Background: Mycobacterium tuberculosis (Mtb) has evolved multiple mechanisms to manipulate its cellular niche for its own advantage. Many efforts have been made to understand basal mechanisms of mycobacterial infections. However, the underlying molecular regulation is not fully understood. Recently, a new class of non-coding, small RNAs, called microRNAs (miRNAs), has emerged as important regulators in biological processes, and their involvement in mycobacterial infection has been identified, thus opening a new field of research. Methods: This study aimed to determine by TaqMan Low Density Array the host genome-wide miRNA expression profile of primary human monocyte-derived macrophages (MDM) infected with two members of the Mtb complex: virulent Mtb H37Rv and the non-virulent vaccine strain Mycobacterium bovis Bacillus Calmette-Guerin (BCG) in comparison with chemically-inactivated Mtb bacilli. Results: The findings of this study showed that infection of MDM with H37Rv or BCG results in a signature of miRNA expression mostly overlapping between the two mycobacteria. A substantially different signature emerged from infection with killed virulent bacilli, suggesting an active influence of live intracellular bacteria on cellular miRNA metabolism. Specifically, Mtb induced miRNA signature is composed of miRNAs well established in immune regulation, miR-155 and miR-146a, as well as a set of miRNAs newly associated with Mtb infection: miR-145, miR-222*, miR-27a and miR-27b. All of these miRNAs are predicted to target important immune-related genes. Conclusions: This study signifies the miRNA host response upon intracellular mycobacterial infection in macrophages, providing new aspects of regulation in host-pathogen interactions, at post-transcriptional levels.

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Mycobacterium tuberculosis remains a major global health problem and is currently killing 1.5 million people every year. One of the most important steps in tuberculosis control is the rapid and accurate laboratory diagnosis. The Xpert MTB/RIF assay is a novel molecular, easy-to-use assay, which can lead to tuberculosis identification in less than 2 h. In this study, the Xpert MTB/RIF assay performance for rapid diagnosis of tuberculosis was evaluated in comparison with conventional culture methods; 361 pulmonary and extrapulmonary patient samples were collected between October 2010 and October 2012 and were analyzed at the National Reference laboratory for Mycobacteria, Zagreb, Croatia. For pulmonary samples the sensitivity and specificity were 86% and 100%, while for extrapulmonary samples the sensitivity and specificity were 75% and 99%, respectively. It was concluded that Xpert MTB/RIF assay has high sensitivity and specificity for both pulmonary and extrapulmonary specimens.

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Background: Fluorescence microscopy (FM) over the years has shown the potential for increasing the performance of microscopy. The present study was aimed to access the performance of the LED microscope for the detection of acid fast bacilli in a tuberculosis (TB) endemic country. Methods: The study was conducted at a National Reference Laboratory (NRL) in New Delhi, India. Sputum samples were collected from suspected TB patients. Each sample was processed with Auramine O and ZN methods. Auramine O stained smears were evaluated using two different excitatory light sources (MVP and LED); and ZN stained smears were examined under light microscope. The mean time required to read the smears with different modalities was recorded. Bacterial cultures provided the reference standard. Results: A total of 200 patients were included in this study. Sensitivity and specificity for the LED assessment, MVP assessment and light microscopy were 83.1% and 82.4%, 78.5% and 87.5% and 81.6% and 83.5%, respectively. Mean reading time was approximately three times faster than ZN microscopy. The mean time to read a negative smear was 2 min with fluorescence microscopy and 5 min with light microscopy with time savings of 60%. Conclusion: Although the use of LED-FM only marginally increased sensitivity, the considerable time saving ability combined with very good acceptance and ease of use makes it a reliable alternative to other conventional methods available.

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Diagnosis of smear-negative tuberculosis (TB), which is frequently seen in HIV-infected patients, is a challenge without conventional culture methods. Since 2007, the WHO (World Health Organization) has endorsed new or improved tests for increased and rapid diagnosis of TB. This study was undertaken in an effort to evaluate the accuracy of two rapid culture methods: the Microscopic Observation Drug Susceptibility assay (MODS) and Nitrate Reductase Assay (NRA), and the molecular based test Xpert ® MTB/Rif (Xpert), for diagnosis of smear-negative TB in HIV patients using the mycobacteria growth indicator tube (MGIT) in the BACTEC^TM MGIT^TM 960 system as the reference test. 430 smear-negative patients with presumptive TB were enrolled in a cross-sectional study at a tertiary care facility in Uganda. Their sputum was tested on MODS, NRA, Xpert and MGIT. Of the 430 patients, 373 had complete results to compute test accuracy. Mycobacterium tuberculosis (MTB) was detected in 43 patients by MGIT. The sensitivity and specificity were 24.4% and 98.1% for MODS, 41.5% and 92% for NRA, 48.8% and 95.1% for Xpert, respectively. The low sensitivity of the tests implies that additional diagnostics such as chest X-ray and conventional liquid culture methods might still be needed to detect TB in smear-negative HIV patients. The high specificity of the tests is useful to confirm TB in HIV patients with symptoms suggestive of TB.

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Starting with 257 outpatients attending the specialized health service for tuberculosis (TB) between 2002 and 2006 in Araraquara, an agro-industrial area with low tuberculosis (TB) incidence in São Paulo state, Brazil, positive mycobacterial cultures were obtained in 130 cases, of which 121 were confirmed as Mycobacterium tuberculosis complex. This report assesses the genetic diversity observed on 69.42% (n = 84) of the clinical isolates, for which both spoligotyping and 12-loci MIRU typing data were fully interpretable. In order to monitor changes in the population dynamics of circulating M. tuberculosis strains over time, spoligotypes were compared from this study (n = 84) with an earlier study from 1998 to 2001 (n = 70 strains); and these two datasets from low-incidence Araraquara area were also compared with a 2-year cohort in the nearby higher-incidence São Paulo city area from 2006 to 2008 (n = 93). The results obtained showed that with 58.3% (49/84) of the strains, the Latin-American-Mediterranean (LAM) was the predominant lineage in the present follow-up study; major patterns being SIT42/LAM9 11.9% (10/84), and SIT20/LAM1 10.7% (9/84). As compared with the 1998–2001 period when 40% (28/70) of the isolates belonged to the ill-defined T family, it was replaced by LAM strains between 2002 and 2006 with a visible shift to a population structure characteristic of the metropolitan São Paulo city. Further typing of the follow-up isolates from 2002 to 2006 using 12 loci MIRUs in conjunction with conventional epidemiology did not link this population structure shift to an increase in ongoing transmission or drug-resistance. Instead, it is most probably linked to movements of the important migrant community of Araraquara to higher TB incidence metropolitan areas such as São Paulo city. This is of particular concern owing to the increment in the global burden of LAM strains and the recent association of certain LAM sublineages with multidrug- and extensively drug-resistant TB. These observations suggest the need for further molecular monitoring of the TB population structure and the evaluation of transmission trends amongst migrant workers and other risk groups, such as persons in homeless shelters, in correctional facilities, drug users, and those with HIV infection, etc.

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Due to difficulties in direct diagnosis of Mycobacterium tuberculosis infection where site-specific specimens are not available, indirect methods of testing for infection are required. M. tuberculosis early secreted antigen target-6 (ESAT6) induced IFN-γ responses are specific, but do not differentiate between latent and active TB. The use of adjunct biomarkers for TB diagnosis has been proposed, such as the chemokines: CXCL9, CXCL10 and CCL2. ESAT6-induced IFN-γ CXCL9, CXCL10 and CCL2 was measured in whole blood cell supernatants of patients with pulmonary tuberculosis (PTB, n = 36) and extrapulmonary TB (ETB, n = 31) and compared with healthy endemic controls (EC, n = 33). ESAT6-induced IFN-γ responses were positive in 32% of TB cases as compared with 15% of EC cases (p = 0.048). ESAT6-induced CXCL9 responses were positive in 42% of TB cases and 15% of EC cases (p = 0.006). ESAT6-induced-CXCL10 and -CCL2 responses did not discriminate between TB and EC groups. Measurement of IFN-γ or CXCL9 together diagnosed TB (53%) cases and was significant as compared with EC (p = 0.014) cases. IFN-γ and CXCL10 together did not increase the number of TB cases diagnosed. Within TB groups, ESAT6-IFN-γ/CXCL9-based detection increased to 53% in PTB (p = 0.031) and 54% in ETB (p = 0.021), with comparable diagnosis in less severe extrapulmonary TB (L-ETB, 55%) and severe disseminated extrapulmonary TB (D-ETB, 50%). Given that 47% of TB cases remained undetected, this study shown that ESAT6-induced IFNγ and CXCL9 can support diagnosis, but must be supported by clinical correlation and other relevant investigations.

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Tuberculosis represents an important clinical and public health problem globally, particularly in low- and middle-income countries. During the last 20 years, two relevant events has changed the epidemiology of the disease: the spread of the TB/HIV co-infection and the emergence and spread of the multi-drug resistance tuberculosis (i.e., tuberculosis caused by strains resistant to at least isoniazid and rifampicin). The latter phenomenon has been generated by the inappropriate management of the anti-tuberculosis drugs. Currently, the World Health Organization estimates at least 600,000 MDR-TB cases worldwide, particularly in China, India, South Africa, and in former Soviet Union countries. Unfortunately, new difficult-to-treat MDR-TB cases have been described, named XDR- or TDR-TB (extensively or totally drug-resistant tuberculosis, respectively). Numerous observational retrospective studies proved the poorer prognostic profile of the MDR-TB cases when compared with drug-susceptible tuberculosis. The clinical management of the patients with an XDR and beyond pattern is complicated owing to the poorest, expensive, and toxic therapeutic options. MDR-TB is currently under-reported because of methodological issues, mainly related to the poor proficiency of laboratory testing. National public health strategies should reduce the increase of tuberculosis cases without therapeutic alternatives. Furthermore, research and development activities, based on continuous and sustained funding, should be improved, together with the implementation and the scale-up of effective infection control measures in healthcare settings and in the community.

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Objectives: To characterize by spoligotyping clinical isolates of Mycobacterium tuberculosis (MTB) collected between July 2003 and October 2005 from all Syrian provinces (muhafazat). Methods: All isolates (n = 96) were cultured and identified by biochemical characteristics. DNA extracts of these samples were amplified by PCR and genotyped by spoligotyping. Results: Twelve patterns were identified: 46.8% of the strains belonged to T 1 family; 20.8% to LAM 9; 10.4% to CAS; 9.3% to Haarlem 3; 4.1% to Haarlem 1; 2.1% to Family 34; and 1% to each of Family 36, EAI 5, LAM 1, LAM 8, T 3, and X 3 families. The noticeable absence of the Beijing family was not consistent with the patterns reported in most neighboring countries. Conclusion: A more inclusive study of the Syrian population is necessary to more accurately identify most of the prevailing families in the country.

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Spontaneous ovine paratuberculosis in an organized farm was diagnosed based on histopathological lesions, demonstration of acid-fast bacilli in different visceral organs, and detection of antibody levels against Mycobacterium paratuberculosis by agar gel immunodiffusion test (AGID). Out of 190 morbid specimens examined histopathologically, 77% of specimens had pathological lesions, which included predominant epithelioid cell formation, infiltration of lymphocytes, monocytes and macrophages. In acid-fast staining of tissue sections, 74% of intestinal specimens and 53% of mesenteric lymph nodes were positive for the presence of acid-fast bacilli. Hematologically, the animals were showing leucopenia, macrocytic and normochromic anemia. Out of 49 animals tested, 28.5% of animals were positive for antibodies against M. paratuberculosis. Local breeds, namely Nilagiri and Sandyno sheep, were found to be more susceptible than exotic breeds, namely Dorset. Females were more susceptible than males. Mortality was noticed in the age group of 4 months to 10 years.

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Actinomycosis is an infectious disease caused by a gram-positive anaerobic or microaerophilic Actinomyces species that causes both chronic suppurative and granulomatous inflammation. The following study reports a 48-year-old Iranian woman presenting with a spontaneous discharging sinus on the hard palate for 8 months. The patient has no past medical history of note. Laboratory findings were unremarkable. The diagnosis was based on history and clinical evidence of the lesion confirmed by histopathological examination. The patient was treated with a regimen of oral ampicillin 500 mg four times a day. She had a marked response to the treatment after 4 weeks, and it was planned to continue the treatment for at least 6 months with regular follow-up. To the best of the researchers’ knowledge, this is the first report of actinomycotic sinus tract of the hard palate in Iran.

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The goal of this study is to describe the experience with smear microscopy examination for acid-fast bacilli (AFB) of spontaneous sputum from children in a district hospital located in a rural zone of Ethiopia. All sputum reports of children were retrospectively reviewed from July 2007 until June 2012. During the period of study, 875 children less than 15 years old were screened and 48 (5.5%, 95% confidence interval [CI]: 4.1–7.3%) were diagnosed with pulmonary tuberculosis sputum smear positive. The mean age of the children with sputum positive for AFB was significantly higher than children with sputum negative for AFB (11.4 versus 10.4) (p =0.001). Only 1 out of 47 (1.3%) children of 6 years or less had sputum positive for AFB. In 13- and 14-year-old children, 8.7% had sputum positive for AFB (20 of 229) (p = 0.03). Spontaneous sputum has a low diagnostic yield in childhood in low-income countries. Alternative methods should be implemented in rural areas to improve diagnosis of pulmonary TB, particularly in children less than 12 years old.

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Multifocal tuberculosis is characterized by the presence of large multifocal tuberculosis areas in the same or different adjacent or distant organs. Primary lesions are usually in the lungs in the majority of patients. Difficulty in confirming multifocal tuberculosis and consideration of other diseases may lead to a delay in diagnosis and thus in initiating treatment. Bone and joint involvement in tuberculosis is uncommon. While osteoarticular tuberculosis most commonly occurs in the vertebral column, less frequently affected sites are hip, knee, and sacroiliac joints. The following is a fascinating case of open pulmonary tuberculosis associated with extensive extra-pulmonary multifocal tuberculosis.

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