Host DNA depletion can increase the sensitivity of Mycobacterium spp. detection through shotgun metagenomics in sputum

Abstract

Identification and phenotypic drug-susceptibility testing for mycobacteria are time-consuming and challenging but essential for managing mycobacterial infections. Next-generation sequencing (NGS) technologies can increase diagnostic speed and quality, but standardization is still lacking for many aspects (e.g., unbiased extraction, host depletion, bioinformatic analysis). Targeted PCR approaches directly on sample material are limited by the number of targets that can be included. Unbiased shotgun metagenomics on direct material is hampered by the massive amount of host DNA, which should be removed to improve the microbial detection sensitivity. For this reason, we developed a method for NGS-based diagnosis of mycobacteria directly from patient material. As a model, we used the non-tuberculous mycobacterium (NTM) Mycobacterium abscessus. We first compared the efficiency of three different DNA extraction kits for isolating DNA (quality and concentration). The two most efficient kits were then used in a follow-up study using artificial sputum. Finally, one extraction kit was selected and further evaluated for DNA isolation from a patients’ sputum mixture spiked with M. abscessus at three concentrations (final concentrations 108, 107, 106 CFU/ml). The spiked sputum samples were processed with and without saponin treatment (ST) in combination with DNAse treatment prior to bacterial DNA extraction to evaluate the recovery of bacteria and depletion of host DNA by PCR and Illumina sequencing.While Ct values of the qPCR targeting mycobacterial ITS DNA remained rather stable, Ct values in the qPCR targeting the human β-actin gene increased by five Ct values in ST samples. In subsequent Illumina sequencing, a decrease of 89% of reads mapped to the human genome was observed in ST samples. The percentage of reads mapped to M. abscessus (108 CFU/ml) increased by 89%, and the sequencing depth increased two times when undergoing ST.In conclusion, the sensitivity of M. abscessus detection in artificial sputum was increased using a saponin pre-treatment step. The saponin followed by the DNase I treatment approach could be efficiently applied to detect and characterize mycobacterial infections, including tuberculosis, directly from sputum.

Unified metagenomic method for rapid detection of bacteria, fungi and viruses in clinical samples

“…Median human DNA depletion in 29 lower respiratory tract samples was ten cycle thresholds (IQR 4-12). Overall, across upper and lower respiratory tract samples the human DNA depletion was eight (IQR [4][5][6][7][8][9][10][11][12] representing an approximate 256-fold reduction (Table S.2 and S.3).…”

“…Depletion of the human DNA is crucial for clinical metagenomics as it enables faster and more sensitive detection of microorganisms directly from clinical samples (4,5). There are many challenges to achieving this objective, given the heterogeneity of human samples and the need to focus on identifying DNA and RNA pathogens that have many different sizes and structures and are often in low abundance compared with host's cells (6).…”

Host DNA depletion assisted metagenomic sequencing of bronchoalveolar lavage fluids for diagnosis of pulmonary tuberculosis

“…Our results highlight the potential use of mNGS-based methods for detecting TB drug resistance gene in inferring antimicrobial susceptibility 22 . However, conventional mNGS has limited effectiveness in detecting pathogen AMRs due to the low abundance of these sequences in respirators or body uids 23 and thus the use of mNGS for this purpose has remained limited 5,[24][25][26][27] . Meantime, our results demonstrated limited coverage and depth of MTB genomic sequencing of conventional mNGS, resulting in very low effectiveness of AMR gene detection, consistent with the results of a previously reported study 3 .…”

“…To tackle this problem, saponin-based host DNA depletion-assisted (HDA) nanopore sequencing was developed to effectively remove large quantities of human DNA from samples, thus reducing high hostrelated metagenomic sequencing output read rates that mask genuine pathogenic signals by increasing base-calling error rates 5,6 . Particularly, HDA-mNGS has been applied to clinical Mycobacterium spp., detection, resulting in improved diagnostic performance and data quality when used to detect mycobacteria in sputum samples 6 .…”

Untitled

“…The relevance of mycobacteriosis was raised after an increase in mortality of AIDS patients with generalised NTMB infection was noted (Ose et al, 2021;Thomas et al, 2023). The increase in the incidence of mycobacteriosis is associated with immunosuppression resulting from glucocorticoid and cytostatic therapy, treatment with tumour necrosis factor α blockers (Taira et al, 2018), a decrease in the spread of tuberculosis (Madzinga et al, 2022), and improved methods of isolation and identification of NTMB (Haworth et al, 2017;Kok et al, 2022).…”