Nuno Rufino de Sousa

Tuberculosis (TB) remains the leading cause of death by a single infectious organism with over 1.3 million people succumbing to the disease every year. Despite efficient antibiotic treatment the disease continues to spread with transmission being the major contributor. TB is caused by the bacterium Mycobacterium tuberculosis (Mtb), an intracellular pathogen the transmits through the air. Importantly, airborne transmission is what makes this disease difficult to study, detect and control. Survival to aerosol transport is critical for the mycobacteria to be able to successfully transmit and infect new individuals. However, little is known on how Mtb survives aerosol transport and its impact on the subsequent ability to establish an infection.The aim of this PhD project was to study Mycobacterium tuberculosis adaptations to aerosol transport and determine how this knowledge can be employed in transmission studies.With this in mind, we sought to create a method by which aerosols carrying Mtb could be studied. In Paper I, we tested the feasibility of using a Coriolis® u air sampler, a wet-walled cyclone sampler designed for collection of viable airborne microorganisms, for the study of Mtb aeromicrobiology. In Paper II, we employed this air sampler together with the Anderssen cascade impactor (ACI), a sampler that impacts aerosols onto agar plates, to investigate the influence of temperature and humidity on the survival of aerosolized Mtb, as well as the changes to its gene expression in response to stress and its recovery capacity after aerosolization. Finaly, in Paper III, we developed a protocol to capture and detect Mtb aerosols using an electrostatic air sampler in combination with the widely used GeneXpert® platform and employed it to collect cough generated aerosols from TB patients.In Paper I, we investigated the sampling efficiency of the Coriolis for aeromicrobiology studies. The sampling protocol suggested by the manufacturer of 1 h continuous sampling with a single collector cone was evaluated. We found this protocol to be detrimental to the collection capacity after 30 min of sampling. From this time point, no further material seemed to be collected and even a decrease in the total material collected observed after 1 h. We then developed a cumulative sampling protocol, where a new collector cone was replaced every 10 min, which improved collection capacity. Furthermore, we detected a loss of material from the collector cone during sampling periods longer than 10 min, meaning that collected material was being re-aerosolized. Indeed, we were able to measure contamination of all the tubing parts of the air sampler following a collection run. With this in mind, we adapted an HEPA filter to the device to eliminate contamination between collection runs and environments. The device was then tested in a real-life scenario during a biological spill simulation in a laboratory setting. Following a spill of one billion microspheres in 500 ml of water, virtually no particles were detected with the Coriolis at bench height. Taken together, the results of this study provide a stable and effective method for collecting airborne particles without cross contamination or losses. Additionally, our simulation experiment suggests that there is no scientific basis for evacuating a laboratory following a spill, on account of aerosol generation. Instead, it may be better to inactivate and clean the spill as soon as possible.In Paper II, we studied how Mtb is able to survive the process of aerosolization and its impact on the bacteria. For this, we aerosolized the H37Rv lab strain of Mtb inside an aerosol-tight, hygrothermally-controlled aerosol chamber in a biosafety level 3 laboratory. Our results demonstrated that Mtb suffers a loss of viability when aerosolized in conditions of lower humidity independent of the temperatures tested. These effects were exacerbated by the time that the bacteria were allowed to dwell in the air, with almost no recovery of culturable Mtb observed after 30 min. We then sought to determine if virulence affected Mtb ability to survive aerosol transport under intermediate and high humidity conditions. The avirulent H37Ra laboratory strain of Mtb exhibited a lower survival compared to its virulent counterpart H37Rv independent of humidity. Aerosol transport also induced the expression of reactive oxygen species (ROS) detoxifying genes. Under intermediate humidity conditions this expression also increased the longer the bacteria remained in aerosol. Mtb also induced the expression of the hspX gene after aerosolization. This gene is known to block Mtb replication. Indeed, when aerosolized Mtb were returned to liquid media, its replication was arrested after 24 h, partly recovering after 48 hours. Altogether these results provide novel insights into how Mtb reacts to aerosolization, suggesting humidity and partly virulence dependent survival, as well as active adaptations in response to ROS damage suffered during the aerosol transport.In Paper III, we set to determine the feasibility of detecting Mtb in respiratory aerosols and its clinical relevance for diagnostics. We used an electrostatic air sampler previously developed in the group to collect aerosols from TB patients. The detection limit of the device was tested by aerosolizing the TB vaccine M. bovis BCG, collecting it on the air sampler and then analyzing detection of bacterial DNA using the Xpert MTB/RIF Ultra cartridge. Our results showed a detection limit of 0.5 genomes/Lair. We deemed that this sensitivity was conductive to the device being tested in a clinical setting. We then proceeded to use the device to detect Mtb DNA in cough aerosols of TB patients in a clinic in South Africa. Overall, the sensitivity of this method was 46%, with sensitivity increasing to 60% in patients with higher Mtb burden in their sputum. We were also able to detect Mtb DNA in the coughs of sputum-negative patients, although...

Tuberculosis (TB) remains the leading cause of death by a single infectious organism with over 1.3 million people succumbing to the disease every year. Despite efficient antibiotic treatment the disease continues to spread with transmission being the major contributor. TB is caused by the bacterium Mycobacterium tuberculosis (Mtb), an intracellular pathogen the transmits through the air. Importantly, airborne transmission is what makes this disease difficult to study, detect and control. Survival to aerosol transport is critical for the mycobacteria to be able to successfully transmit and infect new individuals. However, little is known on how Mtb survives aerosol transport and its impact on the subsequent ability to establish an infection.The aim of this PhD project was to study Mycobacterium tuberculosis adaptations to aerosol transport and determine how this knowledge can be employed in transmission studies.With this in mind, we sought to create a method by which aerosols carrying Mtb could be studied. In Paper I, we tested the feasibility of using a Coriolis® u air sampler, a wet-walled cyclone sampler designed for collection of viable airborne microorganisms, for the study of Mtb aeromicrobiology. In Paper II, we employed this air sampler together with the Anderssen cascade impactor (ACI), a sampler that impacts aerosols onto agar plates, to investigate the influence of temperature and humidity on the survival of aerosolized Mtb, as well as the changes to its gene expression in response to stress and its recovery capacity after aerosolization. Finaly, in Paper III, we developed a protocol to capture and detect Mtb aerosols using an electrostatic air sampler in combination with the widely used GeneXpert® platform and employed it to collect cough generated aerosols from TB patients.In Paper I, we investigated the sampling efficiency of the Coriolis for aeromicrobiology studies. The sampling protocol suggested by the manufacturer of 1 h continuous sampling with a single collector cone was evaluated. We found this protocol to be detrimental to the collection capacity after 30 min of sampling. From this time point, no further material seemed to be collected and even a decrease in the total material collected observed after 1 h. We then developed a cumulative sampling protocol, where a new collector cone was replaced every 10 min, which improved collection capacity. Furthermore, we detected a loss of material from the collector cone during sampling periods longer than 10 min, meaning that collected material was being re-aerosolized. Indeed, we were able to measure contamination of all the tubing parts of the air sampler following a collection run. With this in mind, we adapted an HEPA filter to the device to eliminate contamination between collection runs and environments. The device was then tested in a real-life scenario during a biological spill simulation in a laboratory setting. Following a spill of one billion microspheres in 500 ml of water, virtually no particles were detected with the Coriolis at bench height. Taken together, the results of this study provide a stable and effective method for collecting airborne particles without cross contamination or losses. Additionally, our simulation experiment suggests that there is no scientific basis for evacuating a laboratory following a spill, on account of aerosol generation. Instead, it may be better to inactivate and clean the spill as soon as possible.In Paper II, we studied how Mtb is able to survive the process of aerosolization and its impact on the bacteria. For this, we aerosolized the H37Rv lab strain of Mtb inside an aerosol-tight, hygrothermally-controlled aerosol chamber in a biosafety level 3 laboratory. Our results demonstrated that Mtb suffers a loss of viability when aerosolized in conditions of lower humidity independent of the temperatures tested. These effects were exacerbated by the time that the bacteria were allowed to dwell in the air, with almost no recovery of culturable Mtb observed after 30 min. We then sought to determine if virulence affected Mtb ability to survive aerosol transport under intermediate and high humidity conditions. The avirulent H37Ra laboratory strain of Mtb exhibited a lower survival compared to its virulent counterpart H37Rv independent of humidity. Aerosol transport also induced the expression of reactive oxygen species (ROS) detoxifying genes. Under intermediate humidity conditions this expression also increased the longer the bacteria remained in aerosol. Mtb also induced the expression of the hspX gene after aerosolization. This gene is known to block Mtb replication. Indeed, when aerosolized Mtb were returned to liquid media, its replication was arrested after 24 h, partly recovering after 48 hours. Altogether these results provide novel insights into how Mtb reacts to aerosolization, suggesting humidity and partly virulence dependent survival, as well as active adaptations in response to ROS damage suffered during the aerosol transport.In Paper III, we set to determine the feasibility of detecting Mtb in respiratory aerosols and its clinical relevance for diagnostics. We used an electrostatic air sampler previously developed in the group to collect aerosols from TB patients. The detection limit of the device was tested by aerosolizing the TB vaccine M. bovis BCG, collecting it on the air sampler and then analyzing detection of bacterial DNA using the Xpert MTB/RIF Ultra cartridge. Our results showed a detection limit of 0.5 genomes/Lair. We deemed that this sensitivity was conductive to the device being tested in a clinical setting. We then proceeded to use the device to detect Mtb DNA in cough aerosols of TB patients in a clinic in South Africa. Overall, the sensitivity of this method was 46%, with sensitivity increasing to 60% in patients with higher Mtb burden in their sputum. We were also able to detect Mtb DNA in the coughs of sputum-negative patients, although...

Background: Non–sputum-based diagnosis of tuberculosis is a public health priority. Little is known about the feasibility of detecting Mycobacterium tuberculosis (Mtb) complex DNA in respiratory aerosols in primary care, its diagnostic value, and clinical and microbiological characteristics associated with detection. Methods: We recruited symptomatic adults self-presenting to South African primary care clinics with a sputum Xpert MTB/RIF Ultra (Ultra) result. Cough aerosols were collected on-site by the TB Hotspot Detector, a novel electrostatic aerosol sampler, and tested by Ultra. Environmental and laboratory controls were collected. Predictors of aerosol Mtb DNA (AMD) detection were assessed. Results: Among 137 participants, 71 (52%) had medium or high sputum Ultra semiquantitative results and 34 (25%) had negative results. When compared with sputum Ultra detection, AMD detection sensitivity and specificity were 46.6% (95% CI, 42.5%–50.7%) and 76.5% (95% CI, 70.4%–82.5%), respectively. Sensitivity was higher in people with a sputum Ultra semiquantitation category of high (56.9%; 95% CI, 51.1%–62.7%). Factors associated with AMD detection were male sex with a sputum Ultra semiquantitative result of medium or greater (adjusted risk ratio, 3.26; 95% CI, 1.11–9.55; P = .024) and a reported fever (adjusted risk ratio, 0.58; 95% CI, .29–1.07; P = .099). Sputum to aerosol ratios were ≥0.75 in 3 participants, suggesting a high capacity to expel Mtb DNA. Despite rigorous decontamination, AMD was detected from 30% of environmental samples, highlighting the TB Hotspot Detector's potent sampling capability and potential nosocomial transmission risks. Conclusions: Electrostatic aerosol sampling is feasible in primary care to detect people with infectious tuberculosis. Deployment of this and other practical aerosol-sampling tools might help to characterize predictors of tuberculosis transmission.

Transmission mechanisms for severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) are incompletely understood. In particular, aerosol transmission remains unclear, with viral detection in air and demonstration of its infection potential being actively investigated. To this end, we employed a novel electrostatic collector to sample air from rooms occupied by COVID‐19 patients in a major Swedish hospital. Electrostatic air sampling in conjunction with extraction‐free, reverse‐transcriptase polymerase chain reaction (hid‐RT‐PCR) enabled detection of SARS‐CoV‐2 in air from patient rooms (9/22; 41%) and adjoining anterooms (10/22; 45%). Detection with hid‐RT‐PCR was concomitant with viral RNA presence on the surface of exhaust ventilation channels in patients and anterooms more than 2 m from the COVID‐19 patient. Importantly, it was possible to detect active SARS‐CoV‐2 particles from room air, with a total of 496 plaque‐forming units (PFUs) being isolated, establishing the presence of infectious, airborne SARS‐CoV‐2 in rooms occupied by COVID‐19 patients. Our results support circulation of SARS‐CoV‐2 via aerosols and urge the revision of existing infection control frameworks to include airborne transmission.

Neuropsychiatric manifestations are common in both acute and post-acute phase of SARS-CoV-2 infection, but the mechanism of these effects is unknown. Here, we derive human brain organoids with innately developing microglia to investigate the cellular responses to SARS-CoV-2 infection on a single cell level. We find evidence of limited tropism to SARS-CoV-2 and observe extensive neuronal cell death that also include non-infected cells. Single cell transcriptome profiling reveals distinct responses in microglia and astrocytes that share features with cellular states observed in neurodegenerative diseases, includes upregulation of genes with relevance for synaptic stripping, and suggests altered blood brain barrier integrity. Across all cell types, we observe a global translational shut-down as well as altered carbohydrate metabolism and cellular respiration. Together, our findings provide insights into cellular responses of the resident brain immune cells to SARS-CoV-2 and pinpoint mechanisms that may be of relevance for the neuropathological changes observed in COVID-19 patients.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is commonly diagnosed by reverse transcription polymerase chain reaction (RT-PCR) to detect viral RNA in patient samples, but RNA extraction constitutes a major bottleneck in current testing. Methodological simplification could increase diagnostic availability and efficiency, benefitting patient care and infection control. Here, we describe methods circumventing RNA extraction in COVID-19 testing by performing RT-PCR directly on heat-inactivated or lysed samples. Our data, including benchmarking using 597 clinical patient samples and a standardised diagnostic system, demonstrate that direct RT-PCR is viable option to extraction-based tests. Using controlled amounts of active SARS-CoV-2, we confirm effectiveness of heat inactivation by plaque assay and evaluate various generic buffers as transport medium for direct RT-PCR. Significant savings in time and cost are achieved through RNA-extraction-free protocols that are directly compatible with established PCR-based testing pipelines. This could aid expansion of COVID-19 testing.

Detecting infectious aerosols is central for gauging and countering airborne threats. In this regard, the Coriolis® µ cyclonic air sampler is a practical, commercial collector that can be used with various analysis methods to monitor pathogens in air. However, information on how to operate this unit under optimal sampling and biosafety conditions is limited. We investigated Coriolis performance in aerosol dispersal experiments with polystyrene microspheres and Bacillus globigii spores. We report inconsistent sample recovery from the collector cone due to loss of material when sampling continuously for more than 30 min. Introducing a new collector cone every 10 min improved this shortcoming. Moreover, we found that several surfaces on the device become contaminated during sampling. Adapting a high efficiency particulate air-filter system to the Coriolis prevented contamination without altering collection efficiency or tactical deployment. A Coriolis modified with these operative and technical improvements was used to collect aerosols carrying microspheres released inside a Biosafety Level-3 laboratory during simulations of microbiological spills and aerosol dispersals. In summary, we provide operative and technical solutions to the Coriolis that optimize microbiological air sampling and improve biosafety.