Context: Calcification of the arterial wall in diabetes contributes to the arterial occlusive process occurring below the knee. The osteoprotegerin (OPG)/receptor activator of nuclear factor κB ligand (RANKL) system is suspected to be involved in the calcification process. Objective: The aim of the study was to investigate whether there is a link between arterial calcification in type 2 diabetes and 1) conventional cardiovascular risk factors, 2) serum RANKL and OPG levels, and 3) neuropathy. Patients and Methods: We objectively scored, in a cross-sectional study, infrapopliteal vascular calcification using computed tomography scanning in 198 patients with type 2 diabetes, a high cardiovascular risk, and a glomerular filtration rate >30 mL/min. Color duplex ultrasonography was performed to assess peripheral arterial occlusive disease, and mediacalcosis. Peripheral neuropathy was defined by a neuropathy disability score >6. RANKL and OPG were measured in the serum by routine chemistry. Results: Below-knee arterial calcification was associated with arterial occlusive disease. In multivariate logistic regression analysis, the variables significantly and independently associated with the calcification score were age (odds ratio [OR] = 1.08; 95% confidence interval [CI] = 1.04–1.13; P < .0001), male gender (OR = 3.53; 95% CI = 1.54–8.08; P = .003), previous cardiovascular disease (OR = 2.78; 95% CI = 1.39–5.59; P = .005), and neuropathy disability score (per 1 point, OR = 1.21; 95% CI = 1.05–1.38; P = .006). The association with ln OPG, significantly associated with calcification score in univariate analysis (OR = 3.14; 95% CI = 1.05–9.40; P = .045), was no longer significant in multivariate analysis. RANKL and OPG/RANKL were not significantly associated with the calcification score. Conclusions: Below-knee arterial calcification severity is clearly correlated with peripheral neuropathy severity and with several usual cardiovascular risk factors, but not with serum RANKL level.

Ethionamide (ETH) is a second-line antituberculosis drug. ETH resistance (ETH-R) is mainly related to the mutations of the monooxygenase-activating ETH (EthA), the ETH target (InhA), and the inhA promoter. Nonetheless, diagnosing ETH-R is still challenging. We assessed the strategy used for detecting ETH-R at the French National Reference Center for Mycobacteria in 497 MDR-TB isolates received from 2008 to 2016. The genotypic ETH’s resistance detection was performed by sequencing ethA, ethR, the ethA-ethR intergenic region, and the inhA promoter in the 497 multidrug-resistant isolates, whereas the phenotypic ETH susceptibility testing (PST) was performed using the reference proportion method. Mutations were found in up to 76% of the 387 resistant isolates and in up to 28% of the 110 susceptible isolates. Our results do not support the role of ethR mutations in ETH resistance. Altogether, the positive predictive value of our genotypic strategy to diagnose ETH-R was improved when only considering the variants included in the WHO catalogue and in other databases, such as TB-Profiler. Therefore, our work will help to update the list of mutations that could be graded as being associated with resistance to improve ETH-R diagnosis.

The MDR/MTB ELITe MGB^® kit (ELITech) carried on the ELITe InGenius^® platform is a new real-time PCR assay allowing automated extraction and detection of DNA of the Mycobacterium tuberculosis complex (MTB) and mutations in the rpoB and katG genes and inhA promoter region (pro-inhA) associated to resistance to rifampicin and isoniazid, the two markers of multidrug-resistant TB (MDR). We assessed the performances of the test on a collection of strains (n = 54) and a set of clinical samples (n = 242) from routine practice, comparatively to TB diagnosis and genotypic drug susceptibility testing (gDST) as references. Regarding the 242 clinical samples, the sensitivity and specificity of MTB detection by ELITe were 90.9% and 97.5%, respectively. For the detection of resistance-conferring mutations on positive clinical samples, we observed perfect agreement with gDST for katG and pro-inhA (κ = 1.0) and two discordant results for rpoB (κ = 0.82). Considering the 54 cultured strains, very good agreement with gDST was observed for the detection of the 25 distinct mutations in rpoB, katG, and pro-inhA, (κ = 0.95, 0.88, and 0.95, respectively). In conclusion, the automated MDR/MTB ELITe MGB^® assay shows great promise and appears to be a valuable tool for rapid detection of pre-MDR- and MDR-TB directly from clinical specimens.

Pulmonary tuberculosis (TB) is the most frequent site of TB and the one leading its spread worldwide. Multiple specimens are commonly collected for TB diagnosis including those requiring invasive procedures. This study aimed to review the sampling strategy for the microbiological diagnosis of pulmonary TB. A retrospective analysis of collected samples from September 1st 2014 to May 1st 2016 in the Bacteriology laboratory of Pitié-Salpêtrière Hospital (Paris, France) was performed. All the samples collected in patients aged over 18 years for the bacteriological diagnosis of pulmonary TB were included. A total of 6267 samples were collected in 2187 patients. One hundred and twenty-six patients (6%) had a culture confirmed pulmonary TB. Among them, multiple sputum collections were sufficient for TB diagnosis in 63.5%, gastric lavages permitted to avoid bronchoscopy in only 7.1%, and bronchoscopy was necessary in 29.4%. The culture positivity of sputa (8.6%) was higher than that of bronchial aspirations (3.1%), bronchiolo-alveolar lavages (BAL) (2.3%) or gastric lavages (4.8%) (P<0.001). From its 70.0% theoretical PPV value, the 46.1% selection in bronchial aspirations allocated to molecular test increased PPV up to 88.9%. Based on our data, we suggest to collect sputum consistently. If smear negative a bronchoscopy should be performed and molecular diagnosis be performed on a subset of bronchial aspirations based on expertise of the bronchoscopist.

Contexte : La tuberculose (TB) demeure une des premières causes de décès à l’échelle mondiale. La dissémination de souches multirésistantes (MDR) est alarmante. Les molécules de 2nde ligne classées dans le groupe A de l’OMS (fluoroquinolones (FQ), bédaquiline (BDQ) et linézolide), sont les molécules clé du traitement. Malheureusement, on assiste ces dernières années à l’émergence de souches ultrarésistantes (XDR) qui sont MDR et résistantes aux FQ et à l’une des autres molécules du groupe A. La lutte contre l’émergence et la dissémination de ces formes résistantes repose sur : (i) le traitement précoce et efficace des patients ; il est ainsi indispensable de diagnostiquer au mieux la résistance aux antituberculeux (antiTB), en optimisant le diagnostic moléculaire et (ii) la compréhension des mécanismes de dissémination des souches MDR. Ce travail répond à ces enjeux avec une approche génomique, par les objectifs suivants : améliorer le diagnostic de la résistance aux FQ (axe 1), et investiguer les mécanismes moléculaires de dissémination d’un cluster particulier de souches MDR (axe 2). Au-delà du travail expérimental nécessaire pour mener à bien ces objectifs, des travaux et publications en lien avec le contexte du sujet et me permettant de bien appréhender mon projet de thèse ont été réalisés. Ces travaux cités dans la partie bibliographique de ce manuscrit de thèse (articles 1 à 6), ne seront pas détaillés ici. Axe 1 : nous avons investigué les mécanismes moléculaires de résistance aux FQ non liés aux mutations dans l’ADN gyrase. Nous avons analysé les génomes de près de 1500 souches de M. tuberculosis (Mtb) résistantes aux FQ sans mutation dans l’ADN gyrase, à la fois de souches cliniques (database du CNR des Mycobactéries et consortium CRyPTIC) et de souches sélectionnées in vivo (modèle murin de TB). L’analyse génomique a mis en évidence : (i) le rôle putatif de l’opéron mfpAE, et (ii) un mécanisme inédit chez Mtb, à savoir la présence de duplications génomiques massives contenant cet opéron. Ces hypothèses in silico ont ensuite été explorées par une approche expérimentale. Axe 2 : Parmi les souches collectées au CNR ces 20 dernières années, un cluster MDR de la lignée 4 se distinguait par une combinaison spécifique de mutations de résistance : KatG-A110V et EthA-Q269* ; les souches ayant peu de mutations d’écart (< 8), bien qu’isolées sur une période de 20 ans, témoignant d’une faible vitesse d’évolution. Nous avons exploré les caractéristiques moléculaires de ce cluster, notamment sur le plan de la virulence pour expliquer cette persistance. L’analyse génomique a mis en évidence une mutation spécifique au niveau d’ESX-1, élément clé de la virulence chez Mtb, en amont d’espK. Afin d’explorer l’hypothèse de la localisation de cette mutation au sein du promoteur d’espK, nous avons réalisé (i) une étude de prédiction du promoteur in silico et (ii) des expériences de qPCR pour évaluer l’impact de cette mutation sur la transcription d’espK, mais aussi d’esxA, qui code le facteur de virulence ESAT-6. Nous avons montré qu’il y aurait une corrélation inverse entre le niveau de transcription d’espK et d’esxA. Conclusion : ce travail de Thèse a ainsi permis (i) d’améliorer les connaissances sur la résistance aux FQ, et (ii) de mieux comprendre les bases moléculaires de la persistance d’un cluster MDR liée à ESX-1. Dans la continuité de ces travaux, je poursuivrai l’étude des mécanismes moléculaires impliqués dans la résistance aux nouveaux antituberculeux (bédaquiline, délamanide et prétomanide).