Currently, one third of the world's population is latently infected with Mycobacterium tuberculosis (MTB), while 8.9–9.9 million new and relapse cases of tuberculosis (TB) are reported yearly. The renewed research interests in natural products in the hope of discovering new and novel antitubercular leads have been driven partly by the increased incidence of multidrug-resistant strains of MTB and the adverse effects associated with the first- and second-line antitubercular drugs. Natural products have been, and will continue to be a rich source of new drugs against many diseases. The depth and breadth of therapeutic agents that have their origins in the secondary metabolites produced by living organisms cannot be compared with any other source of therapeutic agents. Discovery of new chemical molecules against active and latent TB from natural products requires an interdisciplinary approach, which is a major challenge facing scientists in this field. In order to overcome this challenge, cutting edge techniques in mycobacteriology and innovative natural product chemistry tools need to be developed and used in tandem. The present review provides a cross-linkage to the most recent literature in both fields and their potential to impact the early phase of drug discovery against TB if seamlessly combined.

Background: Buruli ulcer (BU) disease, a skin condition caused by Mycobacterium ulcerans (M. ulcerans) is endemic in remote rural areas. Disease diagnosis on clinical basis alone can be misleading, requiring definitive diagnosis based on laboratory tests. Resource constraints in BU endemic areas make microscopy for the detection of acid fast bacilli (AFB) an important and useful method. It is rapid, user-friendly, convenient and cheap. Despite its usefulness, its performance is relatively low. This study investigated modifications of the current method aimed at improving its performance. Forty (IS) 2404 polymerase chain reactions (PCR) positive BU samples were processed by eight physical (centrifugation and overnight sedimentation) and chemical (phenol ammonium sulphate and sodium hypochlorite) modifications of the current direct method. Assessments were based on standard AFB evaluation coupled with in house criteria; positivity (P), clarity and contrast (C) release of bacilli from specimen (R). Overall AFB positivity rate was 64% (409/640). Each protocol had 80 smears. The percentage positivity (P) for the conventional method was 58% (46/80) smears. The highest positivity rate of 57/80 (%) was by protocol 7 (5% phenol in 4% ammonium sulphate (PhAS) and concentrated by overnight gravitational sedimentation). The least positivity rate at 35% (28/80) was by protocol 1 (smears from direct application of swab tips). The differences in performance between the two chemical tested; 5% phenol in 4% ammonium sulphate (PhAS) and 3.5% NaHOCl was significant (p < 0.05). The differences between the two physical methods were however not significant (p > 0.05). This study concluded that BU samples treated with a solution of 5% phenol in 4% ammonium sulphate and concentrated by either centrifugation or overnight sedimentation is useful for maximizing AFB detection by bright field microscopy. This can be useful in rural health facilities with resource constraints.

Tuberculosis (TB) control remains an important public health priority for Bulgaria. The population structure of Mycobacterium tuberculosis is clonal and certain genetic families of this species (e.g., Latin-American-Mediterranean [LAM]) have attracted more attention due to their global dissemination and/or particular pathogenic properties, e.g., association with multidrug resistance (MDR). The aim of this study was to evaluate the prevalence of the M. tuberculosis LAM family in Bulgaria based on the use of different molecular markers. A total of 101 previously spoligotyped M. tuberculosis strains were studied by LAM-specific PCR assay to detect an insertion of IS6110 in the specific genome region. On the whole, clear-cut results were obtained for most strains; spoligotype-based family was reassigned in some of them. At the same time, double bands were amplified in some cases and warrant further validation studies of this method. The higher MDR rate among LAM versus other genotype isolates was observed (P = 0.04). In conclusion, these results suggest a low (<4%) prevalence rate of LAM in Bulgaria (that is similar to its Balkan neighbors) and highlight the importance of using robust markers for correct detection of the LAM family.

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%. Tuberculin skin test (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% of cases, 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.

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.

Mycobacterium aurum (M. aurum) is an environmental mycobacteria that has previously been used in studies of anti-mycobacterial drugs due to its fast growth rate and low pathogenicity. The M. aurum genome has been sequenced and assembled into 46 contigs, with a total length of 6.02 Mb containing 5684 annotated protein-coding genes. A phylogenetic analysis using whole genome alignments positioned M. aurum close to Mycobacterium vaccae and Mycobacterium vanbaalenii, within a clade related to fast-growing mycobacteria. Large-scale genomic rearrangements were identified by comparing the M. aurum genome to those of Mycobacterium tuberculosis and Mycobacterium leprae. M. aurum orthologous genes implicated in resistance to anti-tuberculosis drugs in M. tuberculosis were observed. The sequence identity at the DNA level varied from 68.6% for pncA (pyrazinamide drug-related) to 96.2% for rrs (streptomycin, capreomycin). We observed two homologous genes encoding the catalase-peroxidase enzyme (katG) that is associated with resistance to isoniazid. Similarly, two emb B homologues were identified in the M. aurum genome. In addition to describing for the first time the genome of M. aurum , this work provides a resource to aid the use of M. aurum in studies to develop improved drugs for the pathogenic mycobacteria M. tuberculosis and M. leprae.

Background: Pulmonary mycobacterial diseases describe both tuberculosis (TB) and nontuberculous mycobacteria (NTM). Few data are available measuring the cost burden of mycobacterial diseases at the national level. The purpose of this study is to evaluate the cost burden and measure emerging trends in hospitalization of pulmonary TB and NTM cases in the United States from 2001 through 2012. Methods: This study is 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. The statistical significance of observed trends of NTM and TB national hospital costs was calculated using Poisson log-linear regression. Results: 20,049 hospital discharges were reported for pulmonary NTM and 69,257 for pulmonary TB in the US from 2001 through 2012. The total associated cost of these discharges was $903,767,292 for pulmonary NTM and $2,078,113,317 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). However, no such increase was found for national hospital costs of pulmonary TB. Conclusions: The national hospital cost of NTM management is increasing. These results emphasize the importance of continued research in pulmonary NTM in order to improve current guidelines in prevention and treatment strategies.

Introduction: The advent of molecular typing using MIRU-VNTR mini-satellites has largely facilitated tuberculosis (TB) molecular epidemiological studies. Apart from detecting the chains of transmission and risk factors, these markers have also allowed to study the phenomena of mixed strain infections versus microevolutionary events. Methods: An initial set of Mycobacterium tuberculosis strains (n = 161) genotyped using spoligotyping and MIRU-VNTRs in Guyana and Suriname was evaluated for indications mixed strain infections (characterized by the detection of double alleles in 2 or more MIRU loci) versus “in-patient” microevolutionary events (characterized by the detection of double alleles in a single locus). Results: The present study hereby reports evidence of microevolution in 3.7% (n = 6/161) of the studied population, vs. 0.6% (n = 1/161) for mixed infection. The strains belonged to three different spoligotyping-based lineages, namely the T (SITs 44, 53, and 1081), Haarlem (SIT47), and EAI (SITs 72 and 349) lineages, while 1 isolate (SIT237) could not be assigned to any lineage. Discussion: By comparing these results on microevolutionary cases (n = 6) to 112,000 strains present in the SITVIT2 database, evidence is presented that in 2/6 cases (each case corresponding to 2 patterns due to MIRU double bands), one of the patterns corresponded to a shared type found exclusively in Suriname or Guyana. Phylogenetic analysis showed that no spoligotyping lineage in particular was more prone to microevolutionary events in this study's sample. Overall, the observations fortify the awareness regarding the existence of microevolution and polyclonal TB infections which has important implications for patient care.

Background: Rapid and accurate techniques are always welcomed for the detection of resistant strains of Mycobacterium tuberculosis MTB. Objectives: The objective of this study is to evaluate the pyrosequencing technology for the detection of MTB resistance to Rifampicin (RIF) and Isoniazid (INH) in Syrian and Lebanese clinical strains; 66 strains resistant to INH, among them 56 resistant also to RIF, were tested. Methods: Four pyrosequencing assays were optimized and applied to the following loci: rpoB rpoB RIF resistance-determining region, katG , the promoter regions of inhA and ahpC - oxyR intergenic region. Results: The prevalence of mutations on codon 315 of the katG gene, inhA and ahpc - oxyR were 42.4%, 21.2% and 9.0%, respectively, which make an overall sensitivity of 72.6% for INH resistance. All RIF-resistant strains contained at least one non-synonymous codon change in the sequenced rpoB region (507–533) relative to the ATCC reference strain. The RIF drug resistance region (RRDR) sequencing identified 96 modified codons representing 34 different mutations. Conclusions: The high sensitivity and the short turnaround time combined with multilocus sequencing of several isolates in parallel make pyrosequencing an attractive method for drug resistance screening for MTB.

Background: Tuberculosis (TB) is a serpent disease with various pulmonary manifestations, and timely diagnosis of the disease is paramount, since delayed treatment is associated with severe morbidity, particularly in intensive care units (ICU). Therefore, it is imperative that intensivists understand the typical distribution, patterns, and imaging manifestations of TB. Aim: To describe different manifestations of pulmonary TB in patients in the ICU. Methods: In a retrospective study, all patients with a clinical and a laboratory-confirmed diagnosis of TB who were admitted to the ICU were entered in the study. All patients had a confirmatory laboratory diagnosis of TB including positive smears. The patterns of parenchymal lesions, involved segments and presence of cavity, bronchiectasis and bronchogenic spread of the lesions with computed tomography (CT) and chest/X-ray (CXR) were recorded and analyzed. Results: Data of 146 patients with TB were entered in the study. The most common finding in CT was acute respiratory distress syndrome (ARDS)-like radiologic manifestations (17.1%), followed by parenchymal nodular infiltration (13.6%) and cavitation (10.9%), consolidation (10.2%), interstitial involvement (9.5%), calcified parenchymal mass (8.3%), ground-glass opacities (7.5%), and pleural effusion or thickening (6.9%). Radiologic evidence of lymphadenopathy was seen in up to 43% of adults. Miliary TB was observed in 2.3% of patients, mostly in those older than 60 years of age. ARDS-like (64.5%) manifestations on CT and miliary TB (85.5%) had the highest mortality rates among other pulmonary manifestations. Conclusion: ARDS, interstitial involvement, and Parenchymal nodular infiltration are the most common manifestations of pulmonary TB. Various features of TB in ICU patients could be misleading for intensivists.

Background: Childhood tuberculosis (TB) has been neglected by national TB programs in sub-Saharan Africa because of the emphasis on adult smear-positive TB cases. About 80,000 HIV children die from TB, and over 550,000 childhood TB cases occur annually, representing 6% of the global TB burden, making TB an important cause of morbidity and mortality in children. Thus, this study assessed the trend of childhood TB cases notified in Lagos, Nigeria from 2011 to 2014. Methods: Retrospective data review of childhood TB cases notified to the Lagos State TB and Leprosy Control Programme (LSTBLCP) between January 1, 2011 and December 31, 2014. Results: A total of 2396 children were treated for all forms of TB representing 6.8% of the total 35,305 TB cases notified during the study period. This constituted 1102 (46%) males and 1294 (54%) females. There was a progressive increase in the proportion of children treated for TB from 495 (5.9%) in 2011, 539 (6.4%) in 2012, 682 (7.2%) in 2013 and 680 (7.6%) in 2014. Of the total childhood TB cases notified, 16.3–20% were new sputum pulmonary smear positive; 68.2–74.6% were new sputum pulmonary smear negative; while extra-pulmonary TB accounted for 6.7–10.6%. The case notification rate (CNR) of childhood TB per 100,000 increased from 13.4 in 2011, 14.3 in 2012, 17.7 in 2013 and 17.2 in 2014. Conclusion: There was an increase in the case notification rate of TB among children between 2011 and 2014. Efforts should be made to sustain this increasing trend.

Background: A concentration of specimen is recommended for the effective recovery of Mycobacterium tuberculosis (MTB), but the bacteriological efficiency is not well evaluated. The present study evaluated the factors contributing to concentration efficiency of centrifugation and bead-based technique (TB-Beads; Microsens, UK) to recover MTB by using simple in vitro specimens. Methods: Four specimens were prepared (6.5 × 10³ ; 8.1 × 10⁴ ; 7.9 × 10⁵ ; and 6.4 × 10⁶ cfu/mL) of different concentrations with or without 5 × 10⁴ of THP-1 cells (RIKEN BRC, Japan). Specimens were subjected to centrifugation at 2000, 3000, and 4000 g for 15 min, and to TB-Beads. The concentration and recovery rate were calculated to evaluate the efficiency of each method. Results: The specimens containing a higher number of bacteria and THP-1 cells had a tendency to yield a higher concentration and recovery rate (P = 0.001–0.083). MTB was recovered more efficiently with THP-1 cells from the 6.5 × 10³ cfu/mL specimen by centrifugation (p ≤ 0.001) than without them; 24.7–54.4% of MTB were recovered with THP-1 cells by centrifugation at 3000 g for 15 min, while the recovery using TB-Beads was a maximum of 12.7%. Conclusions: The efficiency of centrifugation depends on the bacterial density and the co-existence of THP-1 cells. The efficiency of TB-Beads was not as high as centrifugation.

Tuberculosis dactylitis is exceptional. We report 4 cases of osteoarticular tuberculous dactylitis in 3 women and 1 man. The diagnosis was suspected on chronic and insidious clinical presentation, and confirmed by histology. Patients were treated by anti-tubercular drugs with good functional and radiological outcome in all cases. Clinical and therapeutic issues are discussed by the authors in the context of an endemic country.

Multifocal tuberculosis (TB) is rare. It occurs especially in immunocompromised patients. Association with skin involvement is rarer, and few cases are reported in the literature. The present study reports 7 cases of multifocal TB with cutaneous localization in immunocompetent patients. Cutaneous forms of TB included in this series are: gummas, scrofuloderma, vasculitis TB and lupus TB. The patients had at least two extra skin locations, namely: osteoarticular, lung, pleural, scrotal, muscular, digestive, laryngeal, nodal and splenic locations. These patients had no context of immunosuppression which is uncommon, but should be kept in mind, especially in endemic countries.