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Table of Contents
Year : 2022  |  Volume : 1  |  Issue : 4  |  Page : 209-216

Methods for bioaerosol sampling in tuberculosis and Coronavirus Disease 2019: Potential tool for disease diagnosis and assessment of infectivity

Department of Tuberculosis, The Foundation for Medical Research, Mumbai, Maharashtra, India

Date of Submission01-Jul-2022
Date of Acceptance05-Sep-2022
Date of Web Publication5-Dec-2022

Correspondence Address:
Ambreen Mohamadmunir Shaikh
The Foundation for Medical Research, Kantilal Sheth Memorial Building, 84-A R G Thadani Marg, Siddharth Nagar, Worli, Mumbai, Maharashtra
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jpdtsm.jpdtsm_84_22

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Respiratory infections such as Tuberculosis (TB) and coronavirus disease 2019 (COVID-19) have tremendously impacted global morbidity and mortality. It is now known that their causative agents have an airborne route of transmission. Clinical diagnosis of these diseases often relies on samples that are difficult to obtain or involve invasive techniques. These limitations have given impetus to research focusing on pathogen detection in bioaerosols. Collection, detection, and quantification of infectious aerosols released by patients can serve as a diagnostic tool while assessing the infectiousness of the pathogen being transmitted. In this review, we have described the various methods of bioaerosol sampling in TB and COVID-19 along with their applications in real-life clinical settings. From aerosol sampling systems and cough chambers to the recent face mask sampling, techniques have advanced over the years moving toward the development of a point-of-care tool for disease diagnosis. Among these, the mask sampling approach has an edge over other methods in terms of convenience and usability. Such sampling techniques, combined with sensitive detection systems have the potential to rapidly detect respiratory pathogens and may ultimately play a role in preventing the spread of these diseases in the community. The review highlights the advances in the application of bioaerosol sampling with a focus on the potential of mask-based bioaerosol sampling method. It also discusses the future research and clinical prospects of bioaerosol sampling.

Keywords: Bioaerosol sampling, diagnosis, infectivity, mask sampling, respiratory diseases

How to cite this article:
Vaswani SR, Shaikh AM. Methods for bioaerosol sampling in tuberculosis and Coronavirus Disease 2019: Potential tool for disease diagnosis and assessment of infectivity. J Prev Diagn Treat Strategies Med 2022;1:209-16

How to cite this URL:
Vaswani SR, Shaikh AM. Methods for bioaerosol sampling in tuberculosis and Coronavirus Disease 2019: Potential tool for disease diagnosis and assessment of infectivity. J Prev Diagn Treat Strategies Med [serial online] 2022 [cited 2023 Jan 29];1:209-16. Available from: http://www.jpdtsm.com/text.asp?2022/1/4/209/362828

  Introduction Top

Respiratory infections such as the recent Coronavirus disease 2019 (COVID-19) pandemic or age-old tuberculosis (TB) are leading infectious killers and causes of global health concern.[1],[2] Routinely, for these diseases, diagnosis and assessing patient infectivity involves analyzing either the burden of Mycobacterium tubeculosis (Mtb) in sputum samples collected from TB patients or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral copies in nasopharyngeal swabs of COVID-19 patients.[3],[4],[5],[6],[7],[8],[9] However, sputum production is variable among TB patients and difficult sample to obtain from populations who cannot produce sputum.[10],[11],[12] In the case of nasopharyngeal swabs, the collection procedure is slightly invasive, uncomfortable, and requires trained staff.[13],[14] It is also important to note that both these sources are largely representative of upper respiratory tract infection.[8],[12],[15],[16] These limitations combined with the well-recognized fact that transmission occurs from sputum smear-negative patients in TB[17],[18] and asymptomatic COVID-19 patients[19],[20],[21],[22] increase the impetus to explore better sampling sources for diagnosis and prediction of infectiousness.

Multiple lines of evidence including historic studies by Riley and Wells on TB and a large compilation of studies on COVID-19 point to the importance of infectious aerosol inhalation as the primary mode of transmission for these diseases.[11],[23],[24],[25],[26],[27],[28] These infectious bioaerosols contain a suspension of pathogens in air particles exhaled/expelled during talking, coughing, sneezing, or breathing by the infected person.[29],[30],[31] Collecting, detecting, and quantifying bioaerosols expelled/exhaled by an infected person could provide insights into individual infectivity and transmission risk. Conventionally, the two main methods of aerosol collection are as follows: (1) Indirect sampling – collecting aerosols from the environmental air in hospitals, public transport, etc.,[32],[33],[34],[35],[36] and (2) Direct sampling – capturing aerosol exhaled by infected individuals while naturally breathing or performing vocal maneuvers such as talking, coughing, and singing.[37],[38],[39] Indirect aerosol collection techniques have been widely used for the detection of influenza and severe acute respiratory syndrome and are well discussed in preexisting literature.[40],[41] Here, we have reviewed the various methods currently available for collecting infectious bioaerosols exhaled by TB and COVID-19 patients and discussed the key findings that have come to light from the application of these methods.

  Cough-Generated Aerosol Sampling System Top

In 2003, Fennelly's group was the first to develop a system for directly capturing and quantifying the Mtb burden in the cough of TB patients by measuring colony-forming units (CFU).[42] The system consisted of two Andersen cascade impactors with six plates each of 7H11 agar media and was termed as cough aerosol sampling system (CASS). Patients were asked to cough into the chamber for 5 min, where samples were collected on the impactors and cough frequency was recorded. The media plates were incubated at 37°C in presence of CO2 and were checked weekly for about 2 months to record the CFU counts. An advantage of CASS is that it can quantify viable bacteria in cough-generated aerosols with particle size measurement in a brief sampling time.[42] CASS had a detection rate of culturable cough aerosols between 25% and 60%[42],[43] and culturable Mtb was present in cough aerosols sized between 0.56 and 4.7 μm.[42]

The CASS has been used in multiple studies of drug-sensitive and resistant TB strains.[12],[17],[42],[43],[44],[45],[46] In their preliminary study, Fennelly's group observed that CFU counts decreased with treatment initiation, indicating that patients post TB treatment had reduced infectivity and suggesting the potential of CASS for monitoring efficiency of treatment regimens.[42] In a clinical setting, using the CASS, Jones-López et al. demonstrated contacts of high aerosol-producing (CFU >10) TB patients had 6.9 times increased odds of developing new infection compared to contacts of low or aerosol negative TB patients, thus providing first-time evidence that exposure to high infectious aerosols increases the risk of transmission in contacts.[17] This contact tracing study and subsequent studies from Brazil and Uganda highlighted that though cough aerosols are produced by a subset of patients, direct measurement of these bioaerosols may better predict infectiousness compared to sputum bacillary burden.[12],[17],[43] Moreover, the Brazilian study also showed that microbiologically proven secondary TB disease in household contacts clustered among contacts of aerosol-positive TB cases.[43] In 2020, Theron et al. using CASS demonstrated that more than half of drug-resistant (DR) TB patients had culturable bacilli in their cough aerosols, thereby questioning the paradigm on infectivity of DR strains.[46] In addition to TB, CASS has been used for detecting Pseudomonas aeruginosa in cystic fibrosis patients and the influenza virus.[45],[47] CASS is widely considered to be the gold standard for direct aerosol capture from pulmonary TB patients, however, it has its limitations like requiring specialized infrastructure for sample collection and Mtb culture, long turnaround times due to prolonged incubation, higher probability of fungal contamination and media and bacilli desiccation due to prolonged sampling.[15],[48] From the patient's perspective, the method is taxing as it requires the patient to cough; and also does not examine the bacillary burden exhaled under natural conditions or expelled due to other vocal maneuvers.[15]

  Respiratory Aerosol Sampling Chamber Top

In 2016, Wood et al. developed a personal clean room-like unit consisting of multiple sampling devices with an aim to capture and study characteristics of bioaerosols exhaled by TB patients. The unit was termed as Respiratory Aerosol Sampling Chamber (RASC).[48] The first model of RASC was a sputum collection booth modified to function as a cleanroom, which facilitated passive sampling of bioaerosols generated by TB patients seated in the chamber for 60 min while performing normal activities such as coughing, breathing, and talking.[48] Bioaerosols were captured by three different collection devices incorporated into the RASC design (liquid impingers, solid impactors, and filters) and within the chamber, temperature, humidity, and C02 levels were constantly monitored. In addition to this, a six-stage Andersen cascade impactor was used to quantify viable organisms by CFU counting on media plates based on particle size.[48],[49] In RASC, sampling begins after C02 levels within the chamber reaches the target concentration, which generally happens after patients have spent 25–30 min in the RASC chamber. After sampling is initiated first 10 min are utilized for bacteriology sample collection, followed by bioaerosol collection for imaging and molecular assays.[48],[49] RASC provides a real-time measurement of the number, particle size distribution, and morphological parameters of bioaerosols generated by patients.[48] The combination of bioaerosol sampling with analysis techniques enables the identification, visualization, phenotyping, genotyping, and determining infectiousness of the airborne TB bacilli isolated in the process.[48],[49] RASC detects Mtb present in the patient bioaerosols but which have aged in the chamber environment, thus closely mimicking the infectious particle involved in disease transmission.[49] In a clinical setting, RASC was able to detect putative Mtb in 77.1% of TB patient aerosol samples, 42.8% of these samples were positive by mycobacterial culture, and 92.96% were positive by molecular confirmation using digital droplet polymerase chain reaction (PCR).[48]

The RASC platform has been applied to demonstrate that viable Mtb could be detected in more than 90% of patients who undertook 60 min of relaxed breathing, suggesting cough is not necessary for Mtb exhalation and a possibility of a noncough transmission by TB patients.[49],[50] The RASC platform was modified to enable liquid capture of exhaled bioaerosols to overcome the limitations of solid media sampling. In order to develop a culture-independent RASC system capable of enumerating viable Mtb, RASC sampling was combined with fluorescence microscopy and detection using mycobacterial cell wall specific viability probe DMN-Trehalose.[51] This advancement in the RASC system was helpful in studying the physiological state of aerosolized TB bacilli and heterogeneity among them. Moreover, the enumeration of viable Mtb was also applied to provide evidence that tidal breathing contributed to 90% of the patient's daily aerosolized Mtb output.[51],[52] From its various applications, it is evident that the RASC system can be used for disease diagnosis, assessing TB patients' infectious inoculum, and studying changes in the Mtb physiology in response to treatment. RASC also faces similar limitations as seen for CASS, like the need for complex infrastructure and trained staff for sample collection, but unlike CASS it has not been used to study infectiousness or transmission among household contacts of TB patients.[15]

  Mask-Based Aerosol Sampling System Top

Face-mask sampling

Although both CASS and RASC are efficient for infectious aerosol collection, due to the requirement of complex set-up, and longer turnaround time, they are difficult to be used as the point of care methods.[15],[42] In 2014, Caroline Williams group, at the University of Leicester, UK, developed a new approach to capture bioaerosols from TB patients using a face mask and detect TB bacilli expelled from patients, termed as face mask sampling.[53] In their pilot study, they evaluated two different types of mask and detection techniques for face-mask sampling (FMS) (1) patients wore standard surgical masks for as long as they were comfortable, performed vocal maneuvers such as talking, coughing, and sneezing; and TB bacilli were detected using a bacteriophage assay and (2) patients wore FFP30 mask with an attached gelatin filter for 1 h, performed similar vocal maneuvers and TB detection was done using the Cepheid GeneXpert MTB/RIF system.[53] By combining both mask sampling methods, Mtb could be detected in the face masks of 65% of confirmed TB patients.[53] In a larger follow-up study, involving 1-h face mask sampling of 192 pulmonary TB patients, mask positivity rate of 85.5% was observed.[18] The face mask assembly was modified by replacing the gelatin filter with a 3D-printed polyvinyl alcohol (PVA) matrix, which was a more stable, economical alternative, devoid of any background DNA with bacterial recovery comparable to that of the gelatin filter.[54] To enhance recovery, the thickness of the PVA matrix was reduced which increased the bacterial detection by 2-fold.[54]

FMS was used for the first time in a clinical setting for longitudinal sampling to provide insights into the daily Mtb aerosol output of TB patients. It was evident from this study that Mtb aerosol production in patients did not follow a diurnal pattern.[18] A household contact study used FMS to demonstrate the association between Mtb aerosols exhaled by TB patients and disease transmission by investigating the relationship between aerosolized Mtb burden of pulmonary TB patients and infections observed in their household contacts.[15] Similar to findings observed from the CASS study, this study also indicated that a small subset of TB patients transmit more than others and showed that only high Mtb aerosol mask output (>20,000 IS6110 copies) was associated with a three-fold increase in household infections.[15] Importantly, findings from FMS studies showed that traditional markers of infectivity (cough frequency, sputum bacillary content, or chest radiographic disease severity) did not associate household infection rate, supporting the finding that aerosol TB burden is a better predictor of infectiousness than sputum bacterial burden.[15] A subsequent pilot study demonstrated the potential application of FMS for prospective active case-finding. In this study, 6/8 presumptive TB cases were face-mask positive and sputum AFB smear-negative at screening, and four of these individuals turned sputum positive after 6 weeks. These findings suggested that FMS could detect active TB infection with greater consistency and at an earlier stage compared to sputum sampling.[18]

With the advent of the COVID-19 pandemic, as the aerosol route of transmission for the disease became evident, FMS was adapted in a clinical setting to measure exhaled SARS-CoV-2 RNA from COVID-19 patients.[16],[55] For COVID-19 sampling, patients wore face mask with attached PVA strips for 30 min. FMS positivity was observed in 38% of the confirmed COVID-19 patients and high FMS viral loads (>10,000 SARS-COV-2 RNA copies/PVA strip) could be seen in the early stage of infection and in patients with active respiratory symptoms.[16] Moreover, the high FMS viral load was associated with high ISARIC mortality and deterioration scores. The study highlighted the potential of FMS in understanding SARS-COV-2 infectivity and in identifying patients who are at risk of developing severe disease.[16] A follow-up study carried out during the alpha wave of infection in the UK demonstrated the potential of FMS in evaluating longitudinal patterns of naturally exhaled SARS-CoV-2 RNA in COVID-19 patients and in understanding the relationship between exhaled SARS-CoV-2 output and household transmission rates.[55] The study findings showed that exhaled RNA viral load peaked during the early stage of infection (first five days) and 75% household transmission was seen only in patients with high FMS viral load, with the strongest association seen between exhaled viral RNA and household transmission on the 3rd day of infection.[55] The logistic regression model for household transmission used in the study highlighted that for every logarithmic increase in peak exhaled viral load in an infected individual the probability of household transmission increased from 5 to 20-fold.[55]

  Modified N-95 Mask Sampling in Tuberculosis and Coronavirus Disease 2019 Top

Studies on aerosol sampling in TB patients with facemasks utilized microbial DNA for detecting the TB bacilli. However, both live and dead bacteria contribute to the total DNA estimated in any molecular assay, whereas microbial RNA is indicative of only live bacilli.[56] In line with this and the preliminary studies conducted by Williams et al., our group checked the feasibility of detecting Mtb RNA in aerosols captured on face masks.[57] We also evaluated two types of mask sampling techniques: (1) standard surgical masks, worn by patients for 1 h and 3 h during their daily routine with intermittent coughing into the mask, and (2) N95 masks with attached cellulose acetate (CA) membrane, with a sampling time of 5–10 min, wherein patients were instructed to perform specific vocal maneuvers such as reading, talking, coughing, and tidal breathing.[57] Post sampling, face masks were transferred into RNAzol solution, processed for RNA isolation followed by detection of TB bacilli, using a quantitative reverse transcriptase (qRT-PCR) examining the expression of Mtb-specific genes (16S, rpoB, sigA, fgd, ppsD, Rv1687c). We observed no significant differences in RNA yields from mask sampling for 1 h and 3 h, and the expression of Mtb-specific genes in RNA isolated from mask samples confirmed that Mtb-specific RNA could be detected in 80% of the samples.[57] However, the use of surgical mask for sampling due to longer collection times had had low acceptability among patients, led to RNA degradation and had low expression levels of Mtb-specific genes. In comparison, a 7.6-fold higher RNA output was recovered from the CA membrane, and expression of Mtb-specific genes could be detected in all confirmed positive samples.[57] Mtb RNA was also detected in mask samples of patients with unproductive sputum, suggesting the potential of this method in diagnosing TB in patients who find it difficult to produce sputum. Eventually, we replaced the CA membrane in the N95 mask with a gelatin filter, as it had the highest viral/bacterial capture efficiency (99.99%) and did not give out any background RNA reads. This updated design was used in all our subsequent studies for the detection of TB and COVID-19.

Further to establishing the feasibility of detecting the bacteria using adapted N95 mask sampling, we evaluated the diagnostic accuracy of the method in a patient cohort of 55 adult pulmonary TB suspects or GeneXpert-positive patients. Our mask sampling and detection method were able to accurately detect Mtb RNA in 96% of confirmed TB cases, which included 10% of patients who had an unproductive cough (unpublished observations). Considering the mask sampling method's ability to detect TB in persons who cannot produce sputum, we tested the potential of using the N95-mask sampling approach in confirmed pediatric TB patients and performed a head-to-head comparison of GeneXpert and RT-PCR in mask samples taken consecutively from the same patients. Our findings show that the detection rates with mask sampling were comparable to detection with routinely used GeneXpert testing in sputum or gastric lavage samples indicating N95-mask samplings' diagnostic value in children (unpublished observation). We have also shown the potential of mask sampling in studying transcriptomic changes in the aerosolized TB bacilli. Combining mask sampling with RNA sequencing has provided us insight into changes in aerosolized bacteria expelled by patients before and after TB treatment initiation and identify critical genes that were dysregulated in aerosolized Mtb when the patient received effective treatment.[58]

We have also used the noninvasive N95-mask sampling method to detect and quantify SARS-CoV-2 RNA expelled by COVID-19 patients and shown the potential of mask sampling in assessing the infectivity of COVID-19 patients and understanding individual transmission risk.[59],[60] In our study, undertaken during the first wave of ancestral SARS-CoV-2 strain in Mumbai, using mask sampling we were able to detect SARS-CoV-2 in 41.9% of patients. On quantifying the exhaled RNA copies, patients could be grouped into low emitters (expelling <100 viral copies) and high emitters (expelling >1000 viral copies), reflecting differences in infectivity and transmission risk among COVID-19 patients.[59] Subsequently, during the second wave of COVID infection dominated by the Delta strain, we used the N95-mask sampling method to demonstrate the impact of circulating SARS-CoV-2 variants and vaccination on the viral load expelled/exhaled by COVID-19 patients. Through this method, we demonstrated (1) increased expulsion of viral RNA copies by patients infected with Delta strain, (2) a higher proportion of high viral RNA emitters during Delta dominated infection wave, (3) a small subset of patients continued to be high emitters even during the late stage of infection, and (4) importantly no high emitters were observed among vaccinated patients with high neutralizing antibody capacity and evidence of past infection.[60] Our findings indicated mask sampling could be a useful tool for understanding the potential of future vaccine candidates, therapeutics or interventions for their ability to block transmission.[60]

Collectively mask-based aerosol sampling studies have demonstrated, i.e., a non-invasive, simple, easy to use and replicate technique, which can be adapted in clinical settings worldwide and at the point of care.[16],[18],[53],[57],[59],[60] Although mask sampling is an efficient technique, there is very little known about the role played by mask assembly design, amount of sample collected, different types of vocal maneuvers, sample processing in the detection of bacterial/viral burden. Few aspects that have not been much explored in mask sampling include the use of genomic sequencing to analyze the bacterial/viral strain of aerosolized pathogen and the possibility of culturing pathogen directly from the mask. However, a recent study has reported the culturability of SARS-CoV-2 captured on face masks.[61]

In addition to the methods mentioned above, [Table 1] provides details of studies conducted by various research groups in capturing infectious aerosols exhaled by TB and COVID-19 patients. From the sampling devices mentioned in the table, G-II system has been effectively used for the detection of patient virus shedding rates during Alpha, Delta, and Omicron waves,[66],[67] whereas micro silicon-based handheld device for bioaerosol capture and SARS-CoV-2 detection is being developed by Imec for commercial use.[68]
Table 1: Additional methods for detection of exhaled aerosols in tuberculosis and coronavirus disease 2019

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  Conclusion and Future Prospects Top

Bioaerosol sampling has utility in both research and clinical settings. From the research point of view, capturing and studying patient-generated bioaerosols could provide insight into the physiological changes that occur in the pathogen due to aerosolization or in response to treatment. Using mask sampling, we were able to study the changes that occurred in aerosolized Mtb transcriptome in response to treatment.[58] Similarly, studies can explore differences in transcriptome of aerosolized culturable versus nonculturable bacilli. An important next step would be to better understand the interactions of the TB bacilli and SARS-CoV-2 virus with each other and with other microorganisms in the host, and how it impacts the transmission of the disease. Future studies coupling bioaerosol sampling with metagenomic analysis to describe an exhaled microbiome can help shed light on this aspect.

Most of the above-reviewed tools and related studies have highlighted that sampling patient-generated bioaerosols might prove more useful in identifying the infectious potential of the patient and transmission risk than traditional upper respiratory sampling.[11],[12],[16],[18],[42],[53],[57],[59] Thus, in a clinical setting, sampling and quantifying of patient bioaerosol incorporated as point-of-care test could aid in the identification of the most infectious patients allowing for more rational, cost-effective use of resources for infection control. If risk of disease infection is modulated by large inhaled dose of infected inoculum,[11] then bioaerosol sampling could be a useful tool in triaging contacts based on their exposure which may help in better guiding the implementation of preventive treatments and monitoring framework. COVID-19 studies have shown that the analysis of infectious bioaerosol output helps understand the potential transmission risk of circulating and new variants/strains,[16],[59],[60] thus, it can be applied during clinical trials to test the effectiveness of interventions, multivalent vaccines, and treatment strategies aiming to suppress transmission.

Bioaerosol sampling using CASS and mask-based methods have demonstrated their potential in detecting TB in patients who do not expectorate sputum[12],[18],[42],[53],[57] and thus could become a very important diagnostic tool for TB detection in populations where sputum collection is challenging, especially children, who often must undergo invasive, less sensitive procedures for sampling. Moreover, mask sampling has shown its ability to detect Mtb and SARS-CoV-2 earlier than sputum and NPS swab,[15],[16] respectively, making it an attractive tool to understand the role played by asymptomatic TB and COVID-19 patients in community transmission. A challenge in using respiratory aerosol analysis as a diagnostic tool is the small amount of pathogenic material contained in the aerosols, leading to difficulty in detecting diseases in patients with low pathogen burden. Thus, future work could concentrate on developing more sensitive detection techniques that can be coupled with aerosol sampling.

Finally, the extensive application of bioaerosol sampling in both TB and COVID-19 highlights the tool's obvious potential for the study of other respiratory pathogens. With regards to this the use of mask sampling for the detection of NTM, Pneumocystis, Streptococcus, and Influenza is being investigated.[15],[69],[70] This also opens up the avenue for developing kits using bioaerosol sampling for simultaneous detection of respiratory pathogens from a single sample. Other than respiratory pathogens, a recent study has also shown the capability of mask sampling to detect antimicrobial resistance genes in exhaled breath of cystic fibrosis patients, making it one of the first studies to indicate the possibility of the spread of AMR through the aerosol route.[71]

Among the bioaerosol sampling techniques reviewed here, mask-based sampling – extensively studied by us and Williams et al.[15],[16],[18],[53],[55],[57],[58],[59],[60] – has certain advantages compared to other direct sampling methods with its major strength being compatibility with clinical practice, no requirement of trained staff, faster detection rates when combined with qRT-PCR and the potential to use it in resource-limited settings. Further development of such sampling techniques coupled with sensitive, rapid detection assays will lay path for patient-generated infectious aerosols to become routine specimens for diagnosis of respiratory infections and assessing patient infectivity.

Financial support and sponsorship

The studies on the use of mask sampling for TB and COVID-19 conducted at the Foundation for Medical Research were supported by India Health Fund (TB), individual donations from members of Harvard Business School Alumni Club of India (COVID-19), and general donations to the Foundation for Medical Research (TB and COVID-19).

Conflicts of interest

There are no conflicts of interest.

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