In-Hospital Source of Airborne Penicillium Species Spores

JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1987, p. 1-4 0095-1137/87/010001-04$02.00/0 Copyright © 1987, American Society for Microbiology

Vol. 25, No. 1

In-Hospital Source of Airborne Penicillium Species Spores ANDREW J. STREIFEL,l POLLY P. STEVENS,'t AND FRANK S. RHAME2 3* Department of Environmental Health and Safety, University of Minnesota Hospital and Clinics,' Infectious Diseases Section, Department of Medicine, and Department of Laboratory Medicine and Pathology, School of Medicine,2* and the Division of Epidemiology, School of Public Health,3 University of Minnesota, Minneapolis, Minnesota 55455 Received 30 December 1985/Accepted 23 September 1986

Since filamentous fungi proliferate in decaying organic debris, it is generally perceived that significant sources of airborne fungal spores are restricted to the outdoors. Protection of people from airborne spores, relevant to the prevention of nosocomial invasive filamentous fungal infection in immunosuppressed patients and allergic manifestations in atopic patients, is felt to be a function of air filtration systems and the prevention of infiltration of unfiltered outside air. Most outbreaks of nosocomial filamentous fungal disease have been attributed to airborne fungi from sources outside of the hospital (8-10, 12, 13). In one outbreak, in which construction within the hospital was implicated (4), the ultimate source of the spores was still probably external to the hospital. Airborne spores entered the hospital, settled, and became airborne again as a result of construction activities. In only one outbreak was a source of fungal growth within the hospital demonstrated (1). A water suspension of cellulose-based fireproofing material was sprayed on girders and concrete. Before the material could dry completely, proliferation and spore formation probably occurred. In contrast to hospitals, in offices and homes there has been greater concern about indoor sources of airborne fungal spores. To reduce the expense of providing heated or cooled air, net air change rates in the home and workplace are decreased by increased weatherproofing. In these settings, concern about indoor air pollution is shifting from outdoor to indoor sources (15). During the course of monitoring airborne fungal spores in our bone marrow transplant unit, a prolonged and marked increase in Penicillium spore levels in corridor air occurred. An in-hospital source was ultimately discovered. We report the details of this fungal spore outburst herein.

MATERIALS AND METHODS

Air sampling. Air sampling at the University of Minnesota Hospital bone marrow transplant unit was carried out between September 1983 and December 1984 as part of research on sources of pathogenic airborne filamentous fungus spores. Reyniers slit air samplers were calibrated at 0.028 m3/min with wet test methods. Simultaneous, 2-hour, 3.4-m3 air samples from outside, a corridor site, and a patient room were obtained one to four times each weekday. Outside air was taken approximately 40 m from the air intake for the air handling system for the bone marrow transplant station. Corridor samples were taken on a platform 1.6 m above the floor in the middle of the bone marrow transplant station, ca. 8.6 m from the medication room. The sampler in the patient room, the door of which was opposite the corridor sampler, was on a platform approximately 84 cm off the ground in the middle of the room. The Falcon sampling plates (150 by 15 mm; BectonDickinson Labware, Oxnard, Calif.) were filled with approximately 70 ml of inhibitory mold agar (BBL Microbiology Systems, Cockeysville, Md.). The sample plates were incubated at 37°C and were evaluated for growth after 48 h. Confirmatory identification of representative fungal isolates was provided by the University of Minnesota Hospital Diagnostic Microbiology Laboratory. Because a variable number of air samples was obtained each day, an arithmetic mean spore content for each day was first calculated and then used to calculate subsequent arithmetic means and standard errors of the mean. Air handling system. The outside air supply intake is at the level of floor 5, facing westward on an internal court. The air is processed through a central 20% efficient (2), roll-type filter and then a point-of-use corridor (high-efficiency particulate air (HEPA) filter (model 105; NuAire Inc., Plymouth, Minn.) with 99.97% reduction of 0.3-,itm particles. The corridor has five supply duct filters, each adjusted to supply 13 m3 fresh air per min and refilter 4 m3 of corridor air per

* Corresponding author. t Present address: University of Minnesota Hospitals, Minneapolis, MN 55455.

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Between 16 July and 1 October 1984, prospectively monitored corridor air samples from a bone marrow transplant station revealed a marked increase in airborne thermotolerant Penicillium spores. Simultaneous cultures of outside air showed lower spore counts, which were unchanged before, during, and after the corridor outburst, establishing that the source was within the hospital. Although the corridor was equipped with recirculating high-efficiency particulate air filtration units which provided 16 air changes per h, the mean corridor air count rose to 64.4 thermotolerant Penicillium CFU/m3 during the outburst period. The in-hospital source was ultimately traced to rotting cabinet wood enclosing a sink with leaking pipes in the medication room. It produced approximately 5.5 x 105 thermotolerant Penicillium CFU/h. In a patient room supplied by corridor air, an in-room recirculating high-efficiency particulate air filter reduced the mean thermotolerant Penicillium count to 2.2 CFU/m3. No patient illness or colonization occurred as a result of this event, although the cabinet wood, after sterilization, was shown to sustain abundant growth of Aspergillusfumigatus and Aspergillusflavus. Wet organic substrates should be avoided in hospital areas with immunosuppressed patients.

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min. The volume of the corridor, including the medication room, is about 327 m3. Thus, the corridor air filters provide a total of 16 filtered-air corridor volumes per h. Barrier doors are located at either end of the bone marrow transplant station, and the air supply is sufficient to pressurize this area. All windows in the station are sealed with duct tape, and the

window air conditioners are operated in the recirculation mode. Patient rooms have no supply air ducts; corridor air makes up for air removed through the bathroom exhaust. The net room air change rate during the period described in this report was 1/h. Within each patient room is a free-standing HEPA filter (model MS685; Dexon, Inc., St. Louis Park, Minn.) operating at 28 m3/min. In the sampled patient room, which had a volume of 35 m3, the HEPA filter provided 30 filtered-air room volumes per h. Medication room sink experiment. To evaluate the medication room for spore sources, we brought into the room a portable HEPA filter and air samplers. The room was sealed with duct tape, and the sink cabinet doors were closed and taped. The filter was operated for approximately 20 room air changes and then turned off. Immediately after reducing the ambient spore levels with the air filtration, a 5-min control air sample was taken. A second sample was taken 30 min later. The experiment was then repeated with the sink cabinet doors opened. Six-stage Andersen air samplers at 0.028 m3/min were used. Positive hole count corrections (3) were made when indicated. Anterior nares cultures. Routine weekly anterior nares swab cultures from all bone marrow transplant patients are plated on Sabouraud dextrose agar and evaluated for the presence of Aspergillus spp. and phycomycetes by the Diagnostic Microbiology Laboratory. Between 6 August and 15 November 1984, all anterior nares cultures were also specifically evaluated for Penicillium spp. RESULTS During the week beginning 16 July 1984, prospectively monitored bone marrow transplant station corridor air counts of thermotolerant fungi abruptly rose and remained elevated (Fig. 1). The mean corridor count during this outburst period was 68.2 CFU/m3 (range, 5.8 to 162.3), of

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Time Period FIG. 2. Mean total thermotolerant fungi and Penicillium species in outdoor and corridor air for the periods before, during, and after the Penicillium outburst. Differences between outdoor and corridor thermotolerant fungi (-) and Penicillium species (0) were significant (t test, P < 0.001) for three time periods: 1, 1 June 1984 to 13 July 1984; 2, 16 July 1984 to 21 September 1984; 3, 24 September 1984 to 1 December 1984.


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which 64.4 CFU/m3 (range, 5.8 to 162.0) were thermotolerant Penicillium spp. Although all the supply air to the corridor was HEPA filtered, infiltration through inadequately sealed corridor windows could have caused the increased levels, but the levels for the corridor air were persistently higher than those for the outdoor air (Fig. 1). Moreover, there was no change in the outside thermotolerant CFU before, during, or after the outburst (Fig. 2A). Likewise, during and after the outburst, when outdoor plates were specifically evaluated for Penicillium spp., the outdoor thermotolerant Penicillium CFU remained constant. The corridor outburst of thermotolerant fungi was entirely due to Penicillium spores (Fig. 2B). While several unproductive hypotheses were being evaluated, a nurse requested repair of a leaking sink in a wooden cabinet in the medication room. Inspection revealed rotting cabinet wood. An experiment was performed in the closed and sealed medication room to characterize spore production from cabinet wood. With the cabinet doors closed and taped, a portable HEPA filter was operated for 20 air changes and then turned off. A base-line level of 88 thermotolerant fungal CFU/m3 was established. After 30 min, it had risen to 1,480 thermotolerant fungal CFU/m3. With the cabinet doors open, the base-line level could be reduced only to 812 thermotolerant fungal CFU/m3, which rose to 8,613 CFU/m3 30 min later. An Andersen air sampler placed in the cabinet revealed a concentration of 7.9 x 104 thermotolerant fungal CFU/m3. Virtually all of the thermotolerant CFU were Penicillium spp. At least six Penicillium species were present. A scanning electron micrograph of the rotting wood (Fig. 3) revealed abundant filamentous fungal growth morphologically consistent with Penicillium spp. When this wood was steam autoclaved and inoculated with Aspergillus fumigatus or Aspergillus flavus, abundant growth occurred. During the outburst interval, 77 simultaneous triplet air samples (2 h each, 270 m3 total at each site) were obtained

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from outside, in the corridor, and in the patient room. The mean corridor total thermotolerant fungal spore count was 68.2 CFU/m3, and the thermotolerant Penicillium count was 64.4 CFU/m3 (Fig. 2B). In the patient room, counts were, respectively, 2.9 and 2.2 CFU/m3. During the outburst interval, 109 nasal swab cultures were obtained from 28 patients on the bone marrow transplant station. The mycology section medical technologists recalled on 6 August no prior isolation of Penicillium CFU from the 24 cultures; from the remaining 85 cultures, they specifically sought, but recovered only 1 CFU from 1 of 6 cultures from one patient. No clinical illness due to Penicillium spp. has ever occurred among our bone marrow transplant patients. A rise in thermotolerant fungal CFU of 7,800 m3 in 30 min in the medication room (volume = 35 m3) indicated a spore production rate of 5.5 x 105 CFU/h. Predictive calculations about spore dissemination may be made by using a formula derived as follows. In a chamber of volume V, where spores are being produced at rate P, with an air change rate A, the formula for the concentration C of spores at time t is described by the following differential equation: dC = PIV C A dt. Integration yields C = (P/AV) - (J/A)e&A,. The expected corridor concentration of thermotolerant Penicillium sp. at steady state (t = c), presuming V = 327 m3, P = 5.5 x 105 CFU/h, and A = 16/h is 108 CFU/m3. The measured value was 68.2 CFU/m3. The rate of introduction, P, of spores into the patient room, calculated from the concentration of spores in the corridor (68.2 CFU/m3) and the bathroom exhaust rate (3.8 m3/h) is 129 CFU/h. The expected patient room concentration at steady state, presuming V = 35 m3, P = 129 CFU/h, and A = 30/h, is 0.15 -

CFU/m3. The measured value was 2.9 CFU/m3.

DISCUSSION Air sampling on our bone marrow transplant station has permitted a detailed description of an outburst of airborne Penicillium spores, which was ultimately traced to rotting wood in a cabinet under a sink with leaking pipework in the medication room. The source produced about 5.5 x 105 thermotolerant Penicillium CFU/h. Airborne spore counts in the corridor 8.6 m from the medication room of the implicated sink averaged 64.4 thermotolerant Penicillium CFU/m3. The expected count, based on the corridor volume, spore production rate, and overall corridor air change rate of 16/h, from a formula which presumes perfect air mixing in the corridor, was 108 CFU/m3. The low observed mean corridor air count probably occurred because there was a recirculating corridor HEPA filter between the sampler and the medication room. In the patient room, with the entrance door near the corridor sampler, the mean thermotolerant Penicillium spore count was only 2.2 CFU/m3, pointing out the beneficial effect of the recirculating in-room HEPA filters. The predicted room spore concentration was 0.15 CFU/m3, well below the observed rate. This probably reflects enhanced corridor-to-room air mixing, resulting from frequent entrances into the patient room by personnel. These visits averaged about 5/h during the day. We verified this mixing effect with a particle counter at a time when the patient rooms had HEPA filters, but the corridor did not. The rise in particle counts was immediate and dramatic after someone entered the patient room, bringing in a wake of corridor air. The increase in Penicillium spores when the medication cabinet doors were shut reflects exposure of the rotting surfaces in the back of the cabinet. The apparent decrease in


FIG. 3. Scanning electron micrograph of wood obtained from the rotting sink cabinet. The fungal organisms are morphologically consistent with Penicillium spp. Original magnification, x 1,000.

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ACKNOWLEDGMENTS We thank the Mycology Section, Diagnostic Microbiology Laboratory, University of Minnesota Hospital, for confirming fungal identities and processing nasal surveillance cultures; the nursing staff of the bone marrow transplant station for facilitating exclusive access to the medication room; Patricia Ferrieri for reviewing the

manuscript; Su Reaney for help in data compilation; and Leesa Schofield for word processing assistance. LITERATURE CITED 1. Aisner, J., S. C. Schimpff, J. E. Bennett, V. M. Young, and P. H. Wiernik. 1976. Aspergillus infections in cancer patients: association with fireproofing materials in a new hospital. J. Am. Med. Assoc. 235:411-412. 2. American Society of Heating, Refrigeration, and Air-Conditioning Engineers. Method of testing air-cleaning devices used in general ventilation for removing particulate matter. Standard 52-76. American Society of Heating, Refrigeration, and AirConditioning Engineers, Atlanta. 3. Andersen, A. A. 1958. New sampler for the collection, sizing, and enumeration of viable airborne particles. J. Bacteriol. 76:471-484. 4. Arnow, P. M., R. L. Anderson, P. D. Mainous, and E. J. Smith. 1978. Pulmonary aspergillosis during hospital renovation. Am. Rev. Respir. Dis. 118:49-53. 5. Arnow, P. M., J. N. Fink, D. P. Schlueter, J. J. Barboriak, G. Mallison, S. I. Said, S. Martin, G. F. Unger, G. T. Scanlon, and V. P. Kurup. 1978. Early detection of hypersensitivity pneumonitis in office workers. Am. J. Med. 64:236-242. 6. Bernstein, R. S., W. G. Sorenson, D. Garabrant, C. Reaux, and R. D. Treitman. 1983. Exposures to respirable, airborne Penicilhum from a contaminated ventilation system: clinical, environmental and epidemiological aspects. Am. lnd. Hyg. Assoc. J. 44:161-169. 7. Hirsch, D. J., S. R. Hirsch, and J. H. Kalbfleisch. 1978. Effect of central air conditioning and meteorologic factors on indoor spore counts. J. Allergy Clin. Immunol. 62:22-26. 8. Kyriakides, G. K., H. H. Zinneman, W. H. Hall, V. K. Arora, J. Lifton, W. C. DeWolf, and J. Miller. 1976. Immunologic monitoring and aspergillosis in renal transplant patients. Am. J. Surg.

131:246-252. 9. Lentino, J. R., M. A. Rosenkranz, J. A. Michaels, V. P. Kurup, H. D. Rose, and M. W. Rytel. 1982. Nosocomial aspergillosis: a retrospective review of airborne disease secondary to road construction and contaminated air conditioners. Am. J. Epidemiol. 116:430-437. 10. Mahoney, D. H., Jr., C. P. Steuber, K. A. Starling, F. F. Barrett, J. Goldberg, and D. J. Fernbach. 1979. An outbreak of aspergillosis in children with acute leukemia. J. Pediatr. 95:70-72. 11. Rippon, J. W. 1974. Medical mycology: the pathogenic fungi and the pathogenic actinomycetes. The W. B. Saunders Co.,

Philadelphia. 12. Rotstein, C., K. M. Cummings, J. Tidings, K. Killion, E. Powell, T. L. Gustafson, and D. Higby. 1985. An outbreak of invasive aspergillus among allogeneic bone marrow transplants: a casecontrol study. Infect. Control 6:347-355. 13. Sarubbi, F. A., Jr., H. B. Kopf, M. B. Wilson, M. R. McGinnis, and W. A. Rutala. 1982. increased recovery of Aspergillus flavus from respiratory specimens during hospital construction. Am. Rev. Respir. Dis. 125:33-38. 14. Solley, G. O., and R. E. Hyatt. 1980. Hypersensitivity pneumonitis induced by Penicillium species. J. Allergy Clin. Immunol. 65:65-70. 15. Spendglove, J. D., and K. F. Fammin. 1983. Source, significances, and control of indoor microbial aerosols: human health aspects. Public Health Rep. 98:229-244.


corridor non-Penicillium thermotolerant spores during the outburst period (Fig. 2B) probably reflects overgrowth of the sampling plates by the Penicillium colonies during the outburst period with artifactual undercounting of the nonPenicillium fungal colonies. A similar effect probably accounts for the apparent higher fraction of total fungal colonies which were Penicillium spp. from the corridor (94%) compared with the patient room (76%). Clinical illness due to proliferating Penicillium spp. is quite rare (11), particularly in view of the ubiquity of spores of this genus. Hypersensitivity pneumonitis due to Penicillium spp. definitely occurs (14). The event described in our report was not recognized as producing invasive disease, hypersensitivity reactions, or nasal colonization. However, in one instance of office hypersensitivity pneumonitis, airborne Penicillium levels above 1,000 CFU/m3 were demonstrated (6). We did, however, demonstrate that the rotting wood involved could support the growth of A. fumigatus and A. flavus. It may have been fortuitous that a less pathogenic species colonized the wood, although it is more likely that Penicillium spp. predominated because of differential abilities to use the substrate, exposure to materials poured down the drain, temperature, any preservatives in the wood, or other unknown factors. We are aware of only one previous demonstration of an in-hospital focus of proliferation of filamentous fungi (1). In that outbreak, water and an organic substrate were also involved. We have found, in another area of our hospital, Aspergillus niger proliferation on the paper surface of rainwater-soaked wallboard. Allergists and students of indoor air pollution have also sought evidence of indoor fungal proliferation. Large-scale ventilation systems with open water sprays for humidification or cooling have been identified as sources for Penicillium spores (5) and other allergens. In homes, however, air conditioners appear to reduce airborne fungal counts as long as windows are kept closed (7). All these experiences together suggest that organic material inside the hospital must be kept dry. Ironically, handwashing sinks may be a particular problem because of splashing. Areas around sinks should be waterproofed. Water leaks, which inevitably occur, require prompt attention. Any resultant moistened organic debris in hospital areas with immunosuppressed patients probably requires special attention. Prompt drying, removal, or application of fungicides, such as copper-8-quinolinolate, should be considered.