Specific detection of Aspergillus species in blood and bronchoalveolar lavage samples of immunocompromised patients by two-step PCR

The increasing incidence of aspergillosis, a life-threatening infection in immunocompromised patients, emphasizes the need to improve the currently Li mited diagnostic tools. We developed a two-step PCR assay that specifically amplifies a region of the 18S rRNA gene that is highly conserved in Asperg illus species. A number of primers with the least homology to equivalent hu man or Candida gene sequences were screened for the pairs that gave the hig hest sensitivity and specificity. No cross-reaction with the wide range of fungal and bacterial pathogens so far tested was observed. This assay allow s direct and rapid detection of down to 10 fg of Aspergillus DNA correspond ing to 1 to 5 CFU per mi of blood. A total of 315 blood and bronchoalveolar lavage samples from 140 subjects, including 93 patients at risk for invasi ve fungal disease, were screened. The result was a 100% correlation between positive histology, culture, or high-resolution computed tomography findin gs and PCR results. The test specificity was 89%. Our data point to the con siderable potential clinical value of this simple, specific, rapid, and ine xpensive PCR assay for improving the means of early diagnosis of systemic a spergillosis in high-risk patients.