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Journal of Clinical Microbiology, April 2008, p. 1565, Vol. 46, No. 4
0095-1137/08/$08.00+0     doi:10.1128/JCM.02471-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

LETTER TO THE EDITOR

Stability of Frozen, Heat-Killed Cultures of Coccidioides immitis as Positive- Control Material in the Gen-Probe AccuProbe Coccidioides immitis Culture Identification Test{triangledown}


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LETTER
 
The requirement set forth in the Federal Register for the possession, use, and transfer of select agents and toxins (final rule) restricts the use of Coccidioides immitis, a select agent, in laboratories (1). This requirement has created a dilemma for diagnostic, clinical, and public health laboratories that need positive-control material for the Gen-Probe AccuProbe Coccidioides immitis culture identification test (Gen-Probe, Inc., San Diego, CA). The Gen-Probe AccuProbe test uses an acridinium ester-labeled DNA probe which hybridizes to specific rRNA sequences that are unique to C. immitis. In an attempt to reduce the hazards of maintaining viable cultures, we published a method in 1997 for maintaining mycelial suspensions of avirulent C. immitis strain ATCC 96907 (acquired from Gen-Probe, Inc.) which were heat killed at 95°C and frozen at –70°C as positive-control material for the Gen-Probe AccuProbe test (3). In that study, we demonstrated the stability of the samples for at least 10 months.

We report here on the stability of this same preparation after approximately 13 years of storage at –70°C. Since that original publication, the same lot of heat-killed, frozen samples of mycelial C. immitis culture in sterile 10% aqueous glycerol, prepared in 1994, was recently tested and found to maintain high positive relative light unit (RLU) activity. Aliquots (0.2 ml) of frozen samples were tested in five separate assays in 2004 and 2005 and again in 2007 and gave average readings of 599,520 RLU, with a range of 156,530 to 802,887 RLU (cutoff value, 50,000 RLU). By comparison, the positive-control C. immitis strain included in each assay gave average readings of 231,532 RLU, with a range of 146,408 to 266,423, whereas the negative-control Trichosporon beigelii strain gave average readings of 8,975 RLU, with a range of 1,010 to 16,307 RLU, for the same assays (cutoff value, <20,000 RLU). Preparation and long-term frozen storage of heat-killed aliquots in 10% aqueous glycerol reduce the hazards of propagating viable cultures and maintain the stability of nucleic acids for genetic probe testing. Recently, McGinnis et al. reported the use of a genetically modified, attenuated, nonvirulent strain of Coccidioides posadasii {Delta}chs5 as positive-control material that was reactive with the AccuProbe test (2). Procurement of this attenuated strain of C. posadasii, which is not a select agent, is an alternative solution to using C. immitis as a positive-control culture for this test.


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FOOTNOTES
 
{triangledown} Published ahead of print on 6 February 2008. Back


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REFERENCES
 
    1
  1. Federal Register. 2005. Possession, use, and transfer of select agents and toxins, final rule. Fed. Regist. 70:13294-13325.
    2. 2
  2. McGinnis, M. R., M. B. Smith, and E. Hinson. 2006. Use of the Coccidioides posadasii {Delta}chs5 strain for quality control in the ACCUPROBE culture identification test for Coccidioides immitis. J. Clin. Microbiol. 44:4250-4251.[Abstract/Free Full Text]
    3. 3
  3. Valesco, M., and K. Johnston. 1997. Stability of hybridization activity of Coccidioides immitis in live and heat-killed frozen cultures tested by AccuProbe Coccidioides immitis culture identification test. J. Clin. Microbiol. 35:736-737.[Abstract]
Miriam Valesco
Public Health Laboratory, Division of Disease Control
Alameda County Health Care Services Agency
1000 Broadway, Suite 500
Oakland, California 94607

Phone: (510) 268-2700, Fax: (510) 268-2709, E-mail: forest.paws{at}gmail.com

Journal of Clinical Microbiology, April 2008, p. 1565, Vol. 46, No. 4
0095-1137/08/$08.00+0     doi:10.1128/JCM.02471-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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