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
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)
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
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
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
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.
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.