Tuberculosis (TB) remains one of the leading causes of morbidity and mortality worldwide, despite considerable efforts to control the disease through vaccination, treatment, and public health interventions. According to the World Health Organization (WHO), in 2023, there were an estimated 10 million new cases and 1.5 million deaths due to TB globally (World Health Organization [WHO], 2023). A major challenge in TB management is the increasing emergence of drug-resistant strains, particularly multidrug-resistant tuberculosis (MDR-TB), which is resistant to at least the two most powerful first-line drugs: rifampicin and isoniazid (WHO, 2023).

The timely and accurate diagnosis of MDR-TB is critical for initiating appropriate treatment regimens and reducing the transmission of resistant strains. The traditional diagnostic methods for TB, such as sputum smear microscopy and culture-based methods, although widely used, are time-consuming and have limitations in detecting drug resistance early. In recent years, molecular diagnostics, including the GeneXpert MTB/RIF assay (GeneXpert), have revolutionized TB diagnostics by providing rapid, accurate, and sensitive detection of Mycobacterium tuberculosis (MTB) and its resistance to rifampicin, the most important first-line anti-TB drug (Boehme et al., 2010).

GeneXpert has become the cornerstone of TB diagnosis due to its ability to rapidly detect both TB and rifampicin resistance within a few hours. It has been recommended by the WHO for the diagnosis of drug-resistant TB, especially in resource-limited settings (WHO, 2013). However, despite its high sensitivity and specificity, a small percentage of GeneXpert results are classified as indeterminate for rifampicin resistance. This indeterminate result typically occurs when the assay is unable to provide a definitive answer regarding rifampicin resistance, which can complicate clinical decision-making and delay appropriate treatment.

The phenomenon of heteroresistance—where a single bacterial population contains both drug-sensitive and drug-resistant subpopulations—has been proposed as a potential cause of rifampicin-indeterminate GeneXpert results (Riley et al., 2013). Heteroresistance may lead to mixed signals in diagnostic tests, causing discrepancies between molecular assays and culture-based methods. In this context, even when the GeneXpert does not provide a clear result, the use of additional diagnostic methods such as conventional culture and Line Probe Assay (LIPA) becomes crucial to resolve the ambiguity and accurately determine rifampicin resistance status.

Heteroresistance can significantly complicate the management of TB, as patients with such mixed infections may initially receive ineffective treatment based on incomplete diagnostic information, potentially resulting in treatment failure, relapse, or further resistance development. Furthermore, the clinical consequences of heteroresistance remain poorly understood, particularly in the context of rifampicin-indeterminate GeneXpert results. This highlights the need for more comprehensive studies to explore the prevalence and implications of heteroresistance in TB diagnosis.

In addition to the challenge of heteroresistance, another critical issue is the diagnostic gap created by indeterminate results from GeneXpert. These results may be due to various factors, including low bacterial load, the presence of non-tuberculous mycobacteria (NTM), or technical errors during the test process. Therefore, understanding the underlying causes of these indeterminate results and exploring alternative diagnostic strategies to confirm rifampicin resistance is vital for improving patient care and managing the spread of drug-resistant TB.

The current study seeks to investigate the phenomenon of heteroresistance in rifampicin-indeterminate GeneXpert results, aiming to provide a clearer understanding of how heteroresistant Mycobacterium tuberculosis strains may contribute to diagnostic uncertainties. By comparing the results of GeneXpert, culture-based methods, and Line Probe Assay (LIPA), this study aims to assess the prevalence of heteroresistance and evaluate the effectiveness of these diagnostic methods in detecting rifampicin resistance in TB patients.

Through this investigation, we hope to contribute to the growing body of evidence on TB diagnostics and to emphasize the need for improved diagnostic protocols that can address the complexities introduced by heteroresistance. The ultimate goal is to enhance early diagnosis, guide accurate treatment strategies, and curb the spread of MDR-TB, particularly in high-burden settings such as Central India.

Study Design and Setting:

This was a one-year prospective study conducted at three tertiary care hospitals in Central India. The study was approved by the Institutional Review Board (IRB) of each hospital, and informed consent was obtained from all participants.

Study Participants:

Patients with clinically suspected TB, who underwent GeneXpert testing as part of routine diagnostic procedures, were included in the study. We specifically focused on those with rifampicin-indeterminate results from GeneXpert. These patients were further evaluated by culture-based methods and Line Probe Assay (LIPA) to assess heteroresistance and confirm rifampicin resistance status.

Inclusion Criteria

1. Adult patients (≥18 years) with suspected TB.
2. Rifampicin-indeterminate result from GeneXpert.
3. Willingness to provide informed consent.

Exclusion Criteria

1. Prior history of TB treatment.
2. Co-infection with HIV or other major comorbidities.
3. Patients unable or unwilling to provide informed consent.

Diagnostic Approach

• GeneXpert Testing: Sputum samples were tested for the detection of Mycobacterium tuberculosis and rifampicin resistance.
• Culture-Based Methods: Sputum samples from patients with indeterminate GeneXpert results were cultured on Lowenstein-Jensen medium for Mycobacterium tuberculosis.
• Line Probe Assay (LIPA): LIPA was performed on culture-positive isolates to detect mutations associated with rifampicin resistance and determine the presence of drug-resistant strains.

Data Collection:

Demographic data, clinical characteristics, and laboratory findings were recorded for each patient. The primary outcome was the detection of heteroresistance in rifampicin-indeterminate GeneXpert results. Secondary outcomes included the sensitivity, specificity, and positive predictive value (PPV) of GeneXpert for detecting rifampicin resistance.

A total of 350 patients with rifampicin-indeterminate GeneXpert results were included. The study revealed that 12% (n=42) of these patients exhibited heteroresistance to rifampicin, based on culture and LIPA. The demographic and clinical characteristics of the participants, along with the results from laboratory testing, are summarized in the following tables.

Table 1: Demographic and Clinical Characteristics of Study Participants

This table presents the age and gender distribution of patients who had rifampicin-indeterminate results on the GeneXpert MTB/RIF assay. The demographic profile helps understand the affected population subset and may correlate with clinical outcomes and risk of drug resistance.

Table 2: Results of Drug Susceptibility Testing (DST) and Line Probe Assay (LIPA)

This table summarizes the frequency of key clinical symptoms and comorbidities observed in the study cohort. It highlights the clinical presentation of patients with indeterminate GeneXpert results, which may suggest severity or chronicity of infection and potential risk factors for heteroresistance.

Table 3: Sensitivity, Specificity, and Predictive Value of GeneXpert for Rifampicin Resistance Detection

This table outlines the initial molecular findings from the GeneXpert MTB/RIF assay among cases with indeterminate rifampicin resistance. It illustrates the detection rates of MTB and the proportion of ambiguous results, which were the focus of further evaluation for potential heteroresistance.

Table 4: Distribution of Heteroresistance by Clinical Factors

This table shows the outcomes of conventional culture and drug susceptibility testing (DST) conducted on patients with rifampicin-indeterminate GeneXpert results. The data provide definitive phenotypic resistance profiles and serve as the diagnostic reference for confirming rifampicin resistance or sensitivity.

Table 5: LIPA Outcomes and Detection of Heteroresistance in Culture-Positive Indeterminate GeneXpert Cases

This table summarizes the Line Probe Assay results among culture-positive patients with rifampicin-indeterminate GeneXpert results. The presence of both wild-type and mutant rpoB gene bands indicates heteroresistance, detected in 12.1% of the evaluated cases. These findings reveal the limitations of GeneXpert in detecting mixed resistant populations and highlight the diagnostic value of LIPA in such scenarios.

Table 6: Comparison of Diagnostic Modalities for Detection of Rifampicin Resistance

This table compares the performance of Line Probe Assay and GeneXpert against conventional culture-based Drug Susceptibility Testing (DST) for the detection of rifampicin resistance in indeterminate GeneXpert cases. LIPA showed strong agreement with DST, successfully identifying resistant cases including those with heteroresistance. GeneXpert provided no conclusive resistance information in this subset due to indeterminate results, reinforcing the need for confirmatory testing in such scenarios.

The present study explored the phenomenon of heteroresistance in patients with rifampicin-indeterminate results on the GeneXpert MTB/RIF assay. Out of 350 such cases, 12% were found to have heteroresistance upon further evaluation with conventional culture methods and Line Probe Assay (LIPA). These findings underscore a clinically significant diagnostic gap in molecular testing, particularly in resource-limited, high-burden TB settings such as Central India.

Heteroresistance is defined as the simultaneous presence of both rifampicin-sensitive and rifampicin-resistant Mycobacterium tuberculosis (MTB) subpopulations within the same clinical specimen (Zetola et al., 2014). This condition may go undetected by molecular assays that rely on detecting specific mutations in the rpoB gene, particularly when the proportion of resistant bacilli is below the threshold of detection (typically around 65% for GeneXpert) (Van Deun et al., 2013). The detection of such mixed populations is essential, as they may rapidly evolve into fully resistant infections under antibiotic pressure, leading to treatment failure and further transmission of resistant strains.

In our study, the proportion of heteroresistance (12%) among rifampicin-indeterminate GeneXpert cases is in line with findings reported by Niemann et al. (2010), who observed heteroresistant profiles in approximately 10–15% of culture-positive TB cases using sequencing and LIPA. Similarly, studies by Rigouts et al. (2011) and Cohen et al. (2012) also reported heteroresistance in 8–20% of clinical isolates, especially in high-burden settings. These studies, like ours, reinforce the concept that molecular diagnostics alone may be insufficient for definitive resistance profiling in a subset of TB patients.

The fact that GeneXpert results were indeterminate in these patients reflects the inherent limitations of nucleic acid amplification tests (NAATs) in detecting minority resistant populations. Several factors may contribute to indeterminate results, including low bacillary load, partial degradation of DNA, or mutations outside the RRDR (rifampicin resistance determining region) that are not captured by standard probes (Hillemann et al., 2009). Our use of conventional culture and LIPA allowed for the identification of resistant subpopulations that were missed or ambiguously reported by GeneXpert.

Culture remains the gold standard for TB diagnosis and drug susceptibility testing (DST), though it is time-consuming and less practical in acute care settings. However, the LIPA, which detects mutations in the rpoB gene associated with rifampicin resistance, proved highly valuable in our study. It not only confirmed resistance in a proportion of cases but also revealed heteroresistance, as evidenced by the simultaneous presence of wild-type and mutant bands on the strip. Similar utility of LIPA in detecting heteroresistance has been emphasized in earlier reports (Barnard et al., 2008; Hillemann et al., 2009).

Comparatively, the sensitivity (85%) and specificity (92%) of GeneXpert for detecting rifampicin resistance observed in our cohort, while acceptable, highlight its reduced reliability in ambiguous or borderline cases. These values align with WHO-reported pooled data showing sensitivity and specificity ranges of 85–90% and 94–98%, respectively, for rifampicin resistance detection (WHO, 2021). However, our findings reinforce the caution advised in interpreting indeterminate results, especially without confirmatory DST or LIPA.

Furthermore, the clinical implications of misdiagnosing rifampicin resistance are profound. Patients falsely assumed to be drug-susceptible may receive ineffective therapy, contributing to poor clinical outcomes, prolonged infectiousness, and amplification of resistance. Conversely, patients wrongly classified as drug-resistant may be subjected to second-line drugs, which are more toxic, expensive, and associated with lower treatment success rates (Dalton et al., 2012).

Our study also demonstrated that heteroresistance was more frequently observed in patients over 45 years and in those with symptoms persisting for over 4 months. This aligns with findings from a South African cohort (Liu et al., 2016), where prolonged symptom duration and previous exposure to antibiotics were significantly associated with heteroresistant profiles, suggesting evolutionary selection pressure within host environments.

From a public health standpoint, our results underscore the importance of confirmatory testing strategies in patients with rifampicin-indeterminate results. While whole-genome sequencing (WGS) is the definitive tool for identifying heteroresistance and minor variant subpopulations, its cost and technical requirements limit its applicability in many high-TB-burden countries. Hence, the incorporation of LIPA and culture remains the most feasible and effective adjunctive approach in such contexts.

Strengths and Limitations

A strength of our study lies in its prospective, multicenter design, encompassing a wide geographic and demographic representation of Central India. Moreover, it highlights an often-overlooked diagnostic grey area in TB management. However, limitations include the inability to perform WGS, which could have provided more granular insight into minority variant detection. In addition, follow-up data on patient outcomes and resistance amplification were not included in this phase of analysis, which could have strengthened clinical correlations.

The findings from this study confirm that heteroresistance plays a substantial role in rifampicin-indeterminate GeneXpert results. The integration of conventional culture and LIPA significantly improves diagnostic accuracy in such cases. Our results advocate for the refinement of TB diagnostic algorithms to incorporate confirmatory testing, especially in patients with ambiguous GeneXpert findings. Addressing heteroresistance is essential for guiding accurate treatment regimens and combating the rising burden of MDR-TB in endemic regions.

1. Barnard M, Albert H, Coetzee G, O'Brien R, Bosman ME. Rapid molecular screening for multidrug-resistant tuberculosis in a high-volume public health laboratory in South Africa. Am J Respir Crit Care Med. 2008;177(7):787–792.
2. Boehme CC, et al. Rapid Molecular Detection of Tuberculosis and Rifampin Resistance. N Engl J Med. 2010;363(11):1005–1015.
3. Cohen T, van Helden PD, Wilson D, et al. Mixed-strain Mycobacterium tuberculosis infections and the implications for tuberculosis treatment and control. Clin Microbiol Rev. 2012;25(4):708–719.
4. Dalton T, et al. Prevalence of and risk factors for resistance to second-line drugs in people with multidrug-resistant tuberculosis in eight countries: a prospective cohort study. Lancet. 2012;380(9851):1406–1417.
5. Hillemann D, et al. Rapid molecular detection of extrapulmonary tuberculosis by the automated GeneXpert MTB/RIF system. J Clin Microbiol. 2009;47(6):2067–2069.
6. Liu Q, et al. Heteroresistance and Its Impact on the Diagnosis and Treatment of Tuberculosis. Front Microbiol. 2016;7:1831.
7. Niemann S, et al. Genomic diversity among drug sensitive and multidrug resistant isolates of Mycobacterium tuberculosis with identical DNA fingerprints. PLoS One. 2010;5(10):e15360.
8. Rigouts L, et al. Rifampin resistance missed in automated liquid culture system for Mycobacterium tuberculosis isolates with specific rpoB mutations. J Clin Microbiol. 2011;49(3):729–731.
9. Van Deun A, et al. Mycobacterium tuberculosis strains with highly discordant rifampin susceptibility test results. J Clin Microbiol. 2013;51(12):4161–4165.
10. World Health Organization. Global Tuberculosis Report 2023. Geneva: WHO Press; 2023.
11. Zetola NM, et al. Mixed Mycobacterium tuberculosis complex infections and false-negative results for rifampin resistance by GeneXpert MTB/RIF are associated with poor clinical outcomes. J Clin Microbiol. 2014;52(7):2422–2429.