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By Mary Jo Vesper and Stephen Vesper
According to the Centers for Disease Control and Prevention(CDC), about 2 million patients develop nosocomial infections each year.Although fungal infections represent a small part of these, they cause adisproportionately high percentage of the fatal infections. For example, Dr.John Perfect and colleagues studied the outcome of aspergillosis cases at 24medical centers. More than half of the patients with invasive aspergillosis died within three months of a positive culture.1The mortality rate due to invasive aspergillosis increased by 357 percent between 1980 and 1997.2
Nosocomial fungal infections present a persistent threat inhospitals. Fungi co-habit indoor environments, yet we often ignore theirpotential to become opportunistic invasive pathogens. The major limitation in controlling hospital fungalenvironments has been monitoring fungi in a real-time manner. Traditionally,fungal monitoring has been performed by plate culturing. This century-oldprocess often takes weeks to complete.
In addition, the standard air sampling method used withculture plates limits the sampling to a few minutes duration, often insufficient to sample the air adequately. Thio et al3clearly demonstrated this in their 2000 study. Plate culturingis also selective for only a few of the fungal species that pose threats; manyfungal species do not grow or do not reproduce in standard culture conditions.Fungal reproductive structures must appear in culture to make positive identification of fungal species. With the limitations of the plate culture method, itis problematic to rely on plate cultures when searching for opportunisticpathogens among indoor fungi.
Recent technological advances now allow us to break themold in detection and identification of fungi in hospital indoorenvironments. The time and sampling limitations of antiquated fungal culturetechniques have been obviated by development of a DNA-based method that can beused for the identification and quantification of more than 100 species offungi, including the pathogenic Aspergillus and Candida species.The new methodology, fungal quantitative polymerase chain reaction (QPCR), issimple, fast, inexpensive, accurate and reproducible.
The first tests of using fungal QPCR in hospital settingswere published recently. Articles appearing in the Journal of HospitalInfection4 and Infection Control and Hospital Epidemiology5documented how QPCR was applied to achieve rapid and accurate fungal detectionand quantification during two construction and renovation projects. With fungalQPCR technology verified for use in hospital settings by these reports,hospital infection control and clinical personnel can now have confidence intheir ability to monitor conveniently many hospital environments for potentialfungal pathogens.
Fungi thrive in a wide array of indoor environmental nichesand are considered natural components of indoor ecosystems. These indoor co-habitants can become dangerous pathogens forcertain patient populations (e.g., immuno-compromised patients). Unlike mostpathogenic bacteria, many pathogenic fungi live and multiply with ease onsubstrates other than the host organism. Yet when encountering an unprotectedhuman host, they can invade and establish their aggressive growth patterns withdisastrous consequences. Hospital environments, therefore, need specialattention for monitoring fungi.
We will review here the two cases recently published; they arenoteworthy because each presents elements of possible quality assuranceprotocols that could be established for environmental monitoring during hospitalconstruction and renovation. The first case illustrates the monitoring forfungal contamination during the construction of a new hospital wing. The second case reviews fungal monitoring during a renovationproject in which large areas of carpeting were replaced within a hospitaldedicated to treating burn patients.
In the first application, construction materials had beenexposed to high moisture levels from rain and were used while still wet. Thispractice is not uncommon for construction projects. In this case, visible fungalgrowth became apparent on some of these materials. This situation had thepotential for significant consequences because the new wing would house a newoperating room (OR) and neonatal intensive care unit (NICU). Hospital officialsknew that patient populations to be treated in the new wing were highlysusceptible to opportunistic fungal infections. Wisely, the consulting engineersremoved By Mary Jo Vesper and Stephen Vesperand replaced the visibly contaminated material; in addition,they also decided to check for hidden contamination. Since it was imperativethat construction be kept to the time schedule, the engineers chose to use thenew fungal QPCR technology for their analysis. Their decision was driven by apowerful threesome: the rapid availability of results (within 24 hours); theyield of quantitative data to show relative abundance; and, the species-specificity of the analysis.
For this project, both air and surface samples were tested forfungal contamination. Air was sampled at multiple locations for three hours withan air sampler pump pulling 3.5 liters per minute, and surface dust samples werealso collected at multiple locations. All three floors of the construction sitewere sampled by these methods, and samples were shipped overnight to acommercial laboratory for fungal QPCR analysis. Results showed hidden contamination to be highly localized tothe second floor, where several Aspergillus species were detected. Theoffending construction material was identified, removed and replaced.Construction then continued uninterrupted and was completed on schedule.
Since the second floor of the new wing would house both ORand NICU suites, consulting engineers decided to perform a QPCR check forresidual Aspergillus spores near the end of construction during the finishing stages. Although workers removed construction materials that weremarkedly contaminated by fungi early in construction, high concentrations ofseveral Aspergillus species, including A. fumigatus and A.niger, were still detected in surface dust samples at the fi nishing stage.
After this discovery, standard disinfection cleanings wereperformed consisting of scrubbing the fl oors and walls using mechanicalfriction with hospital-grade quaternary ammonia disinfectant. QPCR was usedduring the disinfection process to monitor the progress of decontamination andto confirm the elimination of Aspergillus. It was likely that spread offungal contamination from the earliest stages of construction impacted the newunit, and fortunately was detected and eliminated.
Alerted by the presence of fungal contamination at two timepoints during the construction process, consulting engineers also chose to usepost-construction, post-finishing monitoring by QPCR throughout the newlyconstructed wing. At this terminal phase, monitoring revealed the OR and NICUsuites were again contaminated with a number of Aspergillus species,including A. fl avus, a species not found earlier on this project. Thenew carpeting was thought the probable source for this post-constructioncontamination. Carpet is a known haven for fungi in indoor environments. 6 Finalcleaning procedures then focused on the carpet and included extensive HEPAvacuuming. This process dramatically reduced the Aspergillus burden, asmonitored by fungal QPCR.
The application of QPRC on this hospital construction projectalso provided insight into the development of methods that might become standardprocedures for monitoring air, surfaces and other media for fungal contaminantsduring the time course of building construction. With the advent of the rapid detection process of QPCR,monitoring for fungal contaminants at critical time points in construction andfinishing has become possible. Surveillance of air and surfaces for fungi canalso be much more thorough than can be obtained with the old standard of platecultures.
The second case study of the application of fungal QPCRinvolves a renovation project. The working environment was a hospital dedicatedto the treatment of burn patients. During renovation, the removal of a10-year-old carpet became necessary. As noted earlier, carpet is known as acommon reservoir for fungi indoors. Suspecting a high load of fungi might befound in the old carpet, hospital officials used every precaution to preventaerosolization of the fungi during the project. Burn patients are highlysusceptible to infection by aerosolized fungi such as Aspergillus.
The hospitals procedures for carpet removal included thefollowing steps: The carpet was thoroughly shampooed using a quarternaryammonium disinfectant cleaner before removal began, and all patients weretemporarily relocated to another fl oor. To determine how long it would bebefore the patients could return safely, workers conducted environmentalmonitoring of the entire area. This hospitals standard procedure formonitoring fungi specified use of settle plates and/or plates from an impingingair sampler. These plates are incubated until fungal species grow, reproduce andsubsequent identification is made. This time period extends as long as twoweeks for slow growing species that do not produce their species-specificreproductive structures in less time. Due to time constraints and the need for ahighly sensitive detection method, fungal QPCR was chosen to overcome thelimitations of the standard plate culture methods. Furthermore, the positivefeatures of QPRC made it possible to use this rapid monitoring method throughoutthe carpet removal process. Prior to the start of the carpet removal, airsamplers were placed around the carpet removal area, and pre-removal air sampleswere taken for baseline determinations. Since the primary concern was foraerosolization of fungi, no surface samples were taken for this study.
In this project, A. niger was the predominant fungalspecies, and tracking its prevalence was easily done with fungal QPCR. Analysisof the air drawn for a baseline study showed only very low levels of A. niger.Air monitoring continued during the carpet removal process, and the fungal QPCRanalysis showed the A. niger concentration jumped nearly 10-fold as thework proceeded. Then, with old carpet gone and the new flooring incomplete,decreasing levels of A niger were tracked in the air samples.
After the new non-carpet flooring was installed and the areacleaned and disinfected according to standard procedures, no Aspergillus sporeswere detected in the air samples. Patients were returned to the renovated areaearlier than anticipated, and the risk of infection from aerosolized fungi inthe environment was greatly diminished.
For both of these hospital projects, QPCR data were availablein a matter of hours after samples were received. Hospital infection controlmanagers had information on fungal presence many days sooner than with thetraditional plate culture methods. With this rapid turnaround in analysis, itbecame possible to monitor for fungal contaminants at the beginning of theproject, at a midpoint (or during various critical points) and at the end as acheck on the clean-up process without delaying project work.
The studies published and described above dealt with air andsurface samples, but fungi lurk in more niches than hospital air and floorsurfaces. Anaisse et al7 showed that hospital water can be a source offungi related to fungal infections, especially aspergillosis. The authorsfound Aspergillus strains in the showerheads of patients rooms, andthese matched the strains isolated from patients Aspergillus infections.The association of indoor fungal growth and moisture is well documented, and itis not surprising that opportunistic pathogens were found among the fungi present. Water samples can easily be analyzed by fungal QPCRas well,8 and monitoring the fungal environments of seriously illpatients can now be considered a quite feasible.
Clinicians are increasingly concerned that someinfections labeled nosocomial may actually originate from home exposures.9With the ease of sample collection involved in fungal QPCR, monitoring the homeenvironment of immuno-compromised patients is now a possibility. This step can beconsidered when patients will be recuperating at home for extended periods.
If fungal QPCR sounds like a good method to add to yourhospital infection control protocols, how can it be implemented? There are twooptions: Set up the analysis in-house, or use a contract laboratory. For thosehospitals interested in developing their own capacity to perform QPCR, thefollowing considerations are offered. QPCR is a DNA-based detection method, andthe sequence detection (SD) instrument critical for the QPCR method is probablythe largest expense to be incurred.
There are several manufacturers of SDs and the instrumentsrange in price from about $40,000 to $80,000.Various SD formats are availablefor running the analyses. The most common format is the 96-well reaction plate;in other words, one can perform 96 analyses in one run. QPCR SD equipment canalso be used in analyses for other pathogens including viruses. New molecularprobes continue to be constructed and added to the lists available from vendorsof these technologies.
Desiring to avoid setting up a QPCR lab and concomitanttraining of staff, hospitals may choose an alternative option (i.e., using apay-per-sample service). There are currently about 15 licensed companies in theU.S. and the E.U. that can provide skilled analyses of samples. Since thetechnology is new, the number of contract laboratories continues to grow. Thesamples from the hospital construction project described here were analyzed byP&K Microbiology Services. For this company and others, with overnightshipping, one can usually count on having results in hand the next day. Thelocation and contact information for the various companies can be found at theWebsite http://www.epa.gov/nerlcwww/moldtech.htm.
Fungal QPCR offers a method of high sensitivity for specificidentification of numerous fungal pathogens in air and other samples. Sensitivity is generally at the level of a single spore. QPCRalso provides rapid analysis results are available in two to three hours.Because of these tremendously beneficial features, fungal QPCR is clearlycreating a new standard in fungal detection and quantitation. With this newstandard in hand, hospitals can consider a wide array of applications, such asimplementation of fungal monitoring as part of standard procedures for infectioncontrol.
Note: Environmental Protection Agency, through its Office ofResearch and Development, collaborated in this research. It has been subjectedto agency review and approved for publication.
Mary Jo Vesper has a PhD in biological sciences and has been auniversity professor and dean. She is currently working under a grant from theEPA as a science writer/editor and science communication specialist.
Stephen Vesper has a PhD in environmental microbiology and isa research scientist with the EPA in Cincinnati. He has been active indeveloping new methods of detection and quantification for indoor.