Persistent Mycobacterium tuberculosis bioaerosol release in a tuberculosis-endemic setting.

Pioneering studies linking symptomatic disease and cough-mediated release of Mycobacterium tuberculosis (Mtb) established the infectious origin of tuberculosis (TB), simultaneously informing the pervasive notion that pathology is a prerequisite for Mtb transmission. Our prior work has challenged this assumption: by sampling TB clinic attendees, we detected equivalent release of Mtb-containing bioaerosols by confirmed TB patients and individuals not receiving a TB diagnosis, and we demonstrated a time-dependent reduction in Mtb bioaerosol positivity during six-months' follow-up, irrespective of anti-TB chemotherapy. Now, by extending bioaerosol sampling to a randomly selected community cohort, we show that Mtb release is common in a TB-endemic setting: of 89 participants, 79.8% (71/89) produced Mtb bioaerosols independently of QuantiFERON-TB Gold status, a standard test for Mtb infection; moreover, during two-months' longitudinal sampling, only 2% (1/50) were serially Mtb bioaerosol negative. These results necessitate a reframing of the prevailing paradigm of Mtb transmission and infection, and may explain the current inability to elucidate Mtb transmission networks in TB-endemic regions.

Aerosolization of viable Mycobacterium tuberculosis bacilli by tuberculosis clinic attendees independent of sputum-Xpert Ultra status.

Potential Mycobacterium tuberculosis (Mtb) transmission during different pulmonary tuberculosis (TB) disease states is poorly understood. We quantified viable aerosolized Mtb from TB clinic attendees following diagnosis and through six months' follow-up thereafter. Presumptive TB patients (n=102) were classified by laboratory, radiological, and clinical features into Group A: Sputum-Xpert Ultra-positive TB (n=52), Group B: Sputum-Xpert Ultra-negative TB (n=20), or Group C: TB undiagnosed (n=30). All groups were assessed for Mtb bioaerosol release at baseline, and subsequently at 2 wk, 2 mo, and 6 mo. Groups A and B were notified to the national TB program and received standard anti-TB chemotherapy; Mtb was isolated from 92% and 90% at presentation, 87% and 74% at 2 wk, 54% and 44% at 2 mo and 32% and 20% at 6 mo, respectively. Surprisingly, similar numbers were detected in Group C not initiating TB treatment: 93%, 70%, 48% and 22% at the same timepoints. A temporal association was observed between Mtb bioaerosol release and TB symptoms in all three groups. Persistence of Mtb bioaerosol positivity was observed in ~30% of participants irrespective of TB chemotherapy. Captured Mtb bacilli were predominantly acid-fast stain-negative and poorly culturable; however, three bioaerosol samples yielded sufficient biomass following culture for whole-genome sequencing, revealing two different Mtb lineages. Detection of viable aerosolized Mtb in clinic attendees, independent of TB diagnosis, suggests that unidentified Mtb transmitters might contribute a significant attributable proportion of community exposure. Additional longitudinal studies with sputum culture-positive and -negative control participants are required to investigate this possibility.

Aerosolization of Mycobacterium tuberculosis by Tidal Breathing.

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 CO2 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.

Capture and visualization of live Mycobacterium tuberculosis bacilli from tuberculosis patient bioaerosols.

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.

Cough-independent production of viable Mycobacterium tuberculosis in bioaerosol.

BACKGROUND: Symptoms of infectious respiratory illnesses are often assumed to drive transmission. However, production and release of Mycobacterium tuberculosis (Mtb) bioaerosols is poorly understood. We report quantitation of Mtb exhaled during specific respiratory manoeuvres. METHODS: Direct capture of nascent bioaerosol particles and indirect collection of aged particles was performed in 10 healthy subjects. Indirect and direct capture of exhaled viable Mtb bacilli was compared in 38 PTB patients and directly captured viable Mtb during cough and bronchiole-burst manoeuvres in 27 of the PTB patients. RESULTS: Direct sampling of healthy subjects captured larger bioaerosol volumes with higher proportions of 2-5 µm particles than indirect sampling. Indirect sampling identified viable Mtb in 92.1% (35 of 38) of PTB patients during 60-min relaxed breathing, median bacillary count 7.5 (IQR: 3.25-19). Direct sampling for 10-min identified Mtb in 97.4% (37 of 38) of PTB patients with higher bacilli counts (p < 0.001), median 24.5 (IQR:11.25-37.5). A short 5-min sampling regimen of 10 coughs or 10 bronchiole-burst manoeuvres yielded a median of 11 (IQR: 4-17) and 11 (IQR: 7-17.5) Mtb bacilli, respectively (p = 0.53). CONCLUSIONS: Peripheral lung bioaerosol released through deep exhalations alone contained viable Mtb suggesting non-cough transmission is possible in PTB.