Phage cocktail to control the exponential growth of normal flora in processed sputum specimens grown overnight in liquid medium for rapid TB diagnosis.

The mechanical pressure exerted during centrifugation and the chemical pressure experienced when sputum specimens are processed, leave the tubercle bacilli in the sputum unsuitable for rapid detection especially in phage based assays. Thus, growing Mycobacterium tuberculosis in broth, at least overnight, is mandatory for allowing the tubercle bacilli to recoup. During this time the surviving colonizing flora grow faster and overgrow tubercle bacilli interfering with TB diagnosis. In the present study normal flora surviving the action of 4% NaOH was isolated and characterized. Phages capable of killing 14 different species representing this normal flora were isolated from soil and sewage samples and characterized. A novel and bio-friendly approach to treat sputum samples with a cocktail of three phages capable of killing most of the 14 representative organisms and not infecting mycobacteria is explored to control the overgrowth of colonizing bacteria in broth culture. While 26 of the 100 sputum samples processed by modified Petroff's procedure showed growth of colonizing flora on blood agar, all of them when grown in broth overnight showed mixed, confluent growth. The addition of phagebiotics controlled them all, showing a significant reduction in colony forming units but resulting in few discrete colonies in 54 samples. Isolation of phages capable of controlling these surviving organisms and including them in the phagebiotics mixture should lead to the control of colonizing bacteria effectively.

A novel approach for reducing toxic emissions during high temperature processing of electronic waste.

A novel approach is presented to capture some of the potentially toxic elements (PTEs), other particulates and emissions during the heat treatment of e-waste using alumina adsorbents. Waste PCBs from mobile phones were mechanically crushed to sizes less than 1mm; their thermal degradation was investigated using thermo-gravimetric analysis. Observed weight loss was attributed to the degradation of polymers and the vaporization of organic constituents and volatile metals. The sample assembly containing PCB powder and adsorbent was heat treated at 600°C for times ranging between 10 and 30min with air, nitrogen and argon as carrier gases. Weight gains up to ∼17% were recorded in the adsorbent thereby indicating the capture of significant amounts of particulates. The highest level of adsorption was observed in N2 atmosphere for small particle sizes of alumina. SEM/EDS results on the adsorbent indicated the presence of Cu, Pb, Si, Mg and C. These studies were supplemented with ICP-OES analysis to determine the extent of various species captured as a function of operating parameters. This innovative, low-cost approach has the potential for utilization in the informal sector and/or developing countries, and could play a significant role in reducing toxic emissions from e-waste processing towards environmentally safe limits.

Biochemical & histochemical changes relating to fibrosis following infection with Mycobacterium tuberculosis in the guinea pig.

Guinea pigs infected with M. tuberculosis were studied for parameters relating to fibrosis following infection. The infected animals were followed up to a period of 44 wk and the changes that occurred in the lung, liver and spleen were studied. Corresponding tissues from animals injected with bleomycin, an anti-mitotic drug which has the ability to produce pulmonary fibrosis, served as positive controls. Tissue collagen, elastin and hexosamines were estimated biochemically. The presence of granuloma and stainable collagen in paraffin sections of these tissues was also studied. Establishment of the infection was assessed bacteriologically by culturing the viable organisms from the spleen. It was observed that a self-limiting infection was established in the guinea pigs and none of the animals died of the infection. In the infected animals, collagen, elastin and hexosamines showed an initial decrease followed by an increase. While the elastin and the hexosamine levels returned to the basal levels in all the three organs, collagen levels increased in the lung and were comparable to those of the bleomycin control. Collagen stainable by Van Gieson's method was found to be increased in the lung from the 4th wk onwards. The present report indicates the potential of adopting this system for studying mechanisms of fibrogenesis in tuberculous infection.

The clearance of tubercle bacilli & mycobacterial antigen vis a vis the granuloma in different organs of guinea pigs.

In the present study, an attempt was made to define the relationship of intact tubercle bacilli and/or their antigenic fragments to a granuloma in the guinea pig in order to distinguish an active from a resolving granuloma. In one set of animals, granuloma was induced in the skin by injecting heat-killed Mycobacterium tuberculosis intradermally and in another set, granuloma was produced in the lung and spleen by injecting live M. tuberculosis intramuscularly. The animals were sacrificed at various time points and skin, lung and spleen from the two groups were subjected to histological examination for the presence of granuloma, bacilli and antigenic fragments. In the dermal lesion, intact acid fast bacilli were cleared first by day 42 followed by the removal of their antigenic fragments by day 63 and finally by day 84, the granuloma had resolved completely. In the guinea pigs infected with live M. tuberculosis, removal of the bacilli followed by the clearance of antigen was observed. Though the granuloma itself did not subside completely in these animals, it was found that there was a reduction in congestion and oedema of the granulomatous area. It is concluded from the results that the demonstration of antigen at the site of lesion may be potentially useful to discriminate between a persisting and a resolving tuberculous granuloma.

Induction of rifampicin metabolism during treatment of tuberculous patients with daily and fully intermittent regimens containing the drug.

Self-induction of rifampicin metabolism during daily and intermittent chemotherapy was studied by monitoring the changes in the serum half-life of the drug over a 4-week period in patients with pulmonary tuberculosis. Rifampicin 450 mg was administered to 8 patients who received treatment daily, 7 on thrice-weekly and 7 others on twice-weekly treatment. Serum half-life was computed from concentrations of the drug determined at 3, 4 1/2 and 6 hours after drug administration, on admission and at 1, 2 and 4 weeks after start of treatment. In the daily series, the mean serum half-life decreased from 4.9 hours on admission to 3.6 hours at 1 week (P = 0.02), and treatment beyond this had no further effect. In the thrice-weekly series, maximal induction was observed at the 2nd week, the mean values on admission and at 2 weeks being 5.8 and 3.7 hours, respectively (P less than 0.01). In the twice-weekly series, maximal induction was observed only at the 4th week, the mean values on admission and at 4 weeks being 4.9 and 3.7 hours, respectively (P less than 0.01). Serum activity of gamma glutamyl transferase was not found to be a suitable in vivo marker to monitor induction of the hepatic microsomal enzymes as no significant changes were observed in the activity of this enzyme in any of the 3 series during the 4-week period.

Nitrogen laser irradiation (337 nm) causes temporary inactivation of clinical isolates of Mycobacterium tuberculosis.

We have investigated the effect of nitrogen laser irradiation (337 nm) on viability of clinical isolates of Mycobacterium tuberculosis. Bacteria were exposed to a nitrogen laser (average power 2.0 mW) in vitro at power density of 70 +/- 0.7 W/m2 for 0-30 min, and the cell viability was determined by luciferase reporter phage (LRP) assay. Immediately after laser exposure, all the clinical isolates investigated showed a dose-dependent decrease in cell viability. However, when the laser-exposed isolates were incubated in broth medium for 3 days, most of these showed significant recovery from laser-induced damage. Addition of 5.0 microg/ml acriflavine (a DNA repair inhibitor) in the incubation medium had no significant effect on recovery. This suggests that DNA damage may not be involved in the cell inactivation. Electron paramagnetic resonance (EPR) studies using 5-doxyl strearic acid (5-DS) as a probe suggest alterations in lipid regions of the cell wall. Implications of these results for understanding therapeutic effect of nitrogen laser on drug-resistant tuberculosis are discussed.

Biochemical and histochemical changes pertaining to active and healed cutaneous tuberculosis.

BACKGROUND: Fibrosis is one of the major causes of post-treatment morbidity in tuberculosis. The molecular basis of fibrosis in active and healed tuberculous lesions is yet to be fully characterized. OBJECTIVES: To measure the tissue levels of collagen, elastin, fibronectin, transforming growth factor-beta (TGF-beta) and zinc in active and healed lesions of cutaneous tuberculosis. PATIENTS AND METHODS: Biopsy lesions obtained from 17 patients with active cutaneous tuberculosis and 12 patients after a 6-month regimen of antituberculous chemotherapy were examined. Collagen, elastin and zinc were estimated biochemically. In addition, the presence of collagen IV, TGF-beta and fibronectin were determined immunohistochemically. RESULTS: It was found that collagen, elastin, fibronectin and TGF-beta levels were higher in the active lesions. The levels of zinc were similar in both active and healed lesions. Clinically, scar tissue or keloid formation was not seen in any of the healed lesions. CONCLUSIONS: Effective antituberculous chemotherapy will lead to a substantial reduction of fibrosis and the consequent disability that can arise in patients with tuberculosis.