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.

Rationale Interrupting tuberculosis (TB) transmission requires an improved understanding of how and when the causative organism, Mycobacterium tuberculosis ( Mtb ), is aerosolized. Although cough is commonly assumed to be the dominant source of Mtb aerosols, recent evidence of cough-independent Mtb release implies the contribution of alternative mechanisms. Objectives To compare the aerosolization of Mtb bacilli and total particulate matter from patients with TB during three separate respiratory maneuvers: tidal breathing (TiBr), FVC, and cough. Methods Bioaerosol sampling and Mtb enumeration by live-cell, fluorescence microscopy were combined with real-time measurement of CO 2 concentration and total particle counts from 38 patients with GeneXpert-positive TB before treatment initiation. Measurements and Main Results For all maneuvers, the proportions of particles detected across five size categories were similar, with most particles falling between 0.5–5 μm. Although total particle counts were 4.8-fold greater in cough samples than either TiBr or FVC, all three maneuvers returned similar rates of positivity for Mtb . No correlation was observed between total particle production and Mtb count. Instead, for total Mtb counts, the variability between individuals was greater than the variability between sampling maneuvers. Finally, when modelled using 24-hour breath and cough frequencies, our data indicate that TiBr might contribute more than 90% of the daily aerosolized Mtb among symptomatic patients with TB. Conclusions Assuming the number of viable Mtb organisms released offers a reliable proxy of patient infectiousness, our observations imply that TiBr and interindividual variability in Mtb release might be significant contributors to TB transmission among active cases.

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