By David Kahane, CIH, MPH
Forensic Science was one of the earliest applied sciences to utilize DNA-based methods to identify biological evidence left behind at crime scenes and individualize that evidence to suspects or victims. It has revolutionized criminal procedure, uncovered limitations of earlier methods of biological evidence analysis, and has essentially become the forensic gold standard after intense scrutiny in the courts.
This gold standard has now made its appearance and use in the world of Environmental Microbiology, compliments of the EPA's National Exposure Research Branch and their technology transfer initiatives. Two researchers, Drs. Stephen Vesper and Richard Haugland have introduced the use of real-time Polymerase Chain Reaction (PCR) techniques to the identification and speciation of over 100 fungi, as well as bacteria associated with water intrusion problems in buildings1. The method has recently been employed in the evaluation of remediation associated with the infamous Cleveland homes2. Further, this group has established licensing agreements with a limited number of laboratories, with the expressed intent of commercializing these methods. The use of real-time PCR methods is already available to the mold market from some of these laboratories.
The underlying concepts of real time PCR are presented elsewhere, and the undeniable fact is that the genetic fingerprinting already adopted in Forensic Science is ready for adoption by all parties in the mold market.
Why PCR?
The most obvious advantages of PCR methods are sensitivity, reproducibility and rapid turnaround. In a world where culture-based laboratory methods force decisions regarding abatement and risk assessment to be put on hold for a week to two, a method that allows for fast speciation in any matrix is desperately needed. Can we replace culture-based (and in some cases non-culture based methods), in the day-to-day real world situations faced by mold practitioners? Without a doubt, here are some of the possibilities.
Air Sampling
Discussions with many experts indicate that spore trapping methods are intensively used for their speed and ease of use, however all parties note the limitation of speciation, causing the necessity for culture samples to be run simultaneously in various situations. These culture samples are run regardless of the concentrations or species that might be found in the sampled environment. While they are run for determination of "viability", most experts admit that they are run to help clarify whether Penicillium or Aspergillus, or other small fungi are present since they cannot be easily identified by direct microscopic methods.
Imagine sampling just with a spore trap, having it run by direct examination initially, and then performing a follow-up analysis for selected species when indicated, on the same sample. As regulations become established, one can expect an OSHA exposure standard for abatement workers just as with other chemical or biological contaminants. Imagine the ability to quantitate an 8-hour time weighted average for fungi. Additionally, clearance air or bulk standards will likely be established by a federal or state agency given their past approaches to other building related contaminants such as asbestos or lead. Sampling for hours, not minutes would likely be part of that standard. These scenarios could never be possible with direct microscopic methods, but are available today with PCR methods.
Bulk, Surface or Micro-vacuum Samples
Some experts enjoy employing surface sampling to establish contamination areas for cleanup, determine historical deposition, evaluate abatement completion, or to assess re-occupancy criteria. The drawback to these methods again reside in the conflict of the superiority of culture based methods for fungal enumeration and identification over direct microscopic methods, versus the seven day minimum waiting time for such data. Using PCR methods will allow for experts to incorporate bulk criteria, a much better predictor than airborne criteria, into their inspection and abatement planning.
Laboratory Traceability
Anyone actively involved in the mold remediation market knows that laboratories are stretched to the limit in meeting present day turnaround times. It is a simple supply and demand problem. Training a microscopist with any science background to accurately identify fungi under the microscope is a time consuming (several months to an entire lifetime) process. Add to that the relatively loose standard methods on the books for the identification of mold. Top this with the extreme variability in sampling, sampling environments, and lab counting techniques, and one has to question how accurate, precise or consistent (particularly between laboratories) optical methods are.
Instrumental methods are far more conducive to quality assurance and control procedures and far more easily measured than are optical methods, which incorporate human bias. A smart attorney will spend all day roasting optical laboratory data, making it essentially useless in legal cases.
State of the Art
The specter of litigation looms on any mold abatement project. Projects that find their way into litigation and those individuals invited to that party will be looking for the best available tactics in which to plead their case. It is assured that attorneys, doctors and other experts will play the PCR card in some fashion and manner in the hopes that their position is bolstered. State-of-the-Art-science, applied correctly or not, and litigation go hand in hand, despite the differing objectives of each profession.
The commercial introduction of PCR methods will mean that professionals within the mold industry will be examining their procedures and approaches to the use of laboratory analysis in the assessment of environmental mold cases. This process is likely to take a little time, but given the potential applicability combined with the creativity of experts in the field, it will not be long before the gold standard in Forensic Science becomes one in Environmental Microbiology.
1. Haugland, RA., Vesper, S.J., and Wymer, L.J., Qualitative
Measurement of Stachybotrys chartarum conidia using
real time detection of PCR products with the TaqMan fl
orogenic probe system. Molecular and Cellular Probes,
1999, 13:329-340
2. Vesper, S.J. et al., Evaluation of Stachybotrys chartarum
in the house of an infant with pulmonary hemorrage:
Quantitative assessment before, during and after remediation.
Journal of Urban Health, Bulletin of the New York
Academy of Sciences, 2000, 77:1, 68-95
For more information please contact Forensic Analytical,
1-800-827-3274, E-mail: dk@forensica.com
