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May 2006 Volume 4 | Issue 5

Hello,

I hope that you are doing well and enjoying an early summer. Below is an article about aerosols of spores and their settling rates by Dr. Harriet Burge and an article about Cryptococcus by Yamile Echemendia. I hope you'll find them both useful and interesting.

With best wishes,
Dave Gallup




How Long Do Spores Stay in the Air?
By Dr. Harriet Burge

Aerosol Events
Many different activities or processes cause fungal spores to enter the air. Once in the air, gravity causes the spores to begin to fall and the spore cloud gradually disappears. Understanding how spore clouds are generated and their fate in the air is important for designing investigative protocols. Aerosol generating activities may include intense activity associated with remediation, or gentle air movements. Clouds may result from crumbling blue cheese, throwing away moldy food, or cleaning mold from window sills or shower curtains. Even gentle air currents can disturb fresh growth sufficiently to cause spores to become airborne.

The Nature of Spore Clouds
Spore clouds are generally composed of several different types of spores, often of variable size and shape. In addition, clouds resulting from indoor aerosolizing events often become airborne in chains or clumps. Thus the cloud will include a broad range of particle sizes and shapes.

Once airborne, the spores are affected by gravity, drag related to the viscosity of the air, and air movements (turbulence). Spores encounter or are attracted to surfaces electrostatically and are removed from the aerosol. All of these factors are influenced by the density of the spore, its diameter and its shape. Gravity tends to pull the spore toward the ground, independent of density. Thus when Newton dropped two large objects from his tower, their differing density did not affect their rate of fall. However, spores are very small compared to (for example) an apple, and spores are strongly affected by the viscosity of the air. This viscosity tends to counteract the action of gravity, an effect that is called drag. Air movements may counteract or enhance the gravitational effect, and their effects are also related to the size, shape and density of the spore.

In order to calculate settling rates for a spore, one must consider a sphere of equivalent diameter and density to the spore, and this estimation is complicated by the aerodynamic shape of the spore. Alternaria spores are streamlined, and probably act as spheres of approximately the width of the spore (~10µm) rather than the length which can vary from 40µm to >100µm. On the other hand, Tetraploa is a plant pathogenic fungus that produces large spores with long spreading appendages. This is not a streamlined spore, and is probably affected by drag in excess of what would be expected from its size (i.e., it would likely fall more slowly than would be predicted by its size). Short ornamentation may also increase the effects of drag so that a rough 2µm Penicillium spore might fall more slowly than a smooth spore of the same diameter.

Table 1: Settling velocities estimated using Stokes' equations, assuming density of 0.9

Particle Diameter (µm) Settling Velocity (m/sec) Time to Fall 1 Meter (minutes) Comments
1.5 7.5E-05 223  
2 1.3E-04 166  
3 2.9E-04 58 Approx. size of Penicillium spores
4 5.0E-04 33  
5 7.8E-04 21  
6 1.1E-03 15 Approx. size of Stachybotrys spores
8 2.0E-03 8  
10 3.16E-03 5 Approx. size of Alternaria spores
15 6.8E-03 2  
20 1.2E-02 1  

In the ideal world, one could look at Table 1 and estimate approximately how long a cloud of a particular spore type would remain airborne. However, given all of the variables discussed above, these settling rates and times are only very rough approximations. Thus, in chamber studies, Penicillium spore clouds tend to fall much faster than would be expected by the table and may disappear completely within 15-20 minutes. This is due, in part, to capture of spores on surfaces either through mechanical impaction or electrostatic attraction. Unfortunately, this kind of table has been used to predict disappearance of spore clouds, often with unfortunate consequences. For example, tossing Botrytis covered strawberries in the trash should (using the table and 5µm as a diameter) result in a cloud that lasts at least 15-20 minutes. However, as in the Penicillium example, the aerosol actually disappears much faster than that.

Practically speaking, air samples should be taken within minutes of aerosolizing activities. If you actually want to know how long the cloud lasts, then take samples every 5 minutes for a total of 20 or so minutes. You can then plot the decay curve, and extrapolate to the time when concentrations should approach zero. For small spores like Penicillium, longer array of sampling events may be necessary, while for spores like Stachybotrys, one might need to sample more often over a shorter period of time. These kinds of experiments are strongly affected by air movements, air exchange rates, and source strengths.

The bottom line: It is dangerous to mentally estimate how long a spore cloud will remain in the air, and in general, people think the spores remain airborne longer than they actually do, especially indoors.




Fungus of the Month: Cryptococcus species
By Yamile Echemendia

The genus Cryptococcus includes more than 37 species with Cryptococcus neoformans as the species with the most significant impact on human health.

C. neoformans is the causative agent of cryptococcosis. It has been frequently referred to as an opportunistic pathogen because people suffering from immunodeficiency (AIDS patients and others) are more susceptible to the infection than immunocompetent individuals. It is generally accepted that the organism enters the host through the respiratory tract and gets into the lungs in the form of dehydrated yeast cells or as basidiospores of less than 3µm in diameter. However, the exact nature of the infectious particles of the fungus has not been firmly established. Fortunately, this infection is rare and is not associated with typical fungal IAQ investigations. According to the Centers for Disease Control, its annual incidence rate is 0.4-1.3 cases per 100,000 people in the general population and 2-7 cases per 100,000 amongst AIDS patients.

The typical vegetative state of C. neoformans is an encapsulated yeast-like form and a cell diameter of 2.5µm to 10µm. On cornmeal Tween 80 agar, C. neoformans produces round, budding yeast cells with no true hyphae visible. It grows well at 25°C as well as 37°C, but its ability to grow at 37°C is one of the features that differentiates C. neoformans from other Cryptococcus species. The sexual state is the basidiomycete Filobasidiella neoformans that produces basidiospores of approximately 1.8µm to 3µm. It is noteworthy that sexual reproduction appears to occur much less frequently in nature than the asexual or vegetative reproduction.

C. neoformans can be identified from culturable air samples by using various selective culture media such as Niger seed agar, Pal's medium, and Birdseed agar. On these media, the fungal colonies can be easily identified by their dark brown color. This coloration is due to the production of a black pigment (melanin) mediated by the activity of a phenoloxidase enzyme present in the fungus. The final identification of the fungus is based on colony and microscopic morphology, carbohydrate assimilation tests, the presence of capsule on India ink preparations, urease production on Christensen medium and the ability to grow at 37°C.

Based on differences in ecology, physiology, clinical manifestations and capsular polysaccharide, C. neoformans was recently divided into three varieties C. neoformans var. gattii, C. neoformans var. grubii and C. neoformans var. neoformans which include different serotypes (A, B, C, D and AD).

C. neoformans var. gattii has a more restricted geographical distribution than the other two varieties. It has been recovered from the environment on the plant debris of some Eucalyptus species such as E. camaldulensis and E. tereticornis.

C. neoformans var. grubii and C. neoformans var. neoformans have been isolated from various sources in nature. Their association with pigeon excreta is known but they have also been isolated from droppings of a large variety of avian species like caged birds including canaries and parrots.

Many studies have been conducted to evaluate environmental samples from different sources to verify the presence of C. neoformans in urban saprophytic sources including pigeon droppings, avian droppings, soil, Eucalyptus barks, leaves and seeds. As a result it was established that the majority of C. neoformans recovered from pigeon droppings, collected on different urban sites such as churches, hospitals, old buildings and downtown streets belonged to C. neoformans var. grubii, the variety responsible for more than 90% of all cryptococcosis cases. Such investigations confirmed that C. neoformans is clearly associated with weathered pigeon excreta implying that this bird's habitats may provide sources of human infection. Pigeons are very common in large cities and many buildings undergo extensive cleanup to remove their droppings and feathers.

This association between C. neoformans and pigeons has been reported frequently. However, pigeons do not acquire cryptococcosis, most likely because the fungus cannot grow at the bird's body temperature of 42°C. An acceptable explanation for this intimate association between pigeon excreta and C. neoformans is that it offers an advantageous ecological niche for the fungi over other competitors.

Despite many studies on the saprophytic sources of C. neoformans in various parts of the world, there are still many unanswered questions concerning the ecology of the fungus as well as the treatment and the epidemiology of the disease.

References:
1. Anastasia P. Litvintseva, et al. 2005. Comparative analysis of environmental and clinical populations of C. neoformans. Journal of Clinical Microbiology 43 (2): 556-564, Feb.

2. Claudia Castello, et al. 2005. Characterization of Cryptococcus neoformans isolated from urban environmental sources in Goiania, Goias State, Brazil. Rev. Inst. Med. trop. S. Paulo 47 (4):203-207, July-August.

3. E. Levetin, et al. 2002. A mycologistís guide to indoor mold investigations. Supplement to Micologia, vol. 53 (6). Newsletter of the Mycological Society of America.

4. L. Curtis, et al. 2002. Pigeons allergens in indoor environments: a preliminary study. Allergy 57: 627-631.

5. Maria Cecilia Bianchi, et al. 2005. Environmental strains of Cryptococcus neoformans variety grubii in the city of Santos, SP, Brazil. Rev. Inst. Med. trop. S. Paulo 47 (1): 31-36, January-February.

6. Tania C. Sorrell and David H. Ellis. 1997. Ecology of Cryptococcus neoformans. Rev. Iberoam Micol, 14: 42-43



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