TEXT

Top Abstract Text References

Aspergillus spp. are ubiquitous, commonly occurring in soil, water, and decaying vegetation. Reservoirs in hospitals from which these fungi have been cultured include unfiltered air, ventilation systems, contaminated dust dislodged during hospital construction, carpeting, food, and ornamental plants (4). Members of the genus Aspergillus are opportunistic pathogens; most infections occur in severely immunocompromised patients. Aspergillus fumigatus is the most common agent of systemic infections, followed by Aspergillus flavus (2). Inhalation of fungal conidia is thought to be the primary means of acquiring aspergillosis. Another proposed route of acquisition of Aspergillus spp., specially A. flavus, is through ingestion of contaminated food (10).

Because Aspergillus spp. are ubiquitous in the environment, the simultaneous isolation of the same species from clinical and environmental specimens collected during epidemiological surveillance is not enough to demonstrate a common origin for the isolates. In these cases, only genotyping data provide conclusive evidence of nosocomiality. Although an ideal genotyping procedure applicable to a wide range of Aspergillus species remains to be determined, randomly amplified polymorphic DNA (RAPD) analysis has been shown to be a rapid and reliable method useful for A. fumigatus, A. flavus, and Aspergillus terreus (1, 7, 8, 9, 10).

Here we describe the isolation of one A. flavus strain causing an aortic prosthesis infection three months after aortic valve replacement and two A. flavus strains recovered from a cooling and heating unit used in the operating room. The RAPD patterns of these isolates were compared to check their genetic relatedness. A random selection of eight unrelated strains was used as a control population.

Case report. A 54-year-old female received an aortic prosthesis in October 1998 because of aortic valve insufficiency. In January 1999, she had to be readmitted and operated on again because of an aortic aneurysm in the area next to the prosthesis. At this time, an A. flavus strain was isolated from the prosthesis. Liposomal amphotericin B (AmBisome; Nexstar Pharmaceuticals, Madrid, Spain) at 1 mg/kg of body weight/day was administered for 10 days. Because of the absence of clinical response, the dose of liposomal amphotericin B was increased to 3 mg/kg/day for another 5 days and then to 5 mg/kg/day. Despite this treatment, the patient died.

Environmental surveillance. Air sampling is routinely performed in the room used for heart surgery in this hospital every 2 months by means of a volumetric sampler (1 m³/10 min). In addition, Sabouraud plates are systematically laid on the exits of the two HEPA filters installed in the room. As mentioned above, the prosthesis implantation was performed in October 1998. The previous and subsequent environmental controls performed in the operating room (September and November, respectively) were negative. This means that both the volumetric samples and the controls for the HEPA filters were negative. When A. flavus was isolated from the patient (January 1999), the environmental controls remained negative. Then epidemiological surveillance was conducted by taking samples with cotton-tipped swabs from the surfaces and equipment within the operating room. Two A. flavus strains were cultured from the grilles of the dual-reservoir cooler-heater (Hemotherm; CincinnatiSubzero, Cincinnati, Ohio) usually employed in heart surgery to maintain the extracorporal blood at the proper temperature. This electric device has internal coils filled with water to cool or heat the ducts with the circulating blood. The water is supplied from water pans placed inside the device. The water inside this reservoir was not cultured. Two A. flavus isolates were separately recovered from the cooling-heating unit, one from the upper part of the grille and the other from the lower part (Fig. 1).

View larger version (55K): [in this window] [in a new window]   FIG. 1.   Dual-reservoir cooler-heater (Hemotherm) employed in heart surgery. The arrows indicate the upper and lower parts of the grille, where the two A. flavus strains were isolated.

Mycology. The sources of the isolates discussed in this study are described in Table 1. Isolates 1, 2, and 3 were epidemiologically related strains: isolates 1 and 2 were isolated from the cooling-heating unit in the operating room. Isolate 3 was found on the cardiac prosthesis of the patient. The eight epidemiologically unrelated strains (no. 4, 5, 8, 10, 12, 13, 16, and 19) belonged to the mold collection at the Mycology Reference Laboratory (Instituto de Salud Carlos III, Madrid, Spain).

Molecular typing. Conidia from each strain were inoculated into 3 ml of GYEP broth (2% glucose, 0.3% yeast extract, 1% peptone) and were grown overnight at 37°C. The mycelium mats were recovered and subjected to a DNA extraction protocol described previously (5). After RNase treatment, the samples were treated with proteinase K (Sigma-Aldrich, Alcobendas, Spain), and the DNA was purified again by extraction with phenol-chloroform and ethanol precipitation.

View larger version (71K): [in this window] [in a new window]   FIG. 2.   DNA band patterns obtained with three different arbitrary primers. (A) Primer R-108; (B) primer R-151; (C) primer AP12h. Lane M, 100 bp ladder (Pharmacia); the size in kilobase pairs of the most intense fragment is shown at the left of each panel. Lane m, kilobase pair ladder (Pharmacia), with sizes noted at the right of each panel. The disposition of the strains is the same in the three panels. Lanes 1 and 2, A. flavus strains isolated from the heat exchanger in the operating room; lane 3, A. flavus strain from the aortic prosthesis of the case patient; lanes 4, 5, 8, 10, 12, 13, 16, and 19, unrelated isolates used as controls; lane W, blank control for each PCR procedure.