Assessment of Aspergillus fumigatus in guinea pig bronchoalveolar lavages and pulmonary tissue by culture and realtime polymerase chain reaction studies

• Published in: International journal of molecular sciences Vol. 13; no. 1; pp. 726 - 736
• Main Authors: Hooper, Dennis G, Bolton, Vincent E, Sutton, John S, Guilford, Frederick T, Straus, David C, Najvar, Laura K, Wiederhold, Nathan P, Kirkpatrick, William R, Patterson, Thomas F
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 01.01.2012; Molecular Diversity Preservation International (MDPI)
• Subjects: Alveoli; Animals; Aspergillosis; Aspergillus fumigatus; Aspergillus fumigatus - genetics; Aspergillus fumigatus - isolation & purification; Automation; Blood culture; Bronchoalveolar Lavage Fluid - microbiology; bronchoalveolar lavages; Bronchus; Data processing; DNA Primers - genetics; DNA Primers - metabolism; DNA, Fungal - analysis; Fungal infections; Guinea Pigs; Hydrolysis; Infection; Laboratories; Lung; Lung - microbiology; Lung - pathology; Male; Polymerase chain reaction; Primers; Probes; Real-Time Polymerase Chain Reaction; Realtime PCR; Spores; Tissue culture; Transplants & implants; United States > US; Texas; Realtime PCR; bronchoalveolar lavages; Aspergillus fumigatus
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms13010726

In this study we pursued a diagnostic target in Aspergillus fumigatus (AF) by using qualitative Realtime PCR combined with proprietary DNA primers and a hydrolysis probe specific for this fungal target. Qualitative Realtime PCR is a diagnostic tool that utilizes Realtime PCR technology and detects the presence or absence target specific DNA within a predetermined detection range. Respiratory tissue and fluids from experimentally infected guinea pigs were tested by extracting DNA from the samples which were amplified and detected using AF specific DNA primers and probe. This study included qualitative evaluations of all specimens for the presence of the DNA of AF. The findings in the tissues after AF infection were compared to the numbers of spores in aerosolized samples used to inoculate the animals. Results demonstrated that the specific probe and primer set could detect the presence or absence of AF DNA in the sample. The qualitative detection limit of the assay ranged from 6 × 10(4) copies to 6 copies. Since blood cultures are rarely positive for Aspergillosis, our data indicate that qualitative Realtime PCR, in combination with the appropriate DNA primers and probe can serve as an effective diagnostic tool in the early detection of fungal infections.