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BMJ Open
BMJ Open
bmjopen
bmjopen
BMJ Open
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BMJ Publishing Group BMA House, Tavistock Square, London, WC1H 9JR

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10.1136/bmjopen-2024-087026
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Protocol
Diagnostics
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Combination of exhaled breath condensate samples with lipoarabinomannan point of care assay for pulmonary tuberculosis (TB) diagnosis: protocol for a diagnostic accuracy study
http://orcid.org/0000-0002-7875-2291
Pouzol Stephane 1stephane.pouzol@fondation-merieux.org

Khaja Mafij Uddin Mohammad 2kmuddin@icddrb.org

http://orcid.org/0009-0007-9479-2860
Islam Ashabul 2ashabul.islam@icddrb.org

Jabin Maha Sultana 2maha.jabin@icddrb.org

Nigou Jérôme 3jerome.nigou@ipbs.fr

Banu Sayera 2sbanu@icddrb.org

Hoffmann Jonathan 1jonathan.hoffmann@fondation-merieux.org

1 Medical and Scientific Department, Merieux Foundation, Lyon, France
2 Infectious Disease Division, International Centre for Diarrhoeal Disease Research Bangladesh, Dhaka, Dhaka District, Bangladesh
3 Institute of Pharmacology and Structural Biology, Toulouse, Midi-Pyrénées, France
StephanePouzol; stephane.pouzol@fondation-merieux.org
None declared.

2024
16 9 2024
14 9 e08702628 3 2024
04 9 2024
Copyright © Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/.

Abstract

Introduction

The WHO estimates a gap of about 30% between the incident (10.6 million) and notified (7.5 million) cases of tuberculosis (TB). Combined with the growing recognition in prevalence surveys of the high proportion of cases identified who are asymptomatic or paucisymptomatic, these data underscore how current symptom screening approaches and use of diagnostic tests with suboptimal performance on sputum miss large numbers of cases. Thus, the development of sputum-free biomarker-based tests for diagnosis is becoming necessary, which the WHO has already identified as a priority for new TB diagnostics.

The objective of this study is to evaluate a combination of exhaled breath condensate (EBC) samples and mycobacterial lipoarabinomannan (LAM) as point-of-care (POC) assays to identify TB patients.

Methods and analysis

This prospective diagnostic accuracy study is conducted at the TB Screening and Treatment Centre of International Center for Diarrhoeal Disease Research, Bangladesh, on a cohort of adults and adolescents >11 years of age. A total of 614 individuals with presumptive pulmonary TB based on TB signs, symptoms and radiography are being recruited from 28 August 2023. Spot sputum is collected for standard reference testing (L-J culture, GeneXpert MTB/Rif, acid-fast Bacilli microscopy) to fine-tune categorisation of TB disease status for each participant, defined as (1) definite TB (at least one positive standard reference test); (2) probable TB (not microbiologically confirmed but under TB treatment); (3) possible TB (no TB treatment but signs, symptoms and radiography suggestive of TB); (4) other respiratory disease (microbiologically not confirmed and no radiography presenting abnormalities compatible with TB); and (5) unknown (no microbiological evidence with normal/no TB abnormalities with radiography). Urine and EBC specimens will be subjected to LAM POC testing and biobanked for further investigation. Statistical analyses will include an assessment of diagnostic accuracy by constructing receiver operating curves and calculating sensitivity and specificity, as well as post-test probabilities.

Ethics and dissemination

The study protocol was approved by the Research Review Committee as well as the Ethical Review Committee of icddr,b and recorded under a protocol reference number, PR-2301. Results will be submitted to open-access peer-reviewed journals, presented at academic meetings, and shared with national and international policymaking bodies.

Tuberculosis
Diagnostic microbiology
Pulmonary Disease
==== Body
pmcSTRENGTHS AND LIMITATIONS OF THIS STUDY

Exhaled breath condensate (EBC) collection is a noninvasive and painless procedure, making it suitable for all categories of participants and can be easily implemented in routine practice.

EBC contains components from the lining fluid of the lower airways, which is difficult to access directly using other methods. This can provide valuable insights into airway inflammation and other processes.

The rigorous classification of patients and sample size calculation will ensure an estimation of test performance with good precision.

The variation in the volume of EBC collected depends on the individual regardless of the time or volume of exhaled air recorded.

This study does not include children who are one of the main target populations for a non-sputum-based diagnostic test.

Introduction

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a curable and preventable disease that affected 10.6 million individuals and caused 1.3 million deaths worldwide in 2022.1 Among the highest TB burden countries, eight countries account for two-thirds of the global total, and Bangladesh alone accounts for 3.6%, with 379 000 reported cases and 42 000 deaths.

There is growing recognition of the high proportion of cases identified in prevalence surveys who are asymptomatic or presymptomatic (presumed subclinical TB), redefining the landscape of the clinical presentation of the disease and underscoring how large numbers will be missed by current symptom screening approaches.2 Thus, 3.1 million TB cases are ‘missed’ each year by health systems and do not get the TB care they need and deserve. Identifying patients at the early phase of the disease to prevent disease progression and subsequent morbidity and mortality is a considered option of the ‘end TB strategy’.3 However, as these individuals cannot produce sputum, the use of rapid, accurate, noninvasive and sputum-free tests for triage and confirmatory diagnosis is becoming necessary.

In recent years, multiple studies and reviews4 5 have been published to assess the current status of biomarkers that can be used to diagnose active, latent and incipient TB. Many biomarkers were derived from profiling the transcriptional response of host peripheral blood immune cells, such as RISK6 6 7or Xpert HR (Cepheid, USA),8 but the cost and optimal placement in clinical algorithms remain unclear. Human exhaled air contains water vapour, aerosols and trace amounts of organic materials, including volatile organic compounds (VOCs), nonvolatile organic compounds (NOCs) and particulate matter including microbes.9 Breath analysis relies on the premise that infections, including Mtb infection, change the host metabolism, producing distinct VOCs that can be detected through electronic nose.10 NOCs in human exhaled air include metabolites, lipids, glycoconjugates and proteins. These compounds can be sampled by collecting exhaled breath condensate (EBC; ie, the liquid phase of the exhaled air sampled by cooling), containing exhaled water vapour and exhaled breath aerosols (EBA)11 in which NOCs are in solution. Mtb DNA and bacilli detection in EBA studies are promising as identification of patients with subclinical disease was reported, and robust sensitivity (up to 86%) was demonstrated in pulmonary TB patients.12 Moreover, fatty acids, oxidative stress and inflammatory mediator profiles in EBC can differentiate TB-infected adults and children from healthy controls.13

Mycobacterial lipoarabinomannan (LAM), a major cell envelope component of Mtb, has been found in sputum, serum and urine in measurable quantities.14 Tests based on the detection of mycobacterial LAM antigen in urine have emerged as point-of-care (POC) tests for TB with endorsement by WHO of the Determine TB-LAM Ag (Abbott, USA) for TB diagnostic in HIV patients,15 and several ultra-sensitive third-generation LAM assays to be used irrespective of HIV status will enter trials in 2023.16 Interestingly, a study demonstrated the identification of LAM in EBC from TB patients through LAM immunoassay using CS-35 anti-LAM antibody.17 The LAM identification in EBC, confirmed by chemical detection of D-arabinose, revealed unsuspected amounts (in the µg/ml range) of the compound in the sample, making further identification of sputum-negative patients and children relevant. If confirmed, this approach would constitute a breakthrough in TB diagnosis and would be of high interest in developing a POC test.

There is a great need to develop new screening tests that are operationally feasible in high-burdened settings and can rapidly and accurately detect active TB disease at a low cost per test. Fondation Mérieux (FMX), in collaboration with icddr,b and IPBS/CNRS, sets up a 12-month diagnostic accuracy study (clinical setting) to bridge the gap in non-sputum-based diagnosis tests for TB disease through LAM testing on EBC specimen.

Methods

Study setting

This observational, prospective, diagnostic accuracy study is conducted at the TB Screening and Treatment Centre (TBSTC) of icddr,b on a cohort of adults and adolescents >11 years old in Dhaka, Bangladesh. Patients visiting this TBSTC were referred mainly through private and public health providers on suspicion of TB disease. Some walk-in clients without a referral history also attended these centres. This advanced facility is experienced with clinical research/surveillance18 and is equipped with state-of-the-art digital chest X-ray (CXR) systems, GeneXpert instruments and spirometry. It also serves as a direct observational therapy centre. A daily average of 150–170 presumptive TB cases visit this clinic where trained study physicians, radiologists, medical technologists and health workers are assigned to evaluate the presumptive cases, diagnose TB and other comorbid conditions, carry out chest X-rays and ensure proper anti-TB treatment. Standard case management adhering to country procedures will be followed accordingly.19 This study is reported in accordance with the Standards for Reporting of Diagnostic Accuracy Studies guidelines.20

Enrolment of the study participants

At the TBSTC, a comprehensive physical examination and assessment of basic clinical parameters will be conducted; the patients who meet the inclusion criteria mentioned in table 1 will be enrolled. From 28 August 2023 to 28 August 2024, this 1 year study will enrol patients with presumptive pulmonary TB with subsequent characterisation based on the standard reference test used for diagnosis. Patients with disease other than respiratory (control group) will also be enrolled as comparators matched to the narrowest window with relation to cases for age distribution. Before any procedure, written informed consent will be obtained from the participant or parent/guardian, and assent will be obtained from minors.

Table 1 inclusion and exclusion criteria for presumptive TB and control individuals

Presumptive TB case inclusion criteria	Presumptive TB case exclusion criteria	
All subjects: male or female, older than 11 years with at least one of the following suggestive symptoms and signs of TB disease:Cough >2 weeks

Fever >38°C

Night sweats

Weight loss

Tiredness and fatigue

Loss of appetite

Swellings in the neck

	Age less than 11 years	
Patients who have not yet started anti-TB treatment	Patients who have already started anti-TB treatment	
Provide written informed parental/guardian consent	Non-consenting people	
Residency in the defined catchment area of the selected health facility	Patients or caregivers with cognitive impairment and those with mental illness with high risk of compromised adherence to the protocol and/or understanding of the consent criteria	
Controls inclusion criteria	Controls exclusion criteria	
Any subjects: male or female, older than 11 years presenting at the facility as an attendant of a patient (who are not subsequently diagnosed with TB disease, such as those in the unlikely group), with no signs or symptoms related to TB or other respiratory illness and no previous contact with TB patient	Age less than 11 years with respiratory symptoms and signs	
Provide written informed parental/guardian consent	Nonconsenting people	
Residency in the defined catchment area of the selected health facility	Patients or caregivers with cognitive impairment and those with mental illness with high risk of compromised adherence to the protocol and/or understanding of the consent criteria	
TBtuberculosis

Study workflow

When participants enrol (figure 1), they will undergo a standard TB work-up, which will include a clinical assessment, a CXR and a microbiological test to detect Mtb from a sputum sample (using GeneXpert MTB/RIF, acid-fast Bacilli (AFB) smear microscopy and L-J culture). The results from sputum tests and CXR will be used to define patient’s status accordingly. Investigational tests will also be performed, which involve Determine TB-LAM Ag assay from EBC and urine samples. Before/after collection of sputum samples, the study participants will undergo EBC sampling through EBC collection device (Medivac). Of note, a subset of presumptive pulmonary TB individuals (n=200) will be subjected to an additional EBC collection process through a prototype device for performance comparison purpose. The order of the EBC device used will be alternated to avoid any bias in the analysis. The results obtained from investigational tests will not interfere in any way with the standard TB diagnosis and treatment plan of study participants. Any leftover EBC and urine samples will be stored for future use.

Figure 1 Study workflow. *exhaled breath condensate (EBC) and urine collection, †sputum testing is performed as soon as possible; most of the time results are available the day after the visit at TB Screening and Treatment Centre (TBSTC), ‡individuals under surveillance. LAM, lipoarabinomannan; ORD, other respiratory disease; TB, tuberculosis.

Patient classification

In addition to clinical symptoms, signs and X-ray interpretation, standard reference tests (GeneXpert MTB/RIF, AFB microscopy and L-J culture) results will give strong evidence for groups’ determination as defined below:

Presumptive TB: a patient suspected by a registered physician to have TB like symptoms including cough for more than 2 weeks or more with or without production of sputum and at least one of the following signs or symptoms: fever >38°C, night sweats, weight loss, tiredness and fatigue, loss of appetite and swelling in the neck (extra-pulmonary TB).

Definite TB: a presumptive TB patient is classified as definite TB when microbiological evidence has been found in any standard reference tests (microbiologically confirmed presumptive TB) followed by a decision to give the patient a full course of TB treatment.

Probable TB: a presumptive TB patient who does not fulfil the criteria for bacteriological confirmation but has been diagnosed with active TB by a registered physician based on strong clinical evidence followed by a decision to give the patient a full course of TB treatment. This definition includes cases diagnosed by clinicians based on X-ray abnormalities compatible with TB and signs and symptoms of TB.

Possible TB: a presumptive TB patient presenting X-ray abnormalities compatible with TB but does not fulfil the criteria for bacteriological confirmation followed by a decision to NOT give the patient a full course of TB treatment. These individuals usually receive antibiotic treatments and are under surveillance (status could be refined with culture result and treatment adjustments).

Other respiratory disease (ORD): a presumptive TB patient who does not fulfil the criteria for bacteriological confirmation and for whom the physician issued a diagnosis of respiratory diseases other than TB, accompanied by a full course of treatment for the diagnosed respiratory illness.

Unknown: a presumptive TB patient who does not fulfil the criteria for bacteriological confirmation with normal X-ray or abnormalities not compatible with TB and for whom the physician has NOT issued a definite diagnosis.

Control group: an individual presenting at TBSTC for consultation without pulmonary symptoms. The purpose of the control group is to define the baseline in TB High endemic country for LAM detection in EBC samples.

EBC collection

For both devices, the exhaled air and EBC sample volume as well as the collection time have been recorded during standardisation phase. Individuals were subsequently instructed to breathe normally for 10 min for EBC sampling.

EBC sampling through DECCS 14 ST (Medivac, Italy)

This pilot study kit (figure 2) consists of a series of standard DECCS 14 ST collection circuits, an aluminium sleeve for cooling the collection tube, an insulating shell for protecting the aluminium block from loss of temperature and a Volmet 20 device that records the volume of air exhaled during EBC collection. The aluminium sleeve is placed in one of the two insulating shells to be previously cooled using an external cooling source, specifically a freezer that reaches at least – 20°C, where it must remain for at least 2 hours. The EBC collection procedure is simple, noninvasive and does not require additional skill. EBC collection is made by tidal breathing into the mouthpiece and transfer to the disposable collection tube. Immediately after collection, EBC sample will be stored at 4°C before being transferred to the lab and subjected to LAM POC testing. The remaining samples will be aliquoted in 31.5-mL tubes and stored at −80°C until further LAM quantitation, biomarker investigation and biobanking.

Figure 2 EBC collections using Medivac DECCS Pilot Study Kit Patient breathes in and out using the mouthpiece of the DECCS Pilot Study Kit. (2) Air enters through the one-way inhalation valve. (3) Medivac Volmet 20 measures the volume of air being exhaled. (4) Air enters a 50-mL centrifuge tube attached to the DECCS system. (5) A pre-cooled aluminium sleeve surrounds the falcon tube, condensing the exhaled air passing through. (6) A sleeve is used to insulate the aluminium block. (7) A thermometer is used to monitor the temperature of the aluminium block. (8) Air exits through the one-way exhalation valve.

EBC sampling through a prototype device

An EBC collector prototype developed by KTH-Sveind Ab (Sweden) and provided by the Foundation for Innovative New Diagnostics (FIND, Switzerland) is also evaluated. Due to the patent process, details of the instrument’s operation cannot be disclosed at this time.

Determine TB-LAM Ag testing

Determine TB-LAM Ag is an immunochromatographic test recommended from urine specimen for the diagnosis of TB among HIV-infected individuals. We decided to use 60 µL of EBC sample (same as urine) due to the uncertain concentration of LAM in this sample. Because LAM POC testing is carried out before the GeneXpert results are available, readers do not know the status of patients. Two independent readings are taken for assay analysis after the incubation period (25 min), plus a photograph for a third independent reading in case of discrepancies. A positive result (a visible purple/grey line) indicates that LAM antigen of mycobacteria is present in the sample at or above the test’s detection limit. In contrast, a negative result (no visible purple/grey line) indicates that it is not present or below the test’s detection limit. A procedural control bar is incorporated in the assay device to ensure assay validity.

Aims and outcome measures

Our main objectives are (1) to evaluate the relevance of using EBC samples for the diagnosis of pulmonary TB in adults and adolescents and (2) to estimate the accuracy of LAM POC assay in EBC samples compared with urine samples. Secondary aims include the following: (1) benchmarking performance of different devices for EBC collection; (2) determining the feasibility of implementing this sampling method in routine practice; (3) estimating the best position of this test with respect to WHO-TPP for non-sputum based test; (4) quantifying LAM in EBC samples; (5) building capacity through the technological transfer of Dot-blot immunoassays for LAM quantification; and (6) biobanking EBC and urine samples for further third-generation LAM POC assays testing as well as biomarker identification or confirmation.

Sample size

Data from the clinical site indicates an average of 12.1% of suspected cases reported as GeneXpert positive and an additional 1.2% clinically diagnosed at the TBSTC. In the systematic review by Zhang et al21, the sensitivities of GeneXpert Ultra ranged between 82% (95% CI, 65% to 93%) and 100% (95% CI, 91% to 100%). In this systematic review, the specificities of GeneXpert Ultra ranged between 96% and 100%, with the lower bound of 95% CI ranging between 88% and 94% and the upper bound of 95% CI ranging between 97% and 100%.

To compute sample size in a general model, we assume a 13% prevalence of the disease in the population study, 90% sensitivity of the GeneXpert MTB/RIF assay, a margin of error of 10% for 95% CI and 70% power for the evaluated test. Sample size should be 584 individuals including 76 definite TB individuals. The number of control individuals has not been computed as the intent purpose of this group is to verify the specificity of the method. We therefore plan to enrol 30 individuals in this group.

Patient and public involvement

Patients were not involved in the design or the planning of this research.

Data analysis

Data will be entered and analysed using the Statistical Package for the Social Sciences (SPSS) software V.20 or higher and R Studio software V.4.0.3 or higher. Epi Core Team (from FMX, icddr,b and IPBS/CNRS) will be in charge of the analysis.

The accuracy of the index test (LAM POC test from EBC) will be measured against different groups defined in the Gold Standard Classification section. The large spectrum of clinical status indicated in the document will be refined with culture results and treatment adjustments, ultimately leading to four groups: definite TB, probable TB, ORD and unknown disease. A subanalysis with other standards such as the Microbiological Reference Standard (MRS) and a Composite Reference Standard (CRS) will also be performed in absence of ‘gold standard’ classification. The MRS requires the presence of Mtb to be confirmed (AFB smear microscopy, culture or GeneXpert MTB/RIF test) from at least one sample, and the CRS adds to this the presence of signs and symptoms suggestive of TB and CXR presenting abnormality compatible with TB disease. MRS is a useful reference to assess the ability of the test to discriminate between TB and non-TB patients. Definite versus non-TB patients (ORD and unknown disease, combined or not) are therefore the most appropriate analysis for performance evaluation. CRS will be used as gold standard to compare definite TB and probable TB versus ORD and unknown disease (combined or not) groups. Results should provide evidence to potentially support clinician in a decision to give treatment. Additional performance comparisons between definite TB (microbiological confirmed TB) versus probable TB (clinical TB) groups as well as probable TB versus ORD and unknown disease (combined or not) groups will provide evidence of the ability of the test to correctly detect the presence or not of the target condition. The intent purpose of the control group is to define the baseline in TB high endemicity country for LAM detection in EBC samples. Normally distributed continuous variables will be analysed with the Student’s t-test, while the Mann–Whitney or Kruskal–Wallis rank-sum test will be used for non-normal distribution. Fisher’s exact test with Bonferroni’s post hoc test will be used to analyse discrete variables. Non-parametric data will be presented as median±interquartile range, and the statistical significance cut-off will be a p value of <0.05. The receiver-operating characteristic curves will be used to evaluate the ability of POC LAM assay results from EBC (and urine) to triage or confirm diagnosis among specific groups, CRS and MRS. Statistical evaluation of diagnostic performance will be done by calculating the area under the receiver operating characteristic curve and associated 95% CI using packages in R. Clinical performance such as sensitivity, specificity, odd ratio or positive/negative predictive value will be assessed through analysis of contingency tables.

Ethics and dissemination

Ethics

The protocol has been approved by the Institutional Review Board of icddr,b and recorded under a protocol reference number, PR-2301. The principles of informed consent in the current edition of the Declaration of Helsinki will be implemented before any protocol-specified procedures or interventions are carried out. Information will be given in both oral and written form. Independent witnesses will be used to attest that illiterate potential participants have understood the contents of the informed consent document.

To maintain confidentiality, all laboratory specimens, evaluation forms, reports and other records on individual subjects will be identified only by a coded number and subject initials. All information obtained will be kept confidential, and study documents will be stored in a locked cabinet.

During and after the programme, all data on subjects will be kept in strict confidence and will not be disclosed to a third party by any study team member. To achieve this, access to programme computers will be restricted using passwords, and study data forms will be kept locked. Confidential information stored on computers and paper forms will only be available to co-investigators and authorised study staff. Personal data relating to persons taking part in research and processed for this purpose may not be kept in the information systems of the controller, the investigating centre or the professional involved in the research until 2 years after the last publication of the results of the research or, in the absence of publication, until the signature of the final research report. They are then archived on paper or electronically for a period of 5 years.

Dissemination

Results testing will not be disseminated to individuals, and clinicians will be blinded to not interfere with the clinical decision on whether to put individuals on treatment. The results of this study will be subjected to a peer-review journal before being shared with National Tuberculosis Control Program and international agencies and presented at national or international workshops/seminars/conferences.

Acknowledgements

The authors are thankful to all staff from Mohakhali TB Screening and Treatment Centre and the laboratory staff for their excellent work on this study. They are also grateful to Foundation for Innovative New Diagnostics (FIND, Switzerland) and KTH-Sveind Ab (Sweden) for the opportunity to evaluate a prototype to collect EBC samples.

Review Process File
16 09 2024

Funding: The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.

Prepublication history for this paper is available online. To view these files, please visit the journal online (https://doi.org/10.1136/bmjopen-2024-087026).

Patient consent for publication: Not applicable.

Provenance and peer review: Not commissioned; externally peer reviewed.

Patient and public involvement: Patients and/or the public were not involved in the design, conduct, reporting or dissemination plans of this research.
==== Refs
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