Dr Ngo Tat Trung, PhD Danang– September 2015 Translating molecular testing into sepsis diagnosis: the challenge in clinical practices. Dr Ngo Tat Trung, PhD (Dept. Molecular biology 108 Military Central Hospital) Danang– September 2015

Sepsis related deaths Hospitalized sepsis patient Sepsis related death Năm US: 750.000 patients/year; 215 000 death/year, death=20-70%; rank 10th; account for 6% death. European135 000 death/ year, rank 3th of death Harrison et al. Critical Care 2006; Nicasio Mancini, 2010

Classical Sepsis detection tools CÁC PHƯƠNG PHÁP CHẨN ĐOÁN NN GÂY NKH Clinical symptoms Immune response monitor Blood culture

Blood culture Classical method, widely implemented but still far from making clinician satisfied because of its intrinsic drawbacks: Impractical for fastidious pathogens Time required for first wave of microbial colonies to appear is too long that might switch patients into worse deleterious situation Large volumes blood is mandatory for proper culturing of aerobic and anaerobic bacteria

Consequence of mis/late detection Risk to be sepsis shock: Reduce chances to survive Increased hospital cost Drug resistance clones emerge → need to develop relevent approaches that work complimentary to blood culture *NicasioMancini et al, 2010

PCR combined mass spectrometric DNA sequencing Nucleic acid test(NAT)

Disadvantage: Supper expensive equipments : including both PCR system and Mass spectrometric installation Well trained personnel

Promising technology, especially for multi-readout assays

Challenges to PCR’s sensitivity An optimized PCR reaction using DNA extracted by Qiagen, Zymo blood extraction kits can only sense pathogen’s ribosomal 16S pieces if the bacterial load exceeds roughly 500 CFU/ml Patients would present sepsis – related clinical symptoms if bacterial load exceed 10- 500 CFU/ml Klouche and Schröder 2008

How the primers mis-pairing to human DNA happens NATURE | VOL 431 | 9 SEPTEMBER 2004

Two aspects must be focused to harness PCR into sepsis diagnosis Virut Two aspects must be focused to harness PCR into sepsis diagnosis Human DNA removal/bacterial DNA enrichment Optimize the conditions for PCR diagnostic algorithm

Our strategic resolution Virut Our strategic resolution

bacterial DNA enrichment Human DNA removal bacterial DNA enrichment Disrupt human blood cells/shear but keep bacterial cells intact Figure 1: Stepwise pre-analytical protocol to remove human DNA from sepsis- suspected- blood samples Spin to pellet bacterial cells Total DNA extraction Input template for PCR assays

Removal of 97-98% of human DNA from blood samples NaOH- SDS NaOH- SDS MCLB1 Beta-globin 6 cycles Figure 3 Efficiency of human DNA removal: The residues of human DNA after MCLB1 treatment was monitored via beta-globin derived amplification assays: upper panel gel based PCR assay target beta-globin gene; lower panel is sybr green based realtime PCR to quantify residual beta globin gene fragment Total DNA prepared by NaOH/SDS DNA processed by MBLC1

Bacterial limits of detection Upon human DNA removal Pseudo sepsis samples (bacterial spiked healthy blood dilution series) Disrupt human blood cells and shear chromatin by basic pH combined polar detergent / Spin to pellet bacterial cells Total DNA extraction Input template for PCR assays

10000 CFU/ml 1000 CFU/ml Blank control 100 CFU/ml 10 CFU/ml 1 CFU/ml E. coli A. baumanii S.aureus P. aeruginosa K. pneumonia S pneumonia Figure 4: Sepsis causative pathogens detection limits after human DNA removal Salmonella P. mirabilis S. pidermidis S. suise Enterococcus sp

Enterobacteriaceae piked healthy blood dilution series

Optimize the conditions for PCR diagnostic algorithm

Input bacterial DNA extracted after human chromatin removal Group specfic screening assays 4h Non-Enterobacteriaceae Gram(-) Enterobactericeae Gram(+) 6h Figure 2 Group specific – screening algorithm: the first three screening taqman PCR reactions that target bacterial ribosome-16S genes to simultaneously differentiatiate Gram-positive and Gram-negative and Enterobacteriaceae Groups. Samples that are positive in the screening assay will be subjected to genus specific realtime PCR reactions to detect 12 most common sepsis causative pathogens P. aeruginosa A. baumannii N. meningitidis H. influenza E. coli K. pneumoniae Salmollela P. mirabilis Staphylococus sp Streptococus sp Enterococcocus sp

114 sepsis suspected blood samples recruited In real clinical diagnosis Group specific screening realtime PCR analysis Genus specific realtime PCR confirmation 1.2 ml for In-house human DNA removal In put DNA 114 sepsis suspected blood samples recruited Suspected blood sepsis Figure 5: Study flowchart to compare sepsis causative pathogens diagnostics from direct DNA extraction samples, human DNA removed samples and blood culture. 10ml for blood culture Bacterial confirmation

108SHPT @Bacterial screen vs culture discrepancy Figure 6: Difference in diagnostics of sepsis causative pathogens either by molecular approach using from direct human DNA removed samples or classical blood culture methodology: upper panel Ven diagram shows the diagnostics overlap between PCR method using human DNA removed input and blood culture, lower panel shows diagnostic details of the two methods

The discrepancy showed by previous studies Blood culture(+) Multiplex PCR (+) 67 (21,5%) 27 (8,7%) 18 (5,8%) 199 (64%) n=311 Blood(-) PCR (-) Frank Bloos et al. 2012

In conclude: Targeted enrichment of bacterial DNA as consequence of human DNA removal significantly enhances the sensitivity of downstream PCR based sepsis diagnostics

Thank you for your attention We would like to acknowledge the funding from Vietnamese Ministry of Science and Technology (Grant: KC-10.43/11-15) for this study Thank you for your attention