PCR-based detection of
            <i>Aspergillus fumigatus Cyp51A</i>
            mutations on bronchoalveolar lavage: a multicentre validation of the AsperGenius assay
            <sup>®</sup>
            in 201 patients with haematological disease suspected for invasive aspergillosis

Chong, Ga-Lai M.; Beek, Martha T. van der; Borne, Peter A. von dem; Boelens, Jerina; Steel, Eva; Kampinga, Greetje A.; Span, L. F. R.; Lagrou, Katrien; Maertens, J; Dingemans, Gijs; Gaajetaan, Giel R.; Tegelen, D.W.E. van; Cornelissen, Jan J.; Vonk, Alieke G.; Rijnders, Bart J. A.

doi:10.1093/jac/dkw323

Cited by 119 publications

(114 citation statements)

References 27 publications

Supporting

Mentioning

104

Contrasting

Unclassified

Order By: Relevance

“…A similar clinical performance for the AsperGenius assay was determined in other studies (33)(34)(35)(36). Chong et al (34) favored a cutoff value of Յ38 to increase sensitivity to 84% without harming specificity, while in another study a cutoff value of 39 was chosen for testing AsperGenius in serum samples (35). Diagnostic performances of Aspergillus PCRs in the literature are often contradictory and difficult to compare, mainly because of differences in the protocols used.…”

Section: Discussionmentioning

confidence: 72%

“…For example, prolonged exposition to azole treatment is associated with selective pressure, resulting in the emergence of azole-resistance mediated by point mutations (10)(11)(12)(13)(14). Furthermore, A. fumigatus azole-resistant strains have also been isolated from azole-naive patients and from the environment due to mutant selection (mainly TR 34 /L98H and TR 46 /Y121F/T289A) caused by the use of fungicides in agriculture, as confirmed by molecular typing (12,15).…”

mentioning

confidence: 97%

See 1 more Smart Citation

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

et al. 2017

Self Cite

View full text Add to dashboard Cite

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 ϭ 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 ϭ 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.KEYWORDS VIPcheck, AsperGenius, cyp51A, cyp51B, hapE A zole resistance in Aspergillus fumigatus is an emerging problem worldwide, with major epidemiological and clinical implications (1-6). Mold active triazoles are commonly used as first line treatment and prophylaxis of invasive aspergillosis (IA) (7). Mutations in the cyp51A gene, which encodes the target of azoles, the lanosterol 14␣-demethylase, represent the most commonly reported mechanism conferring azole resistance and consequently treatment failure in A. fumigatus (8-10). The most prevalent mutation (TR 34 /L98H) involves the insertion of a 34-bp tandem repeat (TR 34 ) in the promoter region of the cyp51A gene and a point mutation within the gene leading to an amino acid substitution (L98H). More recently, an additional cyp51A-mediated resistant genotype (TR 46 /Y121F/T289A) has been reported: a 46-bp tandem repeat (TR 46 ) in the promoter of cyp51A gene and two point mutations within the gene

show abstract

Section: Discussionmentioning

confidence: 72%

mentioning

confidence: 97%

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

et al. 2017

Self Cite

View full text Add to dashboard Cite

show abstract

“…The only strain in the panel with the G448S mutation was also properly identified. Strains in the panel that were carrying the TR (TR 34 [n ϭ 14], TR 46 [n ϭ 2], or TR 53 [n ϭ 2]) were specifically identified.…”

Section: Resultsmentioning

confidence: 99%

“…To address all of these challenges, we have developed a single HRM assay to detect each of the most frequent mutations related to azole resistance (changes at G54, Y121, M220, or G448 and promoter insertions of variable sizes [TR 34 , TR 46 , or TR 53 ]). We were able to clearly differentiate the azole-resistant isolates from the WT isolates by HRM analysis, showing the reliability of this diagnostic tool for the rapid identification of known azole resistance mechanisms in both clinical and environmental A. fumigatus strains.…”

Section: Discussionmentioning

confidence: 99%

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

Bernal‐Martínez

Gil

Rivero-Menéndez

et al. 2017

Antimicrob Agents Chemother

View full text Add to dashboard Cite

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 TR 34 , TR 46 , and TR 53 . 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.

show abstract

“…Nonetheless, clinicians in the United States are likely to include PCR assays in their diagnostic repertoire, since DNA detection can exceed culture in sensitivity, PCR of blood or BAL specimens has a relatively high negative predictive value for invasive aspergillosis (12), and its lower specificity may be offset when combined with GM. In addition, new PCR assays have the ability to detect specific strains of Aspergillus and common azole-resistance mutations in the Cyp51A gene (13). The combination of GM and PCR may be particularly useful for earlier diagnosis of invasive aspergillosis (14,15).…”

Section: Commentarymentioning

confidence: 99%

Updated guidelines for the diagnosis and management of aspergillosis

Misch¹,

Safdar²

2016

J. Thorac. Dis.

View full text Add to dashboard Cite

PCR-based detection of Aspergillus fumigatus Cyp51A mutations on bronchoalveolar lavage: a multicentre validation of the AsperGenius assay ^® in 201 patients with haematological disease suspected for invasive aspergillosis

Abstract: The AsperGenius assay had a good diagnostic performance on BAL and differentiated WT from Aspergillus fumigatus with RAMs, including in culture-negative BAL samples. Most importantly, detection of RAMs was associated with azole treatment failure.

Cited by 119 publications

References 27 publications

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

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

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

Updated guidelines for the diagnosis and management of aspergillosis

Contact Info

Product

Resources

About

PCR-based detection of Aspergillus fumigatus Cyp51A mutations on bronchoalveolar lavage: a multicentre validation of the AsperGenius assay ® in 201 patients with haematological disease suspected for invasive aspergillosis

Abstract: The AsperGenius assay had a good diagnostic performance on BAL and differentiated WT from Aspergillus fumigatus with RAMs, including in culture-negative BAL samples. Most importantly, detection of RAMs was associated with azole treatment failure.

Cited by 119 publications

References 27 publications

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

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

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

Updated guidelines for the diagnosis and management of aspergillosis

Contact Info

Product

Resources

About

PCR-based detection of Aspergillus fumigatus Cyp51A mutations on bronchoalveolar lavage: a multicentre validation of the AsperGenius assay ^® in 201 patients with haematological disease suspected for invasive aspergillosis