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Environmental Analysis

Chain of Custody Form

Published: July 17th, 2009

Revised: February 13th, 2014

The Chain of Custody form ensures that every step with regards to our analysis process is reliable, confidential and secure.

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Swab/ wipe, semiquantitative fungal culture [FC-S]

Published: July 8th, 2009

Revised: March 27th, 2019

[details to follow…]

Fungal DNA barcode sequence identification [FDNA]

Published: July 8th, 2009

Revised: September 17th, 2019

This test provides a low-level identification of a single fungal isolate by sequencing of portions of the nuclear ribosomal gene, specifically the internal transcribed spacer (ITS) region. DNA-based sequence identification is the gold-standard for microbial identification, since it is capable of identifying rare or atypical fungal isolates irrespective of their responses to diagnostic culture media or biochemical tests.

DNA is isolated from cells using standard procedures. The nuclear ribosomal ITS 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 one or more fungal DNA sequence database tools. This approach to fungal identification is unmatched in its ability to accurately identify troublesome isolates, or to ascertain the taxonomic position of novel strains.

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Avian fungal pathogen screen in air | bulk (Histoplasma, Cryptococcus & Chlamydophila by PCR) [AFPS]

Published: July 8th, 2009

Revised: January 23rd, 2019

The fungi Histoplasma capsulatum and Cryptococcus neoformans are two agents of serious disease in otherwise healthy, immunologically normal humans and animals. Both agents are endemic in Canada, and both occur in the droppings of certain bird species in addition to other materials. Additionally, the bacterium Chlamydophila psittaci is the causative agent of psittacosis and is associated with bird feces (although in Canada this agent is predominantly restricted to captive parrots).

Histoplasma capsulatum is the causative agent of histoplasmosis, a non-communicable infectious disease that is mostly acquired by inhalation of environmental particulates. Greater than 95% of infections are clinically silent. These cases are detected sporadically by later serological tests or by the observation of small lung calcifications by x-ray. Clinically significant pulmonary histoplasmosis, in general, is often related to inhalation of a large inoculum load, or to the presence of an underlying immunodeficiency in the patient. Secondary blood-borne spread occurs in a small subset of cases, typically in people with serious pre-existing immune dysfunction. When treated, histoplasmosis generally responds well to systemic antifungal drugs. Histoplasma capsulatum occurs in accumulations of bat, pigeon or starling guano, particularly where those materials have come in contact with moist soil, where it grows as a cottony white mould until changing to a budding yeast-like phase in human or animal tissue. Only a few parts of Canada lie within the endemic distribution of H. capsulatum. Scattered cases and moderate to low levels of subclinical exposure are only found in southern areas of Quebec, Ontario, and Manitoba, as well as a few parts of Alberta, New Brunswick and Nova Scotia.

Members of the C. neoformans species complex are widely distributed basidiomycetous yeasts (more closely related to mushrooms than moulds) that occur in bat guano and bird droppings, particularly those of pigeons and chickens. Cryptococcus neoformans is an occasional cause of pneumonia and meningitis in people who inhale typically large quantities of infectious material. Immunocompromised people are more susceptible to cryptococcal disease, developing it occasionally following contact with birds and bird nesting materials.

The recognition and elimination of infective reservoirs is central to the prevention of both histoplasmosis and cryptococcosis. This test uses an extremely sensitive and specific PCR-based method to detect both agents in bulk material samples or air samples collected on polycarbonate membrane filters. Bulk samples should be collected dry in a clean, new, food-grade, zippered freezer bag. Damp or moist samples should be refrigerated after collection prior to delivery to the laboratory. Air samples should be collected on a 25 or 37 mm 0.4 um membrane filter. A minimum of 1 cubic metre of air should be collected. Samples should be placed individually in a clean, new, food-grade, zippered freezer bag, and delivered to the laboratory within 24 hr of collection.

The detection of H. capsulatum is accomplished using primer sets that target portions of a catalase gene known as the “M-antigen”. Cryptococcus neoformans group is detected by primer sets that target a gene involved in capsular synthesis, CAP59. This method cannot distinguish between living and dead cellular material. A negative result does not necessarily refute the presence of potentially infectious material in the area tested, since only a small amount of material is subjected to the test. The testing of multiple replicate samples is helpful to establish confidence on the generalizability of results. A positive result, however, may provide information that is useful in public health interventions.

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Opportunistic fungal pathogen screen (semiquantitative) [OFPS]

Published: July 8th, 2009

Revised: January 29th, 2019

FusariumSerious infections caused by filamentous or dimorphic fungi are rare in immunologically normal people and normally only caused by one of several pathogenic species. In people with varying degrees of immune dysfunction, however, serious fungal infections are common, and may be caused by a much larger set of fungi many of which are not pathogenic under normal circumstances. These fungi are sometimes called “opportunistic pathogens”, in reference to their proclivity to invade tissues given the opportunity presented by immune compromise. While not typically a problem in the broad community, opportunistic fungal pathogens can be important agents of serious and even fatal disease in health care institutions.

Culture-based methods are the current gold-standard for the detection of opportunistic fungal pathogens (Mayhall 2003). This test is read rapidly (at 48 hr) by culture-dependent evaluation of the presence of the most commonly occurring agents of opportunistic fungal disease on bulk, swab or wipe samples. Results are reported on the same day that the samples are read.

Field sampling method

Bulk samples should be collected individually and sealed in an envelope of plastic pouch. Damp samples should not be placed in plastic in order to prevent microbial growth post sampling. Swab and wipe samples can be collected using typical protocols (e.g., Bulk/ swab/ wipe, semiquantitative fungal culture [C111]).

Results reporting

Results of this test are reported semiquantiatively as 1+, 2+, 3+, etc., where the numeral preceding the plus sign indicates the order of magnitude of colones observed, broken down according to taxon identified. The following taxa are specifically assayed by this test:

  • Absidia (sensu Lichthemia) species (including A. corymbifera)
  • Acremonium species
  • Aspergillus species complexes (including A. flavus, A. fumigatus, A. lentulus, Neosartorya species, Emericella/ Aspergillus nidulans, A. niger, A. terreus, and A. ustus)
  • Cunninghamella species (including C. bertholletiae)
  • Dematiaceous fungi with opportunistic potential (including Curvularia species and relatives, Cladophialophora species and others)
  • Exophiala species
  • Fusarium species complexes (including F. solani, F. dimerum, F verticillioides, and F. oxysporum)
  • Mucor species (including M. circinelloides and M. indicus)
  • Paecilomyces species (including P. lilacinus and P. variotii species complexes)
  • Phaeoacremonium species
  • Phialemonium species
  • Rhizomucor species
  • Rhizopus species (excluding R. stolonifer)
  • Scedosporium species (including S. apiospermum and S. prolificans species complexes)
  • Trichoderma species (including T. citrinoviride/ longibrachiatum complex)
  • Trichosporon species

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