Use of Serial Quantitative PCR of the vapA Gene of Rhodococcus equi in Feces for Early Detection of R. equi Pneumonia in Foals

Abstract

Background: Current screening tests for Rhodococcus equi pneumonia in foals lack adequate accuracy for clinical use. Real-time, quantitative PCR (qPCR) for virulent R. equi in feces has not been systematically evaluated as a screening test.

Objective: The objective of this study was to evaluate the accuracy of qPCR for vapA in serially collected fecal samples as a screening test for R. equi pneumonia in foals.

Animals: One hundred and twenty-five foals born in 2011 at a ranch in Texas.

Methods: Fecal samples were collected concurrently with thoracic ultrasonography (TUS) screening examinations at ages 3, 5, and 7 weeks. Affected (pneumonic) foals (n = 25) were matched by age and date-of-birth to unaffected (n = 25) and subclinical (ie, having thoracic TUS lesions but no clinical signs of pneumonia) foals (n = 75). DNA was extracted from feces using commercial kits and concentration of virulent R. equi in feces was determined by qPCR.

Results: Subsequently affected foals had significantly greater concentrations of vapA in feces than foals that did not develop pneumonia (unaffected and subclinical foals) at 5 and 7 weeks of age. Accuracy of fecal qPCR, however, was poor as a screening test to differentiate foals that would develop clinical signs of pneumonia from those that would remain free of clinical signs (including foals with subclinical pulmonary lesions attributed to R. equi) using receiver operating characteristic (ROC) methods.

Conclusions and clinical importance: In the population studied, serial qPCR on feces lacked adequate accuracy as a screening test for clinical R. equi foal pneumonia.

Keywords: Feces; Horse; Pneumonia; Rhodococcus equi.

Figure 1

Boxplot of fecal concentration of vapA by sample‐time (1, 2, or 3, corresponding to ages 3, 5, and 7 weeks, respectively), by group (NotRE = unaffected or subclinical pneumonia foals; RE = foals that developed R. equi pneumonia). The triangles in the boxes represent the median value; the bottom and tops of the boxes represent the 25th and 75th percentiles of the data, respectively; the thin horizontal lines from the top of the boxes extend to the 97.5th percentile. Boxes with different superscripted letters differ significantly (P < .05). Although the fecal concentrations of vapA did not increase for foals that remained free of disease (ie, subclinical and unaffected groups), fecal concentrations of vapA was significantly increased with age among foals that subsequently developed disease.

Figure 2

ROC curve for fecal vapA concentration at 5 weeks of age for predicting development of pneumonia during the interval between 5 and 7 weeks of age among 10 foals that developed R. equi pneumonia during the interval and 115 foals that did not. The thin diagonal line represents area under the ROC curve of 50% (a nondiscriminating test). The solid circle symbol represents the optimal cutoff value15 and the value beneath it external to the parentheses is the actual copy numbers represented at this point of the curve (ie, 0.8 copies for this curve). The values inside the parentheses are the estimated specificity and sensitivity, respectively; this order is the default for the software program).

Figure 3

ROC curve for fecal vapA concentration at 7 weeks of age for predicting development of pneumonia after 7 weeks of age among 15 foals that developed R. equi pneumonia during that interval and 100 foals that did not. These data exclude the 10 foals that developed R. equi pneumonia between 5 and 7 weeks of age. The thin diagonal line represents area under the ROC curve of 50% (a nondiscriminating test). The solid circle symbol represents the optimal cutoff value15 and the value beneath it external to the parentheses is the actual copy numbers represented at this point of the curve (ie, 220 copies for this curve). The values inside the parentheses are the estimated specificity and sensitivity, respectively; this order is the default for the software program).