In this study, we aimed to gain further insight into the airborne phase of TB and establish the bacillary concentration in exhaled bio-aerosols. We used the respiratory aerosol sampling chamber (RASC) 16 , 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. The resulting ‘dried residua’ 17 , formed from larger respiratory droplets, are predicted to mimic more closely the putative infectious particle.

Of particular interest, a proof of concept study 12 and subsequent feasibility study in Uganda 14 sampled cough-generated aerosols from pulmonary TB patients. Coughing directly into a sampling chamber equipped with two viable cascade impactors resulted in positive cultures from more than a quarter of participants despite their having received 1–6 days of chemotherapy. A follow-up work employing the same apparatus found that participants with higher aerosol bacillary loads could be linked to greater household transmission rates 14 and development of disease 15 findings which suggest that quantitative airborne sampling may serve as a clinical relevant measure of infectivity.

Empirical studies to characterise airborne infectious particles have been sparse. Two major difficulties plaguing investigation are the purportedly low concentrations of naturally produced Mtb particles, and the complication of environmental and patient-derived bacterial and fungal contamination of airborne samples 5 . There have nonetheless been a number of attempts at airborne detection. 6 13 .

The capacity for airborne transmission of Mtb bacilli was first demonstrated in an elegant series of experiments by Richard Riley and colleagues nearly seventy years ago 4 . Venting exhaled air from pulmonary TB patients over a guinea pig facility resulted in infection of the animals leading to the concept of infectious ‘quanta’ (the dose of infectious air required to cause an infection). Notably, these pioneering studies indicated that quanta production was extremely infrequent and definitively attributable to only a small minority of patients. Furthermore, the quantitative relationship between airborne infectious particles and quanta remains unclear.

Tuberculosis (TB) has surpassed HIV/AIDS as a global killer with more than 4000 daily deaths 1 . The rate of decline in incidence remains inadequate at a reported 1.5% per annum 1 and it is unlikely that treatment alone will significantly reduce the burden of disease 2 . In communities with highly prevalent HIV, Mycobacterium tuberculosis (Mtb) genotyping studies have found that recent transmission, rather than reactivation, accounts for the majority (54%) of incident TB cases 3 . Therefore, interruption of transmission would likely have a rapid, measurable impact on TB incidence. The physical process of TB transmission remains poorly understood and the application of new technologies to elucidate key events in infectious aerosol production, release, and inhalation, has been slow.

Methods

Ethical statement Ethics approval was obtained from the University of Cape Town Faculty of Health Sciences Human Research Ethics Committee (HREC/REF: 680/2013). Written informed consent for publication of the participants details was obtained from the participants. Sampling took place on the same day as treatment initiation with a typical delay of 1–2 hours to complete the study protocol.

Subject recruitment Participants who had tested positive for drug-sensitive pulmonary TB by GeneXpert were recruited prior to initiation of chemotherapy from a peri-urban township 40km south of Cape Town Baseline patient data were collected from the clinical records and a chest X-ray was taken approximately seven days after the start of treatment. The presence of lung cavitation was scored by one of the authors (BP) based on the chest X-ray and this score was compared to a radiologist report for agreement.

Respiratory Aerosol Sampling Chamber protocol The Respiratory Aerosol Sampling Chamber (RASC) has previously been described in detail16. 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 Woodet al.16 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). If the target is not reached after 30 minutes have elapsed, the sampling phase is started at a lower exhaled air proportion. After sampling, the chamber is again purged to remove residualMtb from the air. Contamination of the sampling chamber was driven primarily by tidal breathing in addition to spontaneous coughing or sneezing. Particles and organisms derived from sources other than breath were minimised by the participant wearing a full-body DuPont Tyvek suit during sampling and an initial purge phase to minimise ambient contamination. Drawing the chamber air over a range of devices allowed mycobacterial detection by microbiological culture or molecular quantitation of genome equivalents.

Particle size measurement Aerosolized particles were monitored from the final minute of the purge phase and throughout the remainder of the experimental protocol via an aerodynamic particle sizer (APS Model 3321, TSI, Shoreview, MN USA). Sound recording. Sounds from the inside of the sampling chamber were recorded by microphone and stored as 44.1 KHz 16-bit WAV files using a custom-built recorder application. The files were securely transmitted to a server where automated cough sound analysis can occur. The cough sound analysis divides the input recording into multiple segments of time, and a machine learning algorithm classifies each segment of time as either a cough or not a cough, using characteristics of the signal at that moment in time such as the overall energy within the signal, the distribution of energy across frequencies and the amount of change in energy within the signal within that segment. These classifications are then merged together in order to identify longer segments in time that are continuously cough or non-cough segments, which are then used to identify periods of coughing. This analysis was used to determine cough frequency and cough length for each participant.

Particle capture for microbiological analysis The sampling phase utilised a six-stage viable Andersen Impactor (Model 10830-EPD, Thermo Scientific, USA) which allowed physical separation of aerosolized particles by size, based on the principle of inertial impaction. These captured particles are incubated to ascertain the number of Mtb bacilli released. The impactor sampled chamber air at a rate of 28 l/min for 10 minutes. Each impactor stage contained a glass Petri dish containing solid Middlebrook 7H10 medium further described below. For participants 14 to 35, a 0.2 µm polycarbonate filter (Sterlitech Corporation, WA USA) of 47 mm diameter was placed on the agar plate abutting the edge, and subsequently removed and analysed using droplet digital PCR (ddPCR). Direct capture took place using a 0.4 µm microporous polycarbonate filter (Sterlitech Corporation, WA USA) positioned above the participant in an open-faced mount with a 20 l/min flow rate run for 10 minutes. The filter was cut with one half analysed by the microbiological culture method and the other half by ddPCR. A gel filter (Model 12602-37-ALK, Sartorius, Goettingen, Germany) was similarly positioned and run at a flow rate of 20 l/min for 10 minutes. A 0.4 µm polycarbonate filter was placed in-line and downstream of the gel filter. An open-faced, polyester felt filter of 47mm diameter and 1.0μm pore size (American Felt and Filter Company, New Windsor, New York; Lockheed Martin, Alexandria, VA, USA) was used to sample at a high flow rate (approx. 300 l/min) for 10 mins at the end of the experiment and was analysed by ddPCR (see below).

Particle capture for imaging A Dekati three-stage impactor (PM10, Dekati, Kangasala, Finland) sampling at 30 l/min for ten minutes was used to separate respired particles according to size onto uncoated aluminium foil discs. Assuming a particle density of 1 g/cm3, the three stages collect particles in the size ranges: >14.1 μm, 14.1 μm - 3.5 μm and 3.5 μm - 1.4 μm respectively. A 0.4 µm polycarbonate filter was placed at the outflow of this impactor to capture aerosols of less than 1.4 µm. The foil discs were air-dried and sterilised by UV-irradiation before imaging, uncoated, by scanning electron microscopy (Zeiss/Leo 1450, ZEISS, Oberkochen, Germany) in secondary electron mode at 10 kv.

Quantification of microbiological specimens For an individual inside the RASC, the ratio of exhaled air volume to chamber volume is equal to the ratio of excess CO 2 (measured CO 2 less ambient CO 2 ) to the CO 2 in exhaled breath (approx. 40,000 ppm). Continuous CO 2 monitoring therefore allowed a close approximation of the proportion of exhaled air volume for each participant in the RASC at any given time. The sampled exhaled air volume was the product of this proportion and the air volume sampled by each detection device. Concentrations of colony forming units (CFU) by unit volume of exhaled air could then be established for any of the sampling devices. [CO2]excess=[CO2]measured−[CO2]ambient ExhaledAirVolume=[CO2]excess[CO2]exhaled×DeviceFlowRate×DeviceSamplingTime CFUperunitVolumeofAir=CFUcountExhaledAirVolume Simultaneous measurement of the particle content of the chamber air at the point of microbiological sampling allowed calculation of an aerosol volume per unit volume of air sampled. From these two measures, an approximateMtb CFU concentration by volume of bio-aerosol was determined (aerosol geometry assumed to be spherical). BioaerosolVolumeSvChamberAir(singlesizebin)=(43π(binsize/2)3×particlecount)5×10* BioaerosolVolumeSvChamberAir(AndersenStage)=∑BioaerosolVolumeSvChamberAir(correspondingsizebins) CFUperVolumeBioaerosol(AndersenStage)=CFUcountatstageSvChamberAir÷BioaerosolVolumeSvChamberAir(AndersenStage) Sv = Volume Sampled *APS sampled at rate of 5L/min; Andersen sampling over 10 minutes Microbiological detection methods Culture Andersen Impactor plates were unloaded in a biosafety cabinet. Filters analysed by culture were placed face-up on solid Middlebrook 7H10 agar supplemented with Glycerol, OADC, and0.05% Tween80, PANTA (BD) antibiotic mixture and incubated at 37°C for 4–6 weeks. The number of CFU consistent with expectedMtb colony morphology and rate of formationin vitro was recorded, and genomic DNA extracted for PCR confirmation using primers RD9F (5’-gtgtaggtcagccccatcc-3’), RD9R (5’-gctaccctcgaccaagtgtt-3’) and RD9Int (5’gctaccctcgaccaagtgtt-3’) using a protocol developed elsewhere.18. Protocol for RD9 confirmation of Mtb Colonies 20 μl reactions containing 2 µl of template DNA are set up with 1x reaction buffer, 200 μM of each dNTP, 0.5–1.0 μM of each primer, 1.5 mM MgCl 2 , 1x GC-rich solution, and 2U/50 μl of DNA polymerase. Thermal cycler parameters used for DNA amplification are as follows: denature at 95°C for 5 min, followed by 40 cycles of denaturation (94°c for 30s), annealing (65°C for 1min), and extension (72°C for 10min), and a final extension at 72°C for 7 min. Droplet digital PCR (ddPCR) Filters were removed from the RASC and transported in 50 ml Falcon tubes. The specimen was processed by vortexing in sterile PBS + 0.05% Tween 80 and centrifuged (3750 rpm for 15 minutes) to harvest the pellet which was lysed for DNA extraction and purification. Quantitative analysis of the RD9 region used a modified protocol and the QIAamp DNA Mini Kit (Qiagen). Primers (RD9/qRTF 5’-tgagtggcgatggtcaacac-3’ and RD9/qRTR 5’-gatggcgttcggaaagaaac-3’) and TaqMan minor groove binder (MGB) probe (RD9/probe 5’-actacgcggcttagtg-3’) were designed using Primer Express software (version 3.0.1). TaqMan MGB probe homologous to the RD9 gene was labelled with 6-carboxyfluorescein (FAM). The ddPCR reaction set-up and the run were performed as described previously19. An evaluable result for TB DNA was assigned when the following conditions were satisfied: (i) the total number of droplets read in the well was greater than 10,000; (ii) positive droplets possessed a fluorescence intensity above a threshold of 3500; (iii) minimal numbers of intermediate droplets (“rain”) were observed between positive and negative values; and (iv) the observed droplet distribution was consistent with a subpopulation of the positive control which comprised known concentrations of genomic DNA extracted fromMtb H37Rv.