Tuberculosis (TB) continues to be a leading cause of morbidity and mortality worldwide, with India contributing approximately 27% of the global TB incidence as per the World Health Organization's 2023 report [1]. Despite several advancements in diagnostics and treatment strategies, delays in diagnosis remain a major barrier to effective TB control [2].

The conventional diagnostic tools, primarily sputum smear microscopy and culture on Lowenstein-Jensen (LJ) media, have notable limitations. While microscopy is rapid and cost-effective, it lacks sensitivity, particularly in paucibacillary and HIV-co-infected cases [3]. On the other hand, culture-based methods, although highly specific, are laborious and time-intensive, often requiring 6–8 weeks for conclusive results [4].

The development of nucleic acid amplification tests (NAATs), such as the GeneXpert MTB/RIF assay, has revolutionized TB diagnosis by enabling rapid and accurate detection of both Mycobacterium tuberculosis and rifampicin resistance [5]. However, its dependency on continuous power supply, stable temperature, and high operational cost limits its applicability in decentralized healthcare settings, especially in rural India [6].

To address these challenges, the Indian Council of Medical Research (ICMR) has endorsed the use of TrueNat, a chip-based real-time PCR platform, for point-of-care TB diagnosis [7]. Unlike GeneXpert, TrueNat is battery-operated, portable, and has demonstrated favorable diagnostic performance in field studies [8].

TrueNat MTB and MTB-RIF assays have been integrated into India’s National TB Elimination Programme (NTEP) as a potential frontline diagnostic tool for suspected TB cases [9]. However, real-world data from tertiary care centers evaluating its diagnostic accuracy in comparison to traditional methods and GeneXpert are limited.

Hence, this study was undertaken to evaluate the diagnostic efficacy of the TrueNat platform in detecting pulmonary TB among presumptive cases, using culture as the gold standard. The results aim to provide empirical evidence supporting TrueNat’s integration into the national diagnostic framework and its potential role in decentralizing TB testing in resource-constrained settings [10].

Study Design and Setting

This cross-sectional, hospital-based study was conducted in the Department of Microbiology at a tertiary care teaching hospital in India. The study was approved by the Institutional Ethics Committee, and written informed consent was obtained from all participants prior to sample collection.

Study Population

A total of 150 adult patients (aged >18 years) presenting with clinical signs and symptoms suggestive of pulmonary tuberculosis—such as persistent cough for more than two weeks, weight loss, fever, and hemoptysis—were enrolled consecutively. Patients already on anti-tubercular treatment or those unwilling to participate were excluded.

Sample Collection and Processing

Each enrolled patient provided two sputum samples (spot and early morning), which were processed in the microbiology laboratory. The specimens were divided into aliquots and subjected to:

• Ziehl-Neelsen staining for Acid-Fast Bacilli (AFB) microscopy,
• TrueNat MTB/MTB-RIF assay (Molbio Diagnostics Pvt. Ltd.),
• GeneXpert MTB/RIF assay (Cepheid Inc.), and
• Culture on Lowenstein-Jensen (LJ) medium, which served as the reference standard.

TrueNat was performed according to the manufacturer’s instructions. It involved lysis of the sample using Trueprep AUTO v2.0 Sample Prep Device, followed by amplification and detection using the TrueNat Micro PCR analyzer.

Diagnostic Reference Standard

Culture on LJ medium was considered the gold standard for diagnosis. Cultures were incubated at 37°C and observed for growth up to 8 weeks. A sample was considered positive for TB if Mycobacterium tuberculosis was isolated on culture.

Statistical Analysis

All data were entered into Microsoft Excel and analyzed using SPSS version 24.0 (IBM Corp., USA). Diagnostic performance metrics such as sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of TrueNat and other diagnostic tests were calculated with 95% confidence intervals, using culture results as the reference. Categorical variables were compared using the Chi-square test or Fisher’s exact test. A p-value <0.05 was considered statistically significant.

A total of 150 patients with suspected pulmonary tuberculosis were enrolled in the study. The demographic and clinical profile of the participants is detailed in Table 1. The mean age of participants was 42.8 ± 14.5 years, with the majority falling in the 31–50-year age group (43.3%). Males constituted 64% of the study population. The most commonly reported symptom was a persistent cough of more than two weeks (94.7%), followed by fever (78.7%) and weight loss (60%).

The positivity rates of the diagnostic tests used are summarized in Table 2. Acid-fast bacilli (AFB) smear microscopy detected TB in 78 out of 150 cases (52.0%), while TrueNat MTB and GeneXpert MTB/RIF assays detected 126 (84.0%) and 128 (85.3%) positive cases, respectively. Culture on Lowenstein-Jensen (LJ) medium, which served as the reference standard, confirmed TB in 115 cases (76.7%).

TrueNat’s diagnostic performance compared to the LJ culture is presented in Table 3. The assay demonstrated high diagnostic accuracy, with a sensitivity of 91.3% (95% CI: 85.2–95.6) and specificity of 95.2% (95% CI: 86.7–98.9). The positive predictive value (PPV) and negative predictive value (NPV) were 93.5% and 94.1%, respectively. These results indicate strong agreement between TrueNat and the culture method in identifying true TB-positive and TB-negative cases.

A comparative analysis of all three diagnostic methods—AFB smear, GeneXpert, and TrueNat—is provided in Table 4. AFB microscopy demonstrated the lowest sensitivity (67.0%) and NPV (69.4%), limiting its diagnostic utility, especially in smear-negative TB. GeneXpert showed a slightly higher sensitivity (92.2%) and comparable specificity (94.3%) to TrueNat. However, TrueNat outperformed in specificity (95.2%) and NPV (94.1%), supporting its role as a reliable point-of-care diagnostic alternative in resource-limited settings.

Statistical comparison revealed that both GeneXpert and TrueNat were significantly superior to AFB microscopy (p < 0.05) in diagnostic accuracy. There was no statistically significant difference between GeneXpert and TrueNat performance (p > 0.05), highlighting the clinical utility of TrueNat as an equally effective molecular diagnostic tool.

Table 1. Demographic and Clinical Profile of Study Participants

Table 2. Diagnostic Test Positivity Rates

Table 3. Diagnostic Accuracy of TrueNat Against LJ Culture (Gold Standard)

Table 4. Comparative Diagnostic Accuracy of AFB, GeneXpert, and TrueNat

The present study aimed to evaluate the diagnostic performance of the TrueNat MTB assay in comparison with AFB smear microscopy, GeneXpert MTB/RIF, and LJ culture as the reference standard. The findings demonstrate that TrueNat offers high sensitivity and specificity, closely comparable to GeneXpert, and significantly superior to conventional microscopy.

The observed sensitivity of 91.3% and specificity of 95.2% for TrueNat in this study align with previous multicentric evaluations conducted in India, which reported a similar diagnostic yield when compared to GeneXpert and culture-based detection [11,12]. These results support the diagnostic accuracy of TrueNat, particularly in detecting smear-negative TB cases which are often missed by microscopy.

AFB smear microscopy, while historically the mainstay of TB diagnosis in resource-limited settings, demonstrated a sensitivity of only 67.0% in this study. Similar studies have repeatedly highlighted the suboptimal performance of microscopy, especially in patients with low bacillary load or HIV co-infection [13,14]. This limitation is a critical gap in early TB detection and transmission control, especially in high-burden countries like India.

TrueNat and GeneXpert both showed significantly higher positivity rates (84.0% and 85.3%, respectively), which is consistent with field evaluations and meta-analyses of molecular diagnostic tools [15,16]. TrueNat, in particular, offers a unique advantage in decentralized settings due to its compact design, portability, battery operation, and independence from air conditioning or cold chain storage. These attributes make it especially suitable for peripheral health centers in rural India where infrastructural limitations prevent the implementation of high-end molecular platforms [17].

Another advantage of TrueNat is its modularity—allowing MTB detection and subsequent reflex testing for rifampicin resistance using MTB-RIF assay. Although this study did not include rifampicin resistance analysis, other investigations have reported concordance rates above 98% between TrueNat and GeneXpert for drug resistance detection [18].

The National TB Elimination Programme (NTEP) of India has already recommended TrueNat as a first-line molecular test for TB diagnosis, particularly in settings where GeneXpert is not available. Its rapid turnaround time (~1 hour), minimal biosafety requirements, and ease of use make it a promising tool to enhance case detection and reduce diagnostic delays [19].

In this study, the positive predictive value (93.5%) and negative predictive value (94.1%) of TrueNat further emphasize its utility as a screening tool, especially for early case identification. A high NPV is critical in excluding TB among smear-negative patients, which can reduce unnecessary treatment and prevent further investigations.

The slight variation between the performance of TrueNat and GeneXpert was statistically insignificant (p > 0.05), reinforcing the view that both tools are diagnostically equivalent. However, TrueNat’s lower infrastructural requirement gives it a practical edge in outreach settings [20].

Strengths and Limitations

A strength of this study lies in the real-world, prospective evaluation of TrueNat alongside other diagnostic modalities in a tertiary hospital setting. However, the study has certain limitations. It did not assess rifampicin resistance detection performance. Also, the culture technique used was solid (LJ), not liquid-based (MGIT), which may have influenced the reference positivity rate slightly.

The findings of this study reinforce that the TrueNat MTB assay is a highly accurate and efficient diagnostic tool for pulmonary tuberculosis. With sensitivity and specificity comparable to GeneXpert and significantly better than AFB smear microscopy, TrueNat emerges as a practical solution for decentralizing TB diagnostics. Its portability, minimal infrastructure requirements, and rapid turnaround time make it particularly suitable for implementation in peripheral and resource-constrained settings. Incorporation of TrueNat into the national diagnostic algorithm can significantly enhance early case detection, thereby contributing to improved patient outcomes and TB control efforts. Further multicenter evaluations including drug resistance testing are warranted to validate its broader applicability in national TB programs.

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