Sensitivity optimisation of tuberculosis bioaerosol sampling.

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 wet-walled 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 CO2 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 (IQR: 0.05-0.10) for RASC-1 (p<0.0001). The detection yield of viable Mtb improved from 43% (median 2 CFU, range: 1-14) to 95% (median 20.5 DMN-Trehalose positive bacilli, range: 2-155). These improvements represent a lowering of the limit of detection in the RASC-2 platform to 0.9 Mtb bacilli per 100L of exhaled air from 3.3 Mtb bacilli per 100L (RASC-1).
CONCLUSION: This study demonstrates that technical improvements in particle collection together with sensitive detection enable rapid quantitation of viable Mtb in bioaerosols of sputum positive TB cases. Increased sampling sensitivity may allow future TB transmission studies to be extended to sputum-negative and subclinical individuals, and suggests the potential utility of bioaerosol measurement for rapid intervention in other airborne infectious diseases.

Introduction

Tuberculosis (TB) remains a serious global public health threat [1]. In 2018, the global incidence was estimated at 10 million with a death toll of 1.5 million [2]. Driving the high burden is on-going transmission of infection, as demonstrated by the 1.1 million cases of childhood TB disease globally. Further evidence for the prominence of transmission comes from molecular epidemiology studies in high-prevalence countries which have highlighted that the majority of TB disease follows from recent Mtb infection [3].

Transmission events are influenced by many factors which relate to the source case, the bacillus, the environment and the host. Fundamental to transmission, however, is the production of aerosolized Mtb: in the absence of aerosol release, no transfer of infectious organisms between individuals can occur. Pioneering experiments performed by Wells and Riley more than 60 years ago utilized the guinea pig as an aerosol sampler, detecting transmission by the demonstration of new infection in the animals [4,5]. Employing a sampling system with physical separation of patients (source case) and guinea pigs (sampler) led to the confirmation of airborne transmission. Moreover, intricate linking of individual patients to specific guinea pig infections by bacillary resistance patterns and timing of entry and departure from the ward allowed for identification of a minority of highly infectious ‘super-spreaders’. The guinea pig model therefore supported the concepts that infectiousness is highly heterogenous and that small bioaerosol–which arise as a consequence of dehydration of buoyant airborne droplets during “aging” following release into the environment–are primarily responsible for remote transmission [6].

Invaluable though this contribution was to the early understanding of TB transmission, the experimental design was not optimised for sensitive sampling [7]. Minimal sensitivity to Mtb production necessitates sampling multiple patients for days to months for both historical and recent in vivo sampling experiments [8-10]. However, some modern approaches with greater proximity between source case and sampler, single patient sampling and viable organism detection have dramatically improved the overall detection sensitivity. Direct detection has identified Mtb bacilli within only minutes to hours of sampling [11-13], generated individual-level information, and found a greater proportion of emitters.

These sensitive techniques have the potential to efficiently answer questions regarding the transmission potential of patient sub-groups and test strategies for transmission mitigation. Further sensitivity improvement may allow investigation of Mtb-containing droplet release from patients across the TB disease spectrum, including sputum smear-negative cases, HIV-coinfected and even subclinical TB cases. Sensitive sampling combined with quantitative forms of detection also provide an infectivity measure. Such a measure could be applied longitudinally to those receiving chemotherapy to more accurately establish time to bioaerosol sterilisation with direct clinical application.

We describe a direct bioaerosol sampling device designed to collect aged aerosol. We have attempted to optimise key components necessary to maximise detection yield through an iterative re-design process. Modifications to both the sampling process and the method of organism detection are described with the results of sampling from both healthy controls and individuals with newly diagnosed pulmonary TB presented to evaluate the improvements.

Materials and methods

The Respiratory Aerosol Sampling Chamber (RASC) is an enclosed, clean space optimised for the sampling of aged bioaerosol from a single individual during a 60-minute period of natural respiratory activity. A previous description of the RASC design, “RASC-1”, has been published [14] in addition to results from a 35-patient pulmonary TB sampling study [11]. Briefly, the RASC is a 1.4m3 chamber in which participants can comfortably sit throughout the study period. The chamber is first sealed and then an air purge phase is performed by drawing external air across HEPA filters for a 10-minute period. The next phase is passive contamination with respiratory bioaerosol as the participant respires. A sampling phase then occurs with various devices drawing volumes of air out of the chamber and extracting the airborne bioaerosol. Finally, a second 10-minute purge phase is performed to remove residual Mtb bioaerosol.

The apparatus and sampling protocols employed for those initial studies is referred to throughout this report as “RASC-1”. The current study describes several design modifications, informed by an iterative series of experiments, which have refined the apparatus and sampling protocol while maintaining the basic structure of the chamber. This new version is referred to as “RASC-2”.

Table 1 summarises the improvements in the sampling and detection systems which are further explained in the following text.

Table 1

Comparison of the features modified from RASC-1 to RASC-2.

Optimisation Parameter	RASC-1	RASC-2	Advantage
Sampler	Primarily the Andersen impactor	Cyclone collector (Coriolis μ air sampler)	Liquid collection may improve in recovery of live Mtb bacilli
Sampling Rate and Time Period (see Fig 1)	28L/min for 10 minutes	250L/min for 60 minutes	Increased exhaled air volume (estimated by CO2. See Fig 2A.)
			Increased bioaerosol content (estimated by aerodynamic particle counting. See Fig 2B).
			Improved regulation of humidity and temperature
Detection Method	Mtb Culture (Middlebrook 7H10 Solid Agar)	DMN-trehalose dye	More rapid result
			Retains high sensitivity (see Fig 4) Indication of viability via requirement for metabolic activity for DMN-tre dye uptake

Sample collection

RASC-1 included an array of aerosol sample collection instruments operated consecutively at the peak of chamber air contamination during the sampling phase. This included polycarbonate, gelatin and felt filters, Dekati and Andersen impactors, and a cyclone collector. The flow rates (chamber outflow) varied between instruments, subject to device specifications. Both impactors and the polycarbonate and gelatin filters sampled with flow rates of 20–30 L/min, whereas the felt filter and the cyclone collector sampled with higher flow rates: 300 and 250 L/min, respectively. Results yielded no positive samples from the felt or gelatin filters. For the rest, no sampling instruments demonstrated significantly higher yield per unit of exhaled air sampled. Therefore, to maximize the exhaled air volume sampled, the sample collection process was simplified to a single cyclone collector for RASC-2 (described below).

Cyclone collector

In RASC-2 a high-efficiency cyclone collector (Coriolis μ air sampler, Bertin, Montigny le Bretonneux, France) was used to collect airborne bioaerosol into 5–10 mL of sterile phosphate-buffered saline (PBS) solution. Mounted within the chamber, the air enters the conical collector through a tangential nozzle. The high flow rate of 250 L/min generates a liquid cyclone and particle inertia leads to deposition of bioaerosol from the airstream onto the wet wall with a high collection efficiency. Collection into liquid has the advantage of minimizing particle bounce, improving the recovery of viable Mtb bacilli and maximizing the air volume sampled over time. (see appendix for schematic diagrams of RASC-1 (modified from ref 14.) and RASC-2).

Sampling protocol

The experimental protocol was modified to continuous sampling of the chamber air via the single high-flow (250 L/min) cyclone collector. This replaces the sequential sampling phases of the RASC-1 protocol. Consequently, there is a lowering of the CO2 set-point throughout the protocol and improved regulation of chamber humidity and temperature, illustrated by the CO2 traces in Fig 1. This maximizes the air volume sampled by reducing the loss of exhaled air at the end of the study protocol. There is an added benefit of improved thermal comfort for participants.

Fig 1

Differing example CO2 traces for the two sampling protocols (A) RASC-1: Initial contamination and short, steady-state sampling (median 3800 PPM CO2). (B) RASC-2: Continuous sampling with a lower steady-state (median 1600 PPM CO2).

Mtb detection

Liquid specimen collection facilitates sample concentration via centrifugation and the application of culture-independent detection techniques. For the RASC-2 protocol, the centrifuged bioaerosol sample pellet was incubated with a novel solvatochromic trehalose analog, DMN-Trehalose, which was incorporated into the cell walls of metabolically active mycobacteria, and subsequently yielding high fluorescence intensity detectable by a fluorescence microscope [15]. 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.

CO2 and bioaerosol monitoring

Continuous CO2 and aerodynamic particle monitoring of the chamber air was performed in both sampling protocols, RASC-1 and RASC-2. Estimates of captured Mtb bacilli per unit volume of exhaled air and per unit volume of respiratory bioaerosol were calculated using the method described in the previous (RASC-1) study [12].

Study participants

All recruited participants were residents from two informal peri-urban settlements outside Cape Town and in close proximity to the study site. Eligible candidates were those with GeneXpert-positive sputum without evidence of drug resistance. Bioaerosol sampling was performed before initiation of anti-tuberculous therapy, as per the approved Ethics protocol (see below). The empty chamber was sampled between human participants to serve as chamber decontamination controls. 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.

Statistical analysis

The performance of the RASC-2 protocol was compared with the original RASC-1 design in terms of the exhaled air volume and bioaerosol volume sampled and Mtb bacilli captured. Comparison between the protocols was made with a Wilcoxon rank sum test. Statistical analyses were performed using R Core Team (2019).

Ethical statement

The patient studies are covered by two separate ethics approvals from the University of Cape Town Faculty of Health Sciences Human Research Ethics Committee (HREC/REF: 680/2013; HREC/REF: 529/2019). Each patient provided written informed consent prior to participation in the study, including consent to publication of the clinical and demographic details.

Results

Patient characteristics

Individuals were consecutively recruited from the same two communities. The RASC-1 protocol was implemented over the time period 2015 to 2017 and RASC-2 from 2018 to 2019. Table 2 compares basic clinical and demographic characteristics, confirming broad equivalence between patients sampled by the two protocols.

Table 2

Baseline demographics and clinical characteristics of RASC-1 protocol and RASC-2 protocol.

	RASC -1	RASC -2	p-value
N	35	21
Age
median [IQR]	32[25,39]	35[31,41]	0.23
Sex
Male (%)	20 (57.1)	14 (77.8)	0.24
HIV Status
Positive (%)	17 (48.6)	6 (28.6)	0.23
CD4+ Count
median [IQR]	122[63,417]	90[49,146]	0.51
Sputum GeneXpert
Positive (%)	35 (100)	21 (100)	N/A
Previous TB
Yes (%)	11 (31.4)	5 (23.8)	0.76
Cavitation*
Present (%)	13 (39.4)	6 (46.2)	0.93

*Chest radiograph performed in 33 patients in RASC-1 and in 12 patients from the RASC-2 group

RASC comparison

The two RASC designs were compared using indicators of sampling efficiency (Fig 2A and 2B) and numbers of Mtb bacilli captured (Fig 2C). The sampled volume of exhaled air was calculated using continuous CO2 measurement to determine exhaled air proportion in chamber air during device sampling. Similarly, chamber bioaerosol concentration was determined by particle counting and volume calculation with the assumption that bioaerosol are spherical. Captured bioaerosol volume was estimated by assuming completely efficient sampling of this concentration. Both these methods have been described previously [11].

Fig 2

Comparative efficiencies of RASC-1 versus RASC-2 in capture of exhaled air (A), collection of bioaerosol volume (B), and capture of Mtb organisms (C).

Note that numbers in (C) were determined by colony forming units (RASC-1) and DMN-Trehalose-positive organisms identified using fluorescence microscopy (RASC-2).

RASC decontamination

As mentioned previously [14], the highly sensitive Mtb detection methodology used in RASC-2 is vulnerable to contamination owing to incomplete sterilization of the chamber. To control for this risk, patient sampling was interspersed with empty RASC sampling. Detected organisms in the control samples suggested a small degree of contamination. Fig 3 demonstrates the different bacillary counts for patient sampling and empty RASC sampling. Control samples remained negative after modification of the collector design and high concentration ozone sterilization of the RASC.

Fig 3

Comparison of total bacillary counts following RASC-2 sampling of sputum GeneXpert-positive TB patients (n = 21) and the inter-patient empty booth RASC sampling controls (n = 22).

P<0.0001.

Limit of detection

A bacillary count per unit volume of exhaled air was calculated and compared between the two sampling protocols. Fig 4 illustrates the range of detectable concentrations across the sampled populations. Notably, by combining the design improvements, the limit of detection has been lowered from approximately 3.3 bacilli per 100 L exhaled air to 0.9 bacillus per 100 L.

Fig 4

Mtb bacillary counts per 100 L exhaled air for RASC-1 versus RASC-2.

Sampled individuals not yielding detectable Mtb bacilli are illustrated by points on the x-axis.

Discussion

This study has shown that, by optimising both bioaerosol collection and Mtb detection, a majority of newly diagnosed GeneXpert sputum positive TB patients can be shown to exhale viable Mtb organisms. The increased numbers of Mtb organisms captured by RASC-2 compared with RASC-1 were associated with an approximate 1 log10 increase in exhaled air sampled and bioaerosol particle volume collected. This is not explained by demographic and clinical characteristics which were broadly equivalent between the patients sampled by the different methods. The resulting lowered limit of detection enabled identification of putative Mtb organisms in 95% of patients whose bacillary concentrations varied over a 2 log10 range from 1 to 100 per 100 L exhaled air. The proportion of patients identified with Mtb positive bioaerosols is therefore a function of lower level of detection. However, exhaled particle volumes varied markedly between individuals, suggesting that infectivity is likely to be impacted by both Mtb concentration of lung fluids and individual bioaerosol particle production.

The current study sampled newly diagnosed TB patients while seated in a small personalized clean room with normal respiration and spontaneous coughing over 60-minutes. Collected bioaerosol were those which remained airborne in the RASC and, at time of collection, were predominantly in the 1–5 μm diameter range. Consistent with prior observations [6], these are the particles thought most likely to reach the peripheral lungs of susceptible individuals sharing an indoor location [17]. It is also probable that these bioaerosols containing viable Mtb organisms might be indicative of a wide range of source infectivity. The investigation of Mtb production and, consequently, potential infectivity during various respiratory manoeuvres is under current investigation in our laboratory.

The sensitive detection of individual Mtb organisms, with DMN-Trehalose staining and subsequent fluorescence microscopy analysis, requires a high degree of sterility to exclude contamination during sampling and isolation. After detailed analysis of the platform and collection equipment, we noted that the wet-walled cyclone contained a dead space which was a potential reservoir for live Mtb cells. Therefore, the device was modified to enable dismantling and autoclaving. The very low numbers of Mtb organisms detected during empty booth sampling could also have resulted from the release of trapped Mtb organisms by the negative pressure inducing retrograde airflow across the exit HEPA filter. Reassuringly, following modification of the collector and the application of combined dry vapour hydrogen peroxide and ozone treatments of the RASC after use, the empty booth controls remained negative. A limitation of this study is that modifications to the sampling protocol and the detection method were evaluated in combination. It is therefore not possible to delineate the extent to which individual improvements impact sensitivity.

Conclusion

The development of a sensitive and reproducible measure of Mtb in exhaled bioaerosols has enabled the demonstration of a wide range of potential infectivity of newly diagnosed TB patients. Bioaerosol assays may offer a useful adjunct to TB transmission studies, enabling demonstration of infectivity of sputum negative individuals and the effect of specific chemotherapies on infectivity. Moreover, the protocols and equipment described here suggest the potential application of this technology to other airborne infectious diseases, especially where rapid interventions such as containment and quarantine are required to curb outbreaks [18].

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11 Jun 2020

PONE-D-20-11317

Sensitivity Optimisation of Tuberculosis Bioaerosol Sampling

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Reviewer #1: The authors describe some changes to an aerosol sampling procedure that appears to improve the sensitivity in detecting sputum-positive TB patients who exhale infectious aerosols. There are some serious problems with the manuscript.

1) The paper compares data from the current study (RASC-2) with a previous study (RASC-1). Data from RASC-1 have already been published (Ref 14) and are repeated here.

2) The two studies cannot be compared. They were conducted 2-3 years apart with different patient groups. More importantly, two different measurements were used to determine the number of mycobacteria in the sampled aerosols – viable bacilli or cfu (RASC-1) and fluorescence microscopy (RASC-2). The authors provide no evidence that the fluorescently-labeled bacilli in RASC-2 were viable.

3) The authors admit (lines 143-144) that the fluorescence microscopy detection assay for detecting mycobacteria in RASC-2 was subjective and dependent upon the skill of the operator. Please provide some data or references to support the validity of this assay (e.g., repeated determinations from the same sample).

4) The overall conclusion of the study is trivial and can be summarized in a single sentence – “Increasing the amount of exhaled air sampled will increase the sensitivity of bacillary detection”. This is intuitively obvious to any intelligent person and does not require data or a manuscript to confirm.

5) The authors have grossly over interpreted their results. They speculate wildly about the possible implications of their aerosol sampling results with no supporting data.

6) The authors utilize jargon that is not defined for the reader. What, precisely, is an “aged” aerosol? What is the difference between “exhaled air” and “respiratory bioaerosol”?

Reviewer #2: In the present study, the authors have presented the method to sample bioaerosol from TB patients in order to determine the infectiousness. The method they described is an improvement of their previous work. However, I find following concerns,

1. Data for TB cases has been given for year 2018, instead of 2019.

2. The technical improvements in the design of RASC has not been elaborately explained. The focus has more been put on the collection outputs of aerosols in results section. I think, it will be better to present collection output in relation to modifications in the design of RASC.

3. In the method section, superficial information has been given. They should describe all the modifications in detail.

4. In the sample collection section, it is difficult to understand the technicalities. The authors should present these elaborately with proper diagrams and flowcharts.

5. In fig3, statistical significance and p value are missing.

6. Figure legends are not self explanatory, more details are required.

Reviewer #3: In the present study, authors employed modified version of Respiratory Aerosol Sampling Chamber (RASC) to incorporate liquid collection of bioaerosol using a high-flow wet-walled cyclone (RASC-2). Authors demonstrated this technical modification increased the utility of bioaerosol sampling and lowered the limit of Mtb detection in RASC-2 platform. Study result is interesting and has future application in characterizing exhaled air and bioaerosol from sputum negative and subclinical individuals. Authors have to address the following comments.

Major Comments

1. Authors estimated the Mtb count per 100 liters of exhaled air as Mtb-CFU for RASC and DMN-Tre stained Mtb bacilli. Since authors employed two different methodology to estimate the lower limit of detection, study result may vary. Please clarify this discrepancy in methodology.

2. This includes patients with and without cavitary TB. Earlier studies have shown that pulmonary TB patients with lung cavitation are the main source of disease transmission. Did you analyze the data such as Mtb count and lower limit of Mtb detection between the patients with and without cavitary TB in both RASC-1 and RASC-2.

3. Is it possible to study the nature of Mtb such as Mtb in the form of singles, small and large clumps in exhaled air and bioaerosol?

4. There is typo in the table 1; the number of patients with lung cavitation in RASC-2 is 6 (28.6%) not 46.2%. Correct the typo.

Reviewer #4: Authors have tried to improvise on a previously established protocol for aerosol sampling from the patients for possible bacterial count. The sensitivity can improvise the testing capability for possible MTB clearance.

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2 Jul 2020

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Reviewers' comments:

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Reviewer #2: Partly

Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #1: I Don't Know

Reviewer #2: No

Reviewer #3: Yes

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Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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5. Review Comments to the Author

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Reviewer #1: The authors describe some changes to an aerosol sampling procedure that appears to improve the sensitivity in detecting sputum-positive TB patients who exhale infectious aerosols. There are some serious problems with the manuscript.

Thank you for your review. The points raised are addressed below:

1) The paper compares data from the current study (RASC-2) with a previous study (RASC-1). Data from RASC-1 have already been published (Ref 14) and are repeated here.

This is true as we make clear by referencing the previous study. To our knowledge this does not contravene the ICMJE recommendations as a new research question is being addressed.

2) The two studies cannot be compared. They were conducted 2-3 years apart with different patient groups.

We have compared baseline characteristics between the two groups of patients and found no significant differences. We are not aware of a biologically plausible reason why there would be a change in Mtb bioaerosol production in TB patients over time between the two sampling periods.

More importantly, two different measurements were used to determine the number of mycobacteria in the sampled aerosols – viable bacilli or cfu (RASC-1) and fluorescence microscopy (RASC-2).

The purpose of this study is to demonstrate the improvement in multiple aspects of the bioaerosol sampling methodology including both the sampling process and the method of organism detection. The following sentence in the final paragraph of the introduction has been amended to clarify this:

“Modifications to both the sampling process and the method of organism detection are described with the results of sampling from both healthy controls and individuals with newly diagnosed pulmonary TB presented to evaluate the improvements.”

A caveat has been added to the discussion section.

“A limitation of this study is that modifications to the sampling protocol and the detection method were evaluated in combination. It is therefore not possible to delineate the extent to which individual improvements impact sensitivity.”

The authors provide no evidence that the fluorescently-labeled bacilli in RASC-2 were viable.

The evidence for viability in fluorescently-labeled bacilli is referred to in ref 15. It’s beyond the scope of this study to re-examine this established dye characteristic.

3) The authors admit (lines 143-144) that the fluorescence microscopy detection assay for detecting mycobacteria in RASC-2 was subjective and dependent upon the skill of the operator. Please provide some data or references to support the validity of this assay (e.g., repeated determinations from the same sample).

Indeed as with auramine staining in clinical practice there is a degree of operator dependence with any fluorescence microscopy detection. However, this does not invalidate the usefulness of this assay.

4) The overall conclusion of the study is trivial and can be summarized in a single sentence – “Increasing the amount of exhaled air sampled will increase the sensitivity of bacillary detection”. This is intuitively obvious to any intelligent person and does not require data or a manuscript to confirm.

We strongly disagree with this overly reductive assessment. Successfully identifying Mtb bacilli in bioaerosol is far from straight-forward and the various sampling methodologies described in the literature vary in the techniques employed and the detection yields as we have highlighted in the introduction. One aspect of the improved sensitivity is the increase in exhaled air volume sampled which we have quantified using CO2 measurements – a technique not used by other groups and not previously discussed as an explanation for low yield. Furthermore, we also describe other critical modifications such as the use of the DMN-trehalose dye for bacillus detection. This is an entirely novel approach with additional benefit in terms of a rapid time to result and assessment of organism viability. If this is a trivial we would invite the reviewer to direct us to a previous aerosol sampling study with a near 100% detection yield.

5) The authors have grossly over interpreted their results. They speculate wildly about the possible implications of their aerosol sampling results with no supporting data.

It is not clear to us what the speculative implications are to which the reviewer is referring. The only speculative statement in the discussion/conclusion is the following:

“Bioaerosol assays may offer a useful adjunct to TB transmission studies, enabling demonstration of infectivity of sputum negative individuals and the effect of specific chemotherapies on infectivity”

This is clearly not a strong (or wild) assertion but offers an indication to the reader of what the future value of this work may be. As is typical for concluding remarks, data supporting these concepts will be presented in forthcoming publications and is beyond the scope of the current paper.

6) The authors utilize jargon that is not defined for the reader. What, precisely, is an “aged” aerosol?

The merriam-webster dictionary defines jargon as “obscure and often pretentious language marked by circumlocutions and long words”. This is not an accurate description of the term highlighted. Furthermore the intended meaning of the word “aged” is made clear in the introduction (lines 84-87)

“The guinea pig model therefore supported the concepts that infectiousness is highly heterogenous and that small bioaerosol – which arise as a consequence of dehydration of buoyant airborne droplets during “aging” following release into the environment –are primarily responsible for remote transmission”

What is the difference between “exhaled air” and “respiratory bioaerosol”?

These are basic aerobiology concepts. Respiratory bioaerosol are liquid droplets suspended in exhaled air.

Reviewer #2: In the present study, the authors have presented the method to sample bioaerosol from TB patients in order to determine the infectiousness. The method they described is an improvement of their previous work. However, I find following concerns,

Thank you for your thoughtful review. The points raised are addressed below:

1. Data for TB cases has been given for year 2018, instead of 2019.

We are not aware that global statistics have been published by the WHO with data from 2019 yet.

2. The technical improvements in the design of RASC has not been elaborately explained. The focus has more been put on the collection outputs of aerosols in results section. I think, it will be better to present collection output in relation to modifications in the design of RASC.

We are comparing the sensitivity improvement following a package of improvements in the entire sampling and detection process i.e. comparing before and after.

“Modifications to both the sampling process and the method of organism detection are described with the results of sampling from both healthy controls and individuals with newly diagnosed pulmonary TB presented to evaluate the improvements.”

A caveat has been added to the discussion section.

“A limitation of this study is that modifications to the sampling protocol and the detection method were evaluated in combination. It is therefore not possible to delineate the extent to which individual improvements impact sensitivity.”

3. In the method section, superficial information has been given. They should describe all the modifications in detail.

Additional description has been added to the methods to

“Briefly, the RASC is a 1.4m3 chamber in which participants can comfortably sit throughout the study period. The chamber is first sealed and then an air purge phase is performed by drawing external air across HEPA filters for a 10-minute period. The next phase is passive contamination with respiratory bioaerosol as the participant respires. A sampling phase then occurs with various devices drawing volumes of air out of the chamber and extracting the airborne bioaerosol. Finally, a second 10-minute purge phase is performed to remove residual Mtb bioaerosol.”

4. In the sample collection section, it is difficult to understand the technicalities. The authors should present these elaborately with proper diagrams and flowcharts.

An original schematic diagram of RASC-1 and a new schematic diagram of RASC-2 have been added as an appendix (referred to on line 160)

5. In fig3, statistical significance and p value are missing.

p-value has been added with the figure caption

“Fig 3. Comparison of total bacillary counts following RASC-2 sampling of sputum GeneXpert-positive TB patients (n=21) and the inter-patient empty booth RASC sampling controls (n=22). P<0.0001.”

6. Figure legends are not self explanatory, more details are required.

The figure legends have all been expanded

Reviewer #3: In the present study, authors employed modified version of Respiratory Aerosol Sampling Chamber (RASC) to incorporate liquid collection of bioaerosol using a high-flow wet-walled cyclone (RASC-2). Authors demonstrated this technical modification increased the utility of bioaerosol sampling and lowered the limit of Mtb detection in RASC-2 platform. Study result is interesting and has future application in characterizing exhaled air and bioaerosol from sputum negative and subclinical individuals. Authors have to address the following comments.

Thank you for your thoughtful and thought-provoking review. The points raised are addressed below:

Major Comments

1. Authors estimated the Mtb count per 100 liters of exhaled air as Mtb-CFU for RASC and DMN-Tre stained Mtb bacilli. Since authors employed two different methodology to estimate the lower limit of detection, study result may vary. Please clarify this discrepancy in methodology.

The purpose of this study is to demonstrate the improvement in multiple aspects of the bioaerosol sampling methodology including both the sampling process and the method of organism detection. The following sentence in the final paragraph of the introduction has been amended to clarify this:

“Modifications to both the sampling process and the method of organism detection are described with the results of sampling from both healthy controls and individuals with newly diagnosed pulmonary TB presented to evaluate the improvements.”

A caveat has been added to the discussion section.

“A limitation of this study is that modifications to the sampling protocol and the detection method were evaluated in combination. It is therefore not possible to delineate the extent to which individual improvements impact sensitivity.”

2. This includes patients with and without cavitary TB. Earlier studies have shown that pulmonary TB patients with lung cavitation are the main source of disease transmission. Did you analyze the data such as Mtb count and lower limit of Mtb detection between the patients with and without cavitary TB in both RASC-1 and RASC-2.

This is an interesting question and one which was explored in the RASC-1 study where we found no association between cavitary disease and presence of culturable aerosol. However, we feel that in the current study the number of patients is too few to further address this question and would not wish to over-interpret our results. The potential to explore the aerosol output from cavitary and non-cavitary TB does highlight the utility of our methodology and the importance of optimizing sensitivity.

3. Is it possible to study the nature of Mtb such as Mtb in the form of singles, small and large clumps in exhaled air and bioaerosol?

Again an interesting suggestion, however, we suspect this is a drawback of the cyclone sampler which may disrupt clumps of bacilli through physical agitation during sample collection.

4. There is typo in the table 1; the number of patients with lung cavitation in RASC-2 is 6 (28.6%) not 46.2%. Correct the typo.

The percentage is correct since not all had a CXR this was only the case in 13 patients. The table has been amended to make this clear.

Reviewer #4: Authors have tried to improvise on a previously established protocol for aerosol sampling from the patients for possible bacterial count. The sensitivity can improvise the testing capability for possible MTB clearance.

Thank you for your comments

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

Submitted filename: ResponseToReviewers.docx

Click here for additional data file.

21 Jul 2020

PONE-D-20-11317R1

Sensitivity Optimisation of Tuberculosis Bioaerosol Sampling

PLOS ONE

Dear Dr. Patterson,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

ACADEMIC EDITOR: The crux of this paper is that an improvised air sample collection system increased the sensitivity of Mtb detection. As Rev#1 mentioned in previous round of review, this could be done without comparing the two methods, since they cannot be compared head-to-head. As can be seen from the instrumentation picture, these two air collection systems are not comparable. The parameters used to collect samples, the pressure of the unit, and the Mtb detection methods are very different. As the authors noted, since multiple parameters are changed, it is hard to define which parameter contributed to the improved sensitivity. Then, a question is "why to compare these two in the first place ?". Thus, i would suggest the authors to present only the features of the latest model (RASC-2), its design, parameters and performance. The previous publication on the RASC-1 model can be discussed in the "Discussion" section, if/when relevant. Alternatively, if the authors still want to compare these two systems head-to-head, then add a table to explicitly mention all the minute differences between the two units on every parameter, such that the reader would understand what "optimization" happened from RASC-1 to become RASC-2.

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==============================

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Academic Editor

PLOS ONE

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Reviewers' comments:

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Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #4: All comments have been addressed

**********

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Reviewer #2: Yes

Reviewer #4: Yes

**********

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Reviewer #1: (No Response)

Reviewer #2: Yes

Reviewer #4: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #4: Yes

**********

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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #4: Yes

**********

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Reviewer #1: Please see the previous review......................................................................

Reviewer #2: (No Response)

Reviewer #4: (No Response)

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28 Jul 2020

ACADEMIC EDITOR: The crux of this paper is that an improvised air sample collection system increased the sensitivity of Mtb detection. As Rev#1 mentioned in previous round of review, this could be done without comparing the two methods, since they cannot be compared head-to-head. As can be seen from the instrumentation picture, these two air collection systems are not comparable. The parameters used to collect samples, the pressure of the unit, and the Mtb detection methods are very different. As the authors noted, since multiple parameters are changed, it is hard to define which parameter contributed to the improved sensitivity. Then, a question is "why to compare these two in the first place ?". Thus, i would suggest the authors to present only the features of the latest model (RASC-2), its design, parameters and performance. The previous publication on the RASC-1 model can be discussed in the "Discussion" section, if/when relevant. Alternatively, if the authors still want to compare these two systems head-to-head, then add a table to explicitly mention all the minute differences between the two units on every parameter, such that the reader would understand what "optimization" happened from RASC-1 to become RASC-2.

Thank you very much for your continued assessment of this paper.

Researchers have been trying to identify Mtb in aerosols since Chausee in 1913 with very gradual increases in the proportion of TB cases identified with Mtb positive aerosols over the last century. Our comparison of sampling and detection systems increased the proportion of patients with detectable airborne Mtb from ~40% to approaching 100% and highlights some of the specific drivers required to improve sensitivity. The lowered limit of detection is a result of the improvements in the entire system (sampler, sampling volume and detection method). Comparing sampling outputs between the two systems - in the form of exhaled air volume and bioaerosol volume (Fig. 2) – demonstrates the sampling component of this improvement. The organism recovery and detection component is demonstrated by plotting organism count per 100L of exhaled air for each patient with each system (Fig. 4). This shows the range of aerosol concentrations detected and highlights the zero-inflation problem of the less sensitive system.

We therefore feel strongly that the comparison is valid and has explanatory value. For further clarity we have added the a table to the manuscript.

Submitted filename: ResponseToReviewers_R2.docx

Click here for additional data file.

12 Aug 2020

Sensitivity Optimisation of Tuberculosis Bioaerosol Sampling

PONE-D-20-11317R2

Dear Dr. Patterson,

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Kind regards,

Selvakumar Subbian, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

21 Aug 2020

PONE-D-20-11317R2

Sensitivity Optimisation of Tuberculosis Bioaerosol Sampling

Dear Dr. Patterson:

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on behalf of

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PLOS ONE