AJVR, Vol 66, No. 5, May 2005 755 R hodococcus equi is a gram-positive, soil-saprophytic bacterium that shares characteristics with members of the genera Mycobacterium and Corynebacterium 1 and has worldwide distribution. Rhodococcus equi is a fac- ultative intracellular pathogen of macrophages and causes severe pneumonia in foals. 2 The bacterium is also an emerging opportunistic pathogen in immuno- suppressed humans. 2,3 In foals, virulence of R equi is associated with an 80- to 90-kbp plasmid containing the gene (vapA) that codes and mediates expression of a 15- to 17-kd virulence-associated protein (VapA) located on the cell wall surface. 4-7 Deletion of the vapA gene attenuates virulence of the bacterium in mice. 8 The VapA surface protein is expressed by R equi isolates recovered from foals with pneumonia, but it is not uni- formly expressed by isolates from other sources. 2 Although the exact function of VapA is not known, expression of the protein increases in response to iron deprivation, suggesting a role in the acquisition and utilization of iron by R equi. 7,9 The quantitation of bacteria in biological speci- mens is typically performed by use of methods that are dependent on microbial culture (ie, determination of CFUs or the most probable number). 10 However, cul- ture-dependent methods for quantitating intracellular bacteria, such as R equi and Mycobacterium tuberculosis, generally underestimate the number of viable organ- isms because of a tendency for the bacteria to clump. 11,12 Alternative methods such as immunofluo- rescence assays, flow cytometry, and radioisotope labeling have been successfully used to quantify R equi. 11,13 Unfortunately, these techniques are laborious and expensive and thus of marginal value for routine clinical and epidemiologic use. The use of quantitative polymerase chain reac- tion (QPCR) assays in the study of infectious disease has greatly increased, and the method has rapidly gained acceptance as an effective and reliable diag- nostic test. 12,14,15 Unlike standard polymerase chain reactions (PCRs), QPCRs can be monitored in real time by use of a fluorescent sequence detection sys- tem that allows the initial quantity of a gene to be determined. The initial copy number is deduced from the threshold cycle (Ct) value, in which the fluorescent signal generated by amplification of the target gene crosses a specified threshold. 16 Monitoring the reaction in real time eliminates the requirement for post-PCR analysis with agarose gel electrophoresis, thereby reducing the potential for contamination. Diagnostic methods that use QPCR have been reported for pathogens that are slow growing and diffi- cult to cultivate, such as Rickettsia rickettsii and M tuberculosis. 12,14 To the authors’ knowledge, use of QPCR assays has not been reported for quantitation of R equi. The objective of this study was to evaluate a Received September 16, 2004. Accepted November 29, 2004. From the Departments of Large Animal Medicine and Surgery (Harrington, Martens, Halbert, Cohen) and Veterinary Physiology and Pharmacology (Golding), College of Veterinary Medicine, Texas A&M University, College Station, TX 77843. Dr. Golding’s present address is Cold Spring Harbor Laboratories, McClintock Building, 1 Bungtown Rd, Cold Spring Harbor, NY 11724. Supported by the Link Equine Research Endowment, Texas A&M University, and the Grayson-Jockey Club Research Foundation. The authors thank Dr. Jim Derr, Michael Grimm, Amanda Jackson, Cody Nerren, Ruth Reitzel, and Joshua Turse for technical assistance. Address correspondence to Dr. Cohen. Evaluation of a real-time quantitative polymerase chain reaction assay for detection and quantitation of virulent Rhodococcus equi Jessica R. Harrington, BS; Michael C. Golding, PhD; Ronald J. Martens, DVM; Natalie D. Halbert, PhD; Noah D. Cohen, VMD, MPH, PhD Objective—To evaluate a real-time quantitative poly- merase chain reaction (QPCR) assay in the detection and quantitation of virulent Rhodococcus equi. Sample Population—1 virulent, 2 intermediately vir- ulent, and 2 avirulent strains of R equi and 16 isolates of bacteria genetically related to R equi. Procedure—The QPCR assay was evaluated for detection and quantitation of the virulence-associated gene (vapA) of R equi in pure culture and in samples of tracheobronchial fluid, which were inoculated with known numbers of virulent R equi. Results were com- pared with those derived via quantitative microbial culture and standard polymerase chain reaction methods. Results—The QPCR assay detected the vapA gene in pure culture of R equi and in tracheobronchial fluid samples that contained as few as 20 CFUs of virulent R equi/mL and accurately quantitated virulent R equi to 10 3 CFUs/mL of fluid. The assay was highly specif- ic for detection of the vapA gene of virulent R equi and was more sensitive than standard polymerase chain reaction for detection of R equi in tracheo- bronchial fluid. Conclusions and Clinical Relevance—The QPCR assay appears to be a rapid and reliable method for detecting and quantitating virulent R equi. The accu- racy of the QPCR assay is comparable to that of quantitative microbial culture. The increased sensitiv- ity of the QPCR method in detection of virulent R equi should facilitate rapid and accurate diagnosis of R equi pneumonia in foals. (Am J Vet Res 2005;66:755–761)