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

Abstract

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

FIG. 1

Locations of primer pairs AFU5S-AFU5AS and AFU7S-AFU7AS used in the two-step PCR to detect Aspergillus DNA. The primers are derived from the 18S rRNA gene of Aspergillus spp. The first PCR step (with AFU7S-AFU7AS) results in amplification of a 405-bp fragment, and the second step (with AFU5S-AFU5AS) amplifies an internal fragment of 236 bp.

FIG. 2

Alignment of DNAs of 18S rRNA genes of A. fumigatus (GenBank accession no. AB008401) and humans (GenBank accession no. M10098) and the corresponding 16S rRNA gene of C. albicans (GenBank accession no. X53497). The locations of primer pairs AFU5S-AFU5AS and AFU7S-AFU7AS used in the two-step PCR to detect Aspergillus DNA are indicated by arrows. Homologous regions are boxed.

FIG. 3

Determination of the sensitivity of the two-step PCR assay with purified A. fumigatus DNA diluted in human DNA (50 ng). The signal derived from 10 fg of Aspergillus template DNA was clearly detectable by ethidium bromide staining of an agarose gel. As a positive control, only DNA extracted from A. fumigatus (10 pg) was used in a single PCR amplification of the 236-bp fragment with the second primer pair (lane +). A negative reagent control amplification without addition of DNA (lane −) as well as purified DNA from a human cell line (T47D) resulted in no bands. The 123-bp ladder (Gibco BRL) was used as molecular size marker (lane M).

FIG. 4

Peripheral blood samples from healthy donors were spiked with defined numbers of conidia from A. fumigatus. The signal derived from 10¹ to 10⁰ CFU per ml of blood were still detectable by ethidium bromide staining of an agarose gel. As a positive control, DNA extracted from A. fumigatus (10 pg) was used for single-step PCR amplification of the 236-bp fragment (lane +). Negative reagent control amplification without conidia (0) resulted in no bands. The 123-bp ladder (Gibco BRL) was used as a molecular size marker (M).