Examination of smears for tubercle bacilli by Fluorescence Microscopy

IN underdeveloped countries, laboratory facilities for the bacteriological diagnosis of tuberculosis are at present, very limited. Cultural methods are unlikely to be used on a large scale for many years to come. It is, therefore, important to investigate the most economical method of examining smears for tubercle bacilli. Fluorescence microscopy was introduced by Hagemann (1937) and has since been described by many authors, including Tanner (1941, 1948), Lind and Shaughnessy (1941), Lempert (1944), Norman and Jelks (1945), Clegg and Foster-Carter (1946), Wilson (1952), Von Haebler and Murray (1954), and Needham (1957). The great advantage claimed for this method is that stained bacilli can be detected using a much lower magnification than with the usual Ziehl-Neelsen method. Considerable time is saved in examining smears and larger areas can be searched. The method has not been widely employed for two reasons. In the first place, the light source must be very bright and many of the optical systems described previously have only supplied sufficient light if the equipment was used in a darkened room. Secondly, some workers (Ritterhoff and Bowman, 1945; Kuster, 1939; Holm and Plum, 1943) consider that false positive results can be obtained, since some smears may contain small naturally fluorescent particles which can be confused with bacilli. Equipment for fluorescence microscopy that can be used in normal daylight has been in use at the Tuberculosis Chemotherapy Centre, Madras, for over two years. When it was first introduced, a comparison between this method and the conventional Ziehl-Neelsen method was undertaken to test their relative sensitivities, and to see whether fluorescence microscopy yielded false positive results. The results of this comparison are described

The Susceptibility to Hydrogen Peroxide of Indian and British Isoniazid-Sensitive and Isoniazid- Resistant Tubercle Bacilli

The present work describes an attempt to modify the method of Kreis and Le Joubioux (1957a) so that it would accurately estimate the relative proportions of catalase-positive and catalase-negative organisms in strains containing mixtures of the two types. A bactericidal test was chosen in preference to a bacteriostatic test, since it is difficult to obtain quantitative measurement with the latter technique. In performing a bactericidal test residual peroxide must be inactivated or removed by dilution so that it does not inhibit the growth of surviving organisms. Knox, Meadow and Worssam (1956) removed peroxide by centrifugation and washing, but this method was considered impracticable if this test were to be used on a large scale, and likely to produce inaccurate counts on the surviving organisms. In the present work the method of removal of peroxide was studied as well as the determination of the optimal peroxide concentration and period of exposure which would kill all catalase-negative organisms, but would leave catalase-positive organisms unaffected. In addition, the method of Kreis & Le Joubioux (1957a) was modified by reducing the inoculum of organisms exposed to peroxide so that catalase-positive bacilli would not be able to destroy peroxide during the test itself. The standardised bactericidal test was then employed in comparing the susceptibility to peroxide of isoniazid-sensitive strains from British and Indian patients, and in investigating the relationship between the peroxide susceptibility and the catalase activity of their isoniazid-resistant mutant strains

The detection of airborne transmission of tuberculosis from HIV-infected patients, using an in vivo air sampling model

Background. Nosocomial transmission of tuberculosis remains an important public health problem. We created an in vivo air sampling model to study airborne transmission of tuberculosis from patients coinfected with human immunodeficiency virus (HIV) and to evaluate environmental control measures. Methods. An animal facility was built above a mechanically ventilated HIV‐tuberculosis ward in Lima, Peru. A mean of 92 guinea pigs were continuously exposed to all ward exhaust air for 16 months. Animals had tuberculin skin tests performed at monthly intervals, and those with positive reactions were removed for autopsy and culture for tuberculosis. Results. Over 505 consecutive days, there were 118 ward admissions by 97 patients with pulmonary tuberculosis, with a median duration of hospitalization of 11 days. All patients were infected with HIV and constituted a heterogeneous group with both new and existing diagnoses of tuberculosis. There was a wide variation in monthly rates of guinea pigs developing positive tuberculin test results (0%–53%). Of 292 animals exposed to ward air, 159 developed positive tuberculin skin test results, of which 129 had laboratory confirmation of tuberculosis. The HIV‐positive patients with pulmonary tuberculosis produced a mean of 8.2 infectious quanta per hour, compared with 1.25 for HIV‐negative patients with tuberculosis in similar studies from the 1950s. The mean monthly patient infectiousness varied greatly, from production of 0–44 infectious quanta per hour, as did the theoretical risk for a health care worker to acquire tuberculosis by breathing ward air. Conclusions. HIV‐positive patients with tuberculosis varied greatly in their infectiousness, and some were highly infectious. Use of environmental control strategies for nosocomial tuberculosis is therefore a priority, especially in areas with a high prevalence of both tuberculosis and HIV infection

The Virulence in the Guinea-pig of Tubercle Bacilli Isolated before Treatment from South Indian Patients with Pulmonary Tuberculosis: 1. Homogeneity of the Investigation and a Critique of the Virulence Test

A series of studies on the virulence in the guinea-pig of tubercle bacilli isolated before treatment from Indian tuberculous patients admitted to a controlled comparison of different regimens of domiciliary chemotherapy has recently been undertaken by the Tuberculosis Chemotherapy Centre, Madras. The main object of these studies was to determine whether the differences in virulence of the tubercle bacilli obtained from Indian patients before the start of chemotherapy were related to the severtiy or type of the patients’ disease at that time and to the subsequent response to treatment. Before these relationships could be‘ investigated, however, it was necessary to find out whether the results of the virulence tests, which were carried out over a period of two-and-a-half years at the Centre and at the Microbiological Research Establishment, Porton, England, could be considered as a unified whole-that, is, as if they had all been done on the same day in the same laboratory. A proportion of the cultures was stored at – 20°C for 44-78 weeks, but this did not affect their virulence. Inter-experimental variation was found to be small in the Porton series of tests and undetectable in the Madras series, and the results in the latter series could be successfully adjusted to those in the former by allowing for differences in the means and standard deviations of the distributions for the two series. The measure of virulence used was found to be reasonably acceptable for the analysis of variance technique. Suggestions are made as to ways of improving the efficiency of the experimental design in future studies

Storage capacity of correlated perceptrons

We consider an ensemble of $K$ single-layer perceptrons exposed to random inputs and investigate the conditions under which the couplings of these perceptrons can be chosen such that prescribed correlations between the outputs occur. A general formalism is introduced using a multi-perceptron costfunction that allows to determine the maximal number of random inputs as a function of the desired values of the correlations. Replica-symmetric results for $K=2$ and $K=3$ are compared with properties of two-layer networks of tree-structure and fixed Boolean function between hidden units and output. The results show which correlations in the hidden layer of multi-layer neural networks are crucial for the value of the storage capacity.Comment: 16 pages, Latex2

Diabetes and disordered eating behaviours in a community-based sample of australian adolescents

Background:People with diabetes have been shown to be at risk for disordered eating compared to their non-diabetic peers. However, the majority of studies have been conducted in relatively small samples drawn from clinical diabetes settings or registries. Community-based samples are required to better understand disordered eating behaviours in this population. In a large community-based population sample of Australian adolescents, this study aimed to (1) investigate disordered eating behaviours in adolescents reporting a diagnosis of diabetes compared to their non-diabetic peers and (2) test associations between disordered eating behaviours and insulin restriction.Methods:Secondary school students (n = 4854; mean (SD) age 14.4 (1.6) years; 47% boys) completed an online survey, including self-reported presence of diabetes, demographics, weight status, substance use, insulin restriction and disordered eating behaviours. Clinically meaningful cut-offs for disordered eating behaviours were generated for analysis.Results:Disordered eating behaviours, specifically self-induced vomiting (diabetes 19.2%, no diabetes 3.3%; p p p p Conclusion:There was a high rate of disordered eating behaviours in adolescents with diabetes compared to their peers without diabetes. The findings of this study may have the potential to inform future health promotion, prevention, and early intervention approaches for those with comorbid diabetes and disordered eating behaviours. Future longitudinal studies are required to evaluate disordered eating behaviours in those with diabetes over time in community-based samples

Examining the basis of isoniazid tolerance in nonreplicating Mycobacterium tuberculosis using transcriptional profiling

Background: Understanding how growth state influences Mycobacterium tuberculosis responses to antibiotic exposure provides a window into drug action during patient chemotherapy. In this article, we describe the transcriptional programs mediated by isoniazid (INH) during the transition from log-phase to nonreplicating bacilli, from INH-sensitive to INH-tolerant bacilli respectively, using the Wayne model. Results: INH treatment did not elicit a transcriptional response from nonreplicating bacteria under microarophilic conditions (NRP2), unlike the induction of a robust and well-characterized INH signature in log-phase bacilli. Conclusion: The differential regulation (between drug-free NRP2 and log-phase bacilli) of genes directly implicated in INH resistance could not account for the abrogation of INH killing in nongrowing bacilli. Thus, factors affecting the requirement for mycolic acids and the redox status of bacilli are likely responsible for the reduction in INH efficacy. We speculate on additional mechanisms revealed by transcriptome analysis that might account for INH tolerance

Quantitative predictions on auxin-induced polar distribution of PIN proteins during vein formation in leaves

The dynamic patterning of the plant hormone auxin and its efflux facilitator the PIN protein are the key regulator for the spatial and temporal organization of plant development. In particular auxin induces the polar localization of its own efflux facilitator. Due to this positive feedback auxin flow is directed and patterns of auxin and PIN arise. During the earliest stage of vein initiation in leaves auxin accumulates in a single cell in a rim of epidermal cells from which it flows into the ground meristem tissue of the leaf blade. There the localized auxin supply yields the successive polarization of PIN distribution along a strand of cells. We model the auxin and PIN dynamics within cells with a minimal canalization model. Solving the model analytically we uncover an excitable polarization front that triggers a polar distribution of PIN proteins in cells. As polarization fronts may extend to opposing directions from their initiation site we suggest a possible resolution to the puzzling occurrence of bipolar cells, such we offer an explanation for the development of closed, looped veins. Employing non-linear analysis we identify the role of the contributing microscopic processes during polarization. Furthermore, we deduce quantitative predictions on polarization fronts establishing a route to determine the up to now largely unknown kinetic rates of auxin and PIN dynamics.Comment: 9 pages, 4 figures, supplemental information included, accepted for publication in Eur. Phys. J.

Ectopic A-lattice seams destabilize microtubules

Natural microtubules typically include one A-lattice seam within an otherwise helically symmetric B-lattice tube. It is currently unclear how A-lattice seams influence microtubule dynamic instability. Here we find that including extra A-lattice seams in GMPCPP microtubules, structural analogues of the GTP caps of dynamic microtubules, destabilizes them, enhancing their median shrinkage rate by >20-fold. Dynamic microtubules nucleated by seeds containing extra A-lattice seams have growth rates similar to microtubules nucleated by B-lattice seeds, yet have increased catastrophe frequencies at both ends. Furthermore, binding B-lattice GDP microtubules to a rigor kinesin surface stabilizes them against shrinkage, whereas microtubules with extra A-lattice seams are stabilized only slightly. Our data suggest that introducing extra A-lattice seams into dynamic microtubules destabilizes them by destabilizing their GTP caps. On this basis, we propose that the single A-lattice seam of natural B-lattice MTs may act as a trigger point, and potentially a regulation point, for catastrophe

Modeling oscillatory Microtubule--Polymerization

Polymerization of microtubules is ubiquitous in biological cells and under certain conditions it becomes oscillatory in time. Here simple reaction models are analyzed that capture such oscillations as well as the length distribution of microtubules. We assume reaction conditions that are stationary over many oscillation periods, and it is a Hopf bifurcation that leads to a persistent oscillatory microtubule polymerization in these models. Analytical expressions are derived for the threshold of the bifurcation and the oscillation frequency in terms of reaction rates as well as typical trends of their parameter dependence are presented. Both, a catastrophe rate that depends on the density of {\it guanosine triphosphate} (GTP) liganded tubulin dimers and a delay reaction, such as the depolymerization of shrinking microtubules or the decay of oligomers, support oscillations. For a tubulin dimer concentration below the threshold oscillatory microtubule polymerization occurs transiently on the route to a stationary state, as shown by numerical solutions of the model equations. Close to threshold a so--called amplitude equation is derived and it is shown that the bifurcation to microtubule oscillations is supercritical.Comment: 21 pages and 12 figure