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.

Reverse-transcription polymerase chain reaction/pyrosequencing to characterize neuraminidase H275 residue of influenza A 2009 H1N1 virus for rapid and specific detection of the viral oseltamivir resistance marker in a clinical laboratory.

Pandemic 2009 H1N1 is normally susceptible to oseltamivir, but variants harboring the H275Y (CAC → TAC) mutation exhibit resistance. We describe the use of a combined reverse-transcription polymerase chain reaction (RT-PCR)/pyrosequencing approach to identify the H275 residue. A total of 223 specimens were tested with this method: 216 randomly selected clinical specimens positive for 2009 H1N1 and 7 cell-culture supernatants from the Centers for Disease Control and Prevention (CDC; 4 resistant, 3 susceptible 2009 H1N1 strains). The assay detected H275Y in 1 clinical respiratory sample (0.5%) and all 4 oseltamivir-resistant strains from the CDC; the remaining 215 clinical and 3 susceptible CDC specimens were wild-type. Sanger sequencing confirmed the results for 50 of 50 selected isolates. The RT-PCR/pyrosequencing method was highly specific, producing no amplicons or valid sequences from samples containing non-H1N1 viruses or bacteria. Our findings suggest that this method provides a rapid tool for H275Y detection, with high sensitivity and potential benefit for patient care.