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The Environmental Reporter
October 2008 Volume 6 | Issue 10

Hello,

I hope you're doing well and enjoying the start of autumn. Below is an article about Polymerase Chain Reaction (PCR) Testing by Dr. Michael Berg and an article about Histoplasma capsulatum by Agner Martinez. I hope you find them interesting and helpful.

With best wishes,
Dave Gallup




Using Polymerase Chain Reaction (PCR) in Indoor Environmental Testing
By Dr. Michael Berg, EMLab P&K Senior Molecular Biologist

We were passing a car dealership with a full lot of brand-new pick-up trucks the other day when a friend of mine said: "Why do people buy those gas-guzzlers? I don't think they should be on the market any more." This converstation reminded me of comments people have made about using PCR testing and the ERMI (Environmental Relative Moldiness Index) in the Indoor Air Quality industry. I asked my friend if he had ever tried to haul furniture on a bicycle or economy car. My point was that vehicles have a purpose and function and while trucks may guzzle gas, they are great for heavy loads. For most applications one or the other equipment or procedure is the most effective and useful... it all depends on what you need and what you use it for.

PCR stands for Polymerase Chain Reaction and describes a procedure that amplifies (multiplies) a specific region of DNA from a target organism. To accomplish the amplification, we add specific starter molecules called "primers" to the reaction. Primers are short fragments of DNA designed to match (or anneal) to specific sequences of the target genome allowing the DNA-Polymerase enzyme to start producing new DNA. This is very important because the amplification only works if we are using two primers that anneal on opposite strands of the target DNA in proximity to each other.

We have to remember that DNA is specific for every individual – this is why we all look different and identical twins look the same. At the same time we can easily differentiate humans from animals, plants or fungi. As morphological differences, increase DNA similarities generally become less. This is also true for molds. Every individual strain has a unique DNA sequence but there are also many similarities between isolates of the same species or the same genus and even between all fungi. The trick with PCR detection of one species, genus or subspecies is to know and compare DNA from a number of isolates and design the starter molecules (primers) appropriately. Molecular biologists have a good idea where to look for conserved or more variable stretches of DNA to make a PCR reaction work and to make it specific for the target. It also helps to have free online support from places like NCBI (National Center for Biotechnology Information) with over 80 million sequence entries in a database and online tools to search and align DNA sequences. The bottom-line is that PCR detection can be much more specific than identification based on morphology or spore characteristics but it depends on the design of the assay. Dr. Steve Vesper and his research group at the EPA developed and designed PCR assays specific for certain mold species, mold groups and/or sub-species (Types).

In my opinion, the question is not if there is a place for mold-specific PCR in the indoor environmental testing but rather when and where to use the additional tool most efficiently. Culture analysis typically allows species or genus identification of the majority of fungi found in IAQ investigations but accurate species identification typically takes at least 5 days and can take several weeks. In contrast microscopic spore analysis is fast but rarely allows for speciation. PCR testing allows excellent identification of many fungi and is fast, but it is more expensive and you have to know in advance what you are looking for. So every method has its pros and cons – the key is to know when to use which one.




Microorganism of the Month: Histoplasma capsulatum
By Agner Martinez, EMLab P&K Analyst

Histoplasma capsulatum is the etiologic agent of histplasmosis, a common granulomatous disease of worldwide distribution. Inhalation of a sufficient amount of conidia can potentially cause an infection in the lungs of a healthy person. In the vast majority of cases the infection is benign, leaving only residual calcifications in the lung and sometimes the spleen. However, it can occasionally progress to a life threatening, disseminated form, particularly affecting the reticuloendothelial system. There are three varieties recognized, depending on the clinical disease: Histoplasma capsulatum var. capsulatum is the most common cause of histoplasmosis; var. duboisii causes histoplasmosis duboisii, common in Africa; and var. farciminosum causes lymphangitis of horses and mules, and is endemic in Asia, Europe, and Africa.

Despite its worldwide distribution, H. capsulatum is most commonly encountered in tropical or subtropical regions, as well as in several large river basins in temperate regions. The most highly endemic areas in the United States are the central and eastern states, especially along the valleys of the Ohio, Mississippi, and St. Lawrence rivers.

Histoplasma capsulatum is a dimorphic (having two forms) fungus that grows as white to brownish mycelium on natural substrates and in culture at temperatures below 35°C. The organism produces characteristic tuberculate (warty), round, or pyriform (pear-shaped) macroconidia (larger spores; 8-16 µm in diameter) and small (2-5 µm in diameter) round, sparse, or abundant microconidia (smaller spores). When inhaled into the alveolar spaces, it is primarily the microconidia that sprout and then transform into small budding yeasts that are 2 to 5 µm in diameter. In culture at a temperature of 37° C, the organism also grows in the yeast-like form. The variety duboisii differs by the production of larger yeast cells, which are 8 to 15 µm in length with thick walls.  Because of close similarity that exists between spores of Histoplasma and the spores produced by many other fungi, identification of this fungus on spore traps is not possible and could be easily placed under Penicillium/Aspergillus type spores. Similarly, identification of this fungus by direct microscopic examination of tapes, bulks, and swabs is problematic. The most effective method for identifying this fungus is by culturing bulk samples.

The saprophytic fungus Sepedonium also produces tuberculate macroconidia, but is usually distinguishable from H. capsulatum by the absence of microconidia and does not convert to the yeast form at 37°C. Chrysosporium species may also resemble isolates of H. capsulatum. The full identification of the organism requires demonstration of the appropriate exoantigen (inducer of antibody formation, separate or separable from its source) and/or conversion to the yeast form at 37°C. Selective media such as mycobiont agar have been used to grow species of Histoplasma. Once the plates are inoculated they are incubated for 3 to 4 weeks.

It is firmly established that H. capsulatum grows in soil with high nitrogen content, generally associated with the guano of birds and bats. The first isolation of the organism from a natural environment was from soil near a chicken house, and since that time it has been recovered on numerous occasions from bat caves, bird roosts, chicken houses, silos inhabited by pigeons, and other such environments. In avian habitats, the organism seems to grow preferentially where the guano is decaying and mixed with soil rather than in nests or fresh deposits.

Anyone working at a job or present near activities where material contaminated with H. capsulatum becomes airborne can develop histoplasmosis if enough spores are inhaled. After an exposure, how ill a person becomes varies greatly and most likely depends on the number of spores inhaled and a person's susceptibility to the disease. Infants, young children, and older persons, in particular those with chronic lung disease, are at increased risk for developing symptomatic histoplasmosis. The disease can also appear as an opportunistic infection in persons infected with Human Immunodeficiency Virus (HIV).

Some occupations and hobbies may be at increased risk for exposure to H. capsulatum such as construction, demolition, chimney cleaning, farming, gardening, restoring of historic or abandoned buildings, roofing, bridge inspection, and cave exploration, among others. Individuals likely to come into contact with contaminated soil, bat droppings, bird manures, or similar materials should take appropriate precautions. As far as we know, at the time of this writing, there are no documented cases of histoplasmosis specifically associated with indoor air quality.

References:
1. Centers for Disease Control


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