9 months of delamanid, linezolid, levofloxacin, and pyrazinamide versus conventional therapy for treatment of fluoroquinolone-sensitive multidrug-resistant tuberculosis (MDR-END): a multicentre, randomised, open-label phase 2/3 non-inferiority trial in South Korea.

BACKGROUND: With the introduction of new anti-tuberculosis drugs, all-oral regimens with shorter treatment durations for multidrug-resistant tuberculosis have been anticipated. We aimed to investigate whether a new all-oral regimen was non-inferior to the conventional regimen including second-line anti-tuberculosis drugs for 20-24 months in the treatment of fluoroquinolone-sensitive multidrug-resistant tuberculosis. METHODS: In this multicentre, randomised, open-label phase 2/3 non-inferiority trial, we enrolled men and women aged 19-85 years with multidrug-resistant tuberculosis confirmed by phenotypic or genotypic drug susceptibility tests or rifampicin-resistant tuberculosis by genotypic tests at 12 participating hospitals throughout South Korea. Participants with fluoroquinolone-resistant multidrug-resistant tuberculosis were excluded. Participants were randomly assigned (1:1) to two groups using a block randomisation, stratified by the presence of diabetes and cavitation on baseline chest radiographs. The investigational group received delamanid, linezolid, levofloxacin, and pyrazinamide for 9 months, and the control group received a conventional 20-24-month regimen, according to the 2014 WHO guidelines. The primary outcome was the treatment success rate at 24 months after treatment initiation in the modified intention-to-treat population and the per-protocol population. Participants who were "cured" and "treatment completed" were defined as treatment success following the 2014 WHO guidelines. Non-inferiority was confirmed if the lower limit of a 97·5% one-sided CI of the difference between the groups was greater than -10%. Safety data were collected for 24 months in participants who received a predefined regimen at least once. This study is registered with ClinicalTrials.gov, NCT02619994. FINDINGS: Between March 4, 2016, and Sept 14, 2019, 214 participants were enrolled, 168 (78·5%) of whom were included in the modified intention-to-treat population. At 24 months after treatment initiation, 60 (70·6%) of 85 participants in the control group had treatment success, as did 54 (75·0%) of 72 participants in the shorter-regimen group (between-group difference 4·4% [97·5% one-sided CI -9·5% to ∞]), satisfying the predefined non-inferiority margin. No difference in safety outcomes was identified between the control group and the shorter-regimen group. INTERPRETATION: 9-month treatment with oral delamanid, linezolid, levofloxacin, and pyrazinamide could represent a new treatment option for participants with fluoroquinolone-sensitive multidrug-resistant tuberculosis. FUNDING: Korea Disease Control and Prevention Agency, South Korea.

PPE39 of the Mycobacterium tuberculosis strain Beijing/K induces Th1-cell polarization through dendritic cell maturation.

In a previous study, we have identified MTBK_24820, the complete protein form of PPE39 in the hypervirulent Mycobacterium tuberculosis (Mtb) strain Beijing/K by using comparative genomic analysis. PPE39 exhibited vaccine potential against Mtb challenge in a murine model. Thus, in this present study, we characterize PPE39-induced immunological features by investigating the interaction of PPE39 with dendritic cells (DCs). PPE39-treated DCs display reduced dextran uptake and enhanced MHC-I, MHC-II, CD80 and CD86 expression, indicating that this PPE protein induces phenotypic DC maturation. In addition, PPE39-treated DCs produce TNF-α, IL-6 and IL-12p70 to a similar and/or greater extent than lipopolysaccharide-treated DCs in a dose-dependent manner. The activating effect of PPE39 on DCs was mediated by TLR4 through downstream MAPK and NF-κB signaling pathways. Moreover, PPE39-treated DCs promoted naïve CD4+ T-cell proliferation accompanied by remarkable increases of IFN-γ and IL-2 secretion levels, and an increase in the Th1-related transcription factor T-bet but not in Th2-associated expression of GATA-3, suggesting that PPE39 induces Th1-type T-cell responses through DC activation. Collectively, the results indicate that the complete form of PPE39 is a so-far-unknown TLR4 agonist that induces Th1-cell biased immune responses by interacting with DCs.This article has an associated First Person interview with the first author of the paper.

Evaluation of a Rapid Molecular Drug-Susceptibility Test for Tuberculosis.

BACKGROUND: Fluoroquinolones and second-line injectable drugs are the backbone of treatment regimens for multidrug-resistant tuberculosis, and resistance to these drugs defines extensively drug-resistant tuberculosis. We assessed the accuracy of an automated, cartridge-based molecular assay for the detection, directly from sputum specimens, of Mycobacterium tuberculosis with resistance to fluoroquinolones, aminoglycosides, and isoniazid. METHODS: We conducted a prospective diagnostic accuracy study to compare the investigational assay against phenotypic drug-susceptibility testing and DNA sequencing among adults in China and South Korea who had symptoms of tuberculosis. The Xpert MTB/RIF assay and sputum culture were performed. M. tuberculosis isolates underwent phenotypic drug-susceptibility testing and DNA sequencing of the genes katG, gyrA, gyrB, and rrs and of the eis and inhA promoter regions. RESULTS: Among the 308 participants who were culture-positive for M. tuberculosis, when phenotypic drug-susceptibility testing was used as the reference standard, the sensitivities of the investigational assay for detecting resistance were 83.3% for isoniazid (95% confidence interval [CI], 77.1 to 88.5), 88.4% for ofloxacin (95% CI, 80.2 to 94.1), 87.6% for moxifloxacin at a critical concentration of 0.5 µg per milliliter (95% CI, 79.0 to 93.7), 96.2% for moxifloxacin at a critical concentration of 2.0 µg per milliliter (95% CI, 87.0 to 99.5), 71.4% for kanamycin (95% CI, 56.7 to 83.4), and 70.7% for amikacin (95% CI, 54.5 to 83.9). The specificity of the assay for the detection of phenotypic resistance was 94.3% or greater for all drugs except moxifloxacin at a critical concentration of 2.0 µg per milliliter (specificity, 84.0% [95% CI, 78.9 to 88.3]). When DNA sequencing was used as the reference standard, the sensitivities of the investigational assay for detecting mutations associated with resistance were 98.1% for isoniazid (95% CI, 94.4 to 99.6), 95.8% for fluoroquinolones (95% CI, 89.6 to 98.8), 92.7% for kanamycin (95% CI, 80.1 to 98.5), and 96.8% for amikacin (95% CI, 83.3 to 99.9), and the specificity for all drugs was 99.6% (95% CI, 97.9 to 100) or greater. CONCLUSIONS: This investigational assay accurately detected M. tuberculosis mutations associated with resistance to isoniazid, fluoroquinolones, and aminoglycosides and holds promise as a rapid point-of-care test to guide therapeutic decisions for patients with tuberculosis. (Funded by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, and the Ministry of Science and Technology of China; ClinicalTrials.gov number, NCT02251327 .).

Does the Effectiveness and Mechanical Strength of Kanamycin-Loaded Bone Cement in Musculoskeletal Tuberculosis Compare to Vancomycin-Loaded Bone Cement.

BACKGROUND: Antibiotic-loaded bone cement (ALBC) is used to deliver antimycobacterial agents into the focal lesion of musculoskeletal tuberculosis. Although kanamycin is currently used as an antimycobacterial agent for the treatment of multidrug-resistant tuberculosis, there is no information about its suitability in ALBC. METHODS: An in vitro experiment was conducted with cylindrical shape of 40 g of bone cement with 1, 2, and 3 g of kanamycin. Eluate (1 mL) was extracted from each specimen to measure the level of elution and antimycobacterial activity on days 1, 4, 7, 14, and 30. The quantity of kanamycin in eluates was evaluated by a liquid chromatography-mass spectrometry system, and the antimycobacterial activity of eluates against Mycobacterium tuberculosis H37Rv was calculated by comparing the minimal inhibitory concentration. The ultimate compression strength was conducted using a material testing system machine (Instron 3366; Instron, Norwood, MA) before and after elution. RESULTS: Eluates from ALBC containing 2 and 3 g of kanamycin had effective antimycobacterial activity for 30 days, whereas eluates from ALBC containing 1 g of kanamycin were partially active until day 30. The pre-eluted compression strength of kanamycin-loaded cement and vancomycin-loaded cement was weaker as they contained a larger amount of antibiotics. There was no statistical difference between the strength of all kanamycin regimens and 1 g of vancomycin in the ultimate compression test. After 30 days of elution, the strength of all kanamycin-loaded cement and vancomycin-loaded cement cylinders was significantly lower than that of initial specimens (P < .05). CONCLUSION: The antimycobacterial activity of ALBC containing more than 2 g of kanamycin was effective during a 30-day period. The ultimate compression strength of bone cement loaded with 1-3 g of kanamycin was comparable with 1 g of vancomycin while maintaining effective elution until day 30.

Construction and Characterization of the Mycobacterium tuberculosis sigE fadD26 Unmarked Double Mutant as a Vaccine Candidate.

Despite the great increase in the understanding of the biology and pathogenesis of Mycobacterium tuberculosis achieved by the scientific community in recent decades, tuberculosis (TB) still represents one of the major threats to global human health. The only available vaccine (Mycobacterium bovis BCG) protects children from disseminated forms of TB but does not effectively protect adults from the respiratory form of the disease, making the development of new and more-efficacious vaccines against the pulmonary forms of TB a major goal for the improvement of global health. Among the different strategies being developed to reach this goal is the construction of attenuated strains more efficacious and safer than BCG. We recently showed that a sigE mutant of M. tuberculosis was more attenuated and more efficacious than BCG in a mouse model of infection. In this paper, we describe the construction and characterization of an M. tuberculosissigE fadD26 unmarked double mutant fulfilling the criteria of the Geneva Consensus for entering human clinical trials. The data presented suggest that this mutant is even more attenuated and slightly more efficacious than the previous sigE mutant in different mouse models of infection and is equivalent to BCG in a guinea pig model of infection.

Urine IP-10 as a biomarker of therapeutic response in patients with active pulmonary tuberculosis.

BACKGROUND: Prior to clinical trials of new TB drugs or therapeutic vaccines, it is necessary to develop monitoring tools to predict treatment outcomes in TB patients. Urine interferon gamma inducible protein 10 (IP-10) is a potential biomarker of treatment response in chronic hepatitis C virus infection and lung diseases, including tuberculosis. In this study, we assessed IP-10 levels in urine samples from patients with active TB at diagnosis, during treatment, and at completion, and compared these with levels in serum samples collected in parallel from matched patients to determine whether urine IP-10 can be used to monitor treatment response in patients with active TB. METHODS: IP-10 was measured using enzyme-linked immunosorbent assays in urine and serum samples collected concomitantly from 23 patients with active TB and 21 healthy adults (44 total individuals). The Mann-Whitney U test and Wilcoxon matched-pairs signed rank test were used for comparisons among healthy controls and patients at three time points, and LOESS regression was used for longitudinal data. RESULTS: The levels of IP-10 in urine increased significantly after 2 months of treatment (P = 0.0163), but decreased by the completion of treatment (P = 0.0035). Serum IP-10 levels exhibited a similar trend, but did not increase significantly after 2 months of treatment in patients with active TB. CONCLUSIONS: Unstimulated IP-10 in urine can be used as a biomarker to monitor treatment response in patients with active pulmonary TB.

A Novel Therapeutic Approach Using Mesenchymal Stem Cells to Protect Against Mycobacterium abscessus.

Recent studies have demonstrated the therapeutic potential of mesenchymal stem cells (MSCs) for the treatment of acute inflammatory injury and bacterial pneumonia, but their therapeutic applications in mycobacterial infections have not been investigated. In this study, we demonstrated the use of MSCs as a novel therapeutic strategy against Mycobacterium abscessus (M. abscessus), which is the most drug-resistant and difficult-to-treat mycobacterial pathogen. The systemic intravenous injection of MSCs not only improved mouse survival but also enhanced bacterial clearance in the lungs and spleen. Additionally, MSCs enhanced IFN-γ, TNF-α, IL-6, MCP-1, nitric oxide (NO) and PGE2 production and facilitated CD4(+) /CD8(+) T cell, CD11b(high) macrophage, and monocyte recruitment in the lungs of M. abscessus-infected mice. To precisely elucidate the functions of MSCs in M. abscessus infection, an in vitro macrophage infection system was used. MSCs caused markedly increased NO production via NF-κB activation in M. abscessus-infected macrophages cultured in the presence of IFN-γ. Inhibiting NO or NF-κB signaling using specific inhibitors reduced the antimycobacterial activity of MSCs. Furthermore, the cellular crosstalk between TNF-α released from IFN-γ-stimulated M. abscessus-infected macrophages and PGE2 produced by MSCs was necessary for the mycobacterial-killing activity of the macrophages. Finally, the importance of increased NO production in response to MSC administration was confirmed in the mouse M. abscessus infection model. Our results suggest that MSCs may offer a novel therapeutic strategy for treating this drug-resistant mycobacterial infection by enhancing the bacterial-killing power of macrophages. Stem Cells 2016;34:1957-1970.

How Long Does Antimycobacterial Antibiotic-loaded Bone Cement Have In Vitro Activity for Musculoskeletal Tuberculosis?

BACKGROUND: Antibiotic-loaded bone cement is accepted as an effective treatment modality for musculoskeletal tuberculosis. However, comparative information regarding combinations and concentrations of second-line antimycobacterial drugs, such as streptomycin and amoxicillin and clavulanic acid, are lacking. QUESTIONS/PURPOSES: (1) In antibiotic-loaded cement, is there effective elution of streptomycin and Augmentin® (amoxicillin and clavulanic acid) individually and in combination? (2) What is the antibacterial activity duration for streptomycin- and amoxicillin and clavulanic acid -loaded cement? METHODS: Six different types of bone cement discs were created by mixing 40 g bone cement with 1 or 2 g streptomycin only, 0.6 g or 1.2 g Augmentin® (amoxicillin and clavulanic acid) only, and a combination of 1 g streptomycin plus 0.6 g amoxicillin and clavulanic acid and 2 g streptomycin plus 1.2 g amoxicillin and clavulanic acid. Five bone discs of each type were incubated in phosphate buffered saline for 30 days with renewal of the phosphate buffered saline every day. The quantity of streptomycin and/or amoxicillin and clavulanic acid in eluates were measured by a liquid chromatography-mass spectrometry system, and the antimycobacterial activity of eluates against Mycobacterium tuberculosis H37Rv, were calculated by comparing the minimal inhibitory concentration of each eluate with that of tested drugs using broth dilution assay on microplate. RESULTS: Streptomycin was detected in eluates for 30 days (in 1 g and 2 g discs), whereas 1.2 g amoxicillin and clavulanate eluted until Day 7 and 0.6 g amoxicillin and clavulanate until Day 3. All eluates in streptomycin-containing discs (streptomycin only, and in combination with amoxicillin and clavulanic acid) had effective antimycobacterial activity for 30 days, while amoxicillin and clavulanate-only preparations were only active until Day 14. The antimycobacterial activity of eluates of 2 g streptomycin plus 1.2 g amoxicillin and clavulanate were higher than those of discs containing 1 g streptomycin plus 0.6 g amoxicillin and clavulanate until Day 3, without differences (Day 3, 1 g streptomycin plus 0.6 g amoxicillin and clavulanate: 17.5 ± 6.85 ug/mL; 2 g streptomycin plus 1.2 g amoxicillin and clavulanate: 32.5 ± 16.77 ug/mL; p = 0.109). After Day 7, however, values of the two combinations remained no different than that of Day 30 (Day 30, 1 g streptomycin plus 0.6 g amoxicillin and clavulanate: 0.88 ± 0.34 ug/mL; 2 g streptomycin plus 1.2 g amoxicillin and clavulanate: 0.59 ± 0.94 ug/mL; p = 0.107). CONCLUSIONS: Streptomycin, in the form of antibiotic-loaded bone cement, had effective elution characteristics and antimycobacterial effects during a 30-day period, whereas amoxicillin and clavulanate only had effective elution and antimycobacterial characteristics during the early period of this study. The two drugs did not interfere with each other during the elution test. CLINICAL RELEVANCE: This research revealed that combinations of streptomycin and amoxicillin and clavulanate mixed with bone cement are effective for 30 days. Further trials to determine various different combinations of drugs are necessary to improve the effectiveness of treatments for musculoskeletal tuberculosis.

Geographic Differences in the Contribution of ubiA Mutations to High-Level Ethambutol Resistance in Mycobacterium tuberculosis.

Ethambutol (EMB) resistance can evolve through a multistep process, and mutations in the ubiA (Rv3806c) gene appear to be responsible for high-level EMB resistance in Mycobacterium tuberculosis We evaluated the prevalence of ubiA and embB (Rv3795) mutations in EMB-resistant strains originating from Africa and South Korea. No differences in embB mutation frequencies were observed between strains from both origins. However, ubiA mutations were present in 45.5% ± 6.5% of the African EMB-resistant isolates but in only 9.5% ± 1.5% of the South Korean EMB-resistant isolates. The ubiA mutations associated with EMB resistance were localized to regions encoding the transmembrane domains of the protein, whereas the embB mutations were localized to regions encoding the extramembrane domains. Larger studies are needed to investigate the causes of increased ubiA mutations as a pathway to high-level EMB resistance in African countries, such as extended EMB usage during tuberculosis treatment.

Essential engagement of Toll-like receptor 2 in initiation of early protective Th1 response against rough variants of Mycobacterium abscessus.

Although Mycobacterium abscessus (M. abscessus) is becoming more prevalent in patients without overt immunodeficiency, little is known about the factors that contribute to disease susceptibility. This study was undertaken to investigate how Toll-like receptor 2 (TLR2) functionally contributes to the generation of protective immunity against M. abscessus in a morphotype-specific manner. We found that Tlr2-/- mice were extremely susceptible to an intravenous (i.v.) model of infection by M. abscessus rough variants, displaying uncontrolled infection in the lungs and a significantly lower survival rate than with wild-type (WT) mice. This uncontrolled infection resulted from failures in the following processes: (i) production of the crucial cytokines gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin 12p70 (IL-12p70); (ii) early infiltration of neutrophils, monocytes, and dendritic cells (DCs) in the lungs of Tlr2-/- mice; (iii) rapid influx of CD4+ and CD8+ T cells; and (iv) the expansion of memory/effector T cells. Notably, systemic administration of M. abscessus culture filtrate-treated syngeneic DCs from WT mice greatly strengthened immune priming in vivo, resulting in a dramatic reduction in bacterial growth and improved long-term survival in Tlr2-/- mice, with a recovery of protective immunity. Our findings demonstrate that TLR2 is an essential contributor to instructive and effector immunity during M. abscessus infection in a morphotype-specific manner.

Fitness costs of rifampicin resistance in Mycobacterium tuberculosis are amplified under conditions of nutrient starvation and compensated by mutation in the ß' subunit of RNA polymerase.

Rifampicin resistance, a defining attribute of multidrug-resistant tuberculosis, is conferred by mutations in the ß subunit of RNA polymerase. Sequencing of rifampicin-resistant (RIF-R) clinical isolates of Mycobacterium tuberculosis revealed, in addition to RIF-R mutations, enrichment of potential compensatory mutations around the double-psi ß-barrel domain of the ß' subunit comprising the catalytic site and the exit tunnel for newly synthesized RNA. Sequential introduction of the resistance allele followed by the compensatory allele in isogenic Mycobacterium smegmatis showed that these mutations respectively caused and compensated a starvation enhanced growth defect by altering RNA polymerase activity. While specific combinations of resistance and compensatory alleles converged in divergent lineages, other combinations recurred among related isolates suggesting transmission of compensated RIF-R strains. These findings suggest nutrient poor growth conditions impose larger selective pressure on RIF-R organisms that results in the selection of compensatory mutations in a domain involved in catalysis and starvation control of RNA polymerase transcription.

Linezolid for treatment of chronic extensively drug-resistant tuberculosis.

BACKGROUND: Linezolid has antimycobacterial activity in vitro and is increasingly used for patients with highly drug-resistant tuberculosis. METHODS: We enrolled 41 patients who had sputum-culture-positive extensively drug-resistant (XDR) tuberculosis and who had not had a response to any available chemotherapeutic option during the previous 6 months. Patients were randomly assigned to linezolid therapy that started immediately or after 2 months, at a dose of 600 mg per day, without a change in their background regimen. The primary end point was the time to sputum-culture conversion on solid medium, with data censored 4 months after study entry. After confirmed sputum-smear conversion or 4 months (whichever came first), patients underwent a second randomization to continued linezolid therapy at a dose of 600 mg per day or 300 mg per day for at least an additional 18 months, with careful toxicity monitoring. RESULTS: By 4 months, 15 of the 19 patients (79%) in the immediate-start group and 7 of the 20 (35%) in the delayed-start group had culture conversion (P=0.001). Most patients (34 of 39 [87%]) had a negative sputum culture within 6 months after linezolid had been added to their drug regimen. Of the 38 patients with exposure to linezolid, 31 (82%) had clinically significant adverse events that were possibly or probably related to linezolid, including 3 patients who discontinued therapy. Patients who received 300 mg per day after the second randomization had fewer adverse events than those who continued taking 600 mg per day. Thirteen patients completed therapy and have not had a relapse. Four cases of acquired resistance to linezolid have been observed. CONCLUSIONS: Linezolid is effective at achieving culture conversion among patients with treatment-refractory XDR pulmonary tuberculosis, but patients must be monitored carefully for adverse events. (Funded by the National Institute of Allergy and Infectious Diseases and the Ministry of Health and Welfare, South Korea; ClinicalTrials.gov number, NCT00727844.).

Evaluation of antigen-specific immunoglobulin g responses in pulmonary tuberculosis patients and contacts.

This study aimed to evaluate the serodiagnostic potential of immunoglobulin G (IgG) responses to Mycobacterium tuberculosis antigens in pulmonary tuberculosis (TB) patients, recent TB contacts with latent TB infection (LTBI), and healthy subjects. Infections were assessed using tuberculin skin tests, QuantiFERON-TB Gold In-Tube tests, drug susceptibility testing, and molecular genotyping of clinical isolates. Serum IgG responses to selective M. tuberculosis antigens, including the 38-kDa and 16-kDa antigens, lipoarabinomannan (LAM), and recombinant early secreted antigen target 6 kDa (ESAT-6) and culture filtrate protein 10 kDa (CFP-10), were determined. We found that the serum IgG responses to all antigens might differentiate between active TB and LTBI, with LAM having the highest diagnostic value (area under the curve [AUC] of 0.7756, P < 0.001). Recurrent TB cases showed significantly higher IgG responses to 38 kDa, CFP-10 (P < 0.01), and LAM (P < 0.05) than new cases, and male patients had higher levels of antigen-specific IgG than females (P < 0.05). Conversely, drug resistance and patient body mass index did not affect IgG responses (P > 0.05). LAM-specific IgG responses differentiated between acid-fast bacillus (AFB) smear-positive and -negative patients (P < 0.01), whereas antigen-specific IgG responses did not vary with the M. tuberculosis genotype (P > 0.05). Significantly higher IgG responses to 38 kDa and 16 kDa were observed in AFB smear-negative patients than in controls. These results suggest that assessment of serum IgG responses to selective purified M. tuberculosis antigens may help improve the diagnosis of active TB, particularly for sputum smear-negative patients or recurrent cases, and these may also help to differentiate between active TB and LTBI.

Discrimination between active and latent tuberculosis based on ratio of antigen-specific to mitogen-induced IP-10 production.

Mycobacterium tuberculosis is the major causative agent of tuberculosis (TB). The gamma interferon (IFN-γ) release assay (IGRA) has been widely used to diagnose TB by testing cell-mediated immune responses but has no capacity for distinguishing between active TB and latent TB infection (LTBI). This study aims to identify a parameter that will help to discriminate active TB and LTBI. Whole-blood samples from 33 active TB patients, 20 individuals with LTBI, and 26 non-TB controls were applied to the commercial IFN-γ release assay, QuantiFERON-TB Gold In-Tube, and plasma samples were analyzed for interleukin-2 (IL-2), IL-6, IL-8, IL-10, IL-13, tumor necrosis factor-alpha (TNF-α), IFN-γ, monokine induced by IFN-γ (MIG), interferon gamma inducible protein 10 (IP-10), interferon-inducible T cell alpha chemoattractant (I-TAC), and monocyte chemoattractant protein 1 (MCP-1) by using a commercial cytometric bead array. The Mycobacterium tuberculosis antigen-specific production of most of the assayed cytokines and chemokines was higher in the active TB than in the LTBI group. The mitogen-induced responses were lower in the active TB than in the LTBI group. When the ratio of TB-specific to mitogen-induced responses was calculated, IL-2, IL-6, IL-10, IL-13, TNF-α, IFN-γ, MIG, and IP-10 were more useful in discriminating active TB from LTBI. In particular, most patients showed higher IP-10 production to Mycobacterium tuberculosis antigens than to mitogen at the individual level, and the ratio for IP-10 was the strongest indicator of active infection versus LTBI with 93.9% sensitivity and 90% specificity. In conclusion, the ratio of the TB-specific to the mitogen-induced IP-10 responses showed the most promising accuracy for discriminating active TB versus LTBI and should be further studied to determine whether it can serve as a biomarker that might help clinicians administer appropriate treatments.

Genotypic susceptibility testing of Mycobacterium tuberculosis isolates for amikacin and kanamycin resistance by use of a rapid sloppy molecular beacon-based assay identifies more cases of low-level drug resistance than phenotypic Lowenstein-Jensen testing.

Resistance to amikacin (AMK) and kanamycin (KAN) in clinical Mycobacterium tuberculosis strains is largely determined by specific mutations in the rrs gene and eis gene promoter. We developed a rapid, multiplexed sloppy molecular beacon (SMB) assay to identify these mutations and then evaluated assay performance on 603 clinical M. tuberculosis DNA samples collected in South Korea. Assay performance was compared to gold-standard phenotypic drug susceptibility tests, including Lowenstein-Jensen (LJ) absolute concentration, mycobacterial growth indicator tubes (MGIT), and TREK Sensititre MycoTB MIC plate (MycoTB) methods. Target amplicons were also tested for mutations by Sanger sequencing. The SMB assay correctly detected 115/116 mutant and mixed sequences and 487/487 wild-type sequences (sensitivity and specificity of 99.1 and 100%, respectively). Using the LJ method as the reference, sensitivity and specificity for AMK resistance were 92.2% and 100%, respectively, and sensitivity and specificity for KAN resistance were 87.7% and 95.6%, respectively. Mutations in the rrs gene were unequivocally associated with high-level cross-resistance to AMK and KAN in all three conventional drug susceptibility testing methods. However, eis promoter mutations were associated with KAN resistance using the MGIT or MycoTB methods but not the LJ method. No testing method associated eis promoter mutations with AMK resistance. Among the discordant samples with AMK and/or KAN resistance but wild-type sequence at the target genes, we discovered four new mutations in the whiB7 5' untranslated region (UTR) in 6/22 samples. All six samples were resistant only to KAN, suggesting the possible role of these whiB7 5' UTR mutations in KAN resistance.

Mycobacterium tuberculosis MmsA, a novel immunostimulatory antigen, induces dendritic cell activation and promotes Th1 cell-type immune responses.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is an outstanding pathogen that modulates the host immune response. This inconvenient truth drives the continual identification of antigens that generate protective immunity, including Th1-type T cell immunity. Here, the contribution of methylmalonate semialdehyde dehydrogenase (MmsA, Rv0753c) of Mtb to immune responses was examined in the context of dendritic cell (DC) activation and T cell immunity both in vitro and in vivo. The results showed that MmsA induced DC activation by activating the MAPK and NF-κB signaling pathways. Additionally, MmsA-treated DCs activated naïve T cells, effectively polarized CD4(+) and CD8(+) T cells to secrete IFN-γ and IL-2, and induced T cell proliferation. These results indicate that MmsA is a novel DC maturation-inducing antigen that drives the Th1 immune response. Thus, MmsA was found to potentially regulate immune responses via DC activation toward Th1-type T cell immunity, enhancing our understanding of Mtb pathogenesis.

Differentiation of antigen-specific T cells with limited functional capacity during Mycobacterium tuberculosis infection.

Despite the generation of Mycobacterium tuberculosis-specific T cell immune responses during the course of infection, only 5 to 10% of exposed individuals develop active disease, while others develop a latent infection. This phenomenon suggests defective M. tuberculosis-specific immunity, which necessitates more careful characterization of M. tuberculosis-specific T cell responses. Here, we longitudinally analyzed the phenotypes and functions of M. tuberculosis-specific T cells. In contrast to the functional exhaustion of T cells observed after chronic infection, M. tuberculosis-specific CD8(+) T cells differentiated into either effector (CD127(lo) CD62L(lo)) or effector memory (CD127(hi) CD62L(lo)) cells, but not central memory cells (CD127(hi) CD62L(hi)), with low programmed death 1 (PD-1) expression, even in the presence of high levels of bacteria. Additionally, M. tuberculosis-specific CD8(+) and CD4(+) T cells produced substantial levels of tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ), but not interleukin 2 (IL-2), upon in vitro restimulation. Among M. tuberculosis-specific CD8(+) T cells, CD127(hi) effector memory cells displayed slower ongoing turnover but greater survival potential. In addition, these cells produced more IFN-γ and TNF-α and displayed lytic activity upon antigen stimulation. However, the effector function of M. tuberculosis-specific CD8(+) CD127(hi) effector memory T cells was inferior to that of canonical CD8(+) CD127(hi) memory T cells generated after acute lymphocytic choriomeningitis virus infection. Collectively, our data demonstrate that M. tuberculosis-specific T cells can differentiate into memory T cells during the course of M. tuberculosis infection independent of the bacterial burden but with limited functionality. These results provide a framework for further understanding the mechanisms of M. tuberculosis infection that can be used to develop more effective vaccines.

Extensive drug resistance acquired during treatment of multidrug-resistant tuberculosis.

BACKGROUND: Increasing access to drugs for the treatment of multidrug-resistant (MDR) tuberculosis is crucial but could lead to increasing resistance to these same drugs. In 2000, the international Green Light Committee (GLC) initiative began to increase access while attempting to prevent acquired resistance. METHODS: To assess the GLC's impact, we followed adults with pulmonary MDR tuberculosis from the start to the end of treatment with monthly sputum cultures, drug susceptibility testing, and genotyping. We compared the frequency and predictors of acquired resistance to second-line drugs (SLDs) in 9 countries that volunteered to participate, 5 countries that met GLC criteria, and 4 countries that did not apply to the GLC. RESULTS: In total, 832 subjects were enrolled. Of those without baseline resistance to specific SLDs, 68 (8.9%) acquired extensively drug-resistant (XDR) tuberculosis, 79 (11.2%) acquired fluoroquinolone (FQ) resistance, and 56 (7.8%) acquired resistance to second-line injectable drugs (SLIs). The relative risk (95% confidence interval [CI]) of acquired resistance was lower at GLC-approved sites: 0.27 (.16-.47) for XDR tuberculosis, 0.28 (.17-.45) for FQ, and 0.15 (.06-.39) to 0.60 (.34-1.05) for 3 different SLIs. The risk increased as the number of potentially effective drugs decreased. Controlling for baseline drug resistance and differences between sites, the odds ratios (95% CIs) were 0.21 (.07-.62) for acquired XDR tuberculosis and 0.23 (.09-.59) for acquired FQ resistance. CONCLUSIONS: Treatment of MDR tuberculosis involves substantial risk of acquired resistance to SLDs, increasing as baseline drug resistance increases. The risk was significantly lower in programs documented by the GLC to meet specific standards.

Sensititre MYCOTB MIC plate for testing Mycobacterium tuberculosis susceptibility to first- and second-line drugs.

For Mycobacterium tuberculosis, phenotypic methods for drug susceptibility testing of second-line drugs are poorly standardized and technically challenging. The Sensititre MYCOTB MIC plate (MYCOTB) is a microtiter plate containing lyophilized antibiotics and configured for determination of MICs to first- and second-line antituberculosis drugs. To evaluate the performance of MYCOTB for M. tuberculosis drug susceptibility testing using the Middlebrook 7H10 agar proportion method (APM) as the comparator, we conducted a two-site study using archived M. tuberculosis isolates from Uganda and the Republic of Korea. Thawed isolates were subcultured, and dilutions were inoculated into MYCOTB wells and onto 7H10 agar. MYCOTB results were read at days 7, 10, 14, and 21; APM results were read at 21 days. A total of 222 isolates provided results on both platforms. By APM, 106/222 (47.7%) of isolates were resistant to at least isoniazid and rifampin. Agreement between MYCOTB and APM with respect to susceptibility or resistance was ≥92% for 7 of 12 drugs when a strict definition was used and ≥96% for 10 of 12 drugs when agreement was defined by allowing a ± one-well range of dilutions around the APM critical concentration. For ethambutol, agreement was 80% to 81%. For moxifloxacin, agreement was 83% to 85%; incorporating existing DNA sequencing information for discrepant analysis raised agreement to 91% to 96%. For MYCOTB, the median time to plate interpretation was 10 days and interreader agreement was ≥95% for all drugs. MYCOTB provided reliable results for M. tuberculosis susceptibility testing of first- and second-line drugs except ethambutol, and results were available sooner than those determined by APM.

Neutrophils Promote Mycobacterial Trehalose Dimycolate-Induced Lung Inflammation via the Mincle Pathway.

Trehalose 6,6'-dimycolate (TDM), a cord factor of Mycobacterium tuberculosis (Mtb), is an important regulator of immune responses during Mtb infections. Macrophages recognize TDM through the Mincle receptor and initiate TDM-induced inflammatory responses, leading to lung granuloma formation. Although various immune cells are recruited to lung granulomas, the roles of other immune cells, especially during the initial process of TDM-induced inflammation, are not clear. In this study, Mincle signaling on neutrophils played an important role in TDM-induced lung inflammation by promoting adhesion and innate immune responses. Neutrophils were recruited during the early stage of lung inflammation following TDM-induced granuloma formation. Mincle expression on neutrophils was required for infiltration of TDM-challenged sites in a granuloma model induced by TDM-coated-beads. TDM-induced Mincle signaling on neutrophils increased cell adherence by enhancing F-actin polymerization and CD11b/CD18 surface expression. The TDM-induced effects were dependent on Src, Syk, and MAPK/ERK kinases (MEK). Moreover, coactivation of the Mincle and TLR2 pathways by TDM and Pam3CSK4 treatment synergistically induced CD11b/CD18 surface expression, reactive oxygen species, and TNFα production by neutrophils. These synergistically-enhanced immune responses correlated with the degree of Mincle expression on neutrophil surfaces. The physiological relevance of the Mincle-mediated anti-TDM immune response was confirmed by defective immune responses in Mincle⁻/⁻ mice upon aerosol infections with Mtb. Mincle-mutant mice had higher inflammation levels and mycobacterial loads than WT mice. Neutrophil depletion with anti-Ly6G antibody caused a reduction in IL-6 and monocyte chemotactic protein-1 expression upon TDM treatment, and reduced levels of immune cell recruitment during the initial stage of infection. These findings suggest a new role of Mincle signaling on neutrophils during anti-mycobacterial responses.