SETTING: A tertiary university medical centre in north-ern Israel.OBJECTIVE: To evaluate the clinical significance of non-tuberculous mycobacteria (NTM) isolated from pulmonary specimens.DESIGN: Clinical and microbiological data were col-lected from patient files. Cases were classified as definite, probable and possible NTM.RESULTS: Between 2004 and 2010, 215 cases with respiratory isolates of NTM were identified. Mycobacterium xenopi was the most common species (n = 84, 39.1%), followed by M. simiae (n = 52, 24.2%). A total of 170 (79.1%) cases were classified as possible and 24 (11.2%) as probable NTM. Only 21 (9.8%) cases were considered definite NTM, the majority of which were M. kansasii and M. avium complex. CONCLUSIONS: M. xenopi and M. simiae are the most prevalent species of NTM isolated from respiratory samples in northern Israel. However, most of these isolates represent colonisation. Of the relatively small number of clinically significant isolates, M. kansasii and M. avium complex were the most common.

The cannabinoid system is known to be involved in the regulation of inflammatory processes. Therefore, drugs targeting cannabinoid receptors are considered as candidates for anti-inflammatory and tissue protective therapy. We demonstrated that the prototypical cannabinoid agonist R(+)WIN55,212-2 (WIN) reduced the secretion of matrix metalloproteinase-9 (MMP-9) in a murine model of cigarette-smoke induced lung inflammation. In experiments using primary cells and cell lines of the monocyte-macrophage-system we found that binding of the cannabinoid-receptor agonist WIN to a stereo-selective, specific binding site in cells of the monocyte-macrophage-system induced a significant down-regulation of MMP-9 secretion and disturbance of intracellular processing, which subsequently down-regulated MMP-9 mRNA expression via a ERK1/2-phosphorylation-dependent pathway. Surprisingly, the anti-inflammatory effect was independent from classical cannabinoid receptors. Our experiments supposed an involvement of TRPV1, but other yet unidentified sites are also possible. We conclude that cannabinoid-induced control of MMP-9 in the monocyte-macrophage system via a cannabinoid-receptor independent pathway represents a general option for tissue protection during inflammation, such as during lung inflammation and other diseases associated with inflammatory tissue damage.

Cutaneous tuberculosis continues to be an important public health problem even with the availability of highly effective anti-tuberculous drugs. It constitutes 0.1% of all cases of extrapulmonary tuberculosis. Lupus vulgaris is the most common form of cutaneous tuberculosis that occurs in previously sensitized individuals with a moderate degree of immunity against tubercle bacilli. The different types of lupus vulgaris include plaque, ulcerative, vegetative, papular and nodular, and tumor forms. A 40-year-old man presented with large multiple plaques over right upper limb, right side of chest and back, and right lower limb for the past 30 years. Histopathology showed numerous noncaseating granulomas with Langhan's type of giant cells. The Mantoux test showed strong positivity and there was excellent response to anti-tuberculous treatment. This case is being reported because of its extreme chronicity of 30 years duration, unusually large size and multiplicity of lesions.

Cryptococcosis is a common opportunistic infection among immunocompromised individuals. Some of the commonly affected sites are respiratory and central nervous system. Lymph node is an unusual site of involvement which could mimic tuberculosis, as seen in our case. We report a 32-year-old male immunocompromised patient presenting with generalized lymphadenopathy who was clinically suspected to have tuberculous lymphadenitis. He was diagnosed to have disseminated cryptococcosis on fine needle aspiration cytology and fungal isolation on culture.

BACKGROUND Microscopy detection of acid fast bacilli (AFB) by Ziehl-Neelsen (ZN) method has many advantages when it comes to speed and feasibility though it has a low sensitivity. If the sensitivity could be improved, it has the potential to become an even more valuable tool for detection of AFB. OBJECTIVES To evaluate the efficacy of bleach concentration method in the cytodiagnosis of tuberculous lymphadenitis in comparison with routine Ziehl-Neelsen method and to compare the positivity in various cytomorphological categories. MATERIALS AND METHODS A total of 112 cases of tuberculous lymphadenitis diagnosed by fine needle aspiration cytology (FNAC) were categorized into six cytomorphological patterns. The acid-fast bacilli positivity by routine staining was correlated with modified bleach methods of ZN staining. Sensitivity of routine ZN and modified bleach concentration was compared. RESULTS The classic cytomorphological pattern of tuberculosis of epithelioid granulomas, langhans giant cells and caseous necrosis was seen in 37.5% of cases. Routine ZN staining detected AFB in 12.5% of cases and the modified bleach method in 60.7%. Modified bleach method showed AFB positivity in additional 54 cases where routine AFB staining was negative. The modified bleach method showed AFB positivity in all cases where routine ZN staining was positive. CONCLUSION The modified bleach method was more sensitive and safer than routine ZN staining. As the background was clear, the bacilli were easily visible and the screening time was shorter.

Multidrug chemotherapy for 6-9-months is one of the primary treatments in effective control of tuberculosis, although the mechanisms underlying the persistence of its etiological agent, Mycobacterium tuberculosis, against antibiotics remain unclear. Ever-mounting evidence indicates that the survival of many environmental and pathogenic microbial species against antibiotics is influenced by their ability to grow as surface-associated multicellular communities called biofilms. In recent years, several mycobacterial species, including M. tuberculosis, have been found to form drug-tolerant biofilms in vitro through genetically controlled mechanisms. In this review, the authors discuss the relevance of the in vitro mycobacterial biofilms in understanding the antibiotic recalcitrance of tuberculosis infections.

We have isolated and identified the biotype of environmental mycobacteria from the expectorate of leprosy patients, their contacts, their drinking water supply and also from the sputa samples of tuberculosis patients. 78% of the isolates from lepromatous leprosy patients and their contacts wereMycobacterium fortuitum- chelonae complex (MFC), 9%Mycobacterium avium complex (MAC), 9%Mycobacterium scrofulaceum and 4% wereMycobacterium smegmatis. Among the isolates from tuberculosis patients 63% belonged toM. fortuitum- chelonae complex, 19% toM. avium complex, 12% toMycobacterium Kansasii and 6% toM. smegmatis. All the isolates were multi-drug resistant when tested for sensitivity total of 21 drugs. TheMycobacterium fortuitum-chelonae complex organisms from leprosy contacts were more sensitive to rifampicin than those isolated from lepromatous leprosy and tuberculosis patients. Among 23 isolates from leprosy patients one isolate was resistant to 20 drugs, one isolate to 17 drugs and another isolate was resistant to 13 drugs. Among the 18 isolates from drinking water supply six showed resistance to more than 12 drugs. Polymerase Chain Reaction (PCR) and subsequent hybridisation with specific probes confirmed all the isolated strains as nontuberculous mycobacteria (Using genus primers and probe sensitivity 100%) and none asM. tuberculosis, suggesting that PCR could be used to rapidly identify mycobacteria at the genus level and to rule out tuberculosis in leprosy patients at an early stage to decide on appropriate course of therapy.

Background. Tuberculosis, a global health problem and highly prevalent in India, has always been a serious problem with respect to definitive diagnosis. Polymerase chain reaction (PCR) techniques are now widely used for early detection and species differentiation of mycobacteria, but mostly with their own limitations. We aim to detect and differentiate Mycobacterium tuberculosis (Mtb) infections by choosing appropriate target sequences, ideally present in all mycobacterial species (MTB complex) and absent in others. Methods. Amplification of three target sequences from unrelated genes, namely, hsp 65 (165 bp), dnaJ (365 bp), and insertion element IS 6110 (541 bp) by PCR was carried out in clinical samples from suspected cases of tuberculosis/ mycobacterioses and healthy controls. Results. The sensitivity of this method ranged from 73.33% to 84.61%, and the specificity was 80%. The PCR method was significantly better (P = 0.03 and P = 0.009) than both smear and culture methods. Conclusion. Our trimarker-based PCR method could specifically detect M. tuberculosis and MTB complex infection from that of major pathogenic NTM and nonpathogenic mycobacteria. This method, by well distinguishing between MTB complex and NTM, presented a fast and accurate method to detect and diagnose mycobacterial infections more efficiently and could thereby help in better patient management particularly considering the increase in mycobacterial infections due to emergence of NTM over the past decades.

Background.  There have been few studies on cutaneous tuberculosis (TB) in Europe in recent years. Objective.  To retrospectively analyse the evolution of the various types of cutaneous TB over the past 30 years in an adult population in Spain. Methods.  Patients with cutaneous TB diagnosed between 1981 and 2011 at Bellvitge Hospital, Barcelona, Spain, were included in the study. Chest radiography was performed for all patients, and the presence of TB elsewhere in the body was excluded when clinically suspected. Results.  In total, 36 patients (15 male, 21 female, mean age 53.72 years) were diagnosed with cutaneous TB. There were 22 patients with lupus vulgaris (LV), 4 with scrofuloderma, 4 with miliary TB, 3 with tuberculous abscess/ulcer, and 1 each with orificial TB, warty TB, and an iatrogenic inoculation from underlying visceral focus. Of the 36 patients, 16 (38.88%) had TB presenting simultaneously in other organs. Mycobacterial culture from skin biopsies was positive for Mycobacterium tuberculosis complex in 17 of the 32 cases tested (53.12%), whereas stains for acid-fast bacilli in skin samples were positive in only 3 of 36 patients (8.33%). Conclusions.  Although the number of cases of cutaneous TB diagnosed yearly in our population has declined over the past 30 years, cutaneous TB still exists in Europe, and its incidence is expected to increase, owing to the increased immigration into the continent in recent years. The most common type of cutaneous TB in our adult population was LV. It should be noted that despite being considered a benign form of TB, cutaneous TB can be accompanied by TB in internal organs, and severe complications can occur, such as the development of squamous cell carcinoma in long-lasting lesions.

We sought to determine tuberculosis (TB) prevalence including multidrug resistant (MDR)-TB among a cohort of high risk patients at two directly observed treatment short course (DOTS) clinics in Delhi, India. We also aimed to compare the sensitivity of acid-fast bacilli (AFB) smear tests for patients with HIV using sputum cultures as the gold standard. A cross-section study was conducted among adult patients (> or = 18 years old) with prolonged cough (greater than two weeks), night sweats, fever, and/or weight loss suspected of pulmonary TB between February and March 2006. Sputum samples were obtained and processed for 165 patients; 53 (32.1%) were culture positive for TB. Patients with TB were predominantly male (92.1%), young (median age of 32 years), and the HIV-seroprevalence was high (41.5%). In the multivariable analysis adjusted for age, HIV infection was significantly associated (POR = 2.0, p < 0.05) with the presence of TB disease. Among Mycobacterium tuberculosis isolates recovered from 53 cases, 25 (47.2%) were resistant to > or = 1 first line anti-TB drugs and 7 (13.2%) were MDR-TB. The sensitivity of AFB smears among HIV negative and positive participants was 35.5% and 18.0%, respectively. Our findings demonstrated that the sensitivity of AFB smears to detect TB among HIV positive patients was low. Additionally, we found that even in regions where population drug resistance estimates are low, sentinel surveillance of MDR-TB in high-risk populations is useful to prioritize target groups in need of additional prevention, monitoring and health outreach.

Bovine tuberculosis (BTB) is a widespread and endemic disease of cattle in Ethiopia posing a significant threat to public health. Regular surveillance by skin test, bacteriology, and molecular methods is not feasible due to lack of resources. Thus, routine abattoir (RA) inspection will continue to play a key role for national surveillance. A cross-sectional study was conducted at Woldiya municipal abattoir from April 1, 2009 to April 5, 2010 to estimate the prevalence of BTB in slaughtered cattle on the basis of detailed abattoir inspection and to compare efficacy of RA inspection with respect to detailed abattoir inspection and isolation and identification of Mycobacterium. Diagnostic accuracies (with corresponding measures of statistical uncertainty) were determined by computing test property statistics (sensitivity and specificity). Agreement between RA and detailed abattoir inspections was measured using kappa statistics. Out of 1,029 slaughtered heads of cattle examined during the study period, 63 (6.12 %) and 15 (1.45 %) were diagnosed with gross tuberculous lesions by detailed abattoir meat inspections and RA meat inspections, respectively, making a prevalence of 6.12 %(95 % CI: 5.2-7.1) on the basis of detailed abattoir inspection. About 59.45 % of tuberculous lesions were observed in the lungs and associated lymph nodes, whereas 35.13 % lesions were from the lymph nodes of the head. From 63 cattle suspected with tuberculosis (TB) based on detailed abattoir meat inspection, nine (19.05 %) were identified as Mycobacterium bovis, while three (4.8 %) as Mycobacterium tuberculosis. The sensitivity of RA meat inspection was 23.8 % in comparison to the detailed abattoir meat inspection and 25 % in comparison to culture, respectively. Poor agreement (k = 0.37) was seen between RA meat examination and detailed abattoir meat examination methods. Similarly, poor agreement (k = 0.013) was seen between RA meat examination and culture results. In conclusion, relatively higher prevalence (6.12 %) was recorded in Woldiya municipal abattoir on the basis of detailed Abattoir inspection and RA meat inspection protocols currently utilized in Ethiopia which are insufficient to detect the majority (76.19 %) of TB lesions at the gross level, which indicates the magnitude of meat borne zoonotic TB as an ongoing risk to public health. Detailed abattoir inspection protocols were demonstrated to improve the detection level by approximately fourfold. In conclusion, routine meat inspections have limitations in detecting BTB-suggestive lesions which indicate the magnitude of meat-borne zoonotic TB as an ongoing risk to public health.

Kawasaki disease (KD) is the most common cause of multisystem vasculitis in childhood. Although cervical lymphadenitis is one of the major symptoms in KD, lymph node biopsy is rarely performed, because KD is usually diagnosed by clinical symptoms. A cervical lymph node biopsy was taken from a girl aged 1 year and 8 months who had suspected lymphoma, but she was diagnosed with KD after the biopsy. The cervical lymph node specimen was analyzed with multivirus real-time PCR that can detect >160 viruses, and unbiased direct sequencing with a next-generation DNA sequencer to detect potential pathogens in the lymph node. Histologically, focal necrosis with inflammatory cell infiltration, including neutrophils and macrophages, was observed in the marginal zone of the cervical lymph node, which was compatible with the acute phase of KD. Multivirus real-time PCR detected a low copy number of torque teno virus in the sample. Comprehensive direct sequencing of the cervical lymph node biopsy sample sequenced more than 8 million and 3 million reads from DNA and RNA samples, respectively. Bacterial genomes were detected in 0.03% and 1.79% of all reads in DNA and RNA samples, respectively. Although many reads corresponded to genomes of bacterial environmental microorganisms, Streptococcus spp. genome was detected in both DNA (77 reads) and RNA (2,925 reads) samples. Further studies are required to reveal any association of microbial or viral infection with the pathogenesis of KD.

Malacoplakia is a rare inflammatory condition characterized by the accumulation of benign macrophages associated with pathognomonic Michaelis-Gutmann bodies (MGBs). It is usually found in the genito-urinary tract, and has been associated with immunocompromised states. In this short report, we present 5 patients with pulmonary nodules clinically suspicious for primary or metastatic lung cancer. The histologic examination of the surgical specimens revealed a nonspecific granulomatous chronic disease, and despite the paucity of classical MGBs, a pulmonary malacoplakia was suspected. In all cases the opportunistic pathogen Rhodococcus equi (R. equi) was identified by 16S rRNA gene sequence analysis, leading to the final pathological diagnosis of malacoplakia. We conclude that pulmonary malacoplakia associated with R. equi is a rare disease affecting also immunocompetent patients. The pathogenesis and the diagnostic problems are discussed. Since infection by R. equi is treatable, the importance of its early recognition should be emphasized.

The purpose of this investigation was to evaluate the usefulness of multiplex polymerase chain reaction (m-PCR) in detecting uterine tuberculosis in women with infertility. In a prospective study, endometrial curetting from 620 females with infertility were investigated using laparoscopy, hysteroscopy, histopathology, smear microscopy, mycobacterial culture in BACTEC MGIT™ 960, and in-house m-PCR. The mean age of the women was 29.75 ± 4.66 years. The majority (596) sought medical attention for infertility; of them, 455 (76.34 %) presented with primary and 141 (23.65 %) with secondary infertility. A total of 158 (25.48 %) women were diagnosed as having uterine tuberculosis by at least one of the diagnostic methods. Among them, laparoscopy was positive in 46 (29.11 %), hysteroscopy in 77 (48.73 %), histopathology in only 8 (5.06 %), smear for acid fast bacilli in 4 (2.53 %), and liquid culture in 24 (15.18 %) patients. The in-house m-PCR was positive in 135 (85.44 %) women. Of these, 129 (95.55 %) samples were positive for Mycobacterium tuberculosis, while 6 (4.44 %) were positive for non-tuberculous mycobacterial DNA. Of the 129 M. tuberculosis PCR-positive women, 112 received anti-tubercular treatment and 23 of these conceived and fell pregnant after the completion of treatment. For the diagnosis of uterine tuberculosis, m-PCR was found to be the most efficient diagnostic tool compared to the other methods.

Members of the Mycobacterium avium complex (MAC) are naturally occurring bacteria in the environment. A link has been suggested between M. avium strains in drinking water and clinical isolates from infected individuals. There is a need to develop new screening methodologies that can identify specific virulence properties of M. avium isolates found in water that predict a level of risk to exposed individuals. In this work we have characterized 15 clinical and environmental M. avium spp. isolates provided by the US Environmental Protection Agency (EPA) to improve our understanding of the key processes involved in the binding, uptake and survival of these isolates in primary human macrophages. M. avium serovar 8 was predominant among the isolates studied. Different amounts and exposure of mannose-capped lipoarabinomannan (ManLAM) and glycopeptidolipids (GPLs), both major mycobacterial virulence factors, were found among the isolates studied. Reference clinical isolate 104 serovar 1 and clinical isolates 11 and 14 serovar 8 showed an increased association with macrophages. Serum opsonization increased the cell association and survival at 2 h post infection for all isolates. However, only the clinical isolates 104 and 3 among those tested showed an increased growth in primary human macrophages. The other isolates varied in their survival in these cells. Thus we conclude that the amounts of cell envelope ManLAM and GPL, as well as GPL serovar specificity are not the only important bacterial factors for dictating the early interactions of M. avium with human macrophages.

SUMMARY A dairy herd (77 cows) from Rio de Janeiro, Brazil, with a history of tuberculosis infection was tested by a comparative cervical test (CCT). Seventeen cows were reactive and seven were inconclusive (swelling ⩾2·0 mm and ⩽3·9 mm, respectively). All of these 24 cows were slaughtered and necropsied; samples from lungs and lymph nodes were collected for multiplex polymerase chain reaction (PCR) and culturing. Infection was confirmed in 23/24 (95·8%) of the slaughtered animals (five by culturing, four by PCR, and 14 by both tests). All cows with inconclusive results at CCT were confirmed as infected. Although slaughter of inconclusive reactor cows is not mandatory in many countries, our study provided evidence to support the slaughter of these cows, at least during an outbreak.

BackgroundDetermination of disease activity of chronic destructive pulmonary tuberculosis (TB) on imaging studies can be difficult because several imaging findings due to disease chronicity such as a residual cavity can be misinterpreted as an active disease.PurposeTo evaluate computed tomography (CT) findings to predict active disease in patients with chronic destructive pulmonary TB.Material and MethodsCT findings of 36 patients with chronic active destructive pulmonary TB and 78 patients with chronic inactive destructive pulmonary TB were reviewed and their patterns of lung lesions were compared. Statistical comparisons were performed using chi-square and Student's T tests for univariate analyses, and a stepwise logistic regression method was used for multivariate analysis.ResultsBased on univariate analyses, cavitary destruction (P = 0.015), non-branching centrilobular nodules (P < 0.001), tree-in-bud pattern (P < 0.001), airspace nodules (P < 0.001), and cavities in other lobes (P = 0.001) were more frequently seen in chronic active destructive pulmonary TB. A stepwise logistic regression analysis demonstrated that tree-in-bud pattern (odds ratio, 52.3; 95% confidence interval, 6.2-437.2; P < 0.001) were significant CT findings associated with active disease.ConclusionTree-in-bud pattern were the most characteristic CT findings to predict active disease in patients with chronic destructive pulmonary TB.

ABSTRACT: BACKGROUND: The genus Mycobacterium (M.) comprises highly pathogenic bacteria such as M. tuberculosis as well as environmental opportunistic bacteria called non-tuberculous mycobacteria (NTM). While the incidence of tuberculosis is declining in the developed world, infection rates by NTM are increasing. NTM are ubiquitous and have been isolated from soil, natural water sources, tap water, biofilms, aerosols, dust and sawdust. Lung infections as well as lymphadenitis are most often caused by M. avium subsp. hominissuis (MAH), which is considered to be among the clinically most important NTM. Only few virulence genes from M. avium have been defined among other things due to difficulties in generating M. avium mutants. More efforts in developing new methods for mutagenesis of M. avium and identification of virulence-associated genes are therefore needed. RESULTS: We developed a random mutagenesis method based on illegitimate recombination and integration of a Hygromycin-resistance marker. Screening for mutations possibly affecting virulence was performed by monitoring of pH resistance, colony morphology, cytokine induction in infected macrophages and intracellular persistence. Out of 50 randomly chosen Hygromycin-resistant colonies, four revealed to be affected in virulence-related traits. The mutated genes were MAV_4334 (nitroreductase family protein), MAV_5106 (phosphoenolpyruvate carboxykinase), MAV_1778 (GTP-binding protein LepA) and MAV_3128 (lysyl-tRNA synthetase LysS). CONCLUSIONS: We established a random mutagenesis method for MAH that can be easily carried out and combined it with a set of phenotypic screening methods for the identification of virulence-associated mutants. By this method, four new MAH genes were identified that may be involved in virulence.

Lymphadenopathy is a rare mode of presentation of cysticercus infestation. Hence, in endemic areas, cysticercosis must be included in the differential diagnosis of superficial palpable swellings in the neck region. We report two cases of cervical lymphadenopathy which were clinically suspected to be of tuberculous etiology. However, fine-needle aspiration cytology (FNAC) revealed features of parasitic lymphadenitis consistent with cysticercosis. Our cases highlight the importance of FNAC as an initial and rapid diagnostic modality for detecting parasitic lesions manifesting as lymphadenitis. Diagnosis by the minimally invasive FNA technique prompted an early therapeutic intervention with good response in our patients.

UNLABELLED ABSTRACT: BACKGROUND This study aims to analyze the structure and quantities of cellular elements in sarcoid granulomas. METHODS We investigated 34 transbronchial lung biopsy samples obtained from 34 sarcoid patients. The quantity and composition of the cellular elements inside a granuloma were determined by the quantitative stereometry method, employing the numerical density as a stereological method. RESULTS A total of 102 sarcoid granulomas were analyzed. The central part of all granulomas was occupied by epithelioid cells. Besides these, giant cells, lymphocytes, macrophages and plasma cells were also seen. The mean numerical density of all the cells in the central part of a sarcoid granuloma was 111,751 mm-3. Lymphocytes prevailed in number, exceeding the total count of all other cells. With a mean numerical density of 74,321 mm-3, lymphocytes were twice as numerous as both epithelioid cells and macrophages with a mean numerical density of 37,193 mm-3. CONCLUSIONS Lymphocytes are the predominant cell type in the central part of a sarcoid granuloma, significantly exceeding both epithelioid cells and macrophages in number, raising the question if the term "epithelioid granuloma", routinely used to designate sarcoid granulomas, is correct, or if it would be more logical to call them "lymphocytic-epithelioid granulomas" instead. TRIAL REGISTRATION This study was supported by the Serbian Ministry of Science and Environmental Protection Grant Number 175006/2011.