Real-Time Investigation of Tuberculosis Transmission: Developing the Respiratory Aerosol Sampling Chamber (RASC)

Abstract

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.

Detection of Mycobacterium tuberculosis bacilli in bio-aerosols from untreated TB patients

“…Culturable Mtb isolated in cough-derived aerosols has been shown to be associated with transmission risk 14 15 . The presence of culturable Mtb organisms in such high concentrations in respirable bio-aerosols does suggest probable host physical or immunologic control to limit Mtb infection becoming widespread throughout the respiratory system.…”

“…The Respiratory Aerosol Sampling Chamber (RASC) has previously been described in detail 17 . The RASC consists of a small personal clean space (1.4 m ) in which a participant is seated and engages passively in an exhaled air sampling protocol.…”

“…Approximately an hour is spent in the chamber following the phases outlined in Wood et al . 15 Briefly, the chamber is sealed and an air purge phase is performed entraining ambient air through high-efficiency particulate arrestance (HEPA) filters for a period of 10 minutes. This is followed by a participant-driven contamination phase in which the chamber is isolated from the external environment and the proportion of exhaled air allowed to rise to a 10% threshold defined by a chamber CO 2 concentration of 4,000 ppm above the ambient level (based on an assumed exhaled air CO 2 concentration of 40,000 ppm).…”

“…We used the respiratory aerosol sampling chamber (RASC) 15 , a novel apparatus designed to optimise patient-derived aerosol sampling, to isolate and accumulate respirable aerosol from a single patient. Environmental sampling detects the Mtb present after a period of ageing in the chamber air.…”

Capture and visualization of live Mycobacterium tuberculosis bacilli from tuberculosis bioaerosols

“…In attempting to address these limitations, we developed a platform for the detection and capture of live Mtb bacilli from TB patients (37). Our Respiratory Aerosol Sampling Chamber (RASC) effectively functions as a personal clean-room, enabling collection and concentration of particulate material released by an individual patient during normal respiratory activity, including from natural (non-induced) cough (29,37). Through multiple iterations informed by our preliminary experimental and clinical observations, we have equipped the RASC to address systematically the comprehensive TB transmission research agenda outlined by others (38).…”