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.

Molecular Detection of Airborne Mycobacterium tuberculosis in South African High Schools.

Rationale: South African adolescents carry a high tuberculosis disease burden. It is not known if schools are high-risk settings for Mycobacterium tuberculosis (MTB) transmission. Objectives: To detect airborne MTB genomic DNA in classrooms. Methods: We studied 72 classrooms occupied by 2,262 students in two South African schools. High-volume air filtration was performed for median 40 (interquartile range [IQR], 35-54) minutes and assayed by droplet digital PCR (ddPCR)-targeting MTB region of difference 9 (RD9), with concurrent CO2 concentration measurement. Classroom data were benchmarked against public health clinics. Students who consented to individual tuberculosis screening completed a questionnaire and sputum collection (Xpert MTB/RIF Ultra) if symptom positive. Poisson statistics were used for MTB RD9 copy quantification. Measurements and Main Results: ddPCR assays were positive in 13/72 (18.1%) classrooms and 4/39 (10.3%) clinic measurements (P = 0.276). Median ambient CO2 concentration was 886 (IQR, 747-1223) ppm in classrooms versus 490 (IQR, 405-587) ppm in clinics (P < 0.001). Average airborne concentration of MTB RD9 was 3.61 copies per 180,000 liters in classrooms versus 1.74 copies per 180,000 liters in clinics (P = 0.280). Across all classrooms, the average risk of an occupant inhaling one MTB RD9 copy was estimated as 0.71% during one standard lesson of 35 minutes. Among 1,836/2,262 (81.2%) students who consented to screening, 21/90 (23.3%) symptomatic students produced a sputum sample, of which one was Xpert MTB/RIF Ultra positive. Conclusions: Airborne MTB genomic DNA was detected frequently in high school classrooms. Instantaneous risk of classroom exposure was similar to the risk in public health clinics.

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.

Environmental air sampling for detection and quantification of Mycobacterium tuberculosis in clinical settings: Proof of concept.

OBJECTIVE: Novel approaches are needed to understand and disrupt Mycobacterium tuberculosis transmission. In this proof-of-concept study, we investigated the use of environmental air samplings to detect and quantify M. tuberculosis in different clinic settings in a high-burden area. DESIGN: Cross-sectional, environmental sampling. SETTING: Primary-care clinic. METHODS: A portable, high-flow dry filter unit (DFU) was used to draw air through polyester felt filters for 2 hours. Samples were collected in the waiting area and TB room of a primary care clinic. Controls included sterile filters placed directly into collection tubes at the DFU sampling site, and filter samplings performed outdoors. DNA was extracted from the filters, and droplet digital polymerase chain reaction (ddPCR) was used to quantify M. tuberculosis DNA copies. Carbon dioxide (CO2) data loggers captured CO2 concentrations in the sampled areas. RESULTS: The median sampling time was 123 minutes (interquartile range [IQR], 121-126). A median of 121 (IQR, 35-243) M. tuberculosis DNA copies were obtained from 74 clinic samplings, compared to a median of 3 (IQR, 1-33; P < .001) obtained from 47 controls. At a threshold of 320 DNA copies, specificity was 100%, and 18% of clinic samples would be classified as positive. CONCLUSIONS: This proof-of-concept study suggests that the potential for airborne M. tuberculosis detection based on M. tuberculosis DNA copy yield to enable the identification of high-risk transmission locations. Further optimization of the M. tuberculosis extraction technique and ddPCR data analysis would improve detection and enable robust interpretation of these data.