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Journal of Clinical Microbiology, January 1998, p. 148-152, Vol. 36, No. 1
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Clinical Application of PCR-Restriction Enzyme Pattern Analysis for Rapid Identification of Aerobic Actinomycete Isolates

Rebecca W. Wilson,1 Vincent A. Steingrube,2,* Barbara A. Brown,2 and Richard J. Wallace Jr.1,2

Center for Pulmonary and Infectious Disease Control1 and Department of Microbiology,2 The University of Texas Health Center at Tyler, Tyler, Texas

Received 9 July 1997/Returned for modification 23 September 1997/Accepted 17 October 1997

The accuracy and practicality of PCR-restriction enzyme pattern analysis (PRA) for routine identification of aerobic actinomycete clinical isolates were evaluated for 299 cultures submitted to the Mycobacteria/Nocardia Laboratory at the University of Texas Health Center at Tyler. PRA identification using an amplified 439-bp segment (amplicon) of the 65-kDa heat shock protein gene was compared to identification by traditional methods, including growth characteristics, susceptibility patterns, biochemical testing, and high-performance liquid chromatography analysis. Microbiological examination of six cultures ruled out aerobic actinomycetes, and they were omitted from the study. Amplicons were analyzed with BstEII, HaeIII, MspI, HinfI, and BsaHI. When necessary, AciI, HhaI, and NarI were also used. From March 1995 through May 1997 (27 months), 274 of the remaining 293 (93.5%) isolates were accurately identified by PRA. Major diagnostic groups included 170 mycobacteria, 93 nocardiae, and 30 other aerobic actinomycetes. Mixed cultures were readily recognized by PRA, including a wound culture that contained two Nocardia taxa that were indistinguishable morphologically. Mycobacterium mucogenicum was identified in three cultures heavily contaminated with gram-positive cocci. The 19 isolates that produced PRA patterns that did not match those in the current PRA database were differentiated into 8 Mycobacterium species and 11 other aerobic actinomycetes by the presence or absence of BstEII recognition sites. Identification of 15 of these 19 isolates was also equivocal by traditional methods. PRA results were reportable within 2 to 5 working days and were as accurate as and faster and less expensive to obtain than those of traditional methods.

* Corresponding author. Mailing address: Department of Microbiology, The University of Texas Health Center at Tyler, P.O. Box 2003, Tyler, TX 75710-2003. Phone: (903) 877-7685. Fax: (903) 877-7652. E-mail: vsteingr{at}UTHCT.edu.

Journal of Clinical Microbiology, January 1998, p. 148-152, Vol. 36, No. 1
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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