Development and evaluation of a novel fast broad-range PCR and sequencing assay (FBR-PCR/S) using dual priming oligonucleotides targeting the ITS/LSU gene regions for rapid diagnosis of invasive fungal diseases: multi-year experience in a large Canadian healthcare zone.

BACKGROUND: This study evaluated the performance of a novel fast broad range PCR and sequencing (FBR-PCR/S) assay for the improved diagnosis of invasive fungal disease (IFD) in high-risk patients in a large Canadian healthcare region. METHODS: A total of 114 clinical specimens (CS) including bronchoalveolar lavages (BALs) were prospectively tested from 107 patients over a 2-year period. Contrived BALs (n = 33) inoculated with known fungi pathogens were also tested to increase diversity. Patient characteristics, fungal stain and culture results were collected from the laboratory information system. Dual-priming oligonucleotide (DPO) primers targeted to the internal transcribed spacer (ITS) (~ 350 bp) and large subunit (LSU) (~ 550 bp) gene regions were used to perform FBR-PCR/S assays on extracted BALs/CS. The performance of the molecular test was evaluated against standard microbiological methods and clinical review for the presence of IFD. RESULTS: The 107 patients were predominantly male (67, 62.6%) with a mean age of 59 years (range = 0-85 years): 74 (69.2%) patients had at least one underlying comorbidity: 19 (34.5%) had confirmed and 12 (21.8%) had probable IFD. Culture recovered 66 fungal isolates from 55 BALs/CS with Candida spp. and Aspergillus spp. being most common. For BALs, the molecular assay vs. standard methods had sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV), and efficiency of 88.5% vs.100%, 100% vs. 61.1%, 100% vs. 88.5%, 61.1% vs. 100%, and 90.2% for both. For other CS, the molecular assay had similar performance to standard methods with sensitivity, specificity, PPV, NPV and efficiency of 66.7%, 87.0%, 66.7%, 87.0% and 81.3% for both methods. Both methods also performed similarly, regardless of whether CS stain/microscopy showed yeast/fungal elements. FBR-PCR/S assays results were reported in ~ 8 h compared to fungal cultures that took between 4 and 6 weeks. CONCLUSIONS: Rapid molecular testing compared to standard methods have equivalent diagnostic efficiency but improves clinical utility by reporting a rapid species-level identification the same dayshift (~ 8 h).

Staphylococcus aureus small colony variants are induced by the endothelial cell intracellular milieu.

Recent studies have reported that Staphylococcus aureus small colony variants (SCVs) can cause highly persistent infections in humans and in cultured endothelial cells. To understand the process by which SCVs of S. aureus appear in subjects who have not received antibiotic treatment, bovine endothelial cells were coincubated with a wild S. aureus strain for 72 h in the presence of lysostaphin. Intracellular bacteria were harvested and screened for stable SCVs. Intracellular bacteria developed the SCV phenotype at a greater rate than control bacteria not exposed to endothelial cells: The intracellular induction rate was approximately 10(-3) versus a spontaneous rate of <10(-7). This observation suggest that SCVs are induced by the intracellular milieu and suggest a possible mechanism for the intriguing pathophysiology of tissue persistence of staphylococci.