Direct detection and identification of acid-fast bacteria from smear-positive broth cultures using a pyrosequencing method

Broth culture is a standard method for detection of acid-fast bacteria (AFB) (e.g., Mycobacterium and Nocardia) from patient specimens. Direct nucleic acid-based identification from smear-positive broths expedites the infectious disease diagnosis. We developed and evaluated the performance of a pyrogram-based technique (direct-broth-pyrosequencing [DBP]) to identify AFB directly from smear-positive broths. One hundred thirteen AFB-positive broths from patient specimens were tested. Bacterial DNA was amplified by polymerase chain reaction and sequenced using the PyroMark ID system. The DBP method correctly identified the AFB species/group in 109 (97%) of the 113 broths, including 15 Mycobacterium species and 4 Nocardia species. Three broths that yielded indeterminate results were found to be AFB-AFB mixed broths and required purified colonies on solid media for definite identification. The 4th broth was repeatedly identified by sequencing to be Mycobacterium intracellulare, even though the organism was not isolated and the AccuProbe was negative. This method did not identify the AFB organisms from broths containing 2 AFB organisms, but did not produce false identification. No cross-reaction was observed when AFB-positive broths were spiked with non-AFB microorganisms, indicating that the DBP method was specific to AFB. The DBP method gives rapid (within 8 h), accurate AFB identification directly from broth cultures and provides another useful AFB identification tool in a clinical laboratory.