Application of the Hollow-Fiber Infection Model to Personalized Precision Dosing of Isoniazid in a Clinical Setting.

BACKGROUND: The hollow-fiber infection model (HFIM) is a valuable tool for evaluating pharmacokinetics/pharmacodynamics relationships and determining the optimal antibiotic dose in monotherapy or combination therapy, but the application for personalized precision medicine in tuberculosis treatment remains limited. This study aimed to evaluate the efficacy of adjusted antibiotic doses for a tuberculosis patient using HFIM. METHODS: Model-based Bayesian forecasting was utilized to assess the proposed reduction of the isoniazid dose from 300 mg daily to 150 mg daily in a patient with an ultra-slow-acetylation phenotype. The efficacy of the adjusted 150-mg dose was evaluated in a time-to-kill assay performed using the bacterial isolate Mycobacterium tuberculosis (Mtb) H37Ra in a HFIM that mimicked the individual pharmacokinetic profile of the patient. RESULTS: The isoniazid concentration observed in the HFIM adequately reflected the target drug exposures simulated by the model. After 7 days of repeated dose administration, isoniazid killed 4 log10 Mtb CFU/mL in the treatment arm, while the control arm without isoniazid increased 1.6 log10 CFU/mL. CONCLUSION: Our results provide an example of the utility of the HFIM for predicting the efficacy of specific recommended doses of anti-tuberculosis drugs in real clinical setting.

Diagnosing adult and pediatric extrapulmonary tuberculosis by MPT64 antigen detection with immunohistochemistry and immunocytochemistry using reproduced polyclonal antibodies.

Diagnosing extrapulmonary tuberculosis (EPTB) is challenging. Immunohistochemistry or immunocytochemistry has been used to diagnose tuberculosis (TB) by detection of MPT64 antigen from various extrapulmonary specimens and has shown good diagnostic performance in our previous studies. The test can distinguish between disease caused by Mycobacterium tuberculosis (Mtb) complex and nontuberculous mycobacteria and can be applied on formalin-fixed paraffin-embedded tissue. As the antibodies previously used were in limited supply, a new batch of polyclonal antibodies was developed for scale-up and evaluated for the first time in this study. Our aim was to assess the diagnostic accuracy of the MPT64 test with reproduced antibodies in the high burden settings of Pakistan and India. Patients were enrolled prospectively. Samples from suspected sites of infection were collected and subjected to histopathologic and/or cytologic evaluation, routine TB diagnostics, GeneXpert MTB/RIF (Xpert), and the MPT64 antigen detection test. Patients were followed until the end of treatment. Based on a composite reference standard (CRS), 556 patients were categorized as TB cases and 175 as non-TB cases. The MPT64 test performed well on biopsies with a sensitivity and specificity of 94% and 75%, respectively, against a CRS. For cytology samples, the sensitivity was low (36%), whereas the specificity was 81%. Overall, the MPT64 test showed higher sensitivity (73%) than Xpert (38%) and Mtb culture (33%). The test performed equally well in adults and children. We found an additive diagnostic value of the MPT64 test in conjunction with histology and molecular tests, increasing the yield for EPTB. In conclusion, immunochemical staining with MPT64 antibodies improves the diagnosis of EPTB in high burden settings and could be a valuable addition to routine diagnostics.

Urinary lipoarabinomannan in individuals with sputum-negative pulmonary tuberculosis.

BACKGROUND OBJECTIVES: Tuberculosis (TB) is a major global cause of ill health. Sputum microscopy for confirmation of presumptive pulmonary TB (PTB) has a reportedly low sensitivity of 22-43 per cent for single smear and up to 60 per cent under optimal conditions. National TB Elimination Programme in India recommends the use of cartridge-based nucleic acid amplification test (CBNAAT) and culture for microbiological confirmation in presumptive PTB individuals with sputum smear negative test. The use of lateral flow urine lipoarabinomannan (LF-LAM) is usually recommended for the diagnosis of TB in HIV-positive individuals with low CD4 counts or those who are seriously ill. The objective of this study was to detect urinary LAM using cage nanotechnology that does not require a physiologic or immunologic consequence of HIV infection for LAM quantification in human urine in 50 HIV-seronegative sputum smear-negative PTB individuals. METHODS: To study the diagnostic value of urinary LAM in sputum smear negative PTB individuals, a cage based nanotechnology ELISA technique was used for urinary LAM in three different groups of participants. Fifty smears negative PTB clinically diagnosed, 15 smear positive PTB and 15 post TB sequel individuals. Sputum was tested by smear, CBNAAT, and culture along with urine LAM before treatment. The results were interpreted by ROC curve in comparison to the standard tests like CBNAAT and culture. RESULTS: The mean urinary LAM value was 0.84 ng/ml in 37 culture-positive [Mycobacterium tuberculosis (M.tb)] and 0.49 ng/ml in 13 culture-negative (M.tb) smear-negative individuals with PTB, respectively. In 47 smear-negative PTB cases with microbiologically confirmed TB by CBNAAT, the mean urinary LAM was 0.76 ng/ml. The mean urinary LAM in post-TB sequel individuals was 0.47 ng/ml. As per the receiver operating characteristic curve, cut-off value of urinary LAM in individuals with smear-negative PTB microbiologically confirmed by: (i) CBNAAT was 0.695 ng/ml and (ii) culture was 0.615 ng/ml. INTERPRETATION CONCLUSIONS: The findings of this study suggest that individuals with smear-negative PTB and a urinary LAM value of >0.615 ng/ml were most likely to have microbiological confirmed TB while those with a LAM value <0.615 ng/ml >0.478 ng/ml are less likely and those with a value <0.478 ng/ml are unlikely to have microbiological confirmed TB.

A protective role for type I interferon signaling following infection with Mycobacterium tuberculosis carrying the rifampicin drug resistance-conferring RpoB mutation H445Y.

Interleukin-1 (IL-1) signaling is essential for controlling virulent Mycobacterium tuberculosis (Mtb) infection since antagonism of this pathway leads to exacerbated pathology and increased susceptibility. In contrast, the triggering of type I interferon (IFN) signaling is associated with the progression of tuberculosis (TB) disease and linked with negative regulation of IL-1 signaling. However, mice lacking IL-1 signaling can control Mtb infection if infected with an Mtb strain carrying the rifampin-resistance conferring mutation H445Y in its RNA polymerase ß subunit (rpoB-H445Y Mtb). The mechanisms that govern protection in the absence of IL-1 signaling during rpoB-H445Y Mtb infection are unknown. In this study, we show that in the absence of IL-1 signaling, type I IFN signaling controls rpoB-H445Y Mtb replication, lung pathology, and excessive myeloid cell infiltration. Additionally, type I IFN is produced predominantly by monocytes and recruited macrophages and acts on LysM-expressing cells to drive protection through nitric oxide (NO) production to restrict intracellular rpoB-H445Y Mtb. These findings reveal an unexpected protective role for type I IFN signaling in compensating for deficiencies in IL-1 pathways during rpoB-H445Y Mtb infection.

A robust computational quest: Discovering potential hits to improve the treatment of pyrazinamide-resistant Mycobacterium tuberculosis.

The rise of pyrazinamide (PZA)-resistant strains of Mycobacterium tuberculosis (MTB) poses a major challenge to conventional tuberculosis (TB) treatments. PZA, a cornerstone of TB therapy, must be activated by the mycobacterial enzyme pyrazinamidase (PZase) to convert its active form, pyrazinoic acid, which targets the ribosomal protein S1. Resistance, often associated with mutations in the RpsA protein, complicates treatment and highlights a critical gap in the understanding of structural dynamics and mechanisms of resistance, particularly in the context of the G97D mutation. This study utilizes a novel integration of computational techniques, including multiscale biomolecular and molecular dynamics simulations, physicochemical and medicinal chemistry predictions, quantum computations and virtual screening from the ZINC and Chembridge databases, to elucidate the resistance mechanism and identify lead compounds that have the potential to improve treatment outcomes for PZA-resistant MTB, namely ZINC15913786, ZINC20735155, Chem10269711, Chem10279789 and Chem10295790. These computational methods offer a cost-effective, rapid alternative to traditional drug trials by bypassing the need for organic subjects while providing highly accurate insight into the binding sites and efficacy of new drug candidates. The need for rapid and appropriate drug development emphasizes the need for robust computational analysis to justify further validation through in vitro and in vivo experiments.

Gut bacterial and fungal dysbiosis in tuberculosis patients.

BACKGROUND: Recent studies have more focused on gut microbial alteration in tuberculosis (TB) patients. However, no detailed study on gut fungi modification has been reported till now. So, current research explores the characteristics of gut microbiota (bacteria)- and mycobiota (fungi)-dysbiosis in TB patients and also assesses the correlation between the gut microbiome and serum cytokines. It may help to screen the potential diagnostic biomarker for TB. RESULTS: The results show that the alpha diversity of the gut microbiome (including bacteria and fungi) decreased and altered the gut microbiome composition of TB patients. The bacterial genera Bacteroides and Prevotella were significantly increased, and Blautia and Bifidobacterium decreased in the TB patients group. The fungi genus Saccharomyces was increased while decreased levels of Aspergillus in TB patients. It indicates that gut microbial equilibrium between bacteria and fungi has been altered in TB patients. The fungal-to-bacterial species ratio was significantly decreased, and the bacterial-fungal trans-kingdom interactions have been reduced in TB patients. A set model including Bacteroides, Blautia, Eubacterium_hallii_group, Apiotrichum, Penicillium, and Saccharomyces may provide a better TB diagnostics option than using single bacterial or fungi sets. Also, gut microbial dysbiosis has a strong correlation with the alteration of IL-17 and IFN-γ. CONCLUSIONS: Our results demonstrate that TB patients exhibit the gut bacterial and fungal dysbiosis. In the clinics, some gut microbes may be considered as potential biomarkers for auxiliary TB diagnosis.

Bacterial diversity dominates variable macrophage responses of tuberculosis patients in Tanzania.

The Mycobacterium tuberculosis complex (MTBC) comprises nine human-adapted lineages that differ in their geographical distribution. Local adaptation of specific MTBC genotypes to the respective human host population has been invoked in this context. We aimed to assess if bacterial genetics governs MTBC pathogenesis or if local co-adaptation translates into differential susceptibility of human macrophages to infection by different MTBC genotypes. We generated macrophages from cryopreserved blood mononuclear cells of Tanzanian tuberculosis patients, from which the infecting MTBC strains had previously been phylogenetically characterized. We infected these macrophages ex vivo with a phylogenetically similar MTBC strain ("matched infection") or with strains representative of other MTBC lineages ("mismatched infection"). We found that L1 infections resulted in a significantly lower bacterial burden and that the intra-cellular replication rate of L2 strains was significantly higher compared the other MTBC lineages, irrespective of the MTBC lineage originally infecting the patients. Moreover, L4-infected macrophages released significantly greater amounts of TNF-α, IL-6, IL-10, MIP-1ß, and IL-1ß compared to macrophages infected by all other strains. While our results revealed no measurable effect of local adaptation, they further highlight the strong impact of MTBC phylogenetic diversity on the variable outcome of the host-pathogen interaction in human tuberculosis.

The role of transcriptional regulators in metal ion homeostasis of Mycobacterium tuberculosis.

Metal ions are essential trace elements for all living organisms and play critical catalytic, structural, and allosteric roles in many enzymes and transcription factors. Mycobacterium tuberculosis (MTB), as an intracellular pathogen, is usually found in host macrophages, where the bacterium can survive and replicate. One of the reasons why Tuberculosis (TB) is so difficult to eradicate is the continuous adaptation of its pathogen. It is capable of adapting to a wide range of harsh environmental stresses, including metal ion toxicity in the host macrophages. Altering the concentration of metal ions is the common host strategy to limit MTB replication and persistence. This review mainly focuses on transcriptional regulatory proteins in MTB that are involved in the regulation of metal ions such as iron, copper and zinc. The aim is to offer novel insights and strategies for screening targets for TB treatment, as well as for the development and design of new therapeutic interventions.

Bacteriological culture and direct PCR for detecting the Mycobacterium tuberculosis complex in the Italian eradication campaign: a decade of experience at the National Reference Laboratory.

AIMS: Our study evaluates the capacity of direct real-time PCR for detecting Mycobacterium tuberculosis complex (MTBC), with a focus on diagnostic performances and the feasibility of implementing this protocol in an eradication campaign. Specifically, we compare the effectiveness of the direct PCR method to various culture systems used by the Italian National Reference Laboratory over the last decade to detect MTBC. METHODS AND RESULTS: Bovine tissue samples were routinely tested and analyzed for bovine tuberculosis (bTB) confirmation using microbiological culture (solid and liquid media), histopathological analysis, and a direct PCR assay targeting IS6110, an insertion sequence specific to the MTBC that is widely used for tuberculosis diagnosis. The direct real-time PCR demonstrated a high concordance (K = 0.871) with microbiological culture, as well as good sensitivity (91.84%) and specificity (95.24%). In contrast, histopathology demonstrated lower concordance (K = 0.746) and performance levels (sensitivity 91.41%, specificity 82.88%). Liquid media promoted faster and more efficient growth of MTBC than solid media. M. bovis and M. caprae had the comparable ability to respond to the direct real-time PCR test and grow on the microbiological medium. CONCLUSIONS: This study confirms that direct real-time PCR can detect MTBC with high diagnostic accuracy within a few days. This study found no significant differences in performance between culture media and direct PCR for M. bovis and M. caprae.

Profile of non-tuberculous mycobacteria amongst tuberculosis presumptive people in Cameroon.

BACKGROUND: Cameroon is a tuberculosis (TB) burden country with a 12% positivity among TB presumptive cases. Of the presumptive cases with a negative TB test, some are infected with Non-tuberculous Mycobacteria (NTM). However, the diagnosis of NTM infections remains difficult due to the lack of tools in many laboratories, particularly in resource limited laboratories and remote setting. The present study was undertaken to determine NTM profile and associated comorbidities among TB presumptive people. METHODS: A retrospective study was conducted from December 2018 to December 2019 in the Tuberculosis-National Reference Laboratory (TB-NRL) for Bacteriological analysis of samples and Jamot Hospital of Yaounde (JHY) for clinical evaluation of confirmed NTM patients. We included in this study data of 5267 TB presumptive people previously diagnosed using three consecutive samples and having culture and SD Bioline results with or without Microscopy and reverse hybridization-based Line Probe Assay(LPA) results. The data on co-morbidities or history of people infected with NTM were then collected from the three participants with available clinical data. RESULTS: We collected data of 5267 presumptive TB people. Among them, 3436 (65.24%), have a positive culture with 3200 (60.75%) isolates belong to Mycobacterium tuberculosis Complex (MBTC) and 236 (4.48%) to NTM. Our results showed that, 123 (52.11%) NTM were isolated from people with negative microscopy and 113 (47.88%) from people with positive microscopy. Among the 236 NTM, 108 (45.8%) isolates were identified using LPA. M. fortuitum was the most represented species (32.41%) followed by M. intracellulare (19.44%). Sputum had the highest proportion of NTM (56%), followed by bronchial aspirations (31%). The extra-pulmonary samples presented lower proportions of isolates compared to pulmonary samples. Some patients affected with NTM presented comorbidities as HIV infection, Pulmonary tuberculosis, Type 2 diabetes, Chronic bronchitis and Alveolar pneumonia. CONCLUSIONS: Our study showed the presence of NTM strains among presumptive TB people with a predominance of M. fortuitum and M. intracellulare. It is important to implement a surveillance system of NTM in TB burden country and also to develop a point-of-care test for NTM identification in limited-resource settings.

The use of Kudoh method for culture of Mycobacterium tuberculosis and Mycobacterium africanum in The Gambia.

BACKGROUND: Mycobacterium tuberculosis culturing remains the gold standard for laboratory diagnosis of tuberculosis. Tuberculosis remains a great public health problem in developing countries like The Gambia, as most of the methods currently used for bacterial isolation are either time-consuming or costly. OBJECTIVE: To evaluate the Kudoh swab method in a West African setting in Gambia, with a particular focus on the method's performance when culturing Mycobacterium africanum West Africa 2 (MAF2) isolates. METHOD: 75 sputum samples were collected in the Greater Banjul Area and decontaminated in parallel with both the standard N-acetyl-L-Cysteine-NaOH (NALC-NaOH) and the Kudoh swab method in the TB diagnostics laboratory in the Medical Research Council Unit The Gambia between 30th December 2017 and 25th February 2018. These samples were subsequently cultured on standard Löwenstein-Jensen and Modified Ogawa media respectively and incubated at 37°C for mycobacterial growth. Spoligotyping was done to determine if the decontamination and culture methods compared could equally detect Mycobacterium tuberculosis, Mycobacterium africanum West Africa 1 and Mycobacterium africanum West Africa 2. RESULT: Among the 50 smear positives, 35 (70%) were culture-positive with Kudoh and 32 (64%) were culture positive with NALC-NaOH, whilst 7(28%) of the 25 smear negative samples were culture positive with both methods (Table 2). There was no significant difference in recovery between both methods (McNemar's test, p-value = 0.7003), suggesting that the overall positivity rate between the two methods is comparable. There were no differences in time-to-positivity or contamination rate between the methods. However, Kudoh yielded positive cultures that were negative on LJ and vice versa. All findings were irrespective of mycobacterial lineages. CONCLUSION: The Kudoh method has comparable sensitivity to the NALC-NaOH method for detecting Mycobacterium tuberculosis complex isolates. It is easy to perform and could be an add on option for mycobacterial culture in the field in The Gambia, since it requires less biosafety equipment.

Application of CRISPR-cas-based technology for the identification of tuberculosis, drug discovery and vaccine development.

Tuberculosis (TB), which caused by Mycobacterium tuberculosis, is the leading cause of death from a single infectious agent and continues to be a major public health burden for the global community. Despite being the only globally licenced prophylactic vaccine, Bacillus Calmette-Guérin (BCG) has multiple deficiencies, and effective diagnostic and therapeutic options are limited. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) is an adaptive immune system that is found in bacteria and has great potential for the development of novel antituberculosis drugs and vaccines. In addition, CRISPR-Cas is currently recognized as a prospective tool for the development of therapies for TB infection with potential diagnostic and therapeutic value, and CRISPR-Cas may become a viable tool for eliminating TB in the future. Herein, we systematically summarize the current applications of CRISPR-Cas-based technology for TB detection and its potential roles in drug discovery and vaccine development.

The structure of Mycobacterium thermoresistibile MmpS5 reveals a conserved disulfide bond across mycobacteria.

The tuberculosis (TB) emergency has been a pressing health threat for decades. With the emergence of drug-resistant TB and complications from the COVID-19 pandemic, the TB health crisis is more serious than ever. Mycobacterium tuberculosis (Mtb), the causative agent of TB, requires iron for its survival. Thus, Mtb has evolved several mechanisms to acquire iron from the host. Mtb produces two siderophores, mycobactin and carboxymycobactin, which scavenge for host iron. Mtb siderophore-dependent iron acquisition requires the export of apo-siderophores from the cytosol to the host environment and import of iron-bound siderophores. The export of Mtb apo-siderophores across the inner membrane is facilitated by two mycobacterial inner membrane proteins with their cognate periplasmic accessory proteins, designated MmpL4/MmpS4 and MmpL5/MmpS5. Notably, the Mtb MmpL4/MmpS4 and MmpL5/MmpS5 complexes have also been implicated in the efflux of anti-TB drugs. Herein, we solved the crystal structure of M. thermoresistibile MmpS5. The MmpS5 structure reveals a previously uncharacterized, biologically relevant disulfide bond that appears to be conserved across the Mycobacterium MmpS4/S5 homologs, and comparison with structural homologs suggests that MmpS5 may be dimeric.

Origin and dispersal of the Mycobacterium tuberculosis Haarlem genotype: Clues from its phylogeographic landscape and human migration.

The Haarlem family belongs to the Euro-American phylogenetic lineage of Mycobacterium tuberculosis and is one of the globally spread genotypes of this important human pathogen. In spite of the sporadic observations on drug resistance and peculiar virulence profile, Haarlem remains in the shade of other M. tuberculosis genotypes. I analyzed genotyping data of the Haarlem genotype in light of its pathogenic properties and relevant human migration, to gain insight into its origin, evolutionary history, and current spread. Central Europe is marked with a very high prevalence of both major Haarlem subclades ancestral H3/SIT50 and derived H1, jointly making 33-41% in Czechia, Austria, and Hungary. There is a declining gradient of Haarlem beyond central Europe with 10-18% in Italy, France, Belgium, 10-13% in the Balkan countries and Turkey. Placing the available genetic diversity and ancient DNA data within the historical context, I hypothesize that M. tuberculosis Haarlem genotype likely originated in Central Europe and its primary long-term circulation occurred within the area of the former Austria/Austria-Hungary Empire in the 14th-19th centuries. The genotype is not highly transmissible and its spread was driven by long-term human migration. The European colonial expansion (when accompanied by a sufficient volume of migration) was a vehicle of its secondary dissemination. I conclude that human migration and its lack thereof (but not strain pathobiology) was a major driving force that shaped the population structure of this global lineage of M. tuberculosis. At the same time, Haarlem strains appear over-represented in some ethnic groups which warrants in-depth experimental research.

State-of-the-art detection of Mycobacterium tuberculosis in blood during tuberculosis infection using phage technology.

Tuberculosis (TB), an aerosol-transmitted infection caused by Mycobacterium tuberculosis (Mtb), remains the commonest cause of death globally, from an infectious bacterial disease. Nine years on from the launch of the World Health Organization (WHO)'s END-TB strategy, disease incidence rates are stubbornly unchanged [1]. While this represents, in part, a reversal of improving trends caused by the COVID-19 pandemic, it also reflects the fragility and inadequacy of healthcare systems to sustain TB control [2]. Although multifactorial, a key reason for this is the ineffectiveness of existing clinical tools to meet the two key objectives of the END-TB strategy-(i) early diagnosis and treatment of TB disease (to limit onward transmission); and (ii) disease prevention through screening for asymptomatic TB infection (TBI). Meeting both objectives will rely on the development of new biomarkers with high accuracy, but the global nature of the TB problem also requires that new tests are rapid, low cost and can be measured in patients by sampling from universally accessible sites. In this review, we will present the accumulating evidence for circulating Mtb in both TB disease and asymptomatic TBI and discuss the potential utility of novel bacteriophage-based technology for blood-based detection of Mtb.

Unlocking InhA: Novel approaches to inhibit Mycobacterium tuberculosis.

Multidrug-resistant tuberculosis continues to pose a health security risk and remains a public health emergency. Antimicrobial resistance result from treatment regimens that are both insufficient and incomplete leading to the emergence of multidrug-resistant tuberculosis, extensively drug-resistant tuberculosis and totally drug-resistant tuberculosis. The impact of tuberculosis on the people suffering from HIV (Human immunodeficiency virus infection) have resulted in the increased research efforts in designing and discovery of novel antitubercular drugs that may result in decreasing treatment duration, minimising the need for multiple drug intake, minimising cytotoxicity and enhancing the mechanism of action of drug. While many drugs are available to treat tuberculosis, a precise and timely cure is still absent. Consequently, further investigation is needed to identify more recent molecular equivalents that have the potential to swiftly remove this disease. Isoniazid (INH), a treatment for tuberculosis (TB), targets the enzyme InhA (mycobacterium enoyl acyl carrier protein reductase), the Mycobacterium tuberculosis enoyl-acyl carrier protein (ACP) reductase, most common INH resistance is circumvented by InhA inhibitors that do not require KatG (catalase-peroxidase) activation, as a result, researchers are trying to work in the area of development of InhA inhibitors which could help in eradicating the era of tuberculosis from the world.

Normalizing granuloma vasculature and matrix improves drug delivery and reduces bacterial burden in tuberculosis-infected rabbits.

Host-directed therapies (HDTs) represent an emerging approach for bacterial clearance during tuberculosis (TB) infection. While most HDTs are designed and implemented for immuno-modulation, other host targets-such as nonimmune stromal components found in pulmonary granulomas-may prove equally viable. Building on our previous work characterizing and normalizing the aberrant granuloma-associated vasculature, here we demonstrate that FDA-approved therapies (bevacizumab and losartan, respectively) can be repurposed as HDTs to normalize blood vessels and extracellular matrix (ECM), improve drug delivery, and reduce bacterial loads in TB granulomas. Granulomas feature an overabundance of ECM and compressed blood vessels, both of which are effectively reduced by losartan treatment in the rabbit model of TB. Combining both HDTs promotes secretion of proinflammatory cytokines and improves anti-TB drug delivery. Finally, alone and in combination with second-line antitubercular agents (moxifloxacin or bedaquiline), these HDTs significantly reduce bacterial burden. RNA sequencing analysis of HDT-treated lung and granuloma tissues implicates up-regulated antimicrobial peptide and proinflammatory gene expression by ciliated epithelial airway cells as a putative mechanism of the observed antitubercular benefits in the absence of chemotherapy. These findings demonstrate that bevacizumab and losartan are well-tolerated stroma-targeting HDTs, normalize the granuloma microenvironment, and improve TB outcomes, providing the rationale to clinically test this combination in TB patients.

Acetic Acid Enables Molecular Enumeration of Mycobacterium tuberculosis from Sputum and Eliminates the Need for a Biosafety Level 3 Laboratory.

BACKGROUND: Improved monitoring of Mycobacterium tuberculosis response to treatment is urgently required. We previously developed the molecular bacterial load assay (MBLA), but it is challenging to integrate into the clinical diagnostic laboratory due to a labor-intensive protocol required at biosafety level 3 (BSL-3). A modified assay was needed. METHODS: The rapid enumeration and diagnostic for tuberculosis (READ-TB) assay was developed. Acetic acid was tested and compared to 4 M guanidine thiocyanate to be simultaneously bactericidal and preserve mycobacterial RNA. The extraction was based on silica column technology and incorporated low-cost reagents: 3 M sodium acetate and ethanol for the RNA extraction to replace phenol-chloroform. READ-TB was fully validated and compared directly to the MBLA using sputa collected from individuals with tuberculosis. RESULTS: Acetic acid was bactericidal to M. tuberculosis with no significant loss in 16S rRNA or an unprotected mRNA fragment when sputum was stored in acetic acid at 25°C for 2 weeks or -20°C for 1 year. This novel use of acetic acid allows processing of sputum for READ-TB at biosafety level 2 (BSL-2) on sample receipt. READ-TB is semiautomated and rapid. READ-TB correlated with the MBLA when 85 human sputum samples were directly compared (R2 = 0.74). CONCLUSIONS: READ-TB is an improved version of the MBLA and is available to be adopted by clinical microbiology laboratories as a tool for tuberculosis treatment monitoring. READ-TB will have a particular impact in low- and middle-income countries (LMICs) for laboratories with no BSL-3 laboratory and for clinical trials testing new combinations of anti-tuberculosis drugs.

Computational drug repositioning identifies niclosamide and tribromsalan as inhibitors of Mycobacterium tuberculosis and Mycobacterium abscessus.

Tuberculosis (TB) is still a major global health challenge, killing over 1.5 million people each year, and hence, there is a need to identify and develop novel treatments for Mycobacterium tuberculosis (M. tuberculosis). The prevalence of infections caused by nontuberculous mycobacteria (NTM) is also increasing and has overtaken TB cases in the United States and much of the developed world. Mycobacterium abscessus (M. abscessus) is one of the most frequently encountered NTM and is difficult to treat. We describe the use of drug-disease association using a semantic knowledge graph approach combined with machine learning models that has enabled the identification of several molecules for testing anti-mycobacterial activity. We established that niclosamide (M. tuberculosis IC90 2.95 µM; M. abscessus IC90 59.1 µM) and tribromsalan (M. tuberculosis IC90 76.92 µM; M. abscessus IC90 147.4 µM) inhibit M. tuberculosis and M. abscessus in vitro. To investigate the mode of action, we determined the transcriptional response of M. tuberculosis and M. abscessus to both compounds in axenic log phase, demonstrating a broad effect on gene expression that differed from known M. tuberculosis inhibitors. Both compounds elicited transcriptional responses indicative of respiratory pathway stress and the dysregulation of fatty acid metabolism.

In silico agent-based modeling approach to characterize multiple in vitro tuberculosis infection models.

In vitro models of Mycobacterium tuberculosis (Mtb) infection are a valuable tool for examining host-pathogen interactions and screening drugs. With the development of more complex in vitro models, there is a need for tools to help analyze and integrate data from these models. To this end, we introduce an agent-based model (ABM) representation of the interactions between immune cells and bacteria in an in vitro setting. This in silico model was used to simulate both traditional and spheroid cell culture models by changing the movement rules and initial spatial layout of the cells in accordance with the respective in vitro models. The traditional and spheroid simulations were calibrated to published experimental data in a paired manner, by using the same parameters in both simulations. Within the calibrated simulations, heterogeneous outputs are seen for bacterial count and T cell infiltration into the macrophage core of the spheroid. The simulations also predict that equivalent numbers of activated macrophages do not necessarily result in similar bacterial reductions; that host immune responses can control bacterial growth in both spheroid structure dependent and independent manners; that STAT1 activation is the limiting step in macrophage activation in spheroids; and that drug screening and macrophage activation studies could have different outcomes depending on the in vitro culture used. Future model iterations will be guided by the limitations of the current model, specifically which parts of the output space were harder to reach. This ABM can be used to represent more in vitro Mtb infection models due to its flexible structure, thereby accelerating in vitro discoveries.