Faculty Opinions recommendation of Novel mixed-format real-time PCR assay to detect mutations conferring resistance to triazoles in Aspergillus fumigatus and prevalence of multi-triazole resistance among clinical isolates in the Netherlands.

Azole resistance inAspergillus fumigatusis increasingly reported. Here, we describe the validation of the AsperGenius, a new multiplex real-time PCR assay consisting of two multiplex real-time PCRs, one that identifies the clinically relevantAspergillusspecies, and one that detects the TR34, L98H, T289A, and Y121F mutations in CYP51A and differentiates susceptible from resistantA. fumigatusstrains. The diagnostic performance of the AsperGenius assay was tested on 37 bronchoalveolar lavage (BAL) fluid samples from hematology patients and 40 BAL fluid samples from intensive care unit (ICU) patients using a BAL fluid galactomannan level of ≥1.0 or positive culture as the gold standard for detecting the presence ofAspergillus. In the hematology and ICU groups combined, there were 22 BAL fluid samples from patients with invasive aspergillosis (IA) (2 proven, 9 probable, and 11 nonclassifiable). Nineteen of the 22 BAL fluid samples were positive, according to the gold standard. The optimal cycle threshold value for the presence ofAspergilluswas <36. Sixteen of the 19 BAL fluid samples had a positive PCR (2Aspergillusspecies and 14A. fumigatussamples). This resulted in a sensitivity, specificity, and positive and negative predictive values of 88.9%, 89.3%, 72.7%, and 96.2%, respectively, for the hematology group and 80.0%, 93.3%, 80.0%, and 93.3%, respectively, in the ICU group. The CYP51A real-time PCR confirmed 12 wild-type and 2 resistant strains (1 TR34-L98H and 1 TR46-Y121F-T289A mutant). Voriconazole therapy failed for both patients. The AsperGenius multiplex real-time PCR assay allows for sensitive and fast detection ofAspergillusspecies directly from BAL fluid samples. More importantly, this assay detects and differentiates wild-type from resistant strains, even if BAL fluid cultures remain negative.

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Multiplex Real-Time PCR for Detection of an Epidemic KPC-Producing Klebsiella pneumoniae ST258 Clone

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00316-12 ◽

2012 ◽

Vol 56 (6) ◽

pp. 3444-3447 ◽

Cited By ~ 37

Author(s):

Liang Chen ◽

Kalyan D. Chavda ◽

José R. Mediavilla ◽

Yanan Zhao ◽

Henry S. Fraimow ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Real Time ◽

Real Time Pcr ◽

Clinical Isolates ◽

Pcr Assay ◽

Content Type ◽

Carbapenem Resistant ◽

Sequence Types ◽

Single Reaction

ABSTRACTWe describe a multiplex real-time PCR assay capable of identifying both the epidemicKlebsiella pneumoniaeST258 clone andblaKPCcarbapenemase genes in a single reaction. The assay displayed excellent sensitivity (100%) and specificity (100%) for identification of ST258 clone andblaKPCin a collection of 75K. pneumoniaeisolates comprising 41 sequence types. Our results suggest that this assay is an effective tool for surveillance of this clone among carbapenem-resistantK. pneumoniaeclinical isolates.

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