It's Time To Break The Mold

It's Time To Break The Mold

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 a disproportionately high percentage of the fatal infections. For example, Dr. John Perfect and colleagues studied the outcome of aspergillosis cases at 24 medical centers. More than half of the patients with invasive aspergillosis died within three months of a positive culture.1 The mortality rate due to invasive aspergillosis increased by 357 percent between 1980 and 1997.2

Nosocomial fungal infections present a persistent threat in hospitals. Fungi co-habit indoor environments, yet we often ignore their potential to become opportunistic invasive pathogens. The major limitation in controlling hospital fungal environments has been monitoring fungi in a real-time manner. Traditionally, fungal monitoring has been performed by plate culturing. This century-old process often takes weeks to complete.

In addition, the standard air sampling method used with culture plates limits the sampling to a few minutes duration, often insufficient to sample the air adequately. Thio et al3 clearly demonstrated this in their 2000 study. Plate culturing is also selective for only a few of the fungal species that pose threats; many fungal 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, it is problematic to rely on plate cultures when searching for opportunistic pathogens among indoor fungi.

Recent technological advances now allow us to break the mold in detection and identification of fungi in hospital indoor environments. The time and sampling limitations of antiquated fungal culture techniques have been obviated by development of a DNA-based method that can be used for the identification and quantification of more than 100 species of fungi, including the pathogenic Aspergillus and Candida species. The new methodology, fungal quantitative polymerase chain reaction (QPCR), is simple, fast, inexpensive, accurate and reproducible.

The first tests of using fungal QPCR in hospital settings were published recently. Articles appearing in the Journal of Hospital Infection4 and Infection Control and Hospital Epidemiology5 documented how QPCR was applied to achieve rapid and accurate fungal detection and quantification during two construction and renovation projects. With fungal QPCR technology verified for use in hospital settings by these reports, hospital infection control and clinical personnel can now have confidence in their ability to monitor conveniently many hospital environments for potential fungal pathogens.

Fungi thrive in a wide array of indoor environmental niches and are considered natural components of indoor ecosystems. These indoor co-habitants can become dangerous pathogens for certain patient populations (e.g., immuno-compromised patients). Unlike most pathogenic bacteria, many pathogenic fungi live and multiply with ease on substrates other than the host organism. Yet when encountering an unprotected human host, they can invade and establish their aggressive growth patterns with disastrous consequences. Hospital environments, therefore, need special attention for monitoring fungi.

We will review here the two cases recently published; they are noteworthy because each presents elements of possible quality assurance protocols that could be established for environmental monitoring during hospital construction and renovation. The first case illustrates the monitoring for fungal contamination during the construction of a new hospital wing. The second case reviews fungal monitoring during a renovation project in which large areas of carpeting were replaced within a hospital dedicated to treating burn patients.

In the first application, construction materials had been exposed to high moisture levels from rain and were used while still wet. This practice is not uncommon for construction projects. In this case, visible fungal growth became apparent on some of these materials. This situation had the potential for significant consequences because the new wing would house a new operating room (OR) and neonatal intensive care unit (NICU). Hospital officials knew that patient populations to be treated in the new wing were highly susceptible to opportunistic fungal infections. Wisely, the consulting engineers removed By Mary Jo Vesper and Stephen Vesper and replaced the visibly contaminated material; in addition, they also decided to check for hidden contamination. Since it was imperative that construction be kept to the time schedule, the engineers chose to use the new fungal QPCR technology for their analysis. Their decision was driven by a powerful threesome: the rapid availability of results (within 24 hours); the yield of quantitative data to show relative abundance; and, the species-specificity of the analysis.

For this project, both air and surface samples were tested for fungal contamination. Air was sampled at multiple locations for three hours with an air sampler pump pulling 3.5 liters per minute, and surface dust samples were also collected at multiple locations. All three floors of the construction site were sampled by these methods, and samples were shipped overnight to a commercial laboratory for fungal QPCR analysis. Results showed hidden contamination to be highly localized to the second floor, where several Aspergillus species were detected. The offending 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 OR and NICU suites, consulting engineers decided to perform a QPCR check for residual Aspergillus spores near the end of construction during the fi nishing stages. Although workers removed construction materials that were markedly contaminated by fungi early in construction, high concentrations of several 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 were performed consisting of scrubbing the fl oors and walls using mechanical friction with hospital-grade quaternary ammonia disinfectant. QPCR was used during the disinfection process to monitor the progress of decontamination and to confirm the elimination of Aspergillus. It was likely that spread of fungal contamination from the earliest stages of construction impacted the new unit, and fortunately was detected and eliminated.

Alerted by the presence of fungal contamination at two time points during the construction process, consulting engineers also chose to use post-construction, post-finishing monitoring by QPCR throughout the newly constructed wing. At this terminal phase, monitoring revealed the OR and NICU suites were again contaminated with a number of Aspergillus species, including A. fl avus, a species not found earlier on this project. The new carpeting was thought the probable source for this post-construction contamination. Carpet is a known haven for fungi in indoor environments. 6 Final cleaning procedures then focused on the carpet and included extensive HEPA vacuuming. This process dramatically reduced the Aspergillus burden, as monitored by fungal QPCR.

The application of QPRC on this hospital construction project also provided insight into the development of methods that might become standard procedures for monitoring air, surfaces and other media for fungal contaminants during 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 and finishing has become possible. Surveillance of air and surfaces for fungi can also be much more thorough than can be obtained with the old standard of plate cultures.

The second case study of the application of fungal QPCR involves a renovation project. The working environment was a hospital dedicated to the treatment of burn patients. During renovation, the removal of a 10-year-old carpet became necessary. As noted earlier, carpet is known as a common reservoir for fungi indoors. Suspecting a high load of fungi might be found in the old carpet, hospital officials used every precaution to prevent aerosolization of the fungi during the project. Burn patients are highly susceptible to infection by aerosolized fungi such as Aspergillus.

The hospitals procedures for carpet removal included the following steps: The carpet was thoroughly shampooed using a quarternary ammonium disinfectant cleaner before removal began, and all patients were temporarily relocated to another fl oor. To determine how long it would be before the patients could return safely, workers conducted environmental monitoring of the entire area. This hospitals standard procedure for monitoring fungi specified use of settle plates and/or plates from an impinging air sampler. These plates are incubated until fungal species grow, reproduce and subsequent identification is made. This time period extends as long as two weeks for slow growing species that do not produce their species-specific reproductive structures in less time. Due to time constraints and the need for a highly sensitive detection method, fungal QPCR was chosen to overcome the limitations of the standard plate culture methods. Furthermore, the positive features of QPRC made it possible to use this rapid monitoring method throughout the carpet removal process. Prior to the start of the carpet removal, air samplers were placed around the carpet removal area, and pre-removal air samples were taken for baseline determinations. Since the primary concern was for aerosolization of fungi, no surface samples were taken for this study.

In this project, A. niger was the predominant fungal species, and tracking its prevalence was easily done with fungal QPCR. Analysis of 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 QPCR analysis showed the A. niger concentration jumped nearly 10-fold as the work 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 area cleaned and disinfected according to standard procedures, no Aspergillus spores were detected in the air samples. Patients were returned to the renovated area earlier than anticipated, and the risk of infection from aerosolized fungi in the environment was greatly diminished.

For both of these hospital projects, QPCR data were available in a matter of hours after samples were received. Hospital infection control managers had information on fungal presence many days sooner than with the traditional plate culture methods. With this rapid turnaround in analysis, it became possible to monitor for fungal contaminants at the beginning of the project, at a midpoint (or during various critical points) and at the end as a check on the clean-up process without delaying project work.

The studies published and described above dealt with air and surface samples, but fungi lurk in more niches than hospital air and floor surfaces. Anaisse et al7 showed that hospital water can be a source of fungi related to fungal infections, especially aspergillosis. The authors found Aspergillus strains in the showerheads of patients rooms, and these matched the strains isolated from patients Aspergillus infections. The association of indoor fungal growth and moisture is well documented, and it is not surprising that opportunistic pathogens were found among the fungi present. Water samples can easily be analyzed by fungal QPCR as well,8 and monitoring the fungal environments of seriously ill patients can now be considered a quite feasible.

Clinicians are increasingly concerned that some infections labeled nosocomial may actually originate from home exposures.9 With the ease of sample collection involved in fungal QPCR, monitoring the home environment of immuno-compromised patients is now a possibility. This step can be considered when patients will be recuperating at home for extended periods.

If fungal QPCR sounds like a good method to add to your hospital infection control protocols, how can it be implemented? There are two options: Set up the analysis in-house, or use a contract laboratory. For those hospitals interested in developing their own capacity to perform QPCR, the following considerations are offered. QPCR is a DNA-based detection method, and the sequence detection (SD) instrument critical for the QPCR method is probably the largest expense to be incurred.

There are several manufacturers of SDs and the instruments range in price from about $40,000 to $80,000.Various SD formats are available for 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 can also be used in analyses for other pathogens including viruses. New molecular probes continue to be constructed and added to the lists available from vendors of these technologies.

Desiring to avoid setting up a QPCR lab and concomitant training of staff, hospitals may choose an alternative option (i.e., using a pay-per-sample service). There are currently about 15 licensed companies in the U.S. and the E.U. that can provide skilled analyses of samples. Since the technology is new, the number of contract laboratories continues to grow. The samples from the hospital construction project described here were analyzed by P&K Microbiology Services. For this company and others, with overnight shipping, one can usually count on having results in hand the next day. The location and contact information for the various companies can be found at the Website

Fungal QPCR offers a method of high sensitivity for specific identification of numerous fungal pathogens in air and other samples. Sensitivity is generally at the level of a single spore. QPCR also provides rapid analysis results are available in two to three hours. Because of these tremendously beneficial features, fungal QPCR is clearly creating a new standard in fungal detection and quantitation. With this new standard in hand, hospitals can consider a wide array of applications, such as implementation of fungal monitoring as part of standard procedures for infection control.

Note: Environmental Protection Agency, through its Office of Research and Development, collaborated in this research. It has been subjected to agency review and approved for publication.

Mary Jo Vesper has a PhD in biological sciences and has been a university professor and dean. She is currently working under a grant from the EPA as a science writer/editor and science communication specialist.

Stephen Vesper has a PhD in environmental microbiology and is a research scientist with the EPA in Cincinnati. He has been active in developing new methods of detection and quantification for indoor.

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