Case Identification and Environmental Surveillance

Recognition of time-clustered cases in 2003 prompted us to do this retrospective study and epidemiologic investigation. Cases of infection caused by A. ustus were identified by review of microbiology and infection control records available from 1993 to 2003. Charts were reviewed for clinical data (demographics, underlying disease, transplantation characteristics, antifungal therapies, radiographic and laboratory studies, and outcome). Cases were classified as proven, probable, or possible according to consensus criteria published by the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (18). The Fred Hutchinson Cancer Research Center institutional review board approved this study.

The hospital is a large tertiary care facility that houses patients with HSCT on the top 2 floors (the northeast wings of the seventh and eighth floors). A spot map depicting case-patient location and timeline relating location to time of diagnosis was created. Information on timing of construction activities and airflow information was obtained from hospital engineering and infection control personnel. An attack rate was estimated among the potentially exposed HSCT patients using as the denominator the number of patients who were admitted for HSCT from July through October 2001 and March through September 2003, the at-risk periods when cases occurred.

Environmental Sampling

Based on the spot map, environmental air sampling of patient hospital rooms was performed, and environmental isolates were obtained. An air particle sampler (SAS Super 100, PBI International, Milan, Italy) was used to collect ambient "dust" to the 0.3-μm size. Samples (0.5 m³) were cultured on inhibitory mold agar plates (Remel IMA plates, Lenexa, KS, USA). Organisms were identified to the species level by using standard morphologic criteria for A. ustus. Isolates were stored at -70°C.

Molecular Typing

Molecular typing of A. ustus clinical and environmental isolates was performed by randomly amplified polymorphic DNA (RAPD) analysis by using A. ustus ATCC 1041, NRRL 275, and Candida parapsilosis for outgroup comparison (19). DNA templates were purified from ≈50 mg cells, resuspended in phosphate-buffered saline (PBS), treated with Lyticase 10 μg/mL (Sigma Chemical Co., St. Louis, MO, USA) for 1 h at 37°C, and then digested with Proteinase K 10 μg/mL (Sigma Chemical Co.). Mixtures were subjected to 3 cycles of freeze-thaw in liquid nitrogen, alternating with vortexing with 0.2 g glass beads. Genomic DNA was isolated with the DNeasy Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The RAPD reactions were run under conditions optimized for each primer (Table 1) by using a PerkinElmer 9700 thermal cycler (PerkinElmer, Cetus, CT, USA). PCR products underwent electrophoresis in 1.8% agarose gels, were stained with ethidium bromide, and images were obtained by using an Alpha Imager (Alpha Innotech Corporation, San Leandro, CA, USA). Only bands that possessed one-tenth the integrated intensity of the 1,650-bp band of the molecular marker (4 ng) (area under the curve [AUC] = 1,132) were defined as positive bands for subsequent band relational analysis. The band patterns from each gel with each primer were analyzed by using tools for population genetics analysis (TFPGA) (unpub. data). Cluster analysis was performed by the unweighted pair group mean with arithmetic average (UPGMA) method (20). Bootstrapping was performed with 1,000 tree comparisons with averages by using TFPGA. Band patterns of >95% similarity were classified as identical.

Antifungal Drug Susceptibility Testing

Antifungal drug susceptibility testing of A. ustus isolates was performed by using a microbroth dilution assay, as described by the National Committee for Clinical Laboratory Standards or the filamentous fungi (M38-A) for itraconazole (Janssen, Titusville, NJ, USA), voriconazole (Pfizer, New York, NY, USA), and amphotericin B (Bristol-Myers Squibb Co., Princeton, NJ, USA) (21). Susceptibility (minimal effective concentration) to caspofungin (Merck Research Laboratories, Rahway, NJ, USA) was determined by using a microbroth dilution assay in antibiotic 3 (AM3) media, as described previously (22).

Outbreak Cases

We identified 2 clusters of A. ustus infection among HSCT recipients in our hospital during the study. The first occurred from July to October 2001 (3 probable lung infections: patients 1, 2, and 3). The second occurred from March to September 2003 (1 proven skin infection [likely disseminated from lung] and 2 probable lung infections: patients 4, 5, and 6) (Table A1); 1 lung transplant recipient was colonized with A. ustus while in the hospital (data not shown).

The median age of patients was 59 (range 29 -63) years; 5 (83.3%) were male; median neutropenia duration 15 (range 4-22) days; 5 (83.3%) patients had graft-versus-host disease that required therapy; 5 (83.3%) patients had received mold-active antifungal drugs prophylactically (itraconazole, n = 4) or for a prior diagnosis (voriconazole, n = 1). The median time of diagnosis after transplantation was 222 (range 60-1,295) days (n = 5). Three (50%) of the 6 patients died, all within 8 days of diagnostic culture collection.

Epidemiologic Investigation

Estimating that 382 patients were admitted for HSCT during the at-risk period, the highest overall attack rate was 1.6%, which is above the baseline rate of infection with A. ustus at our institution (0%). No changes in laboratory processing or mold identification methods occurred during the study. Of note, construction of a new surgery pavilion occurred outside our hospital building beginning in July 2001 and ending in December 2003. Airflow to hospital rooms in which the patients resided passed through multiple filters (blanket filter, pre-filter, 95% filter, HEPA filter).

Figure 1

Figure 1. Spot map illustrating case-patient location on the northwestern wing of the eighth floor (8NE) and the seventh floor (7NE) from the 2001 (left panel) and 2003 outbreaks (right panel) at the...

The spot map and time line showed that cases clustered mainly along 2 corridors on 2 floors, 1 directly above the other, around the time of diagnosis. In the 2001 and 2003 outbreaks, all case-patients resided in the same or adjacent rooms before diagnosis (Figure 1).

Environmental air sampling performed 2 months after the last case occurred in 2003 found no A. ustus isolates in the rooms of HSCT patients. One environmental A. ustus isolate was obtained from the carpeted floor of the hall near the room in which the colonized lung transplant recipient resided. The same bronchoscope was used to evaluate each patient; however, it was cleaned after each examination. Also, several patients who were not found to have A. ustus on bronchoalveolar lavage underwent bronchoscopic examination before case-patients, suggesting that cross-contamination was unlikely.

Analyses of Isolates

Figure 2

Figure 2. Molecular typing of Aspergillus ustus isolates by using random amplification of polymorphic DNA. The isolate from patient 3 was not viable on subculturing and, as such, was not available for molecular...

Eleven A. ustus isolates were available for analysis. One patient (patient 3) did not have a viable isolate stored, and 1 patient (patient 5) had 3 isolates recovered during the course of infection. A total of 73 bands were resolved from the 11 A. ustus isolates (Figure 2). The isolates recovered from the 5 HSCT were genetically similar. Three isolates from patient 5 were genetically most similar to the isolate from patient 2. At the time of his diagnosis and death, patient 2 resided in a room directly adjacent to and above the room of patient 5, albeit 2 years earlier (Figure 1). Similarly, the isolate from patient 1 was genetically most similar to that of patient 4; patients 1 and 4 resided in adjacent rooms, also separated by a period of 2 years. Of note, the lung transplant patient appeared to be colonized with a strain of A. ustus that was genetically as distant from the patient isolates as the wild-type ATCC strain. Antifungal drug susceptibility testing of clinical isolates demonstrated relatively high MICs to all antifungal drugs tested (Table 2).