-resistant isolates and detectable in 5/12 (42%) patients with CF. CONCLUSIONS: In our specialist cardiothoracic centre, the prevalence of triazole-resistant A. fumigatus is alarmingly high (13.2%). The majority of azole-resistant isolates were from patients with CF. We found a higher prevalence of the environmentally driven mutation TR34/L98H in our A. fumigatus isolates than in published UK data from other specialist respiratory centres, which may reflect differing patient populations managed at these institutions.
Santiago V, Rezvani K, Sekine T, et al., 2018, Human NK cells develop an exhaustion phenotype during polar degranulation at the aspergillus fumigatus hyphal synapse, Frontiers in Immunology, Vol: 9, ISSN: 1664-3224
Pulmonary aspergillosis is an opportunistic fungal infection affecting immunocompromised individuals. Increasing understanding of natural killer (NK) cell immunobiology has aroused considerable interest around the role of NK cells in pulmonary aspergillosis in the immunocompromised host. Murine studies indicate that NK cells play a critical role in pulmonary clearance of A. fumigatus. We show that the in vitro interaction between NK cells and A. fumigatus induces partial activation of NK cell immune response, characterised by low-level production of IFN-γ, TNF-α, MIP-1α, MIP-1β, and RANTES, polarisation of lytic granules and release of fungal DNA. We observed a contact-dependent down-regulation of activatory receptors NKG2D and NKp46 on the NK cell surface, and a failure of full granule release. Furthermore, the NK cell cytokine-mediated response to leukaemic cells was impaired in the presence of A. fumigatus. These observations suggest that A. fumigatus-mediated NK cell immunoparesis may represent an important mechanism of immune evasion during pulmonary aspergillosis.
Nwankwo L, Periselneris J, Cheong J, et al., 2018, Impact of an antifungal stewardship programme in a tertiary respiratory medicine setting: a prospective real-world study, Antimicrobial Agents and Chemotherapy, Vol: 62, ISSN: 0066-4804
There has been an increase in fungal infections in patients with chronic lung disease over the past decades, which is associated with rapidly increasing costs to healthcare systems.An antifungal stewardship team was introduced to a tertiary cardiopulmonary hospital, consisting of a medical mycologist and pharmacy support providing weekly stewardship ward rounds, twice monthly multidisciplinary team meetings and a dedicated weekly outpatient clinic. A database was set up to record the activity of the stewardship team.During the first eighteen months of implementation the antifungal stewardship team had reviewed 178 patients, with 285 recommendations made to inpatients, and 287 outpatient visits. The commonest diagnoses treated were allergic bronchopulmonary aspergillosis and chronic pulmonary aspergillosis. Cystic fibrosis was the largest patient group treated followed by asthma and interstitial lung disease. There was a significant, sustained reduction in monthly antifungal expenditure (p=0.005) by £130,000 per month. There was also a significant reduction in antifungal use measured as Defined Daily Dose/100 bed days (p=0.017). There were no significant changes in expenditure on diagnostic tests. There has been a trend toward more patients having therapeutic levels of voriconazole (p=0.086) and a significant increase in therapeutic levels of posaconazole (p<0.0001).This study shows that an effective antifungal stewardship programme can significantly reduce expenditure in a specialist respiratory service.
Abdolrasouli A, Petrou MA, Park H, et al., 2018, Surveillance for azole-resistant Aspergillus fumigatus in a centralized diagnostic mycology service, London, United Kingdom, 1998-2017, Frontiers in Microbiology, Vol: 9, ISSN: 1664-302X
Background/Objectives: Aspergillus fumigatus is the leading cause of invasive aspergillosis. Treatment is hindered by the emergence of resistance to triazole antimycotic agents. Here, we present the prevalence of triazole resistance among clinical isolates at a major centralized medical mycology laboratory in London, United Kingdom, in the period 1998–2017.Methods: A large number (n = 1469) of clinical A. fumigatus isolates from unselected clinical specimens were identified and their susceptibility against three triazoles, amphotericin B and three echinocandin agents was carried out. All isolates were identified phenotypically and antifungal susceptibility testing was carried out by using a standard broth microdilution method.Results: Retrospective surveillance (1998–2011) shows 5/1151 (0.43%) isolates were resistant to at least one of the clinically used triazole antifungal agents. Prospective surveillance (2015–2017) shows 7/356 (2.2%) isolates were resistant to at least one triazole antifungals demonstrating an increase in incidence of triazole-resistant A. fumigatus in our laboratory. Among five isolates collected from 2015 to 2017 and available for molecular testing, three harbored TR34/L98H alteration in the cyp51A gene that are associated with the acquisition of resistance in the non-patient environment.Conclusion: These data show that historically low prevalence of azole resistance may be increasing, warranting further surveillance of susceptible patients.
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