Detection of Aspergillus fumigatus‐specific DNA, (1–3)‐β‐d‐glucan and galactomannan in serum and bronchoalveolar lavage specimens of experimentally infected rats

Abstract

The aim of this study was to detect Aspergillus fumigatus-specific DNA by nested PCR (nPCR) in serum and bronchoalveolar lavage (BAL) specimens of experimentally infected rats and compare the results with (1-3)-beta-D-glucan (BDG) and galactomannan (GM) detection. Sixty Wistar rats, immunosuppressed with an intraperitoneal injection of cyclophosphamide (70 mg kg(-1)) were infected with 1 x 10(6)A. fumigatus conidia. The rats were killed on days 1, 3, 5, 7 and 9 postinfection in groups of six each and their BAL, blood and lungs were cultured. The A. fumigatus-specific DNA, BDG and GM in serum and BAL were detected by nPCR, Fungitell kit and Aspergillus Platelia kit respectively. Base line values were obtained by using sera from six healthy rats. Except the lungs, blood and BAL specimens of all the infected rats were negative for A. fumigatus culture. The BDG, GM and nPCR positivity in serum specimens was 80%, 77% and 63% respectively. The sensitivity of GM and nPCR tests in BAL specimens was 77% and 70% respectively. The data suggest that BDG and GM appear early in the course of infection, and have similar kinetics (r = 0.483, P = 0.007). Hence, their combined detection could be useful in the early diagnosis of invasive aspergillosis.

Strain-Dependent Variation in 18S Ribosomal DNA Copy Numbers in Aspergillus fumigatus

“…Finally, even though qPCR is not the first choice for clinical diagnosis of IA, it has proven useful for quantitating Aspergillus spp. from a variety of patient specimens (2,26,38) and has proven extremely useful as a secondary assay for comparative purposes during assay development (8,23).…”

Simple, Low-Cost Molecular Assays for TR 34 /L98H Mutations in the cyp51A Gene for Rapid Detection of Triazole-Resistant Aspergillus fumigatus Isolates

“…DNA from the isolates was prepared and the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) was amplified with AFUF2 and AFUR2 primers for the identification of A. fumigatus isolates as described previously (17). The presence or absence of TR 34 in the promoter region was determined by PCR amplification by using AFCYPPF (5=-AATAATCGCAGCACCAC TTC-3=) and AFCYPPR (5=-TGGTATGCTGGAACTACACCTT-3=) primers.…”

Development and Evaluation of a Loop-Mediated Isothermal Amplification Method for Rapid Detection of Aspergillus fumigatus

“…In addition, fungi are common laboratory contaminants, so the specificity of the culturing method is not satisfactory for the identification of the target microbes. Modified PCR-based techniques such as nested PCR and real-time quantitative PCR are usually complicated and require a high-precision thermal cycler, laboratory-scale instrumentation, and skilled personnel (9,31). In contrast, the LAMP assay does not require sophisticated and expensive equipment; most importantly, maintenance of a constant temperature of 60 to 65°C for 1 h is sufficient for the amplification reaction (14).…”