Development of a nested PCR for the diagnosis of Rhodococcus equi infection

Polymerase chain reaction (PCR) is widely recognized as a valuable tool in the diagnosis of Rhodococcus equi infection as only a brief period of time is required in order to identify virulent strains. This report describes a nested PCR protocol that was developed in order to increase analytical sensitivity and specificity of the test and as a result, the clinical validity of the procedure in comparison with the standard single cycle protocol. In this study, 36 isolates of R. equi, 31 clinical samples in which R. equi was isolated and 14 foal fecal samples taken from an endemic farm were analyzed using both single and nested PCR protocols. The use of nested PCR increased the sensitivity 100-fold, permitting the identification of 7 positive clinical samples (22%) and 3 positive foal fecal samples, that had resulted negative using standard single cycle PCR. The possibility of direct application of nested PCR on clinical samples, coupled with an increased analytical and diagnostic sensitivity of the techniques, render this method very useful in difficult cases, when diagnosis is not possible using routine analyses, or in epidemiological studies.