Definitive and rapid diagnosis of extrapulmonary tuberculosis is challenging since conventional techniques have limitations. We have developed a universal sample processing (USP) technology for detecting mycobacteria in clinical specimens. In this study, this technology was evaluated blindly on 99 extrapulmonary specimens collected from 87 patients. USP-processed specimens were submitted to smear microscopy for detection of acid-fast bacilli (AFB), culture, and two PCR tests targeting devR (Rv3133c) and IS6110 gene sequences. On the basis of clinical characteristics, histology and cytology, conventional microbiology results, and response to antitubercular therapy, 68 patients were diagnosed with tuberculosis. Although USP smear and culture were significantly superior to conventional microbiology, which was not optimized (P < 0.0001), these approaches fell short of PCR tests (P < 0.0001). The low yields by smear and culture are attributed to the paucibacillary load in the specimens. The highest sensitivity in PCR was achieved when devR and IS6110 test results were combined; the sensitivity and specificity values were 83 and 93.8%, 87.5 and 100%, and 66.7 and 75%, respectively, in pleural fluid, needle-biopsied pleural tissue, and lymph node specimens. In conclusion, the application of USP technology, together with clinicopathological characteristics, promises to improve the accuracy and confidence of extrapulmonary tuberculosis diagnosis.Significant mortality and morbidity is caused by tuberculosis in developing countries, including India (31). Tuberculous pleural effusion is the most common exudative pleural effusion prevalent in India in contrast to the west, where malignant effusions are more frequent (27). However, the disease most often remains undiagnosed and, even worse, untreated. The chief difficulty with extrapulmonary specimens is that they yield very few bacilli and consequently are associated with low sensitivity of acid-fast bacillus (AFB) smear and culture. Acid-fast staining was positive in fewer than 10% of patients in most reports, whereas pleural fluid cultures for M. tuberculosis were positive in up to 12 to 70% of cases and pleural biopsies revealed granulomas in 50 to 97% of patients with tuberculous pleural effusion (1). The role of PCR in the diagnosis of tubercular pleural effusion has been evaluated extensively as an alternative diagnostic tool and has yielded variable results, with sensitivities ranging between 42 and 100% and specificities ranging between 85 and 100% using various PCR targets such as IS6110, 65kDa, TRC4, GCRS, etc. (9,10,22,23,26,29,30). The most common form of extrapulmonary tuberculosis is tuberculous lymphadenopathy (2,17,20), and its diagnosis remains a challenge since granulomatous lymphadenopathy has an extensive differential diagnosis. Several conditions, including sarcoidosis, fungal infections, and other inflammatory conditions, can present the same cytology and/or histopathology as tuberculous lymphadenopathy. A diagnosis of tuberculosis is then confirmed by ...

The DevR-DevS two-component system of Mycobacterium tuberculosis mediates bacterial adaptation to hypoxia, a condition believed to be associated with the initiation and maintenance of dormant bacilli during latent tuberculosis. The activity of the Rv3134c-devRS operon was studied in M. tuberculosis using several transcriptional fusions comprised of promoter regions and the gfp reporter gene under inducing and aerobic conditions. Aerobic transcription was DevR independent, while hypoxic induction was completely DevR dependent. The hypoxia transcriptional start point, T H , was mapped at ؊40 bp upstream of Rv3134c. In contrast, the divergently transcribed Rv3135 gene was not induced under hypoxic conditions. DNase I footprinting and mutational analyses demonstrated that induction required the interaction of DevRϳP with binding sites centered at bp ؊42.5 and ؊63.5 relative to T H . Binding to the distal site (D) was necessary to recruit another molecule of DevRϳP to the proximal site (P), and interaction with both sequences was essential for promoter activation. These sites did not bind to either unphosphorylated or phosphorylation-defective DevR protein, which was consistent with an essential role for DevRϳP in activation. Phosphorylated DevR also bound to three copies of the motif at the hspX promoter. The Rv3134c and hspX promoters have a similar architecture, wherein the proximal DevRϳP binding site overlaps with the promoter ؊35 element. A model for the likely mode of action of DevR at these promoters is discussed.Mycobacterium tuberculosis is a remarkable human pathogen whose success is attributed in no small measure to its ability to establish an asymptomatic latent infection. Latently infected individuals comprise approximately one-third of the world's population and serve as a vast reservoir of bacilli for future reactivation of disease. Existing therapies are not very effective against metabolically sluggish dormant bacilli, and prolonged treatment is required to virtually eradicate them. One of the important aspects of understanding latent tuberculosis is to learn about the molecular basis of adaptation of tubercle bacilli to a dormant state in response to environmental stresses. In mice, latent tuberculosis is believed to result from bacterial adaptation to a dormant state in response to hypoxia and nitric oxide (18,35). Both of these stimuli induce the expression of M. tuberculosis devR (sometimes called dosR) and its target genes referred to as the dormancy regulon (21, 23, 27, 32). Induction of some or all of these genes also occurs in an in vivo dormancy model and in gamma-interferon (IFN-␥)-activated mouse macrophages (11,26). A recent report demonstrated the generation of a robust IFN-␥ response in latently infected individuals to dormancy regulon antigens, suggesting that these antigens may contribute to the control of latent infection (15).The most well-characterized in vitro model of latency and persistence was pioneered by Wayne et al., who used hypoxic culture conditions to transform growing ...

Two-component systems play a central role in the adaptation of pathogenic bacteria to the environment prevailing within host tissues. The genes encoding the response regulator DevR (Rv3133c/DosR) and the cytoplasmic portion (DevS 201 ) of the histidine kinase DevS (Rv3132c/DosS), a putative two-component system of Mycobacterium tuberculosis, were cloned and the protein products were overexpressed, purified and refolded as N-terminally His 6 -tagged proteins from Escherichia coli.