Background: DNA sequencing is a powerful tool for bacterial detection and identification. We sought to evaluate the utility of direct DNA sequencing from various clinical specimens compared to routine work-up and determine if positive Gram stains were likely to yield positive sequencing results.
Methods: This is a retrospective review of clinical specimens that were submitted for direct DNA sequencing over a 2-year period from 11/2012 -1/2015. Clinical specimens were sent to the clinical microbiology laboratory where microscopy and culture were performed according to standard operating procedures. Clinical specimens were also submitted to reference laboratory for direct sequencing: 16S r-RNA gene for detection of bacteria, 28S and ITS sequence for detection of fungi.
Results: A total of 76 specimens from 45 patients were submitted for DNA sequencing. The specimens comprised of 37 body fluids (49%), 29 fresh tissues (38%) and 10 formalin-fixed paraffin embedded samples (13%). A total of 14 (18%) samples were positive by either sequencing, culture or histopathology; 11 bacteria, 7 fungi and 1 parasite (Table 1). Sequencing detected organisms in 4 culture-negative specimens; 2 Streptococcus pneumoniae and 2 Aspergillus species. 5/14 (36%) of specimens were positive by sequencing and culture, and concordance was found in only 2 samples; 1 Legionella species and 1 Candida lusitaniae. Histopathology identified fungal elements in 5 specimens and only 3 (60 %) yielded positive sequencing results. 71 Gram stain results were reviewed, and organisms was seen in 4 (6%), all of which yielded negative sequencing results. Of the 9 sequencing positive specimens, no organisms were seen in 9/9 and moderate to many WBC were seen in 3/9 cases, 2 of which were S. pneumoniae empyema.
Conclusion: Direct specimen sequencing is a diagnostic alternative to culture to assist in identification of pathogens in culture-negative infections. Compared to culture and histopathology, sequencing provided useful diagnostic input in 5 additional cases; pleural empyema and presence of fungal elements on histopathology. There was a poor correlation between presence of organisms in Gram stain and sequencing. The likelihood of a positive sequencing result in the absence of WBC is low. Rather, sequencing may be warranted in the presence of moderate or many WBC in Gram stain.