Drug resistance among tubercle bacilli from pulmonary tuberculosis cases in central India

An estimate of drug resistance is extremely important in the epidemiology and control of tuberculosis. Data on drug resistance among mycobacterial isolates from sputum samples analysed at Microbiology dept. of Choithram Hospital and Research Centre, Indore, M.P. is presented here. Drug sensitivity testing was carried out on 1426 Mycobacterial isolates by the method of proportion using critical concentration in Lowenstein Jensen medium. Resistance for Isoniazid, streptomycin, and pyrazinamide was found to be high (54.2%, 41.5% and 50% respectively) and was followed by resistance to rifampin (25%) and ethambutol (22%). Resistance for kanamycin, p-aminosalicylic acid, thiacetazone and ciprofloxacin was much lower (18%, 13%, 6.5% and 3.6% respectively). Only 12% of the isolates were sensitive to all the anti-TB drugs while resistance to two, three, and four or more drugs was in the range of 20-25%. Pattern wise, simultaneous resistance to INF and Rifampin with or without resistance to other drugs was observed in 8.1% while resistance for Isoniazid + pyrazinamide and Isoniazid + streptomycin was 11.9 and 11.5% respectively. Resistance for Isoniazid + ethambutol was the lowest (5.1%). Growing multiple drug resistance among tubercle bacilli warrant urgent attention in tuberculosis control programme.     

Cavitary tuberculosis produced in rabbits by aerosolized virulent tubercle bacilli.

Liquefaction of solid caseous tuberculous lesions and the subsequent cavity formation are probably the most dangerous processes in the pathogenesis of human pulmonary tuberculosis. In liquefied caseum, the tubercle bacilli grow extracellularly for the first time since the onset of the disease and can reach such large numbers that mutants with antimicrobial resistance may develop. From a cavity, the bacilli enter the bronchial tree and spread to other parts of the lung and also to other people. Of the commonly used laboratory animals, the rabbit is the only one in which cavitary tuberculosis can be readily produced. This report is the first to describe and analyze the complete course of cavitary tuberculosis, produced by aerosolized virulent bovine-type tubercle bacilli in commercially available New Zealand white rabbits. After the inhalation of 220 to 880 bacillary units, all of the rabbits were overtly well until they were sacrificed at 33 weeks. After the inhalation of 3,900 to 5,800 bacillary units, half of the rabbits died of progressive tuberculosis between 5 and 9 weeks and the other half lived until they were sacrificed at 18 weeks. Pulmonary cavities developed in both low- and high-dose groups, some beginning as early as 6 weeks. Bacilli from primary cavities sometimes caused nearby secondary cavities, but more frequently, they ascended the bronchial escalator, were swallowed, and caused secondary tubercles in the lymphoid tissue of the appendix and ileocecal junction. Histologically, and by culture, the number of bacilli found in the liquefied caseum varied from many to comparatively few. Strong tuberculin reactions at 4 weeks after infection were associated with fewer primary lesions, while strong tuberculin reactions at 33 weeks were associated with more cavitary lesions. In the tuberculous granulation tissue surrounding caseous and liquefied pulmonary foci and cavities, we found many mature epithelioid macrophages that contained high levels of the proteinase cathepsin D. Therefore, cathepsin D probably plays a major role in the liquefaction of solid caseous material and in the subsequent cavity formation.     

Examination of operation specimens from patients with spinal tuberculosis for tubercle bacilli

Operation specimens from 52 Hong Kong patients considered to have tuberculosis of the spine were sent at -78 degrees C to London where they were cultured on two Löwenstein-Jensen medium slopes, a slope of 7H11 medium made selective with antibiotics and in two bottles of selective liquid Kirchner medium. Cultures of M tuberculosis, usually with only scanty colonies, were obtained from 37 of the patients. Pus and caseous matter yielded more positive cultures and more numerous colonies than other specimens, but eight of the 37 patients yielded positive cultures only from tissues lining abscess walls or from bone or intervertebral disc specimens. Cultures in Kirchner medium were never contaminated and twice as many were positive as cultures on the slopes; nine patients had cultures positive only on Kirchner medium. It is recommended that (i) a variety of operation specimens, always including pus or caseous material in volumes of at least 1 ml, should be sent for bacteriology; (ii) specimens should be cultured in selective Kirchner medium, with or without Löwenstein-Jensen slopes; (iii) cultures should be incubated for a minimum of 8-9 wk.     

Improved polyacrylamide-based artificial sputum with formalin-fixed tubercle bacilli for training of tuberculosis microscopists

Sputum smear microscopy is an easy, inexpensive, and rapid method for detecting tubercle bacilli when there are more than 10,000 bacilli/ml in the original sputum. Furthermore, because the microscopic method provides not only quantitative, but also qualitative information, such as the shape of bacilli, it has remained significant. We have previously developed and reported panel test slides made from polyacrylamide-based artificial sputum (PBAS) mixed with both cultured THP-1 cells and nonpathogenic mycobacteria. In this paper, we report an improved preparation method for PBAS for panel test slides that provides a simplified method and enhanced availability with high consistency in each grade and in which only negative PBAS is prepared from polyacrylamide and cultured THP-1 cells and mixed with graded formalin-fixed Mycobacterium tuberculosis solution (FFTBS) containing oral flora and Pseudomonas aeruginosa on the slides. In the smears prepared using this improved method, the numbers (average ± standard deviation [SD]) of acid-fast bacilli (AFB) in 300 fields (2- by 3-cm smear) in eight smears of each grade ranged from 5 to 9 (6.4 ± 1.4), from 59 to 88 (74.6 ± 10.0), from 503 to 912 (705.0 ± 145.7), and from 1,819 to 3,256 (2133.3 ± 478.0) in ±, +, ++, and +++ smears, respectively. In addition, this preparation method provided high similarity to the microscopic appearance of bacilli and background seen in the actual patient sputum, with high feasibility. These results revealed that our new PBAS had high authenticity in the appearance and consistency in each grade, which could make it valuable as a reliable artificial sputum for the training of microscopists.     

Comparative genomics of the leprosy and tubercle bacilli

To achieve the quantum leap in understanding required to overcome two major human diseases, leprosy and tuberculosis, systematic and comparative genome analysis has been undertaken. New insight into the biology of their causative agents has been obtained and the principle findings are reported here.     

Immunobiology of tubercle bacilli and prospects of immunomodulatory drugs to tackle tuberculosis (TB) and other non-tubercular mycobacterial infections

The COVID-19 pandemic has set back progress made on antimicrobial resistance (AMR). Without urgent re-focus, we risk slowing down drug discovery and providing treatment for drug resistant Mycobacterium tuberculosis. Unique in its immune evasion, dormancy and resuscitation, the causal pathogens of tuberculosis (TB) have demonstrated resistance to antibiotics with efflux pumps and the ability to form biofilms. Repurposing drugs is a prospective avenue for finding new anti-TB drugs. There are many advantages to discovering novel targets of an existing drug, as the pharmacokinetic and pharmacodynamic properties have already been established, they are cost-efficient and can be commercially accelerated for the new development. One such group of drugs are non-steroidal anti-inflammatory drugs (NSAIDs) that are originally known for their ability to supress the host proinflammatory responses. In addition to their anti-inflammatory properties, some NSAIDs have been discovered to have antimicrobial modes of action. Of particular interest is Carprofen, identified to inhibit the efflux mechanism and disrupt biofilm formation in mycobacteria. Due to the complexities of host-pathogens interactions in the lung microbiome, inflammatory responses must carefully be controlled alongside the in vivo actions of the prospective anti-infectives. This critical review explores the potential dual role of a selection of NSAIDs, as an anti-inflammatory and anti-tubercular adjunct to reverse the tide of antimicrobial resistance in existing treatments.     

Role of cytokines in immune response to pulmonary tuberculosis

Immunopathogenesis of tuberculosis needs to be explored in search of a proper vaccine as well as for adjunctive immunotherapy particularly in patients with drug resistant tuberculosis. In tuberculosis, IFN-gamma, a product of T lymphocytes, contributes to protective immunity against M. tuberculosis by activating macrophages to a more effective elimination of these organisms. Interleukin-12 and interleukin-18 are macrophage products that favor the development of Th1 type of protective immune response. Production of these cytokines may not only facilitate granuloma formation and bacillary elimination but may also cause local tissue necrosis and systemic effects such as fever and wasting, due to the release of TNF-alpha into the circulation. The production of anti-inflammatory cytokines such as IL-10, TGF-beta and IL-4 in response to M. tuberculosis may down regulate the immune response and limit tissue injury by inhibiting excessive inflammatory response. These cytokines, if produced in excess, may result in failure to control infection resulting in widely disseminated tuberculosis. It is the balance between the inflammatory and protective immune response that determines the outcome of tuberculosis infection. In that context, increased IFN-y as against reduced TNF-alpha probably suggests a better outcome. Similarly, an effective vaccine has to stimulate a precise combination of T cells and cytokines needed for the many aspects of immune response and a potent immunotherapeutic agent may require to encompass the multiple parameters to be of therapeutic relevance.     

Factors influencing response to treatment of pulmonary tuberculosis

We analyzed 150 patients with pulmonary tuberculosis from 1990 to 1996 (i) to evaluate the frequency of drug resistance, (ii) to elucidate factors influencing the response to chemotherapy, and (iii) to attempt to improve the therapeutic approach. Multidrug-resistant tuberculosis strains were not found. By univariate analysis, there were 8 factors associated with an increased sputum conversion time: male gender, prior treatment, complications, progressive chest radiographic findings, a high Ziehl-Neelsen stain score, lymphocytopenia, a high erythrocyte sedimentation rate (ESR), and hypoproteinemia. Complications, prior treatment, a high Ziehl-Neelsen stain score, and a high ESR were independent predictive factors in a Cox proportional hazard model. Recursive partitioning and amalgamation (RPA) defined 3 subgroups that responded to treatment. In order to reduce the time to sputum conversion, poor responders according to the RPA should be treated with a 4-drug regimen containing pyrazinamide.     

Host genetic studies in adult pulmonary tuberculosis

Early observations, candidate gene studies and, more recently, genome-wide association studies have shown that susceptibility to tuberculosis has a host genetic component. Because the value of candidate gene studies has been doubted due to major limitations such as lack of sufficient power and small study groups, lack of reproducibility in independent groups and, often, ambiguous or even contrasting results in attempts of replication, much hope and expectancy has been put on the progress the genome-wide association approach has created. However, much less than initially expected became clear by the results obtained in genome-wide studies, emphasizing the need of increasing sample sizes, e.g. through meta-analyses, and of increasing the density of genetic variants studied across the human genome. A further reason why a rather low number of associated genetic variants were identified to date in infectious diseases in general and tuberculosis in particular might be the fact that selection acts strongly in diseases that affect the reproductive success. As in most genome-wide association studies performed so far, significant signals, often most likely surrogate marker only, have been found in non-coding regions of genomes, the identification of truly causative genetic variation and of the functionality of associated factors needs urgent attention. In the following we briefly discuss genetic studies in tuberculosis and describe new technologies that are currently employed in the search for responsible genetic elements involved in tuberculosis susceptibility.     

Survival of tubercle bacilli in heat-fixed sputum smears.

Tubercle bacilli, which survived heat fixation, were detected with a slide culture technique which allowed the entire smear to be examined. Both conventional flame fixation and the use of a controlled hot-plate failed to render tuberculous sputum smears safe for further handling. Smears which were stained with the phenol-auramine method failed to yield growth on culture. If delay between preparation and staining is unavoidable, it is recommended that smears are given additional treatment to prevent the survival of tubercle bacilli.     

Universal occurrence of antibodies to tubercle bacilli in sera from non-tuberculous and tuberculous individuals

Studies were carried out to detect and quantitate humoral antibodies to components derived from Mycobacterium tuberculosis. Using both quantitative and qualitative primary tests, binding was observed by all sera obtained from normal as well as from tuberculous subjects. There was a significant difference, however, in the amount of binding by sera from controls. There was a high incidence of both IgG and IgM antibodies in all sera.

The universal occurrence of humoral antibodies demonstrated in this study suggests that most, if not all, persons have been sensitized to the tubercle bacillus to one degree or another. Evidence is presented that the immunogens involved may be derived from saprophytic and/or avirulent mycobacteria.

     

Selective media in the isolation of tubercle bacilli from tissues

Direct culture on Lowenstein-Jensen slopes and on three media made selective for tubercle bacilli by the addition of four antibacterial agents was compared with guinea-pig inoculation on 490 tissue specimens. Tubercle bacilli were obtained from 15 specimens by culture and 14 by guinea-pig inoculation; only one specimen was positive by guinea-pig and not by culture. The most efficient culture medium was a selective 7H11 slope. Routine guinea-pig inoculation has been replaced by a wider range of culture procedures.