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  • Author or Editor: Natalie D. Halbert x
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Abstract

Objective—To evaluate sensitivity and specificity of a multiplex polymerase chain reaction (PCR) assay for simultaneous detection of Rhodococcus equi and differentiation of strains that contain the virulence-associated gene (vapA) from strains that do not.

Sample Population—187 isolates of R equi from equine and nonequine tissue and environmental specimens and 27 isolates of bacterial species genetically or morphologically similar to R equi.

Procedure—The multiplex PCR assay included 3 gene targets: a universal 311-bp bacterial 16S ribosomal RNA amplicon (positive internal control), a 959-bp R equi-specific target in the cholesterol oxidase gene ( choE ), and a 564-bp amplicon of the vapA gene. Duplicate multiplex PCR assays for these targets and confirmatory singleplex PCR assays for vapA and choE were performed for each R equiisolate. An additional PCR assay was used to examine isolates for the vapB gene.

Results—Results of duplicate multiplex and singleplex PCR assays were correlated in all instances, revealing high specificity and reliability (reproducibility) of the vapA multiplex assay. Of the pulmonary isolates from horses with suspected R equi pneumonia, 97.4% (76/78) yielded positive results for vapA. Seven of 50 (14%) human isolates of R equi yielded positive results for vapA. Six human R equi isolates and 1 porcine isolate yielded positive results for vapB. No isolates with vapA and vapB genes were detected.

Conclusions and Clinical Relevance—The multiplex PCR assay is a sensitive and specific method for simultaneous confirmation of species identity and detection of the vapA gene. The assay appeared to be a useful tool for microbiologic and epidemiologic diagnosis and research. (Am J Vet Res 2005;66:1380–1385)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To evaluate a real-time quantitative polymerase chain reaction (QPCR) assay in the detection and quantitation of virulent Rhodococcus equi.

Sample Population—1 virulent, 2 intermediately virulent, 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 compared with those derived via quantitative microbial culture and standard polymerase chain reaction methods.

Results—The QPCR assay detected the vapAgene 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 103 CFUs/mL of fluid. The assay was highly specific 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 tracheobronchial fluid.

Conclusions and Clinical Relevance—The QPCR assay appears to be a rapid and reliable method for detecting and quantitating virulent R equi. The accuracy of the QPCR assay is comparable to that of quantitative microbial culture. The increased sensitivity 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)

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in American Journal of Veterinary Research

Abstract

Objective—To determine the prevalence of antimicrobial resistance to macrolide antimicrobials or rifampin in Rhodococcus equi isolates and to describe treatment outcome in foals infected with antimicrobial-resistant isolates of R equi.

Design—Cross-sectional study.

Sample Population—38 isolates classified as resistant to macrolide antimicrobials or rifampin received from 9 veterinary diagnostic laboratories between January 1997 and December 2008.

Procedures—For each isolate, the minimum inhibitory concentration of macrolide antimicrobials (ie, azithromycin, erythromycin, and clarithromycin) and rifampin was determined by use of a concentration-gradient test. Prevalence of R equi isolates from Florida and Texas resistant to macrolide antimicrobials or rifampin was determined. Outcome of antimicrobial treatment in foals infected with antimicrobial-resistant isolates of R equi was determined.

Results—Only 24 of 38 (63.2%) isolates were resistant to > 1 antimicrobial. Two isolates were resistant only to rifampin, whereas 22 isolates were resistant to azithromycin, erythromycin, clarithromycin, and rifampin. The overall prevalence of antimicrobial-resistant isolates in submissions received from Florida and Texas was 3.7% (12/328). The survival proportion of foals infected with resistant R equi isolates (2/8 [25.0%]) was significantly less, compared with the survival proportion in foals that received the same antimicrobial treatment from which antimicrobial-susceptible isolates were cultured (55/79 [69.6%]). Odds of nonsurvival for foals infected with resistant R equi isolates were 6.9 (95% confidence interval, 1.3 to 37) times the odds for foals infected with susceptible isolates.

Conclusions and Clinical Relevance—Interpretation of the results emphasized the importance of microbiological culture and antimicrobial susceptibility testing in foals with pneumonia caused by R equi.

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in Journal of the American Veterinary Medical Association