Tuberculosis (TB) remains one of the leading infectious causes of morbidity and mortality worldwide. According to the World Health Organization (WHO) Global Tuberculosis Report 2024, an estimated 10.6 million people developed TB globally in 2023, corresponding to an incidence rate of approximately 133 cases per 100,000 population [1]. Despite global control efforts, TB caused about 1.3 million deaths among HIV-negative individuals and an additional 300,000 deaths among people living with HIV, highlighting its continued public health significance [1].

India carries the highest TB burden globally. As per WHO estimates, India accounted for nearly 27% of global incident TB cases, with approximately 2.8 million new cases annually. The estimated TB incidence rate in India in 2023–2024 was about 187 cases per 100,000 population [1,2]. According to the India TB Report 2024, TB mortality remains substantial despite improved treatment access and expanded diagnostic services under the National TB Elimination Programme (NTEP) [2,15]. The transition from the Revised National Tuberculosis Control Programme (RNTCP) to NTEP has strengthened case detection, standardized treatment protocols, drug-resistance surveillance, and decentralized molecular diagnostics across the country [2,15].

The emergence of drug-resistant tuberculosis has further complicated TB control efforts. Globally, over 4,10,000 individuals developed drug-resistant TB in 2022, with India contributing a significant proportion of the global burden [1,2]. India accounts for nearly one-fourth of the global multidrug-resistant TB (MDR-TB) burden, with MDR-TB prevalence estimated at 2.5–3.9% among new cases and 13–16% among previously treated cases [2,13]. Rifampicin-resistant TB (RR-TB) is defined as resistance to rifampicin detected by phenotypic or genotypic methods, with or without resistance to other anti-TB drugs [13]. Multidrug-resistant TB (MDR-TB) refers to resistance to isoniazid and rifampicin [3,13], while extensively drug-resistant TB (XDR-TB)  strains are MDR-TB  and involves additional resistance to fluoroquinolones and  at least one  of the three second line injectable drugs (capreomycin, kanamycin and amikacin).  [4,13].

The emergence of MDR-TB and RR-TB is largely attributed to inappropriate treatment regimens, poor drug adherence, substandard drug quality, and premature interruption of therapy, leading to increased community transmission [13,14]. Drug-resistant TB is associated with prolonged therapy, higher treatment costs, increased toxicity, and lower treatment success rates compared to drug-susceptible TB [3,4].

Rifampicin acts by inhibiting bacterial RNA polymerase and is highly effective against both intracellular and extracellular bacilli. Mutations in the rpoB gene lead to rifampicin resistance, making it a reliable surrogate marker for MDR-TB in most clinical settings [4,11]. Conventional culture-based drug susceptibility testing (DST), though considered the reference standard, requires several weeks to yield results [5,12].

Sputum smear microscopy, though widely used in India, has limitations including low sensitivity and the requirement of a high bacillary load (approximately 10,000 bacilli/ml) for detection. The advent of rapid molecular diagnostic techniques such as TrueNat and CBNAAT has significantly improved TB detection and rifampicin resistance identification, even in low bacillary load specimens [6,7,10].

TrueNat MTB/RIF is a chip-based real-time PCR platform developed in India, capable of detecting Mycobacterium tuberculosis complex DNA and rpoB gene mutations within 1–2 hours [6]. The World Health Organization endorsed TrueNat for TB diagnosis and rifampicin resistance detection in 2020, and it has been incorporated into the National TB Elimination Programme for decentralized use [7,16]. Its portability, minimal biosafety requirements, and suitability for peripheral laboratories make it particularly valuable in resource-limited settings [6,7,16].

In view of the persistent TB burden and the growing concern of drug resistance, assessing the prevalence of rifampicin resistance among suspected pulmonary TB patients in tertiary care settings is essential for understanding local resistance patterns and guiding effective treatment strategies aligned with national TB elimination goals.

MATERIALS AND METHODS

Study Design and Setting

This is a prospective observational study conducted in the Department of Microbiology in collaboration with Department of Pulmonology, Viswabharathi Medical College and General Hospital. The study was done between April 2024 to January 2026 after obtaining approval from the Institutional Ethics Committee.

Study Population

The study population comprised of patients with clinical features suggestive of pulmonary tuberculosis such as cough> 2 weeks, fever, weakness, weight loss, hemoptysis in accordance with national tuberculosis diagnostic guidelines. [17] All the suspected patients were advised to collect two sputum samples and undergo radiological examination.

 Patients with signs and symptoms of extrapulmonary tuberculosis, already receiving anti-tubercular therapy for more than two weeks prior to sample collection, Patients who were unable to provide an adequate specimen were excluded from the study.

Sample Collection and Processing

Sputum samples were collected following standard recommendations under the National Tuberculosis Elimination Programme (NTEP) guidelines.[17] Patients were instructed to provide two sputum samples, one at the spot and an early morning sample. Samples were collected in sterile, wide-mouthed, leak-proof containers. A minimum volume of 2–3 mL was considered adequate. Care was taken to ensure that the specimen contained mucopurulent material rather than saliva.

Specimens were transported to the microbiology laboratory within one hour of collection. In case of delay, samples were stored at 2–8°C and processed within 24 hours as per WHO laboratory biosafety recommendations.[18] All handling of specimens was performed under appropriate biosafety precautions.

Ziehl–Neelsen (ZN) Staining for Acid-Fast Bacilli

Direct smear microscopy was performed using the Ziehl–Neelsen (ZN) staining method for detection of acid-fast bacilli (AFB) following standard WHO and RNTCP/NTEP guidelines. [19]

A thin smear was prepared on a clean glass slide, air-dried, and heat-fixed. The smear was flooded with strong carbol fuchsin and gently heated until steaming for five minutes. After rinsing with water, decolorization was carried out using 20% sulfuric acid until the smear appeared pale pink. The slide was then counterstained with methylene blue for one minute, washed, air-dried, and examined under oil immersion (100× objective).

Smears were graded according to standard WHO grading guidelines. [19] The presence of red, slender, beaded bacilli against a blue background was considered positive for AFB.

Detection of Mycobacterium tuberculosis and Rifampicin Resistance by TrueNat

All   sputum samples from suspected case of pulmonary tuberculosis were subjected to molecular testing using the TrueNat MTB/MTB-RIF assay for rapid detection of Mycobacterium tuberculosis complex and rifampicin resistance as recommended by national guidelines. [20]

Principle of TrueNat

TrueNat is a chip-based real-time polymerase chain reaction (RT-PCR) assay. The test involves automated DNA extraction from processed sputum samples followed by amplification of MTB-specific genetic targets. Rifampicin resistance is detected by identifying mutations in the rpoB gene, which encodes the β-subunit of RNA polymerase. [21]  Amplification and detection occur on a microchip platform with real-time fluorescence monitoring. Results are generated within approximately 1–2 hours and reported as MTB detected/not detected and rifampicin sensitive/resistant. [20]

Comparison of TrueNat with CBNAAT

TrueNat offers several operational advantages compared to Cartridge-Based Nucleic Acid Amplification Test (CBNAAT), including portability, battery-operated functionality, lower infrastructure requirements, suitability for decentralized settings, and reduced turnaround time. It can be deployed in peripheral laboratories with minimal biosafety infrastructure. [22]

However, certain limitations exist. TrueNat requires sequential testing (initial MTB detection followed by rifampicin resistance testing). Sensitivity may be reduced in specimens with very low bacillary load. Additionally, confirmatory culture and drug susceptibility testing remain necessary in selected cases. [17]

Importance of the Present Study

Rifampicin resistance is a critical marker of multidrug-resistant tuberculosis and has significant implications for patient management and public health. Early detection facilitates timely initiation of appropriate treatment regimens and reduces community transmission of resistant strains.

Tertiary care hospitals serve as referral centers for complicated and previously treated cases, making them important sites for surveillance of drug resistance patterns. Determining the prevalence of rifampicin resistance among suspected pulmonary tuberculosis patients in these settings provides essential epidemiological data and supports evidence-based treatment strategies aligned with national TB elimination goals.

RESULTS

A total of 2527 samples were screened during the study period from April 2024 to January 2026 as shown in Table 1.

Table 1: Number of samples screened year wise

Year-wise distribution of test results (Table 2) revealed that  during the study period, 2527 samples were screened. 184 sputum samples were positive by TrueNat assay, 163 sputum samples were positive by acid fast staining and 22 patients showed the radiological evidence of tuberculosis.

Table 2: Year wise Distribution of all test results

Rifampicin resistance detected by Truenat assay (Table 3) showed that the majority of cases were Rifampicin-sensitive (179 cases, 97.3%), while only 5 cases (2.7%) were found to be Rifampicin-resistant. Rifampicin resistance was seen in all newly diagnosed cases of tuberculosis.

Table 3: Rifampicin resistance detected by Truenat Assay

Gender-wise distribution showed a male predominance, with 101 males (54.9%) and 83 females (45.1%) as shown in table 4.

Table 4: Gender wise distribution of positive samples

DISCUSSION

Tuberculosis continues to remain a major public health problem globally including India despite sustained control efforts [1,2]. In the present study, 2527 samples were screened over 21 months, of which 184 (7.28%) were positive. This positivity rate aligns with findings from other tertiary-care-based Indian studies reporting rates between 5–15% [8].

Male predominance (54.9%) observed in this study is consistent with global epidemiological patterns [1,2,9]. Biological, behavioral, and socio-environmental factors have been implicated in higher TB prevalence among males [9].

Rifampicin resistance was detected in 2.7% of confirmed cases, which corresponds with national estimates of 2–4% among new TB cases in India [2,13]. Early identification of rifampicin resistance is critical under current DR-TB management guidelines to ensure timely initiation of appropriate second-line regimens [13,14]. Mutations in the rpoB gene serve as a surrogate marker for MDR-TB, allowing rapid detection through molecular methods [4,11].

TrueNat has demonstrated high sensitivity and operational feasibility in decentralized settings [6,10]. WHO endorsement and incorporation into national TB diagnostic algorithms have strengthened India’s strategy toward universal drug susceptibility testing [7,16].

CONCLUSION

The present study demonstrated a tuberculosis positivity rate of 7.28% among clinically suspected pulmonary TB patients in a tertiary care hospital setting. A clear male predominance was observed, consistent with national epidemiological trends. Rifampicin resistance was detected in 2.7% of confirmed cases, indicating a relatively low prevalence of drug resistance in the study population. The use of TrueNat enabled rapid detection of both Mycobacterium tuberculosis and rifampicin resistance, facilitating timely initiation of appropriate therapy. Continuous surveillance using rapid molecular diagnostics is essential to strengthen TB control efforts and support national TB elimination goals.

REFERENCES

1. World Health Organization. Global tuberculosis report 2024. Geneva: WHO; 2024.
2. Central TB Division, Ministry of Health and Family Welfare, Government of India. India TB Report 2024: National Tuberculosis Elimination Programme Annual Report. New Delhi: MoHFW; 2024.
3. World Health Organization. Guidelines for the programmatic management of drug-resistant tuberculosis. Geneva: WHO; 2011.
4. World Health Organization. WHO consolidated guidelines on tuberculosis. Module 4: treatment – drug-resistant tuberculosis treatment. Geneva: WHO; 2022.
5. Canetti G, Rist N, Grosset J. Measurement of sensitivity of tuberculous bacilli to antibacterial drugs. Bull World Health Organ. 1963;29:565–78.
6. Nikam C, Jagannath M, Narayanan MM, et al. Rapid diagnosis of Mycobacterium tuberculosis with Truenat MTB: a near-care molecular assay. J Clin Microbiol. 2013;51(7):2131–2136.
7. World Health Organization. Truenat MTB, MTB Plus and MTB-RIF Dx assays: WHO policy statement. Geneva: WHO; 2020.
8. Sharma SK, Mohan A, Kadhiravan T. Tuberculosis: Control in India—progress, challenges and the way forward. Indian J Med Res. 2017;145(6):696–708.
9. Horton KC, MacPherson P, Houben RMGJ, White RG, Corbett EL. Sex differences in tuberculosis burden and notifications in low- and middle-income countries: a systematic review and meta-analysis. PLoS Med. 2016;13(9):e1002119.
10. Penn-Nicholson A, Gomathi SN, Ugarte-Gil C, et al. Diagnostic accuracy study of Truenat MTB-RIF. Lancet Infect Dis. 2021;21(10):1448–1457.
11. Lange C, Chesov D, Heyckendorf J, et al. Drug-resistant tuberculosis: an update. Respirology. 2018;23(7):656–673.
12. Siddiqi SH, Rüsch-Gerdes S. MGIT procedure manual. Geneva: Foundation for Innovative New Diagnostics; 2006.
13. Central TB Division. National guidelines for management of drug-resistant tuberculosis in India. New Delhi: MoHFW; 2021.
14. Central TB Division. Guidelines for Programmatic Management of Drug Resistant TB (PMDT) in India. New Delhi: MoHFW; 2017.
15. Directorate General of Health Services. National Tuberculosis Elimination Programme (NTEP). MoHFW, Government of India.
16. Indian Council of Medical Research. WHO endorsement of Truenat for TB diagnosis. New Delhi: ICMR; 2020.
17. Central TB Division, Ministry of Health and Family Welfare, Government of India. National Guidelines for Management of Drug-Resistant Tuberculosis in India. New Delhi: MoHFW; 2025.
18. World Health Organization. Laboratory biosafety manual. 4th ed. Geneva: WHO; 2020.
19. World Health Organization. Laboratory services in tuberculosis control: Part II – Microscopy. Geneva: WHO; 2013 update.
20. Central TB Division, MoHFW. Guidelines on the use of Truenat for diagnosis of TB under NTEP. New Delhi: MoHFW; 2021.
21. Telenti A, Imboden P, Marchesi F, et al. Detection of rifampicin-resistance mutations in Mycobacterium tuberculosis. Lancet. 1993;341:647–650.
22. World Health Organization. WHO consolidated guidelines on tuberculosis: Module 3 – Diagnosis – Rapid diagnostics for tuberculosis detection. Geneva: WHO; 2023 update.