Use of the Coccidioides posadasii chs5 Strain for Quality Control in t
http://www.100md.com
《微生物临床杂志》
Clinical Microbiology and Immunology Laboratories, Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555
ABSTRACT
Coccidioides posadasii chs5 is a strain that is excluded from the select agent list. Sixteen assays using test reagents from three different ACCUPROBE Coccidioides immitis culture identification test lots had an average of 132,998 relative light units (RLU), which is well beyond the 50,000-RLU positive cutoff value for the test. Coccidioides posadasii chs5 is a satisfactory quality control isolate in the ACCUPROBE culture identification test for Coccidioides immitis.
TEXT
Coccidioidomycosis is a serious fungal infection that requires prompt identification of the etiologic agent. Because Coccidioides immitis morphologically resembles species of Malbranchea, molecular probes are often used to identify this pathogen (3). The use of molecular probes has two potential problems. First, it has been suggested that C. immitis consists of two distinct, divergent, genetically recombining, monophyletic clades or populations (2). Based upon phylogenetic analysis using single-nucleotide polymorphisms, genes, and microsatellites, a second species of Coccidioides, C. posadasii, was proposed. Coccidioides posadasii has been referred as non-California C. immitis, which is a misnomer, as the species occurs in California and elsewhere in the Americas. Phenotypically, C. immitis and C. posadasii are indistinguishable. Recognizing the significance of the two clades within Coccidioides, Cox and Magee considered the two clades to be separable at the variety level rather than at the species level and proposed the names C. immitis var. immitis and C. immitis var. posadasii (1).
The final rule that addresses select agents and toxins as set forth in reference 1b implements the provisions of the Public Health Security and Bioterrorism Preparedness and Response Act of 2002 and stipulates the requirements for possession, use, and transfer of C. immitis. Inclusion of C. immitis in this act as a select agent has resulted in problems for clinical laboratories that keep isolates for use as controls in the molecular identification of clinical isolates of C. immitis. The Secretary of the Department of Health and Human Services (HHS) and the Secretary of the U.S. Department of Agriculture (USDA) have, however, determined that the attenuated strains Coccidioides posadasii chs5 (effective 14 October 2003) and C. posadasii cts2/ard1/cts3 (effective 3 March 2006) are excluded from the select agent list (http://www.cdc.gov/od/sap/sap/exclusion.htm). Even though they are excluded from the select agent list (HHS and USDA select agents and toxins [1a and 1b]), they must be handled with appropriate safety procedures for C. immitis.
We obtained a culture of C. posadasii chs5 from Gary Cole while he was at the Medical College of Ohio via a material transfer agreement. This isolate was used in all of our studies conducted over several weeks. Coccidioides posadasii chs5, as well as C. immitis quality control (QC) isolate ATCC 28868, were maintained in culture on potato glucose agar at 30°C. Three lots (C1402416, 402416, and 500990) of the ACCUPROBE Coccidioides immitis culture identification test (GEN-PROBE, San Diego, CA) were tested according to the manufacturer's instructions. The ACCUPROBE test uses a single-stranded DNA probe with a chemiluminescent label that is complementary to C. immitis rRNA. The labeled DNA probe combines with the extracted rRNA to form a stable DNA-RNA hybrid. The selection reagent allows for the differentiation of nonhybridized and hybridized probes. The labeled DNA-RNA hybrid is measured using a GEN-PROBE Leader luminometer; a positive result is equal to or greater than 50,000 relative light units (RLU).
The negative control recommended by the manufacturer, Blastomyces dermatitidis ATCC 60916, had an average RLU value of 734 (range, 563 to 952 RLU), whereas the positive C. immitis QC strain had an average of 278,519 RLU (range, 118,554 to 406,703 RLU). The 16 assays for C. posadasii chs5 had an average of 132,998 RLU (range, 91,165 to 184,213 RLU). These results indicate that strain C. posadasii chs5, which is excluded from the select agent list, can be used as a QC strain in the ACCUPROBE Coccidioides immitis culture identification test.
Coccidioides posadasii chs5 has the advantage that it has been genetically modified to be nonvirulent. It can be safely handled in the laboratory under biosafety level 2 conditions. The use of C. posadasii chs5 as a positive QC isolate indicates that the ACCUPROBE test does not distinguish between C. posadasii and C. immitis. This conclusion is supported by the study of Padhye et al. (3), who previously reported that the ACCUPROBE test correctly identified 72 C. immitis isolates originating from throughout the Americas, some of which would be C. posadasii isolates based upon the definition of C. posadasii described previously by Fisher et al. (2). Coccidioides posadasii was established to accommodate isolates of C. immitis found in geographic regions other than California. Because the ACCUPROBE test system apparently does not differentiate between C. immitis and C. posadasii, we suggest that positive results be reported as C. immitis complex. The C. immitis complex consists of both Coccidioides species, which are known pathogens, and provides the clinician with valuable information needed for the diagnosis and management of coccidioidomycosis.
ACKNOWLEDGMENTS
We thank Garry Cole, Department of Biology, University of Texas at San Antonio, the Medical College of Ohio for C. posadasii chs5, and Jude Segerson, GEN-PROBE, for two lots of test kits.
FOOTNOTES
Corresponding author. Mailing address: Department of Pathology, University of Texas Medical Branch, 5.177 John Sealy Annex, 301 University Blvd., Galveston, TX 77555-0740. Phone: (409) 747-0603. Fax: (409) 772-5683. E-mail: mmcginni@utmb.edu.
REFERENCES
Cox, R. A., and D. M. Magee. 2004. Coccidioidomycosis: host response and vaccine development. Clin. Microbiol. Rev. 17:804-839.
Federal Register. 2005. Agricultural Bioterrorism Protection Act of 2002: possession, use, and transfer of biological agents and toxins, final rule. Fed. Regist. 70:13242-13292.
Federal Register. 2005. Possession, use, and transfer of select agents and toxins, final rule. Fed. Regist. 70:13294-13325.
Fisher, M. C., C. L. Koenig, T. J. White, and J. W. Taylor. 2002. Molecular and phenotypic description of Coccidioides posadasii sp. nov., previously recognized as the non-California population of Coccidioides immitis. Mycologia 94:73-84.
Padhye, A. A., G. Smith, P. G. Standard, D. McLaughlin, and L. Kaufman. 1994. Comparative evaluation of chemiluminescent DNA probe assays and exoantigen tests for rapid identification of Blastomyces dermatitidis and Coccidioides immitis. J. Clin. Microbiol. 32:867-870.(Michael R. McGinnis, Michael B. Smith, a)
ABSTRACT
Coccidioides posadasii chs5 is a strain that is excluded from the select agent list. Sixteen assays using test reagents from three different ACCUPROBE Coccidioides immitis culture identification test lots had an average of 132,998 relative light units (RLU), which is well beyond the 50,000-RLU positive cutoff value for the test. Coccidioides posadasii chs5 is a satisfactory quality control isolate in the ACCUPROBE culture identification test for Coccidioides immitis.
TEXT
Coccidioidomycosis is a serious fungal infection that requires prompt identification of the etiologic agent. Because Coccidioides immitis morphologically resembles species of Malbranchea, molecular probes are often used to identify this pathogen (3). The use of molecular probes has two potential problems. First, it has been suggested that C. immitis consists of two distinct, divergent, genetically recombining, monophyletic clades or populations (2). Based upon phylogenetic analysis using single-nucleotide polymorphisms, genes, and microsatellites, a second species of Coccidioides, C. posadasii, was proposed. Coccidioides posadasii has been referred as non-California C. immitis, which is a misnomer, as the species occurs in California and elsewhere in the Americas. Phenotypically, C. immitis and C. posadasii are indistinguishable. Recognizing the significance of the two clades within Coccidioides, Cox and Magee considered the two clades to be separable at the variety level rather than at the species level and proposed the names C. immitis var. immitis and C. immitis var. posadasii (1).
The final rule that addresses select agents and toxins as set forth in reference 1b implements the provisions of the Public Health Security and Bioterrorism Preparedness and Response Act of 2002 and stipulates the requirements for possession, use, and transfer of C. immitis. Inclusion of C. immitis in this act as a select agent has resulted in problems for clinical laboratories that keep isolates for use as controls in the molecular identification of clinical isolates of C. immitis. The Secretary of the Department of Health and Human Services (HHS) and the Secretary of the U.S. Department of Agriculture (USDA) have, however, determined that the attenuated strains Coccidioides posadasii chs5 (effective 14 October 2003) and C. posadasii cts2/ard1/cts3 (effective 3 March 2006) are excluded from the select agent list (http://www.cdc.gov/od/sap/sap/exclusion.htm). Even though they are excluded from the select agent list (HHS and USDA select agents and toxins [1a and 1b]), they must be handled with appropriate safety procedures for C. immitis.
We obtained a culture of C. posadasii chs5 from Gary Cole while he was at the Medical College of Ohio via a material transfer agreement. This isolate was used in all of our studies conducted over several weeks. Coccidioides posadasii chs5, as well as C. immitis quality control (QC) isolate ATCC 28868, were maintained in culture on potato glucose agar at 30°C. Three lots (C1402416, 402416, and 500990) of the ACCUPROBE Coccidioides immitis culture identification test (GEN-PROBE, San Diego, CA) were tested according to the manufacturer's instructions. The ACCUPROBE test uses a single-stranded DNA probe with a chemiluminescent label that is complementary to C. immitis rRNA. The labeled DNA probe combines with the extracted rRNA to form a stable DNA-RNA hybrid. The selection reagent allows for the differentiation of nonhybridized and hybridized probes. The labeled DNA-RNA hybrid is measured using a GEN-PROBE Leader luminometer; a positive result is equal to or greater than 50,000 relative light units (RLU).
The negative control recommended by the manufacturer, Blastomyces dermatitidis ATCC 60916, had an average RLU value of 734 (range, 563 to 952 RLU), whereas the positive C. immitis QC strain had an average of 278,519 RLU (range, 118,554 to 406,703 RLU). The 16 assays for C. posadasii chs5 had an average of 132,998 RLU (range, 91,165 to 184,213 RLU). These results indicate that strain C. posadasii chs5, which is excluded from the select agent list, can be used as a QC strain in the ACCUPROBE Coccidioides immitis culture identification test.
Coccidioides posadasii chs5 has the advantage that it has been genetically modified to be nonvirulent. It can be safely handled in the laboratory under biosafety level 2 conditions. The use of C. posadasii chs5 as a positive QC isolate indicates that the ACCUPROBE test does not distinguish between C. posadasii and C. immitis. This conclusion is supported by the study of Padhye et al. (3), who previously reported that the ACCUPROBE test correctly identified 72 C. immitis isolates originating from throughout the Americas, some of which would be C. posadasii isolates based upon the definition of C. posadasii described previously by Fisher et al. (2). Coccidioides posadasii was established to accommodate isolates of C. immitis found in geographic regions other than California. Because the ACCUPROBE test system apparently does not differentiate between C. immitis and C. posadasii, we suggest that positive results be reported as C. immitis complex. The C. immitis complex consists of both Coccidioides species, which are known pathogens, and provides the clinician with valuable information needed for the diagnosis and management of coccidioidomycosis.
ACKNOWLEDGMENTS
We thank Garry Cole, Department of Biology, University of Texas at San Antonio, the Medical College of Ohio for C. posadasii chs5, and Jude Segerson, GEN-PROBE, for two lots of test kits.
FOOTNOTES
Corresponding author. Mailing address: Department of Pathology, University of Texas Medical Branch, 5.177 John Sealy Annex, 301 University Blvd., Galveston, TX 77555-0740. Phone: (409) 747-0603. Fax: (409) 772-5683. E-mail: mmcginni@utmb.edu.
REFERENCES
Cox, R. A., and D. M. Magee. 2004. Coccidioidomycosis: host response and vaccine development. Clin. Microbiol. Rev. 17:804-839.
Federal Register. 2005. Agricultural Bioterrorism Protection Act of 2002: possession, use, and transfer of biological agents and toxins, final rule. Fed. Regist. 70:13242-13292.
Federal Register. 2005. Possession, use, and transfer of select agents and toxins, final rule. Fed. Regist. 70:13294-13325.
Fisher, M. C., C. L. Koenig, T. J. White, and J. W. Taylor. 2002. Molecular and phenotypic description of Coccidioides posadasii sp. nov., previously recognized as the non-California population of Coccidioides immitis. Mycologia 94:73-84.
Padhye, A. A., G. Smith, P. G. Standard, D. McLaughlin, and L. Kaufman. 1994. Comparative evaluation of chemiluminescent DNA probe assays and exoantigen tests for rapid identification of Blastomyces dermatitidis and Coccidioides immitis. J. Clin. Microbiol. 32:867-870.(Michael R. McGinnis, Michael B. Smith, a)