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However, delayed and missed diagnoses, and the six-month duration of standard chemotherapy, contribute to failure of this approach to control the TB epidemic [ 2 ]. The increasing prevalence of drug-resistant TB, compounded by evidence of the transmission of multi-drug resistant MDR Mtb strains, further undermines this approach [ 3 ].
Together, these factors have focused attention on blocking Mtb transmission as a critical area for novel interventions [ 4 ].
Reconstruction of transmission chains has traditionally required genetic fingerprinting of Mtb strains isolated from active TB cases and their diseased contacts. Despite advances in molecular epidemiology, this has proven enormously challenging even in low-incidence settings [ 6 ]. Consequently, the host and mycobacterial factors which ensure successful transmission remain obscure [ 7 ].
Since the interval between the time of infection and diagnosed disease varies, analyses are necessarily retrospective, making intervention impossible [ 8 ]. Moreover, targeting surveillance to active disease means that the potential contribution of asymptomatic transmitters is overlooked [ 9 ]. Direct study of aerosolized Mtb is equally complicated: issues such as timing of sampling, the small numbers of bacilli released in exhaled air and sputum, and the presence of environmental and patient-derived contaminating microorganisms and particulate matter impose profound technical and analytical challenges [ 11 , 12 ].
Decontamination, too, undermines assessments of bacillary load and physiological state. Where excessive, it depletes the number and viability of Mtb bacilli in the sample [ 15 ]; where inadequate, it risks overgrowth by contaminants, obscuring the signal. Molecular methods have enabled detection of Mtb DNA in bioaerosols [ 16 ], however they do not distinguish live from dead organisms, and even protocols which target RNA [ 17 ] require extraction of intracellular nucleic acid, obviating the potential to investigate the physiological and metabolic state s of aerosolized bacilli.
The method of bacillary capture is also important: approaches based on cough assume symptomatic spread, ignoring the possibility of Mtb transmission during normal respiratory activity and, therefore, potentially not capturing other natural transmission events [ 18 ], especially those from sub-clinical infections [ 19 ]. Face-mask and equivalent sampling methodologies either render live-cell analyses impossible or in vitro propagation via microbiological culture unavoidable [ 20 ]. In addition, many methods are unable to determine whether bacilli derive from small buoyant or large aerosol droplets.
This is a critical flaw given the likelihood that size determines aerosol longevity and ability to access lung alveoli, key elements of infectiousness [ 21 ]. To address these challenges, we sought to develop a method for culture-independent detection, quantification, and visualization of live bacilli in bioaerosols captured using the Respiratory Aerosol Sampling Chamber RASC , a small personal clean-room enabling capture of particulate material released by an individual patient during normal respiratory activity, including natural non-induced cough.
In previous work, we demonstrated the potential for liquid capture of aerosolized Mtb in the RASC, eliminating the dependency on solid culture-based techniques for bacillary detection [ 11 , 12 ].
This was a key innovation since it opened the possibility for detection, isolation, and manipulation of live bacilli for downstream phenotypic and genomic studies. Here, we advance this technology in a new cohort of recently diagnosed TB patients, incorporating methodologies for the specific labelling and enumeration of low numbers of live Mtb bacilli from aerosol samples in a format which enables detection via live-cell fluorescence microscopy of single bacilli arrayed in a nanowell device.
Moreover, by assigning localization co-ordinates to individual fluorescent organisms in the nanowells, we demonstrate the capacity to extend microscopic analyses to live cells for prolonged durations under incubation. The fluorescent trehalose analogue, 4- N , N -dimethylsamino-1,8-napthalimide-trehalose DMN-trehalose , enables rapid labelling and microscopic detection of Mtb in sputum or liquid medium [ 22 ].
Since active membrane biosynthesis is necessary for DMN-trehalose incorporation, this probe possesses the advantage of labelling live, metabolically active organisms only. A Photograph and B schematic of a nanowell-arrayed microscope slide. C 3D scan of a Each device 25 mm x 75 mm consists of two rows of eight round microwells machined from cast acrylic. The microwells are 6 mm in diameter and 2 mm deep.
The nanowell film, which is bonded to the superstructure with UV-curing adhesive, is made from embossed COC film. In contrast, there was a small but statistically significant difference in average cell width Fig 2B , with bacilli entering early stationary phase 0. However, the average differences observed in mycobacterial cell width were unable to differentiate log from stationary phase bacilli owing to the largely overlapping distributions.
Comparisons between log green and stationary blue phase Mtb according to A cell length, B width, and C polarity index. D Average DMN-trehalose profile for log and stationary phase bacilli, with single-cell examples in both E log and F stationary phase. Polarity index for each cell was calculated as the median fluorescence intensity at region 1 divided by the median fluorescence intensity at region 2 in panel D.
The metabolic state of aerosolized Mtb remains unknown. Therefore, we investigated the utility of cytological profiling via DMN-trehalose staining to differentiate bacilli broadly as either slow or fast growers based on their growth phase. Substantial differences in DMN-trehalose uptake and distribution along the cell length were observed between log and stationary phase bacilli Fig 2C—2F.
The polarity index, a simple metric of the relative brightness of the pole compared to the mid cell, was greater in log phase 1. As such, cells identified as putative Mtb based on cell width and DMN-trehalose positivity could be characterized broadly into at least two categories based on DMN-trehalose staining.
DMN-trehalose is not specific for Mtb : all bacteria within the Actinomycetales which encode homologs of the mycobacterial antigen 85 complex possess the capacity to incorporate the fluorescent label. Among these, Corynebacteria are a common constituent of the oral microbiome and were previously identified in TB sputum samples [ 22 ].
Following DMN-trehalose labelling, C. Thirty-one individuals with GeneXpert-positive, drug-susceptible TB were recruited into a pilot study Table 1 for the capture and single-cell detection of live, Mtb bacilli in patient bioaerosols Fig 3.
Columns represent 3 different patients. Bacilli not matching inclusion criteria are excluded from subsequent analysis. The median count in the captured air from TB-positive patients was 14 range 0—36 versus 1. When comparing the basic morphological characteristics of aerosolized Mtb to those observed in log-phase, it was apparent that they were significantly shorter Fig 4B but with the same width on average Fig 4C.
Comparing distributions of B cell lengths and C cell widths in putative Mtb bacilli detected within bioaerosols of TB patients red to Mtb H37Rv cultured within the lab green. Representative D images and E plots of the three distinct, exemplar cytological profiles from three patients in which putative Mtb were detected.
F Average plots and idealised drawings indicating the different staining patterns of all bacilli detected in these three patients. H Polarity index and I length of the bacilli detected within these patients.
Further investigation into the profiles of DMN-trehalose incorporation led to the observation of three distinct labelling patterns Fig 4D—4F , namely: polar labelling sample TRDS, top row , diffuse labelling sample TRDS, middle row , and a patchy labelling pattern sample JM9, bottom row. Both polar and diffuse labelling have been previously observed in log and stationary phase bacilli, respectively Fig 2D.
Like the in vitro cultured organisms, no significant differences in cell length were observed for the different labeling patterns in the bioaerosol samples Fig 4H.
However, more prominent differences were seen in labelling pattern of DMN-trehalose Fig 4I , highlighting the potential utility of the trehalose probe in indicating underlying metabolic states of aerosolized bacilli.
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