Heteroresistance of Mycobacterium tuberculosis (MTB) is defined as the coexistence of susceptible and resistant organisms to anti-tuberculosis (TB) drugs in the same patient. Heteroresistance of MTB is considered a preliminary stage to full resistance. To date, no mechanism causing heteroresistance of MTB has been proven.Clinical specimens and cultures from 35 TB patients from Tashkent, Uzbekistan, were analysed using the Genotype MTBDR assay (Hain Lifescience, Nehren, Germany), which is designed to detect genetic mutations associated with resistance to rifampin and isoniazid. Cases of heteroresistance were further subjected to genotyping using mycobacterial interspersed repetitive unit-variable-number tandem repeat typing, spoligotyping and IS6110 fingerprinting.Heteroresistance to rifampin and/or isoniazid was found in seven cases (20%). In five of them, heteroresistance was caused by two different strains and in two by a single strain of the Beijing genotype. The latter cases had a history of relapse of their TB.For the first time, two different mechanisms of heteroresistance in tuberculosis have been proven using a stepwise molecular-biological approach: 1) superinfection with two different strains, which is of interest for clinical infection control practitioners; and 2) splitting of a single strain into susceptible and resistant organisms. The latter mechanism is most likely to be related to poor treatment quality and could serve as a quality marker for tuberculosis therapy programmes in the future.

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Wolfram syndrome, an autosomal recessive disorder characterized by diabetes mellitus and optic atrophy, is caused by mutations in the WFS1 gene encoding an endoplasmic reticulum (ER) membrane protein, Wolframin. Although its precise functions are unknown, Wolframin deficiency increases ER stress, impairs cell cycle progression and affects calcium homeostasis. To gain further insight into its function and identify molecular partners, we used the WFS1-C-terminal domain as bait in a yeast two-hybrid screen with a human brain cDNA library. Na+/K+ ATPase beta1 subunit was identified as an interacting clone. We mapped the interaction to the WFS1 C-terminal and transmembrane domains, but not the N-terminal domain. Our mapping data suggest that the interaction most likely occurs in the ER. We confirmed the interaction by co-immunoprecipitation in mammalian cells and with endogenous proteins in JEG3 placental cells, neuroblastoma SKNAS and pancreatic MIN6 beta cells. Na+/K+ ATPase beta1 subunit expression was reduced in plasma membrane fractions of human WFS1 mutant fibroblasts and WFS1 knockdown MIN6 pancreatic beta-cells compared with wild-type cells; Na+/K+ ATPase alpha1 subunit expression was also reduced in WFS-depleted MIN6 beta cells. Induction of ER stress in wild-type cells only partly accounted for the reduced Na+/K+ ATPase beta1 subunit expression observed. We conclude that the interaction may be important for Na+/K+ ATPase beta1 subunit maturation; loss of this interaction may contribute to the pathology seen in Wolfram syndrome via reductions in sodium pump alpha1 and beta1 subunit expression in pancreatic beta-cells.

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We established protocols for DST of delamanid in the MGIT and REMA, confirming their feasibility in routine TB diagnostics, utilizing the same discriminative concentration for both methods. Moreover, taking advantage of WGS analysis, we identified polymorphisms potentially associated with resistance in two genes involved in delamanid activation.

Two new drugs, delamanid and bedaquiline, have recently been approved for treatment of multidrug-resistant and extensively drug-resistant (XDR) tuberculosis. Here, we report a case of clofazimine, bedaquiline, and low-level delamanid resistances acquired during treatment of a patient with XDR tuberculosis.

QuantiFERON-TB Gold IT analyses interferon-γ release from CD4(+) T cells after stimulation with specific tuberculosis (TB) antigens. Its sensitivity is approximately 80% for active TB. A new test generation (QFTGplus) also analyses the response of CD8(+) T cells. We investigated both test generations in a direct head-to-head comparison in a German pulmonary hospital. Sensitivity rates for active TB were identical, no matter whether diagnosis was bacteriologically confirmed or not.

bThe Abbott RealTime MTB (RT MTB) assay is a new automated nucleic acid amplification test for the detection of Mycobacterium tuberculosis complex (MTBC) in clinical specimens. In combination with the RealTime MTB INH/RIF (RT MTB INH/RIF) resistance assay, which can be applied to RT MTB-positive specimens as an add-on assay, the tests also indicate the genetic markers of resistance to isoniazid (INH) and rifampin (RIF). We aimed to evaluate the diagnostic sensitivity and specificity of RT MTB using different types of respiratory and extrapulmonary specimens and to compare performance characteristics directly with those of the FluoroType MTB assay. The resistance results obtained by RT MTB INH/RIF were compared to those from the GenoType MTBDRplus and from phenotypic drug susceptibility testing. A total of 715 clinical specimens were analyzed. Compared to culture, the overall sensitivity of RT MTB was 92.1%; the sensitivity rates for smear-positive and smear-negative samples were 100% and 76.2%, respectively. The sensitivities of smear-negative specimens were almost identical for respiratory (76.3%) and extrapulmonary (76%) specimens. Specificity rates were 100% and 95.8% for culturenegative specimens and those that grew nontuberculous mycobacteria, respectively. RT MTB INH/RIF was applied to 233 RT MTB-positive samples and identified resistance markers in 7.7% of samples. Agreement with phenotypic and genotypic drug susceptibility testing was 99.5%. In conclusion, RT MTB and RT MTB INH/RIF allow for the rapid and accurate diagnosis of tuberculosis (TB) in different types of specimens and reliably indicate resistance markers. The strengths of this system are the comparably high sensitivity with paucibacillary specimens, its ability to detect INH and RIF resistance, and its high-throughput capacities. R apid and accurate diagnosis of tuberculosis (TB) and fast detection of drug resistance are essential to ensure early initiation of appropriate antituberculotic treatment, adequately manage the disease, and control further transmission. Worldwide, one-third of all TB cases and almost three-quarters of the 480,000 cases of multidrug-resistant (MDR; defined as resistance toward rifampin [RIF] and isoniazid [INH]) TB are not reported, with the vast majority of them occurring in high-burden countries (1). Molecular tests are the most promising tools to close this diagnostic gap. Consequently, nucleic acid amplification tests (NAATs), such as PCR assays that allow for the fast and accurate detection of Mycobacterium tuberculosis complex (MTBC) DNA directly in clinical specimens, have become an indispensable tool in TB diagnostics over the last several decades. Most commercial tests show excellent specificity and sensitivity rates with smear-positive specimens while sensitivity rates range from 49% to 78% with smearnegative samples (2-7).Particularly in regions with high prevalences of MDR-TB, the molecular detection of genetic markers of resistance directly in the clinical specimen is playing a pivotal role in early not...