Multidrug-resistant tuberculosis (MDR-TB), caused by drug resistant strains of Mycobacterium tuberculosis, is an increasingly serious problem worldwide. In this study, we examined a dataset of 5,310 M. tuberculosis whole genome sequences from five continents. Despite great diversity with respect to geographic point of isolation, genetic background and drug resistance, patterns of drug resistance emergence were conserved globally. We have identified harbinger mutations that often precede MDR. In particular, the katG S315T mutation, conferring resistance to isoniazid, overwhelmingly arose before rifampicin resistance across all lineages, geographic regions, and time periods. Molecular diagnostics that include markers for rifampicin resistance alone will be insufficient to identify pre-MDR strains. Incorporating knowledge of pre-MDR polymorphisms, particularly katG S315, into molecular diagnostics will enable targeted treatment of patients with pre-MDR-TB to prevent further development of MDR-TB.

The ultrastructure of the cell wall of extensively drug-resistant (XDR), multidrug-resistant (MDR) and susceptible tuberculosis (TB) bacilli was viewed under transmission electron microscopy (TEM). Under the TEM, marked differences were observed in the thickness of their cell wall: 20.2 ±1.5 and 17.1 ±1.03 nm for the XDR and MDR TB bacilli, respectively, and 15.6 ± 1.3 nm for the susceptible isolates (p < 0.05). In MDR bacilli, thickening of the cell wall was observed in the intermediate electron-transparent layer (ETL) and outer electron-opaque layer, whereas in XDR TB cells the basal peptidoglycan layer was denser and almost fused with the ETL. Five to seven percent of XDR TB bacilli had the appearance of the stationary phase with cell wall thickness ranging from 21 to 26 nm (p < 0.001). Information provided in this study is of significant importance in terms of drug selection for effective treatment of resistant strains.

Introduction: This study aimed to determine first-line anti-tuberculosis drug resistance rates in new and previously treated cases and to identify risk factors associated with multidrug resistant tuberculosis (MDR-TB) at the National Reference Tuberculosis Laboratory of Iran. Methodology: This was a retrospective analysis of all confirmed TB patients from December 2000 to June 2005. Drug susceptibility testing to isoniazid, rifampicin, streptomycin, ethambutol and pyrazinamide was performed on Löwenstein-Jensen (LJ) medium according to the proportion method. Results: Mycobacterium tuberculosis strains were isolated from 1,742 patients with TB, of whom 935 (53.7%) were male. The mean age of patients was 44.2 ± 17.4 years (SD). A total of 1,074 patients were native Iranians while 668 (38.3%) were immigrant patients. Out of 1,139 (65.4%) new cases, 340 (29.9%) had at least one drug resistance. Of 603 (34.6%) previously treated cases, 416 (69.0%) had resistant strains. There were 263 patients (15.1%) with MDR-TB, 72 of whom were new (6.3% of all new cases) and 191 were previously treated (31.7% of all previously treated cases). Factors associated with MDR-TB included age under 45 years, male sex, previous TB treatment, immigration, poor living conditions, and unemployment. Conclusions: The high rate of initial resistance in MDR-TB cases and the high rate of MDR-TB in a young age group were indicators of recent transmission. Therefore, closer monitoring of transmission trends of drug resistant strains should be considered as priority, to ensure a successful TB control programme.

BackgroundTuberculosis (TB) remains a significant global health concern and its diagnosis is challenging due to the limitations in the specificity and sensitivity of the current diagnostic tests. Exosomes are bioactive 30–100 nm vesicles produced by most cell types and are found in almost all human body fluids. Exosomal microRNAs (miRNAs) can transfer biological information between cells and tissues and may act as potential biomarkers in many diseases. In this pilot study, we assessed the miRNA profile of exosomes released from human monocyte-derived macrophages upon infection with Mycobacterium bovis Bacillus Calmette–Guerin (BCG).MethodsHuman monocytes were obtained from the peripheral blood of three healthy subjects and driven to a monocyte-derived macrophage (MDM) phenotype using standard protocols. MDMs were infected with BCG or left uninfected as control. 72 h post-infection, exosomes were collected from the cell culture medium, RNA was isolated and RNA-seq performed. The raw reads were filtered to eliminate adaptor and primer sequences and the sequences were run against the mature human miRNA sequences available in miRBase. MicroRNAs were identified using an E value <0.01. miRNA network analysis was performed using the DIANA miRNA tool, miRDB and functional KEGG pathway analysis.ResultsInfection of MDMs with BCG leads to the release of several exosomal miRNAs. These included miR-1224, -1293, -425, -4467, -4732, -484, -5094, -6848-6849, -4488 and -96 all of which were predicted to target metabolism and energy production-related pathways.ConclusionsThis study provides evidence for the release of specific exosomal miRNAs from BCG-infected MDMs. These exosomal miRNAs reflect host-pathogen interaction and subversion of host metabolic processes following infection.

Introduction: Several species of non-tuberculosis mycobacteria (NTM) can affect humans and can cause either symptomatic or asymptomatic infection. This study aimed to determine the clinical and radiological manifestation, as well as the treatment, of M. simiae in patients in Masih Daneshvari Hospital, a TB referral hospital in Iran. Methodology: This retrospective study involved all patients presenting to our referral center from 2002 to 2009, with confirmation of M. simiae pulmonary infection. For all patients, sputum smear and culture for identification was performed, as was drug susceptibility testing. Additionally, PCR identification methods for NTM, and high-resolution CT scan were conducted. All patients were treated according to American Thoracic Society recommendations. Results: In total, 26 cases of M. simiae were identified in our center. The mean age of the patients was 58.23 ± 16.9years. Only one patient was HIV positive, and all but one were Iranian. The most frequent symptom was coughing (92.3%), and 100% of the patients had nodular lesions. In addition, bronchiectasis and cavitation were present in 84.6% and 88.5% respectively. All the patients were resistant to every firstline drug. Two patients failed the treatment, and twenty-four were cured, after which no recurrence of the disease was observed. Conclusion: M. simiae may present with clinical and radiological manifestations consistent with tuberculosis, and be resistant to anti-TB agents. A more efficient treatment for NTMs such as M. simiae is needed, to shorten the period of treatment and proved fewer adverse effects than current therapies.

The malachite green microtube (MGMT) susceptibility assay was performed directly on sputum specimens (n ‫؍‬ 80) and indirectly on Mycobacterium tuberculosis clinical isolates (n ‫؍‬ 60). The technique is based on the malachite green dye, which changes color in response to M. tuberculosis growth. The MGMT assay is simple and rapid and does not require expensive instruments.Recent advances in technology have introduced many rapid and reliable methods to differentiate between susceptible and resistant Mycobacterium tuberculosis strains (7,10,12,16). However, due to their high cost and equipment requirement, these new methods are not feasible in the clinical laboratories of developing countries in the diagnosis of tuberculosis (TB). Instead, these countries use the proportional method, which is very time-consuming (2, 11). Consequently, physicians base their diagnosis of TB on microscopy results. Therefore, supplementary rapid and reliable methods are highly needed for clinical laboratories with limited resources. In 1995, Yajko et al. (15) used an oxidation-reduction indicator, Alamar blue, which changes color in response to the growing bacteria. In 2004, we demonstrated the viability of M. tuberculosis in sputum specimens of TB patients using malachite green indicator dye, a compound routinely used in Löwenstein-Jensen (LJ) medium (3, 4). Malachite green is a triphenylmethane dye and has a dark green color, which becomes colorless during M. tuberculosis metabolism (6).In the present study, the malachite green indicator dye was used to assess the susceptibility of M. tuberculosis clinical isolates against first-and second-line anti-TB drugs. The test was also performed directly on sputum specimens of patients with pulmonary TB. In total, 80 sputum specimens from TB patients and 30 sputum specimens from patients without mycobacterial infection (e.g., with lung cancer or asthma) were collected. Furthermore, the malachite green microtube (MGMT) assay was tested indirectly on 60 M. tuberculosis clinical isolates and the M. tuberculosis H 37 R V ATCC 27294 reference strain (American Type Cell Culture Collection, Rockville, MD). The accuracy and feasibility of the MGMT assay were compared to those of the standard LJ culture method for drug susceptibility testing.Sputum specimens were digested and decontaminated by the Petroff method with a 2% final concentration of NaOH.

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BACKGROUND: Tuberculosis (TB) preventive therapy (TPT) decreases the risk of developing TB disease and its associated morbidity and mortality. The aim of these clinical standards is to guide the assessment, management of TB infection (TBI) and implementation of TPT.METHODS: A panel of global experts in the field of TB care was identified; 41 participated in a Delphi process. A 5-point Likert scale was used to score the initial standards. After rounds of revision, the document was approved with 100% agreement.RESULTS: Eight clinical standards were defined: Standard 1, all individuals belonging to at-risk groups for TB should undergo testing for TBI; Standard 2, all individual candidates for TPT (including caregivers of children) should undergo a counselling/health education session; Standard 3, testing for TBI: timing and test of choice should be optimised; Standard 4, TB disease should be excluded prior to initiation of TPT; Standard 5, all candidates for TPT should undergo a set of baseline examinations; Standard 6, all individuals initiating TPT should receive one of the recommended regimens; Standard 7, all individuals who have started TPT should be monitored; Standard 8, a TBI screening and testing register should be kept to inform the cascade of care.CONCLUSION: This is the first consensus-based set of Clinical Standards for TBI. This document guides clinicians, programme managers and public health officers in planning and implementing adequate measures to assess and manage TBI.