Optimization of Mycobacterium tuberculosis DNA processing prior to whole genome sequencing.

The process of whole genome sequencing of the Mycobacterium tuberculosis complex is dependent on complete the inactivation of the strain and subsequent DNA extraction. The objective of this study was to optimise the two steps. Firstly, the efficacy of Triton X-100 as a solvent for the inactivation step was evaluated. This solvent has been demonstrated to be effective in killing bacteria and is less toxic than the previously employed chloroform. For the extraction step, two lysis methods were evaluated: enzymatic (B1 protocol) and mechanical (B2 protocol). For whole genome sequencing, the Nextera XT DNA library preparation protocol was performed for both the B1 and B2 protocols. Subsequently, each library was subjected to whole-genome sequencing. The results demonstrated that heat lysis inactivation with Triton was effective, with no bacteria remaining viable following this treatment. The enzymatic and mechanical extraction protocols yielded comparable results in terms of DNA quantity and quality. The sequencing results showed that there was no significant difference in read depths between the two protocols. In conclusion, for MTBC strains, we recommend the use of our Triton inactivation method, which meets biosafety expectations.

Assessment of adherence to the 2020 Surviving Sepsis Campaign guidelines for fluid resuscitation in children with suspected septic shock in paediatric emergency departments: a prospective multicentre study.

BACKGROUND: Paediatric sepsis is the leading cause of death in children under 5 years. No studies have evaluated the application of the Surviving Sepsis Campaign 2020 (SSC-2020) guidelines in paediatric emergency departments (PEDs). OBJECTIVE: To assess physician adherence to the SSC-2020 fluid resuscitation guidelines in children with suspected septic shock in PEDs. METHODS: This was a prospective multicentre observational study conducted in 21 French hospitals over 5 sequential weeks, between November 2021 and March 2022. Children with suspected septic shock and who received antimicrobial therapy within 72 hours were included. Primary outcome was SSC-2020 fluid resuscitation guidelines adherence (low 0-24%; moderate 25-74%; high 75-100%) according to: bolus volume of 10-20 mL/kg each, exclusive administration of balanced crystalloids at 1 and 24 hours of management, and initiation of fluid resuscitation within 1 hour of septic shock recognition. RESULTS: 63 children were included. 10 (16%) children had severe sepsis and 2 (3%) met the definition of septic shock. Compared with the SSC-2020 guidelines, 43 (68%) patients received boluses of 10-20 mL/kg; fluid resuscitation was initiated within 1 hour of septic shock recognition in 42 (76%) cases; balanced crystalloids were the only fluids administrated in 35 (56%) and 34 (55%) children at 1 and 24 hours of management, respectively. Main barriers reported by physicians were difficult intravenous access (43%), lack of team training (29%), workload constraints (28%), and absence or out-of-date protocols (24%). CONCLUSIONS: This study found high adherence for fluid resuscitation initiation but moderate adherence for bolus volume and fluid choice. TRIAL REGISTRATION NUMBER: NCT05066464.

Performances of bioinformatics tools for the analysis of sequencing data of Mycobacterium tuberculosis complex strains.

Whole-genome sequencing of Mycobacterium tuberculosis complex (MTBC) strains is a rapidly growing tool to obtain results regarding the resistance and phylogeny of the strains. We evaluated the performances of two bioinformatics tools for the analysis of whole-genome sequences of MTBC strains. Two hundred and twenty-seven MTBC strains were isolated and whole-genome sequenced at the laboratory of Avicenne Hospital between 2015 and 2021. We investigated the resistance and susceptibility status of strains using two online tools, Mykrobe and PhyResSE. We compared the genotypic and phenotypic resistance results obtained by drug susceptibility testing. Unlike with the Mykrobe tool, sequencing quality data were obtained using PhyResSE: average coverage of 98% and average depth of 119X. We found a similar concordance between phenotypic and genotypic results when determining susceptibility to first-line anti-tuberculosis drugs (95%) with both tools. The sensitivity and specificity of each tool compared to the phenotypic method were respectively 72% [52-87] and 98% [96-99] for Mykrobe and 76% [57-90] and 97% [94-99] for PhyResSE. Mykrobe and PhyResSE were easy to use and efficient. These platforms are accessible to people not trained in bioinformatics and constitute a complementary approach to phenotypic methods for the study of MTBC strains.

Use of Whole-Genome Sequencing to Explore Mycobacterium tuberculosis Complex Circulating in a Hotspot Department in France.

The Seine-Saint-Denis is the French metropolitan department with the highest incidence of tuberculosis (TB). Our aim was to explore epidemiological and phylogenetic characteristics of TB strains in this hotspot department. We performed WGS on 227 strains of Mycobacterium tuberculosis complex isolated from patients at the Avicenne Hospital from 2016 to 2021 and randomly selected to represent the clinical diversity of French TB localization. Clinical and demographic data were recorded for each TB patient. The mean age of patients was 36 years old. They came from Africa (44%), Asia (27%), Europe (26%) and America (3%). Strains isolated from extrapulmonary samples were associated with Asian patients, whereas strains isolated from pulmonary samples were associated with European patients. We observed a high level of lineage diversity in line with the known worldwide diversity. Interestingly, lineage 3 was associated with lymph node TB. Additionally, the sensitivity of WGS for predicting resistance was 100% for rifampicin, isoniazid and ethambutol and 66.7% for pyrazinamide. The global concordance with drug-susceptibility testing using the phenotypic approach was 97%. In microbiology laboratories, WGS turns out to be an essential tool for better understanding local TB epidemiology, with direct access to circulating lineage identification and to drug susceptibilities to first- and second-line anti-TB drugs.