## 1 Introduction  
Tuberculosis (TB), caused by _Mycobacterium tuberculosis_ (Mtb), remains one of the leading infectious causes of death worldwide and a major public health concern. Each year, about 10 million new TB cases and -1.5 million deaths due to TB are reported around the globe [1]. The increasing prevalence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB presents a serious threat to global TB control efforts. These resistant forms are associated with prolonged treatment, poorer outcomes, and limited therapeutic options. Pyrazinamide (PZA), an important first-line anti-TB drug, is a key component of treatment regimens for both drug-susceptible and MDR TB. Its unique sterilizing activity against non-replicating, persistent Mtb significantly shortens treatment duration and reduces relapse rates [2]. Recent studies, including our evaluation of the subapyridine + clofazimine + TB47 (SCT) regimen combined with PZA (SCTZ) in a well-established murine TB model, demonstrated superior efficacy compared to the standard BPaL regimen. Notably, SCTZ achieved complete sterilization within two months without relapse [3]. Despite its efficacy, the global rise in PZA resistance is concerning, with over 60% of MDR-TB cases exhibiting concurrent resistance to PZA [4]. The emergence of drug resistance in Mtb is mainly caused by a combination of factors [5; 6; 7; 8; 9; 10]. These include genetic mutations within the bacteria, which can alter drug targets and render the antibiotics ineffective [10]. Diagnostic inaccuracies such as false-negative results, delayed detection of resistance, or misclassification of susceptibility can lead to inappropriate or ineffective therapy. This exerts selective pressure on Mtb populations, fostering the survival and amplification of drug-resistant strains, which potentially increases the risk of MDR and XDR TB [1; 8; 11]. In addition, undiagnosed or misdiagnosed cases can facilitate the transmission of resistant strains within communities, worsening the public health burden [6]. Accurate and timely diagnostics, including molecular assays such as whole-genome sequencing (WGS), are essential for detecting resistance-associated mutations and guiding effective treatment regimens [7; 8; 9]. Moreover, the use of inadequate therapeutic regimens, through incorrect combinations, dosages, or durations, further contributes to the development of resistance [5]. Finally, patient non-adherence to prescribed treatments enables the persistence and evolution of resistant strains [7; 8; 9]. Addressing these factors is crucial for effective TB control. Although PZA's mechanism of action is complex and not fully elucidated, it is known to target multiple pathways essential for the survival of persistent Mtb. PZA is a prodrug that needs to be converted to its active form, pyrazinoic acid (POA), by pyrazinamidase (PZase), encoded by the \(pncA\) gene. Mutations in \(pncA\) or its regulatory region, leading to PZase inactivation or reduced expression, are the most common mechanism of PZA resistance [12; 13]. However, other genes have also been implicated in PZA resistance. These include \(rpsA\), encoding ribosomal protein S1, an essential component of the trans-translation system that binds POA and is critical under stress conditions [14]; \(panD\), encoding aspartate decarboxylase involved in CoA biosynthesis [15]; and \(Rv2783c\), a bifunctional enzyme implicated in (p)ppGpp metabolism and nucleic acid hydrolysis [16]. Moreover, some earlier studies have highlighted the role of ClpC1, an ATP-dependent protease, in PZA resistance [17]. However, data on ClpC1 and its association with PZA resistance in clinical isolates of Mtb remain limited, underscoring the need for updated investigations to refine potential resistance markers for molecular diagnosis. Notably, most molecular diagnostic methods for detecting PZA resistance focus solely on mutations in \(pncA\).