Fungi are ubiquitous throughout nature and exist fundamentally as saprophytes.  Their primary role is to act as decomposers.  They are in essence, the garbage men of the world.  Of the several hundred thousand species, less than two hundred are known to be regularly involved in human and animal mycoses.  But the list of clinically relevant fungi continues to grow at an alarming rate.  This can be attributed mainly to the increasing number of immunocompromised patients, including cancer chemotherapy patients, organ transplant patients, those with AIDS and those who are hospitalized.  Organisms once thought to be common contaminants are now being confirmed as true pathogens in the immunocompromised patient. 

            One can begin to appreciate the mycology laboratorian needing a system that goes beyond the culture-based method if you consider the following phenomenon: A convergence of the ever growing list of true and potential pathogens with the explosive growth of the Indoor Air Quality industry.  This convergence plus the potential litigious aspect of the IAQ industry has placed a premium on the need to speciate more fungal isolates. 

            Until recently, identification of filamentous fungi has been performed by macroscopic and microscopic techniques.  This approach has often been referred to as the classical approach to mycological identification. 

            A well trained laboratorian can couple such macroscopic features as color of colony growth, texture of the aerial mycelium, rates of growth on respective media, temperature differentials and topography of colony with microscopic features such as segmented or nonsegmented hyphae, length and shape of conidiophores, conidial size, shape, color and many other microscopic features considered unique to both the genus and species level.

            With these two subsets of data, which can comprise a large number, protocols have been developed with fairly modest objectivity to rule out all but a handful of fungal candidates.  Subjectivity has and always will play a role in the traditional methods of fungal identification.  This is why the field of mycology has often been considered as much an art as it has been a science. 

            Without question, the diagnostic mycology laboratory, must supplement the more traditional approaches with the emerging technologies that are often much quicker, more specific and in some instances more cost effective.  Of all the emergent technologies, molecular methods are receiving the most fanfare.  Undoubtly, molecular microbiology has arrived and is here to stay.  In many clinical microbiology laboratories, molecular diagnostics has become the new gold standard for the diagnosis of several infectious diseases. 

In 1985, Kary Mullis and his colleagues reached a milestone in biotechnology by the development of Polymerase Chain Reaction (PCR).  Other nucleic acid amplification and nonamplification methods are being developed and are having a positive impact on the clinical microbiology laboratory.  But it is with PCR, where many are placing their future goals.  But is PCR the inevitable path for all future fungal identification?

            Quite simply put, PCR is a chemical reaction allowing for the synthesis of limitless quantities of a targeted DNA sequence.  Under proper conditions, a copy of the original targeted sequence can be made. Each copy or amplification constitutes a cycle.  In theory, at the end of each cycle, the targeted DNA is doubled.  At the end of two cycles, the original sequence has now quadrupled.  Consequently, in a matter of a few hours, the original targeted DNA sequence has now increased a billion times to attain analytical quantities. 

            The DNA sequencing approach to microbial identification does have many obvious benefits: 1) Rapidity of results. 2) Can be very sensitive and specific. 3) Able to work with small quantities of material. 4) In some instances, can be easy to perform. 5) Is not an antigen- based test. 6) Appears to have unlimited prospects for future growth and applications. 

            However, at the moment, there are some serious obstacles that PCR-based test will have to overcome before they can become more widely used in the mycology laboratory: 1) The initial startup cost can be prohibitively expensive, particularly for smaller laboratories.  Only a high volume of work can justify the initial cost. 2) Currently, there are only a few FDA-approved PCR based diagnostic assays available. 

Like most governmental agencies, waiting for FDA approvals can and will continue to be lengthy. 3) PCR is a patented technology as well as many of the primers used.  Like all patents, if there is any financial gain, an arrangement must be made between the patent holder and the end user.

            The royalties and up-front cost have seriously restricted the development and introduction of new PCR-based diagnostics. 4) PCR technologies have the potential to be very easily contaminated.  As little as one molecule can have deleterious effects. 5) In order to develop new PCR-based assays for fungal identification, sequencing information must be made available.  However, in the case of fungal identity sequences, the data is limited and may remain so for some time.  If the market does not provide an impetus to provide incentives for additional fungal sequences for the more esoteric fungal isolates, who or what will?

            PCR technologies could actually be considered a genetic profiler.  This means that the entire identification process is based on the genetic information obtained from a selected specimen.  The exclusively genetic approach has provided enormous advances in molecular taxonomy and new fungal classifications.  However, must we inevitably rely exclusively on genotyping (a genetic portrait)?  Is there not room for a phenotypic (physical portrait) approach, which could integrate the advances of science, mathematics and computers with the ever growing list of discovered fungal phenotypes?

            Of the chemical based test on the market, Biolog, with its state-of-the-art phenotype of testing system is by far the leader.  In the year 2000, Biolog, introduced the FF (Filamentous Fungi) MicroPlate.  The FF Database currently has identification patterns for over 618 Taxa from 120 plus genera. 

It also provides a macroscopic and microscopic photo library for many of the species.  Biolog’s identification system is based on the fungal isolates ability to use a specific carbon source.  A fungal isolate, in pure culture, is suspended into an inoculation fluid attaining an inoculum turbidity of 75% transmittance. 

            The suspension is then pipetted into a 96 well microtitre plate, which contains 95 different carbon sources, as well as a negative control.  Each well contains a different carbon source. 

            The microtitre plate is then incubated at 26 degrees Celsius and has the capability to read the plate at 24, 48, 72, 96 hours and at 7 days. Once the desired time period is selected the plate is subsequently read by an automated plate reader.  When a particular carbon source is utilized, either or both of the following changes occur: 1) Increased mitochondrial activity, leading to a reddish-orange color change. 2) Increased growth, leading to turbidity.  Turbidity is an important indicator fungal growth. The MicroPlate(s) are read at both 490nm and 750nm in order to detect and quantify both the color and the turbidity reactions.  There are two sets of data points generated; 96 color data points and 96 turbidity data points for a total of 192 points of data per microplate per reading.  The resulting data, which is a series of positive and negative reactions is interpreted by the Biology software and if an adequate match is found, an identification is called.

            The software will display the species identification in ranked order of the closet match.  Identification scores of percent probabilities, similarity index value and the distance between the MicroPlate results and the database pattern for that species are listed.  You can then verify the match with the fungal isolate tested by comparing the macroscopic and microscopic morphologies against the Biolog photo library. 

It is important to look at the top three choices displayed.  There are a few restrictions which should be noted: 1) A pure culture also must be used – there is no room for a mixed culture.  Fungal isolation from an originally mixed culture can be tedious and time consuming.

 2) Start up cost can be significant but are not as high the start up cost for PCR. 3) Because Biolog’s system is based on a pure and viable culture, the total identification process can take up to two weeks, if not a little longer depending on laboratory work flow.

            All existing methods have both pros and cons, Biolog is no exception.  However, their positive attributes far outweigh their negatives: 1) Biolog’s fungal database is considerably larger than PCR and continues to grow. 2) Biolog’s photo library is a true asset, displaying both macroscopic and microscopic features at the touch of a finger. 3) The ability to compare visually your well reactions with known values from a Bergey’s manual is an additional plus.  When your PCR-based system fails to give you identification, you do not have the luxury of double-checking the data.  You have no choice but to start over or rely on a culture based method. 4) Biolog’s microplate generates 192 points of data.  Just think of how many profile numbers that can be potentially created.  Currently, PCR is restricted by the primer(s) used. 

            Biolog’s advance system by phenotypic testing is a natural progression from the traditional approaches to mycological identification.  In the mycology laboratory, there is no reason why phenotype testing should trail behind genotype testing.  Both systems can and should complement one another.  Both have found their niche and their futures remain bright. 

Advances in molecular biology and automation will eventually make it more and more feasible for the diagnostic mycology laboratory to incorporate more advanced approaches to fungal identification.  And in many instances, nucleic acid amplification tests will be used in the future as primary tools for the diagnosis of fungal infections. 

            But if and when these events occur, I would like to defer to a highly respected mycologist who quipped “that it will be some time before molecular methods allow the differentiation between Exophiala jeanselmei and Exophiala jeanselmei variety lecanii-corni.”

Carlton Woods is the laboratory technical manager for Advanced Scientific Laboratories. He has over twenty years experience in environmental and clinical microbiology and holds a state supervisors license for clinical microbiology. He can be reached by e-mail at carlton@ascientificlab.com or by phone at 954-786-9331.

In our earth’s tropospheric boundary layer, we are bombarded everyday with a constant stream of bioaerosols that are ubiquitous on this planet. Bioaerosols are comprised of a vast array of airborne particles, large molecules or volatile compounds that are defined as bioaerosols. They are composed differently and behave differently. Bioaerosols include fungi, bacteria and viruses to name a few. Fungi or mold particles fall under the category of bioaerosols and based on this an understanding of the composition and behavior is required if we want to sample them. Many believe in this industry that sampling bioaerosols is a simple procedure of taking a pump and running it for a predetermined period of time. In a lot of cases, no thought process is completed regarding how to best sample an indoor environment to ensure a complete investigation. I have determined this based on recommendations present in some reports I have observed and instances when there are recommendations made in the complete absence of a report. Concerns relating to these points are highlighted in this article.

Certain disciplines involved in Indoor Air Quality (IAQ) have a lot of misconceptions, one such discipline being mold testing. Consider first this industry and the problems that we encounter. There are no controlling government bodies or any entity that can conclusively state the correct procedures that must be followed. This leaves an area in which individual professionalism, interpretation and direction becomes the master and this I feel is where the problems arise.

We have home and mold inspectors working in this field procuring IAQ samples and the differences in their approaches are astronomical. We have home and mold inspectors that take such care and work to an ethic that enables them to be truly professional in what they do and it is very encouraging. Unfortunately, we also have the flip side and these individuals do not even take the time to fully understand the basic principles in the procedures that they undertake. Reasons for this may be a due to a lack of knowledge or to a lack of desire or professionalism. There are some individuals in our profession who believe air sampling for mold involves taking a sampling pump and turning it on. A lot of these individuals may not know otherwise based on lack of training or knowledge. Some justify it using statements explaining that their level of investigation does not require this level of knowledge. Some feel that based on the fee they charge it only merits a brief walkthrough and a few random samples.

It is my feeling that if you are a home inspector involved in mold testing then you should be able to take a mold investigation to its logical conclusion as you would a home inspection. To highlight this point I propose this analogy.

For those that observe or participate in a sport that they really enjoy, how committed is the participation? Is it a general awareness without knowing the rules, strategies involved or is it full participation understanding the fundamentals of what is involved in the sport.

My guess is considerable knowledge with the sport is present based on the formula that there is the interest, there has been an exposure to it for a significant period of time and time has been taken to learn about it. The approach in professional practices must be the same.

In this field regardless of the level of investigation you are involved in, their must be a clear understanding of what is involved. No one day or weekend classes can offer you absolute knowledge regarding this field of work. I am not trying to convince anyone that this is rocket science but there is a degree of complexity to this field. I have heard it said that mold testing can offer a home inspector considerable increases in revenue and I would agree but what is sometimes missing is the statement regarding a commitment. This commitment is one to education and one that must be somewhat ongoing. To this end this article serves the purpose to identify some key areas that should be known and made aware.

When dealing with bioaerosols (mold being one of many types considered as a bioaerosol) we must first define them. The ACGIH (American Conference of Governmental Industrial Hygienists) defines bioaerosols as “airborne particles, large molecules or volatile compounds that are living, contain living organisms or were released from living organisms. The size of a bioaerosol particle may vary from100 microns to 0.01 of a micron. The behaviors of bioaerosols are governed by the principles of gravitation, electromagnetism, turbulence and diffusion”.

 Again for the purpose of this article to determine if procuring a sample is just a matter of “sucking air into a cassette” we shall use the field of mold testing as our scope of application. As fungal particles are considered bioaerosols then they too are governed by the principles of gravitation, electromagnetism, turbulence and diffusion.

If we look at each of the principles individually we can observe each of there potential effects on a fungal spore.

By definition it is a movement downwards resulting from gravitational attraction. With bioaerosols ranging in size, particles that are larger are influenced greater by gravitation. Factors affecting gravitation with relation to fungi are size and density.

The greater the size and density the more it is affected by gravity. We can now take this one step further with spherical particles. Their settling rates due to gravitation are dependent on its terminal velocity.

Terminal velocity is when an object is falling and accelerates so much it cannot go any faster. It can be shown the following way:

Particle falling + gravitational force = opposite drag of atmosphere + air resistance

Particle behaviors in this instance are influenced through processes such as electric charges, changes of direction, light absorption and the ability to scatter light. In other words these influences may cause the particle to increase in speed, change direction or move in different motions.

It is the instability in the atmosphere. This affects particles as low turbulence may increase settling rates while high turbulence may keep particles suspended longer.

It is the spreading of something such as particles. Spreading occurs for particles if laminar airflow (non-turbulent streamline flow in parallel lines) occurs because there is a greater chance the particle may break the lines of airflow and deposit elsewhere. Turbulent flow keeps the particles active and deposition is less likely.

With these influences present the realization of the potential affects relating to where and how a sample is taken should set in. If we take a situation where an air sample is procured, the position of the sampling cassette (if a countable sample is sought after) now becomes more important. In the event that a sample is taken too low thus not in the airflow stream then there is the chance that you possibly are only going to sample those spores more greatly influenced by gravitation and diffusion. On the other hand, if the sample is taken too close to the source of the air stream then the potential for the sample to be influenced by turbulence is present. The reason is the more movement of the spore in the air due to turbulence the less likely the spore will be captured in the cassette. 

If we continue to explore bioaerosols after identifying their composition and behavior with our primary focus on mold we again should consider the mind set of those that feel that air sampling in an indoor environment is just a matter of turning on an air pump. IAQ investigations no matter what level investigators want to delve into must be aware of three basic approach criteria:

1. Hypothesis Testing
   

The first criterion is the ability to formulate a hypothesis. A hypothesis is a proposal intended to explain certain facts or observations. The idea of walking into a home or building and procuring a sample with no prior thought is simply not a favorable one.

Investigators regardless of the level of testing or report detail are compelled to produce a hypothesis and then check their assumptions by designing a sampling plan to prove or disprove theories produced in their hypothesis.

1. Sample Collection

The second criterion in an investigators approach is sample collection. An investigator should select a sampling method(s) based on the information they have gathered themselves. Their choice of sampling should never be determined solely on the basis of the following points.

1.      What they were exposed to in a one day/weekend class.

2.      What the laboratory instructs them to do.

3.   What everybody else that they know in the industry does.

The three examples may all play a role in the overall decision but each one should not be the only exclusive determinate. Obtaining the correct knowledge by learning and researching will enable the investigator to make a properly informed decision in most cases.  

As discussed previously sample collection choices should ensure that the data produced will aid in answering any questions raised in their hypothesis. For this reason the investigator is solely responsible for the final decision. The final decision for an investigator for the purpose of bioaerosol sampling should always include the following considerations:

1.      What is the biological agent(s) of interest?

2.      The type and duration of sampling involved.

3.      The required laboratory analysis involved.

Based on these considerations one would make the assumption that the investigator once the biological agent of interest is identified would have a comfortable understanding of the agent. Why would anyone working in the indoor air quality industry for example, lack the necessary working knowledge of the organisms they are trying to detect? The same assumption should be made relative to the equipment used for procuring a sample. Having knowledge regarding what type of sample should be taken and how and when it is taken is very important and not enough investigators in my opinion truly know this. The same applies to sampling media. A lot of investigators know one or two different types and stick with them no matter the situation. The serious question begs as to the number of investigators that actually have a good comprehension of the principals of the tools they work with.

Take a spore trap for example. The frequency of investigators that change the flow rate from the specified requirement of 15 liters is quite high. This should never occur and if the investigator was knowledgeable to this fact then the realization on their part would be present, identifying that spore traps are designed to function at a certain flow rate to optimize certain capture rate efficiencies. This last underlined term describes how efficient spore traps are at capturing particles at certain size ranges from the air.

Studies of certain spore traps tested using various particle sizes, observed that in some cases particles <3 microns had lower capture rate efficiencies than those particles between 3 and 5 microns. Another observation was that particles with different designs (round compared to slightly irregular shapes) were also affected by changes in flow rate with relation to capture rate efficiencies. To this end it must be stated that any deviations from the flow rates suggested by the manufacturer will ultimately affect the efficiency of the sampler to collect particles (i.e mold spores) from the air.

The final consideration from the three listed previously relating to sample collection is one that requires knowledge of the different types of laboratory analysis that can be completed on the sample that was taken. Understanding what the appropriate tests are, what information will be extracted based on the choice of test as well as an overview of the laboratory procedure is a very proactive approach to aid in one’s endeavor to complete a thorough investigation.

3.   Data Interpretation

The third and final criterion in an investigation is one relating to data interpretation to reach a conclusion. It is often obvious from reports (in the event that one is actually produced) that investigators have problems interpreting the data. In other words investigators are unable to draw clear conclusions from environmental data due to the results of poor sampling design and sample collection methods.

To illustrate this point, consider the following: In many cases an investigator will receive a call from a homeowner complaining of mold. The investigator will set up an appointment and visit the home. A lot of times one or two samples will be taken randomly inside the house and one outside the house. Based on the results of these tests the investigator will determine whether a mold problem exists or not. Now I realize that this may be an oversimplified example but the reality of what actually occurs in some cases does not deviate too far from this example.

If we use the above example then ultimately certain problems can surface. Consider a 2000 square foot house and the potential volume of air that is present in the house at any given time. An investigator enters the house and uses for example an Air O Cell spore trap (sampling criteria is 15 liters flow rate for 5 minutes) or a Micro-5 spore trap (sampling criteria is 5 liters flow rate for 5 minutes). The total volume of air sampled is 150 liters and 25 liters respectively.

From the volumes of air illustrated above an investigator will make some form of decision on the condition of this home based on the data produced.Ideally to evaluate a home properly with respect to air sampling one would need to take considerably more samples but with a homeowner’s budget this may not be feasible. The best method to counteract this problem is to use the skill and knowledge that one should have, to ensure representative samples are procured. If the understanding of bioaerosol behavior is clear then the samples that are taken will be representative of the environment sampled to aid in producing data that will ensure informed conclusions.

Informed investigators with limited sample allowances on a project can use their knowledge, say for instance on gravitational or turbulence effects on particles to aid in their sampling strategy. It may help the investigator for example to sample closer to the air vents to ensure that settling of spores due to gravitational influences does not negatively affect the number of particles captured on the spore trap. The same example can be used for turbulence effects, as sampling too close to the air vent with air passing will cause the particles to travel in increased movements thus the potential for capture reduction on the spore trap. These are just two small examples and there are many more examples of situations that must be considered.

The last few paragraphs have dealt with the interpretation of data for the purpose of developing a report. Some reading this may think that these concerns do not apply to them as they do not write reports. It is my feeling that even if an investigator feels that there input on an investigation is minimal based on no clear obvious findings and that they will reference someone more experienced if a situation arises, they should still produce a report indicating their limited findings. The investigator originally was hired to determine if a mold problem exists not to hear that they took the project but could not determine this and pass it onto someone else.

In the event that an investigation does not generate a report due to the potential risk of liability then I feel it is proper that they do not get involved at any level in this field. One point of note is equipping one self with the knowledge, working with a logical mind and doing due diligence can significantly reduce the potential for liability.

Finally to link another situation with the previous paragraph detailing what I feel should never occur is the handing of laboratory results to the homeowner without some interpretation from the investigator. It is not up to the homeowner to interpret the results. The reason that this is not a favorable practice is that some laboratories include in their results, information about the organisms and their symptoms and health effects based on exposure. They reference material that is present in literature that has been documented over time and may be based on isolated cases. If the information is not put into the proper context by the investigator the laboratory results may be interpreted incorrectly and the homeowner may panic. Similar problems may occur if homeowners are left to interpret laboratory results that make statements such as “elevated mold condition”. This interpretation is based on the data and in most cases may be accurate but the laboratories were not in the field and did not observe the building and so the data alone may not represent actual conditions.

It is for this reason that the onus is on the investigator to observe, evaluate and interpret the data and put it into its proper context based on the overall findings.

For many reading this article, redundancy may be a term that comes to mind as the practices described may be common sense. Unfortunately, this is not the case for others. It is for these reasons that all of us involved in this industry should always strive to improve ourselves and others to ensure the longevity and integrity of this industry.