scholarly journals Culture-Based Methods and Molecular Tools for Azole-Resistant Aspergillus fumigatus Detection in a Belgian University Hospital

2017 ◽  
Vol 55 (8) ◽  
pp. 2391-2399 ◽  
Author(s):  
I. Montesinos ◽  
M. A. Argudín ◽  
M. Hites ◽  
F. Ahajjam ◽  
M. Dodémont ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Lavage Fluid ◽  
University Hospital ◽  
Azole Resistance ◽  
Content Type ◽  
Routine Surveillance ◽  
Improve Patient Management ◽  
Detection Of Mutations ◽  
Improve Patient ◽  
Prevalent Mutation

ABSTRACT Azole-resistant Aspergillus fumigatus is an increasing worldwide problem with major clinical implications. Surveillance is warranted to guide clinicians to provide optimal treatment to patients. To investigate azole resistance in clinical Aspergillus isolates in our institution, a Belgian university hospital, we conducted a laboratory-based surveillance between June 2015 and October 2016. Two different approaches were used: a prospective culture-based surveillance using VIPcheck on unselected A. fumigatus ( n = 109 patients, including 19 patients with proven or probable invasive aspergillosis [IA]), followed by molecular detection of mutations conferring azole resistance, and a retrospective detection of azole-resistant A. fumigatus in bronchoalveolar lavage fluid using the commercially available AsperGenius PCR ( n = 100 patients, including 29 patients with proven or probable IA). By VIPcheck, 25 azole-resistant A. fumigatus specimens were isolated from 14 patients (12.8%). Of these 14 patients, only 2 had proven or probable IA (10.5%). Mutations at the cyp51A gene were observed in 23 of the 25 A. fumigatus isolates; TR 34 /L98H was the most prevalent mutation (46.7%), followed by TR 46 /Y121F/T289A (26.7%). Twenty-seven (27%) patients were positive for the presence of Aspergillus species by AsperGenius PCR. A. fumigatus was detected by AsperGenius in 20 patients, and 3 of these patients carried cyp51A mutations. Two patients had proven or probable IA and cyp51A mutation (11.7%). Our study has shown that the detection of azole-resistant A. fumigatus in clinical isolates was a frequent finding in our institution. Hence, a rapid method for resistance detection may be useful to improve patient management. Centers that care for immunocompromised patients should perform routine surveillance to determine their local epidemiology.

scholarly journals Development and Validation of a High-Resolution Melting Assay To Detect Azole Resistance in Aspergillus fumigatus

2017 ◽  
Vol 61 (12) ◽  
Author(s):  
L. Bernal-Martínez ◽  
H. Gil ◽  
O. Rivero-Menéndez ◽  
S. Gago ◽  
M. Cuenca-Estrella ◽  
...  
Keyword(s):  
High Resolution ◽  
Aspergillus Fumigatus ◽  
Tandem Repeats ◽  
High Resolution Melting ◽  
Screening Method ◽  
Melting Curve ◽  
Health Concern ◽  
Azole Resistance ◽  
Content Type ◽  
Selection Of

ABSTRACT The global emergence of azole-resistant Aspergillus fumigatus strains is a growing public health concern. Different patterns of azole resistance are linked to mutations in cyp51A. Therefore, accurate characterization of the mechanisms underlying azole resistance is critical to guide selection of the most appropriate antifungal agent for patients with aspergillosis. This study describes a new sequencing-free molecular screening tool for early detection of the most frequent mutations known to be associated with azole resistance in A. fumigatus. PCRs targeting cyp51A mutations at positions G54, Y121, G448, and M220 and targeting different tandem repeats (TRs) in the promoter region were designed. All PCRs were performed simultaneously, using the same cycling conditions. Amplicons were then distinguished using a high-resolution melting assay. For standardization, 30 well-characterized azole-resistant A. fumigatus strains were used, yielding melting curve clusters for different resistance mechanisms for each target and allowing detection of the most frequent azole resistance mutations, i.e., G54E, G54V, G54R, G54W, Y121F, M220V, M220I, M220T, M220K, and G448S, and the tandem repeats TR34, TR46, and TR53. Validation of the method was performed using a blind panel of 80 A. fumigatus azole-susceptible or azole-resistant strains. All strains included in the blind panel were properly classified as susceptible or resistant with the developed method. The implementation of this screening method can reduce the time needed for the detection of azole-resistant A. fumigatus isolates and therefore facilitate selection of the best antifungal therapy in patients with aspergillosis.

scholarly journals Aspergillus fumigatusAfssn3-Afssn8Pair Reverse Regulates Azole Resistance by Conferring Extracellular Polysaccharide, Sphingolipid Pathway Intermediates, and Efflux Pumps to Biofilm

2018 ◽  
Vol 62 (3) ◽  
Author(s):  
Nanbiao Long ◽  
Liping Zeng ◽  
Shanlei Qiao ◽  
Lei Li ◽  
Guowei Zhong
Keyword(s):  
Amino Acids ◽  
Aspergillus Fumigatus ◽  
Biofilm Formation ◽  
Efflux Pump ◽  
Extracellular Polysaccharide ◽  
Azole Resistance ◽  
Drug Penetration ◽  
Reverse Genetic ◽  
Content Type ◽  
Barrier Layers

ABSTRACTAntifungal treatment is often ineffectual, partly because of biofilm formation. In this study, by using a combined forward and reverse genetic strategy, we identified that nucleus-localized AfSsn3 and its partner AfSsn8, which constitute a Cdk8-cyclin pair, are required for azole resistance inAspergillus fumigatus. Deletion ofAfssn3led to increased absorption and utilization of glucose and amino acids. Interestingly, absorption and utilization of glucose accelerated the extracellular polysaccharide formation, while utilization of the amino acids serine, threonine, and glycine increased sphingolipid pathway intermediate accumulation. In addition, the absence ofAfssn3induced the activity of the efflux pump proteins. These factors indicate the mature biofilm is responsible for the major mechanisms ofA. fumigatusresistance to azoles in the ΔAfssn3mutant. Collectively, the loss ofAfssn3led to two “barrier” layers between the intracellular and extracellular spaces, which consequently decreased drug penetration into the cell.

scholarly journals High Prevalence of Azole-Resistant Aspergillus fumigatus in Adults with Cystic Fibrosis Exposed to Itraconazole

2011 ◽  
Vol 56 (2) ◽  
pp. 869-874 ◽  
Author(s):  
Pierre-Régis Burgel ◽  
Marie-Thérèse Baixench ◽  
Michaël Amsellem ◽  
Etienne Audureau ◽  
Jeanne Chapron ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Aspergillus Fumigatus ◽  
Allergic Bronchopulmonary Aspergillosis ◽  
Previous Treatment ◽  
University Hospital ◽  
Clinical Implications ◽  
Content Type ◽  
Microdilution Method ◽  
Broth Microdilution Method ◽  
High Prevalence

ABSTRACTAspergillus fumigatusis the most frequent fungus found in the sputum of cystic fibrosis (CF) subjects. Itraconazole is prescribed for allergic bronchopulmonary aspergillosis (ABPA) orAspergillusbronchitis in CF subjects. We hypothesized thatA. fumigatusisolates in the sputum of CF subjects with previous exposure to itraconazole was associated with higher prevalence of azole resistance. From June 2010 to April 2011, sputum samples from adult CF subjects at Cochin University Hospital (France) were examined systematically for the detection ofA. fumigatus. MICs ofA. fumigatusisolates against azoles were screened using Etest, and reduced susceptibility to azoles was confirmed using the CLSI broth microdilution method.A. fumigatuswas isolated from the sputum of 131/249 (52.6%) adult CF subjects, and 47/131 (35.9%) subjects had received previous treatment with itraconazole. ReducedA. fumigatussusceptibility to itraconazole (MIC, ≥2 mg/liter) was confirmed in 6/131 (4.6%) subjects. All 6 isolates also had reduced susceptibility to posaconazole (MIC, ≥0.5 mg/liter), and 3/6 isolates had reduced susceptibility to voriconazole (MIC, ≥2 mg/liter). Mutations in thecyp51Agene were detected at positions previously implicated to cause resistance in 5 isolates. Azole-resistantA. fumigatusisolates were found in 5/25 (20%) subjects exposed to itraconazole within the previous 3 years. High rates of azole-resistantA. fumigatusisolates were present in adult CF subjects and were associated with recent itraconazole exposure. Although the clinical implications of these findings will require further studies, the cautious use of itraconazole in adult CF subjects can be recommended.

scholarly journals Multiplecyp51A-Based Mechanisms Identified in Azole-Resistant Isolates of Aspergillus fumigatus from China

2015 ◽  
Vol 59 (7) ◽  
pp. 4321-4325 ◽  
Author(s):  
Musang Liu ◽  
Rong Zeng ◽  
Lili Zhang ◽  
Dongmei Li ◽  
Guixia Lv ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Clinical Isolates ◽  
Resistant Isolate ◽  
Azole Resistance ◽  
Content Type ◽  
Resistant Strains

ABSTRACTSeventy-twoA. fumigatusclinical isolates from China were investigated for azole resistance based on mutations ofcyp51A. We identified four azole-resistant strains, among which we found three strains highly resistant to itraconazole, two of which exhibit the TR34/L98H/S297T/F495I mutation, while one carries only the TR34/L98H mutation. To our knowledge, the latter has not been found previously in China. The fourth multiazole-resistant isolate (with only moderate itraconazole resistance) carries a new G432A mutation.

scholarly journals Azole Resistance in Aspergillus fumigatus Isolates from the ARTEMIS Global Surveillance Study Is Primarily Due to the TR/L98H Mutation in thecyp51AGene

2011 ◽  
Vol 55 (9) ◽  
pp. 4465-4468 ◽  
Author(s):  
Shawn R. Lockhart ◽  
João P. Frade ◽  
Kizee A. Etienne ◽  
Michael A. Pfaller ◽  
Daniel J. Diekema ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Surveillance Study ◽  
Azole Resistance ◽  
Content Type ◽  
First Time

ABSTRACTWe surveyed 497 isolates ofAspergillus fumigatuscollected from 2008 to 2009 as part of the ARTEMIS global surveillance study for elevated MIC values to itraconazole, voriconazole, and posaconazole. Sequencing of thecyp51Agene revealed that 8/29 isolates with elevated MIC values to one or more triazoles, all originating in China, contained the TR/L98H mutation associated with resistant European isolates ofA. fumigatus. This is the first time the TR/L98H mutation has been identified outside Europe.

scholarly journals First Detection of TR34 L98H and TR46 Y121F T289A Cyp51 Mutations in Aspergillus fumigatus Isolates in the United States

2015 ◽  
Vol 54 (1) ◽  
pp. 168-171 ◽  
Author(s):  
Nathan P. Wiederhold ◽  
Veronica Garcia Gil ◽  
Felipe Gutierrez ◽  
Jonathan R. Lindner ◽  
Mohammad T. Albataineh ◽  
...  
Keyword(s):  
United States ◽  
Middle East ◽  
Aspergillus Fumigatus ◽  
East Africa ◽  
The United States ◽  
Azole Resistance ◽  
Content Type

Azole resistance inAspergillus fumigatusis an increasing problem. The TR34 L98H and TR46 Y121F T289A mutations that can occur in patients without previous azole exposure have been reported in Europe, Asia, the Middle East, Africa, and Australia. Here, we report the detection of both the TR34 L98H and TR46 Y121F T289A mutations in confirmedA. fumigatusisolates collected in institutions in the United States. These mutations, other mutations known to cause azole resistance, and azole MICs are reported here.

scholarly journals Molecular Characterization of a Voriconazole-Resistant, Posaconazole-Susceptible Aspergillus fumigatus Isolate in a Lung Transplant Recipient in the United States

2015 ◽  
Vol 60 (2) ◽  
pp. 1129-1133 ◽  
Author(s):  
Jose A. Vazquez ◽  
Elias K. Manavathu
Keyword(s):  
United States ◽  
Aspergillus Fumigatus ◽  
Molecular Characterization ◽  
Lung Transplant ◽  
Transplant Patient ◽  
The United States ◽  
Azole Resistance ◽  
Content Type ◽  
High Level

ABSTRACTMolecular characterization ofcyp51Afrom the azole-resistantAspergillus fumigatusisolate 50593 from a lung transplant patient showed Y121F/T289A changes coupled with a 46-bp tandem repeat (TR46) on the promoter, whereascyp51Afrom the pretherapy isolate,A. fumigatus47381, showed no changes. This is the first reported case ofA. fumigatusazole resistance due to Y121F/T289A/TR46 in the United States, suggesting that multiple mutational alterations ofcyp51Aresulting in high-level azole resistance could occur during prolonged antifungal therapy.

scholarly journals Single-Center Evaluation of an Agar-Based Screening for Azole Resistance in Aspergillus fumigatus by Using VIPcheck

2017 ◽  
Vol 61 (12) ◽  
Author(s):  
J. B. Buil ◽  
H. A. L. van der Lee ◽  
A. J. M. M. Rijs ◽  
J. Zoll ◽  
J. A. M. F. Hovestadt ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Sensitivity And Specificity ◽  
Antifungal Susceptibility ◽  
Screening Method ◽  
Point Mutations ◽  
Azole Resistance ◽  
Minimal Growth ◽  
Content Type ◽  
Mean Sensitivity ◽  
Selection Of

ABSTRACT Antifungal susceptibility testing is an essential tool for guiding therapy, although EUCAST and CLSI reference methods are often available only in specialized centers. We studied the performance of an agar-based screening method for the detection of azole resistance in Aspergillus fumigatus cultures. The VIPcheck consists of four wells containing voriconazole, itraconazole, posaconazole, or a growth control. Ninety-six A. fumigatus isolates were used. Thirty-three isolates harbored a known resistance mechanism: TR34/L98H (11 isolates), TR46/Y121F/T289A (6 isolates), TR53 (2 isolates), and 14 isolates with other cyp51A gene point mutations. Eighteen resistant isolates had no cyp51A-mediated azole resistance. Forty-five isolates had a wild-type (WT) azole phenotype. Four technicians and two inexperienced interns, blinded to the genotype/phenotype, read the plates visually after 24 h and 48 h and documented minimal growth, uninhibited growth, and no growth. The performance was compared to the EUCAST method. After 24 h of incubation, the mean sensitivity and specificity were 0.54 and 1.00, respectively, with uninhibited growth as the threshold. After 48 h of incubation, the performance mean sensitivity and specificity were 0.98 and 0.93, respectively, with minimal growth. The performance was not affected by observer experience in mycology. The interclass correlation coefficient was 0.87 after 24 h and 0.85 after 48 h. VIPcheck enabled the selection of azole-resistant A. fumigatus colonies, with a mean sensitivity and specificity of 0.98 and 0.93, respectively. Uninhibited growth on any azole-containing well after 24 h and minimal growth after 48 h were indicative of resistance. These results indicate that the VIPcheck is an easy-to-use tool for azole resistance screening and the selection of colonies that require MIC testing.

scholarly journals Epidemiology and Molecular Characterizations of Azole Resistance in Clinical and Environmental Aspergillus fumigatus Isolates from China

2016 ◽  
Vol 60 (10) ◽  
pp. 5878-5884 ◽  
Author(s):  
Yong Chen ◽  
Zhongyi Lu ◽  
Jingjun Zhao ◽  
Ziying Zou ◽  
Yanwen Gong ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Antifungal Susceptibility ◽  
Public Health Problem ◽  
Wide Distribution ◽  
Resistance Mechanisms ◽  
Azole Resistance ◽  
Common Mutation ◽  
Content Type ◽  
Environmental Resistance ◽  
Resistant Strains

ABSTRACTAzole resistance inAspergillus fumigatushas emerged as a worldwide public health problem. We sought here to demonstrate the occurrence and characteristics of azole resistance inA. fumigatusfrom different parts of China. A total of 317 clinical and 144 environmentalA. fumigatusisolates from 12 provinces were collected and subjected to screening for azole resistance. Antifungal susceptibility,cyp51Agene sequencing, and genotyping were carried out for all suspected azole-resistant isolates and a subset of azole-susceptible isolates. As a result, 8 (2.5%) clinical and 2 (1.4%) environmentalA. fumigatusisolates were identified as azole resistant. Five azole-resistant strains exhibit the TR34/L98H mutation, whereas four carry the TR34/L98H/S297T/F495I mutation in thecyp51Agene. Genetic typing and phylogenetic analysis showed that there was a worldwide clonal expansion of the TR34/L98H isolates, while the TR34/L98H/S297T/F495I isolates from China harbored a distinct genetic background with resistant isolates from other countries. High polymorphisms existed in thecyp51Agene that produced amino acid changes among azole-susceptibleA. fumigatusisolates, with N248K being the most common mutation. These data suggest that the wide distribution of azole-resistantA. fumigatusmight be attributed to the environmental resistance mechanisms in China.

“That was a good shift”

2017 ◽  
Vol 31 (4) ◽  
pp. 471-486 ◽  
Author(s):  
Anya Johnson ◽  
Helena Nguyen ◽  
Sharon K. Parker ◽  
Markus Groth ◽  
Steven Coote ◽  
...  
Keyword(s):  
Situation Awareness ◽  
Patient Management ◽  
Clinical Decision Making ◽  
Interprofessional Collaboration ◽  
Well Being ◽  
Junior Doctors ◽  
Content Type ◽  
Ward Rounds ◽  
Improve Patient Management ◽  
Improve Patient

Purpose The purpose of this paper is to investigate a boundary spanning, interprofessional collaboration between advanced practice nurses (APNs) and junior doctors to support junior doctors’ learning and improve patient management during the overtime shift. Design/methodology/approach A mixed methods evaluation of an intervention in an adult tertiary referral hospital, to enhance interprofessional collaboration on overtime shifts. Phase 1 compared tasks and ward rounds on 86 intervention shifts with 106 “regular” shifts, and examined the effect on junior doctor patient management testing a model using regression techniques. Phase 2 explored the experience of the intervention for stakeholders. 91 junior doctors participated (89 percent response rate) on 192 overtime shifts. Junior doctors, APNs and senior medical professionals/administrators participated in interviews. Findings The intervention was associated with an increase in self-initiated ward rounds by junior doctors, partially explained by junior doctors completing fewer tasks skilled nurses could also complete. The intervention significantly reduced doctors’ engagement in tasks carried over from day shifts as well as first year (but not more experienced) junior doctors’ total tasks. Interviews suggested the initiative reduced junior doctors’ work pressure and promoted a safe team climate, situation awareness, skills, confidence, and well-being. Originality/value Junior doctors overtime shifts (5 p.m. to 11 p.m.) are important, both for hospitals to maintain patient care after hours and for junior doctors to learn and develop independent clinical decision making skills. However, junior doctors frequently report finding overtime shifts challenging and stressful. Redesigning overtime shifts to facilitate interprofessional collaboration can improve patient management and junior doctors’ learning and well-being.

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