Legionella pneumophila Rapid Screen [M200]
Published: July 8th, 2009
Revised: March 15th, 2012
Background
The Gram negative bacterium Legionella pneumophila is a common environmental cause of pneumonia, often associated with institutional outbreaks and environmental exposures. Legionella pneumophila is an endosymbiont of single celled protozoan that live in warm, stagnant water, particularly around temperatures of 30-40 °C. Building systems such as humidifiers and evaporative coolers may become contaminated by Legionella species including L. pneumophila if maintenance practices lapse or in the event of mechanical failures. Certain types of domestic hot water systems may also become affected. Most susceptible are systems in which untreated cold water is mixed with hot water downstream of the heating unit, usually as a means to prevent scalding at the faucet.
Analysis
Most methods for testing fluids for L. pneumophila rely on culture. Although culture-based methods remain the current gold-standard means of determining the presence of this bacterium, the time-consuming nature of the method makes is poorly suited to informing urgent public health decisions. In response to this need, we provide a PCR-based genetic test to rapidly rule-out the presence of L. pneumophila. Using this method, negative results (N) can be confidently reported in as little as 4 hr (our standard turn-around is 48 hr, but please call if a shorter turn-around is required). Because the test cannot differentiate between active colonization from residual DNA or non-living cells, all positive results (P) must be confirmed by further culture testing, requiring the normal turn-around of 7-14 days. This approach has been extensively validated, and has been used widely in Europe for several years. It has also been advocated by the Ontario Ministry of Health and Long-Term Care as a rapid screening method to rule out the presence of L. pneumophila on an urgent basis (Legionella, Biofilms and More, November 21, 2011, Toronto).
Sample collection procedure
- Collect water (ideally a 1-liter sample, but 200 mL at minimum) in sterile, screw-top bottles. For water sources expected to contain disinfectant chemicals such as chlorine, the collection container should include a suitable preservation buffer (the US-CDC recommends sodium thiosulfate to a final concentration of 0.1 M). We are pleased to provide sample collection containers to meet your needs. Please contact us prior to sampling to make arrangements.
- Collect culture swabs of internal surfaces of faucets, aerators, and shower heads in a sterile, screw-top container (e.g., 50 mL plastic centrifuge tube, with or without preservation buffer, according to the above guidance). Submerge each swab in approximately 5 mL of sample water taken from the same device from which the sample was obtained.
- Transport samples to the laboratory as soon as possible after collection. Samples may be transported at room temperature but must be protected from temperature extremes. Samples not processed with 24 hours of collection must be refrigerated.
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Endotoxin [B200]
Published: July 8th, 2009
Revised: October 4th, 2009
Endotoxin is a lipopolysaccharide found in the cell membranes of Gram-negative bacteria. Endotoxin is a strongly proinflammatory material because of its interactions with receptors that stimulate the innate immune system. Exposure to endotoxin will initiate a strong inflammatory response in anyone and does not require allergic sensitization or individual sensitivity. Inhalation exposure to endotoxin is thought to be a contributor to a number of inflammatory airways diseases such as Farmer’s Lung Disease and other hypersensitivity pneumonitides.
Endotoxin exposures typically arise where fluids contaminated with Gram negative bacteria become nebulized, such as spray-mist type humidification systems, hot tubs. In industry, sewage treatment processes and synthetic and semisynthetic machining coolants are a common source of endotoxins.
This test measures endotoxin content of air (air samples for endotoxin must be collected using special endotoxin-free polycarbonate filter membranes), bulk or TefTex wipe samples using the Limulus Amoebocyte Lysate (LAL) assay. This sensitive bioassay measures endotoxin by a reaction that uses highly sensitive endotoxin recombinant receptors cloned from blood cells of the horseshoe crab (genus Limulus). This is the gold standard method of measuring endotoxin, and it is used in a wide range of applications including the testing of medical devices and pharmaceutical preparations.
We reported levels in endotoxin units (EU) per standard unit of material tested (e.g. EU/mg, EU/m³, etc.). This measure can be converted to gravimetric units (e.g. ng) if required. In the case of air samples, endotoxin levels in a test area are usually compared to background levels in a non-complaint area. Airborne endotoxin greater than 30 times above background has been suggested as an action-level (ACGIH 1999).
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Bacterial DNA barcode sequence identification [M220] NEW!
Published: July 8th, 2009
Revised: October 4th, 2009
This test provides a low-level identification of a single bacterial isolate by sequencing of portions of the 16S ribosomal small subunit gene region. DNA-based sequence identification is the gold-standard for microbial identification, since it is capable of identifying rare or atypical isolates of bacteria irrespective of their responses to diagnostic culture media or biochemical tests.
In this test, we carefully cultivate the bacterial isolate in a broth medium, typically Tryptic Soy Broth (TSB). Log-phase cultures are harvested by centrifugation and DNA is isolated from cells using standard procedures. A diagnostic portion of the ribosomal small subunit gene is amplified by PCR and sequenced in both directions. A consensus sequence of both forward and reverse strands is constructed, and this sequence is used to query the Ribosomal Database Project (RDP), an online data analysis and alignment tool consisting of annotated Bacterial and Archaeal small-subunit 16S rRNA sequences. This approach to bacteria identification is unmatched in its ability to accurately identify troublesome isolates, or to ascertain the taxonomic position of novel strains.
Results of this test are reported as an identification or series of putative identifications with corresponding confidence measures. A phylogram generated using the UPGMA algorithm indicating the phylogenetic placement of the isolate relative to closely related taxa will is also provided.
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Thermophilic actinomycetes [C226]
Published: July 8th, 2009
Revised: October 4th, 2009
This test determines the presence of culturable thermophilic actinomycetes in bulk and swab samples. Test results are provided semiquantitatively as 1+, 2+. 3+, etc., where the number preceding the “plus sign” indicates the order of magnitude of colonies observed.
Actinomycetes are a large and diverse group of Gram-positive bacteria that have growth habits similar to fungi in that they produce branching networks of filaments. Thermophilic actinomycetes occur in a wide range of habitats including common materials such as self-heating plant matter and manure composts. They are also known from highly specialized habitats such as birds’ nests, volcanic vents and hot springs. The term thermophilic refers to the fact that the actinomycetes in question grow optimally at temperatures above 40 °C. Other groups of actinomycetes are known to occur at moderate or extreme cold temperatures.
Actinomycetes are notable as prolific producers of microbial volatile organic compounds (mVOCs). Indeed in this regard, they tend to be much more active than most fungi. Some of the mVOCs commonly produced by actinomycetes are responsible largely for the mouldy or musty odours associated with soils as well as damp basements. Actinomycetes are also extremely active producers of antimicrobial chemicals. Many of the naturally produced antimicrobial drugs in common use today are derived from actinomycetes (e.g. streptomycin, nystatin, and tetracycline). Inhalation exposure to cells and other materials colonized by actinomycetes has been associated with respiratory diseases such as hypersensitivity pneumonitis. This is particularly evident in agricultural settings where actinomycete exposures are known to play a role in the disease known as Farmer’s Lung.
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Psychrophilic actinomycetes [C225]
Published: July 8th, 2009
Revised: October 4th, 2009
This test determines the presence of culturable psychrophilic actinomycetes in bulk and swab samples. Results of this test are provided semiquantiatively, as 1+, 2+, 3+, etc., where the numeral preceding the “plus sign” indicates the order of magnitude of colonies recovered in culture.
Actinomycetes are a large and diverse group of Gram-positive bacteria that have growth habits similar to fungi in that they produce branching networks of filaments. Psychrophilic actinomycetes grow optimally at low temperatures, particularly below 15°C. They occur in a variety of habitats, particularly soils. They are also known from highly specialized habitats such as glaciers, antarctic rocks, and penguin rookeries. Other groups of actinomycetes are known to occur at moderate temperatures or in the presence of extreme heat.
Actinomycetes are notable as prolific producers of microbial volatile organic compounds (mVOCs). Indeed in this regard, they tend to be much more active than most fungi. Some of the mVOCs commonly produced by actinomycetes are responsible largely for the mouldy or musty odours associated with soils as well as damp basements. Actinomycetes are also extremely active producers of antimicrobial chemicals. Many of the naturally produced antimicrobial drugs in common use today are derived from actinomycetes (e.g. streptomycin, nystatin, and tetracycline). Inhalation exposure to cells and other materials colonized by actinomycetes has been associated with respiratory diseases such as hypersensitivity pneumonitis. This is particularly evident in agricultural settings where actinomycete exposures are known to play a role in the disease known as Farmer’s Lung.
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