Bacterial DNA enrichment for low-inoculum fracture-related infection diagnostic using high-throughput sequencing.

One of the main barriers for the implementation of metagenomic sequencing in routine diagnosis of infectious diseases is the presence of host DNA. While several enrichment methods are likely to overcome this issue, their effectiveness for specimens such as bone in the case of chronic infections remains to be determined. We compared the relevance of two methods for bacterial DNA enrichment when compared to a reference protocol during pretreatment of bone samples from fracture-related infections before HTS by both Illumina Miseq and Oxford Nanopore Technology (ONT). The bacterial/human DNA ratio was higher for either protocols than the reference technique (p = 0.00012), without any significant difference between them. HTS sensitivity over culture ranged from 21.7 % to 85 %. The ability of the studied protocols to improve the bacterial/human DNA ratio depends on the sequencing technique employed. In this context, there is room for improvement in enhancing the sensitivity of HTS for diagnostic purpose.

SARS-CoV-2 quasi-species analysis from patients with persistent nasopharyngeal shedding.

At the time of a new and unprecedented viral pandemic, many questions are being asked about the genomic evolution of SARS-CoV-2 and the emergence of different variants, leading to therapeutic and immune evasion and survival of this genetically highly labile RNA virus. The nasopharyngeal persistence of infectious virus beyond 17 days proves its constant interaction with the human immune system and increases the intra-individual mutational possibilities. We performed a prospective high-throughput sequencing study (ARTIC Nanopore) of SARS-CoV-2 from so-called "persistent" patients, comparing them with a non-persistent population, and analyzing the quasi-species present in a single sample at time t. Global intra-individual variability in persistent patients was found to be higher than in controls (mean 5.3%, Standard deviation 0.9 versus 4.6% SD 0.3, respectively, p < 0.001). In the detailed analysis, we found a greater difference between persistent and non-persistent patients with non-severe COVID 19, and between the two groups infected with clade 20A. Furthermore, we found minority N501Y and P681H mutation clouds in all patients, with no significant differences found both groups. The question of the SARS-CoV-2 viral variants' genesis remains to be further investigated, with the need to prevent new viral propagations and their consequences, and quasi-species analysis could be an important key to watch out.