Retrospective and prospective evaluation of the FluoroType®-Mycobacteria VER 1.0 assay for the identification of mycobacteria from cultures in a French center.

PURPOSE: Rapid, reliable identification of mycobacteria from positive cultures is essential for patient management, particularly for the differential diagnosis of Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM) species. The aim of the present study was to evaluate a new "In-Vitro-Diagnostic"-certified PCR kit, FluoroType®-Mycobacteria VER 1.0 (Hain Lifescience GmbH) for NTM and MTBC identification from cultures. METHODS: Mycobacteria identification isolated from positive cultures during routine practice at the Lyon university hospital mycobacteria laboratory obtained by hsp65 amplification/sequencing were compared retrospectively and prospectively to those obtained by and the FluoroType®-Mycobacteria VER 1.0 kit. RESULTS: The overall agreement between hsp65 amplification/sequencing and the FluoroType®-Mycobacteria VER 1.0 kit was 88.4% (84/95); 91.2% (52/57) for the retrospective period and 84.2% (32/38) for the prospective period. There were 9 (9.5%) minor discrepancies (species in the FluoroType®-Mycobacteria VER 1.0 database and identified at genus level): 4 during the retrospective period, 5 during the prospective period; and 2 (2.1%) major discrepancies (species in the FluoroType®-Mycobacteria VER 1.0 database and identified incorrectly to species level): 1 during the retrospective period (M. kumamotonense identified as M. abscessus subsp massiliense by the kit) and 1 during the prospective period (M. chimaera identified as M. smegmatis by the kit). Including concordant results at genus level and minor discrepancies, 17.9% (17/95) of strains were identified as Mycobacterium sp. by the FluoroType®-Mycobacteria-VER 1.0 kit. CONCLUSION: The good performance of the FluoroType®-Mycobacteria-VER 1.0 kit with few major discrepancies could enable its use for first-line identification of positive mycobacteria cultures. However, an alternative identification method at least for reference laboratories is needed owing to the non-negligible proportion of NTM strains were identified at genus level.

Rifampicin exposure reveals within-host Mycobacterium tuberculosis diversity in patients with delayed culture conversion.

Mycobacterium tuberculosis (Mtb) genetic micro-diversity in clinical isolates may underline mycobacterial adaptation to tuberculosis (TB) infection and provide insights to anti-TB treatment response and emergence of resistance. Herein we followed within-host evolution of Mtb clinical isolates in two cohorts of TB patients, either with delayed Mtb culture conversion (> 2 months), or with fast culture conversion (< 2 months). We captured the genetic diversity of Mtb isolates obtained in each patient, by focusing on minor variants detected as unfixed single nucleotide polymorphisms (SNPs). To unmask antibiotic tolerant sub-populations, we exposed these isolates to rifampicin (RIF) prior to whole genome sequencing (WGS) analysis. Thanks to WGS, we detected at least 1 unfixed SNP within the Mtb isolates for 9/15 patients with delayed culture conversion, and non-synonymous (ns) SNPs for 8/15 patients. Furthermore, RIF exposure revealed 9 additional unfixed nsSNP from 6/15 isolates unlinked to drug resistance. By contrast, in the fast culture conversion cohort, RIF exposure only revealed 2 unfixed nsSNP from 2/20 patients. To better understand the dynamics of Mtb micro-diversity, we investigated the variant composition of a persistent Mtb clinical isolate before and after controlled stress experiments mimicking the course of TB disease. A minor variant, featuring a particular mycocerosates profile, became enriched during both RIF exposure and macrophage infection. The variant was associated with drug tolerance and intracellular persistence, consistent with the pharmacological modeling predicting increased risk of treatment failure. A thorough study of such variants not necessarily linked to canonical drug-resistance, but which are prone to promote anti-TB drug tolerance, may be crucial to prevent the subsequent emergence of resistance. Taken together, the present findings support the further exploration of Mtb micro-diversity as a promising tool to detect patients at risk of poorly responding to anti-TB treatment, ultimately allowing improved and personalized TB management.

Rapid, Easy, and Reliable Identification of Nocardia sp. by MALDI-TOF Mass Spectrometry, VITEK®-MS IVD V3.2 Database, Using Direct Deposit.

The reference methods for Nocardia identification are based on gene sequencing. These methods are time-consuming and not accessible for all laboratories. Conversely, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is easy to use and widely available in clinical laboratories, but for Nocardia identification, the VITEK®-MS manufacturer recommends a tedious step of colony preparation that is difficult to integrate into a laboratory workflow. This study aimed to evaluate Nocardia identification by MALDI-TOF VITEK®-MS using direct deposit with the VITEK®-PICKMETM pen and a formic acid-based protein extraction directly onto the bacterial smear on a 134 isolates collection; this identification was compared to the results from molecular reference methods. For 81.3% of the isolates, VITEK®-MS delivered an interpretable result. The overall agreement with the reference method was 78.4%. Taking only the species included in the VITEK®-MS in vitro diagnostic V3.2 database into account, the overall agreement was significantly higher, 93.7%. VITEK®-MS rarely misidentified isolates (4/134, 3%). Among the 25 isolates that produced no result with the VITEK®-MS, 18 were expected, as Nocardia species were not included in the VITEK®-MS V3.2 database. A rapid and reliable Nocardia identification using direct deposit by VITEK®-MS is possible by combining the use of the VITEK®-PICKMETM pen and a formic acid-based protein extractiondirectly onto the bacterial smear.

Importance of the 2,6-Difluorobenzamide Motif for FtsZ Allosteric Inhibition: Insights from Conformational Analysis, Molecular Docking and Structural Modifications.

A conformational analysis and molecular docking study comparing 2,6-difluoro-3-methoxybenzamide (DFMBA) with 3-methoxybenzamide (3-MBA) has been undertaken for investigating the known increase of FtsZ inhibition related anti S. aureus activity due to fluorination. For the isolated molecules, the calculations reveal that the presence of the fluorine atoms in DFMBA is responsible for its non-planarity, with a dihedral angle of -27° between the carboxamide and the aromatic ring. When interacting with the protein, the fluorinated ligand can thus more easily adopt the non-planar conformation found in reported co-crystallized complexes with FtsZ, than the non-fluorinated one. Molecular docking studies of the favored non-planar conformation of 2,6-difluoro-3-methoxybenzamide highlights the strong hydrophobic interactions between the difluoroaromatic ring and several key residues of the allosteric pocket, precisely between the 2-fluoro substituent and residues Val203 and Val297 and between the 6-fluoro group and the residues Asn263. The docking simulation in the allosteric binding site also confirms the critical importance of the hydrogen bonds between the carboxamide group with the residues Val207, Leu209 and Asn263. Changing the carboxamide functional group of 3-alkyloxybenzamide and 3-alkyloxy-2,6-difluorobenzamide to a benzohydroxamic acid or benzohydrazide led to inactive compounds, confirming the importance of the carboxamide group.

Heterogeneous vancomycin resistance in Staphylococcus aureus does not predict development of vancomycin resistance upon vancomycin pressure.

BACKGROUND: It is unclear whether Staphylococcus aureus with heterogeneous intermediate vancomycin resistance (hVISA) can develop vancomycin resistance faster than vancomycin-susceptible S. aureus (VSSA) strains. METHODS: We compared the kinetics of vancomycin MIC increase for 15 days of sustained in vitro vancomycin exposure for clinical hVISA (n = 12) and VSSA (n = 24) isolates, as well as for reference strains Mu3 (hVISA) and ATCC 29213 (VSSA). Clinical isolates were categorized as hVISA using the population analysis profile method. MICs were monitored for 15 days and the rate of MIC increase under exposure, for each strain, was evaluated in a linear regression model relative to time. RESULTS: All isolates acquired vancomycin resistance upon exposure. Vancomycin MICs increased faster for VSSA compared with hVISA isolates (P < 0.01). CONCLUSIONS: The hVISA phenotype does not correspond to an enhanced adaptation potential to in vitro vancomycin pressure.

Development, Evaluation, and Implementation of a House-Made Targeted Next-Generation Sequencing Spoligotyping in a French Laboratory.

Epidemiological studies investigating transmission chains of tuberculosis are undertaken worldwide to tackle its spread. CRISPR locus diversity, called spoligotyping, is a widely used genotyping assay for Mycobacterium tuberculosis complex (MTBC) characterization. Herein, we developed a house-made targeted next-generation sequencing (tNGS) spoligotyping, and compared its outputs with those of membrane-based spoligotyping. A total of 144 clinical MTBC strains were retrospectively selected to be representative of the local epidemiology. Data analysis of a training set allowed for the setting of "presence"/"absence" thresholds for each spacer to maximize the sensibility and specificity related to the membrane-based spoligotyping. The thresholds above, in which the spacer was considered present, were 50 read per millions for spacers 10 and 14, 20,000 for spacers 20, 21, and 31, and 1000 for the other spacers. The confirmation of these thresholds was performed using a validation set. The overall agreement on the training and validation sets was 97.5% and 93.8%, respectively. The discrepancies concerned six strains: Two for spacer 14, two for spacer 31, and two for spacer 32. The tNGS spoligotyping, whose thresholds were finely-tuned during a careful bioinformatics pipeline development process, appears be a technique that is reliable, inexpensive, free of handling errors, and automatable through automatic transfer into the laboratory computer system.

Structural Variations in the Central Heterocyclic Scaffold of Tripartite 2,6-Difluorobenzamides: Influence on Their Antibacterial Activity against MDR Staphylococcus aureus.

Five series of heterocyclic tripartite 2,6-difluorobenzamides, namely 1,2,3-triazoles, 1,2,4- and 1,3,4-oxadiazoles, analogs of reported model anti-staphylococcal compounds, were prepared. The purpose was to investigate the influence of the nature of the heterocyclic central scaffold on the biological activity against three strains of S. aureus, including two drug-resistant ones. Among the 15 compounds of the new collection, a 3-(4-tert-butylphenyl)-1,2,4-oxadiazole linked via a methylene group with a 2,6-difluorobenzamide moiety (II.c) exhibited a minimal inhibitory concentration between 0.5 and 1 µg/mL according to the strain. Subsequent studies on II.c demonstrated no human cytotoxicity, while targeting the bacterial divisome.

Population Pharmacokinetic Modeling and Dosing Simulations of Tobramycin in Pediatric Patients with Cystic Fibrosis.

Initial dosing and dose adjustment of intravenous tobramycin in children with cystic fibrosis (CF) is challenging. The objectives of this study were to develop nonparametric population pharmacokinetic (PK) models of tobramycin in children with CF to be used for dosage design and model-guided therapeutic drug monitoring. We performed a retrospective analysis of tobramycin PK data in our children's CF center. The Pmetrics package was used for nonparametric population PK analysis and dosing simulations. Both the ratios of maximal concentration to the MIC (Cmax/MIC) and daily area under the concentration-time curve to the MIC (AUC24/MIC) were considered efficacy targets. Trough concentration (Cmin) was considered the safety target. A total of 2,884 tobramycin concentrations collected in 195 patients over 9 years were analyzed. A two-compartment model including total body weight, body surface area, and creatinine clearance as covariates best described the data. A simpler model was also derived for implementation in the BestDose software to perform Bayesian dose adjustment. Both models were externally validated. PK/pharmacodynamics (PD) simulations with the final model suggest that an initial dose of tobramycin of 15 to 17.5 mg/kg/day was necessary to achieve Cmax/MICs of ≥10 for MICs up to 2 mg/liter in most patients. The AUC24/MIC target was associated with higher dosage requirements and higher Cmin. A daily dose of 12.5 mg/kg would optimize both efficacy and safety target attainment. We recommend performing tobramycin therapeutic drug monitoring (TDM), model-based dose adjustment, and MIC determination to individualize intravenous tobramycin therapy in children with CF.

Prognostic factors of severe community-acquired staphylococcal pneumonia in France.

PURPOSE: Staphylococcus aureus causes severe forms of community-acquired pneumonia (CAP), namely staphylococcal pleuropneumonia in young children and staphylococcal necrotising pneumonia in older patients. Methicillin resistance and the Panton-Valentine leukocidin (PVL) toxin, as well as less specific factors, have been associated with poor outcome in severe CAP, but their roles are unclear. METHODS: A prospective multicentre cohort study of severe staphylococcal CAP was conducted in 77 paediatric and adult intensive care units in France between January 2011 and December 2016. After age-clustering, risk factors for mortality, including pre-existing conditions, clinical presentation, laboratory features, strain genetic lineage, PVL, other virulence factors and methicillin resistance were assessed using univariate and multivariable Cox and LASSO (least absolute shrinkage and selection operator) regressions. RESULTS: Out of 163 included patients, aged 1 month to 87 years, 85 (52.1%) had PVL-positive CAP; there were 20 (12.3%) patients aged <3 years (hereafter "toddlers"), among whom 19 (95%) had PVL-positive CAP. The features of PVL-positive CAP in toddlers matched with the historical description of staphylococcal pleuropneumonia, with a lower mortality (three (15%) out of 19) compared to PVL-positive CAP in older patients (31 (47%) out of 66). Mortality in older patients was predicted by PVL-positivity (hazard ratio (HR) 1.81, 95% CI 1.03-3.17) and methicillin resistance (HR 2.37, 95% CI 1.29-4.34) independently from S. aureus lineages and the presence of other determinants of virulence. CONCLUSION: PVL was associated with staphylococcal pleuropneumonia in toddlers and was a risk factor for mortality in older patients with severe CAP, independently of methicillin resistance, S. aureus genetic background and other virulence factors.

Long-term use of liposomal nebulized amikacin and tedizolid for the treatment of disseminated nocardiosis after allogeneic hematopoietic stem cell transplantation.

Nocardiosis is a life-threatening opportunistic infection in immunocompromised patients. Herein, we present successful adjunctive use of liposomal nebulized amikacin and tedizolid in a recipient of allogeneic hematopoietic stem cell transplantation infected with Nocardia nova complex who presented multiple complications to conventional therapeutic options.