Midtveisevaluering - Ruben Dyrhovden

Candidate: Ruben Dyrhovden

Main Supervisor: Øyvind Kommedal

Introduction
Complete microbial characterization of polymicrobial invasive infections by deep sequencing may improve our knowledge of the etiology and microbial dynamics of the infection. Such knowledge may lead to better and more targeted treatment of the infections as well as better strategies for preventing the infections. This PhD project investigates the use of amplicon deep sequencing in microbiological diagnostics of polymicrobial invasive infections. Three different types of infections are investigated by use of deep sequencing; Pleural empyema, acute cholecystitis and acute cholangitis.

Methods
Paper one is a retrospective study including 64 patients with pleural empyema. Pleural empyema samples were characterized with, culture, Sanger-based 16S sequencing, 16S rRNA deep sequencing and for selected samples rpoB-gene deep sequencing. Comparative analysis with brain abscesses was performed using metagenomic data from a national Norwegian study. Paper two is a prospective study including bile samples from 36 patients with either moderate or severe acute cholecystitis. Bile samples from the included patients were analyzed using both culture and deep sequencing of bacterial 16S rRNA and rpoB genes and the fungal ITS2-segment. Paper three is a prospective study including 41 bile samples from patients with either acute cholangitis (15) or bile duct stone without infection (26). Samples are analyzed with deep sequencing of bacterial 16S rRNA and rpoB genes and the fungal ITS2-segment, and findings in and discrepancies between the two groups are discussed in a clinical and etiologic perspective. This paper also includes an investigation of the reliability of amplicon deep-sequencing in polymicrobial samples with varying amounts of bacterial DNA and provides evidence-based suggestions for how to handle background contamination.

Results
In paper one we found that of the 385 bacterial detections done by deep sequencing, conventional culture and Sanger-based sequencing only identified 10% and 23% respectively. We also found that a major subgroup of the pleural empyema appeared to be caused by a set of bacteria not normally considered to be involved in pneumonia, and that such empyemas had a similar microbial profile to oral/sinus-derived brain abscesses. We argue that our findings support spread of bacteria to the pleura from the oral cavity, most likely hematogenously.

In paper two we found that culture identified only 40 (38%) of the 106 microbes identified by sequencing in acute cholecystitis bile samples. In none of the 15 polymicrobial samples did culture detect all present microbes. Frequently identified bacteria often missed by culture included oral streptococci, anaerobic bacteria, enterococci and Enterobacteriaceae other than Klebsiella spp. and Escherichia coli. We conclude that culture techniques display decreased sensitivity for the microbial diagnostics of acute cholecystitis, leaving possible pathogens undetected.

Collection and laboratory work for paper three is finished, and the data and results are currently being analyzed. Paper three is planned to be ready for publication first half of 2020.