Detection of Aspergillus fumigatus in Bronchoalveolar Lavage Fluid by a Novel Semiquantitative System in Experimental Invasive Pulmonary Aspergillosis

T. J. WALSH1, A. FRANCESCONI1, M. KASAI1, J. PALMER2, V. PETRAITIS1, R. PETRAITIENE1, K. A. ORLE2

Author address: 

1NCI, NIH, Bethesda, MD, 2Roche Molecular Systems, Alameda, CA.

Abstract: 

Background. Invasive pulmonary aspergillosis (IPA) is a frequently lethal infection in immunocompromised patients that is difficult to diagnose. Current methods for detection of Aspergillus spp. by bronchoalveolar lavage (BAL) vary in sensitivity and specificity. We developed a semiquantitative PCR assay incorporating a magnetic bead extraction procedure for the detection of IPA from BAL fluid. We further investigated this system in the detection of experimental IPA in persistently neutropenic rabbits. Methods. DNA was extracted from Aspergillus fumigatus by a magnetic bead protocol and primers directed to the 18S ribosomal RNA subunit were used for amplification. PCR signal was analyzed semiquantitatively by horseradish peroxidase conjugate and read at 450nm; copy number of amplicon was determined by serial dilution. BAL fluid from normal control rabbits (NCRs) (n=13), untreated controls with IPA (n=12), and animals with IPA treated with amphotericin B 1 mg/kg/day (n=12) was analyzed by PCR and quantitative culture. Results. Semiqauntitative PCR performed on BAL samples from NCRs and untreated animals with IPA yielded a sensitivity of 100% and specificity of 100%. The mean ± SEM signal from BAL of NCRs was 0.245 ± 0.085 versus 3.12 ± 0.062 for untreated animals with IPA (p
2001

abstract No: 

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Full conference title: 

ICAAC 41st
    • ICAAC 41st