Abstract
16S rDNA PCR and sequencing are powerful tools for bacterial detection and identification, although their routine use is not currently widespread in the field of clinical microbiology. The availability of pyrosequencing now makes 16S rDNA assays more accessible to routine diagnostic laboratories, but this approach has had limited evaluation in general diagnostic practice. In this study we evaluated a real-time 16S rDNA PCR and pyrosequencing assay for use in a routine microbiology laboratory, by retrospectively testing joint fluid and joint tissue specimens received for conventional culture. We found that use of the real-time 16S rDNA assay was clinically valuable in this specimen type because it enabled us to identify a small number of culture-negative infections. Although faster and less labour-intensive, we found that the utility of pyrosequencing for pathogen identification is still hampered by shorter read lengths compared to conventional (Sanger) sequencing. Combining results from both molecular and conventional culture methods, bacteria were only detected in 11.8% specimens in this study. However, the detection rate was increased to 18.6% if specimens were only included from patients with a documented clinical suspicion of infection. In conclusion, while pyrosequencing had significant advantages in speed and ease-of-use over conventional sequencing, multiple reactions will be required to deliver comparable species-level identification, thus negating many of the benefits of using the technique. We found that 16S rDNA PCR and sequencing should be rationally targeted on the basis of good clinical information in the routine diagnostic setting, and not used as a general screening test for the exclusion of bacterial infection in joint specimens.
Highlights
Diagnosis of bacterial infection is routinely made through the microbiological culture of clinical specimens
In this study we evaluated a real-time 16S rDNA PCR and pyrosequencing assay for use in a routine microbiology laboratory, by retrospectively testing joint fluid and joint tissue specimens received for conventional culture
Pyrosequencing was rapid to perform, and the ease of use of the small PyroMark Q24 instrument made it well-suited to a routine laboratory, read lengths achieved by de novo sequencing were insufficient for species discrimination
Summary
Diagnosis of bacterial infection is routinely made through the microbiological culture of clinical specimens. Despite overt clinical signs of infection, these specimens may be falsely negative due to the presence of nutritionally fastidious organisms or the prior use of antibiotics. The development of improved bacterial diagnostic assays is integral to efforts to improve antibiotic stewardship. Amplification and sequencing of regions of the panprokaryotic 16S rRNA gene has been helpful in identifying culture-negative bacterial infections in a number of studies [1,2,3]. Conventional PCR, gel-based detection and sequencing methodologies mean that routine testing is labour-intensive and requires considerable financial investment and technical expertise [4]. A number of large clinical microbiology laboratories have made this investment, and may offer testing as a reference service to other laboratories. The ability to perform such testing in-house in the local diagnostic laboratory would be of significant benefit in reducing costs and turn-around-times
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