Translating molecular testing into sepsis diagnosis the challenge

Translating molecular testing into sepsis diagnosis: the challenge in clinical practices. Dr Ngo Tat Trung, Ph. D (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 10 th; account for 6% death. European 135 000 death/ year, rank 3 th of death Harrison et al. Critical Care 2006; Nicasio Mancini, 2010

Classical Sepsis detection tools CÁC PHƯƠNG PHÁP CHẨN ĐOÁN NN G Y 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: 1. Impractical for fastidious pathogens 2. Time required for first wave of microbial colonies to appear is too long that might switch patients into worse deleterious situation 3. 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 *Nicasio. Mancini et al, 2010

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

Disadvantage: 1. Supper expensive equipments : including both PCR system and Mass spectrometric installation 2. 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 16 S 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

Virut Two aspects must be focused to harness PCR into sepsis diagnosis 1. Human DNA removal/bacterial DNA enrichment 2. Optimize the conditions for PCR diagnostic algorithm

Virut Our strategic resolution

Human DNA removal bacterial DNA enrichment Disrupt human blood cells/shear but keep bacterial cells intact Spin to pellet bacterial cells Total DNA extraction Input template for PCR assays

Removal of 97 -98% of human DNA from blood samples M Na OH CL B 1 Beta-globin 6 cycles Total DNA prepared by Na. OH/SDS DNA processed by MBLC 1 - S DS

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 p. H combined polar detergent / Spin to pellet bacterial cells Total DNA extraction Input template for PCR assays

l k co nt ro 1 l C FU /m l 10 00 C FU /m 10 l 00 0 C FU /m Bl an E. coli A. baumanii S. aureus P. aeruginosa K. pneumonia 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 4 h Group specfic screening assays 6 h Non-Enterobacteriaceae Gram(-) Enterobactericeae P. aeruginosa A. baumannii N. meningitidis H. influenza E. coli K. pneumoniae Salmollela P. mirabilis Gram(+) Staphylococus sp Streptococus sp Enterococcocus sp

In real clinical diagnosis 1. 2 ml for In-house human DNA removal Suspected blood sepsis 10 ml for blood culture In put DNA Group specific screening realtime PCR analysis Genus specific realtime PCR confirmation 114 sepsis suspected blood samples recruited Bacterial confirmation

108 SHPT @Bacterial screen vs culture discrepancy

The discrepancy showed by previous studies Blood culture(+) Multiplex PCR (+) 18 (5, 8%) 27 (8, 7%) 67 (21, 5%) n=311 199 (64%) 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

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