Aspergillus fumigatus isolate AF293 (NCPF 7367)

Aspergillus fumigatus isolate AF293 (NCPF 7367)

Clinical aspects

AF293 was isolated from lung tissue taken at autopsy from a patient who had developed a low white cell count after gold therapy for rheumatoid arthritis. She developed a systemic sepsis syndrome and died after a few days with pulmonary shadows that at autopsy were shown to be invasive pulmonary aspergillosis. The microscopic pathology showed tissue and blood vessel invasion by septate hyphae in a pattern typical of invasive aspergillosis.

Taxonomic and phenotypic aspects

The culture was originally grown from the lung biopsy by the Public Health Laboratory of The Royal Shrewsbury Hospital, Shrewsbury, UK, in 1993 (ref. no. R1693). It was sent to the Department of Microbiology at Hope Hospital, Salford, UK for fungal susceptibility testing (received 20/12/93) and assigned the numbers AF293 and FA/01153. It was sent for accession to the National Collection of Pathogenic Fungi at the Mycology Reference Laboratory, Bristol, UK in July 1998 (ref. no. NCPF 7367), where it was confirmed to be Aspergillus fumigatus Fresenius by Dr Colin Campbell. It was in addition, sent to the Centraal Bureau voor Schimmelcultures (CBS), Baarn, the Netherlands, for taxonomic study. It was identified as Aspergillus fumigatus Fresenius, but as an atypical strain because its stipes were flexuous. It has been shown by us that it can grow at 50 °C on PDA. Four subcultures were obtained from single spores to check the genetic homogeneity of the culture (see below).

Susceptibility testing gave the following results:

  MIC (mgl-1) MFC (mgl-1)
Amphotericin B 1 >8
Itraconazole 0.5 >8
Voriconazole 4 >8

Genetic aspects

Nucleic acid was extracted from a culture grown after one subculture from our oldest frozen stock and from the four subcultures obtained from single spores. The parental culture and three of the four single-spore cultures contained 'plasmid-like' RNA molecules which ran as four sharp bands on standard agarose gels. All the DNA samples were typed by RAPD using primer R108 and gave essentially identical patterns. In addition, the banding pattern was typical for Aspergillus fumigatus. The library is being constructed from one of the single-spore cultures (no.1).

Molecular phylogeny

Single spore culture no. 1 was sent to David Geiser who confirmed that the sequence of its asp f 212 and rodA12 regions were typical for A. fumigatus. For the allergen gene, asp f 2, five strains of A. fumigatus and one strain of Neosartorya fischeri were compared. Amongst the five strains of A. fumigatus, there were a total of two differences in 427 bp. The largest number of differences between any two strains was two. For the hydrophobin gene, rodA, three strains of A. fumigatus and one strain each of A. fumigatus var. ellipticus and N. fischeri were compared. There was only one difference between the three A. fumigatus strains. To see the rodA phylogenetic tree, please click here.

Animal virulence

The isolate was grown for 14 days on Sabouraud dextrose agar and the conidia were harvested. Male CD1 mice were immuno-suppressed with cyclophosphamide and infected by either intravenous or intranasal routes with suspensions of the conidia.

Intravenous injection of 1.1x105 conidia caused 10% mortality, 6.4x105 conidia caused 60% mortality and 1.3x106 conidia caused 80% mortality. The isolate would probably therefore have an LD90 of approximately 1.4x106 in this model. This value is slightly higher than the mean value of 10 other isolates used in this animal model (mean LD90 5.0x105).

Intranasal inoculation of 9.0x106 conidia caused 10% mortality, 3.0x107 conidia caused 20% mortality and 9.9x107 conidia caused 70% mortality. It was impossible to determine the LD90 with this isolate as conidial suspensions of counts in excess of 9.9x107 conidia were impossible to introduce into the nostrils due to high viscosity. Two other isolates have had LD90s assessed by this method and their LD90s were 1.5x107 and 9.0x106.

Prepared and updated by Michael Anderson and David W Denning, June 2001.