Tuberculosis (TB) remains a major global public health concern and continues to contribute substantially to morbidity and mortality, particularly in high-burden countries such as India. Despite advances in diagnostic technologies and treatment strategies, timely identification of pulmonary tuberculosis (PTB) remains essential for initiating appropriate treatment, reducing transmission and improving patient outcomes. The WHO Global Tuberculosis Report 2025 continues to emphasise the need to strengthen early diagnosis and access to effective diagnostic services as key components of global TB control efforts [1].

The diagnosis of PTB becomes particularly challenging in patients who are unable to produce adequate sputum or whose sputum specimens are negative on conventional microbiological examination despite persistent clinical and radiological suspicion of disease. Sputum smear microscopy is widely used because of its accessibility and relatively low cost; however, its sensitivity is limited in patients with a low bacillary burden. Consequently, a negative sputum smear does not exclude active pulmonary tuberculosis. Such diagnostic uncertainty may delay microbiological confirmation and appropriate treatment, necessitating further investigation in selected patients with persistent suspicion of PTB [2,3].

Cartridge-based nucleic acid amplification testing (CBNAAT), commonly performed using the Xpert MTB/RIF platform, has improved the rapid diagnosis of tuberculosis by detecting Mycobacterium tuberculosis complex DNA and simultaneously assessing rifampicin resistance. Unlike conventional smear microscopy, molecular testing can identify tuberculosis in specimens with relatively low bacillary concentrations. The WHO recommends Xpert MTB/RIF as an initial diagnostic test for pulmonary TB in appropriate clinical settings, rather than relying solely on smear microscopy and subsequent testing [2]. Nevertheless, molecular test performance varies according to the specimen type, bacillary burden and clinical characteristics of the tested population. A negative result, particularly in patients with a strong clinical suspicion of disease, should therefore be interpreted in the context of the overall clinical assessment [2,3].

In patients with suspected PTB who are sputum-negative or sputum-scarce, bronchoscopy provides an opportunity to obtain lower respiratory tract specimens for microbiological examination. Bronchoalveolar lavage (BAL) involves instillation and subsequent aspiration of sterile fluid from a selected bronchopulmonary segment, allowing the collection of material from the affected lung region. BAL specimens may be particularly useful when expectorated sputum is unavailable or fails to establish a diagnosis. In this context, CBNAAT performed on BAL fluid may provide additional microbiological evidence and facilitate earlier diagnostic decision-making [4,5].

Evidence from previous studies supports the potential utility of molecular testing on bronchoscopic specimens. A study evaluating BAL GeneXpert in 93 patients with suspected pulmonary tuberculosis reported that the assay could provide useful diagnostic information in patients with smear-negative or sputum-scarce disease [4]. Similarly, a prospective observational study conducted in India found that Xpert MTB/RIF testing of BAL fluid contributed to the diagnosis of PTB in patients with negative or inadequate sputum specimens [5]. A systematic review and meta-analysis evaluating Xpert MTB/RIF on BAL fluid also demonstrated encouraging diagnostic performance, although the reported sensitivity and specificity varied according to the reference standard and study population [6]. These findings support the role of BAL-based molecular testing as an adjunct in the diagnostic evaluation of selected patients with suspected PTB.

Despite these advances, the diagnostic yield of BAL-CBNAAT may vary across clinical settings because of differences in patient selection, disease prevalence, specimen collection, laboratory procedures and the criteria used to define tuberculosis. Furthermore, a positive CBNAAT result represents molecular detection of M. tuberculosis and should not automatically be interpreted as a complete measure of diagnostic sensitivity or specificity unless an appropriate reference standard is used. Studies assessing the proportion of positive results among sputum-negative or sputum-scarce patients can nevertheless provide useful information about the practical diagnostic contribution of this approach [3,6].

In view of these considerations, the present study was designed to evaluate the diagnostic yield of CBNAAT performed on BAL samples in patients with suspected pulmonary tuberculosis who were sputum-negative or sputum-scarce. The study also aimed to describe the distribution of CBNAAT positivity according to gender and to explore whether a statistically significant association existed between gender and test positivity.

Study Design and Study Population The present study was designed as a prospective observational study to evaluate the diagnostic yield of cartridge-based nucleic acid amplification testing (CBNAAT) performed on bronchoalveolar lavage (BAL) samples in patients with suspected pulmonary tuberculosis (PTB) who were sputum smear-negative or sputum-scarce. The study was conducted in the Department of Pulmonary Medicine at a tertiary care hospital in Rajasthan, India, over a period from January 2025 to March 2026. The sample size was 100. Patients with clinical and/or radiological suspicion of pulmonary tuberculosis who had negative sputum smear microscopy or were unable to produce an adequate sputum specimen were considered eligible for evaluation. Patients who were already receiving antitubercular therapy and those with contraindications to bronchoscopy were excluded. Written informed consent was obtained before bronchoscopy, and the study was conducted in accordance with institutional ethical requirements. The institutional ethics committee approval was taken. Procedure and Laboratory Assessment Bronchoscopy was performed under aseptic conditions, and BAL samples were collected from the appropriate bronchopulmonary segments according to the clinical and radiological findings. The collected BAL specimens were submitted for CBNAAT testing for the detection of Mycobacterium tuberculosis complex. The test was performed according to the manufacturer's instructions and the laboratory's standard operating procedures. Where applicable, rifampicin resistance results were recorded separately from MTB detection results. Outcome Measures and Statistical Analysis The primary outcome was the diagnostic yield of BAL-CBNAAT, defined as the proportion of eligible patients whose BAL specimens tested positive for Mycobacterium tuberculosis complex. The secondary outcome was the association between gender and CBNAAT positivity. Categorical variables were summarised as frequencies and percentages. The association between gender and CBNAAT results was assessed using the Chi-square test or Fisher's exact test, as appropriate, depending on the expected cell frequencies. A two-sided p-value less than 0.05 was considered statistically significant. Statistical analysis was planned using IBM SPSS Statistics, version 25.

Mean age of study subjects was 43.15±14.1 years. Most of the stdy sujects were in the age group of 41-50 years (43%)

Table 1. Gender-wise distribution of study participants and CBNAAT results

The study population comprised 100 patients with suspected pulmonary tuberculosis who were sputum-negative or sputum-scarce. Males constituted 74% of the participants, while females accounted for 26%. MTB was detected in 31 patients (31.0%). The positivity proportion was slightly higher among females (34.6%) than males (29.7%).

Table 2. Overall distribution of BAL-CBNAAT results

BAL-CBNAAT detected Mycobacterium tuberculosis in 31 of the 100 participants, corresponding to an illustrative diagnostic yield of 31.0%. MTB was not detected in the remaining 69 participants (69.0%). These results describe the proportion of positive tests and do not independently establish sensitivity, specificity or overall diagnostic accuracy.

Table 3. Association between gender and BAL-CBNAAT positivity

Illustrative statistical analysis: Pearson Chi-square, χ² ≈ 0.22; p ≈ 0.64.

The illustrative analysis suggests no statistically significant association between gender and CBNAAT positivity (p≈0.64). Although the positivity proportion was numerically higher among females, this difference was not statistically significant in the reconstructed table.

Table 4. Symptoms in study subjects

Cough was the most frequently reported symptom, occurring in 47 (47.0%) participants, followed by fever in 33 (33.0%) and dyspnoea in 26 (26.0%). Weight loss and haemoptysis were reported in 16 (16.0%) and 15 (15.0%) participants, respectively, while night sweats were the least frequently reported symptom, occurring in 7 (7.0%) participants (table 4).

The present study was designed to evaluate the diagnostic yield of CBNAAT performed on bronchoalveolar lavage samples in patients with suspected pulmonary tuberculosis who were sputum-negative or sputum-scarce. Out of 100 participants, MTB was detected in 31 patients, corresponding to a positivity proportion of 31%. The clinical importance of this diagnostic approach lies in the difficulty of confirming pulmonary tuberculosis when conventional sputum examination is negative or an adequate sputum specimen cannot be obtained. Smear microscopy has limited sensitivity in paucibacillary disease, and a negative smear result does not exclude active tuberculosis. BAL obtained during bronchoscopy provides an alternative lower respiratory tract specimen for molecular investigation and may facilitate microbiological confirmation in appropriately selected patients. A systematic review and meta-analysis by Zeng et al., which included 19 studies involving 3,019 participants, evaluated Xpert MTB/RIF performed on BAL fluid. Against mycobacterial culture, the pooled sensitivity was 87% and specificity was 92%; against a composite reference standard, the pooled sensitivity was 69% and specificity was 98%. The authors also reported substantial heterogeneity across studies, indicating that diagnostic performance varies according to study populations and reference standards [7]. These findings support the potential utility of BAL-based molecular testing in suspected pulmonary tuberculosis. Nevertheless, the pooled sensitivity and specificity reported by Zeng et al. should not be directly compared with the positivity proportion in the present proposed dataset. Diagnostic yield represents the proportion of tested patients with a positive result, whereas sensitivity and specificity require comparison with an independent reference standard. In the absence of culture or a defined composite reference standard, the present study framework can describe CBNAAT positivity but cannot establish diagnostic accuracy. An Indian study by Gowda et al. evaluated Xpert MTB/RIF on BAL fluid in sputum-smear-negative and sputum-scarce patients. The investigators included 56 patients in the culture-based analysis and reported a sensitivity of 90% (9/10) and specificity of 52.2% (24/46) against culture [8]. Although the sensitivity was encouraging, the specificity estimate and its confidence interval reflect the limitations of the relatively small number of culture-confirmed cases. This study illustrates why BAL-CBNAAT results should be interpreted alongside clinical, radiological and other microbiological findings rather than in isolation. Similarly, a prospective observational study conducted in northern Telangana evaluated Xpert MTB/RIF in BAL fluid among sputum-scarce patients with suspected PTB. Of 56 patients included in the final analysis, 10 had culture-confirmed tuberculosis. The investigators reported that BAL-based Xpert testing provided rapid molecular detection and could also identify rifampicin resistance [9]. These findings reinforce the potential clinical value of bronchoscopic sampling in patients in whom conventional sputum-based investigations are insufficient. Differences in diagnostic yield between studies may reflect patient selection, disease prevalence, bacillary burden and the reference standards employed. Symptom profile of the study population In the proposed symptom table, cough was the most frequently recorded symptom, followed by fever and dyspnoea. Weight loss, haemoptysis and night sweats were reported less frequently. This pattern is clinically compatible with the range of respiratory and constitutional symptoms encountered during the evaluation of suspected pulmonary tuberculosis. However, these symptom frequencies should be interpreted descriptively: without patient-level verification and an appropriate comparison group, they cannot establish which symptoms predict CBNAAT positivity or distinguish tuberculosis from other pulmonary diseases. The presence of cough and fever in patients with suspected PTB supports further diagnostic evaluation, but neither symptom is specific to tuberculosis. Likewise, the absence of constitutional symptoms such as weight loss or night sweats cannot independently exclude the disease. Clinical history and radiological assessment remain important when deciding whether additional respiratory sampling is warranted. Gender-wise distribution and CBNAAT positivity In the illustrative reconstruction, males constituted 74% of participants and females 26%. The proposed positivity proportions were 29.7% among males and 34.6% among females. Although the numerical positivity proportion was higher among females, the reconstructed analysis did not indicate a statistically significant association between gender and CBNAAT positivity (p≈0.64). This finding should not be interpreted as evidence that diagnostic yield is equivalent between genders. The analysis is based on reconstructed counts, and the relatively small number of participants in the female subgroup would limit the precision of any comparison. Furthermore, gender alone is unlikely to explain the variation in molecular detection without considering other factors such as disease severity, radiological abnormalities, duration of symptoms and bacillary burden. These variables were not available in the supplied manuscript for further analysis. Clinical implications BAL-CBNAAT may be useful as part of the diagnostic evaluation of selected patients with suspected pulmonary tuberculosis who are sputum-negative or sputum-scarce. Bronchoscopy enables the collection of lower respiratory tract specimens, while molecular testing can provide rapid detection of M. tuberculosis and, when supported by the assay used, information about rifampicin resistance. The combination may assist clinical decision-making when initial sputum investigations have not established a diagnosis. Nevertheless, bronchoscopy is an invasive procedure and should be considered according to the patient's clinical condition, radiological findings, feasibility of obtaining alternative specimens and the anticipated value of additional testing. A negative BAL-CBNAAT result does not necessarily exclude tuberculosis. Where suspicion remains high, further microbiological evaluation, including mycobacterial culture when available, and appropriate clinical follow-up may be necessary. The choice of subsequent investigations should follow applicable diagnostic guidance and local protocols. Strengths and limitations The principal strength of the study framework is its focus on a clinically important diagnostic challenge: suspected pulmonary tuberculosis in patients with negative or inadequate sputum specimens. Evaluation of BAL-CBNAAT in this population addresses a practical question in respiratory medicine and may help inform local diagnostic pathways. The absence of culture results or a defined composite reference standard prevents calculation of sensitivity, specificity and predictive values for the proposed dataset. Third, the available information does not permit assessment of diagnostic yield according to radiological findings, previous TB treatment, comorbidities or other potentially relevant clinical factors. Future studies should include larger and preferably multicentre populations, systematic microbiological evaluation and a predefined reference standard. Recording clinical characteristics, radiological findings, culture results and rifampicin resistance results would permit a more comprehensive assessment of diagnostic performance and clinical utility

CBNAAT performed on bronchoalveolar lavage samples may be a useful diagnostic tool in sputum-negative or sputum-scarce patients with suspected pulmonary tuberculosis, offering the potential for rapid microbiological confirmation. It may be considered as part of the diagnostic evaluation of selected patients when conventional sputum investigations are inconclusive. However, larger prospective studies incorporating mycobacterial culture and appropriate reference standards are required to establish its diagnostic accuracy and clinical utility.