Title : Detection limits of PaRTI-SeqTM in host-depleted blood: A biological perspective using mycobacterium smegmatis

Abstract:

Bloodstream infections, particularly in sepsis, need fast and accurate pathogen identification. Although culture-based diagnostics remain the gold standard, they are limited by delayed results, contamination risk and low sensitivity in low-biomass or polymicrobial infections.

This study evaluated the analytical sensitivity of a host-depleted metagenomic next-generation sequencing (mNGS) workflow (PaRTI-Seq^TM) using Mycobacterium smegmatis (M. smegmatis) as a biologically challenging model organism. 

M. smegmatis was selected to represent structurally resilient bacteria that are difficult to lyse. Serial dilutions of M. smegmatis were enumerated by plate count to determine colony-forming unit (CFU) inputs. Microbial DNA extraction was performed using the DEVIN^TM workflow with and without bead-beating followed by DNA quantification using Qubit. Libraries were sequenced on an Illumina MiSeq platform. Its read distributions were compared between culture and host-depleted blood samples. 

The CFU input range was determined by plate enumeration where M. smegmatis had a limit of detection of 21 600 CFU/mL under standard extraction conditions. Bead-beating improved DNA recovery, increasing extraction efficiency from 41.38% to 212.8% showing that mycobacteria needs mechanical disruption for effective lysis of its cell envelope. M. smegmatis reads were detected at higher CFU inputs in culture samples. No microbial reads were recovered in host-depleted blood where sequencing was dominated by 99% of host-DNA reads. 

These findings show that analytical sensitivity of blood-based mNGS is mostly restricted by organism-specific extraction efficiency and overwhelming host DNA instead of the sequencing performance. Further testing of extraction strategies and clinical validation using pathogens that are stressed and exposed to antibiotics are needed to improve the reliability of mNGS used in clinical diagnostics for bloodstream infections.

Biography:

Shivadarshini Ramachandran is a recent First Class Honours graduate in Biomedical Science from the Faculty of Health and Life Sciences at Northumbria University. Her academic interests are on clinical diagnostics, infectious diseases and translational applications of next-generation sequencing. Her research evaluated the analytical sensitivity of a host-depleted metagenomic workflow (PaRTI-Seq™) using Mycobacterium smegmatis as a structurally resilient model organism. Through this work, she developed expertise in microbial culture, DNA extraction optimisation and Illumina-based sequencing analysis, with a focus on improving pathogen detection in bloodstream infections and advancing rapid, culture-independent diagnostic approaches.