Mycobacterium tuberculosis residing within pulmonary granulomas and cavities represents an important reservoir of persistent organisms during human latent tuberculosis infection. We present a novel in vivo model of tuberculosis involving the encapsulation of bacilli in semidiffusible hollow fibers that are implanted subcutaneously into mice. Granulomatous lesions develop around these hollow fibers, and in this microenvironment, the organisms demonstrate an altered physiologic state characterized by stationary-state colony-forming unit counts and decreased metabolic activity. Moreover, these organisms show an antimicrobial susceptibility pattern similar to persistent bacilli in current models of tuberculosis chemotherapy in that they are more susceptible to the sterilizing drug, rifampin, than to the bactericidal drug isoniazid. We used this model of extracellular persistence within host granulomas to study both gene expression patterns and mutant survival patterns. Our results demonstrate induction of dosR (Rv3133c) and 20 other members of the DosR regulon believed to mediate the transition into dormancy, and that rel Mtb is required for Mycobacterium tuberculosis survival during extracellular persistence within host granulomas. Interestingly, the dormancy phenotype of extracellular M. tuberculosis within host granulomas appears to be immune mediated and interferon-γ dependent.

SummaryWidespread resistance to first-line TB drugs is a major problem that will likely only be resolved through the development of new drugs with novel mechanisms of action. We have used structure-guided methods to develop a lead molecule that targets the thioesterase activity of polyketide synthase Pks13, an essential enzyme that forms mycolic acids, required for the cell wall of Mycobacterium tuberculosis. Our lead, TAM16, is a benzofuran class inhibitor of Pks13 with highly potent in vitro bactericidal activity against drug-susceptible and drug-resistant clinical isolates of M. tuberculosis. In multiple mouse models of TB infection, TAM16 showed in vivo efficacy equal to the first-line TB drug isoniazid, both as a monotherapy and in combination therapy with rifampicin. TAM16 has excellent pharmacological and safety profiles, and the frequency of resistance for TAM16 is ∼100-fold lower than INH, suggesting that it can be developed as a new antitubercular aimed at the acute infection.PaperClip

Persistent Mycobacterium tuberculosis (MTB) likely encounters a phosphate-limited environment within macrophage phagosomes. We studied MTB growth, antibiotic susceptibility, and gene expression during phosphate limitation. With use of MTB mutants deficient in phosphate-related genes, we assessed bacillary survival under phosphate-limited conditions and in mouse and guinea pig lungs. Phosphate limitation restricted MTB growth in a dose-dependent manner, and phosphate-starved bacilli became phenotypically tolerant to isoniazid. The MTB genes ppk1 and relA were upregulated significantly after phosphate starvation, consistent with inorganic polyphosphate accumulation and MTB stringent response induction. The phosphate-specific transport operon pstS3-pstC2-pstA1 was induced during phosphate starvation and its expression was dependent on the 2-component regulatory system SenX3-RegX3. The MTB gene regX3 appears to be essential for bacillary survival during phosphate limitation and in mammalian lungs. Our data suggest that MTB encounters phosphate-limited conditions during mammalian lung infection and that expression of the phosphate starvation response (PSR) is important for MTB persistence.

SUMMARY The aim of this review is to present the current state of knowledge on human latent tuberculosis infection (LTBI) based on clinical studies and observations, as well as experimental in vitro and animal models. Several key terms are defined, including “latency,” “persistence,” “dormancy,” and “antibiotic tolerance.” Dogmas prevalent in the field are critically examined based on available clinical and experimental data, including the long-held beliefs that infection is either latent or active, that LTBI represents a small population of nonreplicating, “dormant” bacilli, and that caseous granulomas are the haven for LTBI. The role of host factors, such as CD4 + and CD8 + T cells, T regulatory cells, tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ), in controlling TB infection is discussed. We also highlight microbial regulatory and metabolic pathways implicated in bacillary growth restriction and antibiotic tolerance under various physiologically relevant conditions. Finally, we pose several clinically important questions, which remain unanswered and will serve to stimulate future research on LTBI.

Background Understanding the factors associated with disease severity and mortality in Coronavirus disease (COVID-19) is imperative to effectively triage patients. We performed a systematic review to determine the demographic, clinical, laboratory and radiological factors associated with severity and mortality in COVID-19. Methods We searched PubMed, Embase and WHO database for English language articles from inception until May 8, 2020. We included Observational studies with direct comparison of clinical characteristics between a) patients who died and those who survived or b) patients with severe disease and those without severe disease. Data extraction and quality assessment were performed by two authors independently. Results Among 15680 articles from the literature search, 109 articles were included in the analysis. The risk of mortality was higher in patients with increasing age, male gender (RR 1.45, 95%CI 1.23–1.71), dyspnea (RR 2.55, 95%CI 1.88–2.46), diabetes (RR 1.59, 95%CI 1.41–1.78), hypertension (RR 1.90, 95%CI 1.69–2.15). Congestive heart failure (OR 4.76, 95%CI 1.34–16.97), hilar lymphadenopathy (OR 8.34, 95%CI 2.57–27.08), bilateral lung involvement (OR 4.86, 95%CI 3.19–7.39) and reticular pattern (OR 5.54, 95%CI 1.24–24.67) were associated with severe disease. Clinically relevant cut-offs for leukocytosis(>10.0 x109/L), lymphopenia(< 1.1 x109/L), elevated C-reactive protein(>100mg/L), LDH(>250U/L) and D-dimer(>1mg/L) had higher odds of severe disease and greater risk of mortality. Conclusion Knowledge of the factors associated of disease severity and mortality identified in our study may assist in clinical decision-making and critical-care resource allocation for patients with COVID-19.

Over 2 billion people are infected with M. tuberculosis (M.tb); however, only 5–10% of those infected will develop active disease. Recent data suggest that containment is controlled locally at the level of the granuloma and that granuloma architecture may differ even within a single infected individual. Formation of a granuloma likely requires exposure to mycobacterial components released from infected macrophages but the mechanism of their release is still unclear. We hypothesize that exosomes, which are small membrane vesicles containing mycobacterial components released from infected macrophages, could promote cellular recruitment during granuloma formation. In support of this hypothesis, we found that C57BL/6 mouse-derived bone marrow macrophages treated with exosomes released from M.tb-infected RAW264.7 cells secrete significant levels of chemokines and can induce migration of CFSE-labeled macrophages and splenocytes. Exosomes isolated from the serum of M. bovis BCG infected mice could also stimulate macrophage production of chemokines and cytokines ex vivo but the level and type differed during the course of a 60 day infection. Interestingly, the exosome concentration in serum correlated strongly with mouse bacterial load, suggesting some role in immune regulation. Finally, hollow fiber-based experiments indicated that macrophages treated with exosomes released from Mtb-infected cells could promote macrophage recruitment in vivo. Exosomes injected intranasally could also recruit CD11b+ cells into the lung. Overall, our study suggests that exosomes may play an important role in recruiting and regulating host cells during an M. tuberculosis infection.

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dIn previous experiments, replacing the 10-mg/kg of body weight daily dose of rifampin with 7.5 to 10 mg/kg of rifapentine in combinations containing isoniazid and pyrazinamide reduced the duration of treatment needed to cure tuberculosis in BALB/c mice by approximately 50% due to rifapentine's more potent activity and greater drug exposures obtained. In the present study, we performed dose-ranging comparisons of the bactericidal and sterilizing activities of rifampin and rifapentine, alone and in combination with isoniazid and pyrazinamide with or without ethambutol, in BALB/c mice and in C3HeB/FeJ mice, which develop necrotic lung granulomas after infection with Mycobacterium tuberculosis. Each rifamycin demonstrated a significant increase in sterilizing activity with increasing dose. Rifapentine was roughly 4 times more potent in both mouse strains. These results reinforce the rationale for ongoing clinical trials to ascertain the highest well-tolerated doses of rifampin and rifapentine. This study also provides an important benchmark for the efficacy of the first-line regimen in C3HeB/FeJ mice, a strain in which the lung lesions observed after M. tuberculosis infection may better represent the pathology of human tuberculosis.T he rifamycins are key sterilizing drugs in the treatment of tuberculosis (TB) (2). Addition of rifampin (RIF, or R) to regimens based on isoniazid (INH, or H) and streptomycin reduced the duration of treatment necessary for cure of human TB from 18 or more months to 9 months, before the subsequent addition of pyrazinamide (PZA, or Z) reduced it to 6 months (3,4,12,13,43). Data from an in vitro hollow fiber model (19), animal models (22,49), and clinical studies (8,23,45) indicate RIF has dose-dependent bactericidal activity that is not optimized at the currently recommended 10-mg/kg of body weight (600 mg maximum) dose for adults (2). It follows that increasing rifamycin exposures may increase bactericidal and sterilizing activities and further shorten the duration of TB treatment (35). To this end, we have previously shown that replacing the 10-mg/kg daily dose of RIF with a 7.5-or 10-mg/kg daily dose of rifapentine (RPT, or P) in combination with INH and PZA reduces the duration of treatment needed to cure TB in mice by approximately 50%, owing to RPT's more potent antituberculosis activity and greater drug exposures obtained at a given dose (in mg/kg) (40, 41). A subsequent dose escalation trial in healthy volunteers showed daily RPT doses as high as 20 mg/kg were well tolerated (9). Daily regimens using RPT doses of 7.5 to 20 mg/kg in place of RIF are now under evaluation for efficacy, safety, and tolerability in at least 3 clinical trials (clinicaltrials.gov identifiers NCT00694629, NCT00728507, and NCT00814671).Unlike RPT, RIF is already in use throughout the world for treatment of TB. Despite 40 years of clinical use, studies to define the highest well-tolerated daily dose are only now under way (clinicaltrials.gov identifiers NCT00760149, NCT01392911, and NCT01408914). To deter...