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[b]Using new technology to improve indoor air quality assessment
The development of Mold Specific Quantitative Polymerase Chain Reaction (MSQPCR) and the Environmental Relative Moldiness Index (ERMISM) allowed the light of illuminating science into the darkness of indoor mold testing. [/b]

[i]BY KING-TEH LIN, PHD., MYCOMETRICS, LLC 11 DEER PARK DRIVE, SUITE 210 MONMOUTH JUNCTION, NJ 08852 AND
RITCHIE C. SHOEMAKER MD, 500 MARKET ST, SUITE 102 POCOMOKE, MD 21851[/i]

Molds and other potentially toxic microbes growing indoors within water-damaged buildings (WDBs) are an escalating problem. Researching what to do about them is a wide-ranging group that includes construction and real estate professionals as well as scientists, physicians and other health professionals. All want to delineate exactly how these microbes harm us, and what we can to stop them from reproducing in our homes.

Perhaps you still think that molds only cause a bit of allergy here and nasal irritation there, but that acquiring a mold or toxin illness is unlikely. Remember the comments from respected authorities just a few years ago about safely occupying WDBs? No one could yet prove that staying in a moldy building was harmful, but no Federal agency or responsible organization was willing to say that staying in one was a good idea, either. Many questioned then whether human illness followed exposure to WDBs, but recent scientific research has confirmed human illness after exposure (1, 2, 3).

In fact, after years of denial and delay, in March 2007, the CDC finally admitted that exposure to mold can cause adverse health effects. A month later, the Minister of Health of Canada chimed in that exposure to wet buildings causes a health risk. In fact, Canadian health officials have made the blanket statement that if there’s a moisture problem, there’s a health problem.

Another sure sign that times have changed: In the abstract of a recent paper on molds in New Orleans, the CDC-based authors said plainly, “Molds, endotoxins and fungal glucans were detected in the environment after Hurricanes Katrina and Rita in New Orleans at concentrations that have been previously associated with health effects (4).” And in the acknowledgments of the same paper: “We are indebted… to the Office of the Inspector General of the US Department of Health and Human Services for ensuring the safety of the sampling teams (4).”
No one who treats mold illness patients questions the fact that mold makes people sick; we see it in our practices every day.

Most patients I’ve seen who have mold illness caused by exposure to their own homes wonder what they should do with perhaps their most valuable asset. Should they hide the fact that the home made them sick, i.e., let the buyer beware? Should they clean the home and confirm with testing that the air is clear? Naturally, some just want to burn the home and start over, and who can blame them? Just try to sell a home with a mold and moisture problem these days now that the American public has heard about toxic mold. Imagine trying to buy a home when you don't know its mold and moisture history.

In the past, when you wanted to investigate whether a place had mold, you’d spend a chunk of cash to bring in an industrial hygienist, who would take a few air samples and maybe a bulk sample or two. Then you’d spend more money on more samples to make sure the results of the first samples were correct, and then you’d have to decide what to do based on tests that sometimes didn’t correlate with each other. Other times you’d have to spring big bucks for mycotoxin testing. But even if you knew to, who’d willingly pay the extra cash to look for bacteria, actinomycetes and the toxins those microbes make?

The problem is, though, if you don’t pay for thorough sampling, the sampling you did commission was a waste of money. Since inadequate sampling is the most common reason for mistakenly claiming that a previously damaged building is safe, industrial hygienists will tell you that you can never have too many samples. On the other hand, with real estate costs these days, spending an extra $5000 for thorough testing that includes testing for bacteria and actinomycetes creates financial pressure to cut corners.

A NEW WAY TO TEST FOR MOLD
But now physicians everywhere can diagnose mold illness easily using readily available, simple tests, and they can treat it with inexpensive medications. The costs are usually covered by health insurance, too. And thankfully, we can sample homes for fungal DNA using recently developed, state-of-the-art forensic methods.

Because people thinking about living in new dwellings still need to know whether their living areas are safe. When you learn about the health threats posed by exposure to WDB, you begin to picture the confusion that might accompany buying or renting new living space: Is that musty smell a warning of hidden mold growth? Can I trust that the joints of the flexible ductwork on the air handler in the crawlspace are airtight? Don’t ask for trouble by buying a home with an unsealed dirt crawlspace or a home that mixes residential air with crawlspace air! And what about that bubbling paint on the living room ceiling? Is the basement playroom next to the crawlspace safe for my children to play in?

Mold illness can result from any indoor environment that’s damaged by water intrusion, whether that water damage was caused by a hurricane, flood, tornado or a simple leak. And yet, despite the frequency of mold contamination, there’s been no standardized, objective methods available to quantify exactly how much indoor mold is in any given home.

Put yourself in the shoes of three actual patients:
• You’re a 35-year-old new home buyer. You have a history of unusual fatigue, some mild cognitive problems and chronic respiratory illness. Your doctor says indoor mold is making you sick. You plan to move, discarding all your porous materials and taking only the non-porous possessions that were cleaned professionally. How can you tell if the beautiful, affordable home across town in the good school district is safe before you put in an offer?
• Now you’re a 55-year-old secretary at a large manufacturing site in Delaware. Your old office had visible mold growth, and a repetitive clinical trial proved that you were made ill from re-exposure to that office. Your employer assures you that the new office they’re moving you into has been cleaned thoroughly and shows you results from two five-minute air samples that say that there were just a few Cladosporium spores. Is it safe for you to work there?
• Now you’re the mother of three kids in a town in Massachusetts facing a river that often floods. Your children’s physician told you they have Lyme disease, but they didn’t get better even after they took tons of antibiotics. A physician in Maryland says your kids are sick from exposure to molds growing in your basement, and the symptoms are identical to those who suffer from Lyme disease. How do you know what they really have and what you should do about it?

Thankfully, for less than $500, in less than 10 days, people can avoid living in environments that pose health risks by using a new technology called Environmental Relative Mold Index (ERMI) testing. Although ERMI testing is easy, it won’t replace industrial hygienists or careful home inspections. But unless ERMI testing is done, a living space hasn’t been tested thoroughly enough.

Just as the science regarding health effects caused by exposure to WDB is mounting rapidly, so too is the science regarding how to assess the potential for exposure to toxigenic fungi found in WDB. In months to come, we’ll probably be using ERMI tests in schools and workplaces with the same assurance that we now use ERMI in homes, but so far, ERMI has only been standardized for homes. While ERMI testing can bring piece of mind to those who want proof that toxigenic fungi aren’t found in their living spaces, it’s best use may be to clearly show the presence of bad-acting fungi inside, even in the absence of visible mold.

WHY DEVELOP ERMI?
Look at what we used to do to decide if a building posed a health threat: Air samples taken for a few minutes were based on the flawed idea that a snapshot in time could actually represent ongoing health risks for occupants of WDB. Some reasonable people actually believe that air samples are accurate indicators of the total health risks posed by exposure to the tested areas. Industrial hygienists compared levels of spores or organisms found indoors to those found outdoors, not bothering to distinguish between exactly which kinds of organisms each were. Some people actually believed that such testing was based on sound science! We know now that some genera of molds won’t cause a biotoxin-associated illness and others will.

Then we tried to establish thresholds for what levels of molds found in indoor air, in carpets or on bulk materials was safe or “normal,” but that didn’t work too well, either, because there were too many variables that impacted sampling, so too often the results couldn’t be reproduced. Just when we thought that spore counts were an important marker, the National Institutes for Occupational Safety and Health (NIOSH) told us that there were fungal toxins on 500 fragments of molds for each toxin molecule found on every spore we found (5). When we tried to incorporate microbiological testing that would isolate certain bugs like actinomycetes and bacteria without overgrowth, we found that the technology wasn’t quite mature yet, and even if it were available, the costs were prohibitive. Why not test for mycotoxins alone? Because that kind of testing has to be thorough, with multiple samples for multiple compounds. Talk about costs!

Thanks to the pioneering work of the Microbial Exposure Laboratories of the Environmental Protection Agency (EPA) in Cincinnati, though, the days of expensive, potentially unreliable indoor testing are over. The idea was simple enough: look for the DNA! And that was made possible by the development of Mold Specific Quantitative Polymerase Chain Reaction (MSQPCR) and Environmental Relative Moldiness Index (ERMI), both of which have brought the light of illuminating science into the darkness of indoor mold testing.

Led by Dr. Stephen Vesper, U.S. EPA scientists developed an objective, standardized DNA–based method to identify and quantify molds. The science behind the breakthrough that led to MSQPCR is now patented (US Patent No. 6,387,652). In 2006, the Department of Housing and Urban Development (HUD) used this technology to complete the American Healthy Homes Survey (AHHS). Based on this national survey and MSQPCR analysis of the settled dust in these homes, a national Environmental Relative Moldiness Index (ERMI) was then developed.
In this survey, scientists collected dust in a nationally representative sampling of 1096 homes by vacuuming two square meters (approximately an area three feet by six feet) in the living room and bedroom for five minutes each with a specially equipped dust sampler-fitted vacuum (Figure 1). The settled dust was collected in a special in-hose device that was sent to a reference laboratory. At the lab, the individual samples were evaluated for quality and reliability against internal standards, and each sample that was deemed methodologically satisfactory was then mixed and sieved through a 300-micron pore nylon mesh screen. The samples were each analyzed by an EPA-licensed laboratory for the DNA of 36 particular mold species, called indicator species because they separate molds found in WDB from molds found in buildings that aren’t damaged by water.

Although it’s impractical and ineffective to measure the DNA of all molds in a home, given the variation in ecological parameters of elevation, climate zone, season, rainfall, prevailing winds and other factors, we have to be able to compare representative levels of mold DNA in homes across the U.S. And although there are species that are unique or important in different climates or locations, these 36 indicator species are common enough to predict the total mold burden discounting all of the above variables. The goal was to measure enough species to allow the laboratory to describe the “relative mold burden” by creating a mold index that has validity anywhere in the country. When they succeeded in doing just that, they’d developed the accurate, low-cost indoor testing everyone’s been waiting for.

WHAT IS THE ERMI?
These 36 species were divided into 26 species/clusters associated with WDB (Group 1) and 10 common species/clusters not associated with WDB, called Group 2. The number calculated as the ERMI is the result of statistically valid manipulations of how much DNA of the different mold species is found. The lab adds up the log-values of the concentration of each species of the selected 36, then subtracts the sum of the logs of Group 2 from the sum of the logs of Group 1. The result is an artificial construct {I think we’ll want to explain what this means…..} called the “mold index.”

You don’t need to get bogged-down in learning what log functions are because the laboratory will report to you the ERMI value and where it falls on the ERMI scale. Of course, the laboratory will also report on a separate table the concentration of each of the 36 species in your sample as well. See sample ERMI analysis in Table 1.

To produce and then validate the ERMI scale, the computed ERMI values for a nationally representative set of 1096 homes were assembled on a continuum from lowest to highest burden of mold. The dark curve line at the arrow shows this assemblage (Figure 2). The scale ranges from about -10 to about 20 or even higher. On the left-hand side of the scale, the quarter of homes from the 1096 that fall into different ERMI percentages is shown. For example, an ERMI of 14 is in the top 25 percent of homes for relative mold burden; an ERMI of -6 would be in the lowest 25 percent of homes for relative mold burden.

There’s a problem with the ERMI being reported as a single number, which is why all of the additional information is supplied. Statisticians tell us that the actual result, based on confidence intervals, must include a standard deviation (SD); for any ERMI value, the standard deviation is plus or minus 3. The ERMI scale isn’t meant to be a method of making fine distinctions. For example, the 95 percent confidence interval for an ERMI of 14 would be from 11 to 17, i.e. 14 plus or minus 3. This means that if a home measures an ERMI of 14, then 95 out of a 100 times, one can be confident that the value of the ERMI would be between 11 and 17. In other words, there’s only a 5 percent chance of misclassifying the home as not having an ERMI of 11-17. An ERMI of 15 would mean that 95 times out of 100, the ERMI will be between 12 and 18. So an ERMI value of 14 isn’t much different from an ERMI value of 15.

USING THE ERMI
The ERMI scale was derived from analyzing the settled dust in the common living room plus bedroom of a home, two areas that were chosen arbitrarily. Even if most of a water-intrusion problem in a home comes from the basement, for example, we won’t suggest sampling the molds in the basement first, as the national standards are derived from sleeping areas and living areas. The ERMI was standardized to vacuum samples from just 18 square feet.

So what should our patients do? The ERMI costs several hundred dollars, but they’re buying essential information that’s far superior to the limited testing done by “experts” that costs 10 times as much.

At our own practice, ongoing research shows us that we can correlate the ERMI with health upon re-exposure to a given environment. We can’t use ERMI to tell us the health of a person who already is ill and untreated, but after treatment, with re-exposure to a building that has water-damage, the C4a results obtained daily for three days after re-entry correlate nicely with ERMI. If ERMI is below 2, rarely does C4a rise much, as it invariably does with re-entry into locations with a higher ERMI. If the ERMI is over 5, don’t go back in!

Our three patients mentioned above each did home samples. The beautiful home across town had a bargain price tag, due to multiple problems with the roof flashing by the chimney and the absence of proper venting of pipes from the bathroom through the roof. An ERMI of 18 saved our compromised-health patient a mountain of trouble by alerting her to too-high mold concentrations, and so she didn’t make an offer. The administrator of the Delaware mega-company found an ERMI of 0.02, moved into her “safe” office and has done well. The Massachusetts Mom found that her home was terribly contaminated, even without visible mold, musty smells or abnormal air sampling from two prior mold inspections. She credits ERMI testing with saving her children’s lives. I’m not sure if that’s true, but her family’s health is stabilized; if they’d relied on results of air samples alone, they would’ve remained ill.

Make no mistake; the presence of health effects shown by a protocol that evaluates health will trump an ERMI every time. {I’m not exactly sure what you’re saying here, could you rephrase? Do you mean nothing can beat a physician using his skills and testing to evaluate/diagnose a patient?} But if you don’t have the data that come from successfully treating a mold illness, followed by a trial where you take the patient off medications and remove them from exposure but the patient doesn’t relapse, but does relapse within three days with re-exposure and no medication, then ERMI should be your first step.

If the ERMI result is elevated, you’re just getting started on finding a solution. Now you have to look for bacteria, actinomycetes and mycobacteria. If the levels are low and there are people in the home with a typical mold illness, consider repeating the ERMI in different areas. If the ERMI is low and no one is ill, your sense of security should increase. (Don’t forget, though, that even if no one in the home is ill, ERMI will help determine if the home is safe for visitors or loved ones who might have a different genetic susceptibility to mold exposure…) If the ERMI value suggests the home is in the upper 25 percent of the scale — an ERMI above 5 — then investigating for water damage is useful and could be health-saving. If the ERMI is in the upper 25 percent, an inspection for water damage is more strongly suggested.

The Institute of Medicine’s report (2004, Damp Indoor Spaces and Health) on dampness and health expressed the opinion that there was scientific evidence linking molds and damp environments with respiratory symptoms (5). The cut-off date for literature to be considered by the IOM for its report was 2003, so you can see that the fast pace of mold illness research has outstripped the earlier IOM recommendations. Still, the IOM is an important milestone in the history of mold illness, even if the conclusions are out-of-date.

It’s important to remember that the ERMI is a mold index, not a health index. Each person varies so much genetically that a level of mold burden for one person may cause asthma symptoms in one person but not affect another at all. Medical questions should always be left to medical professionals. The ERMI value is a tool, just one more piece of information that a physician can use to help in making a diagnosis.
In our Pocomoke, Md., clinical research facility, ERMI values are correlated with multiple laboratory assays, symptoms, neurotoxicological studies and measurements of brain metabolites, especially lactate (indicating capillary hypoperfusion) and ratios of glutamate to glutamine (indicating the balance of excitation versus inhibition of neurotransmission). Of great interest is the clear association of elevated ERMI with elevated levels of lactate measured by magnetic resonance spectroscopy (MRS) in memory centers of the hippocampus and acquisition centers of the frontal lobes, together with reduction of normal levels of excitation to inhibition in the brain. Stated another way, an elevated ERMI is more closely linked to brain fog, memory deficits and abnormalities in executive cognitive function than any other environmental test. (Data presented at the 8th International Association for Chronic Fatigue Syndrome (IACFS) Conference held in Fort Lauderdale, Fla., Jan. 10-14, 2007, sponsored in part by the CDC.)
Do high levels of mold translate in genetically susceptible patients into inflammation that reduces blood flow in particular parts of the brain so that the brain doesn’t work properly? Yes! Even more helpful is the finding that changes in brain chemistry produced by treatment cause a reduction of the MRS abnormalities seen in mold patients, and that the benefit of treatment isn’t lost when patients re-occupy the home if the post-remediation and post-cleaning ERMI is less than 2.

ERMI IS USEFUL IN CLINICAL STUDIES
In a study conducted on the homes of asthmatic children in Cleveland by Case Western Reserve Medical School, remediating water-damaged, moldy homes significantly reduced the asthmatic child’s need for medical intervention to treat their asthma (6). In a prospective study of atopic infants (7), measuring the mold burden with MSQPCR was a better predictor for the development of wheeze/rhinitis in these infants than a home inspection.

Traditional air sampling has never been standardized, so interpreting the results of any air testing is always problematic. The major problem with traditional air samples is that they are necessarily of short duration. Often air samples are taken only for a few minutes because the recovery source, whether a Petri dish or a sticky slide, is quickly over-loaded. However, air samples can be useful and, if properly taken, they can also be analyzed by MSQPCR.

For example, air samples can be useful in hospitals or in an effort to pinpoint the location of a hidden mold problem. In order to take air samples for MSQPCR analysis, the filter used should be made of 25-37-mm diameter polycarbonate, with either a 0.45- or 0.8-micron pore size. The flow rates range from 2–16 liter/minute. The holder for the filter can be a button sampler or cassette. In MSQPCR analysis, the filter cannot be overloaded, so extended air samples can be taken for many hours, even days. The holder containing the filter is then sent to the laboratory for analysis. However, there’s no ERMI scale for air samples, because air samples only represent a “snap-shot” in time, whereas the ERMI represents the extended mold burden that has accumulated as dust. That’s why dust is the preferred sample.

HOW DO WE USE THE ERMI KIT?
First, locate the sofa or the most commonly used chair in the living room. Using a yardstick or tape measure and masking tape, mark a 3-foot by 6-foot rectangular sampling area on the floor immediately next to the selected sofa or chair. If the sample location can’t accommodate a sample area of those dimensions, sample a total of 18 square feet as close to the selected furniture as possible. Record or photograph what these dimensions were and where you took them for later comparison.
Next, go to the main bedroom and repeat the same steps on the floor immediately next to the bed. Use a different shape, approximating 18 square feet, if a 3-foot by 6-foot area isn’t possible.

Don’t forget to follow the instructions on how to use the filter cartridge. Make sure the filter is inserted in the dust sampler before inserting the sampler inside the vacuum cleaner hose. Take off the protective caps on the sampler ends. Use a separate dust sampler to vacuum each 18 square foot area. When you’ve vacuumed the entire area, you’re done sampling. Put the caps back on the dust sampler and place the sampler in a sealable plastic bag. Lastly, send to an EPA-licensed ERMI laboratory and request the ERMI analysis.

Any basement or crawlspace could be a common source of microbial growth, including molds, and a sample can be taken there, remembering that the ERMI wasn’t standardized for basements. If you decide to remediate the high-ERMI home, your contractor will likely use additional testing methods. You should ask for a repeat ERMI, taken in the same spots as before remediation, to ensure that all toxigenic fungi have been adequately cleared.

Help is always just a few clicks away at http://www.mycometrics.com, where trained staff members are happy to assist you. You’ll also find a long list of answers to frequently asked questions (FAQ). Ask about discounts on shipping and handling.

WHERE DO YOU DO NON-RESIDENTIAL ERMI SAMPLING?
Good question. It may be that multiple samples will be required. The answers are likely to come from ongoing research. Do you sample every conference room in a 30-story building, every room in a hotel, every duct in a high-rise condominium? We don’t know. In cases like this, we use a trusted, experienced inspector who looks at the HVAC system and suggests sampling sites based on airflow patterns. If resources permit, sampling upper-level shelves and other duct collectors that haven’t been moved for months is a good idea, especially if the office building has no carpets. We’ve used this approach in two government-owned facilities that re-circulate air, obtaining results that mirror the residential ERMI clinically and with MRS. We believe that an office with an ERMI of 14 will soon be shown to be equivalent to a home with an ERMI in the top 25 percent of homes in the U.S. for relative mold burden, but we haven’t proven that hypothesis yet. We’re working on it.

No amount of sampling can replace the wisdom of an experienced mold inspector or industrial hygienist in finding and dealing with mold problems in buildings. While ERMI is a helpful tool, using it is still new. Further research will refine ERMI’s proper, best uses and applications, particularly in regards to using ERMI as a way to assess the potential for health problems, and we’ll soon be better able to tell you how ERMI testing can and should be used.

ERMI has another role that will require more clarification: How can we tell that cleaning is complete after remediation? Taking a sample for ERMI analysis should help. We expect that the ERMI value will be lower after the remediation process. Please note that a problem with post-remediation ERMI measures could be caused by ERMI’s extreme sensitivity to the presence of fungal DNA. It may take a number of months for the ERMI value in the home to stabilize to its final post-remediation level. We don’t yet know the minimum time after remediation is completed before the ERMI is confirmed to equal final post-remediation levels, but we’re continuing to study it. Because the cost of ERMI isn’t astronomical, I suggest that the sampling be done one week and again three months after remediation (naturally, only if the water intrusion problem is fixed first).

SUMMARY
All homes contain mold, but not all homes contain the same molds and definitely not at the same concentrations. You need not fear molds found inside buildings without water intrusion problems or excessive moisture, but in WDB, molds are proven to effect health. Identifying the species present, as well as how much of each, can be done with state-of-the-art fungal DNA analysis—the MSQPCR, an automated analysis that provides reproducible results that can be reliably interpreted. If you need immediate data, ERMI results can be obtained quickly. For patients in need of sensitive analysis, and for prospective home sellers and buyers, industrial hygienists and mold remediators, ERMI results can be invaluable.





References
1. RCS Johanning
2. RCS NTT1
3. RCS NTT2
4. AEM Rao 3/07
5. S. J. Vesper et al., J. Occup. Environ. Med. 48, 852 (2006).
6. S. J. Vesper et al., J. Occup. Environ. Med. 46, 596 (2004).
7. T. Meklin et al., J. Environ. Monitor. 6, 615 (2004).
8. R. A. Haugland, M. Varma, L. J. Wymer, S. J. Vesper, Syst. Appl. Microbiol. 27, 198
(2004).
9. Institute of Medicine, National Academies of Science. Damp Indoor Spaces
and Health. The National Academies Press. 2004: p. 355.
10. C. M. Kercsmar et al., Environ. Health Perspect. 114,1574 (2006).
11. S. J. Vesper et al., J Exposure Anal. Environ. Epidemiol. Available on-line: Oct 11, 2006.




FIGURE 1. DUST COLLECTOR
FIGURE 2. ERMI SCALE.