Microbial community profiling by next-generation DNA sequencing of adenocarcinoma of the prostate with evidence of ochratoxin A producing fungi.

BACKGROUND: Prostatic carcinomas are a leading cancer and leading cause of mortality in the developed world. The etiology is diverse with underlying patient genetics, environmental factors, and microbial associations. Sequencing DNA for microbes allows the detection of potential disease relationships. OBJECTIVE: Targeted 16S (prokaryotic) and 18S (eukaryotic) rDNA sequencing was performed to map the tumor microbial flora. DESIGN: Twelve patients undergoing elective laparoscopic prostatectomy for biopsy proven adenocarcinoma of the prostate were enrolled. PCR and amplicon based sequencing was conducted; a portion of the sequencing results were confirmed by special stains. SETTING: Patients were recruited by the urologist were prospectively scheduled for radical prostatectomy by 'Da Vinci' robotically assisted procedure in an outpatient setting. Samples were portioned in the hospital surgical suite at the time of prostatectomy. PARTICIPANTS: Male patients were requested to enter the study on a first come basis. OUTCOME MEASUREMENT AND STATISTICAL ANALYSIS: Average age of the 12 participants was 64.3 years. RESULTS AND LIMITATIONS: DNA reads were detected and by 'best match' were identified belonging to Perkinsus, Hydrurus, Diversispora and Funneliformis genera, few samples displayed bacteria. Out of the 12 total patients, 11 patients had detectable DNA sequences matching arbuscular mycorrhizal fungi in the Glomeromycetes Class; Funneliformis mosseae and Diversasporum versiformis. Specific PCR for arbuscular mycorrhizal fungi failed to confirm Glomeromycetes Class; in-depth taxonomic analysis suggests a newer fungal grouping, not falling within an accepted Phylum of fungi. Calcoflour white staining of histological sections confirmed potential fungal markers in all 12 cases. Ochratoxin A antigen was identified by immunofluorescence in all 12 patient samples. The study was limited by the low sample volume and disease free normal controls. CONCLUSIONS: Fungi may play a significant role in adenocarcinoma of the prostate.

Evidence for polymicrobial communities in explanted vascular filters and atheroma debris.

RATIONALE: Microbial communities have been implicated in a variety of disease processes and have been intermittently observed in arterial disease; however, no comprehensive unbiased community analysis has been performed. We hypothesize that complex microbial communities may be involved in chronic vascular diseases as well and may be effectively characterized by molecular assays. OBJECTIVE: The main objective is to survey vascular debris, atheroma, and vascular filters for polymicrobial communities consisting of prokaryotic and eukaryotic microbes, specifically eukaryotic microbes. METHODS AND RESULTS: We examined vascular aspirates of atheromatous debris or embolic protection filters in addition to matched peripheral blood samples, from fifteen patients, as well as three cadaveric coronary arteries from two separate patients, for microbial communities. General fluorescence microscopy by Höechst and ethidium bromide DNA stains, prokaryotic and eukaryotic community analysis by Next Generation DNA Sequencing (NGS), and a eukaryotic microbial 9 probe multiplexed quantitative PCR were used to detect and characterize the presence of putative polymicrobial communities. No prokaryotes were detected in peripheral blood; however, in 4 of 9 sequenced filters and in 2 of 7 sequenced atheroma debris samples, prokaryotic populations were identified. By DNA sequencing, eukaryotic microbes were detected in 4 of 15 blood samples, 5 of the 9 sequenced filters, and 3 of the 7 atheroma debris samples. The quantitative multiplex PCR detected sequences consistent with eukaryotic microbes in all 9 analyzed filter samples as well as 5 of the 7 atheroma debris samples. Microscopy reveals putative polymicrobial communities within filters and atheroma debris. The main contributing prokaryotic species in atheroma debris suggest a diverse and novel composition. Additionally, Funneliformis mosseae, an arbuscular mycorrhizal fungus in the Glomeraceae family, was detected in the coronary hard plaque from two patients. Well studied biofilm forming bacteria were not detectable in circulating peripheral blood and were not universally present in atheroma or filters. Analyses of the sequenced eukaryotes are consistent with a diverse of array poorly studied environmental eukaryotes. In summary, out of 15 patients, 6 exhibited molecular evidence of prokaryotes and 14 had molecular evidence of eukaryotic and/or polymicrobial communities in vivo, while 2 post-mortem coronary plaque samples displayed evidence of fungi. CONCLUSION: Prokaryotes are not consistently observed in atheroma debris or filter samples; however, detection of protozoa and fungi in these samples suggests that they may play a role in arterial vascular disease or atheroma formation.

Rapid infectious disease identification by next-generation DNA sequencing.

Currently, there is a critical need to rapidly identify infectious organisms in clinical samples. Next-Generation Sequencing (NGS) could surmount the deficiencies of culture-based methods; however, there are no standardized, automated programs to process NGS data. To address this deficiency, we developed the Rapid Infectious Disease Identification (RIDI™) system. The system requires minimal guidance, which reduces operator errors. The system is compatible with the three major NGS platforms. It automatically interfaces with the sequencing system, detects their data format, configures the analysis type, applies appropriate quality control, and analyzes the results. Sequence information is characterized using both the NCBI database and RIDI™ specific databases. RIDI™ was designed to identify high probability sequence matches and more divergent matches that could represent different or novel species. We challenged the system using defined American Type Culture Collection (ATCC) reference standards of 27 species, both individually and in varying combinations. The system was able to rapidly detect known organisms in <12h with multi-sample throughput. The system accurately identifies 99.5% of the DNA sequence reads at the genus-level and 75.3% at the species-level in reference standards. It has a limit of detection of 146cells/ml in simulated clinical samples, and is also able to identify the components of polymicrobial samples with 16.9% discrepancy at the genus-level and 31.2% at the species-level. Thus, the system's effectiveness may exceed current methods, especially in situations where culture methods could produce false negatives or where rapid results would influence patient outcomes.