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We report the emergence of an atpE mutation in a clinical Mycobacterium tuberculosis strain. Genotypic and phenotypic bedaquiline susceptibility testing displayed variable results over time and ultimately were not predictive of treatment outcome. This observation highlights the limits of current genotypic and phenotypic methods for detection of bedaquiline resistance.

Pyrazinamidase (PncA) activates the first-line antituberculous drug pyrazinamide into pyrazinoic acid. The crystal structure of the Mycobacterium tuberculosis PncA protein has been determined, showing significant differences in the substrate binding cavity when compared to the pyrazinamidases from Pyrococcus horikoshii and Acinetobacter baumanii. In M. tuberculosis, this region was found to hold a Fe(2+) ion coordinated by one aspartate and three histidines, one of them corresponding to His57 which is replaced by Asp in Mycobacterium bovis, a species naturally resistant to pyrazinamide. The binding cavity also contains a Cys138-Asp8-Lys96 motif evocating a cysteine-based catalytic mechanism. Mutants have been constructed and investigated by kinetic and thermal shift assays, highlighting the importance of protein folding and thermal stability in the pyrazinamidase activity.

The GeneLEAD VIII (Diagenode, Belgium) is a new, fully automated, sample-to-result precision instrument for the extraction of DNA and PCR detection of Mycobacterium tuberculosis complex (MTBC) directly from clinical samples. The Deeplex Myc-TB ® assay (Genoscreen, France) is a diagnostic kit based on the deep sequencing of a 24-plexed amplicon mix allowing simultaneously the detection of resistance to 13 antituberculous (antiTB) drugs and the determination of spoligotype. We evaluated the performance of a strategy combining the both mentioned tools to detect directly from clinical samples, in 8 days, MTBC and its resistance to 13 antiTB drugs, and identify potential transmission of strains from patient-to-patient. Using this approach, we screened 112 clinical samples (65 smear-negative) and 94 MTBC cultured strains. The sensitivity and the specificity of the GeneLEAD/Deeplex Myc-TB approach for MTBC detection were 79.3% and 100%, respectively. One hundred forty successful Deeplex Myc-TB results were obtained for 46 clinical samples and 94 strains, a total of 85.4% of which had a Deeplex Myc-TB susceptibility and resistance prediction consistent with phenotypic drug susceptibility testing (DST). Importantly, the Deeplex Myc-TB assay was able to detect 100% of the multidrug-resistant (MDR) MTBC tested. The lowest concordance rates were for pyrazinamide, ethambutol, streptomycin, and ethionamide (84.5%, 81.5%, 73%, and 55%, respectively) for which the determination of susceptibility or resistance is generally difficult with current tools. One of the main difficulties of Deeplex Myc-TB is to interpret the non-synonymous uncharacterized variants that can represent up to 30% of the detected single nucleotide variants. We observed a good level of concordance between Deeplex Myc-TB-spoligotyping and MIRU-VNTR despite a lower discriminatory power for spoligotyping. The median time to obtain complete results from clinical samples was 8 days (IQR 7–13) provided a high-throughput NGS sequencing platform was available. Our results highlight that the GeneLEAD/Deeplex Myc-TB approach could be a breakthrough in rapid diagnosis of MDR TB in routine practice.

Background We report a case of subdural empyema in a homeless patient caused by Bartonella quintana . B. quintana is a facultative intracellular bacteria for which bacterial growth is fastidious. The molecular biology approach has been a real help in establishing the diagnosis. Case report A 59-years old homeless patient, with a history of chronic alcohol abuse, was brought to the emergency department with a massive subdural empyema. Extensive microbiological evaluation didn’t reveal any pathogen in the pus collected before antibiotic treatment. B. quintana was detected in the pus from the empyema using a 16S rRNA-based PCR. Histology of intraoperative samples was consistent with the diagnosis and a serological assay was positive. The patient responded well to a treatment that included craniectomy with drainage of the loculated pus, total removal of the infected capsule and a combination of antibiotics. Conclusion This unique case of B. quintana -related empyema illustrates the risk of secondary infection of subdural hematoma with B. quintana since such infections have recently reemerged, predominantly among the homeless populations. Patients with subdural empyema in at-risk populations should be systematically evaluated for B. quintana with an appropriate diagnostic approach involving molecular biology.

Disseminated infections due to Bacillus Calmette-Guérin (BCG) are unusual and occur mostly in patients with inborn error of immunity (IEI) or acquired immunodeficiency. However, cases of secondary BCGosis due to intravesical BCG instillation have been described. Herein, we present a case of severe BCGosis occurring in an unusual situation. We report one case of severe disseminated BCG disease occurring after hematological malignancy in a 48-year-old man without BCG instillation and previously vaccinated in infancy with no complication. Laboratory investigations demonstrated that he was not affected by any known or candidate gene of IEI or intrinsic cellular defect involving IFNγ pathway. Whole genome sequencing of the BCG strain showed that it was most closely related to the BCG Tice strain, suggesting an unexpected relationship between the secondary immunodeficiency of the patient and the acquired BCG infection. This case highlights the fact that, in addition to the IEI, physicians, as well as microbiologists and pharmacists should be aware of possible acquired disseminated BCG disease in secondary immunocompromised patients treated in centers that administrate BCG for bladder cancers.

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.

Tuberculosis (TB) is one of the major public health problems in Congo. However, data concerning Mycobacterium tuberculosis drug resistance are lacking because of the insufficient processing capacity. So, the aim of this study was to investigate for the first time the resistance patterns and the strain lineages of a sample of M. tuberculosis complex (MTBC) isolates collected in the two main cities of Congo. Over a 9-day period, 114 smear-positive sputa isolated from 114 patients attending centers for the diagnosis and treatment of TB in Brazzaville and Pointe Noire were collected for culture and drug susceptibility testing (DST). Detection of mutations conferring drug resistance was performed by using line probe assays (GenoType MTBDRplus and MTBDRsl) and DNA sequencing. Strain lineages were determined by MIRU-VNTR genotyping. Of the 114 sputa, 46 were culture positive for MTBC. Twenty-one (46%) were resistant to one or more first-line antiTB drugs. Of these, 15 (71%) were multidrug resistant (MDR). The most prevalent mutations involved in rifampin and isoniazid resistance, D516V (60%) in rpoB and S315T (87%) in katG respectively, were well detected by MTBDRplus assay. All the 15 MDR strains were susceptible to fluoroquinolone and injectable second-line drug. No mutation was detected in the rrs locus involved in resistance to amikacin and capreomycin by both the MTBDRsl assay and DNA sequencing. By contrast, 9 MDR strains belonging to the same cluster related to T-family were identified as being falsely resistant to fluoroquinolone by the MTBDRsl assay due to the presence of a double substitution T80A-A90G in GyrA. Taken together, these data revealed a possible spread of a particular MDR clone in Congo, misidentified as fluoroquinolone resistant by MTBDRsl assay. Thus, this test cannot replace gold-standard culture method and should be interpreted carefully in view of the patient's native land.

Multidrug resistance, which is acquired by both Gram-positive and Gram-negative bacteria, causes infections that are associated with significant morbidity and mortality in many clinical settings around the world. Because of the rapidly increasing incidence of pathogens that have become resistant to all or nearly all available antibiotics, there is a need for a new generation of antimicrobials with a broad therapeutic range for specific applications against infections. Aedesin is a cecropin-like anti-microbial peptide that was recently isolated from dengue virus-infected salivary glands of the Aedes aegypti mosquito. In the present study, we have refined the analysis of its structural characteristics and have determined its antimicrobial effects against a large panel of multidrug resistant bacterial strains, directly isolated from infected patients. Based the results from nuclear magnetic resonance spectroscopy analysis, Aedesin has a helix-bend-helix structure typical for a member of the family of α-helix anti-microbial peptides. Aedesin efficiently killed Gram-negative bacterial strains that display the most worrisome resistance mechanisms encountered in the clinic, including resistance to carbapenems, aminoglycosides, cephalosporins, 4th generation fluoroquinolones, folate inhibitors and monobactams. In contrast, Gram-positive strains were insensitive to the lytic effects of the peptide. The anti-bacterial activity of Aedesin was found to be salt-resistant, indicating that it is active under physiological conditions encountered in body fluids characterized by ionic salt concentrations. In conclusion, because of its strong lytic activity against multidrug resistant Gram-negative bacterial strains displaying all types of clinically relevant resistance mechanisms known today, Aedesin might be an interesting candidate for the development of alternative treatment for infections caused by these types of bacteria.

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.

Molecular detection of rifampin resistance (rpoB analysis) in Mycobacterium leprae was determined for 49 patients who experienced relapse of multibacillary leprosy and for 34 untreated patients. Molecular detection of ofloxacin resistance (gyrA analysis) was determined for the 12 patients who experienced relapse and who had received ofloxacin. Results of molecular tests were compared with the reference susceptibility test in the mouse footpad. Overall, the efficiency of molecular detection—that is, positive DNA amplification—was 95%, whereas that of the in vivo test was 55% (P > .001). Results of molecular detection and in vivo test were fully concordant when both were available—that is, for 35 rifampin-sensitive cases of leprosy (no rpoB mutation), 4 ofloxacin-sensitive cases (no gyrA mutation), 11 rifampin-resistant cases (rpoB missense mutations), and 1 ofloxacin-resistant case (gyrA mutation). rpoB and gyrA analysis appears to be an effective method for detection of rifampin and ofloxacin resistance in patients with leprosy.