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.

Neurodegenerative diseases are estimated to afflict hundreds of thousands of Americans with vastly more worldwide. The etiologies of amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS) have yet to be established. Previous studies have suggested an association of these diseases with viruses, bacteria, and eukaryotic microbes, no new therapies have been forthcoming. High-throughput DNA sequencing has enabled the comprehensive analysis of microbial DNA profiles in diseased populations. To date, no amplicon-based next-generation DNA sequencing prokaryotic and eukaryotic community profiling studies have been completed for these diseases. Analysis of peripheral blood samples from control participants as well as ALS and MS participants was used to characterize the hematologic population of microbial DNA. Categorical and multivariate analysis with control for multiple comparisons and aged matched controls revealed differences in microbial DNA contribution in ALS patients compared to others. Notably, sequences that belonging to Ochrophyta were enriched in ALS patient samples. Mechanisms underlying this association, the role of microbial DNA sequences, and the development or progression of ALS may become a fertile subject of inquiry.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is estimated to afflict hundreds of thousands, if not millions, of Americans with vastly more impacted individuals worldwide; however, the etiology of this disease has not been well established. Based on the features of ME/CFS, we hypothesized an unrecognized vascular infection may be involved. To evaluate this possibility, we performed a blinded pilot study of 30 ME/CFS patients meeting the Fukuda criteria and 48 normal controls. A community-wide analysis using next-generation DNA sequencing methods detected prokaryotic and eukaryotic populations in the peripheral blood of both ME/CFS patients and normal controls. Analysis of the prokaryotic portion of the samples revealed that organisms belonging to the Pseudomonas asplenii species, Pseudomonadaceae family, Pseudomonadales order, and γ-proteobacteria class are inversely correlated with RAND-36 scores, a quality of life metric that is reduced in ME/CFS patients. In addition, analysis of the detected eukaryotic species suggests that the Perkinsus genus is also inversely associated with RAND-36 scores. The most frequently observed eukaryotic DNA was for Funneliformis mosseae, an arbuscular mycorrhizal fungus, which was in both ME/CFS and normal control samples. A multivariate composite score consisting of the Perkinsus genus, Spumella genus, and β-proteobacteria class displays an inverse relationship to RAND-36 scores. Lastly, the combined measurements of several taxa allow for a retrospective categorical sorting of ME/CFS patients from normal control samples. These results suggest that microbial DNA signatures, including those from poorly understood eukaryotes, may be differentially detectable in ME/CFS and normal control samples.

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 poly microbial 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 Hoechst 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. (C) 2017 Elsevier Ltd. All rights reserved.

The advent of high throughput human DNA sequencing capability has allowed a crossover for sequencing infectious diseases. The same technologies that allow us to query the human genome for cancer mutations, pharmacogenomics, and inherited genetic errors now allow a more in-depth analysis of human samples for evidence of infectious disease. Next Generation DNA sequencing (NGS) for infectious disease holds the promise of accuracy with greater sensitivity and specificity than culture, serologic and PCR methods. NGS allows for better discrimination between strains, species, detection of novel variants and new organisms, detection of an ever-growing array of uncultivable organisms, and the ability to detect eukaryotes that were previously undetectable. NGS also may soon provide the ability to determine drug resistance and sensitivity information. The following describes the Rapid Infectious Disease Identification System (RIDI™) and its practical use. Application of the RIDI™ system is discussed in four case reports with patients suffering from chronic malaise, rheumatoid arthritis, osteoarthritis, and chronic fatigue syndrome. Keywords: next-generation sequencing for infectious disease, RIDI™, chronic fatigue syndrome, osteoarthritis, rheumatoid arthritis, Funneliformis mosseae, Saccharomyces cerevisiae, Toxoplasma gondii

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.

Background: Evidence that vascular inflammation is an important mechanism involved in all stages of atherogenesis continues to accumulate. We hypothesize that common complex microbial involvement may be present in atheromatous debris removed from treated lesions. We evaluated this debris with metagenomic analysis. Methods: After informed consent, fifteen cases of vascular aspirate or explanted embolic filters were examined. Thirteen cases were in patients undergoing carotid stenting (3 MoMa, 10 embolic filters). One case was in a saphenous vein graft intervention (aspirate and filter) and one case was after SFA orbital atherectomy (aspirate and filter). Fluorescence microscopy, bacterial and protozoan metagenomic analysis using the RIDI™ Next Generation Sequencing (NGS) analysis system, and specific protozoan multiplex PCR probes were used to assess the presence and composition of biofilm populations. Results: Bacteria were not detected in peripheral blood; however, 4 of 12 filters and 2 of 5 atheroma debris samples had identified bacterial populations (2 patients had atheroma debris and filter evaluated). Evidence of protozoan populations was obtained in 4 of 15 peripheral blood samples, 11 of 12 filters and 4 of 5 atheroma debris samples. Microscopy illustrated a complex composition of biofilm communities in blood, devices, and atheroma debris samples. The identified bacterial taxa in atheroma debris suggest a diverse and novel population composition. Biofilm dwelling bacteria, while present in several atheroma or filter samples, were not detectable in peripheral blood and were not universally present in atheroma or filter. Taxonomic comparisons of sequenced protozoa are consistent with a diverse array of organisms similar to poorly characterized environmental protozoa. Conclusion: Of 15 patients, 6 patients had evidence of bacteria and 13 had evidence of protozoa in debris and 14 exhibited evidence of complex biofilm communities. This data suggests that biofilm forming protozoa may play a key role in arterial vascular disease.

Objectives: New technologies, particularly in the field of clinical metagenomics, have opened up new avenues to explore microbial involvement in illnesses where disease initiation is poorly understood. Here we present a series of six patients suffering from diseases with potential microbial involvement: systemic lupus erythematosus, chronic fatigue syndrome, fibromyalgia, multiple sclerosis, amyotrophic lateral sclerosis, and arteriosclerosis. Material and methods: Peripheral blood (first five cases) and suction debris from carotid percutaneous angioplasty (arteriosclerosis) were refrigerated and transported to the site of analysis. DNA was extracted using the standard QIAamp DNA Blood Mini and Protozoal Extraction kits. Bacterial-specific DNA sequences were amplified and subsequently barcoded for DNA sequencing with 16S rRNA-directed primers flanking the variable regions 1 and 2 and variable regions 4 and 5, respectively. Protozoal-specific DNA sequences were amplified and subsequently barcoded for DNA sequencing using low stringency conditions and primers directed to variable regions in the 18S rRNA gene. Results: We found previously unreported mixed bacterial and protozoal biofilm communities in all samples. In all six disease types we revealed protozoa, being close relatives to known aquatic-based protozoa, such as: Oblongichytrium, Chrysocapsa vernalis, Ochromonas, and Perkinsus qugwadi. In a blood sample from the patient with lupus erythematosus DNA stains and culture studies revealed an unknown protozoan exhibiting profound biofilm-forming capability: candidatus Protomyxzoa rheumatica. Conclusions: Discovery of these potentially pathogenic protozoa in human blood may be of great clinical relevance. However, our preliminary results should be confirmed by independent researchers, and the role of biofilm-forming protozoa in human disease should be elucidated.