Background: Limited evidence showed the persistence of Mycobacterium tuberculosis (MTB) in soil and water of TB sanatoriums or hospitals, but no reports suggest the occurrence of MTB in nature. Here, this study investigates the probability of isolating MTB from the environment with no clear source of contamination. To further highlight the transmission vehicle from human to nature and vice versa, the typing patterns of isolated MTB strains from the environment was compared with clinical MTB isolates. Additionally, the viability of MTB after 3 and 6 months of contamination was investigated. Methods: The soil (n = 700) and water (n = 800) from different locations of suburban towns of Tehran was examined for the presence of MTB. The digested and decontaminated samples were inoculated in three LJ (Lowenstein Jensen) mediums and incubated at 37 °C, 25 °C and 42 °C for 12 weeks. Spoligotypes and MIRU-VNTR typing methods were performed on DNA extracted from single colonies. Thereafter, the obtained typing patterns were compared with the genotyping of TB patients that were residents of Robat Karim (n = 25), Firuzkuh (n = 10), Shahr-e-Ray (n = 20) and Tehran (n = 413). Result: MTB accounts for 14.4% of total mycobacterium that was isolated from soil and water in suburban towns of Tehran. Based on polymerase chain reaction (PCR) typing methods, T family (56/82; 68%) followed by Delhi/CAS (11/82; 13.4%) were the most frequent MTB super families in both water and soil samples. Overall, 27.7% of isolates in clusters were intra-connected strains, i.e., found in different regions; however, no similar typing pattern between soil, water and clinical isolates was observed. The most frequent super family of MTB in clinical isolates was Delhi/CAS (142; 30.3%) followed by NEW-1 (127; 27.1%). The environment and clinical isolates had a minimum of 2 and a maximum of 10loci difference (5–7 difference on average). The bacilli in contaminated soil (36%) and damp water (8.4%) remained alive for a considerable period of time, i.e., up to 9 months. Conclusion: Persistence of MTB in soil and water highlights the risk of transmission, which needs further investigation. This study was supported by the National Research Institute of Tuberculosis and Lung Disease of Iran, Iran.

Aims and objectives: Resistant-, multidrug resistant- (MDR) and even extensively drug resistant- (XDR) Mycobacterium tuberculosis complex (MTBC) strains have emerged worldwide and represent a serious challenge for global tuberculosis (TB) control. Interestingly, high rates of MDR TB have been associated with particular phylogenetic lineages of the MTBC such as the Beijing lineage and the strong clonal expansion of particular MDR strains in Eastern Europe. However, a systematic investigation of the evolution and cross border transmission of particular highly transmissible MDR strains in Eastern Europe has not been performed so far. Methods: To address this question, an in-depth analysis was performed of the population structure (based on 24 loci MIRU-VNTR-typing and spoligotyping) of strains from different settings in Eastern Europe, such as Uzbekistan, Turkmenistan, and Kazakhstan. Results: The results showed that two major clones of the Beijing lineage are the drivers of the MDR epidemic in Eastern Europe. In addition, retrospective investigation of an MDR strain collection from Germany using MIRU-VNTR and IS6110 RFLP patterns revealed that both clones were circulating for more than 15 years. In-depth analysis of genome characteristics confirmed the longitudinal transmission of the two major MDR clones and allowed the identification of particular beneficial combinations of mutations potentially compensating for the fitness defect of the MDR/XDR strains. Moreover, these allele combinations coincide with an enhanced transmissibility compared with other circulating MDR/XDR strain types and promote the selection of extremely drug resistant but also highly virulent MTBC strain variants. Conclusion: Taken together, these data argue strongly for an expansion of particular MDR Beijing clones in Eastern Europe that started at least 15 years ago. Evolution and selection over the past years has resulted in “hyper transmissible” MDR-TB strains that have the potential to spread rapidly and, thus, further complicate TB control.

The Algerian National TB program was first implemented in Algeria in 1965. Since 1965, the Ministry of Health has endorsed many instructions which have given important improvements in the fight against tuberculosis (TB) in the country. The government has actively participated in the fight against TB as expressed in its endorsement of many decisions to this end, such as the withdrawal of TB medications from the private pharmacies, the free-of-charge diagnostics and the treatment for TB patients until the completion of their treatment, and the creation of the National TB Laboratory, as well as many other tasks to control the TB epidemic in the whole country. According to this policy, the surveillance of TB drug resistance has been followed continuously since 1965 up to the present. The National TB Laboratory is a unique laboratory performing the DST for TB strains and is also able to conduct the national drug resistance surveys. The different steps will be presented of the development of the National TB program in Algeria between 1964 and 2014, and in the same way the variations of the prevalence rate of TB drug resistance to demonstrate that the drug resistance surveillance is an acceptable indicator of the performance of TB control program in the country.

Aim and objective: Multidrug-resistant Mycobacterium tuberculosis (MDR-TB) is an increasing public health problem, causing significant difficulties and costs for TB control programs. In some settings, as many as each second infectious TB case has MDR-TB. Reliable and timely detection of these cases is urgently needed. Unfortunately, globally most MDR-TB patients are not identified and will thus not receive appropriate therapy. They are clearly at risk to develop even more resistant TB and to transmit increasingly resistant M. tuberculosis strains. With today's knowledge, available diagnostic tools and epidemiological situation, it cannot be considered acceptable to wait for 8–10 weeks to know if a clinical isolate is drug susceptible or not. Thus, the generally used algorithm of isolation on solid media followed by subsequent DST on solid media must today be seen as obsolete. The objective of this presentation is to discuss the possibilities and limitations of more rapid alternatives. Methods: Different, more rapid alternatives to the time-consuming classical laboratory tests to detect drug-resistant TB were compared. These include automated liquid culturing systems and molecular techniques. Results and discussion: The most rapid and promising techniques are based on molecular detection of resistance-related mutations, especially when the assay is used for direct testing of a smear-positive sputum sample. In this case MDR-TB patients can be detected in 1–2 days which makes early initiation of effective drug combinations possible. Besides a number of locally developed assays, there are today two major commercial alternatives: GeneXpert from Cepheid and the line probe assays (LPA) from Hain Life Science. The Xpert system is the most rapid technique, and it was developed to be easy to use. It offers the simultaneous detection of M. tuberculosis and resistance to rifampicin, which is seen as a proxy for MDR-TB. Rifampicin is, however, not everywhere an applicable MDR marker. For example, in Iran, the prevalence of rifampicin mono-resistant M. tuberculosis is high. The LPA investigates resistance to both rifampicin and isoniazid and is thus more informative. It is, however, somewhat more time-consuming and laborious to perform. Both assays have demonstrated excellent specificity and sensitivity. An exception to the high sensitivity is, however, seen in so-called hetero-resistant TB, where phenotypic DST assays are shown to be more sensitive to detect a small proportion of drug-resistant bacteria. Conclusions: Reliable and timely detection of drug-resistant TB is needed, which is best achieved with molecular assays. In this author's opinion, rapid detection of resistance to isoniazid should be included with rifampicin resistance examination. In MDR, timely detection of the XDR defining agents and PZA is urgently needed. Development and validation of such tests should be a priority, as well as establishing QMS for the implementation and routine use of molecular rapid diagnostics. Each country should develop national diagnostic algorithms for how, when and where rapid molecular assays should be used for early detection of drug-resistant TB.

Understanding environmental mycobacteria and the diseases they cause is woefully lacking. The number of species, their ubiquity in nature and the heterogeneity of their effects on their human hosts have made this a formidable task. Diagnosis is difficult. Standard laboratory testing is controversial, and treatment lacks trial-based evidence. Uncertainty in epidemiology, pathogenesis, diagnosis, and treatment results in inconsistent clinical care for these diseases. The lack of tools to understand the organisms and diseases has stunted research in this field. Knowledge of nontuberculous mycobacteria pales compared to that of tuberculosis. Tuberculosis research and understanding have surged in recent years leading to better understanding, containment, and control of this disease. At the same time, diseases caused by nontuberculous mycobacteria have increased. The field of nontuberculous mycobacteria must learn from developing countries that have used technology to “leap-frog” over other technology that took decades to attain. “Omics” technology could be the key to raising the knowledge of nontuberculous mycobacteria to that of tuberculosis. Genome-based technology could replace the microbiology lab for diagnosis and drug sensitivity identification. It could vastly strengthen epidemiology, better define pathogenesis, effectively guide treatment, and accurately prognosticate outcomes. Genomics are already being used to track mycobacterial infections. Bacterial gene sequencing in patients with cystic fibrosis infected with Mycobacterium abscessus has shown the first human-to-human spread. Sequencing studies have separated out M. avium and M. intracellulare and have shown differences in pathogenicity. Studies on environmental isolation could yield clues to controlling these diseases by system modifications. Genomic approaches to pathogenesis have largely involved M. smegmatis, which has been used as a surrogate for M. tuberculosis in learning about the gene and protein changes that occur when bacteria go into a dormant state. Studies on M. ulcerans have identified factors related to its virulence. The mycobacterial diagnostic lab has used gene-based tests for several years, and there is a growing array of products available. Sequencing of the genes encoding the highly variable regions of bacterial DNA is rapid and accurate. Genotyping already can identify pathogenicity and response to antibiotics in a few instances; the future goal should be to identify sensitivity to a wide variety of organisms and antibiotics. This information will require clinical trials and time to validate it. An interferon gamma release assay for infection with nontuberculous mycobacteria is desperately needed. Success with tuberculosis was attained by finding bacterial antigens associated with virulence and invasive disease. This may be difficult because the damage caused by environmental mycobacteria tends to be epithelial and subepithelial. The cavities may be bronchiectatic in origin and deeper invasion may result from host reactions. Serologic testing has not yielded important clinical help for diseases caused by either M. tuberculosis or the nontuberculous mycobacteria, possibly because good bacterial or host response proteins have not been identified. This could change with a metabolomics approach. Genomic studies that give incremental gains may set the stage for major breakthroughs, but require coordination with good phenotyping. Tools are in place to open an exciting new era for understanding and control of mycobacteria.

Aims and objectives: Bone and joint infections due to environmental mycobacteria are rare and can develop very slowly. The source for these outbreaks is generally tap water supplies. In the present review, the focus is on a large outbreak of Mycobacterium xenopi spinal infections in patients who had undergone surgical microdiscectomy for disc hernia in a French hospital. Surprisingly, a patient was diagnosed with a M. xenopi discitis with secondary extension to the sacroiliac joint 15 years after spinal surgery. The findings of a national investigation launched by health authorities to determine the number of the bone and joint infections due to opportunistic mycobacteria are described in this study. Methods: National health authorities launched a retrospective investigation in patients who were exposed to M. xenopi contamination in that hospital. Moreover, a national survey was conducted across all French laboratories to collect information on bone and joint cases due to opportunistic mycobacteria. The National Reference Center for Mycobacteria investigated hospital tap water supplies and developed a species-specific probe for the rapid identification of M. xenopi. Results: Bone and joint infections, with the exception of the episode of the clinic where the large M. xenopi outbreak occurred, are rare in France. A very small number of cases, all sporadic, were detected and linked to an invasive procedure. In addition to M. xenopi, mycobacterial species involved are Mycobacterium marinum, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium avium and Mycobacterium kansasii. Despite awareness of laboratories to mycobacterial infections, no significant increase in iatrogenic infections has been demonstrated in care facilities. The source of infection of the large outbreak of spondylitis due to M. xenopi was traced to deficient hygiene practices and a high concentration of M. xenopi in the hospital tap water. Conclusions: Failures in hygiene practices could result in an uncontrolled outbreak of nosocomial infection. Patients who have been exposed to an iatrogenic infectious hazard should be screened promptly when symptoms develop.

Background: Pulmonary tuberculosis (TB) is an important disease with various manifestations in intensive care units (ICU). Despite the availability of effective treatments for TB, the mortality for patients admitted with TB to an ICU remains high. Additionally, the history of exposure to TB may not be present, and evidence of active TB is present in less than 50% of cases. Therefore, understanding the typical distribution, patterns, and imaging manifestations of TB is crucial. Methods: In this retrospective study, all patients admitted to ICU with clinical and laboratory-confirmed TB were enrolled. The classic information, i.e., chest X-ray (CXR) and computed tomography (CT), for each patient was analyzed. Likewise, the presence of a cavity, involved segments and patterns of parenchymal lesion were assessed. Finally, tentative diagnosis and disease activity, bronchogenic spread of the lesion with CT and bronchiectasis were recorded. Results: Among the studied cases, 146 were laboratory-confirmed TB patients. The majority of patients had acute respiratory distress syndrome (ARDS) (16.0%, n = 24), followed by interstitial involvement (13.0%, n = 19), parenchymal nodular infiltration (12.0%, n = 18), alveolar consolidations (11.6%, n = 17), cavitary TB (11.0%, n = 16), pleural effusion (10.0%, n = 15), calcified parenchymal masses (9.0%, n = 13), ground glass opacities (8.0%, n = 12) and other manifestations (8.0%, n = 12). Radiographic evidence of lymphadenopathy was seen in up to 43% of adults and 96% of children. In the 73% of cases with parenchymal infiltration, more than one pulmonary segment was involved. Miliary TB was also observed in 5% of studied patients. Conclusion: Different features of TB patients in the ICU may be easily misled, and internists should have a comprehensive knowledge of various radiologic manifestations of TB in order to use this information and not ignore it.

Background: Mycobacterium ulcerans (MU) produces mycolactone toxin when infected Aims and objectives: The aim is to document the clinical and epidemiological features of Mycobacterium ulcerans disease in the Middle Belt of Ghana, and the outcome of treatment. Patients and Methods: Patients with lesions suspected to be MU disease were screened by community workers. Lesions were confirmed by any of the following: direct smear examination, culture, polymerase chain reaction (PCR), or histopathology. Patients were treated with rifampicin (10 mg/kg orally) and streptomycin (15 mg/kg im) combination for 8 weeks. Patients selected for surgical treatment included cases where medical treatment had failed, cases presenting late with complications, and recurrent cases. Results: 258 patients were seen in the Ahafo Ano, Amansie Central, Amansie West, Asunafo, Asutifi, and Upper Denkyira districts of Ghana between 2005 and 2012. Their ages ranged from 1 year 3 months to 98 years, with a mean age of 29.8 (SD 20.4). The clinical forms of MU disease seen were: papule (0.74%), nodule (1.48%), chronic osteomyelitis (1.48%), contracture (1.48%), oedematous lesion (2.69%), and ulcer (91.85%). Uncommon complications include subluxation of knee joint, salivary gland fistula and Marjolin's ulcer. The lesions were distributed as follows: head and neck (6.2%), upper limb (23.1%), trunk (1.5%), and lower limb (69.2%). Conclusion: The use of antibiotics for MU disease has controlled most lesions; however, rare complications requiring reconstructive surgery are emerging.

Objectives: The global increase in drug resistance in Mycobacterium tuberculosis (MTB) strains increases the focus on improved molecular diagnostics for MTB. Extensively drug-resistant (XDR) – TB is caused by MTB strains resistant to rifampicin, isoniazid, fluoroquinolone and aminoglycoside antibiotics. Resistance to anti-tuberculous drugs has been associated with single nucleotide polymorphisms (SNPs), in particular MTB genes. However, there is regional variation between MTB lineages and the SNPs associated with resistance. Therefore, there is a need to identify common resistance conferring SNPs so that effective molecular-based diagnostic tests for MTB can be developed. This study investigated used whole genome sequencing (WGS) to characterize 37 XDR MTB isolates from Pakistan and investigated SNPs related to drug resistance. Methods: XDR-TB strains were selected. DNA was extracted from MTB strains, and samples underwent WGS with 76-base-paired end fragment sizes using Illumina paired end HiSeq2000 technology. Raw sequence data were mapped uniquely to H37Rv reference genome. The mappings allowed SNPs and small indels to be called using SAMtools/BCFtools. Results: This study found that in all XDR strains, rifampicin resistance was attributable to SNPs in the rpoB RDR region. Isoniazid resistance-associated mutations were primarily related to katG codon 315 followed by inhA S94A. Fluoroquinolone resistance was attributable to gyrA 91–94 codons in most strains, while one did not have SNPs in either gyrA or gyrB. Aminoglycoside resistance was mostly associated with SNPs in rrs, except in 6 strains. Ethambutol resistant strains had embB codon 306 mutations, but many strains did not have this present. The SNPs were compared with those present in commercial assays such as LiPA Hain MDRTBsl, and the sensitivity of the assays for these strains was evaluated. Conclusions: If common drug resistance associated with SNPs evaluated the concordance between phenotypic and genotypic testing, the results would be rifampicin (100%), isoniazid (89%), fluoroquinolones (95%), aminoglycoside (81%) and ethambutol (61%). This work highlights the importance of expanded targets for drug resistance detection in MTB isolates.

The aim of this study is to develop an innovative and alternative nucleic acid-based method for multidrug-resistant tuberculosis (MDR-TB) and extensively drug resistant tuberculosis (XDR-TB) diagnostics, such as the Cepheid GeneXPert or the Hain–Line Probe Assay methods, to simultaneously identify MDR-TB (patient benefit) and to provide clues as to MDR-TB transmission (community benefit) at a reasonable cost to the national public health programs. The DPO (Dual-Priming Oligonucleotide) principle was used to initially develop a 4-Plex multiplexed polymerase chain reaction (PCR) that simultaneously amplifies: (1) the CRISPR; (2) the rpoB hotspot, the katG and the inhA genes. The current maximal version of the assay allows the characterization of up to 68 markers, 59 of which are used routinely in one step (for the Luminex 200) or two-step methods (for the MagPix). Spoligotyping requires 43 markers, and rpoB516, 526, 531, katG315, inhA-8, -15, gyrA94, rrs1401, 1402, 1484 requires 25 markers. For each primer couple, one is biotinylated. Hybridization is performed after a PCR reaction on a microbead-based suspension array device with individually detectable oligonucleotide-coupled beads (Luminex 200 or MagPix systems) and detection proceeds through Streptavidin–Phycoerythrin labelling of the biotinylated-hybridized PCR products. This study shows that this technique provides 100% specificity and sensitivity for rpoB and 100% specificity and 90% sensitivity for isoniazid resistance on DNA extracted from cultures, compared with phenotypic DST. The method was fully validated against sequencing. The method is currently in the experimental validation phase on DNA extracted from biological material and preliminary results will be shown. This technique was recently upgraded to detect fluoroquinolone and aminoglycoside resistance by including 2 new PCR primer couples and 9 more probes on the rrs and gyrA genes. It is suggested that population studies using this laboratory method could provide a simpler and cheaper way to perform national TB-resistance surveys and would also allow to delineate more precisely, without further enquiries, MDR-TB transmission risks. In conclusion, these methods appear to be both economically and technically very innovative. The extension from a Research Use Only to a CE-marked In-Vitro Diagnostics assay is in progress.

Introduction: Diagnosing extra-pulmonary tuberculosis continues to be a challenge for both infectious disease specialists and microbiologists. Objectives: This prospective study was done to evaluate the performance of two DNA-based methods for the detection of extra-pulmonary tuberculosis in comparison with the conventional culture technique. Methods: All extra-pulmonary specimens received by the Kuwait National Tuberculosis Reference from October 2011 until August 2013 were included in the study. Smears were stained by Ziehl Neelson (Merck, Germany) followed by inoculation of the specimens into GeneXpert MTB/RIF assay (Cepheid, USA), ProbTec ET PCR (Becton Dickinson) and MGIT 960 (Becton Dickinson). Urine was inoculated into Lowenstein Jensen media (MAST). Results: A total of 1674 extra-pulmonary specimens (pleural fluid 553, ascetic fluid 194, cerebrospinal fluid [CSF] 85, Urine 67, other sterile body fluids 153, fine needle aspirates [FNA] 301, pus 181, tissue 102, swabs 27 and stool 11) were evaluated. Out of 155 extra-pulmonary specimens that grew Mycobacterium tuberculosis (MTB) by culture, 143 were positive by GeneXpert compared with 128 by ProbTec with a sensitivity of 92% and 83%, respectively. Out of 1517 specimens that did not grow by culture, 52 were detected by GeneXpert while 46 were detected by ProbTec with specificity of 96.5% and 96.9%, respectively. All the 4 smear-negative CSF samples which grew MTB were positive by GeneXpert with a sensitivity of 100% compared with only 2 detected by ProbTec with a sensitivity of 50%. Additionally, all CSF specimens that did not grow by culture were negative by both the molecular methods showing 100% specificity. Of the 3 smear-positive urine specimens that grew by culture, all were positive, and of the 64 samples that did not grow by culture, all were negative by both the molecular methods with a sensitivity and specificity of 100%. For other sterile body fluids the sensitivity and specificity of both the methods were 68% and 99%, respectively. Finally, for FNA, pus and tissue, the sensitivity of GeneXpert was 97% compared with 86% for ProbTec. Conclusion: DNA-based technology looks promising for the rapid diagnosis of extra-pulmonary tuberculosis with an overall better performance of GeneXpert over ProbTec.

During recent years, the implication of nanoparticles (NPs) as drug delivery systems has gained much scientific attention. As drugs do not deliver themselves, a nanoparticle can act by optimizing drug delivery in the right place, at the right time and at the right dosage. In previous years, this research successfully developed and evaluated the ChPL-NPs nanoparticles (chitosan protein–lipid) [US patent pending 20140370500]. In the present investigation, rifampicin (RIF) ChPL-NPs nanoparticles were designed and developed. Consequently, the in vitro release of RIF ChPL-NPs nanoparticles was investigated. Material and Methods: Briefly, chitosan powder (90 KD and 90% degree of deacetylation) was dissolved (1% acetic-acid) and mixed with gelatin (3%). Then, the lipid and rifampin in ether was precipitated by rotary vacuum evaporator. Under high speed homogenizer (12,000rpm), both solutions (chitosan–gelatin & rifampin–lipid) were mixed [US patent pending 20140370500]. The obtained RIF ChPL-NPs were put into a dialysis bag with cut-off 14 KD in phosphate buffer solution (pH=7.4). The release of RIF was obtained by reverse phase HPLC using C18 (250 × 4.6 mm, 5 μm) column. The mobile phase consisted of 50:50 v/v acetonitrile and 10 mm potassium dihydrogen phosphate (pH=3.2) and flow rate 1 ml/min. The column temp was maintained at 25 °C with UV detection at 335nm. Results and conclusions: The average size of RIF ChPL-NPs was about 50–250nm. The release of RIF from the dialysis bag started after 30 min which was 2400ng/ml; after 16 h the release of RIF was 15,000ng/ml; and at 40 h the concentration reached to 19,600ng/ml. Therefore, these results showed a slow release of RIF from ChPL-NPs. Basically, the intensity of the surface charges in nanoparticle is important as it determines their interaction with bioactive compound. In RIF ChPL-NPs, lipid had negative charges, whereas chitosan and gelatin had positive charges. The electrostatic interaction between oppositely charged ions would ultimately cause an effective system drug delivery. RIF ChPL-NPs is not only suitable for intravenous administration, but it can be used as an inhalation aerosol, because this nanoparticle has a capacity to adhere to mucosal surfaces and transiently open the tight junction.

To control tuberculosis (TB), it is necessary to detect the disease and provide effective treatment to avert its progression and to interrupt any onward transmission from infectious pulmonary cases. Traditional diagnostic methods rely on the detection of the Mycobacterium tuberculosis (MTB) bacilli in clinical samples such as sputum. Testing is usually done on patients that have been coughing for some time, when transmission is likely to have already occurred. The impact of the GeneXpert MTB/RIF assay has so far had a limited impact on TB control because of its high cost and limited accessibility. A number of follow-up nucleic acid amplification technologies are being evaluated, but they also rely on testing for the presence of the bacteria in sputum. Diagnosing extra-pulmonary disease and TB in children remains very difficult and tests are needed that detect all forms of the disease. For developing countries with a high burden of TB, technology is needed that is robust, affordable and will avoid diagnostic delay; the tests must be readily accessible to the community without waiting for patients to self-refer to a TB testing clinic. Compromises may be needed between accuracy and access where a rapid imperfect test that can be used to test people in their community will have more benefit than a more perfect test that is not accessible. Traditional serological tests have failed for TB because of the complexity of the disease, but by testing for multiple markers, sensitivity and specificity can be improved and it is possible to differentiate latent infection and active disease. Examples of new assays that have the potential to change TB control shall be provided. The challenge now is to develop, manufacture, and distribute detection platforms that are safe, robust and affordable. Recent studies have revealed the market for TB diagnostics to be fragmented and small. Consultation with manufacturers has revealed that access to finance, management of intellectual property, regulatory acceptance and in some countries the lack of distribution networks are significant barriers to innovation. The lack of financial incentives and expectation of poor returns on investment discourages commercial companies, and there is a need for the not-for-profit and academic sectors to expand their role in product development and delivery. Biologists and technologists must work with manufacturers and healthcare providers to overcome the economic and political barriers to product development, manufacture and distribution. To this end, guidelines and a ‘road map’ for taking new in vitro diagnostic medical devices to market have been developed to raise awareness and sensitize the TB research community for the need for innovation in product delivery.

Objective: Mycobacterial disease is still an important cause of morbidity and mortality in the world. Personalized medicine is a rapidly advancing field of medicine. It uses all available omics in order to make accurate decisions about prevention, diagnosis, and treatment of disease. Personalized medicine may be helpful to design more efficient strategies for prevention of mycobacterial diseases and for offering better treatment options. Methods: A literature search was conducted using search keywords “personalized medicine„, “individualized”, “nontuberculous mycobacteria”, “mycobacterium tuberculosis”, “tuberculosis”, “genetic susceptibility”, “genomics”, “side effects”, “treatment”, “prevention” and “diagnosis” from studies that have been published by July 2014. PubMed, Cinahl, Scopus, Embase and the Cochrane Library were searched. Results: The advances in personalized medicine for diagnosis, treatment and prognosis of mycobacterial diseases were addressed. A need assessment for individuating the mycobacterial diseases was performed. Finally, the proposed approach to personalized medicine in mycobacterial diseases was discussed. Conclusions: Moving toward personalized medicine in mycobacterial diseases has already started, but needs further works to make it applicable for patient care. It will help us to improve diagnostic and treatment strategies and possibly to deliver a better quality of healthcare to patients.

Aims: To assess the definitions used by authors for pulmonary and extrapulmonary tuberculosis, definitions which are ostensibly standardized in the literature by the World Health Organization (WHO), Centers for Disease Control and Prevention (CDC) and American Thoracic Society (ATS) definitions. Methodology: Thirty-seven papers used for the study of extrapulmonary tuberculosis, identified by PubMed and Google Scholar searches identified through an earlier study on extrapulmonary tuberculosis and updated, were analyzed for specifics regarding how extrapulmonary, pulmonary, pleural and disseminated tuberculosis were defined. Data were tabulated and analyzed using STATA 11. Results: Thirty-one (84%) of the papers provided data on the numbers of pulmonary and extra-pulmonary cases. The papers were from 34 institutions in 20 nations. Only 14 (38%) of the series reported the number of patients with combined pulmonary and extrapulmonary disease. Among all patients, in only four were the combined patients analyzed as a completely separate category with combined cases otherwise excluded (11), counted as extrapulmonary (7), as pulmonary (3) or both extrapulmonary and pulmonary (1) tuberculosis or unclearly documented (11). Pleural disease was included as extrapulmonary in 25 patients (68%), but as pulmonary in 4 (11%), and there was no criterion in the remaining 8 (22%). In 18 of the studies where disseminated or miliary disease were defined, 5 of the disseminated were categorized otherwise (e.g., along with combined pulmonary and extrapulmonary cases). Conclusion: There is much confusion in the categorization of clinical tuberculosis. The standardized WHO, CDC, and ATS definitions are not always adhered to in collating and analyzing tuberculosis data by authors studying extrapulmonary tuberculosis. The recommendation that pleural disease be considered extrapulmonary is not adhered to in a sizable percentage (32%) of studies and the exclusion of disseminated or miliary disease in a subset of patients is also inconsistent. More restrictive guidelines are needed in the definitions used for tuberculosis so that studies and meta-analyses can be performed with greater validity.

Background: Tuberculosis (TB) infection and spread are preventable, and TB disease is curable depending on the individual and community knowledge of causes of the disease, mode of prevention and cure. Objective: Following a previous program carried out in 2006 in Akwa Ibom State (AKS) of Nigeria that focussed on creating awareness about TB and educating the communities on appropriate care-seeking attitudes, an intervention to reduce the burden of the disease in 18 communities of the State was carried out over a period of one year (2010–2011). The program was phased and was comprised of three components: educational intervention, TB case detection and integration into the State National Tuberculosis and Leprosy Control Programme (NTBLCP), as well as laboratory capacity building. Methods: Standard pretested questionnaires were administered to community residents to test their knowledge, attitudes and practices concerning TB. Information about causes, symptoms and prevention of TB was disseminated in community town halls, churches, markets and schools. Individuals who were coughing for three weeks or more were investigated for TB following clinical examination by a physician. Three sputum samples (spot-morning-spot) were obtained from each individual and examined microscopically for the presence of acid-fast bacilli (AFB) using the Ziehl-Neelsen staining technique. Those with positive AFB results were integrated into the existing NTBLCP TB treatment facilities for immediate commencement of Directly-Observed Therapy Short Course (DOTs). Treatment outcome was monitored by retesting patients’ sputum after 2, 5 and 7 months. Two new laboratories were facilitated while existing laboratory capacity was built by providing higher resolution microscopes, power-generating plants, refrigerators and locally-fabricated incinerators. The program was facilitated by a public–private partnership. Effective Health Care Alliance Research Programme (Nigeria), in collaboration with Nigeria National Petroleum Cooperation and Mobil Producing Nigeria Unlimited (NNPC/MPN) Joint Venture, utilized health personnel from the Akwa Ibom State NTBLCP who conducted laboratory testing and supervised the treatment. Results: The 916 responses to the questionnaires showed that 549/841 (65%) correctly identified that TB is airborne, and 759/871 (86%) were aware that TB could be cured by anti-TB medication. Responses to care-seeking attitudes were provided by 123 respondents. Of this number, fear of stigmatization was the reason for 31% (38) seeking care in unorthodox facilities, while 43% (53) did not believe that orthodox medicine could cure their symptoms. Of the 374 detected cases, 9 did not commence treatment. Hence, 365 cases were placed on DOTs; 36 defaulted while 11 died or failed to convert after the seventh month. At the end of month 8, cure was achieved for 318 (87.1%) of the cases. Conclusion: Though the previous intervention might have helped to increase the knowledge of the respondents about TB in the community and helped to improve their care-seeking attitudes, sustaining active case finding through Public–Private Partnership can go a long way to reduce TB burden, especially in rural communities where health care systems are generally weak or inadequate.

Introduction: Tuberculosis (TB) remains an important public health issue for Bulgaria. Although a number of new cases are showing a steady decline (44/100,000 in 2000, 40/100,000 in 2005), the TB incidence rate in Bulgaria is still sufficiently high (26.7/100,000 in 2013). The current population structure of Mycobacterium tuberculosis is clonal. Certain genetic families of this species have justly attracted more attention due to their global dissemination and/or remarkable pathogenic properties. Beijing, Haarlem, and LAM are the most known examples. The latter family LAM (Latin–American–Mediterranean) was shown in an increasing number of studies to possess increased transmissibility, hence the importance of its rapid detection and correct estimation of its prevalence in a population. Spoligotyping signature of LAM is absence of signals 21–24 and 33–36, although abridged spoligo-profiles with long blocks of deleted spacers result in an uncertain definition of such strains. The use of other molecular markers may be helpful. This study aimed to evaluate the prevalence of LAM strains among M. tuberculosis strains in Bulgaria based on the use of different molecular markers. Materials and Methods: M. tuberculosis isolates were randomly selected among M. tuberculosis strains isolated from newly diagnosed, adult, pulmonary TB patients in different regions of Bulgaria from December 2004 to March 2006. Spoligotyping was used to analyze a variation in the DR locus. The spoligotyping patterns were compared with the international database SITVIT_WEB (Institut Pasteur de Guadeloupe). Analysis of the IS6110 element specific for the LAM genetic family was performed as described previously (Marais et al., 2006). Results: A study sample included 133 strains from different regions of the country and characterized in previous publications (Valcheva et al., 2008, 2010). Application of the published rules for the definition of the major spoligotype clades and comparison with SITVIT_WEB global database permitted this study to assign most of the 133 strains to the known spoligotype families. All available strains (n = 101) were tested for LAM-specific IS6110 insertion. Comparison of results by different methods identified 3 groups of strains. The first group included 11 strains with 2 amplified bands which present an apparent discrepancy; further study is warranted. The second group included 86 strains with a single amplified band, specific for the absence of the IS6110 insertion in the target locus, hence indicative of the other than LAM family. The third group included 4 strains with a LAM-specific band only. Conclusions: Application of the LAM-specific PCR revealed double-sided discrepancies when the obtained results were compared with those obtained by spoligotyping. As a whole, a phylogenetic family of 38 strains was revised or questioned: 27 strains were shown not to belong to LAM, while 11 more strains showed apparently discrepant results that question the global utility of such PCR or at least highlight an importance of using multiple markers for molecular detection of the LAM family of M. tuberculosis. Acknowledgements: Dr. Nadya Markova, Prof. Olga Narvskaya, and Prof. Nalin Rastogi are gratefully acknowledged for kind support and guidance.

The largest global outbreak of extensively drug-resistant (XDR) tuberculosis (TB) was identified in Tugela Ferry, KwaZulu-Natal (KZN), South Africa in 2005. The antecedents and timing of the emergence of drug resistance in this fatal epidemic XDR outbreak are unknown, and it is unclear whether drug resistance in this region continues to be driven by clonal spread or by the development of de novo resistance. A whole genome sequencing and drug susceptibility testing (DST) was performed on 337 clinical isolates of Mycobacterium tuberculosis (M.tb) collected in KZN from 2008 to 2013, in addition to three historical isolates, one of which was isolated during the Tugela Ferry outbreak. Using a variety of whole genome comparative approaches, 11 drug-resistant clones of M.tb circulating from 2008 to 2013 were identified, including a 50-member clone of XDR M.tb that was highly related to the Tugela Ferry XDR outbreak strain. It was calculated that the evolutionary trajectory from first-line drug resistance to XDR in this clone spanned more than four decades and began at the start of the antibiotic era. It was also observed that frequent de novo evolution of MDR and XDR was present, with 56 and 9 independent evolutions, respectively. Thus, ongoing amplification of drug-resistance in KwaZulu-Natal is driven by both clonal spread and de novo acquisition of resistance. In drug-resistant TB, isoniazid resistance was overwhelmingly the initial resistance mutation to be acquired, which would not be detected by current rapid molecular diagnostics that assess only rifampicin resistance.

Background: The evolutionary changes in Mycobacterium tuberculosis (MTB) have been phenotypic as well as genotypic by diversifying into various lines of descent or lineages. ,Methodology and findings: A total of 538 MTB isolates obtained from different parts of India were included in the study. Spoligotyping and drug susceptibility testing was performed on all the MTB isolates. Major cities and parts of India from where these isolations were made include: Delhi, Assam, Punjab, Maharashtra (Nagpur, Mumbai), Tamilnadu (Chennai), Uttar Pradesh (Agra), and West Bengal (Kolkata). Spoligotyping analysis detected 93 distinct spoligo-patterns. A total of 440 (81.7%) isolates could be grouped into 77 SITs which matched the pre-existing database, whereas 16 SITs were newly created for 51 (9.7%) isolates and for 47 (8.7%) isolates no SIT number could be assigned and these were grouped as ‘orphans’. Overall, CAS family was predominant, comprised of 37.9% isolates, followed by EAI 23.6%, Beijing in 13.9%, Manu in 6.8% and T in 4.6% isolates. Other families were rare. Drug susceptibility testing (DST) identified 361 (67.1%) isolates as pan-sensitive to all 4 first-line drugs, 83 (15.4%) were resistant to any one or more drugs, and 94 (17.4%) were identified as multidrug-resistant (MDR). Out of the 94 MDR isolates, the majority of isolates (32; 34%) were Beijing, 31 (32%) CAS, 11 (11.7%) unknown, and 6 (6.3%) each of EAI and T lineages. Among the Beijing lineage, SIT621 was more associated with MDR-TB compared with SIT1. Conclusions: The study provides important baseline data to understand and monitor the molecular epidemiology of MTB in India. The CAS lineage was the most common and widely prevalent across India, while EAI lineage was more common in south and eastern India. The Beijing lineage was significantly (p-value <0.0001) associated with MDR-TB. Funding: This work was supported by the Department of Biotechnology, Ministry of Science and Technology and Department of Health Research (ICMR), Ministry of Health & Family Welfare, Government of India.

Multidrug- (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) present a challenge to disease control and elimination goals. Lisbon, Portugal, has a high TB incidencerate and unusual and successful XDR-TB strains that have been found in circulation foralmost two decades. For the last 20 years, a continued circulation of two phylogenetic clades, Lisboa3 and Q1, which are highly associated with MDR and XDR, have been observed. In recent years, these strains have been well characterized regarding the molecular basis of drug resistance and have been inclusively subjected to whole genome sequencing (WGS). Researchers have been studying the genomic diversity of strains circulating in Lisbon and its genomic determinants through cutting-edge next generation sequencing. An enormous amount of whole genome sequence data are now available for the most prevalent and clinically relevant strains circulating in Lisbon. It is the persistence, prevalence and rapid evolution towards drug resistance that has prompted researchers to investigate the properties of these strains at the genomic level and in the future at a global transcriptomic level. Seventy Mycobacterium tuberculosis (MTB) isolates, mostly recovered in Lisbon, were genotyped by 24-loci Mycobacterial Interspersed Repetitive Unit – Variable Number of Tandem Repeats (MIRU-VNTR) and the genomes sequenced using a next generation sequencing platform – Illumina HiSeq 2000. The genotyping data revealed three major clusters associated with MDR-TB (Lisboa3-A, Lisboa3-B and Q1), two of which are associated with XDR-TB (Lisboa3-B and Q1), whilst the genomic data contributed to elucidating the phylogenetic positioning of circulating MDR-TB strains, showing a high predominance of a single SNP cluster group 5. Furthermore, a genome-wide phylogeny analysis from these strains, together with 19 publicly available genomes of MTB clinical isolates, revealed two major clades responsible for MDR/XDR-TB in the region: Lisboa3 and Q1. The data presented by this study contributes to the expanding knowledge of MTB genomic diversity yielding insights on microevolution and identification of novel compensatory mutations. Additionally, the analysis of nonsynonymous/ synonymous ratios revealed heterogeneities across the chromosome, genotype and Clusters of Orthologous Groups, highlighting possible and different evolution strategies. Overall, the results that are presented support the notion of an increasing genomic diversity that may support both setting and host adaptation.

Aim: Pyrazinamide (PZA) is a first-line key agent in the effective treatment of tuberculosis (TB), including PZA susceptible multidrug-resistant tuberculosis (MDR-TB). Occasionally, TB patients might have mixed infections with both drug-sensitive and -resistant strains. This is termed heteroresistance. If 10% of the bacterial population is resistant to PZA, there is an increased risk for poor treatment outcome. The aim of this study is to evaluate the ability of the three established drug susceptibility testing (DST) techniques – BACTEC MGIT 960, Wayne's pyrazinamidase test and Sanger sequencing of the pncA gene – to detect 10% PZA heteroresistance. Methods: Mixed cultures of the fully drug susceptible Mycobacterium tuberculosis H37Rv reference strain and two laboratory-generated isogenic H37Rv mutants (with C475G and T254C pncA mutations, respectively) were made in proportions of 100%, 10%, 5% and 1% of the PZA-resistant (PZA-R) strain. Corresponding mixed cultures were also made using one drug-susceptible and one PZA-resistant MDR clinical isolate with the T62G pncA mutation, both belonging to one specific MIRU cluster. Additional mixes of 50%, 75%, 90% and 99% of the PZA-R strains were prepared for the Wayne's test. Tests were for all methods performed in duplicates at two separate occasions. Results: Using the MGIT system, the in vitro-generated PZA-R strains were generally detected at a 5% proportion while the clinical PZA-R isolate only was detected at the critical 10% proportion, except for one test occasion. Sanger sequencing was unable to detect 10% PZA heteroresistance. Wayne's test also failed to detect the critical level of 10% PZA resistance; instead it displayed misguiding results determining highly resistant samples as susceptible. Conclusion: Heteroresistance is caused by present drug-resistance development and/or dual infections with one resistant and one susceptible strain. Mixed infections with resistant strains may occur in up to 20% of all TB cases in high burden areas, according to limited data. This study showed that only the phenotypic BACTEC MGIT system was capable in determining the critical proportion of 10% PZA resistance, whereas neither the Sanger nor the Wayne's test were successful in this respect. This indicates a need for diagnostic tools with increased sensitivity to determine heteroresistance in M. tuberculosis.

Mycobacterium bovis (M. bovis) is the causative agent of bovine tuberculosis, a global disease that in Argentina affects approximately 5% of cattle, particularly dairy cattle. Several genotyping methods that distinguish between different isolates of M. bovis have been reported. Among them, the most convenient are spoligotyping (a reverse line probe assay) and genotyping by Variable Number of Tandem Repeat (VNTR). In Argentina, unlike what happens in other countries in the region, there is a highly predominant spoligotype (SB0140), but the reason for this dominance is still unknown. In five countries of Latin America, a total of 244 different spoligotypes were found: 8.4% of isolates had orphan spoligotypes, whereas 91.6% formed clusters. Only 1 spoligotype was common to Argentina Brazil and Chile. However, there were 10 spoligotypes shared between Argentina and Brazil; 2 between Argentina and Chile; and 1 between Chile and Brazil. In conclusion, despite the diversity of spoligotypes found in the five countries, there are major patterns predominating in Argentina, Brazil and Chile. These clusters may reflect a long-lasting active transmission of bovine tuberculosis. The other goal of this study was to investigate whether there are M. bovis genotypes with differential virulence properties. While this is a topic already elucidated for other pathogenic bacteria, the properties for M. bovis are still unknown. First, the virulence of M. bovis isolates were tested in a mice model of progressive disease. Balb/c mice infected with the M. bovis reference strain AN5 showed a 50% survival after four months of infection, with a moderate number of lung bacillary counts. Two weeks after inoculation, it induced a strong inflammatory response with numerous granulomas and progressive pneumonia. In turn, the strain M. bovis 04-303, isolated from a wild boar, was the most lethal, and its most striking feature was sudden pneumonia development with extensive necrosis. Strain 04-302 induced a similar pathology, although to a lesser extent. In contrast, strains 534, V2 (both from cattle) and 02-2B (from humans) were less virulent, permitting higher survival after four months of infection, Thus, as reported with Mycobacterium tuberculosis clinical isolates, M. bovis also showed virulence variability. This variability can be attributed to the induction of different immune response profiles. The virulence of the most virulent strain was also evaluated in guinea pigs and cattle showing the same results. On the other hand, the genome of this strain was sequenced showing that the genome encodes for 3988 protein and 49 RNA genes. Some particularities of the genome sequence and a deduced proteome will be shown. A methodology was designed to establish whether a relationship exists between the genotype of M. bovis and the degree of pathogenicity it causes in cattle. The essence of this methodology is to establish a correlation between the genotype of M. bovis, the degree of progression of tuberculosis and animal age. To this aim, three correlates were used: (1) The genotype was based on the spoligotype; (2) The degree of bovine tuberculosis lesions were quantified with a score based on clinical observations in slaughterhouses, such as the number and location of granulomas, and the histopathologic features; and (3) These data were stratified by the approximated age of the animal, determined by the category (calf, steer, heifer, etc). Thus, the virulence was studied in infected cattle, and not in laboratory animals, through a detailed analysis of the lesions found in the slaughterhouses. The lesions were processed for histopathology and culture. As a control for genetic uniformity, the alleles of BoLA DRB3 were sequenced. Lineages with the highest pathogenicity scores were of low abundance and were found in the south of Cordoba province.

Introduction: Bovine tuberculosis (BTB) caused by Mycobacterium bovis is considered as one of the most important diseases of cattle. Identifying and culling infected animals following positive tuberculin test detection is one of the fundamental control strategies of tuberculosis in cattle in the world. Passing five decades after implementation of such a control program from 1963, now, Iran experiences an admirable drastic decreased rate of 0.18% in comparison with a previous 5% at the beginning of the program. Materials and methods: A total of 31 lymph nodes of positive-tuberculin cattle referred to an Alborz Province abattoir and 70 farm samples obtained in the Tehran Province during the years 1390–1391 Hijri were sent to Razi Institute. Passing standard preparation procedures, the isolates were obtained after 8 weeks at 37 °C. Then their DNA was extracted using van Solingen's method. Employing PCR-RFLP schemes, 13 M. bovis isolates were confirmed. Qualitative and quantitative RFLP evaluation, exploiting PGRS and DR probes for hybridization were performed. Results: Digestion by PvuII enzyme followed by hybridization employing separate probes PGRS and DR resulted in three genetic detection types. Also, the combination of the two probes provided four different patterns. Discussion: The data obtained from this study compared with the national surveillance carried out in 1385, showed similarly a consistent pattern of M. bovis BCG as the predominant isolates found in most of the provinces, particularly in Tehran.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is an important zoonotic disease with global distribution. While M. bovis is inherently resistant to pyrazinamide, human cases of infection with M. bovis might experience serious cure failures if no correct identification of the pathogen is achieved. Bovine TB was initially reported in Iran by a French veterinarian in local breeds of cattle. An official attempt to control the disease was started in the 1940s, which runs today on a national scale. This mini-review addresses a variety of different epidemiological issues in bTb control in the world and in Iran from an immunologist's eye to find the cure for human cases infected with M. bovis. In addition, the benefits and drawbacks of this control scheme are discussed.

Nontuberculous mycobacteria (NTM) are environmental, opportunistic pathogens found in soil and water. NTM are adapted for residence in drinking water distribution systems as they are disinfectant-resistant, surface adherent, and able to grow on low concentrations of organic matter. Reports of NTM infections have been increasing over the past two decades. Of the >150 NTM species reported in the literature, some 25 species have been strongly associated with a variety of human diseases, of which the pulmonary NTM disease (PNTM) is the most frequent. The distribution of NTM species differs strongly by region and it is generally accepted that NTM species differ in their clinical relevance. Further, NTM differ strongly in their growth rate, temperature tolerance, and drug susceptibility, making the correct species identification a very important step in the process of diagnosis. Because NTM are environmental bacteria, the diagnosis of PNTM is complex and requires good communication between clinicians, radiologists, and microbiologists. Extensive microbial resistance, often misleading in vitro drug susceptibility patterns, and complicated treatment regimens are just some of the factors adding to the frustration of the clinical management of NTM diseases. To prevent unwarranted diagnoses and treatment of NTM disease as well as unnecessary diagnostic delay, it could be helpful to use separate, more stringent criteria for species of low relevance, and less stringent criteria for species considered to be of high clinical relevance in the local setting, namely: isolation of Mycobacterium kansasii (worldwide) and Mycobacterium malmoense (north-western Europe) from pulmonary specimens usually indicates disease, whereas Mycobacterium gordonae and Mycobacterium simiae typically represent contamination. This approach requires complete and up-to-date insight in locally prevalent NTM and their clinical relevance. In Croatia, all strains of NTM isolated in any laboratory are sent to the National Reference Laboratory for identification. NTM strains have been systematically recorded and reported since 1982. In the last decade, two retrospective analyses were done to assess the clinical relevance of different NTM species and the burden of pulmonary NTM disease. Over the past decade, a 30-fold increase in overall NTM isolation rates and a 4-fold increase in PNTM incidence were observed. Mycobacterium xenopi was the most frequently seen causative agent of pulmonary NTM disease, but the degree of clinical relevance (i.e., percentage of patients meeting the diagnosis criteria, per species) was higher for isolates of MAC (66.5%) and M. kansasii (57.2%). Only about 30% of the M. xenopi isolates represented true disease. Further, interesting regional differences were observed. Clinically relevant NTM isolates were significantly more often found in the coastal region of Croatia, and the average annual incidence of the PNTM was twice as high in coastal compared with the continental region. The overall burden of PNTM in Croatia is still low compared with tuberculosis. This can, in part, explain the observed lack of knowledge of NTM infections among respiratory specialists. Since these pathogens are increasingly common worldwide, especially in countries where the incidence of tuberculosis is declining, a constant rising of awareness and knowledge is necessary.

Objectives: Nontuberculous mycobacterial (NTM) infections are frequently reported as a cause of bone and soft-tissue infection of the upper extremity, primarily in the hand and wrist. Limited information exists on clinical characteristics and treatment outcomes of patients with NTM infection of the upper extremity. The clinical and radiological characteristic of these infections in a large group of patients with upper extremity NTM infections are described herein. Methods: A retrospective analysis was conducted of NTM infections of the upper extremity at Mayo Clinic Florida from December 2000 through December 2012. Only patients with positive mycobacterial cultures from the upper extremities were included. Data collection included demographics, radiologic and clinical characteristics, mycobacterial culture results, time to diagnosis and treatment outcomes. Results: Forty-two patients were included; 71% were male and the mean age was 60±18 years. Eighteen (43%) patients were diagnosed with skin/soft tissue infections and 15 (36%) had tenosynovitis. Twenty-six (62%) patients were immunosuppressed. The most common underlying medical conditions were rheumatologic disorders (40%) and diabetes mellitus (17%). Sixteen (38%) patients were taking glucocorticosteroids, and 11 (26%) patients were receiving tumor necrosis factor alpha (TNF-α) inhibitors at the time of diagnosis. Injuries to the affected extremity were reported in 62% of patients. Fishing (21%) and gardening (14%) were the most common reported exposures. Signs and symptoms of infection were localized to the skin in 62% and extended to the joints in 52%. Mycobacterium marinum (36%) and Mycobacterium chelonae/abscessus (33%) were the two most commonly identified organisms. Radiologic studies were available in 30 (71%) patients. Average time to clinical evaluation from onset of symptoms was 2.6 (±3.5) months and time to diagnosis from initial clinical evaluation was 4.5 (±4.5) months. Forty-one (98%) patients were treated. Twenty-eight (68%) patients were treated with both antimicrobial and surgical debridement, nine (21%) with antimicrobial treatment alone, and four (10%) with surgical debridement alone. Twenty-six (62%) patients were cured, four (9%) relapsed after the first round of treatment, two (5%) failed all treatment modalities, and three (7%) patients died. Conclusion: Diagnosis of NTM infection of the upper extremity is often delayed due to its indolent presentation and lack of clinical suspicion. Healthcare professionals should be aware of the increasing incidence of soft tissue NTM infection after percutaneous injury, especially in immunosuppressed patients, to improve diagnostic promptness and treatment outcome.

Background: The evidence of increase in the prevalence of non-tuberculosis mycobacteria (NTM) is being reported around the world. In Shanghai, China, it rose from 4% to 6% in the years between 2005 and 2008. Cambodia is one of the 22 high–tuberculosis (TB) burden countries. The NTM isolation rate among pulmonary smear-positive previously treated TB and new smear-positive non-converter at months 2 or 3 was 25% in 2011. Objective: To determine the trend of the NTM isolation rate from presumptive multidrug-resistant tuberculosis (MDR-TB) cases during the period 2011–2013 and their characteristics. Methods: A retrospective cross-sectional study which included all presumptive MDR-TB patients whose samples reached two main mycobacterial culture laboratories of the National TB Program during the period 2011–2013. Each of the two samples were examined by smear microscopy with Ziehl Neelsen, cultured with Lowenstein Jensen and BACTEC MGIT 960, and identified for mycobacteria with ICA test. Possible cases were defined as a single positive NTM isolate, and definite cases were defined as two positive NTM isolates. The NTM isolation rate and the relationship of NTM and smear result were analyzed. Results: A total of 6115 sputum samples of 3,338 patients were cultured, of which 32.3% (n = 1079) of the patients have at least one positive culture with a median age of 51 years (IQR: 40–62) and 59.5% were males. Out of these, 36.9% (n = 398) were NTM isolates with median age of 56.5 years (IQR: 46–65) and 51.0% were males. Of these, 39.7% (n = 158) were defined as NTM cases. The isolation rate of NTM among culture-positive of presumptive MDR-TB patients were 26.1%, 31.5%, and 46.9% in the years 2011, 2012, and 2013, respectively. This isolation rate was strongly correlated with a grade of smear result, but not TB treatment history. The proportion of NTM by grade of smear results were 62%, 53%, 27%, 15%, and 6% among smear-negative, scanty, 1+, 2+, and 3+, respectively, and the proportion of NTM by type of TB patients was 66.7%, 53.0%, 38.5%, 34.4%, 30.9%, 29.4%, and 2.7% among pulmonary TB smear-negative previously treated cases, non-converter of new smear-positive cases, symptomatic close contacts of known MDR-TB patient, failure, HIV/TB new smear-positive, relapse, and return after default, respectively. Conclusions: The isolation rate of NTM in Cambodia among presumptive MDR-TB patients was found to be remarkably high and increasing over the last 3 years and strongly correlated with the grade of smear result. Further studies and appropriate managements should be done for those patients.

The isolation rate of nontuberculous mycobacteria (NTM) species and the prevalence of NTM-associated diseases are on the rise worldwide; however, the species distribution of NTM isolates and the types of diseases caused by NTM species vary from region to region. Treatment of a NTM disease is complicated, and there is no comprehensive guideline regarding the in vitro susceptibility of each antimicrobial agent against NTM. Therefore, appropriate anti-NTM treatment can only be recommended based on individual NTM species and local surveillance studies of anti-NTM resistance. Previous studies on the in vitro susceptibility of Mycobacterium avium complex (MAC) to clarithromycin in some Asian countries have revealed a low rate of resistance to that antimicrobial agent. Thus, a clarithromycin-based anti-MAC regimen should be effective for MAC infections. However, clarithromycin resistance due to the mutation of the 23S rRNA gene in MAC strains has been detected in many countries. Therefore, physicians should avoid monotherapy with clarithromycin and consider the possibility of clarithromycin resistance in patients who do not respond to clarithromycin-based regimens. Rifampicin is the critical component of successful management of Mycobacterium kansasii diseases. Although most M. kansasii isolates are susceptible to rifampicin in Western countries and in Japan, this agent may not work well in Taiwan. Rapidly growing mycobacteria (RGM) is a prevalent NTM group worldwide, particularly in Asia; however, each NTM species in this group may have its own distinct antibiotic susceptibility pattern, and close monitoring of the antibiotic-resistance patterns of RGM is necessary. Most important of all, the in vitro susceptibility may not represent the in vivo activity until the confirmation of the clinical study. Therefore, further investigation of the clinical effectiveness of the anti-NTM agents is warranted.

Background: Mycobacterium abscessus is an opportunistic organism common in the environment. This species rarely cause infections in immunocompetent individuals. Certain patient groups, however, can get severe infections, e.g., in skin, soft tissues or lungs, as well as disseminated infections. During the last years, this organism has emerged as a major pathogen in patients with cystic fibroses. M. abscessus exhibits two different colony types: one with smooth and shining morphology, the other with rough and dry morphology. The smooth strains are considered wild-types, which become rough due to mutation. These features make the M. abscessus variants particularly interesting for scientific studies, since the results of such analyses can be a basis for creating theories also concerning other mycobacteria. Aim; The aim of this study is to analyze potential differences between smooth and rough strains of M. abscessus concerning human host response. Methods: M. abscessus strains isolated at the Mycobacterial Laboratory in Gothenburg, Sweden were analyzed morphologically. The ability of monocytes to internalize the two colony variants of M. abscessus was studied, as well as the cytokine response. Results: The analyses showed that isolates from the wound mainly exhibit smooth colony formation, while those from airways generally are of the rough phenotype. Human monocytes easily internalized the smooth varieties of M. abscessus, whereas the rough ones rarely were internalized. Differences between the two types, concerning the capacity to induce cytokines, were also revealed. Discussion and conclusions: The wound isolates of M. abscessus were mainly smooth, while the lung isolates were rough. The rough variants that lack certain surface glycolipids are likely to be more hydrophobic and aerosol transmissible than the smooth strains, which might explain why the rough ones are more often associated with infections in the airways than are the smooth ones. Studies on other mycobacteria have also shown a link between hydrophobicity and aerosol transmission. These studies showed that human monocytes easily internalize smooth M. abscessus cells, but rarely the rough ones. This capacity of the rough cells to avoid internalization is in all probability also due to absence or reduction of certain surface glycolipids. The lack of these surface lipids makes the rough bacteria stick to each other forming so-called cords, which are aggregates of many cells and thus difficult to internalize. The absence of glycolipids exposing other surface structures of the bacteria cell may also contribute to prevent internalization. Even if intracellular survival is regarded as the most important virulence factor of mycobacteria, it is nevertheless likely that the capacity to avoid internalization can contribute to pathogenesis at several mycobacterioses. It is possible that this phenomenon is important at tuberculosis, since Mycobacterium tuberculosis cells form rough colonies.

Pyrazinamide (PZA) is a prodrug that is converted to the active compound pyrazinoic acid by the pyrazinamidase enzyme (encoded by the pncA gene in Mycobacterium tuberculosis) at low pH. The phenotypic approach for testing pyrazinamide sensitivity is technically demanding (often associated with poorly reliable results) and expensive. For these reasons, sensitivity of PZA is rarely tested and, if tested, when the sensitivity pattern becomes available to clinicians, the drug regimen of the patients is not modified based on the results. The molecular approach to drug resistance identification has considerably changed the capacity to identify and appropriately treat TB cases resistant to Rifampicin, Isoniazid and to some extent Fluoroquinolones and injectables. The identification of mutations on the pncA gene has the potential for rapid detection of PZA-R. However, the genetic variants are highly variable (not all associated to drug resistance) and scattered over the full length of the pncA entangling the development of a molecular test. The TBPannet consortium performed a large study assessing the pncA sequence variations in 1950 clinical isolates, including 1142 MDR and 483 fully susceptible strains. The sequencing analysis identified 280 different mutants. The presence of different mutations was correlated with phenotype, enzymatic activity, structural data, and phylogenetic data. Using an algorithm taking into account all the parameters evaluated, four classes of genetic variants were identified: (1) very high confidence resistance mutations (85% of the genetic variants examined) – always associated with phenotypic resistance, absence of enzymatic activity and protein structure predictive for a lost function; (2) high confidence resistance mutation – highly associated with phenotypic resistance (but not only, probably due to imperfect phenotypic test), absence of enzymatic activity and protein structure predictive for a lost function; (3) mutations with an unclear role found; and (4) mutations not involved in phenotypic resistance (10%). Any future molecular diagnostic assay should be able to target and identify at least the very high and high confidence genetic variants markers of PZA-R. An assay of this kind will have a diagnostic accuracy in the range of 90–99%. This work was supported by the European Community's Seventh Framework Programme (FP7/2007–2013) under grant agreement FP7-223681 to DMC (www.tbpannet.org).

While pulmonary tuberculosis (PTB) remains an important public health issue worldwide, there is an emerging interest in non-tuberculous mycobacteria (NTM) which is responsible for opportunistic infections of the respiratory tract as well as other anatomical sites in both developed and developing countries. In this context the one goal of the clinical mycobacteriology laboratories is to provide physicians with an accurate identification of the mycobacterium as rapidly as possible. During the last ten years, several lines of laboratory tools have been developed in order to speed the isolation and identification of mycobacteria from clinical specimens. Chiefly, the composition of culture medium was renewed along with the protocol of incubation in order to recover Mycobacterium tuberculosis (MTB) micro-colonies as soon as 48 h after the inoculation of the specimen. MALDI-TOF rapid identification is clearly the tool to be implemented in the laboratory for the rapid identification of the micro-colonies. Also, molecular tools and genomics are necessary in order to depict new mycobacteria species, including those of the Mycobacterium abscessus complex and the Mycobacterium avium complex. All these tools and their connections will be presented during this conference.

Aims and objectives: In Lebanon, the trend of tuberculosis (TB) incidence had been declining until the year 2011. In 2012, the National TB Program (NTP) observed that 48% of all notified cases were among the Syrian refugee population. As of August 2013, according to the NTP, 100 Syrian refugees have been diagnosed with TB in Lebanon, including 3 cases of MDR (multidrug-resistant) TB. Resistance-associated point mutations have already been described for commonly used anti-TB drugs. The widespread use of Isoniazid (INH), a cornerstone drug for treating TB, has seen treatment failures due to increasing resistance to the drug. Clinical resistance to INH is widely known to be caused by mutations within katG, inhA and ahpC genes. The objective of this study is to determine by pyrosequencing the prevalence of mutations on the codon 315 of the katG gene, on the inhA promoter and on the ahpC-oxyR intergenic region in 14 and 52 INH-resistant MTB isolates recovered from TB patients in Lebanon and Syria, respectively. Methods: The clinical isolates were provided by the Medical Biotechnology Section of the National Commission for Biotechnology in Syria and the Health and Environment Microbiology Laboratory at the Azm Center for Research in Biotechnology at the Lebanese University. The isolates were derived from 52 Syrian and 14 Lebanese patients between July 2003 and October 2005 from all Syrian and Lebanese provinces. The identification of point mutations on katG, inhA and ahpC-oxyR intergenic region was performed by pyrosequencing, and sequences from clinical isolates were compared with that of wild-type MTB ATCC 25177. Results: The results showed that among the 52 Syrian isolates, 22 (42.3%) had the aa 315 mutation in the katG gene, while 6 of the 14 Lebanese strains (42.8%) had this mutation. The most common mutation was Ser315Thr (92.8%). Mutations in the inhA promoter region were responsible for INH resistance in 14 Syrian strains (26.9%) of the isolates. The most common inhA promoter mutation was −15 C-T and was present in 13 of the14 inhA mutations. Screening for mutations on the ahpC-oxyR intergenic region revealed the presence of 6 mutated Syrian strains (11.5%) with 46 G-A the most common mutation (4 of 6 strains). It is interesting to note that 4 strains had mutations in katG in addition to ahpC-oxyR mutations and 1 strain had both katG and inhA mutations. None of the Lebanese strains had mutations on inhA or ahpC-oxyR implying that these mutations do not contribute to the detection of INH-resistant MTB in the Lebanese strains. Conclusions: This study showed that the pyrosequencing applied to katG, inhA promoter and ahpC-oxyR intergenic region was able to detect a relatively large proportion of Syrian INH-resistant MTB isolates (80.7%) in Syria. This strategy may be inappropriate for Lebanese strains, as the genetic mechanisms of resistance remain unidentified for approximately half of the isolates, so it is quite possible to detect the presence of other mechanisms of resistance.

Introduction: Fluoroquinolones (FQ) are an essential component of current and new regimens for the treatment of tuberculosis (TB). The 2014 Global TB report indicates a FQ resistance rate of 17% amongst multidrug-resistant (MDR) strains of Mycobacterium tuberculosis (MTB) tested in 2013. There is, however, a paucity of FQ-resistance data from high burden countries. In this study the trend of FQ-resistance amongst MDR–MTB and non-MDR–MTB is analyzed over a four-and-a-half-year period (January 2010–July 2014). Methods: This study was conducted at the Aga Khan University laboratory, a technical partner of the Pakistan National TB Program and part of the World Health Organization (WHO) Supra-national Laboratory Network for TB. The laboratory receives specimens from across the country through its peripheral collection units. MTB was isolated using standard methods. Susceptibility testing was performed using the agar proportion method with drug concentrations as recommended by Clinical Laboratory Institute Standards (CLSI). FQ susceptibilities were determined using ofloxacin (2 μg/ml). MTB H37Rv was used as a control with each batch of susceptibility testing. MDR was defined as resistance to both isoniazid (0.2 μg/ml) and rifampicin (1.0 μg/ml). Results: During the study period 14,711 MTB strains were isolated. Of these, 6403 (43.5%) were MDR and 8308 were non-MDR. FQ resistance in MDR strains ranged between 54% and 58%. Amongst non-MDR MTB strains, FQ resistance increased from 214/2059 (10.3%) in 2010 to 180/1049 (17.1%) in 2014. The proportion of FQ mono-resistant TB strains averaged at 10.5% of the non-MDR isolates during this period. Conclusions: FQ resistance in non-MDR–MTB strains with a considerable proportion of FQ mono-resistant strains in Pakistan is alarming. These data highlight the limited potential of empirical FQ usage for TB treatment in both MDR and non-MDR cases and the need to implement regular surveillance for FQ-resistance in MTB in the country. High FQ resistance amongst MTB isolates further emphasizes the importance of stewardship and the responsible use of FQs in particular, and antimicrobials in general in the country.

Aims and objectives: Tuberculosis (TB) is one of the major public health problems in Armenia. In 2009, TB incidence in Armenia was reported to be as high as 45.5 per 100,000 population, and TB mortality was 3.9 per 100,000 population. Only 35% of estimated new sputum smear-positive pulmonary TB patients are notified annually. From 1729 TB cases notified in 2010, only 329 patients were sputum smear-positive. The treatment success rate of new sputum smear-positive pulmonary TB patients was 76%, which is below the WHO target of 85%. Poor treatment outcome is partially explained by the high prevalence of drug resistant forms of TB. According to the 2007 drug resistance survey, MDR-TB among never-treated patients was 9.4% and 43.2% among previously treated cases with 4% of XDR cases. This represents an enormous public health challenge for Armenia; early identification and effective treatment of patients with MDR-TB is crucial to prevent further spread of the disease. Methods: A total of 583 specimens are sent to the NRL Armenia being either acid-fast bacterium positive or negative, but culture positive. Rifampin and isoniazid resistance was performed on 77 out of the 583 specimens of DR TB clinical isolates to analyze resistance for FQL, AM/CP and EMB. The DNA preparation method used was as described and recommended by the manufacturer (MTBDR plus version 1.0 and MTBDR sl version 2.0 Hain Lifescience, Nehren). For 99 (88.4%) and 160 (76.2%) strains, the mutations causing rifampicin and isoniazid resistance were located in the codon of rpoB S531L and katG S315T 1, respectively. Regarding isoniazid, 14 (6.6%) strains have a mutation in the inhA regulatory region (C15T) and 31 (14.7%) in both katG and InhA. Mutations detected in the FQ, EMB and rrs-resistant respectively are: 13 (16.9%) strains with the majority of mutations (10 [13%]) in the codon of 94 of the gyrA gen; 20 (26%) strains, with the majority of mutations (7 [9.1%]) in the codon of 306 of the EMB gen; and 13 (16.8%) strains with the majority of mutations (9 [11.6%]) in the codon of 1401 of the rrs gen. Comparing with phenotypically DST sensitivity and specificity for RMP was 97.6% (69 strains) and 100%, respectively. False resistance was detected for 2 strains containing missed bands of rpoB (W6, 7, 8) and 1 strain W7. Sensitivity and specificity for INH was 98.33%, 87.5% (69 strains). From 2 strains, 1 was false resistant (missing of WT 1, 2 bands) and 1 false sensitive results for INH. The sensitivity and specificity for AM/CM was 80%, 100%, and for the FQL 85.7%, 96.72%, respectively (69 strains). Conclusion: Rapid and accurate detection of resistance to first- and second-line anti-TB drugs is the key to successful therapy and interruption of the transmission chain of MDRTB strains. In summary, this data represent an important addition to the rare epidemiological data concerning resistance patterns of MTB in Armenia and showed that the application of the GenoType MTBDRplus and GenoType MTBDRsl assays might be useful additional tools to allow for a rapid and safe diagnosis of MDR and XDR TB. In mutations associated with katG, high dosages of isoniazid in future will be considered for treatment.

Background: By using whole genome sequencing (WGS), researchers are beginning to understand the genetic diversity of Mycobacterium tuberculosis (MTB) and its consequences for the diagnosis of multidrug-resistant tuberculosis (MDR–TB) on a genomic scale. The Global Consortium for Drug-resistant TB Diagnostics (GCDD) conducted a genome scale variant analyses of 366 clinical MTB genomes (mostly MDR/XDR [extensively drug resistant]) from four countries in order to inform the development of rapid molecular diagnostics. This project has been extended by performing an evolutionary analysis of isoniazid (INH)-resistant isolates for prognostic purposes. Methods: 151 (130 INH^R , 21 INH^S) clinical MTB isolates from India (19: 17 INH^R, 2 INH^S), Moldova (48: 42 INH^R, 6 INH^S), the Philippines (26: 20 INH^R, 6 INH^S), and South Africa (58: 51 INH^R, 7 INH^S) were included in this study. INH drug susceptibility was determined by using MGIT 960 and WHO (World Health Organization)-recommended critical concentration of 0.1 mg/L. Isolates were sequenced using PacBio RS WGS platform. A genome-wide variant analysis was conducted using a proprietary pipeline (PacDAP) developed at San Diego State University. To infer the amino acid changes in katG that confer resistance, PAML was utilized to detect sites in silico that are under positive selection. The dN/dS method was used in combination with Bayes empirical Bayes to determine sites under positive selection and Chi-Squared analysis to determine the significance of the selected sites. Results: PacDAP variant analysis revealed 22 novel catalase-peroxidase (katG product) mutations. Of these, 14 were single nucleotide polymorphisms, while 8 novel mutations appeared in combination with katG S315T and/or with inhA promoter C-15T. These SNPs have not been previously reported. Additionally, 11 previously observed, but uncommon, katG mutations were also observed in these clinical isolates. These results suggest that 17 amino acids in the enzyme are under positive selective pressure; most significantly in South Africa and the Philippines. No selective pressure on codons other than 315 was observed in isolates from Moldova. Due to the low number of isolates from India, the significance of the sites under positive selection was low and no prediction for India could be made based on this study. Conclusions: Eleven of the 14 SNPs are resistance conferring, and it is believed that the remaining 8 combinatorial mutations are either compensatory in nature or, in combination with known SNPs, could increase resistance levels. Positive selection results indicate a diversifying evolutionary path to resistance more in line with long tail statistics and therefore indicate a departure from the traditional point mutation (or “hotspot”) model that current molecular diagnostics are based on. Positive selection pressures indicate a future with elevated diagnostic and prognostic significance of the “long tail” (i.e., alternative mechanisms of resistance) and potentially diminishing significance of the canonical mutations (especially in South Africa and the Philippines), which could have significant future implications on narrowly targeting molecular diagnostics.

Introduction: Drug resistance in Mycobacterium tuberculosis (MTB) is caused by many mechanisms. Integrons are genetic units characterized by their ability to capture and incorporate bacterial genomes by recombination and may contain resistance-related genes. Integrons have an integrase gene (int). The aim of this work is to report a new integrase gene that was not reported in the GeneBank earlier. Materials and Methods: Susceptible, drug-resistant clinical isolates and H37Rv strain underwent DNA extraction. Integron of Mycobacterium abscessus structure was used as a template. The needed primers were designed for a walking method in polymerase chain reaction (PCR). Resulted fragments were sequenced for confirmation of the fragments. Results: Results of the sequencing method revealed that the newfound integrase is not in the GeneBank and was not reported earlier. Its sequence differed from former reported integrases like PhiRv1 integrase (Rv2659c), RVBD_2646 integrase, Rv2309c, CCDC5180_0965 integrase, Rv2894c, etc. Conclusion: This study reports a novel integrase. These studies need to be continued for probable relationship between the whole fragment and resistance genes in the bacterium.

Background: Tuberculosis (TB) remains a major cause of mortality and morbidity all over the world. Multi drug-resistant tuberculosis (MDR-TB) is an important cause for mortality among TB patients, and management of MDR-TB remains a major challenge for national TB programs. Early detection of MDR-TB provides better treatment outcomes and reduces the transmission of MDR. Nucleic acid amplification test (NAAT) like Line Probe Assays have been recently endorsed for use in low-income settings and can be used to screen smear-positive sputum specimens for diagnosing of resistance to rifampicin and isoniazid in 1–2 days. Objective: Overall objective of the study was to evaluate performances of Line Probe Assay GenoType® MTBDRplus (Hain Life science, GmbH, Nehren, Germany) to move forward its introduction into routine diagnostics. Secondary objective was to determine the type of mutations in rpoB, katG and inhA genes from culture specimens isolated from Iraqi selected MDR-TB patients. Materials and Methods: The DNA was extracted by CTAB method from 51 clinical isolates which were previously characterized as MDR strains in reference laboratory/center of Chest and Respiratory Diseases obtained from patients living in Baghdad/Iraq (2010–2011). GenoType® MTBDRplus ver. 2.0 (HainLifescience, GmbH, Nehren, Germany) was performed at the Supra-national TB Reference Laboratory in Milan (Italy) as well as interpretation of mutations involved in drug resistance to rifampin and Isoniazide according to manufacturer's instructions. Results and conclusion: Line Probe Assays were performed on 51 MDR of Mycobacterium tuberculosis strains; 6 strains were excluded from the analysis due to un-interpretable results; 5 strains were identified as sensitive to both rifampicin (RIF) and isoniazid (INH) by MTBDR plus assay. The most common genetic mutation conferring RIF resistance was S531L of rpoB gene which was detected in 33 (82.5%) resistant strains. Other mutations in this gene were D516Vand H526Y which were detected in a single strain (2.5%) for each. Also, there were 5 (12.5%) RIF-resistant strains with mutations that could not be identified specifically in this assay. Isoniazid (INH) resistance due to katG mutation S315T1 was found in 17 (42.5%) strains of INH-resistant M. tuberculosis. The second most common site of mutation was in the upstream promoter sequence of inhA, which was found in 15 (37.5%) strains, 14 (35%) strains of which carried a C → T transition at the −15 position, while single strains (2.5%) carried a T → A mutation at the −8 position. The result showed 8 (20%) strains missing a band on the wild-type region of these strains, but no hybridization with mutation probes on this gene suggested resistance due to mutations other than those included in this assay. Regardless the type of mutations detected, GenoType® MTBDR plus ver. 2.0 showed an overall accuracy of 95.5% (C.I. 95% 89.9–98.1) in detecting MDR strains. Despite the relatively low number of MDR strains tested, they show very common mutations patterns being the majority mutated at the codon 531 and 315 of rpoB and katG gene. In such setting, GenoType® MTBDR plus ver. 2.0 could be a great tool to rapidly screen for MDR-TB and has the potential to substantially reduce the turnaround time of drug sensitivity test (DST) results.

Background: The tuberculosis (TB) situation in Russia is aggravated by the emergence and the spread of multidrug-resistant strains, HIV co-infection and drawbacks in the health control system. The Mycobacterium tuberculosis population structure in Russia has been defined by the remarkable mass migration in the 20th century, the variable genetic background of different ethnic groups inhabiting Russia, and by the pathobiology of circulating strains. Here, I will review the phylogeography and pathobiology of: (i) the dominating Beijing family; (ii) the Latin-American Mediterranean (LAM) family, the second largest in Russia and MDR-associated in some regions; and (iii) the Ural family, endemic in Russia and thought to be less transmissible and drug resistant. Review and analysis: M. tuberculosis variant Beijing B0/W148 is regarded as a successful clone of M. tuberculosis widespread in the former Soviet Union. However, a closer look reveals a peculiar clinal gradient of its geographic distribution; it peaks in Siberia and, to a lesser extent, in the European part of the former USSR. In contrast, its rate is sharply decreased in the Asian part of the former Soviet Union, and it is absent in the autochthonous populations elsewhere in the world. Two interdependent hypotheses will be put forward. First, B0/W148 likely originated in Siberia and its primary dispersal was driven by a massive population outflow from Siberia to European Russia in the period 1960–1980. Second, a historically recent phylogenetically demonstrated successful dissemination of the Beijing B0/W148 strain was triggered by an advent and wide use of the modern anti-TB drugs and was due to its remarkable capacity to acquire drug resistance. Robust phylogenetic markers were used to study the evolution of LAM and its major sublineages in Russia and its neighboring countries. A total of 250 M. tuberculosis isolates were assigned to LAM based on analysis of LAM-specific SNP in Rv3062 and Rv0129c. The family status was rectified for 121 isolates mis-assigned by spoligotyping to non-LAM families (T1 or T5-RUS1). The re-estimated LAM rate increased twofold in Russia and Kazakhstan and fourfold in Belarus. The majority (>90%) of LAM isolates from all three countries belonged to the LAM-RUS sublineage. In contrast, Ibero-American LAM RD-Rio sublineage was identified in only 7 Russian isolates. These findings and further analysis suggest a monophyletic origin of the LAM-RUS subfamily that is endemic in Russia. In contrast, rare LAM RD-Rio isolates were likely brought to Russia through occasional human contact. The analysis of the Ural family showed its highest prevalence in the North/East Pontic (Black Sea) area that may have been an area of its origin and primary dispersal. Ural family strains are not marked by increased pathogenic capacities, association with drug resistance (although there is a trend towards MDR Ural strains in the European part of the former USSR) or increased transmissibility. This reflects their basically low contagiosity which is why the Ural family is still moderately widespread in central Eurasia. Large-scale SNP or WGS population-based studies targeting strains from indigenous populations and, eventually, analysis of ancient DNA will better test these hypotheses. Host genetics factors likely play the most prominent role in the differential dissemination of particular M. tuberculosis genotypes.

Aims and objectives: This study evaluates the response of TB patients to treatment with autologous dendritic cell (DC) vaccine. Methods: There were 25 patients with MDR\XDR pulmonary TB included in the study. DCs were obtained both from peripheral blood monocytes and bone marrow isolated stem cells using standard protocols. DCs were primed with either autologous M. tuberculosis lysates or CFP-10 peptides and cultured with maturation inducers. DCs were tested for immuno-phenotype, viability and sterility and injected into the patients subcutaneously three times at 2- to 3-week intervals. Clinical monitoring, sputum assessments, chest X-rays and immune status were performed before and 2–3 months after the treatment. The control group (C) consisted of 25 patients with MDR\XDR TB matched by sex and age. Results: DCs obtained from all patients were sterile, viable, morphologically intact and phenotypically mature (expression of CD83 and CD86 being >80%). The number of injected DCs averaged 10.2 (range: 8.3–15.6) × 10⁶. Treatment of TB patients with autologous DCs was safe and well tolerated. No significant side effects which required medical aid were noted during the study. The combination of standard treatment and DC-vaccination promoted the decrease or complete clearance of mycobacteria tuberculosis from the sputum (DC-treated patients – 69.23±12.8% versus 30.76±12.8% in C, p<0.05) and X-ray improvement (69.23±12.8% versus 30.76±12.8% in C, p<0.05). Treatment with DCs was also associated with the significant increase of antigen-specific T-cells in the blood, which reflects the accumulation of antigen-specific T-cells in peripheral blood and indicates the restoration of immune response against mycobacteria tuberculosis. Conclusions: DC-based treatment may become an effective valuable method of cellular immunotherapy for MDR- and XDR-TB patients.

Epidemiological studies have demonstrated that there is an association between increases in air pollution and cardiopulmonary mortality and morbidity. Multi-center studies in North America and Western European countries reported that an increase in the levels of particulate matter (PM), ozone, nitrogen oxides (NO_x) and sulphur dioxide (SO₂) leads to increases in prevalence, emergency room visits and hospitalization due to chronic respiratory diseases such as asthma and chronic obstructive disease (COPD). Furthermore, it has been shown that increased levels of air pollutants are associated with increased prevalence of respiratory infections, including pneumonia. Although studies of indoor air pollution clearly showed a significant association with tuberculosis (TB), studies investigating the role of outdoor air pollution are lacking. However, it has recently been reported that increased ambient levels of air pollutants such as PM≤2.5 μm and SO₂ were associated with increased risk of TB. In vivo human studies demonstrated that exposure to ozone, NO2 and diesel exhaust (DE) leads to irritation in the mouth and nose, dyspnoea, wheezing, chest tightness, decreases in lung function tests, and increased airway hyper-responsiveness. There were also increased levels of neutrophils, lymphocytes and inflammatory mediators in bronchial lavage and bronchial biopsies of subjects exposed to ozone and DE. Furthermore, SO₂ and NO₂ increased airway response of allergic asthmatic subjects to allergens, such as house dust mite allergen. In vitro studies reported that DE particles (DEP) induce inflammatory mediator expression and synthesis of IgE specific to allergens in human B cells. Studies of human macrophages demonstrated that fine carbon black (CB) induced inflammatory cytokines and impaired phagocytosis of pneumococci and mycobacteria TB with a reduction in the oxidative burst capacity of these cells. This study has demonstrated that DEP, which constitute an important fraction of PM pollution, can decrease ciliary beat frequency (CBF), and induce the release of inflammatory mediators such as interleukin (IL)-8, granulocyte macrophage-colony stimulating factor (GM-CSF), normal T-cell expressed and secreted (RANTES) and soluble intercellular adhesion molecule (sICAM)-1 from primary bronchial epithelial cells (BECs). More recently, this study found that DEP can decrease airway epithelial cell viability and modify the cell cycle progression and apoptosis by inducing oxidative stress-related pathways, such as activator protein (AP)-1 and nuclear factor (NF)-κB, and an increased expression of apoptosis and cell cycle regulating proteins such as p21, p27, p53, cyclin E, c-myc, and cyclin-dependant kinase 2 (CDK2). Interestingly, serum, as can be seen in the inflamed airways of patients with chronic airway diseases such as asthma and COPD because of extravasation, enhanced the detrimental effects of DEP. Furthermore, these studies showed that gaseous air pollutants such as ozone and NO2 induce permeability of BEC cultures with an increase in the release of inflammatory mediators. More importantly, this study demonstrated that BECs from patients with chronic airway diseases such as asthma and COPD are more susceptible to deleterious effects of air pollutants. In conclusion, air pollutants can induce respiratory mortality and morbidity by leading to airway and lung inflammation and impairing the airway defence system against noxious agents and microorganisms such as mycobacteria TB.

Mycobacterium tuberculosis is a traditional example of bacteria that uniquely adapted to evade the host immune system and establish persistent infection. One of the major stresses encountered by M. tuberculosis is the host oxidative immune response. Studies on other bacteria have suggested that treatment with bactericidal antibiotics may lead to increased oxidative stress. Moreover, certain studies have shown that oxidative stress may sensitize bacteria to antibiotics. In addition, increased antioxidant capabilities of bacteria may therefore protect them from both antibiotics as well as the host immune response. In multi-drug resistant (MDR)-M. tuberculosis isolates, the antioxidant capability is weakened by decreased or even abolished catalase activity. In this presentation, the main focus is directed to the evidence that host oxidative immune responses could be exploited for better treatment results of MDR tuberculosis (TB) cases. Some evidences are generated from these experiences and others are based on others’ experimental studies conducted on M. tuberculosis and other bacterial species.

Background: Tuberculosis (TB) remains a major health challenge in the sub-Saharan African countries; Nigeria being one of the most affected countries. A number of interventions have been employed to reduce the scourge of the disease; however, the burden of the disease remains of public health dimensions. This study seeks to provide insight into the factors that may be affecting access to TB services by exploring the perception of the TB disease among the general population and how this affects health-seeking behavior. The specific perceptions addressed in this study are the causes of TB and whether there is a cure for TB. Methods: Qualitative methodology using in-depth interviews and focus group discussions (FGDs) were employed. This was done as part of a knowledge, attitude and practice survey. The survey was conducted in six States, namely: Akwa Ibom, Ebonyi, Gombe, Katsina, Benue and Ondo States. Results: Community key informants and FGD participants identified financial capability, knowledge about orthodox medicine, fear of stigmatization and the influence of religious leaders as factors that determine the choice of treatment. Across the six States, the general thought is that people first consult the chemist, then traditional healers/faith-based healers before visiting the hospital because it is cheaper. It was found that persons who believe that the disease is caused by germs usually seek health care in the formal health settings, while those who believe that TB is caused by supernatural forces, such as ancestral curses and witchcraft, usually seek help at the herbalists/traditional/unorthodox health settings. Also, people who believe that TB can be cured are more likely to seek medical care. Conclusion: Specific information on TB, such as the fact that TB is curable and caused by a germ, if well disseminated at the population level to the point where the information is understood and accepted to be true, is able to change the health-seeking behavior of the population such that the population seek care for TB at the formal health clinics.

Tuberculosis (TB), the infectious disease caused by the bacterium Mycobacterium tuberculosis (MTB), remains a significant global public health threat. TB is the worldwide leading cause of death from a bacterial disease and the second leading cause of death from an infectious disease after Human Immunodeficiency Virus (HIV) infections. Drug-Resistant TB (DRTB) is a growing concern around the world, but the magnitude of the disease burden is not well defined. A series of workshops (Program Chair, G.H. Cassell) was held by the Institute of Medicine (IOM) of the National Academy of Sciences in the United States of America in 2008 and, subsequently, in 2010–2013 in four of the highest burden countries (South Africa, Russia, India and China) to assess the reality of the challenge of DRTB and the gaps in knowledge required to address the threat. The issues discussed ranged from biology, epidemiology and surveillance to diagnosis, treatment and infection control, as well as the drug supply chain and the needs of vulnerable populations. Through these meetings, a new image has emerged that dictates dramatic and radical policy changes in the approach to TB if policy makers are to prevent TB from becoming once again an incurable disease. The findings were similar in each country and were equally concerning: (1) the magnitude of the problem of multi-drug and extensively-drug resistant TB is underestimated; (2) the number of patients receiving treatment is small and ineffective; (3) over 90% of patients are treated without drug susceptibility data; and (4) drug-resistant strains are spread from human to human far more commonly than previously appreciated. To date, programs have largely been based on an approach that emphasizes treatment of drug-sensitive cases. If these programs provided comprehensive care for both drug-sensitive and drug-resistant TB, this would be encompassing the entire TB epidemic and would be addressing the growing threat of the unchecked growth of resistant strains. In the current presentation, Dr. Gail Cassell, chair of the planning committee convening the IOM workshops, will present the themes, challenges and opportunities emerging from the IOM initiative and discuss potential global actions and next steps to combat DRTB.

More than 150 different mycobacterial strains are known, of which only a few are considered pathogens in humans. The most pathogenic strains are the mycobacteria from the Mycobacterium tuberculosis complex which cause the globally spread disease tuberculosis and the Mycobacterium leprae and Mycobacterium lepromatosis strains that cause leprosy. In addition to these well-known mycobacteria, there are many non-tuberculous mycobacteria (NTM) that are present in the environment and that seldom cause severe disease in healthy individuals. NTM infection can lead to severe disease in individuals with a failing immune system due to a primary or secondary immunodeficiency. Mendelian susceptibility to mycobacterial disease (MSMD) is a rare primary immunodeficiency characterized by a predisposition to severe, sometimes lethal disease caused by otherwise poorly virulent NTM, as well as the vaccine strain Mycobacterium bovis BCG. In these patients the mycobacterial infection is often disseminated to various parts of the body and is difficult to treat. Interestingly, MSMD patients do not, in general, develop pulmonary tuberculosis. MSMD is usually caused by mutations in genes involved in the IL-12/IFN-γ pathway, such as: IL12RB1 which encodes the IL-12Rβ1 chain of the IL-12 and the IL-23 receptor, IL12B which encodes the IL-12p40 subunit of IL-12 and IL-23, IFNGR1 and IFNGR2 which encode the two chains of the IFN-γ receptor, and STAT1 which encodes one of the proteins that signal in response to IFN-γ. The aim of our research is to identify the immunological and genetic defects in MSMD patients. Hereto the integrity of the IL-12/IFN-γ pathway is analysed in blood and isolated cells from the patients, as well as expression of receptor chains on the membrane of the cells. Mutations are subsequently identified by sequencing the genes involved. The effect of novel mutations that lead to amino acid changes can be analysed in retroviral expression models, as we have done for amongst others IL12RB1, IFNGR1 and IFNGR2. To facilitate the analysis of variations identified by researchers around the world, databases have been set up that contain all reported MSMD patients and mutations (see for instance: www.lovd.nl/IL12RB1). Thus far, just over 400 patients have been reported worldwide with MSMD and this is probably only the tip of the iceberg. Also, other genes are still expected to be found to cause MSMD; no genes have been reported so far in which mutations specifically lead to susceptibility to tuberculosis.

Objectives: HIV patients are prone to tuberculosis (TB) disease, and screening these patients for TB is important. The aim of this study is to analyze the prevalence of active and latent TB and the sensitivity, specificity, negative (NPV) and positive predictive value (PPV) of clinical signs and symptoms for the diagnosis of active TB in HIV-infected subjects. Method: From April 2008 to March 2011, 154 consecutive HIV-infected patients attending the HIV clinic at Masih Daneshvari Hospital were enrolled in the study. For the diagnosis of active TB, two sputum samples (one on presentation and another early morning) were collected from each subject and examined by Ziehl–Neelsen (ZN) microscopy for identification of acid-fast bacilli (AFB). Mycobacterial culture sputum specimens were inoculated on Lowenstein–Jensen (LJ) slants for 4–8 weeks to detect colonies. In those patients with a negative sputum sample for AFB, a polymerase chain reaction (PCR) was performed. Active TB was defined as positive sputum smear or culture for mycobacterium TB or positive polymerase chain reaction (PCR). Also, patients with signs and symptoms compatible with TB who responded to anti-tuberculous medications were classified as having active TB. Results: The mean of age was 36±8 (ranged, 22–62) and 127 (82%) were male. The antiretroviral therapy (ART) had been started in 40 (26%) patients, with 15 (10%) receiving trimethoprim/sulfamethoxazole as a prophylaxis; 119 (77%) were intravenous drug users. Among these patients, 58 (38%) individuals were diagnosed with active TB, of which 48 (83%) had smear-positive pulmonary TB. The mean of the baseline CD4 cell count in HIV patients with and without active TB was 67cells/ μl and 180cells/ μl, respectively (P-value=0.018). The multivariable regression analyses found that CD4<100cells/ μl (OR=2.67; 95% CI 1.23–5.78; P-value=0.013) and smoking (OR=13.4; 95% CI 3.03–59.4; P-value=0.001) were the only significant variables associated with TB in this study. Among the 96 patients who were not diagnosed with active TB, 8 (8%) had a positive tuberculin skin test (TST) and isoniazid prophylaxis was initiated. The presence of any one of six clinical features (cough, sputum, fever, night sweating, weight loss and loss of appetite) had sensitivity (89.6%) and specificity (45.8%) with a PPV of 50% and a NPV of 88%. Conclusion: Due to the high rate of active TB, careful screening of patients with signs and symptoms, X-ray and sputum examination must be performed.

Reactivation tuberculosis (TB) and Mycobacterium avium complex (MAC) disease are significant causes of morbidity in HIV infected patients, especially in resource-constrained settings. These diseases are the most common AIDS-presenting illnesses in some countries. Although morbidity and mortality have significantly decreased with the advent of Highly Active Antiretroviral Therapy, significant challenges exist in treating patients, among them overlapping medication toxicities, drug-drug interactions and the risk of developing Immune Reconstitution Inflammatory Syndrome. Mycobacterial resistance to existing antimicrobials continues to rise, further complicating the management of these patients and presenting a public health challenge. Solid organ and hematopoietic stem cell transplant recipients are also at increased risk of developing TB and MAC disease. In addition, although patients with cellular immune defects are perceived to be at higher risk for non-tuberculous mycobacterial infection, limited data exist on the frequency of these infections in this patient population. Incidence may be influenced by the degree of immunosuppression and the types of immunosuppressants used. Diagnosis is sometimes challenging, and the clinician needs to keep a high index of suspicion to correctly diagnose the syndromes caused by these bacteria. The author will review the epidemiology, clinical presentation, diagnostic methods and principles of treatment of the most common mycobacteria that cause disease in HIV and transplant recipients, and will discuss some of the nuances in the management of these patients.

Introduction: Tuberculosis (TB) is considered a major worldwide health problem with 10 million new cases diagnosed each year. The present understanding of TB immunology has become greater and more refined since the identification of Mycobacterium tuberculosis (MTB) as an etiologic agent of disease and the recognition of new signaling pathways modulating infection. Understanding the mechanisms through which the cells of the immune system recognize MTB can be an important step in designing novel therapeutic approaches, as well as improving the limited success of current vaccination strategies. A great challenge in chronic disease is to understand the complexities, mechanisms and consequences of host interactions with pathogens. Innate immune reactions, along with the involvement of distinct inflammatory cytokines and cells, have been shown to play an important role in the host defense against MTB. Several classes of pattern recognition receptors (PRRs) are involved in the recognition of MTB, including Toll-Like Receptors (TLRs), C-type lectin receptors (CLRs) and Nod-like receptors (NLRs) linked to inflammasome activation. Among the TLR family, TLR1, TLR2, TLR4 and TLR9 and their downstream proteins play critical roles in the initiation of the immune response in the pathogenesis of MTB. Materials and results: In this study, the expression of TLR 2 and 4 was tested in PBMC from a TB patient's blood by staining cells for flow cytometry. The results showed that infection with MTB causes up-regulation of TLR2 and 4, but not TLR8. Conclusions: Understanding cross-talk between these signaling pathways in the pathophysiology of TB has an impact in designing novel strategies and in the development of vaccinations and immunotherapy regimes for this disease. Defects in TLR signaling pathways regulated by TB may affect the pathogenesis of MTB and need to be elucidated in future studies.

Mycobacteria can diminish allergic and asthmatic manifestations. This means that mycobacteria could offer therapeutical opportunities as an ‘anti-allergic’ vaccine. In humans, the genetic background and the environment probably contribute to the development of allergies. Over the last 20 years, a popular explanation for the increase in allergies is the ‘hygiene hypothesis’. This hypothesis argues that there might be a misbalance in T-helper-type responses or a misbalance in regulatory immune responses due to less microbial stimulation. It is clear that the hygiene hypothesis should involve the genetic and the environmental background of the individual. Up until now, no specific infectious factor has been found that could explain the hygiene hypothesis. However, interesting data have been obtained in animal models that could support the hygiene hypothesis. These studies also support that mycobacterial treatment results in regulatory mechanisms that restored the immune balance. In this presentation, the most recent basic and clinical findings concerning mycobacterium and allergic diseases will be highlighted.

Introduction: The aim of this study is to compare characteristics of non-cystic fibrosis bronchiectasis (NCFBE) patients with chronic infections with non-tuberculous mycobacteria (NTM) versus those with Pseudomonas aeruginosa or other colonizations. Methods: This was an observational, perspective study of consecutive NCFBE adult patients attending the outpatient bronchiectasis clinic at the San Gerardo Hospital in Monza, Italy, during 2012 and 2013. Patients with a chronic infection were included in the study and divided into three groups: those with NTM (Group A); those with P. aeruginosa (Group B); and those with other pathogens (Group C). Patients with both NTM and another pathogen were included in Group A. Comparison among the three study groups was performed using X² or Fisher exact test for categorical variables or Kruskal–Wallis or Mann–Whitney test for continuous variables. Results: A total of 146 patients (median age 67 years, 40% males) were enrolled: 19 belonged to Group A, 34 to Group B and 93 to Group C. Within group A, 6 patients had only NTM isolation, 7 patients had NTM and P. aeruginosa co-infection and 6 patients had NTM plus another pathogen. The most common isolated pathogens among NTM was Mycobacterium avium complex (15 patients, 79%). A total of 4 patients (21%) with NTM were on active treatment. Patients affected by NTM pulmonary infection had a significantly less severe clinical, functional and radiological involvement compared with patients colonized by P. aeruginosa, see Table. Conclusions: Colonization with P. aeruginosa seems to have the highest impact on the clinical, functional and radiological status of patients with NCFBE. No specific characteristics may help to identify NTM versus other pathogen colonizations. Thus, diagnostics for atypical mycobacteria should be performed on all patients with NCFBE, as suggested by recent international guidelines. [INLINE:1]

Aims and objectives: MicroRNAs are critical regulators of the mammalian immune system through the fine-tuning of gene expression. Mycobacterium tuberculosis (Mtb) can persist alive and replicate into the host due to its ability to interfere with macrophage antimicrobial mechanisms. Recent studies indicate that genetic diversity among Mtb Complex (MTBC) lineages is reflected in different clinical outcomes and epidemiological success. In this study we hypothesized that the success of modern lineages could rely, at least in part, upon their ability to alter host immune responses through the modulation of intracellular microRNAs expression. Methods: Selected strains belonging to both ancient (EAI) and modern (Haarlem and Beijing) lineages were characterized for their ability to enter, survive and replicate in human monocyte-derived macrophages (MDM). Their capacity to modulate the inflammatory immune response was analyzed in terms of cytokine secretion by means of Fluorokine MAP suspension Array System and their influence on cellular microRNAs expression by, TaqMan Low Density Arrays. Results: Infection of human differentiated MDM by modern strains was characterized by variable phagocyte uptake, but higher intracellular growth and higher associated cytotoxicity. The release of several proinflammatory cytokines in response to strains belonging to modern lineages was significantly lower compared to strains from ancient lineages. Finally, we identified a group of microRNAs which are commonly regulated by all clinical strains and which are involved in the regulation of critical functions in host immune response; and a set of microRNAs specifically related to latency, lipid metabolism and proinflammatory cytokine release and responsiveness differentially modulated by modern versus ancient lineages. Conclusion: In this study it was observed that the genetic diversity among Mtb strains and, in particular between ancient and modern strains, reflects on several aspects of host-pathogen interaction. In particular, the modulation of specific cellular microRNAs upon MTBC infection suggests a potential role for these microRNAs in the outcome of infection and, to a major extent, to the different epidemiological success of Mtb strains.

Tuberculosis (TB) has previously been linked to acute respiratory distress syndrome (ARDS). Here this study investigates the link between inflammation and TB in ARDS by measuring inflammatory cytokine and chemokine levels in bronchoalveolar lavage (BAL) from 90 patients with TB or ARDS alone and in patients with TB-induced ARDS (ARDS+TB). BAL was collected by fiber-optic bronchoscopy, and the concentrations of interleukin (IL)-6, CXCL8, TNF α and IL-1β were measured by ELISA. CXCL8 levels in BAL were significantly higher in the ARDS+TB group compared with TB and ARDS alone groups. Disease severity in the ARDS+TB group as determined by Murray score correlated with BAL CXCL8 and neutrophils, but not with IL-6, IL-1β and TNF α concentrations. In addition, CXCL8 levels and neutrophils were increased in non-miliary TB versus miliary TB. This difference in CXCL8 was lost in the presence of ARDS. It was concluded from this study that CXCL8 may play an important role in the pathogenesis of this form of ARDS. This further suggests that CXCL8 inhibitors or blockers may be useful to control the onset and/or development of these combined diseases.

Aim and objectives: To evaluate the implementation of an outpatient TB-clinic in a low-endemic setting. Background: In May 2014 the World Health Organization (WHO) approved the post-2015 global tuberculosis (TB) strategy including the plan for elimination of TB in low-endemic countries. Sweden is a low-endemic country, but accepts large numbers of immigrants (e.g. 116,000 in 2013) and a majority from countries where TB is common. In order to prevent TB and ensure accessibility a new outpatient clinic for adults was initiated at a specialist hospital in a suburb of Gothenburg, Sweden, in February 2014. The population living in this suburd is overrepresented among the active cases of TB and in some subgroups the incidence is equal or even higher than in high-endemic countries. The objective is early detection and treatment of active disease and latent TB infection (LTBI) in a social medical perspective. Methods: A descriptive approach made from the experiences of the authors. Results: The TB-care in Gothenburg has, before 2014, been centralized to the university hospital. A political decision was taken in 2012, in line with the global TB strategy, to start an outpatient clinic where TB is overrepresented in the population. The decision was to be implemented through collaboration with all healthcare providers. The new clinic was aiming to promote redistribution of patients between the hospitals with an emphasis on accessibility for the patient. The decision was made without the involvement of management and clinical staff. As a result the clinic has solely been able to focus its care on finding LTBI and spreading knowledge and awareness of TB in high-risk groups. Conclusion: A new approach of specialist healthcare is decentralization with emphasis on accessibility, improved preventive possibility and knowledge about the setting. This approach is well suited for TB elimination in order to target high-risk groups. The transition does not occur automatically, and commitment and knowledge is needed on every level in the healthcare system. A political basis is necessary, but for success and change the decision has to be established and anchored among management and healthcare staff. To treat TB-patients at an outpatient specialist clinic is economically beneficial and above all causes a positive effect on the patient's psychosocial well-being and adherence to treatment. Unfortunately, the present infrastructure of TB-care in Sweden, as well as the will and knowledge of the staff to adapt and change according to the political decision, is preventing optimal care for TB-patients based on the global TB strategy for elimination. Acknowledgement: None to declare.

Testing for tuberculosis (TB) is difficult because there are so many tests available. Except for the culture-based tests, all others are for screening or add-on tests. On the other hand, there is an urgent need to develop a rapid TB diagnostic test with good sensitivity and specificity. The introduction of liquid culture medium in 1980 (BACTEC 460 radiometric) and later MGIT was a great improvement in a definitive diagnosis, but still results are not as fast as needed. After the discovery of IS6110 sequence and the whole genome sequence of Mycobacterium tuberculosis (MTB) many rapid diagnostic tests have been developed based on the molecular platform. This brought hope that these molecular tests will be the answer to the need for an ideal rapid test for diagnosis and drug susceptibility testing (DST). Among these notable molecular tests are Hain's LPA and Cepheid's GeneXpert–GeneXpert being the leading one. Time to results for detection and DST, which used to take 6–8 weeks with solid media, was reduced to 4 weeks by the BACTEC method and now to 2 h by the GeneXpert. Despite significant improvement in the time to report, molecular-based tests still face many challenges. Sensitivity and specificity of a diagnostic test is critical in infectious diseases. For molecular tests lack of sensitivity in smear-negative TB cases is a great concern. In case of DST it is important to have a test which can detect resistance to first-line and most of the second-line drugs with high sensitivity and specificity. At present the genotypic tests are applicable for only a few drugs, while GeneXpert detects only Rifampicin resistance. Because of these limitations, genotypic tests are considered as good screening tests. Until the molecular tests are made more sensitive for the detection of TB and more knowledge is acquired on molecular mechanisms of drug resistance, these tests will remain as screening tools. Sustainability is another issue. Presently, funding agencies are supporting the high cost of the genotypic tests, but what if these funds dry out? This overall situation has created a dilemma of what should be the right option in choosing a TB diagnostic test. Efforts are being made to improve the sensitivity and specificity of molecular tests. Until then, it appears that phenotypic tests, especially using liquid medium, still remain as the only one offering a comprehensive solution in TB diagnosis and DST. There is still a need for a diagnostic test which is simple, rapid and affordable for the low-income, high-TB prevalence countries, and as comprehensive, sensitive and specific as the liquid culture for TB diagnosis and DST.

Background: The World Health Organisation (WHO) estimates that 3 million cases of tuberculosis (TB) are missed every year. Identification and treatment of these are critical to achieving global TB control. Patients with sub-clinical TB, extra-pulmonary TB, and drug-resistant TB are difficult to diagnose and may be missed at all points of healthcare. An autopsy study was conducted to ascertain the burden of TB at post-mortem in adults who died in the inpatient general medical wards at a tertiary care referral center in Lusaka, Zambia. Methods: Complete whole body autopsies were performed on 125 adult inpatients. Pathological examination involved two stages: (1) Gross pathology was recorded, and samples were taken from all organs for histopathology and cryopreservation; and (2) Histopathological examination of tissue after appropriate staining. Specific pathology and diseases identified on examination were recorded. Lung tissues were processed using the GeneXpert MTB/RIF Assay. Primary outcome measures were specific diseases stratified by HIV status. Secondary outcomes were missed TB and drug-resistant TB cases. Findings: Of 125 adults, median age 35 years (IQR: 29-43), 80 (64%) were male and 101 (80.8%) were HIV-positive. Tuberculosis was the most common finding at autopsy with 78/125 cases (62.4%), of which 66/78 (84.6%) were HIV-infected. There were 35/78 cases (44.9%) with extra-pulmonary TB, the odds of which were higher among HIV-infected cases (aOR 5.14 (95% CI: 1.04–25.4), p = 0.045); 25.6% (20/78) of the TB cases were not diagnosed ante-mortem; and 13/78 (16.7%) of the TB cases had undiagnosed MDR-TB. Other autopsy findings included: pyogenic pneumonia 36.8% (46/125); bacterial meningitis 7.2% (9/125); cardiac failure 7.2% (9/125); and malignancies 8.8% (11/125). Prevalence of HIV did not differ between TB and non-TB cases (84.6% vs. 74.5%: p = 0.163). Interpretation: TB remains an important cause of death in adult inpatients. A substantial number of inpatients with TB and MDR-TB are not diagnosed by the current cascade of healthcare. Inpatient settings in high TB endemic countries should be included in WHO ‘high risk’ groups, and heightened clinical awareness and more proactive screening for TB and MDR-TB in all inpatients should be required.

Phylogenetic knowledge of the genus Mycobacterium is based on comparative analysis of their genetic sequences. The 16S rRNA has remained for many years the only target of such analyses, but in the last few years, other housekeeping genes have been investigated and the phylogeny based on their concatenated sequences become a standard. It is now clear that the robustness of the phylogenetic analysis is strictly related to the size of the genomic target used. Whole genome sequencing (WGS) is nowadays becoming widely accessible and comparatively cheap. It was decided, therefore, to use this approach to reconstruct the ultimate phylogeny of the genus Mycobacterium. Over 50 types of strains of the same number of species of Mycobacterium were sequenced using the Illumina HiSeq platform. The majority of the strains of which the whole sequence was already available in GenBank were excluded from this panel with the aim of maximizing the number of the species with genome available. Following assembling and annotation with proper software, the phylogenetic analysis was conducted with PhyloPhlAn and the pan-genome analysis pipeline. The phylogenetic three which emerged was characterized by a clear-cut distinction of slowly and rapidly growing species with the latter being more ancestral. The species of the Mycobacterium terrae complex occupied an intermediate position between rapid and slow growers. Most of the species revealed clearly related and occupied specific phylogenetic branches. Thanks to the WGS technology, the genus Mycobacterium is finally approaching its definitive location.

Background: Non-tuberculous mycobacteria (NTM) are ubiquitous environmental organisms. Data about NTM in patients with non-CF bronchiectasis are limited. Methods: In a retrospective study, data was collected on patients with bronchiectasis and patients who were diagnosed with NTM were identified. All patients diagnosed with bronchiectasis (code 494) using the International Classification of Diseases, ninth revision (ICD-9), between 1999 and 2006, were identified. Clinical data including radiology findings, lung function, progression of disease, and the presence of NTM in sputum were collected for those who met the study criteria. Results: 300 patients with ICD code for bronchiectasis were found in the data. Of these, 182 patients were enrolled in the study after confirmation of the radiology findings of bronchiectasis by CT scanning. Patients were divided into two groups: bronchiectasis with NTM isolates (n = 68) and bronchiectasis without NTM isolates (n = 114). The clinical data was reviewed to define the clinical characteristics of these patients; 12% of the patients were found to have NTM from their sputum or BAL. Of the total patients, 55 (30%) of these met the American Thoracic Society criteria for the diagnosis of NTM disease. Mycobacterium avium complex (MAC) was the most common isolate. It was found that the probability of NTM isolation was significantly higher in elderly female patients (p = 0.04) with a low body mass index (BMI) (p = 0.002). Additionally, it was found that gram-negative rods were frequently isolated in these patients, and the frequency of exacerbations was significantly higher in patients with NTM infections. Conclusions: NTM infections are common in non-CF bronchiectasis. MAC is the most frequently isolated NTM in these patients. Elderly female patients with a low BMI are a high-risk group for NTM infection in non-CF bronchiectasis. Furthermore, these patients are prone to secondary infections and frequent exacerbations. This study suggests a strong correlation between non-CF bronchiectasis and NTM. Thus, these patients should be routinely screened for NTM infections.

TB incidence and mortality in Belarus has been progressively falling from 54 to 41 and from 12.1 to 6.8 per 100000, accordingly, for the last 7 years. Nevertheless, the number of HIV/TB co-infected rises dramatically. According to a recent nationwide drug resistance survey, MDR-TB was found in 32.3% of new and in 75.6% of previously treated TB cases. Results of treatment of MDR-TB are far from satisfactory (51% treatment success, 11% deaths). Risk factors for MDR-TB acquisition and unfavorable treatment outcome are the following: young age, drug and alcohol abuse, HIV, history of imprisonment, unemployment, and homelessness. Most of such events as treatment failure and loss to follow-up occur at the outpatient treatment phase, particularly in patients from the above-mentioned risk groups. One of the key elements of outpatient care strengthening is social support. Two operational research studies were conducted. One study with 40 MDR-TB patients included showed that treatment cost can be reduced by more than 5200 US dollars per patient using outpatient care with social support instead of existing care based on long-term hospitalization. Another study showed that MDR-TB patients with social support (n = 1057) had better treatment outcomes compared with conventionally managed patients (n = 3514): treatment success 70% vs. 53%; mortality 2% vs. 6%; treatment failure 27% vs. 40%. Along with adequate diagnostics and therapy, outpatient care strengthening and social support are important elements in achieving good treatment outcomes and reducing the cost of treatment of MDR-TB.

Aims and objectives: With the relentless increase in multidrug- and extensively-drug resistant tuberculosis (MDR/XDR-TB), new treatment strategies are necessary. Favorable results have been reported by combining a β-lactam antibiotic and a β-lactamase inhibitor. The β-lactamase encoded by the blaC gene of Mycobacterium tuberculosis (MTB) is the major mechanism of resistance to β-lactam antibiotics (e.g., penicillin). Meropenem, a β-lactam antibiotic of the carbapenem group, is a relatively weak substrate for the β-lactamase of MTB. The β-lactamase inhibitor clavulanate irreversibly inactivates the β-lactamase encoded by the blaC gene, thus making the combination of meropenem and clavulanate an interesting treatment alternative for MTB. However, very few isolates of MTB have been tested for this drug combination and few clinical reports exist. Thus, the present study investigates the in vitro activity of meropenem-clavulanate for drug-resistant MTB isolates, including MDR/XDR-TB. Methods: The minimum inhibitory concentration (MIC) distribution of meropenem-clavulanate was determined using Middlebrook 7H10, including MDR and XDR strains of MTB (n = 68). Meropenem was prepared in a stock solution with a final concentration range of 0.002–512 mg/L. Clavulanate was added at a fixed concentration of 64 mg/L, to avoid a decline of the β-lactamase to insufficient levels during the experiment. All isolates were evaluated after three weeks of growth. The pan-susceptible strain H37Rv was used as a control. Results: There was a Gaussian MIC-distribution between 0.125 and 2 mg/L of meropenem-clavulanate (expressed as the concentration of meropenem), but four isolates had very high MIC levels (16 and 32 mg/L), which is likely to be out of reach in clinical doses ([Figure 1). The susceptibility of the isolates to meropenem-clavulanate was not correlated to the level of resistance to first- or second-line anti-tuberculous drugs. The MIC of the pan-susceptible control strain H37Rv was 1 mg/L of meropenem, when combined with clavulanate.{Figure 1} Conclusions: The present study shows that meropenem-clavulanate has low MICs against MTB in vitro, including MDR and XDR-TB isolates. Meropenem has good tissue penetration and low protein-binding, but requires an intravenous access and is relatively expensive. Meropenem-clavulanate may be a treatment option in selected cases of MDR/XDR-TB, although further clinical studies are warranted.

Aims and objectives: Multidrug-resistant tuberculosis (MDR-TB) has become a major public health problem worldwide, which lays a heavy burden on low- and middle-income countries. Compared with newly diagnosed tuberculosis (TB) patients, patients with a history of anti-TB treatment have increased mortality and a higher prevalence of drug resistance. However, these patients are neglected by both global and national TB control programs and have met barriers to access to medical care for potential drug resistance. This study aims to understand the drug-resistance status of patients with previous treatment history and to determine the factors associated with the accessibility of bacteriological-based TB diagnosis. Methods: A cross-sectional study was conducted in 8 county/district TB clinics in three provinces (Jiangsu, Shandong and Sichuan) of China, where a global fund-supported MDR-TB control project had been initiated. TB patients who had at least one previous treatment episode were interviewed using a structured questionnaire. Information on demographics, socioeconomic status of patients, and their experiences on seeking TB diagnosis and treatment were collected. Results: A total of 196 smear-positive TB patients were recruited, of which 78% (153/196) were male and the average age of these patients was 57 years old. About 70% (137/196) of the subjects were farmers, and 63% (121/195) had less than 6 years’ education. Among these patients, 120 cases (61.22%) received culture-based TB diagnosis in this treatment episode, while patients from Jiangsu province (versus Shandong/Sichuan aOR: 8.10/36.42, 95% CI: 2.70–24.25/12.20–108.66) and patients with a higher annual income per capita (aOR: 3.35, 95% CI: 1.26–8.92) were found to be more likely to reach culture tests after adjusting for gender and age. MTB complex and non-tuberculosis mycobacteria (NTM) strains were isolated from 92 and 10 patients, respectively, and the remaining 18 patients were negative to bacteria culture. Drug sensitivity testing (DST) for first-line anti-TB drugs, and kanamycin and ofloxacin, was completed for the 92 MTB isolates. The total proportion of drug-resistant tuberculosis was 52.48% (47/92), while the prevalence for resistance to a single drug, multidrug-resistant and other combinations of drug resistance were 16.30% (95% CI: 9.78–23.91%), 30.43% (95% CI: 21.74–40.22%), and 4.35% (95% CI: 1.09–8.70%), respectively. Resistance to isoniazid, rifampicin, streptomycin, ethambutol, kanamycin and ofloxacin was 36.96%, 35.87%, 23.91%, 10.87%, 4.35% and 6.52%, respectively. Conclusions: Patients with a previous treatment history are at extremely high risk of drug-resistant tuberculosis in China. The poor accessibility of bacteriological-based diagnosis may aggravate the difficulty of detection and control for drug-resistant TB patients. The next step of the anti-TB strategy should be focused on how to make bacteriological-based diagnosis cheaper, safer and more maneuverable and on how to assure the DST-guided treatment for these high-risk TB patients.

Introduction: The emergence and spread of multidrug-resistant and, more recently, extensively-drug resistant tuberculosis (MDR-TB and XDR-TB) is a major concern of public health that hampers efficient TB control. The Mycobacteriology Laboratory in the St. Petersburg Research Institute of Phthisiopulmonology serves as a reference center for northwest Russia (11 provinces; 1,700,000sq.km; population of 13.5 million); additionally, the laboratory receives strains from other regions across Russia. This study aimed to perform a molecular characterization of XDR Mycobacterium tuberculosis isolates recovered from TB patients in northwestern Russia during the time period of 2011–2013. Materials and Methods: M. tuberculosis isolates from mainly pulmonary TB patients (extrapulmonary TB, n = 7) were identified and characterized using traditional biochemical methods, including susceptibility testing for 11 anti-TB drugs (streptomycin, isoniazid, rifampin, ofloxacin, kanamycin, amikacin, capreomycin, ethambutol, ethionamide, pyrazinamide, and PAS acid). Drug susceptibility testing (DST) was done using a method of absolute concentrations according to the guidelines of the Russian Ministry of Health (order No. 109 of 21 March 2003) and/or BACTEC MGIT 960 system according to the manufacturer's recommendations. Extensive drug resistance was defined as recommended by WHO (World Health Organization) as resistance to isoniazid and rifampin (MDR) plus resistance to one of the fluoroquinolones and one of three injectable drugs (kanamycin, amikacin, capreomycin). The isolates were further subjected to spoligotyping followed by comparison with SITVIT_WEB and MIRU-VNTRplus databases. LAM family was defined by Rv0129c SNP analysis. Results: A total of 115 XDR M. tuberculosis isolates were included in this study. They presented 41 different patterns of drug resistance. XDR was complemented with resistance to streptomycin (n = 114), and/or ethambutol (n = 93), ethionamide (n = 80), pyrazinamide (n = 43), or PAS acid (n = 40). Of the three injectable drugs, XDR was mainly due to kanamycin resistance alone (n = 39, 34%), followed by combined kanamycin and amikacin resistance (n = 13), amikacin and capreomycin resistance (n = 11), kanamycin and capreomycin resistance (n = 9). Spoligotyping assigned the isolates to 3 genetic families: Beijing (98; 87%), LAM (SIT42, n = 6; SIT252, n = 2; SIT496, n = 2) and Ural (SIT262, n = 7). SIT1 was predominant among Beijing isolates. The proportion of isolates resistant to any of 5 drugs (including, streptomycin, isoniazid, rifampin, ofloxacin and/or kanamycin, amikacin, and capreomycin) was 1.7% (n = 2), to 6 drugs – 7.8% (n = 9), to 7 drugs – 19.1% (n = 22), to 8 drugs – 26.9% (n = 31), to 9 drugs – 25.2% (n = 29), to 10 drugs – 12.1% (n = 14). Eight isolates of the Beijing genotype were resistant to all 11 tested drugs. Conclusions: XDR M. tuberculosis population in northwestern Russia is heavily dominated by Beijing genotype isolates (87%). Acknowledgements: Russian Science Foundation (Grant agreement 14-14-00292).

Aims and objectives: People living in contact with smear-positive pulmonary tuberculosis (PTB) patients are highly exposed to TB infection, including children who are a major risk group. TB disease, among those who become infected, manifests itself mainly in the two years (90%) after the identification of the index case. The latent TB infection can be detected by means of two tests: the tuberculin skin test (TST) and the IGRAs test – QuantiFERON-TB Gold in Tube (QFT-G). The latter showed greater specificity in the prediction of latent TB infection in an adult population, but not in a child population. The main objective of this study is to estimate the incidence of TB disease in children living in contact with an M+ Pulmonary tuberculosis index case (PTM+) according to the positivity of the Quantiferon test and/or the tuberculin skin test (TST), during the two years following exposure. The results of this study will eventually rehabilitate the national guidelines on individual contacts. Methods: It is a descriptive study, cohort-type, prospective, multicenter, conducted amongst children in contact with a PTM+ case, aged between 6 months and 15 years, and followed for two years in order to estimate the incidence of TB disease in different groups defined by the results of the Quantiferon test and the TST. The recruitment of children will be based on the PTM+ index case diagnosed and taken in charge by the Services of Control of Tuberculosis and Respiratory Diseases (SCTMR) located in the Wilaya of Algiers, diagnosed in 2014. Seven centers were selected. Results: This initial work will describe the clinical characteristics of the index case diagnosed during 2014 (December 2013–October 2014) and the results of the TST and Quantiferon test carried out in children contacts at the time of their recruitment. At the time, 396 children living in contact with M+PT have been tested with Tuberculin and QFT-G. They are maintained under control during at least two years.

Background: Continuous understanding of the epidemiology of drug resistant TB, especially for multidrug/extensively drug resistant TB (MDR/XDR-TB) is essential to develop the appropriate strategies for preventing its epidemic. The present study aimed to investigate the longitudinal trend of the rates and dynamic change of MDR/XDR M.Tuberculosis and its transmission pattern to adjust the TB control activities in eastern rural China. Methods: The data presented here spanned 8 years and was derived from three population-based studies of drug-resistant TB in two eastern rural counties: DQ and GY in terms of socio-demographic characteristics of drug-resistant cases, as well as the microbiological and molecular features of their infected M. Tuberculosis strains in eastern rural China. Results: Of the 990 smear-positive patients within the studied period, 413 (41.7%) cases showed resistance to any of the detected first- (INH, RIF and EMB) and second-line (OFL, LEV, KAN, AMK, and CAP) anti-TB drugs: 89 with MDR (9.0%). Of these MDR-TB isolates, 34 cases (38.2%) showed resistance to any of the second-line anti-TB drugs: 25 (28.1%) with resistance either to FQs or second-line injective drugs (Pre-XDR) and 9 (10.1%) showed simultaneous resistance to the two categories (XDR). Molecular analysis of pncA gene revealed 185 M. Tuberculosis cases contained the mutation associated with PZA resistance, with a significantly higher proportion in MDR compared with the others (35% vs. 16.8%, p = 0.04). For the longitudinal perspective, MDR-TB (from 9.1% to 14.5%, p = 0.001), Pre-XDR-TB (from 2.5% to 4.5%, p = 0.01) and XDR-TB (from 0.9% to 3.6%, p = 0.04) exhibited an upward trend, while there was a significant decreasing change in the proportion of totally first-line drug sensitivity (p = 0.047). Non-linear regression analysis showed that MDR-TB, Pre-XDR-TB and XDR-TB increased significantly over the study period with a double time of 4.8 year (95% CI: 3.69–5.92), 3.2 year (95% CI: 2.16–5.94) and 3.3 year (95% CI: 1.099–8.32). Being clustered, sputum smear result and previously treated history were factors increasing the trending of M/XDR-TB. The MIRU24 loci genotyping revealed the 21 clusters involving 48 (28.4%) drug-resistant cases in DQ and 28 clusters involving 59 (32.1%) drug-resistant cases in GY. Of these clusters, 11 were related to MDR/XDR-TB and 8 of these clustered genotypes were observed more than 5 years of the study period. Conclusion: These results show that active transmission of MDR/XDR-TB is taking place and that the increasing trend in the observed rate of MDR/XDR-TB tends to be due to the variation in the distribution of patients with different socio-demographic features and the continued transmission of particular genetic clusters. Continuous surveillance of clinical isolates of M. Tuberculosis is needed to identify M/XDR-TB, especially in patients who have a history of TB and have received prior anti-TB treatment.

Aims and objectives: This study aims to identify common mutations leading to Isoniazid (INH), Rifampin (RMP) and Etambutol (EMB) resistance using Multiplex Allele-Specific Polymerase Chain Reaction (MAS-PCR). Method: In a cross-sectional study during 2012–2013, 257 patients with smear-positive pulmonary tuberculosis residing in five frontier west and north-west provinces of Iran were evaluated in respect of common point mutations leading to resistance to tree first-line drugs. Results: The overall frequency of mutations was 37 out of which 8 mutations were related to katG 315, 26 mutations pertained to rpoB 516, 526 and 531 and 3 mutations related to emb B. The rpoB single, double and triple mutations were found in 45.3%, 42.3% and 15.4% of rpoB, respectively. Frequency of patients with mutation to katG and at least one rpoB codon was 7cases (2.7%) at the same time. In this study 60.0% of INH-resistant and 83.3% of RMP-resistant isolates were detected by MAS-PCR technique. Mutation odds were higher in females and in patients with a history of anti-TB drug use. Conclusion: The MAS-PCR is a relatively rapid, sustainable, efficient and accurate technique for detection of drug resistance in tuberculosis. This highlights also the role of mutation at inhA, ahp and oxy R genes in the creation of IHN resistance which may be the causative factor in the remainder of cases.

Background and aim: Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB), does not retain any bacteriological stain due to the high lipid content in its wall. Classic methods like acid-fast staining or Ziehl–Neelsen staining are used for the diagnosis. Nucleic acid amplification techniques, such as polymerase chain reaction (PCR), are very useful in the rapid diagnosis of infections by MTB, but the sensitivity of PCR is considerably lower because of the presence of inhibitory substances in tissues. The aim of this study is to improve the sensitivity of PCR in inhibiting tissue samples. Method and material: Thirty-six lymph nodes isolated from inoculated guinea pigs with BCG vaccine were used. After grinding and suspending samples in sterile distilled water, they were cultured on Lowenstein–Jensen media. DNA extraction was done after 5 days by cetyltrimethylammonium bromide (CTAB) method. Finally, PCR was done for each extracted DNA. Result: This study began with 40 guinea pigs; 4 died before the fourth week. Thirty-six lymph nodes were sent to PRIC lab; all 36 samples were positive in culture and ZN stain. All specimens were assessed by two methods: PCR before and after 5 days of culture. 27.8% (10 samples) and 69.4% (25 samples) were positive PCR before and after brief-culture, respectively. Conclusion: In summary, the aim of this study was to demonstrate the importance of optimal DNA extraction and brief-culture for elevating the efficacy and accuracy of PCR for the rapid detection of lymph node TB.

Aims and objectives: Copper poisoning in macrophages plays an important role in immunity against invading pathogens. Many bacteria, including Mycobacterium tuberculosis (Mtb), have evolved mechanisms to combat copper-derived innate immune responses. Copper homeostasis in Mtb consists of several components, including a multi-copper oxidase, copper efflux pump, cytoplasmic metallothionein and copper-sensing transcriptional regulators. However, components involved in copper uptake are unknown, which prompted this study to investigate the possible role of porins in copper uptake in mycobacteria. Methods: Mycobacterium smegmatis porin mutants were created and tested for their ability to grow under copper-reduced or copper-rich conditions. The M. smegmatis porin gene mspA was expressed in Mtb, and its copper susceptibility profile was investigated in the presence of different copper concentrations. The expression level of a copper detoxifying protein, mycobacterial multi-copper oxidase (MmcO), was monitored by western blot to assess intracellular copper content. Results: Deletion of porin genes from M. smegmatis caused a severe growth defect on trace copper medium. Copper supplementation alleviated this phenotype. The inability to acquire copper in sufficient amounts due to lack of porins can explain this phenomenon. Moreover, porin mutants showed elevated tolerance to copper at concentrations that were toxic for wild-type strains, indicating that the lack of porins protects these strains from copper poisoning. On the other hand, heterologous expression of mspA in Mtb significantly impaired growth at 2.5 μM copper and eliminated growth at 15 μM, while wild-type Mtb eventually reached its normal cell density at this copper concentration. Consistent with a role of porins in copper uptake, expression levels of MmcO in Mtb expressing the M. smegmatis porin mspA was above wild-type levels, indicating that cytoplasmic copper-sensing transcriptional regulators respond by derepressing the expression of copper resistance genes. Moreover, the polyamine spermine, a known inhibitor of porin activity in gram-negative bacteria, increased the tolerance of wild-type Mtb for copper suggesting that endogenous outer membrane proteins with channel-forming activity exist and contribute to copper acquisition and toxicity in Mtb. Conclusions: It was concluded from these results that porins are involved in copper uptake in mycobacteria. Moreover, the outer membrane of Mtb was found to be an important barrier against copper intoxication so that permeabilization of this barrier (e.g., by porins) renders Mtb extremely vulnerable to copper. Consequently, copper homeostasis of Mtb provides a promising drug target for the development of a new class of anti-tuberculosis compounds that can induce a copper hypersensitivity phenotype in Mtb.

Objectives: Tuberculosis (TB) still remains an important medical problem due to high levels of morbidity and mortality worldwide. Tuberculous pleurisy is the most common form of extrapulmonary TB. Since tuberculous pleural effusion usually contains a low number of mycobacteria, the diagnostic sensitivity of both direct microscopy and pleural fluid cultures is relatively low. Adenosine deaminase (ADA) is an essential enzyme in the metabolism of purine nucleosides. For an adequate interpretation of ADA, it is important to highlight the fact that ADA is represented by two isoenzymes: ADA1 and ADA2. The ADA1 isoenzymes are found in all cells, with the highest activity in lymphocytes, whereas ADA2 isoenzymes appear to be found only in antigen-presenting cells. The aim of this study is to evaluate the ADA and ADA1 and ADA2 isoenzyme activity in patients with pulmonary TB and tuberculous pleurisy. Methods: This study included 25 patients with pleural effusions (11 with pleural TB and 14 with nontuberculous etiologies), 35 patients with pulmonary tuberculosis (PTB) and 42 healthy subjects. Total ADA activity level in pleural fluid and blood plasma was measured by the “ADA-test kit” developed in the Republican Scientific and Practical Center for Epidemiology & Microbiology (Minsk, Belarus). The principle “ADA-test” is a colorimetric method in which adenosine is deaminated by ADA and the free ammonia is estimated. Estimated ADA2 activity was calculated from the ADA and 2’-deoxyadenosine deaminase activities using the affinity of ADA2 for the two substrates. Results: The mean plasma ADA level in patients with PTB was 19.2±3.3U/L that was significantly higher than the control group (10.4±0.6U/L). The difference between patients with pleural TB and the controls is also significant (18.2±2.5U/L versus 10.4±0.6U/L, p<0.05). A significantly higher activity of ADA1 and ADA2 in the blood plasma was observed in patients with PTB and pleural TB than those of the corresponding controls (p<0.05). With diagnostic thresholds of 12.5 and 16U/L respectively, the sensitivities of plasma ADA for PTB and pleural TB were 54% and 64%; their specificities 68% and 78%; AUC – 0.67 and 0.73, respectively. The ADA level in the pleural fluid was significantly higher in patients with TB pleural effusion. It was found that the mean ADA level in the pleural fluid was 74.6±7.9U/L in cases of pleural TB, as compared with 21.9±4.3U/L in non-tuberculosis effusion (p<0.05). These results indicate that pleural fluid ADA1 and ADA2 levels were higher in patients with pleural TB as compared with non-tuberculosis effusion. An analysis of the ROC curve for pleural fluid ADA activity showed that at the most accurate cut-off level of 53.4U/L, the sensitivity of the test for tuberculous pleurisy was 91% and the specificity was 93%. Conclusion: This study concluded that pleural fluid ADA analysis could be an easy, cheap and highly sensitive and specific test for the diagnosis of pleural TB.

Introduction: Pulmonary tuberculosis (TB) is an old disease that has been consistently considered as an important cause of inability and mortality. TB is a major human killer that is progressing as before. Pediatric TB causes 5% to 15% of clinical TB. This study investigated the relative prevalence of infection in the 15- to 25-year-old family members or relatives who were in constant, domestic contact with smear-positive and -negative pulmonary TB patients. Methods: This is a descriptive, cross-sectional study conducted on individuals having household contact with negative and positive pulmonary tubercular patients during 2011. After identifying positive and negative smear TB in the Tuberculosis Center of Birjand, all 15- to 25-year-old household contacts were identified. After a physical exam, and in order to rule out, a PPD test was conducted with 0.1cc of the 5 unit PPD solution. The test result was measured and recorded between 48 and 72 h later by a scaled ruler. Induration >5 mm was considered a positive tuberculin test. None of the case contacts has had a BCG vaccination in the last 7 years. In all, CXR was normal. The results were analyzed with the Fisher test. Results: Of 126 case contact with 75 smear-positive and -negative TB, 102 cases had contact with smear-positive TB and 24 cases had contact with smear-negative TB. Of 102 cases that had contact with smear-positive TB, 17 cases have positive PPD (PPD>5 mm), and 85 cases have negative PPD. All of the household contacts with negative smear TB have negative PPD; 60% of PPD positive subjects were male and 40%were female. Individuals with positive PPD included 15 children and 2 relatives of a TB patient. The average size of PPD was 7.2±1.4 mm. All contacts with positive PPD had normal CXR. Thus, 13.5% of all case contacts and 16.7% of contacts with smear-positive TB were infected with MTB. There was statistically significant differences between the two groups (p = 0.04), but there was no significant difference between the sexes. Conclusions: The most important way to prevent TB is omission of the disease transmission sources (TB patients) by anti-TB treatment. Extensive studies are needed to ensure that contacts of patients with pulmonary TB are identified and appropriately screened.

Aims and objectives: Tuberculosis (TB) remains a major global health problem. The incidence of multidrug-resistant tuberculosis (MDR-TB) has increased in recent years. To combat the disease, novel intervention strategies effective against drug-resistant and susceptible subpopulations of Mycobacterium tuberculosis are urgently required as adducts in present treatment regimen. This study has employed a proteomic approach, which is a direct method, to identify proteins from resistant M. tuberculosis isolates compared with sensitive isolates. Methods: The clinical isolates M.tuberculosis strains were provided by the TB bank of Pasteur Institute of Iran. The bacilli were cultured on 7H9 broth with ADC enrichment at 37 °C for 3–4 weeks (late log phase). Cells were washed with PBS, suspended in sonication buffer (10 mM DTT, 1 μg/ml DNAse, 100 mM Glycerol, 10 mM Triton X100, 20 mM EDTA, 50 mM Tris-Hcl, 1 mM PMSF, 0.02% sodium azide, pH 7.4) and then were subjected to sonication for 1 h at 50Hz, in ice, and subsequently centrifuged at 5000rpm for 45 min at −4 °C. Proteins were precipitated by adding refrigerated ethanol or saturated ammonium sulfate to the supernatant. Pellet resuspended in PBS and then dialyzed for 24 h against PBS pH 7.4. IEF was carried out using the Ettan IPGphor 3 isoelecteric focusing system. Immobilized pH gradient IPG strips of pH 4–7 and length 11cm were rehydrated overnight at 20 °C with 500 μg protein which was mixed with rehydration buffer. Proteins were separated in second dimension on 12% SDS-PAGE in a vertical electrophoretic dual gel. 2D gels were analyzed using Image Master, Melanie 7.0 software. Mass spectrometry was performed using Autoflex II TOF/TOF. Protein sequences were retrieved from Tuberculist server hosted by Pasteur Institute, Paris. Results: Mass spectrometry and bioinformatic characterization of both drug-resistant and sensitive isolates of M. tuberculosis showed that the majority of commonly expressed/upregulated proteins belonged to the cellular metabolism and respiration category (Rv0866, Rv3057c, Rv3248c, Rv1133c, Rv0462, and Rv1876). One hypothetical protein (Rv2744c) and two membrane and cell wall fraction proteins (Rv0379, Rv1886c) were identified. Protein spot 1 was 60kDa chaperonin-2/GroEl-2 (Rv0440). This essential gene prevents misfolding and promotes the refolding and proper assembly of unfolded/misfolded polypeptides generated under stress conditions. Further, another spot Rv2013c (HSP16.3/HSPX) has been shown to be induced under oxygen-deficient conditions. Its role in maintenance of long-term viability during latent, asymptomatic infections and in replication during initial infection has also been proposed. Conclusion: Such information could be helpful for the development of newer therapeutic agents or of diagnostic markers for better treatment or diagnosis of TB. This study extends the list of the potential determinants of differences in virulence between the two isolates (MDR and susceptible TB) and adds to the current understanding of MTB pathogenesis.

Introduction: Mycobacterium avium subsp. paratuberculosis causes paratuberculosis (Johne's disease), a systemic infection and chronic inflammation of the intestine that affects many species, including bovine. Infection is widespread in livestock, and human populations are exposed. A possible association between MAP infection and Crohn's disease in humans has been also described. Effective control of paratuberculosis has hampered due to lake of rapid and accurate diagnostic test. Range of diagnostic tests is available, but all have inborn limitations. The present study was designed to develop a loop-mediated isothermal amplification (LAMP) assay for the rapid and simple detection of Mycobacterium avium subsp. paratuberculosis (MAP). Materials and Methods: Six primers were specially designed for recognizing eight distinct sequence of insertion sequence 900 (IS900). To determine the sensitivity of the LAMP assay, 10-fold serial dilutions were made from 431ng/ μl MAP stock solution and compared with Nested-PCR results obtained using similar templates at identical concentrations. Detection limit of the LAMP was defined as the last positive dilution and the reactions were performed four times to examine the reproducibility of the test. The specificity of the assays were evaluated by testing three Gram-positive bacteria including Mycobacterium bovis AN5, Mycobacterium tuberculosis DT and Mycobacterium avium avium. Results: Sensitivity of this assay for detection of DNA of MAP was 4fg/ μl and the specificity was 100%. This assay successfully detected MAP not only in the bacterial cultures but also in clinical fecal samples and the specificity of both PCR was 100%. This LAMP method is performed under isothermal conditions and no special apparatus is needed. In addition, its reactivity is directly observed with the naked eye without electrophoresis either as turbidity or in the form of a color change when SYBR Green 1, a fluorescent dsDNA intercalating dye, is employed. Conclusions: This assay is rapid which requires nearly 1 h for detection of MAP, low in cost and simple to perform, sensitive and practical tool for the detection of MAP and will be useful in facilitating the early diagnosis of paratuberculosis (Johne's disease) caused by the organism.

Background: The problem of high incidence and prevalence rates of tuberculosis (TB) in Iraq is an issue of concern to the national health authorities and international parties, WHO and UNDP. Tremendous efforts have been made in Iraq to control the problem. However, progress is very slow in controlling it. One of the major obstacles that stand against the eradication of this disease is the multiple drug resistance in TB patients. Aim: The current study was carried out to quantify this factor in tuberculous patients in Basrah Province, the capital of the south of Iraq. Method: A total of 2246 presumptive TB patients were referred to and examined at the Respiratory and Chest Disease Centre, the only health center that deals with this health problem in the Province. Infected persons were investigated for Rifampicin resistance using the GeneXpert test. Results: It has been found that about 26% of the examined presumptive patients were tuberculous. Out of those, about 2.9% were found to be Rifampicin resistant. Conclusions: The present study emphasizes the high incidence and prevalence rates of TB in Iraq. The use of modern techniques in the identification of Mycobacterium tuberculosis resistance to antibiotics is a very important necessity, in particular molecular detection using the GeneXpert device given that it is faster and more sensitive compared with other methods.

Aims and objectives: This study evaluated the performance of GeneXpert (GX) for direct detection of MTB in extra-pulmonary specimens and assessed the ability of the assay to detect resistance to RIF. Methods: 104 clinical samples from a tertiary hospital in Sfax, Tunisia were analyzed. Specimens were processed using GX, Ziehl Neelsen and auramine smear, conventional culture on LJ and MGIT >60 media, and drug phenotypic susceptibility testing on LJ and MGIT 960. The diagnosis was made based on clinical, radiological, microbiological, pathological and therapeutic criteria. Results: In total, 51 patients were considered tuberculous. The PCR result obtained in less than two hours was positive for 46 samples and rifampicin was sensitive to all these cases. The sensitivity, specificity, positive predictive value and negative predictive value of GX were 90%, 60.6%, 19.6% and 98.3% compared with direct examination, 84.8%, 74.6%, 60.9% and 91.4% compared with culture and 84.3%, 94.3%, 93.4%, 86.2% compared with final diagnosis. The sensitivity of the GXt was 86% in biopsies and 75% in pus, collections and in biological fluids. Conclusion: The GX is a simple, rapid technique for real-time PCR and has increased the sensitivity of detection of Mycobacterium tuberculosis complex. It must be part of the diagnostic arsenal of tuberculosis without replacing conventional microbiological tools and allow early diagnosis and appropriate treatment.

Aims and objectives: Early detection of multidrug-resistant tuberculosis (MDR-TB) is essential to prevent its transmission in the community and to initiate an effective anti-TB treatment regimen. The objective of this study is to evaluate molecular technique high resolution melting curve (HRM) assay to rapidly detect resistance-conferring mutations in rpoB and katG genes. Methods: HRM analysis was used to screen 95 Mycobacterium tuberculosis (MTB) clinical isolates including 20 rifampin resistant (RIF-R), 21 isoniazid resistant (INH-R), and 54 fully susceptible (S) isolates determined by proportion method of drug susceptibility testing. 19 MTB isolates with known drug susceptibility genotypes were used as control strains for initial validation of the assay. Polymerase chain reaction (PCR) amplicons from rpoB and katG genes were sequenced to investigate the frequency and type of mutations and to confirm HRM results. Results: All RIF-S and INH-S isolates generated wild-type HRM curves and were accurately identified as susceptible by this method. Similarly 19 out of 20 RIF-R and 18 out of 21 INH-R isolates correctly exhibited mutant-type HRM curves. These results were similar to those obtained by direct sequencing. However, 1 RIF-R and 3 INH-R isolates were falsely identified as susceptible by HRM assay. These strains were confirmed as having no mutation in their target regions by sequencing. The main mutations involved in RIF and INH resistance were found at codons rpoB531 (60% of RIF-R isolates) and katG315 (85.7% of INH-R isolates), respectively. Conclusions: HRM was found to be a reliable, rapid and low-cost method to characterize drug susceptibility of clinical TB isolates in resource-limited settings.

Introduction: Diagnosis of tuberculosis (TB) in children is usually based on chest radiography, tuberculin skin testing (TST), and conventional methods such as acid fast staining and culture. However, the efficiency of current TB diagnostic criteria in children has not yet been analyzed in Iran. Material and Methods: In this cross-sectional study, a total of 525 children (aged 1–15 years) were investigated. Among these cases, 198 were diagnosed as TB. The classic information, i.e., chest radiographs, age, sex, nationality, place of birth and laboratory examinations, was collected. Diagnostic and demographic characteristics of the studied children were analyzed by the available criteria. Results: Among the studied cases, 13.1% had extra-pulmonary TB, 72.7% had pulmonary TB, and the remaining 14.1% had both pulmonary and extra-pulmonary involvement. The male to female ratio was 38.9%/61.1%. The majority of patients were Afghanis (65.2%), and the remaining were Iranians (34.8%). About 82.8% of cases had a history of close contact. The incidence of TST, radiograph findings, history contact, clinical symptoms, and microbiology as diagnostic methods was 79.3%, 83.8%, 83.8%, 85.9%, and 58.1%, respectively. In this case study, 90.4% of patients fulfilled the criteria. Conclusions: Acid fast staining and culture analysis of gastric aspirates was determined as a high diagnostic value for the diagnosis of TB in children. However, this could not be considered as the gold standard for the diagnosis of TB in children, and other methods should be taken into consideration. Additionally, the results of analysis of the current diagnostic criteria indicated a significant accuracy and efficacy of the available criteria.

Background: Infants and young children are particularly susceptible to developing tuberculosis (TB) following exposure and infection, and isoniazid preventive therapy can substantially reduce this risk. This evidence-base provides the rationale for the policy and guidelines to screen and manage children that are household contacts of TB cases, prioritizing those with sputum smear-positive TB. The guidelines have been in place for decades, but are rarely implemented. IPT programs in high-TB burden countries are rare and fraught with low uptake and low rates of therapy completion. Methods: This study describes the demographics and outcomes of contact management and the IPT program for <5 years old at the Indus Hospital in Karachi, Pakistan from 2011 to 2014. Children <5 years old in contact with newly diagnosed sputum smear-positive pulmonary TB patients were referred by the counselor for contact evaluation. All children underwent a history, physical, CXR and TST. All contacts were started on IPT based on WHO recommendations after confirming that they had no TB disease. Outcomes were defined as children who were enrolled, started on IPT but never returned for follow-up (primary default), children who had one or more follow-up visits but did not complete treatment (default) and children who completed IPT (treatment completed). Results: A total of 240 children were enrolled in the Indus IPT program from 2011 to 2014. Of these, data from 184 contacts was analyzed in detail as the remainder were still on IPT. All children enrolled were less than 5 years of age (mean age 3 years) and 96 (52%) were males. Of all the enrolled children, 76/240 (31.6%) were <5% weight for their age (underweight), of these 52% were female children. A symptom of either cough or fever was reported by 29/184 (15.7%) children; however, all responded to routine antibiotics and did not have CXR findings suggestive of TB disease. Of the enrolled who had a TST done, 12/209 (5.8%) had a positive result. Analysis of outcomes revealed that only 60/184 (32.6%) completed 6 months of IPT (with no gender predisposition). Among those who completed therapy, none developed TB disease during follow-up. Outcome trends revealed an increase in completion rate (40% in 2013 compared with 26% in 2012), which may reflect improvement in counseling services in 2013. Children who had an initial symptom or were underweight at the start of IPT were more likely to complete treatment (p<0.01). Conclusion: Despite a large cohort of TB patients in the Indus TB program-5487 SS+ patients registered during the study period – this IPT program enrollment has been low. A high rate of patient default after the first visit indicates a lack of understanding about the benefit and safety of preventive therapy in young children among families of TB patients, and awareness enhancing efforts by community field teams will help improve outcomes. It is vital that the National TB Program strengthens and expands contact management with a community-based approach and incentives.

Background and objective: During recent years, the World Health Organization (WHO) proposed new software for improving the tuberculosis (TB) laboratory services. The protocol is known as “quality stepwise implementation tool” and is based on enforcement of quality assurance services through accreditation by the International Organization for Standardization (ISO) 15189. As a national reference TB laboratory (NRL) of Iran, the benefit and challenges of implementing this standard were analyzed. Material and Methods: The investigation was a cross-sectional study and was underlined by the committee of the Quality Management System (QMS) in the NRL. The required information was collected in two steps: first, the QC form that contains 334 quality questions (designed by WHO) was filled out. These questions determine 12 main organizational blocks of laboratory (facilities and safety¹, organization and personnel², documents and records³, management reviews⁴, client management and customer service⁵, equipment⁶, internal audit⁷, purchasing and inventory⁸, information management⁹, process control and internal / external quality management¹⁰, corrective actions occurrence¹¹, and incident management and process improvement¹²). The minimum rating of laboratories is stated to be from 55% to a maximum of 95%. In the second step, using the quality stepwise implementation tool, these blocks were in four phases, and 41 steps were analyzed. The data were compared using Fisher's Exact test and Pearson Chi-Square. Results: In the period 2013–2014, the rate of OMS in three focal points (pre-analytical, analytical, and post-analytical) in the NRL was in the range of 80–100%; whereas in previous years the rate ranged from 48% to 79%. Therefore, through QMS the NRL scores had reached a higher level. Based on available results, the NRL-assigned score was a 5-star rating. Conclusions: As the internal score of NRL reaches 5, an international evaluation of accreditation scheme is sought. This is the first investigation that used the quality stepwise implementation tool in Iran.

Background and objective: Nontuberculosis mycobacteria (NTM) are free-living saprophytes that are found in water, soil, animals and dairy products. During recent years, advances in molecular biology improved the recovery and accuracy of NTM identification. In the present study, similar approaches were used to differentiate environmental NTM. For this reason, the prevalence of environmental NTM in soil and water in two densely populated areas of Tehran were investigated. Material and Methods: A total of 2,540 samples were collected from soil (n = 1237) and water (n = 1303) resources. For the soil sample: digestion and decontamination were performed using modified Engbaeks method, and for the water sample: decontamination followed with ethylpyridinium chloride (CPC; final concentration of 0.05%) and standard protocol. The phenotypic and genotyping tests were performed on all positive cultures. For molecular tests, both 16S-23S RNA and hsp65 genes spacer PCR-RFLP were used. Results: Of the 2,540 collected samples, 285 (12%) were positive for mycobacterium. One hundred and sixty-nine (169; 60%) belonged to a slow growing mycobacteria (SGM) and 116 (41%) were rapid growing mycobacteria (RGM). The most frequent NTM were identified as Mycobacterium farcinogenes (51/485; 10.5%) and Mycobacterium fortuitum (38/485; 7.8%). However, M. farcinogenes is found to be dominant in both studied areas, but the frequency of RGM was different. In Shahre-ray, Mycobacterium senegalense was identified as the most frequent RGM (22/65; 34%), while in Varamin, M. fortuitum (20/51; 40%) had the most frequency. Conclusion: The molecular genotyping methods facilitate the rapid identification of environmental NTM. As the higher prevalence of NTM within a region increases the risk of human infection, this study outlines the importance of further investigation.

Aims and objectives: Current methods for drug susceptibility testing (DST) of Mycobacterium tuberculosis (MTB) are either costly or slow. As the prevalence of multidrug-resistant (MDR) strains increases, the need for fast, reliable, and inexpensive methods is obvious. This study evaluated a rapid colorimetric nitrate reductase assay (NRA) for direct DST of MTB directly from clinical sputum samples. Methods: A total of 111 sputa with positive microscopy results for acid-fast bacilli (AFB) with more than 10 AFB per high-power field were used in the study. The samples were decontaminated using the modified Petroff method. The NRA results were compared with the reference indirect proportion method. Results: The sensitivity and the specificity of the direct NRA were 90% and 97.3%, 92.6% and 98.2%, 52.9% and 100%, and 28.6% and 100% for rifampin, isoniazid, streptomycin, and ethambutol, respectively. The results were in most cases available in 28 days (84.3%). Conclusions: The direct NRA could be used as a rapid, inexpensive, and accurate method to determine rifampin and isoniazid susceptibility directly from sputum. The technique might become a valid alternative to traditional methods, especially in low-income countries.

Introduction: The four basic or “first-line” TB drugs are Isoniazid, Rifampicin, Pyrazinamide and Ethambutol. For the treatment of drug-resistant TB, the current TB drugs are grouped according to their effectiveness and experience of use, such as Streptomycin, pyrazinamide, rifabutin, kanamycin, amikacin, ofloxacin, etc. The drug susceptibility test (DST) is a time-consuming and costly method. Rapid molecular tests may be used by detection of related mutations. The aim of this study is to design and evaluate the quickest methods of detection. Materials and Methods: 120 resistant and susceptible clinical isolates of Mycobacterium tuberculosis (MTB) were evaluated for probable mutations in resistance-related genes. Molecular methods of polymerase chain reaction (PCR)-RFLP, AS-PCR and MAS-PCR as nested or semi-nested forms were used for mutation detection in katG, rpoB, pncA, rpsL, gyrA,inh, rrs, inhA and embA. Evaluation of ethA and pncA genes in the isolates was accomplished by sequencing. Furthermore, the sequencing method was used for all the genes as the golden standard. Results: 88% prevalence of the katG315 mutation was detected in INH-resistant isolates by AS-PCR and 95.6% by PCR-RFLP. In 93% of rifampin-resistant isolates point mutation at codons 516, 526 or 531 were detected by MAS-PCR method and 75% by AS-PCR method. In rapid detection of resistance to injectable drugs, the sensitivity and specificity of PCR-RFLP method for mutation detection in rrs gene by BSTFNI enzyme were 95/65% and 70/83%, by AJIi enzyme were 60% and 90/62% and by MAS-PCR method were 50% and 70/58%, respectively. Ofloxacin resistance was detected in 84.6% of resistant isolates by 4 endonuclease enzymes in PCR-RFLP method and sensitivity and specificity of a MAS-PCR method were 86/11% and 100%, respectively. Sensitivity and specificity of a MAS-PCR method for pncA were 66% and 90% and for PCR-RFLP method for rpsL were 90% and 95%, 36.5% (CI:0.09–0.45) and 100% only for one resistance-related codon for emb gene, respectively. Ethionamide and Pyrazinamide resistance in resistant isolates was proved by 100% sensitivity by the sequence method. Conclusion: Molecular methods of PCR-RFLP, MAS-PCR and sequencing were successfully used for rapid detection of first- and second-line antimycobacterial drugs.

Introduction: In 2008, the World Health Organization (WHO) approved a rapid molecular test known as Line Probe Assay (LiPA) for the diagnosis of multidrug-resistant tuberculosis (MDR-TB) in pulmonary specimens. Due to lack of available data, its use in extra-pulmonary tuberculosis (EPTB) specimens has not been determined yet. Recommendations for the use of Xpert MTB/RIF in EPTB have been issued, but alternative rapid molecular tests for EPTB diagnosis need to be evaluated. Method: LiPA (GenoType® MTBDRplus 2.0 Hain Lifescience) was performed on 97 specimens of extrapulmonary origin at the clinical laboratory at Aga Khan University (Dec 2012–Jan 2014). The results were compared with TB cultures in solid and liquid medium. Results: 97 specimens were tested simultaneously for culture and LiPA, including pleural fluid (35), CSF (22), pus (17), tissue (10) and urine (3). Concentrated smear was positive for 7 while 14 were culture positive for MTB. All 7/7 smear-positive specimens were LiPA positive, while 6/7 were culture positive. Amongst the smear-negative specimens, 8/90 were culture positive and 9/90 were LiPA positive. The overall sensitivity and specificity of LiPA for the detection of MTB in the EPTB specimens was 71.4% (95% CI 41.9–91.4) and 92.8% (95% CI 84.9–97.3), respectively. The highest sensitivity (100%) was seen in urine as 2 of 3 which were culture positive were also LiPA positive. Pus samples showed sensitivity of 85.7% and specificity of 70%. Conclusion: The study shows that LiPA has good overall sensitivity and specificity compared with culture. Although the number of samples was very small, the applicability appears to be most useful in urine and pus specimens and should be explored further as a diagnostic tool in these cases.

Background: Pakistan has an annual tuberculosis (TB) incidence rate of 233 per 100,000 population and ranks sixth among the high TB burden countries worldwide. Tuberculous pleural effusion (TPE) occurs in up to 25% of TB patients. Owing to the pauci-bacillary nature of the pleural fluid, the diagnosis of TPE is a challenge. Objective: The aim of the present study is to evaluate the performance of MTBDRplus line probe assay in terms of its sensitivity and specificity for the rapid TB diagnosis in TPE and frequency of multidrug-resistant TB (MDR-TB) in TPE through the detection of rifampicin (Rif) and isoniazid (INH) resistance. Methods: This study is an ongoing prospective cross-sectional study being performed at Aga Khan University Hospital. Patients aged 18 years or more with suspected TPE were asked to participate. In addition to a preformed questionnaire which gathered information regarding clinical history and previous laboratory investigations, already tapped pleural effusion samples are tested for pleural fluid composition, TB smear and culture, MTBDRplus line probe assay and ADA analysis. Results: So far, 22 patients with a mean age of 49.81±21.75 (range: 18–86; IQR: 29.25, 68.00) years have been enrolled. Almost two-thirds are males (n = 15, 68.2%). The majority of the patient population belongs to Karachi (n = 18, 81.8%), followed by Quetta (n = 2, 9.1%), Faisalabad (n = 1, 4.5%) and Sukkur (n = 1, 4.5%). Half of the patients had a history of TB contact (n = 11, 50.0%), whereas only two (9.1%) had a past history of TB. Fifty percent (n = 11) had a right-sided pleural effusion. Effusion size was moderate in 36.4% (n = 8), mild in 27.3% (n = 6) and massive in 22.5% (n = 5). Pleural fluid MTBDRplus positively detected TB in 9.1% (n = 2) of cases. INH resistance was detected in one of these two cases. Pleural fluid culture was positive in two cases. Pleural fluid AFB smear was negative in all cases. Conclusion: Given the study is still under way, it is hoped that the results of this study may investigate the relevance, sensitivity and specificity of MTBDRplus line probe assay as a tool in the rapid detection of MTB in TPE and the early detection of MDR-TB through the detection of Rif and INH resistance. It may also provide an estimate of prevalence of drug resistant strains in TPE, which would be useful for guiding empirical anti-tuberculous therapy in TPE.

Introduction: GeneXpert MTB/RIF is a fully-automated diagnostic molecular test which simultaneously detects tuberculosis (TB) and Rifampicin (RIF) drug resistance. The purpose of this study is to evaluate the accuracy of the GeneXpert MTB/RIF test for the detection of Mycobacterium tuberculosis complex (MTBC) in lymph node specimens. Materials and Methods: This study was conducted simultaneously in Abderrahmen Mami hospital, the National Reference Laboratory for Mycobacteria in Tunisia and Hedi Chaker Bacteriological Laboratory of Sfax, from January to December 2013. In total, 160 lymph node specimens (tissues and aspirates) were split and processed simultaneously for auramine fluorescent staining and immunohistochemical staining. In addition, conventional culture on both Lowenstein–Jensen and liquid medium (Bactec MGIT 960 BD system) and the new molecular-based GeneXpert MTB/RIF assay system were performed. Positive cultures were confirmed after the detection of the MPT64 antigen (SD BiolineTBAg MPT64 Rapid) and molecular identification (Genotype MTBC Hain Lifescience). Results: Among the 160 samples tested, the GeneXpert detected the DNA of MTC in 120 samples (75%). Standard bacteriological assays including AFB microscopy and culture were positive, respectively, in 37 (23.13%) and 74 (46.25%) specimens. Mycobacterium bovis was isolated in 67.4% of positive cultures. No Rifampicin resistance was detected. GeneXpert sensitivity and specificity results were assessed according to smear and culture results, clinical and histological findings. The sensitivity and specificity of the Xpert assay was 91.5% (118/129) and 70.4% (19/27), respectively. When compared with bacteriological findings, the concordance between the GeneXpert and bacteriological results was 69.4%. Conclusion: The implementation of the GeneXpert MTB/RIF assay may dramatically improve the rapid diagnosis of lymph node TB. This rapid TB test may complete usual methods (conventional microscopy, culture and histopathology).

Background: Drug resistance is the major problem in tuberculosis (TB) treatment and control. In the present study, it was decided to consider the prevalence and distribution of multi-drug resistance Mycobacterium tuberculosis (MDR-TB) in Iran using meta-analysis based on systematic review of articles published, which will provide more detailed information to clearly overview the status of TB. Methods: All original articles published in literature database including PubMed, Sciencedirect, Web of Science, Google Scholar, Biological abs, Iranmedex, and SID systematically reviewed the prevalence of MDR-TB. The summarized data has been analyzed statistically. Results: Final analyses included 39 samples that have been selected from 535 studies. Overall MDR-TB prevalence in Iran was estimated to be 0.15 (95% CI=0.14–0.16). Corresponding estimates by city were Mashhad 0.22 (95% CI=0.18–0.27), Tehran 0.16 (95% CI=0.15–0.17), and Zahedan 0.10 (95% CI=0.07–0.15). In 2003 there was a high report of MDR-TB 0.88 (95% CI=0.75–0.96). Conclusions: According to the results, prevalence of MDR-TB in Iran is lower than worldwide WHO report. Effective control program is needed in order to control and eradicate the spread of drug resistance strains.

Introduction: The tuberculin test is the most common method used for the detection and control of bovine tuberculosis (TB). Specificity and sensitivity of this tuberculin test is low, so it is better to perform another test in parallel for investigation of the disease. The ELISA test is user friendly, so it can be an appropriate test for performing in parallel with the tuberculin test for an accurate detection. The aim of this study is the development of an ELISA test (kit) based on proteins smaller than 10kDa (specially ESAT-6) for the detection of TB in cattle. Materials and Methods: Proteins lower than 10kDa M.W. were isolated from sonicated bacteria and were precipitated with ammonium sulfate. Protein assay was done after dialysis. Then a checkerboard was done for the determination of the best concentration of antigens and antibodies. There were 114 serum samples that were isolated; 100 cows that three continual tuberculin tests were negative and 14 serum samples were positive (in the tuberculin test). Culture and PCR were done from lymph nodes (or tissues) of tuberculin positive cows. Stability was investigated on days 0, 1, 3, 5 and 7 in 37 °C. Results: The best concentration of antigens and antibodies were acquired 200ng and 1/100 respectively. All of the 100 samples that were negative in the tuberculin tests were negative in this ELISA kit as well, but out of 14 samples that were positive in the tuberculin test, just 8 samples were positive in the ELISA kit. Meanwhile, the results of culture and PCR were compatible with the ELISA kit's results. There was a variation of less than 10% between days 0 and 7 at 37 °C that shows at least 1 year stability of this kit in refrigerator temperatures (4–8 °C). Conclusion: Performing the ELISA test based on ESAT-6 antigen shows that this test can be a suitable way for screening beside the tuberculin test for accurate detection. It is noticed that the specificity of the ELISA test was determined more than the tuberculin test, especially since the culture and PCR are gold standards. Therefore, incorrect sampling can change the specificity of the tuberculin test. The results of this kit can encourage designing of an ELISA kit for the detection of human TB.

Objectives: Drug-resistant tuberculosis (TB) is a major challenge in controlling TB. HIV-positive patients are vulnerable to TB 100 times more than the general population. Drug-resistant TB leads to high morbidity and mortality in this population. In this study, the outcome of treatment of drug-resistant TB among HIV positive patients from 2003 to 2013 in a tertiary center in Iran will be reviewed. Materials and Methods: All HIV-positive patients with any drug-resistant TB from 2003 to 2013 were selected. The outcome of treatment was extracted from patients’ charts. Results: Out of 269 TB-HIV patients, 34 patients were recruited. All patients were male and the mean age was 37.38±7.03. Isoniazid (INH) resistant, rifampin (RIF) resistant and multi-drug resistant (MDR) was diagnosed in 11 (32.4%), 7 (20.6%) and 16 (47.1%), respectively. Mean CD4 count was 91.61±23.55. Outcome of treatment in the INH-resistant cases was cured in 5 (45.5%), failure in 2 (18.2%) and death in 4 (36.4%). In the RIF-resistant group, outcome was as follows: cured 5 (71.4%) and failure in 2 (28.6%). In the MDR-TB patients’ group, cured, failure and death were 12 (75%), 2 (12.5%) and 2 (12.5%), respectively. Conclusion: Treatment of drug-resistant TB can be achieved despite considerable mortality.

Background: Mycobacterium tuberculosis (MTB) PE_PGRS genes belong to the PE multi-gene family. Although the function of the members of the PE_PGRS multi-gene family is not yet known, it is hypothesized that the PE_PGRS genes may be associated with genetic variability. Material and Methods: Whole genome sequencing analysis was performed on (n = 37) extensively drug resistant (XDR) MTB strains from Pakistan which included Central Asian (n = 23), East African Indian (n = 2), X3 (n = 1), T group (n = 3) and Orphan (n = 8) MTB strains. Results: By analyzing 42 PE_PGRS genes, 111 SNPs were identified, of which 13 were non-synonymous SNPs (nsSNPs). The nsSNPs identified in the PE_PGRS genes were as follows: 6, 9, 10 and 55 present in each of the CAS, EAI, Orphan, T1 and X3 XDR MTB strains studied. Deletions in PE_PGRS genes: 19, 21 and 23 were observed in 7 (35.0%) CAS1 and 3 (37.5%) in Orphan XDR MTB strains, while deletions in the PE_PGRS genes: 49 and 50 were observed in 36 (95.0%) CAS1 and all CAS, CAS2 and Orphan XDR MTB strains. An insertion in PE_PGRS6 gene was observed in all CAS, EAI3 and Orphan, while insertions in the PE_PGRS genes 19 and 33 were observed in 19 (95%) CAS1 and all CAS, CAS2, EAI3 and Orphan XDR MTB strains. Conclusion: Genetic diversity in PE_PGRS genes contributes to antigenic variability and may result in increased immunogenicity of strains. This is the first study identifying variations in nsSNPs, Insertions and Deletions in the PE_PGRS genes of XDR-TB strains from Pakistan. It highlights common genetic variations which may contribute to persistence.

Aims and objectives: The aim of this work was to highlight macro- and micro-geographical cleavages and phylogeographical specificities of circulating Mycobacterium tuberculosis complex (MTBC) clones worldwide to underline the demographic, epidemiological and drug resistance characteristics of major genotypic lineages. Methods: MTBC genotyping data was retrieved from an international TB genotyping database (SITVIT2) developed at Institut Pasteur de la Guadeloupe, with data on 111,635 MTBC clinical isolates from 169 countries of patient origin – almost twice as compared with the previous SITVITWEB version released in 2012. Strains typed by the most commonly used methodology for TB genotyping today were retained, i.e., spoligotyping based on the polymorphism of the direct repeat (DR) locus, and MIRU-VNTR minisatellites used in 12-, 15-, or 24-loci formats; or a combination of two methods which constitutes the gold-standard for optimal TB surveillance at the national, regional, and global levels. In-depth phylogenetical analysis of circulating strains was performed, followed by data-mining in conjunction with their geographical distribution, drug-resistance, demographic and epidemiologic characteristics. Statistical analyses were used to pinpoint geographical specificities of circulating MTBC lineages. Results: In the present work, the latest 6th version of this database was described. This study shows how the addition of new tools and supplementary information available in the SITVIT2 allowed global mapping of combined search results on MTBC genotypes, and underlined important micro- and macro-geographical cleavages for all major MTBC lineages. As an example, these results relating to the Beijing lineage (n = 10,850 strains) underlined certain specificities, e.g., (i) Japan was unique in having a majority of Beijing patients >60 years of age, while Russia was unique in having a very high proportion of male patients (male to female sex ratio of 4.93); (ii) while modern Beijing strains have spread worldwide, ancient Beijing sublineage was endemic in Japan and to a lesser extent in China; (iii) MIRU-based geo-specificity suggested probable transmission routes among certain countries; (iv) current hotspots for emergence/reemergence of Beijing strains are not necessarily countries with its highest prevalence (e.g., Japan), but rather the ones where this genotype is associated with a younger age of patients, high demography/population density, and drug-resistant TB (such as China, Indian sub-continent, and South-East Asian countries), etc. The specificities for other lineages will be further emphasized as well. Conclusions: This research was focused to improve the in-depth phylogenetic characterization of MTBC lineages in conjunction with epidemiological analysis of circulating clones to generate evidence-based geographical mapping of predominant clinical isolates of tubercle bacilli causing the bulk of the disease both at the country and regional levels. Further superimposition of these maps with socio-political, economical, and demographical characteristics available through Geographic Information Systems (GIS) allows access to a precise view of prevailing disparities as seen at the level of the United Nation's sub-regional stratification. An in-depth comprehension of these disparities and drawbacks is important to take appropriate actions by decision-makers and public health authorities alike, in order to better monitor, understand and control the tuberculosis epidemic worldwide.

Aims and objectives: Tuberculosis (TB) is considered one of the most important pathogens in the world. Iran has a moderate TB incidence, but borders two high-burden TB countries to the east and one high-burden multidrug-resistant (MDR)-TB country to the north. Limited information is available on the genetic diversity and transmission dynamics of MTB in Iran. To determine the MTB genotypes and their transmission patterns among patients, a collection of isolates from different parts of Iran were genotyped. Methods: Genotypes were generated by means of standard 15-locus variable number tandem repeat (VNTR) for 121 MTB clinical isolates collected from three provinces of Iran, including Sistan–Baluchestan (southeast province of Iran, with the highest rate of TB), Kermanshah (western part of Iran with high TB/human immunodeficiency virus [HIV] cases) and Tehran (the capital of Iran). Results: Sixty-six distinct mycobacterial interspersed repetitive unit (MIRU)-VNTR patterns were detected among 121 isolates. Seventy-five strains were grouped into 20 clusters, and 46 isolates were unique. The genetic diversity of strains from Sistan–Baluchestan was higher than that in the other province. All isolates from Tehran or Kermanshah that were grouped into clusters shared more identical patterns with Sistan–Baluchestan. The Hunter-Gaston discriminatory index (HGDI) was 0.972, indicating a high power of discrimination for MIRU-VNTR typing. The MIRU 16, ETR-A, ETR-E, MTUB04 loci were designated as highly discriminative. Conclusions: MIRU-VNTR typing showed a high genetic diversity and suggests the possibility of transmission from Sistan–Baluchestan to other provinces of Iran. This method could be considered a suitable tool for studying the transmission routes of TB and leading to more appropriate measures for TB control. MIRU-VNTR typing leads to a much better understanding of the bacterial population structure and phylogenetic relationships between strains of MTB in different regions of Iran.

Aims and objectives: Molecular methods in bacteriology showed Mycobacterium tuberculosis (MTB) to have families, such as Beijing, Haarlem, Africa, East-African-Indian, Latin American and T. Each year, tuberculosis (TB) causes more than 2 million deaths in the world. The aim of this study is to determine the prevalence of the MTB T family in different countries using a systematic review and meta-analysis. Methods: Data sources of this study are comprised of 151 original articles (2000–2012) that were published in the literature databases: PubMed) www.ncbi.nlm.nih.gov/pubmed, the years of coverage of this web site is 1955). The prevalence of MTB T family in different continents, including America, Europe, Asia and Africa, were studied. Inclusion criteria were: research articles with full text, and articles with abstracts in English. Excluded studies were: review articles, congress abstracts, studies that reported in languages other than English and studies that were not available in abstract or full text, studies that their sampling location was uncertain, studies that locations of sampling were performed at the same time, and studies that their data were not clear. These data were analyzed using meta-analysis and random effects models with the software package Meta R, Version 2.13 (p<0.10) Confidence Interval (CI: 95%). Results: 44 of 151 articles were applied; the prevalence of MTB T family in different continents was in America (Spain; 80% in 2009), Europe (Sough; 48.7% in 2008), Africa (South Africa; 23% in 2011) and Asia (Iran; 32.30% in 2010). The highest and lowest occurrence rate of MTB T family was Spain 80% in 2009 and in South Korea 2.1% in 2010, respectively. Pooled estimation of MTB T family samples was 19,873. Also, fixed effects for years showed that CI was between 5%–40%. Conclusion: Different research was studied on the prevalence of MTB T family in various countries. In this study, Spain with 80% in 2009 had more prevalence compared with other countries. The presence of MTB T family in studies indicate effective programs and management are required to control and prevent the spread of MTB, especially the T family.

Background: Tuberculosis (TB) disease caused by Mycobacterium tuberculosis (MTB) is still an important cause of morbidity and mortality. Latin American and Mediterranean (LAM) is one of a family of phylogenic MTB, and its name is derived from the geographical area that has been isolated. The objective of this study is to determine the prevalence of MTB LAM family worldwide by meta-analysis and a systematic review. Methods: Data sources for this study are comprised of 70 original articles (2001–2013) that were published in the literature in several databases, including PubMed, Science Direct, Google Scholar, Biological abstracts, ISI Web of Knowledge and IranMedex. Data were analyzed using meta-analysis (Meta R, Version 2.13 package [p<0.10], CI: 95%). Results: The highest and lowest occurrence rate of the LAM family in MTB was in Europe and Asia. Totally, the prevalence of the LAM family in Europe, Africa, America and Asia was 32.10%, 29.20%, 14.4% and 0.2%, respectively. Conclusions: This study shows that MTB LAM family is prevalent in European countries. LAM sub-lineage is a major focus of studies carried out in different countries. Proper technique for prevention of transmission of MTB is necessary.

Aims and objectives: Tuberculosis (TB) continues to be an important public health problem in the world. Genotyping of Mycobacterium tuberculosis MTB isolates will contribute to understanding and controlling the spread of the strains. This study aimed to determine the epidemiology of tuberculosis in Iran and to analyze the relationship between bacterial genotype and drug resistance. Methods: Spoligotyping and proportion method of drug susceptibility testing (DST) were used to determine genetic diversity and drug susceptibility of 291 MTB isolates respectively. Results: Spoligotyping produced 75 distinct patterns. 251 isolates (86.2%) were grouped in 35 clusters, while the remaining isolates showed a unique pattern. Ural (34.3%), Central Asian strain (CAS) (24%), T (18.2%), MANU2 (7.5%) and Latin American-Mediterranean (LAM) (6.1%) were found to be five common lineages of the studied population. The five largest clusters were Ural/SIT127 (15.8%), CAS1/SIT26 (9.2%), T1/SIT53 (6.1%), T1/SIT284 (5.4%) and CAS1/SIT25 (4.4%). According to spoligotyping data, 74.2% of infections were attributed to recent transmission. About 5% of isolates had multi-drug resistance (MDR) which was significantly associated with the Beijing genotype. Despite the high prevalence, the Ural family was not characterized with MDR. Conclusions: The present study highlights dominance of Ural, CAS and T families in Iran. The high rate of clustering obtained by this method suggests that recent transmission probably contributes more to TB infections rather than reactivation in this area. Bio-geographical specificity of CAS and T families to border provinces of Iran including Sistan-Baluchestan and Kermanshah, respectively, suggested that these family strains might be transmitted from these regions to other provinces of the country.

Aims and objectives: Clustering of and determining Mycobacterium tuberculosis (MTB) strains is of great concern in control programs of tuberculosis (TB). Identification of transmission type and tracking the infection source is also highly necessary. The aim of the present study is to track and determine the type of MTB infection, as well as its relationship with demographic factors using PGRS-RFLP. Methods: In this cross-sectional study, 84 smear-positive patients from 5 frontier provinces (East Azerbaijan, West Azerbaijan, Ardebil, Kurdistan, and Kermanshah) were investigated according to PGRS-RFLP. Demographic data were collected using a questionnaire. The results were analyzed by SPSS-18 and G-Box. Result: Based on clustering, recent transmission was 66%. Most clusters were obtained from Kurdistan and Kermanshah. Vaccination record (p = 0.49) and treatment group (without previous treatment) (p = 0.004) had a significant relationship with clustering. Other demographic factors including age, gender, religion, drug abuse, smoking, history of migration, and marital status did not show a significant relationship with clustering. Conclusion: Genetic variation of MTB is high in this region. The rate of recent transmission based on clustering was unexpected (global average is 30–40%). Recent transmission was more dynamic in west Iran than northwest Iran. The strong relationship between treatment group 1 (without previous treatment) and clustering based on PGRS-RFLP can demonstrate the high correlation between molecular and classic information. In addition, the significant relationship between vaccination record and clustering highlights the necessity to conduct more extensive studies.

Tuberculosis (TB) remains a common health problem in the State of Qatar. Qatar has a population of around 2.2 million; about 25% are Qatari Nationals. Most of the population reside in the capital Doha. The incidence rate of TB was 41/100,000 in 2012. 90% of the patients are non-Qataris. Most of the TB patients are among male laborers from Asian high TB endemic areas. The size of the labor force in Qatar increased by fourfold during the last decade. Qatar has one National TB Reference Laboratory, which covers all country sectors, public (primary health centers and all public hospitals) and private hospitals and clinics. Qatar has a highly effective National TB Control Program. The Qatar National TB Reference Laboratory does a full range of laboratory diagnosis, microscopy, culture (GMIT 960 and LJ media), identification by immuno-chromogenic (BD MGIT TBc identification test) and molecular methods (Xpert MTB/RIF and Hain System). First-line drug susceptibility testing is done for all MTBC positive patients and repeated after 6 months if the results are still culture positive. Second-line drug susceptibility test for MDR is done at the Mayo Clinic Reference Laboratory. The lab receives an average of 70 samples daily, the majority for culture. Xpert MTB/RIF is performed on all extra-pulmonary samples directly and is also performed on at least one sample for all TB patients to speed up the identification and Rifampicin susceptibility testing. Smear positivity rate ranges from 50% to 80%. In the near future, molecular typing will be performed on all MTBC isolates from the patients. Hamad Medical Corporation is CAP accredited, and the lab is also CAP accredited. External quality assurance covers microscopy, culture and drug susceptibility testing. All TB care, including care for MDR-TB and latent TB infection, are available free of charge for Qatari and non-Qatari residents.

Aims and objectives: The aim of this study is to determine the prevalence of pyrazinamide (PZA) resistance among multidrug-resistant Mycobacterium tuberculosis (MDR-TB) strains isolated in Sweden between 2003 and 2013 and to study the correlation between phenotypic PZA susceptibility testing and the presence of pncA mutations. Methods: A total of 117 clinical TB isolates defined as MDR were tested for PZA resistance in the Bactec MGIT 960 system and subsequently screened for mutations within the pncA gene using Sanger sequencing. Results: PZA is a first-line key drug in the treatment of TB, including MDR-TB susceptible to PZA, and the phenotypic test showed that 53% of the MDR isolates from Sweden were PZA resistant. Preliminary sequencing results show that 95% of the PZA-resistant strains had a mutation in the pncA gene or its putative promoter. Of the PZA-susceptible strains, 73% had a wild type pncA, a proportion which increases to 89% if the silent mutation Ser65Ser (TCC65TCT) is included in this group. Moreover, the sensitivity and specificity of pncA sequencing, using the Bactec MGIT 960 system as the gold standard, was determined to be 95% and 89%, respectively. The results also showed an increase of PZA resistance among the Swedish MDR isolates during the studied period. Between 2003 and 2008, the prevalence of PZA resistance was 44% (n = 48), while in the latter period (2009–2013) the prevalence had increased to 59% (n = 69). No major difference in PZA resistance was seen among MDR patients of various geographical origins. Conclusions: The results from the last 11-year period demonstrate an increase of PZA resistance among Swedish MDR cases. Additionally, the detection of pncA gene mutations, or their absence, was confirmed as a useful method for predicting PZA resistance.

Aims and objectives: Tuberculosis (TB) in children is an important predictor of ongoing transmission within a population. Pakistan has a high TB and multidrug resistant (MDR) TB burden, putting children at high risk for acquiring the disease. Despite this high MDR burden status, MDR TB rates have not been reported in children from Pakistan. Methods: Aga Khan University mycobacteriology laboratory TB culture and susceptibility records have been reviewed for samples received from children 0–14 years of age from all over Pakistan. Specimens were further segregated by age, site (pulmonary vs. extrapulmonary), and drug resistance profile. Results: Of 50,048 specimens received for culture between 2009 and 2012, only 2.1% (n = 1059) were obtained from children. Culture positivity rate among children was 1.5 times higher than that seen in adults (37.7% vs. 24.2%). Of 399 positive cultures, 72.9% (n = 291) were pulmonary, while 37.1% (n = 108) were extrapulmonary, and 73.9% (n = 295) were obtained from females, while 26.1% (n = 104) were obtained from males. Susceptibility testing revealed 41.4% (n = 164) of isolates to be MDR, 89.1% (n = 147) of which had a pulmonary source (i.e., gastric aspirates, bronchial washings, or sputa). MDR TB also showed an older-age predominance where 89.1% (n = 147) of MDR was seen in the 10–14 age group. About half (50.9%; n = 203) of all Mycobacterium tuberculosis isolates from children were isoniazid mono-resistant. Conclusions: Although pediatric specimens comprise only a small proportion of specimens received for mycobacterial culture, the culture positivity rate is much higher among children than in adults, reflecting a need to encourage culture testing of suspected pediatric TB patients. Predominance of positive cultures among female children and MDR TB in the older age groups highlights the need for focus on control efforts among this demographic.

Introduction: In many developing countries, like Iran, newborns receive the BCG vaccination for prophylaxis against mycobacterial infections. Although in many children or infants BCG infection after BCG vaccination is rare, children with primary immunodeficiency are at high risk of developing BCG-osis. According to many studies, some cytokines such as IL-12 play a major role in immunological responses in dealing with mycobacterial infections. Therefore, this research studies the relationship between polymorphism of genes encoding this cytokine and BCG-osis in Iranian children. Method: Thirty-four children up to 7 years old with BCG-osis and 34 healthy children vaccinated with BCG during the first two days of life were chosen for this analysis. DNA samples were extracted from both groups. Single nucleotide polymorphisms (SNPs) in IL-12 (at +705, +1158, +1196 and +1637) genes were assessed using PCR-RFLP. Allele and genotype frequencies in cases and controls were compared using the Pearson Chi-Square test. Result: The prevalence of IL-12 705 AG (heterozygote mutant) genotype was 50% in patients, but in the control group this rate was 38.2%. Likewise, AA (frequent hemozygote) genotypes were 52.9% and 38.2% in patients and control groups, respectively (p-Value >0.05). It was observed only homozygote alleles for 1158, 1196, 1637 and 1664 positions in both patients and control groups. However, there was no significant association between case and control groups. Conclusions: BCG vaccination in infants can cause protective immunity, and IL-12 plays an important role in immune responses. However, this study did not show any significant association between polymorphisms in IL-12 (at +705, +1158, +1196 and +1637) and risk of developing BCG-osis.

Background and objective: Diabetes mellitus (DM) has an effect on many aspects of tuberculosis (TB). The aim of this study is to determine the impact of DM on anti-tuberculosis drug resistance in new cases of TB patients. Materials and Methods: A case-control study was conducted on all newly diagnosed pulmonary TB adult patients with DM as cases and without DM as controls who were hospitalized and treated at the National Research Institute of Tuberculosis and Lung Disease (NRITLD) from May 2013 to October 2013. A molecular resistance test for rapid detection of resistance to isoniazid and rifampin was done for all smear-positive TB patients. A multivariate analysis was performed to determine the impact of DM on any anti-TB drug resistance. Results: 45TB cases with DM and 45TB cases without DM were included. TB cases with DM were more likely to be older (61 vs. 47 years, p = 0.001). Two TB–DM patients had multidrug-resistant TB (MDR-TB) (4.4%) compared with zero cases of MDR-TB in the control group, and TB–DM cases were resistant to at least one drug (11.1% vs. 4.4%, p = 0.43). DM remained significantly associated with any drug resistance (OR: 6.32, 95% CI: 1–40.72) in multivariate analysis. Conclusion: New TB patients with DM are at increased risk of anti-TB drug resistance. More studies are needed to confirm these results.

Introduction: Opportunistic fungal organisms, such as Aspergillus and Candida species, tend to cause diseases in tuberculosis (TB) patients. Respiratory tract mycosis has clinical and radiological characteristics which are very similar to TB, thereby making the disease easily misdiagnosed. TB can be a predisposition to serious opportunistic fungal infections. Prolonged use of anti-TB drugs promotes the growth and reproduction of opportunistic fungi and, in turn, aggravates the course of the underlying process in the lung tissues. The study objective is to determine fungal coexistence with Mycobacterium tuberculosis (MTB) in patients attending the Central Tuberculosis Reference Laboratory of the Health Center in Ghaemshahr city, Iran. Material and Methods: Twenty-five participants were recruited into the study during two months. For each patient, three successive morning samples during one month were collected into sterile wide-neck universal sputum mugs. Specimen collection was taken for analysis at the Laboratory. None of the participants in this study had been placed on anti-fungal therapy. Sputa samples were studied for fungal elements and MTB. Direct microscopy by three stainings – KOH+Calcofluor white, Gomori-methenamine-silver and Giemsa – and fungal culture were done on Sabouraud Dextrose agar. Analysis for acid-fast bacilli (AFB) was done by the Ziehl–Neelsen technique. Results: A total of 75 fresh sputa samples were collected. Branching hyphae in 10 samples of 5 (20%) patients and pseudo-hyphae and blastoconidia in 12 samples of 5 (20%) patients were seen together with TB. Aspergillus flavus and Candida albicans were identified in the culture method and mycological exams. Discussion: Mycotic disease is an important coexistence in patients with TB. Although active mycosis may be an independent marker of advanced immunosuppression, it may also act as a co-factor in accelerating and amplifying the clinical course of TB disease. It is necessary in TB patients to increase the cure rate considering coexistencing opportunistic infections.

Background and objective: One third of the world population is currently infected with Mycobacterium tuberculosis (MTB), but only between 5% and 10% of them show signs of tuberculosis (TB). Factors such as aging, drinking alcohol, and smoking can be involved in contributing to TB progress, but host genetic factors have the most important role in the progression of this disease. Cytokine polymorphisms are among the most important genetic factors influencing an individual's susceptibility and development of the disease. The present study is designed to evaluate the association of interferon-γ (IFN-γ) and p₂x₇ polymorphisms in pulmonary TB patients. Material and Methods: Single-nucleotide polymorphisms in IFN-γ and p₂x₇ genes in patients [n = 100] were analyzed and compared with controls [n = 100]. The genotypes of the above-mentioned genes were investigated by the PCR-RFLP method. Statistical analysis was performed using chi-square test to determine statistical associations between cases and controls. Results: The results indicated that there is a significant association between control and patient in IFN-γ −611 allele (p<0.05). Furthermore, there was a significant association between the two groups in p₂x₇ −1513 and −762 alleles (p<0.05). However, in allele 2109 of IFN-γ gene there was not any association (p > 0.05). Discussion: The presence of polymorphisms in the region-611 of IFN-γ-R1 gene and p₂x₇ 1513/−762 alleles may increase the host susceptibility to MTB infections. Consequently, genotype determination of these special alleles can be regarded as important tools to identify infected persons at high risk of TB.

Introduction: Necrotizing sarcoid granulomatosis (NSG) is a rare syndrome with unknown etiology. The disease is frequently confused with sarcoidosis and other granulomatous diseases. Diagnosis is made based on typical histologic criteria. No specific laboratory finding can confirm NSG diagnosis. The gender ratio of women to men has been reported as being as high as 4:1 and has a good prognosis. Methods and Results: In this report, the clinical and genetic features were surveyed of a 36-year-old male with extra-pulmonary NSG with unique manifestations, such as inguinal mass with positive smear and negative culture for the infection of Mycobacterium tuberculosis (MTB), which was not responsive to the first-line TB treatment and was characterized as a multidrug-resistant (MDR) tuberculosis (TB). Later on, he was admitted for the MDR cure, and he did not react to the gold standard of MDR treatment. Finally, he presented with a huge lymphoid granuloma with massive ascites that was diagnosed as an NSG by IHC. He cured well with prednisolone and all symptoms of the disease were gone. At the hospitalization time, all laboratory experiments were well planned, such as a workup for the detection of defects of loop IL-12/IFN-γ, HLA-DR typing, and immunologic workup by flow-cytometry analysis. Conclusion: This is the first case report from patients with unique features of NSG combined with MTB.

Objective: China has the second highest burden of tuberculosis (TB) worldwide, and it is also facing a remarkable increase of Diabetes Mellitus (DM) in the population. DM patients are at high risk of contracting TB and patients with DM–TB comorbidity usually present a relatively poor disease progress. To achieve the goal of TB control on incidence and mortality, it is of great importance to understand the epidemiology of DM–TB comorbidity in the Chinese population. This study aims to understand the prevalence of DM–TB comorbidity in newly diagnosed active TB patients and its demographic distribution in eastern rural China. Methods: A population-based cross-sectional study was carried out in four counties in eastern rural China, two in Jiangsu Province (GDP per capita in 2013: 74699.37 yuan, prevalence of DM: 8.50%) and two in Jiangxi Province (GDP per capita in 2013: 31835.53 yuan, prevalence of DM: 7.63%). All newly diagnosed and registered active TB patients from April 2013 to March 2014 were investigated using a structured questionnaire. Fasting blood glucose levels were tested to screen potential DM patients. Confirmation of diagnosis was made by glycosylated hemoglobin A1c (HbA1C). Results: In total, 1406TB patients from four counties were recruited with 447 (33.8%) being smear-positive TB. The average age of patients was 41 years, and the male to female ratio was 1019 (76.2%) to 319 (23.8%). Overall, 71 DM–TB comorbidity patients were detected; of them 52 (73.24%) were from Jiangsu and 19 (26.76%) from Jiangxi. The prevalence of DM–TB comorbidity was 5.05% of the 1406TB patients. The prevalence of DM–TB in Jiangsu (9.29%) was 4.13 times higher than Jiangxi Province (2.25%, p<0.01). It was found that ethnicity, family history of diabetes, diet habit and Province were associated with the prevalence of DM in TB patients. Compared to patients from Jiangxi Province and Han ethnicity patients, TB patients from Jiangsu Province (60.2% vs. 39.8%, OR: 24.17, 95% CI: 4.94–118.32) and minority patients (1.4% vs. 98.6%, OR: 24.29, 95% CI: 1.69–349.11) had a higher prevalence of DM–TB comorbidity. It was found that patients with a family history of DM had an increased possibility of DM–TB comorbidity (2.6% vs. 97.4%, OR: 6.46, 95% CI: 1.05–39.70), as well as those having a diet consisting of salty food (25.2% vs. 74.8%, OR: 3.41, 95% CI: 1.25–9.29). Conclusion: DM–TB comorbidity is more prevalent in areas with better economic status and higher prevalence of DM. Patients with a DM family history might be at high risk of DM–TB comorbidity. A TB control program should be integrated with DM screening and management program in eastern rural China.

Introduction: This study was conducted to assess the rate of human P2X₇ and TNF-α gene polymorphisms among Iranian pulmonary tuberculosis (TB) cases. P2X₇ gene is highly polymorphic, and it is up-regulated by an inflammatory cytokine. Recently, a correlation between the expression of the P2X₇ receptor and the elevated levels of TNF-α have been shown, but the apparent clinical correlation could not be demonstrated. Methodology: This study included 80 confirmed pulmonary tuberculosis (PTB) cases. Fifty control subjects were selected from the TB patients (clinical and laboratory) who had positive tuberculin tests (10–15 mm) and showed no sign of diseases (laboratory, radiological and clinical parameter). Single nucleotide polymorphisms (SNPs) in P2X₇ (+1513, −762) and TNF-α (at −238, −308, −244, −857, −863) genes were assessed using PCR-RFLP and allele-specific PCR. Thereafter, haplotype and diplotype variability were compared and analyzed. Results: For 1513 loci, the heterozygosity was higher in patients (35%; 44.3%) than control subjects (12%; 24%) [(p = 0.026) ORS; 2.45 CI 95% (1.13–5.33)]. For −762 loci, the frequency of mutant alleles between patients and controls were not statistically significant. No statistical difference was observed in allele frequencies of TNF −308 and −857. However, the frequency of −238 allele were more in TB cases (72.1%) (p = 0.000)[ORs: 5.85(2.70–12.64). Data analysis showed more frequencies of haplotypes, i.e., TGGA-CA and CGGA-TA in patients (21.5%; 14.6%) than the control group (2.0%; 6.0%), respectively. Additionally, the diplotype “CCGGGGGG-CCAA” was significantly associated with susceptibility to PTB (1.9 [0.08–48.3]). Conclusions: In the studied population, polymorphisms in P2X₇ (1513) and TNF-α (−238) gene was associated with the risk of developing PTB. Additionally, distribution of haplotype and diplotype variables did appear to be more specific than SNPs.

Aims and objectives: The transmission pattern of drug resistant Mycobacterium tuberculosis (MTB) might vary due to the differences in geographic features, socio-economic development and TB epidemic in specific areas and populations. This might also reflect how effectively a TB control program would work. This study aimed to investigate the transmission pattern of drug resistant TB, especially multidrug-resistant (MDR)-TB, and to discuss its implications for an effective TB control program in rural China. Methods: Applying a combination of conventional epidemiology with MIRU-VNTR-based genotyping and DNA sequencing, a descriptive cross-sectional study was performed in two Chinese rural counties: DQ, a long-term DOTS-covered county, and GY, where the DOTS program was launched 11 years later than DQ. Results: In total, 238 bacteriologic confirmed pulmonary TB patients from DQ and 393 from GY diagnosed between 2008 and 2011 were recruited in the study. Of the 631 isolates, 220 (34.9%) were resistant to at least one anti-TB drug, including 95 (15.1%) simultaneously resistant to isoniazid and rifampicin or MDR, albeit with the similar distribution between DQ and GY (32/238 vs. 63/393; p, 0.378). The MIRU-VNTR genotyping revealed 35 isolates from DQ and 86 from GY exhibited 15 and 32 clustering patterns with four patterns shared between two counties. Compared with GY county, DQ had a significantly lower clustering proportion in MTB isolates susceptible to first-line drugs (25/167 vs. 46/198; p, 0.047) and total drug resistant TB isolates (12/71 vs. 44/149; p, 0.044), but a similar clustering proportion in MDR-TB isolates (8/32 vs. 18/63; p, 0.712). A significant higher clustering proportion was observed in the previously treated patients in both counties, but in the sputum smear-positive patients with cavitaries only in GY. Comparing the previously treated patients between the two counties, the proportion of MDR-TB and clustering proportion exhibited a similar distribution, while the average age of previously treated patients in DQ is significantly older than that in GY. Conclusions: A lower proportion of recent transmissions was observed in the county with long-term DOTS implementation. However, DOTS itself might not have worked enough on blocking the recent transmission of MDR-TB. This observation suggests the urgent needs of implementing the Stop-TB strategies; in particular, accelerating the use of rapid molecularbasedTBdiagnosisand drug susceptibility testing, providing active case findings in a high risk population of MDR-TB and enhancing infection control in high MDR-TB burden countries.

The Bacillus Calmette-Guérin (BCG) vaccine is administered to all newborns within the first days of life for the prevention of tuberculosis (TB) in many of the developing countries, including Iran. BCG vaccine is prepared from a special strain of Mycobacterium bovis. This vaccine is safe, but local and systemic side effects are associated with BCG vaccine. Severe adverse effects of BCG vaccination are extremely rare in immunocompetent children, but lymphadenitis, which is the most common side effect of BCG vaccination in children, is seen with high frequency in Iran. Different studies have shown that the vaccine strain and its genetic variations are important in inducing these side effects. Therefore, in this study, the aim is to analyze the genetic characterizations of vaccine strains used in Iran. Methods: The samples were collected from infants showing lymphadenitis induced by BCG vaccination. After acid-fast staining and culture of samples on Lowenstein–Jensen medium, DNA was extracted from samples using Phenol–chloroform method. Using primer for 16sRNA gene, the genus of the isolates was characterized. Then using RD1, RD14 and DU1 primers and followed by the next step using RD9, RD4 Deleted, RD4 Present and RD1 Deleted primers, the species and strains of the isolates were characterized. Results: All 30 samples of acid-fast bacilli that were isolated from infants with BCG complications confirmed as Mycobacterium genus using 16sRNA PCR and using RD1, RD14, DU1, RD9, RD4 Deleted, RD4 Present and RD1 Deleted primers confirmed that all isolates were M. bovis BCG strain Pasteur. Conclusion: As all of the strains isolated from the patients were confirmed as M. bovis BCG strain Pasteur, the other possible factors causing BCG complications, including BCG overdose, faulty intradermal technique, and disturbance of cellular immunity, must be considered.

Aims and objectives: The aim of this study is to investigate and detect the prevalence of Mycobacterium bovis subtypes (Mycobacterium bovis subtype bovis and Mycobacterium bovis subtype caprae) in humans and compare the genetic diversity of Mycobacterium bovis in humans and cattle with spoligotyping methods, as well as pyrazinamide susceptibility study of subtypes. Methods: Examining these subtypes with molecular epidemiology techniques is particularly important due to different treatment of M. bovis diverse subtypes in humans. Culture tests were performed on clinical samples that were isolated from Lowenstein-Jensen culture medium. Identification tests were performed to differentiate Mycobacterium bovis from Mycobacterium tuberculosis. DNA was extracted and spoligotyping (spacer oligonucleotide typing) was performed using the DRb and DRa primers. Results: The results were analyzed with the SPOLDB4 site. PCR-RFLP method of pncA gene was used to evaluate the resistance to pyrazinamide and pncA gene polymorphism. Conclusions: Mycobacterium bovis subtype bovis in the Iranian population was reported with a frequency of 0.6% which was below the average of the previous reviews. In this study, all the strains were M. bovis subtype bovis resistant to pyrazinamide. No Mycobacterium bovis subtype caprae was detected. The only shared ST between humans and cattle was ST694. Mycobacterium bovis subtype bovis with ST 595 was reported as human bovis index.

Introduction: As a rule in any Bovine tuberculosis (BTB) control program, recognition of infection source is of great importance. Hence in this study, isolation and evaluation of Mycobacterial genomic patterns obtained from rodents of infected farms by RFLP method was conducted. Material and Method: Fifteen mice submitted from infected cattle farms with BTB that were collected in four provinces of IRAN were used in this study. Culture of Mycobacterium and RFLP–PGRS and RFLP–DR analysis of bovine isolates from the same areas were performed in the usual manner. Following the detection of different patterns between mice and cattle isolates, identification by PCR of IS6110 and RV0577, RD typing and sequencing of 16SrRNA and rpoB was conducted. Result: Three isolates were obtained out of the 15 mice samples cultured from 2 out of the 4 separate provinces. RFLP analysis with PGRS and DR probes identified 2 different patterns from 5 cattle isolates and 1 single pattern from 2 mice isolates located at Ahwaz province, and 2 different patterns from 9 bovine isolates and 1 pattern from 1 mouse isolates located at West Azerbaijan were identified. Two mice isolates from Ahwaz province were negative at the above PCRs and sequencing of 16SrRNA and rpoB revealed that these isolates are Mycobacterium kansasii. Another mouse identified as belonging to the Mycobacterium tuberculosis complex by PCRs and RD typing revealed that this isolate must be Mycobacterium microti. Discussion: This study was unable to track tangible evidence of tuberculosis transmission by mice. Hence to prove this hypothesis, further studies are advised. However, it was found that mice are potentially a reservoir of zoonotic pathogens, and therefore its importance in this regard must be considered as an effective element of any controlling program.

Aims and objectives: The Mycobacterium avium subsp avium (MAA) is a slow-growing, frequently-encountered mycobacterium in the environment that causes tuberculosis mainly in birds and sometimes in farm animals. As a favorite pet bird, the pigeon is extensively kept for homing and/or racing purposes in Iran, therefore, the main objective of this study was to investigate dissemination of M. avium subsp avium (MAA) in pigeon aviaries in Tabriz, North-western Iran. Methods: From a total of 140 birds collected from private flocks (n = 3), The pathologically changed lungs and the lymph node were examined histologically by staining with Ziehl–Neelsen (Z–N) and hematoxylin–eosin; 39 were subjected to bacterial culture, out of which 34 mycobacterial isolates were recovered. Mycobacterial DNA was isolated according to the previously described method (Van Soolingen et al., 1993). Applying a five-PCR diagnostic algorithm targeting short, but definitive stretches of 16S rRNA and RV0577 genes, IS6110, IS901 and IS1245 genomic loci, all the isolates were identified as MAA. PvuII-IS901 RFLP typing was also performed on all isolates. Results: They were either IS901+/IS1245+ (n = 22) or IS901+/IS1245 (n = 12). In IS901-RFLP strain typing of a subset of the isolates (n = 22), they were classified into five distinct multi-banded but similar patterns, namely PA (n = 13), PB (n = 5), PC (n = 2), PD (n = 1) and PE (n = 1). No correlation between IS901-RFLP genotype and presence/lack of IS1245 was noted as isolates both holding and lacking IS1245 were found to share PA and PB genotypes. Whilst no case of mixed infection with more than one strain was detected in any single bird, it was not possible to extend this observation to the aviary level as original colonies of birds were not recorded. When four healthy cattle sensitized against Mycobacterium bovis AN5 and M. avium D4 were tuberculinated, the results confirmed the observed skin reactions against bovine tuberculin in animals sensitized with M. avium were large enough to complicate test interpretation. Conclusions: It is believed that the extent of such epidemiological impact deserves further investigation if progress in the control of bovine tuberculosis is intended. This indicates the importance of identification of the causative agent before any conclusions are made, based solely upon the results of the skin test and histopathological examination.

Introduction: Tuberculosis (TB) remains a major cause of death in many countries. Bovine tuberculosis caused by Mycobacterium bovis is one of the key members of the Mycobacterium tuberculosis complex. Current methods need long incubation times, which is in contrast to the necessity for rapid and accurate identification and isolation. Molecular techniques, such as RFLP fingerprinting, for accurate identification and differentiation of M. tuberculosis isolates are a more desirable approach. Materials and Methods: In a 12-month study plan, which began in November 2011, lymph nodes obtained from 92 samples of dairy cattle yielded tuberculin-positive marks that were collected from 10 different regions of the province; 43 isolates were identified by acid-fast culture. Using biochemical tests along with IS6110 specific fragment search, colonies were proved to belong to M. tuberculosis complex and more precisely M. bovis strains. PVUII enzymatic digestion of their DNA was followed by Southern blotting and hybridization with DR and PGRS markers. Finally, RFLP fragment hybridized with substrate BCIP and NBT was carried out, and their results were evaluated by GelproAnalyser software. Results: All 43 isolates out of the 92 samples that were analyzed by RFLP with two probes of DR and PGRS, interestingly, proved the circulation of an identical strain of M. bovis throughout the whole province. Discussion and conclusion: Assessing the genomic patterns of all the strains and comparing the results with previous studies conducted in the area revealed that, while they represent the circulation of a common ancestral clone, as a preliminary successful achievement in industrial and semi-industrial dairy farms located in the zone of the tuberculosis control program, they still have a long way to go to meet the ultimate eradication goals.

Khuzestan is a southwestern province of Iran with proximity to the Persian Gulf and the international border with Iraq where harsh climate seriously affects this oil-rich region. In a search for causative agents of bovine tuberculosis (bTB), slaughterhouse specimens (lymph nodes) from 32 tuberculin-positive cows originating from 17 farms were cultured on Lowenstein–Jensen slopes. This was further extended with bacterial culture of postmortem material from 6 trapped feral mice straying on the same farm premises. Twenty-five bovine and 2 murine acid-fast isolates were consequently obtained with all of them confirmed as Mycobacterium tuberculosis (MTB) complex bacteria by IS6110-PCR experiment. Spoligotyping and RD4 typing of the 2 murine and some of the collected bovine isolates left no doubt that Mycobacterium bovis is the principle and possibly the single culprit in bTB in this region. It does not come as a surprise as previous exhaustive works have shown in the Iranian environment other members of MTB complex than M. bovis are very unlikely to have any role in the epidemiology of bTB.

Background: Tuberculosis (TB) is one of the most widespread, infectious, zoonotic diseases and continues to be a leading cause of death. TB is a predominant public health problem in the world. Mycobacterium tuberculosis (MTB) is the most common cause of human TB. M. bovis is the causative agent of this disease in cattle. In this study the human results revealed that from a total 1244 cases, 123 (10%) of the specimens were positive upon direct examination, 173 (14%) by cultivation on LJ medium, and the rest were negative. Fifty positive samples were chosen for direct smear and culturing and were followed-up from the first step of diagnosis to molecular assay; these specimens were taken from 33 (66%) males and 17 (34%) females; fifty positive cases were detected by direct smear. Furthermore, 50 positive cases were also noticed using culturing on Lowenstein–Jensen (LJ) media and 23 positive cases by PCR. The bovine milk sample results showed that 3 positive cases were detected by direct smear and 7 positive cases were observed using culturing on LJ media with sodium pyruvate; the results of culturing methods were confirmed by PCR. Objective: Diagnosis of TB based on phenotypic and genotypic methods using routine methods and PCR by amplification of nucleic acid of MTB. Material and Methods: During the study period of February 2009 to June 2009, the Institute of Chest and Respiratory Diseases – Baghdad received 1244 suspected cases of TB from different Iraqi governorates; 759 (61%) males and 485 (39%) females ranging in age from 2 months to 90 years. Only 50 positive sputum samples were chosen from 173 positive cases for this study and were detected by routine and molecular methods, including polymerase chain reaction. In addition, 68 bovine milk samples were taken from Al-Fthelia – Baghdad during the study period. The results of routine methods were confirmed by PCR. Results: The human samples: The results revealed that from a total 1244 cases, 123 (10%) of the specimens were positive by direct examination and 173 (14%) by cultivation; the rest were negative. The age range of patients was 2 months to 90 years. Distribution of patients among age groups showed that 32 (13.3%) patients were in the <20 years age group, 79 (45.7%) were in the 21–40 years age group, 52 (30%) were in the 41–60 years age group and 19 (11%) in the >60 years age group. It is obvious that positive male cases had the highest number (113/173) throughout the study period. Fifty positive samples for direct smear and culturing were chosen and followed-up from the first step of diagnosis to molecular assay. These specimens were taken from 33 (66%) males and 17 (34%) females with a range of ages in both sexes; such a selection aimed to represent a variety of backgrounds. The results indicated that all the 50 samples were positive for direct smear (100%) and 50 positive in culture (100%), while 23 (46%) cases were positive for PCR and 27 (54%) cases were negative for PCR. The bovine samples: From a total of 68 milk samples, the positive rate for direct smear was 3 (4.4%), while the positive rates for culture were 7 (10.2%). The positive samples were cultured on LJ slant for 3–6 weeks. The appearance of colonies was typical cream-colored, buff and rough colonies against the green egg-based medium. The isolated colonies were identified as M. bovis by five biochemical tests.

Objectives: Avian tuberculosis is one of the most important infections affecting most species of birds. Several mycobacterial species have been identified causing avian tuberculosis, but the organisms confirmed most frequently are Mycobacterium avium and Mycobacterium genavense. All species of birds can be infected with M. avium. M. avium cannot only infect all species of birds, but can also infect some domesticated mammals to cause the disease, usually with localized lesions. Disseminated tuberculosis caused by M. avium has also been reported in rabbits and swine. In immune-competent humans, M. avium complex)MAC(isolates produce localized soft tissue infections, including chronic pulmonary infections in the elderly and cervical lymphadenitis in children, but rarely any disseminated disease. In HIV infected and AIDS patients, or in other immuno-compromised persons, MAC isolates frequently cause severe systemic infections. The most crucial aspect of control and eradication of the disease is identification of infection sources and transmission routes. The use of serotyping for epidemiological studies of virulent M. avium subsp. avium isolates in birds is limited by the prevalence of only three serotypes and untypable auto-agglutinating isolates. Molecular techniques, such as restriction fragment length polymorphism and pulse field gel electrophoresis, have been shown to be much more discriminatory and therefore suitable for use in the epidemiological study of M. avium complex infections. Materials and Methods: Eighty suspected pigeons with avian tuberculosis based on their clinical signs were subjected to the study. Forty M. avium subsp. avium isolates out of a total of 51 identified isolates were subjected to the test. Results and discussion: Clinical signs, necropsy findings, ZN staining and molecular identification confirmed that the pigeons were infected with M. avium subsp. avium. IS901-RFLP using PvuII was successfully conducted on 40 isolates and produced 7 patterns. The majority of isolates (60%) were RFLP type PI.1 and in comparison this type was the most similar type to the standard strain; however, all the patterns obtained in this study were different from the standard strain (M. avium subsp. avium D4 strain, ATCC number 35713). This is probably due to these isolates belonging to this region. In addition, no common pattern between this study and the only other similar study in Iran was found, and it indicates that the sources of their infection are not same. In conclusion: It is suggested that more DNA fingerprinting tests for non-tuberculous Mycobacteria, particularly M. avium complex isolated from infected birds and humans, be conducted to find the source of their infections.

Background: The infections due to rapidly growing mycobacteria (RGM) are becoming an important health problem in many countries in the world. Globally, an increase in RGM infections is being reported from several regions worldwide. However, there is limited information about the prevalence of these kinds of organisms in Iran. Methods: The relevant data of the prevalence of RGM were retrieved by searching several databases, such as PubMed, Web of Science, Cochrane Library, Embase, Scopus, Iranmedex, and Scientific Information Database. Meta-analysis was performed by Comprehensive Meta-Analysis (V2.0, Biostat) software. Results: The meta-analyses showed that the Mycobacterium fortuitum (22.7% [95% CI 16.1–30.9]), Mycobacterium abscessus (14.0% [95% CI 6.4–27.8]) and Mycobacterium chelonae (7.6% [95% CI 2.8–18.8]) were the most prevalent RGM among the conducted studies in Iran. Conclusions: The relatively high prevalence of RGM underlines the need for greater enforcement of infection control strategies. Establishment of appropriate diagnostic criteria and management guidelines for diseases caused by RGM and expanding the number and quality of regional reference laboratories may facilitate more accurate action for prevention and control of this kind of bacteria.

Introduction: Nontuberculous mycobacterium (NTM) has clinical and radiological manifestations that are indistinguishable from Mycobacterium tuberculosis (MTB). In an endemic area for tuberculosis (TB), limited data about prevalence and outcome of treatment of these patients is available. In this study the prevalence of different types of mycobacterium and response to treatment in a tertiary referral center in Iran will be evaluated. Materials and method: All NTM cases from 2004 to 2013 at the National Research Institute of Tuberculosis and Lung Diseases (NRITLD) in Iran were extracted from the database. All NTM patients who were treated for NTM diseases entered this study, and the outcome of treatment was evaluated. Results: A total of 104 cases were detected. The mean age was 56.34±15.77 years. Half of the patients were male. Most of the patients had a history of prior TB treatment. The most common types of NTM were Mycobacterium simiae (44 [42.3%]), Mycobacterium kansasii (18 [17.3%]), Mycobacterium abcessus (15 [14.4%]), and Mycobacterium chelonea (14 [13.5%]), respectively. The outcome of treatment was as follows: cure 61 (58.7%), failure 17 (16.3%), relapse 3 (2.9%), default 13 (12.5%) and death in 10 (9.6%) patients. Conclusion: Treatment of NTM had a low cure rate despite low mortality.

Objective: The purpose of this study is to estimate the frequency of Mycobacterium chelonae among tuberculosis suspected patients in Basra Governorate and to evaluate the antimicrobial susceptibility. Methods: After isolation of M. chelonae from 100 samples from patients admitted to the RPDC, smears were stained with the Ziehl Neelsen technique. Specimens were inoculated on Lowenstein Jensen medium. Identification to species level was achieved on the basis of the growth characteristics. Drug susceptibility was tested with antibiotics (Rifampicin, Isoniazid, Ethambutol, Pyrazinamide and Streptomycin) using proportional method. Results: From the 100 sputum samples among tuberculosis suspected patients, 16 samples (16%) were Non-tuberculosis mycobacterium (NTM), 4 (4%) were M. chelonae. Drug susceptibility results showed all isolates resistant to rifampicin, while one isolate showed intermediate resistance to ethambutol. Also, all isolates of M. chelonae were sensitive to pyrazinamide, isoniazid and streptomycin. Conclusion: The results of this study emphasize that M. chelonae presents with high frequency, especially among tuberculosis suspected patients, which requires confirmation on a follow-up visit, along with the examination of patterns of sensitivity, and it is an absolute necessity in addition to the approximate hour patients typically spend in health centers in Iraq.

Aim and objective: Tuberculosis (TB) is a chronic lung infection that contaminated 1/3 of the people in the world and that causes 2 million deaths and 9 million infected with the disease. Sputum smear and Ziehl-Neelsen staining are the best methods for diagnosis of TB, but chest X-ray is another method that can help diagnose pulmonary TB, and it is especially helpful for smear-negative patients. The aim of this study is to show that radiologic findings can assist in diagnoses in pulmonary TB smear-negative groups. Methods: In this descriptive analytical study, 100 patients with smear-negative pulmonary TB during the time period 2010–2011 were enrolled. The standard World Health Organization (WHO) definition of smear-negative was used in this study. All patients had a chest X-ray (PA and lateral). Data were analyzed with cisquire and T test with the use of SPSS and p < 0.05 was significant. Results: Of the 100 patients, 63.5% were female and 97% Iranian; 25% of patients had ESR more than 50 frequency. Calcification, Hilar adenopathy, incomplete pulmonary destruction and bronchiectasis was 27%, 21%, 14% and 11%, respectively, but frequent reticulonodular infiltration and pulmonary fibrosis (41% and 23%, respectively) were not significant (p = 0.2). Conclusion: Radiologic findings for pulmonary TB cannot be used to diagnose, but are rather suggestive.

Introduction: Nontuberculous mycobacteria (NTM) are opportunistic pathogens that have increased in recent years. The distribution of NTM species is not similar, and it seems to depend on geographic and environmental regions. In this study, the distribution of NTM species from environmental and clinical samples in the Middle East was reviewed. Methods: To provide an overview of NTM, all studies addressing NTM in the Middle East were searched from 1984 to 2014; 111 articles were found describing the prevalence of NTM in this region. Results: A total of 1994 and 886 NTM were included in the clinical and environmental samples, respectively. Mycobacterium fortuitum was the predominant NTM identified among the rapidly growing mycobacteria (RGM) in both environmental (32.6%) and clinical (53.1%) samples. The most common slowly growing mycobacteria (SGM) were MAC 297 (25.3%) in clinical cases. Among SGM in environmental samples, the most common species was Mycobacterium MAC 49 (11%) which reported from Iraq, Iran and Saudi Arabia. Conclusion: This is the first study reporting NTM species in the Middle East. In summary, this review demonstrates that the frequency of NTM has increased in this region. In addition to the limitations of the NTM study in some countries of the Middle East, there is an urgent need to develop laboratory methods for NTM identification to prepare a comprehensive report for this region.

Tuberculosis (TB) is one of the most common infections world-wide, and in 2012, an estimated 8.6 million people developed TB and 1.3 million died from the disease (including 320,000 deaths among HIV-positive people). Mycobacterium tuberculosis (MTB) is an intracellular pathogen capable of infecting and surviving within the hosts mononuclear cells, particularly macrophages. This involves sequestration of MTB within organized granulomas. Nontuberculous mycobacteria refers to all the species in the family of Mycobacteria that may cause human disease, but does not cause TB. Every year in the world approximately 2 people per 100,000 population develop infections caused by these lesser-known “cousins” of TB and leprosy. In this study, the focus is on a rare case of a patient with chronic granulomatous disease presenting with both MTB and Nontuberculous mycobacteria. As far as this research is concerned, this is the first report of a carrier patient with chronic granulomatous disease combined with MTB and Nontuberculous mycobacteria. The presented information may help to improve the diagnosis and open a new light in the investigation of susceptibility of patients to Mycobacterium infections.

Aim: To evaluate patients’ profiles, demographics, clinical and therapeutic approaches and strategies in patients with tuberculous lymphadenitis (TBG). Patients and methods: A retrospective study of all TBG-confirmed cases admitted in a tuberculosis specific health care facility between 1 January 2009 and 16 June 2013. Results: A total of 181 clinical files were examined. Mean age was 32 years old; the female/male ratio was 1.78 to 1. Raw milk consumption was noted in 1/3 of patients. Most cases involved the head and neck region (83.4%), nodes involvement, including axillary (12 cases), and mediastinal (9 cases). Clinical symptoms were present in only 55.2%. TST was conducted with 82.6% positive responses. Diagnostics confirmation was done with anatomical pathology in most of the patients; only 56 of them had any microbiology analysis done. Demonstration of acid-fast bacilli in microscopy from either fine-needle aspirates or biopsies was done in 17.5%, and cultures yielded positive results in 27%. Treatment duration was varied. Paradoxical reactions were noted in 12% and persistent lymphadenopathy after treatment completion was noted in 10% of cases. Conclusions: TBG remains a disease of interest. Today, its diagnosis and management is still a problem despite its increasing worldwide incidence, and especially in this study area. Disease control should be strengthened in this country.

Introduction: In the early 1930s when Tehran Veterinary College – the first modern Iranian veterinary school – was established, the existence of bovine tuberculosis (BTB) in Persian cattle had already been reported by French scientists. Small-scale test-and-slaughter control plans have been employed since then by private and State veterinarians, mostly concentrated in cattle farms of the capital city of Tehran and its suburbs. It was in the 1960s when these discrete operations were scaled up and turned into the present-day national scheme. Materials and Methods: Since 2000, in addition to the traditional bacterial culture, genotyping (e.g., RFLP, spoligotyping and MIRU-VNTR typing) has been employed on a relatively large number of Mycobacterium bovis isolates from across Iran. The present work concentrates on RFLP findings using two markers of PGRS and DR. Altogether, 17 RFLP patterns (identified mutually by either of the two markers) have been found in Iran. Results and discussion: The pattern representing the M. bovis BCG strain, so-called “BCG-like”, is the commonest type being found almost everywhere in the country. Some of the remaining patterns, however, seem to have been geographically isolated over time. What is the current trend in the population of M. bovis in Iran? Are the localization of strains or expansion of them being observed? How extensive is the impact of veterinary measures in this context? These are some of the major questions that authors of this work have tried to raise.

Aims and objectives: This research aims to identify non-tuberculosis mycobacterium (NTM) species by conventional biochemical and genetic methods. Methods: A total of 150 sputum samples were collected from suspected tuberculosis (TB) patients who attended the center of Chest and Respiratory Diseases in the Basrah Governorate during the period from 01/01/2013 to 1/10/2013. All specimens were stained by the Ziehl-Neelsen method, and all negative and positive samples for this method were cultured on Lowenstein-Jensen medium slant, Middlebrook 7H10, 7H11 agar-serum based and Middlebrook 7H9 broth. Drug susceptibility was tested with antibiotics (Rifampicin 1 μg/ml, Ethambutol 2 μg/ml, Pyrazinamide 0.25 μg/ml, Isoniazid 0.2 μg/ml and Streptomycin 2 μg/ml). Results: Thirty-nine positive samples for Ziehl-Neelsen and growth on Lowenstein-Jensen medium were identified by biochemical tests, including: Niacin accumulation, Nitrate reduction, Catalase, Iron uptake, Aryl sulfatase, Hydrolysis of Tween 80, Growth in 5% NaCl, Tellurite reduction, Growth on MacConkey, Pyrazinamidase, Urease and pigmentation. The current results showed that 39 samples (26%) were positive for acid fast bacilli and were identified as Mycobacterium, and 32 of those samples (82.05%) were identified as slow growing mycobacteria, of which 23 (58.97%) samples were identified as M. tuberculosis, 4 (10.25%) as M. avium complex, 2 (17.94%) as M. flavescens, 2 (17.94%) as M. simiae, and 1 (2.56%) as M. kansasii. The remaining 7 samples (17.94%) were rapid growing mycobacteria, of which 4 samples (10.25%) were identified as M. chelonae, 2 samples (5.12%) as M. abscessus, and 1 sample (2.56%) as M. smegmatis. All samples were identified using PCR-based tests for genetic markers. Conclusions: This study emphasizes that NTM is present at high frequency, especially among TB-suspected patients, and this requires confirmation on a follow-up basis, along with the examination of patterns of sensitivity, and is an absolute necessity rather than the current hour in a health center in Iraq.

Introduction: Nontuberculous mycobacteria (NTM) are ubiquitous in the environment and exist as important causes of pulmonary infections in human. Pulmonary involvement is the most common disease manifestation of NTM and the incidence of NTM is growing in the North America. Susceptibility to NTM infection is incompletely understood and, therefore preventative tools are not well defined. Treatment of pulmonary nontuberculous mycobacterial (NTM) infection is difficult and entails multiple antibiotics and an extended treatment course. Also, there is a considerable variation in treatment management that should be considered before initiating treatment. Material and Methods: A literature search was conducted using search keywords “nontuberculous mycobacteria”, “MAC”, “M. abscessus”, “epidemiology”, “treatment”, “North America”, “mortality”, “cystic fibrosis”, “transplantation”, “prevention”, and “diagnosis” from studies that have been published between the years 2009 and 2014. PubMed, Cinahl, Scopus, Embase, and the Cochrane Library were searched. A total of 382 articles were reviewed from which 65 papers met our selection criteria. Titles of interest were further reviewed by all authors. Reference lists of relevant studies were hand-searched in order to identify other potentially relevant articles. Studies included in this review met the following criteria: (i) study populations included patients with NTM; (ii) articles were full reports, case reports or reviews; (iii) articles were in English and published from the US based institutes; (iv) articles were published in peer-reviewed journals. Results: Nontuberculous mycobacteria (NTM) are an important cause of morbidity and mortality, often in the form of progressive lung disease. Few reports are accessible on NTM disease prevalence in the United States; however based on the recent data the incidence and mortality rates of pulmonary NTM have been reported to be rising in North America. We highlight the new findings in the epidemiology, diagnosis and treatment of mycobacterial infections. We debate new advances regarding NTM infection in cystic fibrosis patients and solid organ transplant recipients. Finally, we introduce a new epidemiologic model for NTM disease based on virulence–exposure–host factors. Conclusion: It is critical that NTM is recognized as an important public health issue with potentially significant consequences for affected patients. Finally, the applicability of the virulence–exposure–host model in NTM disease should be investigated.

Introduction: Pulmonary mycobacterial diseases describe both tuberculosis (TB) and nontuberculous mycobacteria (NTM). TB is an established public health issue and a reportable disease. Efforts in treatment and surveillance have resulted in the incidence of TB to decrease in recent years. However, the incidence of NTM is increasing, classifying NTM as an emerging public health problem in the US. Despite the increasing importance of pulmonary mycobacterial diseases, few data are available measuring the cost burden of mycobacterial diseases at the national level. The purpose of this study was to evaluate the cost burden and measure emerging trends in hospitalization of pulmonary TB and NTM in the US from 2001 through 2012. Methods: This study was a retrospective community-based cost analysis of hospitalized patients with a principal diagnosis of pulmonary mycobacterial diseases from 2001 through 2012. Data for pulmonary TB and NTM were retrieved from the Healthcare Cost and Utilization Project (HCUP), US Department of Health and Human services. Data included in this analysis were national hospital costs, payer sources, hospital lengths of stay, in-hospital mortality, and discharge information. The statistical significance of observed trends of NTM and TB national hospital costs were calculated using Poisson log-linear regression. National hospital costs for NTM and TB were projected in relation to health care inflation for each year. A regression model was applied to test the correlation between historic and projected national hospital costs. Results: From a total of 36,484,846 admissions, 20,049 hospital admissions were reported for pulmonary NTM and 69,257 for pulmonary TB in the US from 2001 through 2012. The total associated costs of these admissions was $1,337,939,745,325, an estimated $970,643,222 of which was directly associated with pulmonary NTM and $3,390,013,793 for pulmonary TB. During the study period, the national hospital costs of pulmonary NTM increased at a statistically significant rate in the US over each year (p = 0.001). A linear regression analysis demonstrated a high degree of correlation between NTM historic and projected national hospital costs (R=0.938, p<0.001). However, no such correlation between historic and projected national hospital costs was found for pulmonary TB (R=0.284, p = 0.372). Conclusions: The total estimated cost of inpatient care of pulmonary NTM in the US during the study period was almost $1 billion. The cost of NTM management year after year is likewise increasingly significantly at a rate consistent with healthcare inflation. In contrast, pulmonary TB national hospital costs were decreasing during the study period. These trends emphasize the considerable and increasing burden of pulmonary NTM in the US. Given that the majority of patients with pulmonary NTM are never admitted to the hospital, the total economic burden of this disease is tremendously higher than measured in this study. These results emphasize the importance of continued research of pulmonary NTM in order to improve current guidelines in prevention and treatment strategies.

Objectives: Pulmonary hypertension is a serious disorder with catastrophic outcomes. This study aimed to evaluate the effect of pulmonary arterial hypertension on the outcome among new cases of pulmonary tuberculosis. Novel modalities are available as therapeutic options, so early diagnosis of pulmonary arterial hypertension may be important. Methods: In a cross-sectional study, 777 new cases of pulmonary tuberculosis were recruited in the National Research Institute of Tuberculosis and Lung Disease, Tehran, Iran. Pulmonary arterial pressure was measured by resting transthoracic echocardiography on the beginning of tuberculosis treatment. Echocardiography was performed by a cardiologist expert in the field. Patients with systolic pulmonary arterial pressure more than 35 mmHg were defined as pulmonary hypertensive. The relationship between pulmonary arterial hypertension and mortality was assessed during six months of tuberculosis treatment. Results: Seventy-four (9.5%) of 777 new cases of pulmonary tuberculosis had pulmonary arterial hypertension. Among them, 10 (13.5%) died during the treatment period. In comparison with 5% mortality among cases without pulmonary arterial hypertension, death was significantly associated with pulmonary hypertension (p = 0.007). Logistic regression analysis showed that pulmonary arterial pressure more than 35 mmHg is an independent predictor of death among tuberculosis patients. Conclusion: Among new cases of pulmonary tuberculosis, a significant association between mortality and pulmonary arterial pressure >35 mmHg was found. Therapeutic intervention may change the outcome of these patients.

Aims and objectives: Multidrug resistant tuberculosis (MDR-TB) management is expensive, prolonged and complicated. After starting anti-tuberculous treatment in MDR-TB patients, microbiological response to therapy is monitored by monthly sputum cultures. Conventional culture methods are costly, leading to poor compliance with this recommendation in resource-limited settings and reliance on less sensitive sputum microscopy. Published data suggests a less favorable outcome with sputum microscopy and stresses upon use of follow-up cultures. Micro-colony liquid culture method is an established method for diagnosis of new TB cases. As anti- tuberculous treatment may produce considerable changes in bacterial morphology, leading to pleomorphic shapes and sizes, the utility of micro colony broth culture for treatment follow-up cases is uncertain. Therefore, this study aims to evaluate the micro-colony liquid culture method in terms of its sensitivity, specificity, rapidity and cost to monitor response to second-line ATT in diagnosed MDR-TB patients. Methods: Prospective cross-sectional study performed at Clinical Microbiology Laboratory of Aga Khan University. During the period of February 2013–September 2014, a total of 139 adult, MDR-pulmonary TB patients were enrolled in this study. For each patient an appropriate sputum specimen was collected for MTB culture initially and then monthly (maximum) up to the next 6 months. Samples were processed using the standard protocol for microscopy, routine MIGIT & LJ culture and micro colony broth culture method. Micro colony broth culture finally evaluated for sensitivity, specificity, rapidity, cost and contamination rate. Results: To date, a total of 502 sputum samples were submitted from 139 enrolled patients. Out of these, 170 were smear-positive, while 310 were smear-negative. Sensitivity, specificity, positive predictive value and negative predictive value of micro colony broth culture for AFB smear positive samples were 100% (95% CI: 97.83–100), 95.45% (95% CI: 77.08–99.24), 99.42% (95% CI: 96.77–99.90), and 100.00% (95% CI: 83.75–100.00), respectively. Sensitivity, specificity, positive predictive value and negative predictive value of micro colony broth culture for AFB smear negative samples was 100.00% (95% CI: 93.78–100.00), 99.60% (95% CI: 97.80–99.93), 98.31% (95% CI: 90.88–99.72) and 100.00% (95% CI: 98.53–100.00), respectively. Average time of positivity of standard cultures for smear positive samples (n = 192) was 26.0 days, while micro colony broth culture average positivity time was 8.8 days. Average time of positivity of standard cultures for smear negative samples (n = 310) was 30.0 days, while micro colony broth culture average positivity time was 11.4 days. Average contamination rate of micro colony broth culture was 4.5% in comparison with 6.1% of routine cultures. Finally, the cost of the micro colony methods was about 35% of the combined BACTEC and LJ media cultures. Conclusions: The preliminary data from this study indicates micro colony broth culture method for MTB detection to be highly sensitive, specific, rapid and cost-effective for treatment monitoring of MDR-TB cases. In resource-limited settings this method can be used safely either alone or along with LJ medium for monitoring of second-line anti-tuberculous treatment. Acknowledgement: This work was supported by a grant from Aga Khan University Research Council.

Aims and objectives: Nontuberculous mycobacterium (NTM) infections are caused by mycobacteria that are found in water and soil. The chances of missing NTM species are higher in tuberculosis endemic countries like Iran, which are poorly equipped and overburdened with other diseases. The aim of this study is to access the diversity of rare, unusual and frequent species in Iranian tuberculosis laboratories. Methods: From 2012–2014 a total of 243 different clinical isolates of NTM were recovered from Mycobacteriology Reference Laboratories across Iran (Ahvaz, Esfahan, Tehran, Gorgan, Kermanshah). The isolates identified by rpoB gene sequencing are useful markers in mycobacterium identification. Results: Based on rpoB gene sequencing, 20 groups do not belong to any of the officially recognized mycobacteria. A total of 210 isolates of NTM were identified to species level, including M. fortuitum (66), M. kansasii (20), M. simiae (20), M. conceptionense (10), M. peregrinum (9), M. thermoresistibile (9), M gordonae (8), M. lentiflavum (8), M. phlei (6), M. intracellulare (6), M. abscessus subsp. abscessus (5), M. chelonae (5), M. abscessus subsp. bolletii (4), M. avium (4), M. flavescents (4), M. nonchromogenicum (4), M. terrae (4), M. branderi (3), M. iranicum (3), M. mucogenicum (3), M. scrofulaceum (3), M. smegmatis (3) and M. triplex (2). Among all isolates, M. fortuitum was identified as the most frequent encounter (27%) of NTM, and M. kansasii and M. simiae were the second most dominant species (8.2%) among the isolates. A total of 33 isolates (13%) were unidentifiable to species level. M. fortutium was the most frequent species in Ahvaz, Esfahan, Tehran and Gorgan, but M. simiae was the most frequent species in Kermanshah. Thirty-three isolates (20 groups) presented unique genetic features. Further molecular tests should be carried out to reliable identification of these isolates to species level. Conclusions: The present study provides evidence for the importance of NTM identification in the clinical setting and the presence of diverse species in clinical samples as a causative agent of disease. The presence of diverse species of NTM and unidentifiable strains in a clinical setting in Iran emphasizes the use of sequence analysis of genes for reliable identification.

Aims and objectives: Sequence-based identification of non-tuberculosis mycobacteria (NTM) has become a method of choice in mycobacteriology laboratories and is accomplished by analysis of several targets, such as 16S rRNA, rpoB, hsp65, sodA, etc. A group of pigmented, sl owl-growing mycobacteria were isolated from clinical samples of four unrelated patients with which were undefined at species level by means of phenotypic tests and hsp65-PRA method. The isolates were subjected to batteries of sequencing tests for identification. Methods: From 2009 to 2014, a group of pigmented, slow-growing mycobacteria were isolated from respiratory tract specimens, soft tissue infection biopsies and abscesses of four unrelated patients across Iran. The isolates were not identified to species level by biochemical tests and hsp65-PRA. Then the isolates were subjected to identification by 16S rRNA, hsp65 and rpoB gene sequencing. Results: Based on 16S rRNA sequencing, the isolates gave a closest match (99.8%) with Mycobacterium lentiflavum ATCC 51985T of 99.4%, 99.2% and 99% with those of Mycobacterium simiae ATCC 25725^T, M. triplex ATCC 700071T and Mycobacterium stomatepiae DSM 45059^T, respectively. The hsp65 gene sequence (356bp) (GenBank accession: FR682913) showed the highest similarities of 98.4%, 98.1% and 97.2% with those of Mycobacterium triplex ATCC 700071^T, Mycobacterium stomatepiae DSM 45059^T and Mycobacterium montefiorense ATCC BAA-256T, respectively; the rpoB gene sequence (619bp) (GenBank accession: FR695853) showed the highest similarities of 95%, 94.3% and 92.7% with M. triplex ATCC 700071^T, M. lentiflavum CIP 105465^T and Mycobacterium sherrisii Fl-94099, respectively. Conclusions: The approaches used in the current study confirm the taxonomic status of this group of isolates as a novel Mycobacterium species. Further analysis is needed to fully characterize the isolates. The presence of unidentifiable NTM strains in the clinical setting emphasizes the need to use sequence analysis of genes for reliable identification.

Introduction: Aspergillus species as cosmopolitan fungi with remarkable virulence factors were found to be agents of pulmonary aspergillosis in patients with impaired immunity. The formed cavity of some previously treated lung diseases, such as tuberculosis, sarcoidosis and pneumoconiosis, is usually predisposed to the development of aspergillosis. Pulmonary aspergillosis (PA) is an uncommon disease which is characterized by hemoptysis, malaise, fever, cough, weight loss and nonspecific radiographic manifestations, including an oval or round mass with a radiolucent halo or crescent of air, a focal consolidation, and cavitary lesions. Case presentation: This study presents the case of a 54-year-old woman with dyspnea alongside a history of treated pulmonary tuberculosis (PTB) by ATT 2 years ago. X-ray confirmed the presence of a rounded mass in a surrounding cavity in the lung. Tracheobronchial and chest CT images of the patient showed cavities with tuberculous nodules. Clinical symptoms of the patient were fever, malaise, anorexia, weight loss, chest pain, cough and dark mucus sputum. Aspergillus sp. was detected primarily as branching hyaline hyphae in direct examination of the sputum by calcofluor-white staining. The sample was positive with culture as well. Aspergillus flavus was identified in culture and confirmed by polymerase chain reaction (PCR) and sequencing of the ITS region of rDNA and β-tubulin of fungus. The patient signed an agreement for reporting her case as a medical document in journals or in conferences. Conclusion: The importance of tuberculosis (TB) in the development of aspergillosis, even after treatment, has been highlighted by multiple studies. Microbiological and molecular evaluation are needed to detect PA quickly and accurately. The WHO reported about 8.8 million new cases of TB in 2010. Therefore, it is essential to focus more on monitoring of diagnosis and treatment of PA.

Background: Scaling-up in treatment for Mycobacterium tuberculosis (MTB) is a health priority. Currently, treatment regimens are long with complications, and default rates are very high. Tuberculosis (TB) patients who do not complete treatment pose a potential public health risk and are categorized as default to follow-up (DFU). Methods: In 2014, an anti-TB drug-resistance survey in Tehran, Iran, enrolled 1718 new and re-treatment patients who were TB, MDR-TB (multidrug-resistant) and old TB. TB was detected in these patients with positive acid fast bacilli (AFB) in smear and culture of sputum. Results: All patients received treatment with standardized first-line or second-line regimens (based on the type of TB infection). Patients are followed from the time of registration and treatment until the completion of treatment. This study reports the treatment outcomes of the retrospective study which was assessed from 2004 up to 2014. The reported cure rate for TB patients was 97.5% among new and old cases; 85.9% of patients (n = 1476) had no relapse; 13.7% of them (n = 236) had two or less than two relapses, and only 6 cases (0.4%) had more than two relapses; 10.5% of patients (n = 183) defaulted from anti-TB treatment and have not come back for follow-up after 52 weeks. Conclusions: These results showed the achievement of the National Tuberculosis Control Program implementation. It can be concluded that the treatment plans and appropriate follow-up of therapy have the greatest success in improving TB treatment outcomes.

Background: Seasonal fluctuations in tuberculosis (TB) notifications have been identified and reported in a number of countries. Nigeria remains one of the 22TB high-burden countries (HBCs) in the world, and the notification of TB cases in the country over the years has shown a definite pattern that suggests seasonal variation. Previous studies conducted in India, Japan, Mongolia, the Netherlands, Russia, Spain, the United Kingdom and the United States have evaluated the seasonality of TB notification. However, in Nigeria, there has been no systematic study to establish that this pattern is not just a myth. This study seeks to establish the seasonal variations suggested by the trend pattern of TB case notification (all forms of TB) in Nigeria over the past ten years. Method: The yearly TB notification data in Nigeria from 2004 to 2013 was examined for seasonal fluctuations by plotting the quarterly notification figures for the years under review. A rapid trend analysis was done based on the amplitude of the fluctuating curves. Standardization was done by zones. Results: The trend analysis showed a spike in the first quarter of the year for the ten-year period studied (with the exception of 2005 and 2011). This quarter is generally characterized by the dusty, dry harmattan wind in most parts of the country, particularly the northern region. The curves generally plummeted in the third quarter and remained in that neighborhood for the rest of the year. The differences in case notification between the first and last quarter for the ten-year period ranged from 347 to 4230 cases notified. The result of this trend analysis when standardized by zones for the six zones of the country was similar to the overall result for the country. Conclusion: According to the results of this study, there is evidence to suggest that there are seasonal variations in notification of TB cases across the four quarters of the year. This has significant implications for TB control strategies. Further investigation of the reasons for seasonal variations may help to identify risk factors. Also, planning and forecast of TB commodities to order cannot be based on experience from the preceding quarters, but must rather be based on reports from the same quarter in the previous year. Allocation of resources may also have to be intensified during the peak periods in order to adequately control the disease at these periods. Footnotes: Further investigation is required to unmask the reasons for the seasonal variations in TB notification in Nigeria.

Objective: Over the last decades, great advances in the treatment and prevention of infectious diseases have fostered complacency about infection in a society which has access to antibiotics. However, infections and also adverse reactions of such drugs remain. Also, due to the increasing demand for less toxic and more potent antibiotics, it is necessary to find new sources. Galbanum has a fresh, earthy, balsamic, woody, spicy scent. It may be used in the aromatherapy treatment of abscesses, acne, boils, bronchitis, cuts, lice, mature skin, muscle aches, poor circulation, rheumatism, scars, sores, stretch marks, and wounds. Method: In this research, antimycobacterial activity of the extract of the root and fruit of Ferula gummosa was investigated and was tested against Mycobacterium bovis. The extract of the fruit was achieved by using the maceration method, and the effect of different concentrations of the extract on the growth of Mycobacterium was compared with the standard 1 MC Farland. The quantity of colonies was investigated by the microscopic method. Streptomycin was used for the control of anti-mycobacterial test systems. Result: The result showed that Galbanum of 0.03 and 0.04g/ml have a protection effect, and the other concentrations of extract have a decreasing effect on the growth of M. bovis. Conclusion: Considering the side effects of antimycobacterial drugs, more research into some natural plants for the treatment of mycobacterial disease is necessary. In this in vitro study, the effect of Galbanum against M. bovis is highlighted. Antibacterial activity of the essential oil from the F. gummosa seed: Eftekhar et al.; Antibacterial activity of F. gummosa essential oil was studied against bacterial laboratory ATCC standards using the disk diffusion method. The results showed activity against Gram (+) bacteria and Escherichia coli. Little antibacterial activity was found against Pseudomonas aeroginosa.

Aim and objective: Infecting around one-third of the world population, Mycobacterium tuberculosis (MTB) is a serious health-threatening pathogen worldwide. Although TB has been a well-known disease since ancient times and despite the advances in medical sciences, large numbers of patients still died because of TB infection. In 2012, 1.1 million people died as a result of that infection. The development of new drugs is critical for the future control of tuberculosis (TB), and a number of promising compounds are currently in the pipeline at various stages of drug discovery and clinical development. Synthetic antibiotics for MTB treatment brought about the emergence of multidrug resistant (MDR) and extensively drug-resistant (XTB). MTB strains that has proved to be a serious challenge to global health; additionally, the long duration of treatment has various side effects. Therefore, the use of herbal medicines as an alternative or compliment to synthetic medicines has a considerable importance. The Miswak plant, which is known as “Salvadora persica” of the “Salvadoraceae” family, is traditionally used to ensure oral hygiene among Muslim people in developing countries where it is growing. The antibacterial properties of S. persica originating from various geographic areas have already been reported. The aim of this study is to detect the aquatic extract of S. persica activity on Mycobacterium bovis. Material and method: Extraction: 100 ml of boiling water was poured on the stem of this plant, then left at room temperature for 4 h, and then filtered. The crystals were put in a bath for 8 h to get the condensed extract. Phenotyping: The measurements of synthesized mycolic acids in Middlebrook culture showed growth of the bacteria. Therefore, even if the antigenic structures were destroyed, the cell wall did not form; hence the colony growth will be inhibited. Micro dilution assay: Using the lyophilization powder of the M. bovis which was provided by the Pasteur Institute, antimicrobial extract susceptibility tests were performed by broth micro-dilution methods. Result: The growth of each sample was examined three times with the following concentrations: 0.5, 1, 2.5, 5, 10, 15, 20, 25, and 30 mg/ml. The minimum inhibitory concentration for each bacteria sample became positive at 10 mg/ml each time, and the results of the first, second and third times are the same. Conclusion: As the incidence of M. bovis is increasing throughout the world, this study shows that S. persica has a high antibacterial effect on M. bovis. Other evaluations considering the effects of various herbal extracts as antibacterial agents, as well as in vivo examination of these extracts, are required to provide a natural, cost-effective and viable alternative for the traditionally less-effective antibiotics which are normally used.

Objective: Infectious diseases such as tuberculosis (TB), typhoid and nosocomial infections, play a great role in our daily life, and antibiotic treatment causes some problems, such as medical resistance and side effects; therefore, the use of herbal products may help greatly to cure infections. Methods: The rose “Golmohammadi,” known under the scientific term of “Rosa damascena” of the Rosaceae Family, is being used in traditional medicine due to its wide cultivation in Iran because of its antibacterial effect. This herb is extracted by distillation, and concentrates of 0.l, 0.3, 0.5, and 0.7 are extracted by the Kirby & Bauer method, the tube method and by the McFarland method. Result: It was found that Pseudomonas aeruginosa, Mycobacterium bovis and Salmonella typhimurium bacteria were inactivated by R. damascena. Conclusion: This study concluded that the extraction of R. damascena as an antibacterial agent can be used in the conquest of P. aeruginosa, M. bovis and Salmonella paratyphi A.

Introduction: Robust and physiologically relevant infection models are required to investigate pharmacokinetic–pharmacodynamic (PK/PD) correlations for anti-tuberculosis agents at preclinical discovery. Material and Methods: This study has validated an inhalation-based rat infection model of tuberculosis (TB) harboring mycobacteria in a replicating state that is suitable for investigating pharmacokinetics and drug action of anti-tubercular agents. A reproducible and actively replicating lung infection was established in Wistar rats by inhalation of a series of graded inocula of Mycobacterium tuberculosis (MTB). Following an initial instillation of ˜20(10)log10CFU/lung, MTB grew logarithmically for the first 3 weeks, and then entered into a chronic phase with no net increase in pulmonary bacterial loads. Dose response of front-line anti-TB drugs was investigated following pharmacokinetic measurements in the plasma of infected rats. Results: Rifampicin, Isoniazid, and pyrazinamide dosed per orally exhibited bactericidality and good dose response with maximal effect of 6.77, 3.55, and 5.901.92log10CFU reductions in the lungs, respectively. In contrast, ethambutol was merely bacteriostatic with 5.90log10CFU/lung reduction and did not reduce the bacterial burden beyond the initial bacterial loads present at the beginning of treatment in spite of high blood ethambutol levels. Rat infection model with actively replicating bacilli provides a physiologically distinct and pharmacologically relevant model that can be exploited to distinguish investigational compounds into bacteriostatic or bactericidal scaffolds. Conclusions: The present study proposes that this rat infection model – although it needs more drug substance – can be used in early discovery settings to investigate the pharmacology of novel anti-tubercular agents for the treatment of active pulmonary tuberculosis.

Introduction: Ethambutol (ETB) is an antimycobacterial agent that is most commonly used in combination with other drugs in the treatment of tuberculosis (TB). There is evidence that the drug exerts its bacteriostatic activity by inhibition of the enzyme that is a constituent of a mycobacterial cell wall. Studies have been shown that primary (pretreatment) resistance rates of Mycobacterium tuberculosis (MTB) to ethambutol vary widely, i.e., from 1% to as high as 14%. In the present study, the aim is to investigate the percent of primary resistance among new pulmonary TB cases. Material and Methods: One hundred sixty-five (n = 165) newly positive TB patients were included in this study. The susceptibility and molecular typing were performed on all culture-positive isolates. Both nested allele-specific PCR and PCR-RLFP were used to identify ETB resistant isolates. Results: Out of 165 newly diagnosed patients, 145 (88%) were sensitive to all drugs tested. In total, 20 drug-resistant isolates (13%) were detected by both methods. Four were MDR-TB (3%) and the remaining were mono-drug resistant (10%). Only 6 isolates showed resistance to ETB (4%). Conclusion: The results showed a lower rate of ETB mono-drug resistance in Iran, but more extensive studies are needed to get a clearer picture. Additionally, it was found out that the molecular methods are useful tools to rapidly identify resistant isolates.

Aim and objectives: To implement a collaborative tuberculosis (TB) project involving a low- and a high-endemic country for improved prevention and treatment of TB in both countries. Methods: Descriptive analyses in Somaliland and in Sweden based on the experiences of healthcare staff. The pattern of resistance of Mycobacterium tuberculosis (MTB) and the treatment outcome in the two countries will be compared. Background: Somaliland has among the highest incidence of TB in the world. It is also a poor country which is why every measure has to be valued depending on its cost-effectiveness. A strict standardized approach for case detection and application of treatment is therefore necessary. Active case-finding focusing on smear positivity and contagiousness is given priority before preventive therapy, though the health authorities aim at ensuring easy access to TB care in all rural areas and detection at an early stage of the disease. The general circumstances and underlying social determinants are, however, of major importance in low-resource settings, though less possible to influence. Sweden has among the lowest incidence of TB in the world, but TB is nevertheless not addressed properly among the most vulnerable and hard-to-reach groups, e.g., the newly arrived immigrants from high-incidence countries. The majority of new cases in Sweden are from the Horn of Africa. Cluster analyses have revealed a spread of TB in Sweden within the risk groups and delayed measures for preventing transmission have been observed. Patients’ delay in seeking treatment is for many reasons common, and since TB is not a generally recognized disease in Sweden, doctors taking time to give a correct diagnosis may also occur. Identified priorities are to provide information about TB, particularly for those at risk and their providers and healthcare staff. In accordance with the recommendations by WHO and the European Respiratory Society, the Swedish healthcare system screens for active and latent TB in the most vulnerable and hard-to–reach groups and have a focus on the special needs of migrants. Discussion and conclusions: Herein this study presents a planned TB project aiming at cooperation between healthcare staff from a low- and a high-endemic country. For such a project, several baseline data are required, e.g., the pattern of resistance of MTB and the treatment outcome in Somaliland, as well as among immigrants in Sweden. The social circumstances for any patient with TB, whether in Somaliland or Sweden, during disease and when recovered is a main issue for health from a holistic perspective. Further, the nutritional status is not satisfactory for TB patients in either country, and a dietary intervention may be of importance in both countries. Baseline data according to the above are necessary for assessment of the interventions and are part of ongoing pre-studies. For the Swedish party the exchange of clinical knowledge is beneficial since TB is rare in Sweden and access to TB research and clinical implantation of new methods will be facilitated and possible through the joint project. Ultimately, an expanded project could curb TB at the source and decrease TB in both countries.

Aims and objectives: The role of CD4 T cells in immuno-pathogenesis of Mycobacterium tuberculosis (MTB) has been demonstrated in mouse models and in humans. However, the role of CD8 T cells in protective immunity against TB needs elucidation. Depletion of cytotoxic CD8 T cells have been found to be associated with reduced immunity to TB in different animal models. Apoptosis and hypo-responsiveness of T cells have been reported in TB patients and correlated with the persistence of infection. In a previous report, MTB specific T cells were observed, as well as bystander T cells’ apoptosis, which is induced by ex vivo infected autologous macrophages. It becomes relevant to find out the effect of infected macrophages on antigen-specific CD8 T cells during infection. Method: Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation from 10 healthy BCG-vaccinated subjects. Monocytes from one aliquot of PBMC matured to macrophages for five days. These macrophages were infected with H37Rv at MOI of 1:10. In parallel, PBMC from a second aliquot were stimulated with CFP/WCL at a concentration of 5 μg/ml in the presence of IL-2 for five days. To study whether cytotoxic CD8 T cells undergo apoptosis in the presence of MTB-infected macrophages, CFP/WCL activated T cells were co-cultured with infected macrophages and uninfected macrophages for a further five days. The parameters studied were the expression of AnnexinV as an apoptosis marker, expression of CD95 to understand the role of Fas-FasL mediated apoptosis, expression of CD45/RO as a memory cell marker, expression of CD25 as activation markers on CD4 and CD8 T cells through flow cytometry at 48 h and 5 days of co-culture. The levels of IFN-γ and TNF-α were studied in the respective supernatant. Results: As compared with unactivated PBMC a significantly high percentage of CD4/CD25+ and CD8/CD25⁺ cells in infected Mφ-CFP/WCL stimulated T cell culture indicate the activation of T cells in response to MTB antigens. A significant percentage of CFP/WCL activated CD8 T cells were found to be AV positive in the infected co-culture assay at the fifth day indicating the apoptosis of antigen activated CD8 T cells. Interestingly, a significant percentage of CFP/WCL activated CD8/RO⁺ memory cells were found at 48 h of co-culture, but this percentage was reduced at the fifth day of co-culture. However, the CD4/RO cells percentage remained constant until the fifth day of co-culture. This reduction in CD8 /RO cells and the increase in CD8/AV⁺ cells suggested apoptosis of memory cells which may be induced by infected macrophages. A comparable percentage of CD8/CD95⁺ cells in infected and uninfected co-culture wells at both 48 h and the fifth day was observed. As compared with unactivated PBMC, the percentage of CD8/CD95⁺ cells was found to be significantly higher in both infected and uninfected co-culture assays, suggesting that observed increased apoptosis in infected co-culture assays was not mediated through Fas-FasL pathway. In the infected co-culture supernatant the IFN-γ concentration declined by the fifth day as compared with the uninfected co-culture; this can be correlated with reducing T cell number during infection. In contrast, a positive correlation was observed between the concentration of TNF-α and the percentage of CD8/AV+ cells by the fifth day in infected co-culture. It indicated the role of TNF-α released by MTB-infected macrophages in mediating CD8 T cell apoptosis. Conclusion: The present data indicates that MTB infection of macrophages could be responsible for apoptosis of CD8 T cells, which may be correlated with impaired immunity against TB.

Aims and objectives: Characterization of proteins associated with the mycobacterial cell wall is critical to understanding bacterial survival and immune modulation in the host. A variety of mycobacterial products are able to recognize and activate mammalian toll-like receptors (TLRs) mediating the secretion of antibacterial effector molecules. Mycobacterium tuberculosis MymA Rv3083 protein is a cell wall-associated protein which is up-regulated upon infection of macrophages. The objective of the present study is to understand the role of Rv3083 protein as a TLR agonist and its contribution in activating macrophages. Methods: The MymA (Rv3083) gene was cloned and expressed. The purified 55.5kDa recombinant protein was used to stimulate phorbol myristate acetate (PMA) differentiated THP-1 macrophage cell line. Cell surface markers of Rv3083 stimulated THP-1 cells were done using flow cytometry for TLR2, TLR4, HLA-DR and co-stimulatory molecules CD40, CD64 and CD80. Cytokines TNF-α, IL-10 and IL-12 were assayed in the culture supernatant using ELISA. Results: Stimulation of THP-1 macrophages for 48 and 72 h with rRv3083 protein resulted in significantly increased expression of TLR2. A significant up-regulation was also seen in the expression of co-stimulatory molecules CD40, CD80 and antigen-presenting molecule HLA-DR on THP-1 cells. These macrophages also produced a significant quantity of proinflammatory T_H1 cytokines TNF-α and IL-12, but not IL-10 at 48 and 72 h post-stimulation. Conclusion: The cell wall-associated M. tuberculosis rRv3083 protein acts as an agonist of TLR2 and thereby activates macrophages to produce a T_H1 immune response. Acknowledgements: The financial support of OSDD-CSIR and the research fellowships of ICMR to I. Saraav and S. Singh is duly acknowledged.

Background and objective: Pakistan is one of the high-tuberculosis (TB) burden countries (HBCs) with a considerable proportion of multidrug-resistant TB (MDR-TB) strains. Recently, high resistance to fluoroquinolone has been reported in both MDR and non-MDR-TB strains from Pakistan. This study aims to evaluate trends of resistance in Mycobacterium tuberculosis (MTB) against injectable second-line drugs (2010–July 2014) from Aga Khan University Laboratory, a technical partner of the Pakistan National TB Program and part of the WHO Supra-national Laboratory Network for TB based in Karachi, Pakistan. Methods: MTB strains were isolated using standard methods. Susceptibility testing was performed using the agar proportion method with drug concentrations as recommended by the Clinical Laboratory Institute Standards (CLSI). MTB H37Rv was used as a control with each batch of susceptibility testing. MDR-TB was defined as resistance to both isoniazid (0.2 μg/ml) and rifampicin (1.0 μg/ml). Results: During the study period, 14,711 MTB strains were isolated. Of these, 43.5% were MDR and 56.5% were non-MDR. Resistance pattern to amikacin, kanamycin and capreomycin over the years is shown in the table below. [INLINE:1] Conclusions: Rising resistance to injectable anti-tuberculous agents in MDR-TB isolates possibly reflects their increasing use in patient management. Presence of capreomycin resistance even amongst non-MDR strains is alarming for a country where fluoroquinolone resistance is also high and patient follow-up and drug compliance is a challenge.

Aims and objectives: Secretory antigens of Ag85 complex and 6-kDa Early Secreted Antigenic Target (ESAT-6) play a role in cell wall synthesis and virulence of Mycobacterium tuberculosis (MTB), respectively. They could induce a potent immune response and showed protective effects in some studies in animal models. So, they are considered as potential candidate antigens in new vaccination strategies against MTB. The aim of this study includes cloning, expression and purification of recombinant Ag85A-ESAT6 as a fusion protein for further studies of vaccine designation. Materials and Methods: A synthetic DNA fragment composed of coding sequences of Ag85A and ESAT6 was cloned into the BamHI and EcoRI restriction site of pET28a plasmid. Then the recombinant plasmid was transformed to Escherichia coli BL21 (DE3) and expressed in optimum condition. The expressed protein was analyzed and confirmed by SDS–PAGE and western blotting using anti-His tag and monoclonal anti-ESAT6 antibodies. Protein purification was performed by Ni–NTA spin column under denaturing conditions. Results: The results of SDS–PAGE and western blotting showed that Ag85A-ESAT6 recombinant fusion protein was successfully cloned and expressed with a His tag in its N-terminal. Considering the expression of this protein as an inclusion body, its purification was done by denaturing protocol. Conclusion: The Ag85A-ESAT6 fusion protein expressed in this study can be combined with a suitable adjuvant to induce the cellular and humoral immune responses, and its protective effects against MTB could be evaluated in subsequent studies.