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

Abstract

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.

Capture and visualization of liveMycobacterium tuberculosisbacilli from tuberculosis bioaerosols

“…We previously described the capacity to enumerate Mtb genomes (via RD9-specific PCR) or Mtb colonies (via growth on solid medium loaded into different impactor devices) captured in bioaerosol material produced by tidal breathing and (non-induced) cough during the ~60 minute isolation of confirmed TB patients in the RASC (29,37). Although instructive, both methods (molecular and microbiological) are characterized by limitations which critically undermine their respective utilities for TB transmission research.…”

“…On the one hand, PCR cannot differentiate live from dead bacilli, thereby rendering any estimate of viable Mtb release uncertain. On the other, culture-based methods are notoriously susceptible to contamination, the complications arising from the presence of so-called "viable but non-culturable" or "differentially detectable" organisms (29,42), and the temporal -and, consequently, genetic (43) and physiological -separation of the (single) transmitted bacillus from the micro-population (~10 6 cells) which eventually manifests as a visible Mtb colony on a plate. For these reasons, we deemed culture-independent detection, quantification and visualization of live organisms a priority.…”

“…Therefore, while the interval between host infection and disease can be variable (21-23) -perhaps involving extended periods of clinical latency in some cases (24)(25)(26)-the biological imperative to effect successful transmission (thus ameliorating the risks of extinction in an individual host) is inexorable and has been proposed to underlie the destructive lung pathology which enables aerosolization and release of Mtb bacilli (21,27,28). Studying aerosolized Mtb is complicated by the low numbers of bacilli produced and the presence of environmental and patient-derived "contaminating" microorganisms and particulate matter (29). While molecular methods have enabled consistent and reliable detection of Mtb DNA in bioaerosols (30)(31)(32), these approaches cannot distinguish live from dead organisms nor do they allow investigations of the physiological state(s) of the aerosolized bacilli.…”

“…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).…”

“…Are some individuals "super-spreaders" with an increased propensity for Mtb bioaerosol production? To date, we have demonstrated the potential for liquid capture of aerosolized Mtb in the RASC, eliminating the dependency on solid culture-based techniques for bacillary detection (29,37). This represents a key development, since it promises the possibility of detecting, isolating, and manipulating live bacilli for downstream phenotypic and genomic studies of transmission.…”