Diagnosis of nocardiosis by polymerase chain reaction: an experimental study in mice

Abstract

In this study, we have established a sensitive semi-nested polymerase chain reaction (PCR) for detection of Nocardia in clinical specimens by first amplifying a 422 bp DNA fragment from the groEL gene, followed by a second amplification of 342 bp DNA by targeting sequences internal to the first amplicon. The semi-nested PCR was evaluated in a murine model of nocardiosis for detection of Nocardia in blood and visceral organs. Healthy BALB/c mice were intravenously infected with 0.2 ml suspension of 2.9 x 10(5)/ml cfu of Nocardia asteroides and N. farcinica. Viable counts and seminested PCR were performed post-infection with samples of blood as well as lung, liver, spleen, kidney and brain at 5 minutes, 3 h, and then every 24 h for 3 days. Of the 20 blood samples tested, 15 (75%) were Nocardia positive by culture and 19 (95%) were positive by semi-nested PCR. Likewise, in case of N. asteroides infection, 46% organ samples were positive by culture and 58% by semi-nested PCR. The positivity of organ samples was higher with N. farcinica, 60% by culture and 72% by PCR, which may be attributed to its increased virulence as compared to N. asteroides. These results demonstrate that semi-nested PCR is a rapid and sensitive method for detection of Nocardia in blood and different visceral organs. The diagnostic application of this method provides an additional advantage over culture techniques, as PCR can also detect L-forms of Nocardia in clinical specimens, which otherwise fail to grow on routine isolation medium.