Detection of Mycoplasma mycoides subsp. mycoides SC in bronchoalveolar lavage fluids of cows based on a TaqMan real-time PCR discriminating wild type strains from an lppQ− mutant vaccine strain used for DIVA-strategies

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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EvaGreen-based Multiplex Real-time PCR Assay for Rapid Differentiation of Wild-Type and Glycoprotein E-Deleted Bovine Herpesvirus-1 Strains

Animal Biotechnology ◽

10.1080/10495398.2016.1268620 ◽

2017 ◽

Vol 28 (4) ◽

pp. 248-252 ◽

Cited By ~ 1

Author(s):

Sachin S. Pawar ◽

Chetan D. Meshram ◽

Niraj K. Singh ◽

Mohini Saini ◽

B. P. Mishra ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Bovine Herpesvirus ◽

Wild Type ◽

Pcr Assay ◽

Bovine Herpesvirus 1 ◽

Glycoprotein E ◽

Herpesvirus 1

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Inter-laboratory comparison of three different real-time PCR assays for the detection of Pneumocystis jiroveci in bronchoalveolar lavage fluid samples

Journal of Medical Microbiology ◽

10.1099/jmm.0.46552-0 ◽

2006 ◽

Vol 55 (9) ◽

pp. 1229-1235 ◽

Cited By ~ 40

Author(s):

Catharina F. M. Linssen ◽

Jan A. Jacobs ◽

Pieter Beckers ◽

Kate E. Templeton ◽

Judith Bakkers ◽

...

Keyword(s):

Bronchoalveolar Lavage ◽

Real Time ◽

Real Time Pcr ◽

Bronchoalveolar Lavage Fluid ◽

Medical Center ◽

Tertiary Care ◽

Lavage Fluid ◽

The Other ◽

Pneumocystis Jiroveci ◽

Pcr Assays

Pneumocystis jiroveci pneumonia (PCP) is an opportunistic infection affecting immunocompromised patients. While conventional diagnosis of PCP by microscopy is cumbersome, the use of PCR to diagnose PCP has great potential. Nevertheless, inter-laboratory validation and standardization of PCR assays is lacking. The aim of this study was to evaluate the inter-laboratory agreement of three independently developed real-time PCR assays for the detection of P. jiroveci in bronchoalveolar lavage fluid samples. Therefore, 124 samples were collected in three tertiary care laboratories (Leiden University Medical Center, Maastricht Infection Center and Radboud University Nijmegen Medical Centre) and were tested by both microscopy and real-time PCR. Of 41 samples positive for P. jiroveci by microscopy, 40 were positive in all three PCR assays. The remaining sample was positive in a single assay only. Out of 83 microscopy-negative samples, 69 were negative in all three PCR assays. The other 14 samples were found positive, either in all three assays (n=5), in two (n=2) or in one of the assays (n=7). The data demonstrate high inter-laboratory agreement among real-time PCR assays for the detection of P. jiroveci.

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