Capture and visualization of live<i>Mycobacterium tuberculosis</i>bacilli from tuberculosis bioaerosols

Dinkele, Ryan; Gessner, Sophia; Koch, Anastasia; Morrow, Carl; Gqada, Melitta; Kamariza, Mireille; Bertozzi, Carolyn R.; Smith, Brian R.; McLoud, Courtney; Kamholz, Andrew Evan; Bryden, Wayne A.; Call, Charles J.; Mizrahi, Valerie; Wood, Robin; Warner, Digby F.

doi:10.1101/2019.12.23.887729

Cited by 7 publications

(9 citation statements)

References 64 publications

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“…Each participant sample was added to a microwell device (Edge Technologies, USA) containing an array of 1600 nanowells measuring 50 x 50 μm. This generated multiple discrete microenvironments for Mtb detection and avoided overgrowth of contaminants affecting the whole sample [16]. Fluorescent microscopy employed a skilled operator to distinguish Mtb on the basis of bacillus morphology and specific staining patterns.…”

Section: Mtb Detectionmentioning

confidence: 99%

Sensitivity optimisation of tuberculosis bioaerosol sampling

et al. 2020

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Introduction Detection of Mycobacterium tuberculosis (Mtb) in patient-derived bioaerosol is a potential tool to measure source case infectiousness. However, current bioaerosol sampling approaches have reported low detection yields in sputum-positive TB cases. To increase the utility of bioaerosol sampling, we present advances in bioaerosol collection and Mtb identification that improve detection yields. Methods A previously described Respiratory Aerosol Sampling Chamber (RASC) protocol, or "RASC-1", was modified to incorporate liquid collection of bioaerosol using a high-flow wetwalled cyclone (RASC-2). Individuals with GeneXpert-positive pulmonary TB were sampled pre-treatment over 60-minutes. Putative Mtb bacilli were detected in collected fluid by fluorescence microscopy utilising DMN-Trehalose. Exhaled air and bioaerosol volumes were estimated using continuous CO 2 monitoring and airborne particle counting, respectively. Mtb capture was calculated per exhaled air volume sampled and bioaerosol volume for RASC-1 (n = 35) and for RASC-2 (n = 21). Empty chamber samples were collected between patients as controls. Results The optimised RASC-2 protocol sampled a median of 258.4L (IQR: 226.9-273.6) of exhaled air per patient compared with 27.5L (IQR: 23.6-30.3) for RASC-1 (p<0.0001). Bioaerosol volume collection was estimated at 2.3nL (IQR: 1.1-3.6) for RASC-2 compared with 0.08nL

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Section: Mtb Detectionmentioning

confidence: 99%

Sensitivity optimisation of tuberculosis bioaerosol sampling

et al. 2020

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“…After overnight incubation in media containing DMN-trehalose, the samples are washed and added to a novel nanowell device described in ref. [9]. The devices are sealed with adhesive film and centrifuged prior to visualization by fluorescence microscopy.…”

Section: Microscopy-based Detection Of Mtbmentioning

confidence: 99%

“…The devices are sealed with adhesive film and centrifuged prior to visualization by fluorescence microscopy. Detection, enumeration and differentiation from debris of fluorescent bacilli is based on dye incorporation patterns and differential bacterial cellular morphology (cell length and width) [9].…”

Section: Microscopy-based Detection Of Mtbmentioning

confidence: 99%

“…A high-flow, wet-walled cyclone collector will be used for both methods, ensuring the capture of a large proportion of the exhaled air volumes with extraction of the bioaerosol into liquid. The liquid specimen will then be subjected to live-cell fluorescence imaging to identify single viable Mtb bacilli based on DMN-trehalose probe incorporation patterns and differential bacterial cellular morphology [9].…”

Section: Introductionmentioning

confidence: 99%

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Bioaerosol sampling of patients with suspected pulmonary tuberculosis: a study protocol

et al. 2020

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Background: Tuberculosis (TB) is transmitted in bioaerosols containing Mycobacterium tuberculosis (Mtb). Despite being central to ongoing TB transmission, no routine diagnostic assay exists to measure Mtb in bioaerosols. Furthermore, published studies of Mtb in bioaerosol samples have been limited to individuals with sputum-positive pulmonary TB. Notably, TB diagnosis is based on clinical symptoms and sputum laboratory findings. This is despite the fact that approximately half of all patients commencing TB treatment are sputum-negative, resulting in a high proportion of presumptive treatments. Here, we propose to use a sensitive air sampling protocol to investigate the prevalence of Mtb-containing bioaerosols in both sputum-positive and sputum-negative TB suspects, at the same time evaluating the potential to identify unrecognized transmitters of TB. Methods: Our parallel-group design will identify viable Mtb in bioaerosols produced by individuals attending a TB clinic in South Africa. Sampling will be performed on eligible individuals presenting with symptoms indicative of TB and repeated at 14 days if initially positive. Participants will be prospectively classified into three distinct groups based on National TB Control Program (NTBCP) criteria: Group A, TB notification with sputum-based laboratory confirmation; Group B, TB notification with empiric diagnosis; and Group C, individuals not notified. Group C individuals with detectable Mtb bioaerosol will be monitored until resolution of clinical and laboratory status. Collection of bioaerosol specimens will be via two consecutive sampling modalities: (1) direct sampling following a specific respiratory manoeuvre; and (2) indirect sampling during passive respiratory activity. Bioaerosol specimens will be analyzed for viable Mtb using DMN-trehalose staining and live-cell fluorescence microscopy. Mtb genomes and mycobacterial and host lipids will be detected using droplet digital PCR and mass spectrometry analyses, respectively. The primary objective is to determine the prevalence of Mtb bioaerosols in all TB clinic attendees and in each of the groups. Secondary objectives are to investigate differences in prevalence of Mtb bioaerosol by HIV status and current isoniazid preventive therapy (IPT) use; we will also determine the impact of anti-TB chemotherapy on Mtb-containing bioaerosol production. Discussion: Respiratory bioaerosol has a potential role in non-invasive TB diagnosis, infectivity measurement and treatment monitoring.

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“…Mtb aggregates have been observed in human lung tissue [7], though the fraction of bacilli in this state is unclear. Additionally, Mtb aggregates have recently been shown to be transmitted in bio-aerosols [14].…”

Section: Introductionmentioning

confidence: 99%

Aggregated Mycobacterium tuberculosis enhances the inflammatory response

Sigal

Rodel

Ferreira

et al. 2021

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Mycobacterium tuberculosis (Mtb) readily aggregates in culture and Mtb aggregates in the lung were observed in experimental Mtb infection. However, the physiological consequences of Mtb aggregation are incompletely understood. Here we examined the human macrophage transcriptional response to aggregated Mtb relative to infection with non-aggregated single or multiple bacilli per host cell. Infection with aggregated Mtb led to an early upregulation of pro-inflammatory associated genes and enhanced TNFα signaling via the NFκB pathway. Both these pathways were significantly upregulated relative to infection with single bacilli, and TNFα signaling was also significantly elevated relative to infection with multiple non-aggregated Mtb. Secretion of TNFα and downstream cytokines were also enhanced. On a longer timescale, aggregate infection led to overall increased acidification per macrophage and a high proportion of death in these cells after aggregate phagocytosis. Host cell death did not occur when Mtb aggregates were heat killed despite such clumps being readily picked up. To validate that Mtb aggregates do occur in the human lung, we document Mtb aggregates surrounding a cavity in a human TB lesion. Aggregates may therefore be present in some lesions and elicit a stronger inflammatory response resulting in recruitment of additional phagocytes and their subsequent death, potentially leading to necrosis and transmission.

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Capture and visualization of liveMycobacterium tuberculosisbacilli from tuberculosis bioaerosols

Cited by 7 publications

References 64 publications

Sensitivity optimisation of tuberculosis bioaerosol sampling

Sensitivity optimisation of tuberculosis bioaerosol sampling

Bioaerosol sampling of patients with suspected pulmonary tuberculosis: a study protocol

Aggregated Mycobacterium tuberculosis enhances the inflammatory response

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