No abstract

Tuberculosis (TB) is predominantly an airborne disease. Background However, quantitative and qualitative analysis of bio-aerosols containing the aetiological agent, , has proven very Mycobacterium tuberculosis (Mtb) challenging. Our objective is to sample bio-aerosols from newly diagnosed TB patients for detection and enumeration of bacilli. Mtb : We monitored each of 35 newly diagnosed, GeneXpert Methods sputum-positive, TB patients during 1 hour confinement in a custom-built Respiratory Aerosol Sampling Chamber (RASC). The RASC (a small clean-room of 1.4m ) incorporates aerodynamic particle size detection, viable and non-viable sampling devices, real-time CO monitoring, and cough sound-recording. Microbiological culture and droplet digital polymerase chain reaction (ddPCR) were used to detect in each of the bio-aerosol collection Mtb devices.: was detected in 27/35 (77.1%) of aerosol samples; 15/35 Results Mtb (42.8%) samples were positive by mycobacterial culture and 25/27 (92.96%) were positive by ddPCR. Culturability of collected bacilli was not predicted by radiographic evidence of pulmonary cavitation, sputum smear positivity. A correlation was found between cough rate and culturable bioaerosol.was Mtb detected on all viable cascade impactor stages with a peak at aerosol sizes 2.0-3.5μm. This suggests a median of 0.09 CFU/litre of exhaled air (IQR: 0.07 to 0.3 CFU/l) for the aerosol culture positives and an estimated median concentration of 4.5x10 CFU/ml (IQR: 2.9x10 -5.6x10 ) of exhaled particulate bio-aerosol.: was identified in bio-aerosols exhaled by the majority of Conclusions Mtb untreated TB patients using the RASC. Molecular detection was more sensitive than mycobacterial culture on solid media, suggesting that further studies are required to determine whether this reflects a significant proportion of Page 1 of 25Gates Open Research 2018, 1:11 Last updated: 09 JUL 2018 Gates Open Research Discuss this article(1) Comments than mycobacterial culture on solid media, suggesting that further studies are required to determine whether this reflects a significant proportion of differentially detectable bacilli in these samples.

Knowledge of the airborne nature of respiratory disease transmission owes much to the pioneering experiments of Wells and Riley over half a century ago. However, the mechanical, physiological, and immunopathological processes which drive the production of infectious aerosols by a diseased host remain poorly understood. Similarly, very little is known about the specific physiological, metabolic and morphological adaptations which enable pathogens such as Mycobacterium tuberculosis (Mtb) to exit the infected host, survive exposure to the external environment during airborne carriage, and adopt a form that is able to enter the respiratory tract of a new host, avoiding innate immune and physical defenses to establish a nascent infection. As a first step towards addressing these fundamental knowledge gaps which are central to any efforts to interrupt disease transmission, we developed and characterized a small personal clean room comprising an array of sampling devices which enable isolation and representative sampling of airborne particles and organic matter from tuberculosis (TB) patients. The complete unit, termed the Respiratory Aerosol Sampling Chamber (RASC), is instrumented to provide real-time information about the particulate output of a single patient, and to capture samples via a suite of particulate impingers, impactors and filters. Applying the RASC in a clinical setting, we demonstrate that a combination of molecular and microbiological assays, as well as imaging by fluorescence and scanning electron microscopy, can be applied to investigate the identity, viability, and morphology of isolated aerosolized particles. Importantly, from a preliminary panel of active TB patients, we observed the real-time production of large numbers of airborne particles including Mtb, as confirmed by microbiological culture and polymerase chain reaction (PCR) genotyping. Moreover, direct imaging of captured samples revealed the presence of multiple rod-like Mtb organisms whose physical dimensions suggested the capacity for travel deep into the alveolar spaces of the human lung.

Background: Tuberculosis (TB) is predominantly an airborne disease. However, quantitative and qualitative analysis of bio-aerosols containing the aetiological agent, Mycobacterium tuberculosis (Mtb), has proven very challenging. Our objective is to sample bio-aerosols from newly diagnosed TB patients for detection and enumeration of Mtb bacilli. Methods: We monitored each of 35 newly diagnosed, GeneXpert sputum-positive, TB patients during 1 hour confinement in a custom-built Respiratory Aerosol Sampling Chamber (RASC). The RASC (a small clean-room of 1.4m ) incorporates aerodynamic particle size detection, viable and non-viable sampling devices, real-time CO 2 monitoring, and cough sound-recording. Microbiological culture and droplet digital polymerase chain reaction (ddPCR) were used to detect Mtb in each of the bio-aerosol collection devices. Results:  Mtb was detected in 27/35 (77.1%) of aerosol samples; 15/35 (42.8%) samples were positive by mycobacterial culture and 25/27 (92.96%) were positive by ddPCR. Culturability of collected bacilli was not predicted by radiographic evidence of pulmonary cavitation, sputum smear positivity, or cough rate. Mtb was detected on all viable cascade impactor stages with a peak at aerosol sizes 2.0-3.5μm. This suggests a median of 0.09 CFU/litre of exhaled air (IQR: 0.07 to 0.3 CFU/l) for the aerosol culture positives and an estimated median concentration of 4.5x10 CFU/ml (IQR: 2.9x10 -5.6x10 ) of exhaled particulate bio-aerosol. Conclusions:  Mtb was identified in bio-aerosols exhaled by the majority of untreated TB patients using the RASC. Molecular detection was more sensitive than mycobacterial culture on solid media, suggesting that further studies are required to determine whether this reflects a significant proportion of differentially detectable  bacilli in these samples.

Tuberculosis remains a global health threat killing over 1 million people per year. Current sputum-based diagnostics are specific but lack sensitivity resulting in treatment of many sputum negative cases. In this proof-of-concept study, we used high-resolution mass spectrometry to identify specific lipids in peripheral lung fluid samples of TB patients and controls, captured using a novel non-invasive sampling system. Exhaled respiratory particles were collected in liquid and after concentration and lipid extraction directly infused into a high-resolution mass spectrometer. High-resolution mass spectrometric data collection was conducted in a dual ion mode and chemical compositions were constructed using accurate mass measurement. Over 400 features with high segregating capacity were extracted and optimized using feature selection algorithm and machine learning, from which the accuracy of detection of positive tuberculosis patients was estimated. This current strategy provides sensitivity offered by high-resolution mass spectrometry and can be readily susceptible for developing a novel clinical assay exploring peripheral lung fluid for the detection of active TB cases.

No abstract

Corona plasma discharge provides a rapid and reliable tool for release of biomarkers from gram negative and positive bacteria, spores and viruses for characterization by mass spectrometry.

Interrupting transmission is an attractive anti-tuberculosis (TB) strategy but it remains underexplored owing to our poor understanding of the events surrounding transfer of Mycobacterium tuberculosis (Mtb) between hosts. Determining when live, infectious Mtb bacilli are released and by whom has proven especially challenging. Consequently, transmission chains are inferred only retrospectively, when new cases are diagnosed. This process, which relies on molecular analyses of Mtb isolates for epidemiological fingerprinting, is confounded by the prolonged infectious period of TB and the potential for transmission from transient exposures. We developed a Respiratory Aerosol Sampling Chamber (RASC) equipped with high-efficiency filtration and sampling technologies for liquid-capture of all particulate matter (including Mtb) released during respiration and non-induced cough. Combining the mycobacterial cell wall probe, DMN-trehalose, with fluorescence microscopy of RASC-captured bioaerosols, we detected and quantified putative live Mtb bacilli in bioaerosol samples arrayed in nanowell devices. The RASC enabled non-invasive capture and isolation of viable Mtb from bioaerosol within 24 hours of collection. A median 14 live Mtb bacilli (range 0–36) were isolated in single-cell format from 90% of confirmed TB patients following 60 minutes bioaerosol sampling. This represented a significant increase over previous estimates of transmission potential, implying that many more organisms might be released daily than commonly assumed. Moreover, variations in DMN-trehalose incorporation profiles suggested metabolic heterogeneity in aerosolized Mtb. Finally, preliminary analyses indicated the capacity for serial image capture and analysis of nanowell-arrayed bacilli for periods extending into weeks. These observations support the application of this technology to longstanding questions in TB transmission including the propensity for asymptomatic transmission, the impact of TB treatment on Mtb bioaerosol release, and the physiological state of aerosolized bacilli.