Mycobacterium tuberculosis: The Mechanism of Pathogenicity, Immune Responses, and Diagnostic Challenges

ABSTRACT

Background

The infection caused by Mycobacterium tuberculosis arises from a complex interplay between the host immune system and the bacteria. Early and effective treatment of this disease is of great importance in order to prevent the emergence of drug‐resistant strains. This necessitates the availability of fast and reliable diagnostic methods for managing affected cases. One reason why this study is significant is the lack of a comprehensive review in this field that thoroughly examines the importance, pathogenesis, and diagnosis of M. tuberculosis . Therefore, the aim of this review is to provide updated information on M. tuberculosis .

Methods

We investigate the virulence factors, pathogenicity, and diagnostic methods of this bacterium, alongside the clinical symptoms and interpretation of different types of tuberculosis, including cerebral, miliary, nerve, and tubercular tuberculosis.

Results

Mycobacterium tuberculosis acts as the causative agent of human tuberculosis and is regarded as one of the most adaptable human pathogens. M. tuberculosis possesses several virulence factors that help the bacterium evade mucous barriers. The rise of multidrug‐resistant tuberculosis (MDR‐TB) in both developing and industrialized countries emphasizes the need for rapid diagnostic methods.

Conclusions

Non‐protein virulence factors play a crucial role in the pathogenicity of Mycobacterium tuberculosis (M. tuberculosis). The bacterial cell membrane contains proteins that modulate the host immune response. For instance, ESAT‐6, either alone or in combination with CFP‐10, reduces immune activity. While molecular techniques—such as DNA microarray, luciferase reporter assay, polymerase chain reaction (PCR), DNA and RNA probes, next‐generation sequencing, and whole‐genome sequencing—offer rapid, sensitive, and specific detection of M. tuberculosis, these methods are expensive and require technical expertise.

Mycobacterium tuberculosis acts as the causative agent of human tuberculosis and is regarded as one of the most adaptable human pathogens. M. tuberculosis possesses several virulence factors that help the bacterium evade mucous barriers. The infection caused by M. tuberculosis arises from a complex interplay between the host immune system and the bacteria. Early and effective treatment of this disease is of great importance in order to prevent the emergence of drug‐resistant strains. This necessitates the availability of fast and reliable diagnostic methods for managing affected cases. One reason why this study is significant is the lack of a comprehensive review in this field that thoroughly examines the importance, pathogenesis, and diagnosis of M. tuberculosis .

1. Introduction

In the latter half of the 19th century, Louis Pasteur and Robert Koch conducted investigations on human infectious diseases using animal models, particularly rodents like mice. Among these diseases, tuberculosis has the oldest known association with humans [1]. Tuberculosis (TB), caused by Mycobacterium tuberculosis ( M. tuberculosis ), ranks as the ninth leading cause of death globally due to a single infectious agent, according to the 2019 report by the World Health Organization (WHO), especially in developing countries [2].

In 2016 alone, this single infectious agent claimed 1.7 million lives. Of those deaths, 22% occurred in individuals co‐infected with HIV, and nearly 5% of the 10.4 million cases involved individuals resistant to at least two first‐line tuberculosis drugs [3]. Additionally, it is estimated that approximately one‐quarter of the global population harbors latent tuberculosis infection (LTBI) [4].

M. tuberculosis is a Gram‐positive bacillus and belongs to the group of acid‐fast bacteria. It is the causative agent of human tuberculosis and is regarded as one of the most adaptable human pathogens. The bacterium employs a unique strategy to persist within the host until an opportunity for transmission arises [5]. This facultative intracellular bacterium exhibits slow growth, which hampers the ability of macrophages to clear infected cells by disrupting the natural maturation process of macrophages [3].

M. tuberculosis possesses several virulence factors that help the bacterium evade mucous barriers. Furthermore, it exploits host macrophages to invade lung tissue and other tissues, leading to long‐term granuloma formation [5]. Infection caused by M. tuberculosis results from a complex interplay between the host immune system and the bacteria. Macrophages, granulomas, and IFN‐γ produced by CD4+ T cells contribute to the protective immune response in primary tuberculosis, often referred to as the central dogma, as well as to the systemic immune response that protects the body against disseminated infection [6].

Indeed, tuberculosis is a pathogenic disease. It begins when infectious particles containing M. tuberculosis bacilli are inhaled from the air, typically originating from an individual with active tuberculosis. The bacilli then migrate to the lower respiratory tract, where they invade the alveoli and are engulfed by alveolar macrophages and other phagocytes. M. tuberculosis subsequently initiates intracellular proliferation within these macrophages [7].

Upon entering the body, M. tuberculosis often establishes a prolonged latent infection that remains asymptomatic and generally non‐transmissible. This latent infection can persist for several decades. However, with advancing age or underlying diseases, it may reactivate, leading to an active infection that presents with clinical symptoms and becomes contagious [8].

The host's resistance to M. tuberculosis infection requires the coordination of both the innate and adaptive immune systems. Various populations of myeloid cells respond to M. tuberculosis by initiating inflammation to protect the host and potentially eliminate the bacteria [9]. The immune response against M. tuberculosis primarily involves pattern recognition receptors (PRRs), which include several classes such as Toll‐like receptors (TLRs), nucleotide‐binding oligomerization domain‐like receptors (NLRs), C‐type lectin receptors (CLRs), and cyclic GMP‐AMP synthase (cGAS)/stimulator of interferon genes (STING). These receptors play a crucial role in recognizing M. tuberculosis [10]. A comprehensive understanding of the complex interactions between tuberculosis and host immunity can inform the development of vaccines and the improvement of tuberculosis treatments [11].

Early and effective treatment of tuberculosis is crucial to prevent the emergence of drug‐resistant strains. This requires the availability of rapid and reliable diagnostic methods for managing affected cases. Common techniques for screening and diagnosing tuberculosis include clinical symptom assessment, microscopic examination, lung radiographic imaging, and bacterial culture. Recent advancements in molecular diagnostic methods, such as MTBDR Plus, isothermal amplification by loop‐mediated isothermal amplification (LAMP), line probe assays (LPA), and the GeneXpert device, have enhanced tuberculosis detection and identification [12, 13, 14].

This study is significant due to the lack of a comprehensive review that thoroughly examines the importance, pathogenesis, and diagnosis of M. tuberculosis . Therefore, the aim of this review is to provide updated information on M. tuberculosis . We have investigated the bacterium's virulence factors, pathogenicity, diagnostic methods, clinical symptoms, and the various forms of tuberculosis, including cerebral, millet, nerve, and tubercular tuberculosis.

2. Virulence Factors of Mycobacterium tuberculosis

Mycobacterium tuberculosis ( M. tuberculosis ) is a member of the Mycobacterium family and is the primary cause of tuberculosis in humans. Other members of the M. tuberculosis complex, such as Mycobacterium africanum ( M. africanum ) and Mycobacterium bovis ( M. bovis ), also cause tuberculosis in humans and contribute to disease progression by suppressing the immune system. According to the latest statistics from the WHO, the mortality rate due to tuberculosis increased in 2022. Virulence factors play a crucial role in the pathogenesis of tuberculosis.

2.1. Non‐Protein Virulence Factors

This category includes lipids, glycolipids, glycans, nucleic acids, and metabolites, all of which can impact diagnostic processes, cell survival, interactions with the host's pathogenic factors, and levels of pathogenicity. The primary non‐protein virulence factors are predominantly located within the lipid‐rich cell membrane and play a significant role in the survival and virulence of M. tuberculosis . The cell wall of M. tuberculosis comprises 60% lipids. Secretory components present on or released by M. tuberculosis cells facilitate interactions with host cells [15].

Examples of non‐protein virulence factors include:

2.1.1. Phosphatidyl‐Myo‐Inositol Mannosides (PIMs)

Phosphatidylinositol mannoside is one of the most prevalent glycolipids in the cell membrane and serves as a precursor for lipomannan (LM) and lipoarabinomannan (LAM). PIMs vary in their mannose units and types of acylation. Pathogenic PIMs, particularly those with 5 or 6 mannose units, bind to the mannose receptor, facilitating macrophage uptake. PIMs with fewer mannose units specifically bind to dendritic cells via a specialized receptor known as DC‐SIGN (dendritic cell‐specific intercellular adhesion molecule‐3‐grabbing non‐integrin). Acylphosphatidylinositol mannoside (AcPIM2) is located in the inner part of the mycobacterial membrane, while AcPIM6 contributes to the maintenance and integrity of the cell membrane [15].

2.1.2. Lipomannan (LM)

Lipomannan (LM) is a polyglycosylated lipid, or polymannose phosphatidylinositol, that serves as the structural foundation of LAM. Its acylation pattern determines its function: tetra‐acylated forms can activate the innate immune response by stimulating macrophages through TLR2 and TLR4 receptors, while di‐acylated forms inhibit the production of nitric oxide (NO) and cytokine secretion in activated macrophages [15, 16].

2.1.3. Lipoarabinomannan (LAM)

Lipoarabinomannan (LAM) is a glycolipid conjugate formed by attaching multiple arabinose units to lipomannan. When lipomannan contains mannose residues, it forms ManLAM. Additionally, lipomannan coated with phosphoinositol is referred to as PILAM, while lipomannan modified with arabinofuranose is known as AraLAM [15, 16].

PIMs, LM, and ManLAM bind to receptors on antigen‐presenting cells, such as MR, DC‐SIGN, and DCAR (dendritic cell‐activating receptor). As a result, the diversity of mannose‐containing cell wall components significantly influences the pathogenicity of this mycobacterium [16].

The lectin C receptor plays a critical role in initiating phagocytosis in macrophages, dendritic cells, and neutrophils. MANLAM from M. tuberculosis protects against phagolysosome destruction in macrophages by inhibiting phagosome maturation and acidification. This process is mediated by the inhibition of phosphoinositide 3‐kinase and delayed fusion of the phagosome and lysosome. Mannan and arabinomannan share structural similarities with LM and LAM. Due to their role in capsule formation through glycans, they interact with host cells earlier than LM and LAM [17].

2.1.4. Phthiocerol Dimycocerosates (PDIM)

Phthiocerol dimycocerosate (PDIM) is located in the outer membrane of the cell and can be secreted or shed by mycobacteria. This factor enables M. tuberculosis to evade detection by TLRs and delay the acquired immune response. PDIM also plays a role in phagocytosis and disrupts both phagosomes and mitochondrial membranes [15]. While damaged phagosomes typically initiate autophagy, PDIM inhibits this process by suppressing MyD88 signaling [17].

2.1.5. Lipids and Glycolipids

Most bacteria shed components of their outer membrane and cell wall, which affect host cell biology. Lipid and glycolipid molecules can be directly transferred to host cells through secretion or membrane vesicles. These molecules help strengthen the bacterial cell wall and inhibit the formation of a host defense barrier [17].

2.1.6. Mycolic Acid

Mycolic acid is either covalently attached to the arabinogalactan layer or exists freely within the capsule. In alveolar epithelial cells, mycolic acid inhibits Toll‐like receptor 2 (TLR‐2) and reduces IL‐8 production. Free mycolic acid can also activate the DAP12‐associated stimulatory receptor on innate immune cells, leading to increased monocyte chemoattractant protein‐1 (MC‐P‐1) production and macrophage recruitment. The regulation of inflammation through IL‐8 and MCP‐1 is one of the key mechanisms by which mycolic acid influences the innate immune system [17, 18].

2.1.7. Trehalose Monomycolate (TMM) and Trehalose Dimycolate (TDM)

TMM and TDM consist of mycolic acid and a disaccharide linked by an ester bond. TMM is localized in the cell membrane by an unknown mechanism, while M. tuberculosis secretes it. Monocyte‐inducible C‐type lectin (MINCLE) functions as a pattern recognition receptor (PRR) in macrophages and dendritic cells, binding to the trehalose motif in TDM. By activating the PI3K‐AKT‐GSK3 signaling pathway, MINCLE induces the production of TNF‐α, IL‐6, MCP‐1, as well as the recruitment of neutrophils and monocytes [17].

2.1.8. Phenolic Glycolipids (PGL)

PGL consists of PDIM bound to a phenol moiety and 1–4 sugars, which are important components of the mycobacterial membrane. PGL triggers the secretion of MCP‐1, also known as CCL2. Through interaction with CCR2, CCL2 promotes macrophage recruitment in tuberculosis. By interfering with T cell receptor signaling, inhibiting inflammatory cytokines, or disrupting TLR2 function, PGL alters the immune system's functionality [17, 19].

2.1.9. Diacyl Trehalose

Diacyl trehalose (DAT) is presented to dendritic cells, leading to a decrease in IL‐12 production and an increase in IL‐10. Both DAT and triacyl trehalose (TAT) reduce the expression of inducible nitric oxide synthase (iNOS), resulting in decreased levels of nitric oxide (NO). By binding to the MINCLE receptor, acyltrehaloses induce the production of TNF‐α and other cytokines. Additionally, DAT disrupts T cell and cytokine production by activating protein kinase C (PKC) and inhibiting MAPK, providing protection against tuberculosis [17, 20].

2.1.10. Phospholipids

SL‐1, one of the most abundant lipids in M. tuberculosis , activates pain neurons in humans, resulting in a cough response. The production of this sulfolipid decreases during latent infection but increases during active infection, suggesting that M. tuberculosis regulates SL‐1 production to facilitate transmission [17, 21].

2.1.11. Glycan

Peptidoglycan and arabinogalactan in the cell membrane play a critical role in the pathogenesis and infection of M. tuberculosis by providing structural integrity and wall impermeability. Additionally, mannan, arabinomannan, and α‐glycan significantly contribute to the pathogenicity of M. tuberculosis [17].

2.1.12. Nucleic Acids

Through the VII secretion system, M. tuberculosis causes damage to the phagosome, facilitating its entry into the cytosol. Mitochondrial DNA serves as a ligand for cGAS, and the activation of STING by cGAS‐derived cGAMP leads to the production of IFN‐β and the induction of autophagy [17, 22].

2.2. Protein Virulence Factors

As an obligate intracellular pathogen, M. tuberculosis possesses specific proteins in its proteome that enhance its survival. In addition to lipids, the cell membrane contains proteins that influence the host's immune response [23]. Proteins such as PE, PEE, and lipoproteins modify the host's immune response through mechanisms including phagosome maturation arrest, phagosome escape, changes in cytokine production, autophagy, and cell death.

The ESX secretion system is crucial for pathogenicity, regulating protein secretion, and transferring proteins from the cytoplasmic membrane to the mycobacterial cell wall. The ESX gene encodes proteins such as EspB, EspA, EspC, EspG, the secreted proteins ESAT‐6 and CFP‐10, as well as PE‐PPE family proteins. It also encodes conserved components, including EccB, EccC, EccD, and MycP. The ESX‐3, ESX‐5, and ESX‐1 secretion systems in the inner membrane of M. tuberculosis facilitate the transfer of PE and PPE proteins [15, 17].

2.2.1. Lipoprotein Virulence Factors

Lipoarabinomannan carrier protein (LprG) in the cell wall of M. tuberculosis plays a crucial role in wall synthesis and the expression of LAM. This protein binds to PIM, LM, and LAM, enhancing their detection by TLRs. Additionally, it inhibits MHC II in humans, thereby affecting antigen processing in macrophages and antigen presentation. PstS1, a lipoprotein known as a phosphate transporter, reduces the production of reactive oxygen species (ROS) and induces the release of TNF and IL‐6 in human monocytes [24].

2.2.2. Kinase and Phosphatase Virulence Factors

SapM and PtpA are phosphatases that induce phagosomal arrest, which is essential for the pathogenicity of M. tuberculosis . PtpA suppresses the immune response, thereby supporting the survival and pathogenicity of Mycobacterium species [15].

Antigen protein complex 85 (Ag85) contains fibronectin‐binding proteins (FnBPs) that are crucial for cell wall adhesion. This complex is also effective in catalyzing the binding of mycolic acid to arabinogalactan. Ag85 facilitates the invasion of host cells by binding to fibronectin, tropoelastin, and elastin, which plays an important role in the intracellular environment, promoting stability and inhibiting phagosome maturation.

The factors associated with surface lipids play a significant role in the initial phase of infection, contributing to the stability of the infection. Accurately defining virulence factors is challenging due to the multitude of factors that influence the survival of M. tuberculosis . Some of these factors are crucial for the survival of tuberculosis in specific environmental conditions.

Among 500 genes, several important genes have been identified through comparative analysis, including those coding for proteins involved in the secretory system as well as lipid stability and metabolism mechanisms. The specialized ESX secretion system of this bacterium is designed to transport substances across the cell wall, which consists of an inner bilayer of phospholipids and an outer membrane known as the mycomembrane. The phospholipid membrane is connected to a polysaccharide called arabinogalactan, which separates the inner and outer membranes. In mycobaceria, five secretory systems have been identified, including ESX‐1 to ESX‐5. Among these, three systems—ESX‐1, ESX‐3, and ESX‐5—are implicated in the virulence of tuberculosis [25].

2.2.3. PE‐PGRS Family Surface Proteins

The PE‐PGRS protein is located on the surface of the Mycobacterium and is transported through the membrane by the ESX‐5 system, contributing to the formation of the type VII secretion system. Although the exact role of this protein has not been precisely identified, it is associated with the pathogenicity, persistence, and chronicity of the infection. At this stage, the protein accumulates in necrotic granulomas, which serve as sites for the activation of inflammation due to interactions with TLRs. PE‐PGRS11 and PE‐PGRS17 influence the maturation process of dendritic cells (DCs) and their ability to proliferate and produce IFN‐γ and IL‐5 in CD4+ T cells. A robust T‐cell response is generated against the epitopes in the PE domain [26].

2.2.4. ESAT‐6

ESAT‐6 (early secreted antigenic target 6) was first discovered in 1995 by Sorensen et al. when they identified a potential T lymphocyte antigen in a short‐term filtered culture medium for M. tuberculosis . This protein is encoded by the RD1 gene, which is present and expressed in M. tuberculosis . ESAT‐6 leads to the suppression and inhibition of T lymphocyte function. A significant increase in the expression of ESAT‐6 has been observed in cells infected with mycobacteria. This protein facilitates the destruction of the phagosome membrane by forming a membrane‐spanning channel.

ESAT‐6 disrupts the plasma membrane of red blood cells and causes the lysis of liposomes. It also interacts with MHC‐I and beta‐2 microglobulin (β2M) molecules in nucleated cells. By activating the STING‐TBK1‐IRF3 inflammatory pathway, ESAT‐6 promotes inflammation and aids in the elimination of M. tuberculosis , thereby enhancing the immune response [27].

As a small and potent pathogenic factor, ESAT‐6 induces an immune response. Initially recognized as a T cell antigen, it is now identified as a pore‐forming toxin, playing a crucial role in the pathogenesis of M. tuberculosis [28].

ESAT‐6 is secreted through the ESX‐1 type secretion system or the type VII secretion system of the bacterium. This antigen facilitates the transfer of M. tuberculosis into the cytoplasmic environment of host macrophages. The pathogenicity of M. tuberculosis is directly related to ESAT‐6, with its activity diminishing upon inactivation of this factor. ESAT‐6, either alone or in combination with CFP‐10, results in a decreased immune response (Figure 1).

FIGURE 1.

ESAT‐6 and CFP‐10 of M. tuberculosis that are present in the bloodstream and lead to a stronger immune response.

2.2.5. CFP‐10

The combination of ESAT‐6 and CFP‐10 activates neutrophils and induces Ca²⁺ release from cells. Both ESAT‐6 and CFP‐10 are encoded by the ESXA (RV3875) and ESXB (RV3874) genes, respectively. These proteins are crucial for the pathogenicity and growth of M. tuberculosis (Figure 1).

When M. tuberculosis infects macrophages, these cells secrete cytokines, including TNF‐α, IL‐12, IL‐6, IL‐1α/β, and the IL‐10 family, which are essential for controlling the infection. The ESAT‐6 and CFP‐10 proteins disrupt the processes of programmed cell death (apoptosis) in macrophages and the maturation of phagosomes. A few days after evading the host's immune defenses, M. tuberculosis multiplies to spread the infection throughout the body. Studies indicate that ESAT‐6 helps M. tuberculosis escape phagocytosis. Notably, ESAT‐6 and CFP‐10 are produced only during the initial stage of infection. These proteins can also stimulate the production of cytotoxic T lymphocytes in response to interferon‐γ (IFN‐γ) production [29].

3. Disease Process

3.1. Entry Mechanism

M. tuberculosis is a facultative intracellular pathogen that requires adhesion to macrophages and subsequent multiplication within them to establish infection [30]. The most fundamental stage of entry and pathogenicity is the host‐pathogen interaction, which demonstrates the specific adhesion components utilized by the pathogen to facilitate bacterial entry into host cells. Many bacteria have evolved specialized adhesion proteins that bind to components typically found on the surface of host cells [31].

Mycobacterium has several fibronectin‐binding proteins (FnBPs) associated with the antigen 85 complex, which consists of three proteins: 85A, 85B, and 85C. As previous studies have shown, the binding of FnBP to fibronectin (Fn) is biologically significant in the phagocytosis of M. tuberculosis [32, 33].

M. tuberculosis primarily enters the human body through respiration. When a person with M. tuberculosis coughs or sneezes, the bacteria are released into the air as small droplets known as aerosols. If a susceptible person inhales these airborne particles, the bacteria can reach the alveolar pathways of the lungs, where they encounter resident macrophages, a type of immune cell. The bacteria are then engulfed by macrophages, allowing them to evade the immune system and establish an infection [34].

Particles containing M. tuberculosis can be categorized into two groups: small and large particles. Larger particles are more likely to be trapped in the proximal airways, while smaller particles can reach the distal regions of the lungs. Although the current paradigm suggests that tuberculosis begins with a primary infection of alveolar macrophages (AMs), alternative routes of M. tuberculosis entry may occur through epithelial cells or mucosa‐associated lymphoid tissue (MALT) [35].

Macrophages play a crucial role in the initial response to M. tuberculosis infection, as they can either contain the bacteria or serve as a site for bacterial proliferation. The bacteria enter macrophages through cell surface molecules, including complement receptors of the integrin family, such as CR1 and CR3 [36, 37].

Several factors contribute to the ability of M. tuberculosis to survive and cause disease within the host. This bacterium can manipulate host cell signaling and metabolism to enhance its survival. For example, it can detoxify reactive oxygen species and alter host signaling through the secretion of various molecules [38].

The findings indicate that M cells serve as the entry point for M. tuberculosis . A decrease in M cells is associated with reduced lymph node proliferation, decreased dermal responses, and lower levels of aerosclerosis, which is also linked to atrophy [39].

Once the tuberculosis bacillus enters the lung alveoli, some bacteria may enter the bloodstream and disseminate throughout the body. Typically, within 2–8 weeks, the immune system intervenes and halts replication, preventing further spread. At this stage, a person with a latent TB infection (LTBI) is classified as such because the immune system has contained the infection and is keeping the TB bacteria under control. Individuals with latent tuberculosis infection do not feel sick, exhibit no symptoms of the disease, and are not considered contagious [40].

Tuberculosis primarily affects the lungs, but it can also impact other parts of the body. The bacteria can spread through the bloodstream or lymphatic system, potentially leading to active tuberculosis. This can affect various organs, including the lymph nodes, bones, kidneys, brain, spine, and skin. The risk of contracting tuberculosis is highest for individuals in close contact with infected persons [41].

Understanding the entry mechanisms of M. tuberculosis is crucial for developing effective strategies to combat tuberculosis. The bacterium's entry into host cells, particularly macrophages, is a critical step in establishing infection. Upon entering host cells, M. tuberculosis employs various survival mechanisms, such as preventing phagosomal maturation and utilizing immune evasion strategies to persist and reproduce. Gaining insight into these mechanisms is essential for developing anti‐tuberculosis agents, vaccines, and new drugs to address this global epidemic that claims millions of lives each year [42].

3.2. Different Phases of the Disease

According to statistics from the World Health Organization (WHO), tuberculosis ranked among the top 10 causes of death and was the leading infectious cause of death worldwide in 2022. In 2019, the WHO reported over 10 million tuberculosis cases and 1.4 million deaths. These figures indicate that M. tuberculosis is the second leading cause of death, following the COVID‐19. The persistent prevalence of tuberculosis and high mortality rates can be attributed to factors such as inadequate control of infection sources, latent tuberculosis infection, and the difficulty in detecting active tuberculosis infection (aTB) as distinct from its latent form [17, 43].

The WHO defines latent tuberculosis infection (LTBI) as the asymptomatic immune response to the pathogen. In this context, “latency” refers to the pathogenic agent being dormant under conditions unfavorable for growth and reproduction. During this phase, LTBI demonstrates resistance to anti‐tuberculosis drugs while maintaining its ability to survive. Even after treatment with high doses of anti‐tuberculosis medications that target dividing and active cells, this infection can remain asymptomatic. Following the initial interaction between M. tuberculosis and host cells, the pathogen can either be eliminated or establish a primary infection [44].

M. tuberculosis can persist for extended periods under unfavorable environmental conditions until it encounters optimal conditions for proliferation and becomes active. Tuberculosis can exist in two stable states: the first is the L‐form, which lacks a cell wall and exhibits metabolic rates, reproduction, and growth similar to those of normal cells. The second state consists of latent cells that reside in low‐oxygen environments alongside a proportion of M. tuberculosis bacilli. The distinction between active and latent tuberculosis depends on the duration of latent tuberculosis infection (LTBI). During this period, M. tuberculosis bacilli slow their reproduction rate and eventually become inactive. Granulomas play a significant role in the inactivation of M. tuberculosis . Despite these conditions and even tuberculosis chemotherapy, these bacilli manage to survive. In LTBI, the number of dividing bacilli is low, but it is sufficient to trigger an active infection [44].

LTBI is characterized by a latent state in which immune responses are confined to granulomas and protected sites. The probability of progression from latent to active tuberculosis within the first 2 years of infection is approximately 5%–15%. Additionally, a weakened immune system—due to factors such as aging or underlying conditions like cancer, HIV, diabetes, kidney failure, or viral infections—can increase this probability by an additional 5%. During latent infection, M. tuberculosis exhibits a reduced mutation rate, production time, and metabolic activity. Moreover, the mutation rate and the likelihood of developing active tuberculosis decrease one to two years after transmission. Furthermore, children under 5 years of age are at a higher risk of progressing from latent to active infection [17, 45].

Latent tuberculosis infection (LTBI) is a subclinical or asymptomatic condition. Tuberculosis represents a complex interaction between the host's immune response and Mycobacterium tuberculosis (commonly known as TB). During this stage, there is a delicate balance between the host's immunity and the aggressive nature of the pathogen, and any disruption to this balance can have significant consequences. Immune responses that target and kill the bacteria play a crucial role in determining whether the disease progresses from the latent to the active phase. Upon inhalation, M. tuberculosis targets alveolar epithelial cells in the respiratory system, which are key players in the pathogenesis and dissemination of the infection. Failure to differentiate between the latent and active phases of TB can hinder efforts to prevent progression to active disease [43, 44].

Multifunctional CD4+ T cells, which express IFN‐γ, TNF‐α, and IL‐2, are associated with protective responses against M. tuberculosis antigens. Memory CD4+ T cells, found in the bronchoalveolar fluid of individuals with latent infection, also contribute to these protective responses. The production of these cytokines promotes phagosome maturation, the release of bactericidal agents, and the induction of autophagy. In latent infection, CD4+ regulatory T cells (Tregs) and TH17 cells respond to M. tuberculosis , while high levels of natural killer (NK) cells are critical for controlling latent TB infection [46].

Active tuberculosis (aTB) presents with clinical symptoms, radiological changes, and microbiological evidence. The severity of the disease is linked to increased symptoms, reduced spontaneous healing, poor immune response, and heightened bacterial shedding [44]. Certain factors, such as underlying illnesses or immunosuppressive drugs, elevate the risk of latent infection progressing to active disease [47].

Immunological tests, such as the tuberculin skin test (TST) and interferon‐gamma release assays (IGRAs), are used to detect both latent and active TB infections. However, these tests are not reliable in predicting the risk of progression to active infection in individuals with latent TB (Figure 2) [17].

FIGURE 2.

The mechanism of infection by Mycobacterium tuberculosis .

3.2.1. Pulmonary Tuberculosis

The lungs are the primary target of TB infection, with an estimated 79%–87% of individuals with active tuberculosis experiencing lung involvement. This estimate is similar in immunodeficient individuals, such as those infected with human immunodeficiency virus (HIV) [48].

Studies on tuberculosis pathogenesis have been conducted using animal models, particularly different strains of rabbits, due to the similarity between tuberculosis in rabbits and humans. Newborns, immunosuppressed patients, and individuals with AIDS are considered to exhibit a form of tuberculosis comparable to that seen in susceptible Lowry rabbits, while the immune response in adult humans is likened to that of resistant Lowry rabbits. Pulmonary tuberculosis progresses through five stages, beginning with primary infection and culminating in the involvement of alveolar macrophages and cavity formation (Figure 2) [49].

Clinical symptoms of pulmonary tuberculosis can be nonspecific and develop gradually. Key symptoms include a persistent cough lasting at least 3 weeks, chest pain, coughing up blood or sputum, shortness of breath, fever, night sweats, weight loss, fatigue, and loss of appetite. If left untreated, pulmonary TB can be life‐threatening, but it is treatable with antibiotics, especially when detected early. Systemic complications of pulmonary tuberculosis may include hyponatremia and glucose intolerance, which might not be immediately apparent to the patient. Extrapulmonary TB, where the infection spreads to other parts of the body, can present with symptoms specific to the affected organ, such as fever, chills, and localized pain [50, 51].

According to an initial investigation, the most common clinical manifestations of pulmonary tuberculosis include fever in 56.3% of patients, cough in 47.2%, weight loss in 45.4%, night sweats in 27.2%, and shortness of breath in 25.4% of patients. The majority of cases presented with pulmonary miliary tuberculosis (MTB) (69.1%), followed by tuberculosis lymphadenitis (38.2%), central nervous system involvement (23.6%), and skeletal involvement (20%). Additionally, gastrointestinal involvement was observed in 9.1% of cases, pleural involvement in 5.5%, and genital warts in 3.6%. The mortality rate among patients with pulmonary tuberculosis was 25.5% [52].

3.2.2. Miliary Tuberculosis

Miliary tuberculosis is a severe and disseminated form of the disease, in which bacilli spread to various parts of the body via the bloodstream. It is characterized by small, round, uniform lesions in the lungs and other organs. Initial clinical symptoms are generally nonspecific and gradually worsen, including fever, weakness, and loss of appetite. Night sweats are also a common symptom [53, 54]. Adrenal insufficiency (Addison's disease) may develop during the onset of symptoms and persist throughout anti‐tuberculosis treatment. Complications of miliary tuberculosis can include myocarditis, congestive heart failure, endocarditis, and kidney damage, though these are rare in children who have received the BCG vaccine [53]. Miliary tuberculosis can affect various organs, including the liver, spleen, bone marrow, lungs, and meninges. One contributing factor to miliary tuberculosis is the inadequate response of T lymphocytes in suppressing bacilli. Although previously considered a childhood disease, miliary tuberculosis has been increasingly observed in adults over the past three decades [54, 55]. In some cases, miliary tuberculosis can lead to acute respiratory distress syndrome (ARDS). Radiology is the most effective diagnostic tool for identifying miliary tuberculosis, though sputum smear, acid‐fast staining, and histopathology are also commonly used for diagnosis [55, 56, 57].

3.2.3. Tuberculosis Cavities

Tuberculous cavities are commonly observed in clinical cases of tuberculosis, making individuals with these cavities highly contagious. There is potential for pathogenic elements, such as ESSX‐1, to contribute to the development and transmission of tuberculous cavities, although further research is needed to confirm this hypothesis [38].

Another clinical finding is caseous ulcers, which may follow one of three paths: they may heal or become stable; they may enlarge and release bacilli into the bloodstream or lymph nodes; or they may liquefy and form tuberculous cavities. The formation of these cavities can lead to rapid and unrestricted bacterial growth, facilitating the entry of bacilli into the bronchial passages and potentially enabling further spread of the infection [58].

It has also been shown that the formation of these cavities is caused by the destruction of collagen in the lungs, leading to the development of tuberculosis cavities. The cavity wall consists of an outer layer of collagen and a soft inner area of caseum. Due to the high oxygen content in this region, bacilli proliferate intensely. Several proteases responsible for pulmonary collagen degradation have been identified, with MMP1 protease and cathepsin being the most important [59].

Observations suggest that bacilli grow within macrophages on the surface of tuberculosis cavities, which contradicts the general belief that bacilli multiply in an extracellular environment [60]. Tuberculosis cavities have a poor prognosis; if visible on a chest radiograph during the first two months of treatment, they are associated with an increased risk of treatment failure and disease recurrence [59]. The relationship between cavity formation and recurrence may be explained by the poor penetration of drugs into the cavities and their limited blood supply [61].

Recent studies have shown that individuals with tuberculosis pose a significant risk to society. A higher bacterial burden has been observed in the sputum samples of those with tuberculous cavities, contributing to increased coughing during treatment. A variety of factors influence the contagiousness and high mortality associated with tuberculosis. The high‐oxygen environment within the center of the cavities provides an ideal condition for bacterial proliferation, resulting in a large volume of bacilli within the cavities. This is a critical factor in the transmission of the disease to others and the risk of treatment failure [61].

3.2.4. Skin (Cutaneous) Tuberculosis

Tuberculosis infection, primarily caused by Mycobacterium tuberculosis , most commonly affects the lungs. When it affects the skin, it is referred to as cutaneous tuberculosis. This is a rare disease, and it's first reported case dates back to 1826 when Laennec described a lesion on his own hand. However, the causative organism was not identified until 1882 when Robert Koch discovered M. tuberculosis . Cutaneous tuberculosis can also be caused by Mycobacterium bovis and the BCG vaccine (bacillus Calmette Guerin) [62, 63].

Cutaneous tuberculosis presents with various clinical manifestations, including inflammatory papules, red plaques, purulent nodules, chronic ulcers, and other atypical lesions such as cellulitis, untreated ulcers, subacute or chronic nodular lesions, abscesses, superficial lymphadenitis, lesions on varicose veins, and other findings. Infections with mycobacteria in the skin and subcutaneous tissue have been associated with deformities and disabilities [64, 65].

Humans come into contact with different types of mycobacteria due to their presence in various environments. In many individuals, pathogenic mycobacterial species evade the first line of defense of the innate immune system, leading to the modulation of phagocyte activation and the development of skin and soft tissue diseases. In some cases, these infections can become disseminated. Cutaneous mycobacterial infections can exhibit a wide range of clinical manifestations, which can be categorized into four main disease types: cutaneous manifestations of M. tuberculosis infection, Buruli ulcer caused by Mycobacterium ulcerans and other slow‐growing mycobacteria, leprosy caused by Mycobacterium leprae , and skin infections caused by the rapid growth of Mycobacterium lepromatosis [65].

Clinical manifestations of skin involvement can occur due to the inoculation of a foreign agent, continuous spread from a nearby focus of infection, or hematogenous spread from a distant focus. In the case of inoculation of the exogenous agent of tuberculosis, such as the entry of Mycobacterium into the skin or mucosa, damage is required because the acid‐fast bacillus (AFB) cannot penetrate the intact natural skin barrier. After 2–4 weeks, as the organism multiplies in the skin, a tuberculous chancre slowly develops, which is initially hard and lumpy [66].

Continuous spread from a nearby focus of infection refers to the development of a skin infection with tuberculosis from continuous involvement of the skin on the subcutaneous surface. This can include tuberculous lymphadenitis, tuberculosis of the bones and joints, or may be secondary, such as tuberculous epididymitis [67].

Hematogenous spread from a distant focus refers to cases where AFB spreads from the primary site of infection to the rest of the body. Most cases of hematogenous cutaneous tuberculosis occur in patients with a background of high susceptibility to tuberculosis. The most common form of this infection is lupus vulgaris, which has a high potential for transformation [68].

Diagnosing these lesions can be challenging due to their similarity to other skin diseases. Treatment usually involves multidrug therapy with antituberculosis drugs such as pyrazinamide, rifampin, ethambutol, and isoniazid. Despite the high prevalence of tuberculosis worldwide, the prevalence of cutaneous tuberculosis is relatively low [69].

3.2.5. Neurological Tuberculosis

Tuberculosis affecting the central nervous system is considered one of the most severe forms of extrapulmonary tuberculosis [70]. Involvement of the central nervous system is less frequent compared to other organs in cases of extrapulmonary tuberculosis. Neurological tuberculosis typically arises in around 10% of patients with tuberculosis due to hematogenous spread from a primary focus [71]. The condition is commonly observed in three states: subacute, acute meningitis, and intracranial tuberculosis [72]. Additionally, depending on the site of infection, tuberculosis can be further categorized as tuberculous meningitis, tuberculous encephalopathy, tuberculous vasculopathy, central nervous system tuberculosis, tuberculous brain abscess, spinal tuberculosis, spinal meningitis, and intraspinal tuberculoma [73]. Individuals with the subacute type generally experience a progressive febrile illness. The disease initially presents with symptoms such as lethargy, weakness, mild fever, periodic headaches, and mood changes. Within 2–3 weeks, the disease enters a new phase characterized by symptoms including mild confusion, vomiting, and prolonged headaches. It is during this phase that the disease can rapidly progress to the paralysis phase, which includes seizures, coma, hemiparesis, hemiplegia, and multiple central nervous system defects [72].

In cases of intraspinal tuberculosis, patients typically exhibit compression myelopathy or Cauda equina lesions without any detectable spinal defects upon examination, leading to their classification as having spinal tumor syndrome [74]. Additionally, individuals with neurotuberculosis may exhibit radiographic evidence of pulmonary tuberculosis even in the absence of pulmonary tuberculosis symptoms. Studies have shown that 10% of individuals with neurotuberculosis have a history of past tuberculosis [70].

In the majority of patients with neurological tuberculosis, there is a period of 2–8 weeks of vague illness before the emergence of more pronounced neurological symptoms [75]. These symptoms often include lethargy, fatigue, anorexia, fever, and headache. As the disease progresses, more neurological symptoms manifest, culminating in a state of deep coma and severe muscle contractions [75].

3.2.6. Cerebral Tuberculosis (Cerebral Meningitis)

Cerebral tuberculosis is the most severe form of tuberculosis, characterized by significant morbidity and mortality, as well as severe neurological complications [76]. The development of cerebral tuberculosis occurs when M. tuberculosis infiltrates the subcranial space, triggering a secretory inflammatory response that is further complicated by the presence of the cerebrospinal fluid barrier [76]. Most patients with cerebral tuberculosis have a history of nonspecific illness preceding the onset of meningitis symptoms by 2–3 weeks. These nonspecific symptoms include malaise, anorexia, fatigue, muscle pain, and headache. Adults with tuberculous meningitis often present with typical symptoms of meningitis, such as fever, headache, neck stiffness, cranial nerve deficits, mood changes, and alterations in consciousness [73]. The clinical presentation and severity of symptoms can vary depending on several factors. Fever and headache are the most significant clinical findings, reported in over 80% of affected patients [77]. In addition, vomiting has been reported as a clinical symptom in adults with tuberculous meningitis [76].

It should be noted that 10% of patients with tuberculous meningitis have a history of tuberculosis, and 30%–50% of them have evidence of active pulmonary tuberculosis based on chest X‐ray findings. The incidence of tuberculous meningitis remains unchanged in patients with HIV. Previous studies and data suggest that approximately 10% of cases of tuberculous meningitis are also affected by spinal tuberculosis [73].

Tuberculous meningitis is the most severe form of extrapulmonary tuberculosis and has a longer duration of clinical symptoms compared to other forms of bacterial meningitis, usually lasting up to one month [76, 77]. Unfortunately, the prognosis is poor if the diagnosis is delayed [68]. Therefore, early diagnosis is crucial to ensure prompt and effective implementation of available treatment options [78].

4. Diagnosis

4.1. Diagnosis of Active Tuberculosis

Despite the complexity of this disease and the lack of an effective vaccine, this bacterium is considered a threat to the health of the world. According to WHO statistics and information, every third person has asymptomatic tuberculosis. Therefore, rapid diagnostic methods can be helpful [79, 80].

4.1.1. Microbial Observation

Cultivation in the right environment and examination with a microscope is the gold standard for TB diagnosis. Examples include sputum or laryngeal swabs from the suspect. For sample decontamination, 7% saline and 4% NaOH are used. Suitable media are Middlebrooks 7H10 and Middlebrooks 7H9. In order to increase sensitivity, sputum can be centrifuged with sodium hypochlorite and ammonium hydroxide. M. tuberculosis is resistant to acid fast staining due to the richness of the cell wall in lipids. Therefore, the most common method is Ziehl‐Neelsen. Another method includes cold coloring, in these 2 coloring methods, basic colors such as Fuchsine, methylene blue, and phenolic compounds are used. This method leads to dye penetration into the bacillus cell wall. Bacilli are seen under the microscope in red color, because the primary color (carbol fuchsia) is kept in the cell wall due to the presence of mycolic acid, but they do not recognize the blue color. The best example for this method is the ileocecal mucosa. If the result is positive, 100% of the person has intestinal tuberculosis. Due to the low sensitivity of this method, the possibility of reporting false negative results is high. Fluorescent microscopes can be used to improve diagnosis [79, 80].

4.1.2. Pulmonary Radiology

M. tuberculosis damages many organs of the body, the most common manifestation of which is the lung. Organs other than the lungs, lymph nodes, heart, and skeletal muscles are also involved. Radiological imaging of these organs is essential. Imaging methods include (CT) scan, (PET‐CT), chest X‐ray, and MRI. Tuberculosis patients suffer from coughing with bloody sputum, and they also suffer from pulmonary emphysema, bronchial deviation, and pleural thickening. X‐ray of the chest is considered as the initial test to check the extrapulmonary cavity and coughs. X‐rays are used for general evaluation of the lungs and pleural membrane. CT is considered as a method to investigate the formation of lesions, pleural effusion, and thickening of the lobular septum position. These lesions disappear in affected people within 5–9 months. With the reactivation of tuberculosis, these lesions are seen followed by fibrosis. This method is a differential method for diagnosing tuberculosis from pneumonia. MRI is a better method than CT because the patient is less exposed to radiation. MRI can detect thoracic lymphadenopathy, necrosis, inflammation of the pleural membrane, and high sensitivity of the chest wall. PET SCAN is used to detect pulmonary and extrapulmonary granuloma in tuberculosis infection. PET and SPECT have been combined with imaging by MRI and CT and have created a non‐invasive diagnostic method. In this method, biochemical changes in tuberculosis are also investigated. Immune cells consume glucose during infection for inflammatory reactions, and this change in glucose consumption can be observed with high sensitivity using 18F‐FDG PET method. This method can accurately predict the possibility of re‐infection with M. tuberculosis investigated as well [79, 81].

4.2. Diagnosis of Latent Tuberculosis

LTBI is a subclinical infection, which may revert to an active state. The lack of differential diagnosis of active and inactive TB is very important to control mortality from latent TB. In the case of treatment with anti‐tuberculosis drugs, the chance of the disease progressing to the active phase decreases [79, 80, 82].

4.2.1. Tuberculin Skin Test (TST)

This test is an example of the response of T cells sensitive to M. tuberculosis antigens. This test is done to check the prevalence of latent tuberculosis. Another name of this test is MANTOUX. The cellular immune response in this test is caused by the presence of antigens of this bacterium in the body. TST is based on delayed skin sensitization (DTH) to pure protein derivative (PPD). Pure protein derivative (PPD) is injected to the patient. Forty‐eight to 72 h after the injection, the person experiences edema and fibrin deposition due to the migration of lymphokine cells. The injection dose of PPD, for diagnosis, is 5 IU (each IU is equal to 0.028 μg of PPD). The results are reported by measuring the induration diameter of the injection area. Five‐ and 10‐mm diameter if the person with M. tuberculosis can be considered positive. The diameter of induration is not related to the chance of developing active TB infection in the future. This test is not specific; therefore, with BCG vaccine and infection with non‐tuberculosis mycobacterium, the test result is falsely reported as positive. These results are due to the cross‐reaction with the homologous antigen caused by the BCG vaccine. Also, this method reports false negative results for patients with suppressed immune system [79, 80, 82].

4.2.2. Interferon Gamma Release (IGRAs)

The basis of this test is the immune response of an individual with a strong immune system and suffering from M. tuberculosis . After encountering the pathogenic agent, until re‐encountering the antigen and activating the infection, memory T cells remain active and T cells process pathogenic antigens. T cells produce lymphokines such as IFN‐γ. ESAT‐6 and CFP‐10 of M. tuberculosis are present in the bloodstream and lead to a stronger immune response. Therefore, it is considered as 2 important targets in measuring interferon gamma release. If the person does not respond to these two antigens, protein C from ESX‐1 (EspC) is used. The performance of this test is affected by several factors such as immune system response disorders. For example, the addition of IL‐7 leads to a positive test. The level of IFN‐γ is measured by absorbance at 450 nm, but the performance and result of IGRA can be affected by the disease. Crohn's disease is an immune‐mediated inflammatory disease (IDIM). In this disease, immune cells are suppressed and have an inhibitory effect on T cells. By detecting transient changes in cellular immunity, it is possible to check the possibility of developing an active infection. According to the studies, if the level of IU in the blood is 1 IU/mL, 5 IU/mL, and 15 IU/mL, the chances of contracting active tuberculosis are 2.9, 10.38, and 21.82 times higher, respectively. Types of IFN‐γ release assays include QuantiFERON‐TB and ELISPOT. QuantiFERON‐TB is the gold test, which is incubated with ESAT‐6 and CFP‐10 antigens. These two antigens are epitopes of CD4+ and CD8+ T cells and lead to the release of IFN‐γ from T cells, especially CD4+ T cells. Lymphocytes release IFN‐γ when exposed to mycobacterial antigens. ELISPOT identifies the person infected with M. tuberculosis. The number of spots in this test is directly related to tuberculosis infection. If the number of spots is low, it indicates latent tuberculosis infection, and conversely, a large number of spots indicate active tuberculosis infection [79, 83].

4.2.3. ELISA

Cellular immunity in humans and a pattern of humoral response against M. tuberculosis is shown. The antibody level is used to differentiate latent and active TB infection. For example, IgG against membrane protein (Rv2626c), IgM against membrane antigen, and IgA against alpha crystallin (Acr) are more in latent TB infection than in active infection [79].

4.2.4. Urinary Lipoarabinomannan Test

Considering the difficulty of accurately diagnosing tuberculosis in children due to nonspecific clinical symptoms, the low accuracy of X‐ray, and the difficulty of sputum sampling, this test is considered the gold standard. LAM is a water‐soluble lipoglycan and a TB biomarker. LAM is excreted in urine due to poor T cell response and IL‐12 in children. According to WHO recommendations, LAM is used for patients with tuberculosis and co‐infection and HIV and children with CD4+ T cell counts less than 100 cells per cubic millimeter. The specificity and sensitivity of this test are high in children infected with tuberculosis and HIV due to kidney disorders and LAM excretion. In TB and HIV‐negative patients, LAM protein cannot be measured because it is hidden due to the formation of an immune complex with non‐LAM proteins. Analyzing and checking the results of this method is done with a mass spectrometer. By consolidating monoclonal antibodies against LAM covered with mannose on a 200 nm thick gold layer, an immunoassay is created by the sandwich method. In the next step, the lipoglycan is characterized by the second antibody through nanoparticle Raman spectroscopy (SERS). Another method for HIV‐infected children with tuberculosis is the use of EBC testing as exhaled air condensate [79, 84, 85].

4.3. Diagnosis of Multidrug‐Resistant Tuberculosis (MDR‐TB)

Multidrug resistance of tuberculosis means the resistance of active tuberculosis infection to rifampin and isoniazid drugs. The increase in resistance is due to the lack of drug sensitivity testing [79, 86].

4.3.1. Phenotypic Drug Sensitivity Test (DST)

It is considered as the gold standard in the diagnosis of drug sensitivity. This test is performed by checking the survival and metabolic inhibition of bacilli in environments containing antituberculosis drugs. Metabolic activity is a good measure, but it takes a long time for a confirmatory test to be performed. Therefore, liquid environments are used more than solid environments. This method increases the possibility of infection with non‐tuberculosis mycobacteria [14, 79].

4.3.2. Molecular Drug Sensitivity Test

This method targets genes causing drug resistance. This test includes LATE PCR (with on and off probes) and ligase chain reaction. LATE PCR is an advanced method, which uses different fluorescent dyes to investigate nucleotide substitutions. In PCR, the temperature decreases and DNA molecules are not accessible to the probe. In cases with mutations, there is a temperature change. Rifampicin drug resistance is measured by this method. Ligase chain reaction can identify single nucleotide polymorphisms (SNPs) responsible for isoniazid and rifampin mutations [79].

4.3.3. Probe‐Based Measurement

It is a revolutionary method in the diagnosis of multidrug‐resistant TB because PCR is used to amplify gene mutations. This method is used only in case of drug treatment failure. Some of the assays to detect mutations in rifampicin and isoniazid and WHO approved include: Line probe assay (LPA), Gene Xpert, and GenoType MTBDR. Gene Xpert is an automated test based on PCR that has high sensitivity and specificity.

Line probe assay (LPA) is a method that extracts the patients' DNA and amplifies the location of the gene mutation using the appropriate primer. The considered genes include rpoB, inhA, and katG. If there is a mutation in these genes, the probe will not bind to the specific region. Xpert MTB/RIF is a commercial form of LPA, which detects extrapulmonary TB in addition to MDR‐TB. In this method, reagent buffer is used to inactivate the bacilli and liquefy the patient's sputum sample. It can detect mutations associated with fluoroquinolones and second‐line injectable drugs, kanamycin, amikacin, and capreomycin. Positive results indicate bacillus resistance to first‐line rifampicin drugs, and negative results are caused by bacillus sensitivity to the drug, and indeterminate results cannot evaluate bacillus sensitivity or resistance [13, 79, 80, 85, 87].

4.3.4. DNA Microarray

This technique involves designing an oligonucleotide sequence complementary to 16S rRNA, enabling the precise identification of 17 different mycobacterial species. Additionally, DNA microarray technology utilizes specific gene sequences at designated nucleotide sites to detect mutations in the inhA, katG, and rpoB genes of Mycobacterium tuberculosis , identifying resistance to INH and/or RFP. Compared to other molecular diagnostic techniques, this method offers a simpler and more cost‐effective approach [79, 88, 89].

4.3.5. Lucifer Reporter Assay

A mycobacteriophage engineered to carry the luciferase gene for infecting M. tuberculosis has been developed. The bacteriophage genome contains both an antibiotic resistance gene and the firefly luciferase gene. As the phages multiply, they also infect M. tuberculosis . The presence of antibiotic resistance is determined by assessing the light emission from the population resulting from the growth of M. tuberculosis . When antibiotics target the cell wall of the bacillus, the luciferase gene is not expressed, preventing the infection of M. tuberculosis and leading to a substantial decrease in light intensity [79].

4.3.6. Loop‐Mediated Isothermal Amplification (LAMP)

This colorimetric method is designed for identifying genes associated with antibiotic resistance to Isoniazid, Rifampicin, Amikacin, and Ciprofloxacin medications. The targeted genes include katG, inhA, rpoB, rrs, gyrA, and gyrB. In this technique, when the designated sequence hybridizes with the specific probe, the DNA strands undergo extension, and the resulting product is displaced by the elongated circular primer. Consequently, the amplified product binds to the biotinylated loop‐mediated isothermal amplification (LAMP). The allele‐specific primer, known as the FIP forward primer, displaces and extends the biotinylated LAMP. Hence, these mutations are identified through the synthesis of LAMP and the introduction of a specific primer tailored to the mutated allel [79].

4.4. Advanced Methods of Tuberculosis Diagnosis

4.4.1. Polymerase Chain Reaction (PCR)

The polymerase chain reaction is a technique employed to produce multiple copies of a specific nucleic acid segment. Central to the PCR process is the essential function of polymerase, responsible for generating new DNA strands that match the template. This method involves three key phases: DNA extraction, amplification, and DNA detection. Multiplex PCR enables the amplification of several gene sequences from the patient sample, thereby decreasing the likelihood of false negative results when contrasted with traditional PCR methodologies [80, 86].

4.4.2. Digital PCR

Polymerase chain reaction on respiratory droplet samples represents the third generation of PCR, accurately quantifying the nucleic acid content in the sample. The collected samples are partitioned into specific chambers containing the target genes, with some cells lacking these targets. Subsequently, respiratory droplets are amplified using fluorescent probes such as fluorescein amidite (FAM), hexachlorofluorescein (HEX), or Eva Green. Following the amplification of approximately 20,000 droplets, they are suspended in a water and oil emulsion. Each droplet may contain multiple genes or none at all. Once gene amplification is completed, the droplets are transferred to an analyzer. The target genes are categorized into positive and negative groups based on the colors emitted by the fluorescent dye. A positive outcome indicates the presence of the desired gene.

Digital PCR is employed to explore the expression of mutated genes and the resistance mechanism to 5′‐fluorouracil (5′FU). Mutations in the upp and pyrR genes lead to resistance to 5′FU. Furthermore, this method is capable of detecting extrapulmonary tuberculosis despite the limited DNA quantity. In contrast to conventional drug susceptibility testing (DST) methods, it offers rapid results within a timeframe of 5 h [79, 87].

4.4.3. Next Gene Sequencing

This technique is considered a sophisticated method for amplifying millions of DNA molecules, enabling the expansion of genes such as rpoB, embB, pncA, rpsA, gyrA, gyrB, rrs, and eis. Its high sensitivity to drug resistance mutations related to isoniazid, streptomycin, ofloxacin, levofloxacin, and moxifloxacin sets it apart. Initially, nucleic acid is extracted from the patient sample using a specialized kit. The extracted nucleic acid is then hybridized to multiple oligonucleotides' complementary sequences on microbeads. Subsequent DNA polymerase action results in the formation of double‐stranded DNA. The amplified DNA sequence is converted into single‐stranded DNA through denaturation and annealing. Rolling circle amplification is employed to detect single nucleotide polymorphisms (SNPs) [79, 87].

4.4.4. Detection of MicroRNA (miRNA)

MicroRNAs are short single‐stranded RNA molecules typically composed of 18 to 25 nucleotides. They play a crucial role in various cellular processes such as cell differentiation and innate and adaptive immunity. The expression of genes in macrophages and NK cells triggers the production of microRNAs. MicroRNAs are also implicated in the regulation of autophagy and apoptosis induced by M. tuberculosis , achieved through the modulation of gene expression, particularly miR‐33, which targets ATG5, LC3B, LAMP, miR‐155, miR‐223, and miR‐17‐92 genes. Furthermore, the production of cytokines and chemokines is influenced by pathways regulated by microRNAs. For instance, miR‐223 plays a key role in enhancing neutrophil production and differentiation during M. tuberculosis infection.

Distinguishing between latent TB infection and active infection can be achieved by assessing the distinct expression patterns of microRNAs. In latent tuberculosis infection, genes like let‐7e‐5p, let‐7d‐5p, miR‐450a‐5p, and miR‐140‐5p are notable, whereas in active tuberculosis infection, up‐regulation of miR‐1246, miR‐2110, miR‐370‐3p, miR‐28‐3p, and miR‐193b‐5p genes is observed [79].

4.4.5. Whole Genome Sequencing

This approach involves the cultivation and extraction of DNA from the patient sample, followed by the isolation of colonies carrying the target gene within a synthetic carrier bacterium. Additionally, this method enables the straightforward detection of single nucleotide polymorphisms and the prediction of drug resistance [79].

4.5. Nanomaterial Detection With Nanoparticles

Nanotechnology is recognized as a rapid and efficient technique for tuberculosis diagnosis. Nanoparticles can be utilized for the detection of both viral and bacterial infections. The interaction between nanoparticles and biomolecules generates a measurable signal, facilitating accurate diagnosis [79].

4.5.1. Biosensor Based on Nano Detection

This method serves as an analytical tool for studying the interactions of proteins, antibodies, enzyme receptors, cells, and tissues with an isolated enzyme through optical or thermal image signals. The optical biosensor designed for detecting mycobacterial DNA in patient samples comprises two components: surface plasmon and enhanced surface Raman. The diagnostic process involving surface plasmon entails visualizing the CFP‐10 protein isolated from the patient sample and it's binding to nanoparticles positioned on the optical sensor's surface. To enhance the diagnostic efficacy, anti‐CFP‐10 is immobilized on the immunosensor's surface, complementing the optical immunosensor system based on Surface Plasmon Resonance (SPR). The correlation between CFP‐10 concentration and SPR signal intensity serves as a key indicator of tuberculosis infection. Many sensors used for detecting mycobacterial molecules rely on antigen–antibody reactions, with antigens crucial for vaccine development also playing a significant role in diagnosis. Notably, antigens such as Ag85B, MPB83, lipoglycan, and lipoarabinomannan are extensively studied. This approach specifically targets two early‐stage disease‐secreted antigens, ESAT‐6 and CFP‐10, which distinguish them from other mycobacterial species. These antigens, recognized by memory T cells, are employed for the detection of latent tuberculosis infection [79, 84].

4.6. CRISPR Cas to Detect Tuberculosis

This technique involves the repetitive occurrence of palindromic sequences within the DNA molecule, leading to the production of a protein known as Cas. CRISPR‐Cas forms a component of the bacterial acquired immune system. Various types of Cas proteins share common nucleotide activator and enzyme domains. For instance, Cas12 protein identifies double‐stranded DNA molecules as activators and occasionally cleaves single‐stranded DNA. The distinctiveness and specificity of Cas proteins are attributed to the Protospacer Adjacent Motif (PAM) motif. As a result, they can effectively detect minute quantities of nucleic acid present in patient samples [79].

4.7. Mass Spectrometry (MS)

This approach relies on the phenotypic, biochemical, and molecular features for identification. However, it lacks the capability to differentiate between species of Mycobacterium other than tuberculosis complex. To address this limitation and enable the specific diagnosis of M. tuberculosis , mass spectrometry (MS) is employed. MS is able to detect and identify the antigenic epitopes, thereby facilitating the accurate differentiation of M. tuberculosis strains [79].

4.8. Surface‐Enhanced Raman Spectroscopy

Surface‐Enhanced Raman Spectroscopy (SERS) is a technique utilized for the precise identification and rapid diagnosis of bacteria, offering high sensitivity and quick analysis. The SERS signal obtained provides valuable information from nucleic acids, proteins, and lipids. The enhancement of SERS is reliant on the interaction between the analyte and substrate molecules with the nanostructure. Nanostructured substrates are categorized into nanocolloids and solid bases, with the latter, comprising conventional nanostructures, significantly improving SERS detection. Research indicates that the surface charge of the bacterial membrane plays a crucial role in the binding affinity of nanoparticles, leading to an increase in the SERS signal. Distinguishing between bacterial species is challenging due to their similar chemical composition. To address this issue, aptamers, single‐stranded DNA molecules, are attached to SERS‐active nanoparticles and form covalent bonds with the target bacteria. The combination of bacterial aptamers and SERS nanoparticles enables the achievement of a highly sensitive diagnostic approach [90].

5. Conclusion

Non‐protein virulence factors play a crucial role in the pathogenicity of Mycobacterium tuberculosis ( M. tuberculosis ). Beyond lipids, the bacterial cell membrane contains proteins that modulate the host immune response. For instance, ESAT‐6, either alone or in combination with CFP‐10, reduces immune activity. Additionally, the interaction between fibronectin‐binding protein (FnBP) and fibronectin (Fn) is essential for the phagocytosis of M. tuberculosis . Understanding the mechanisms by which M. tuberculosis enters host cells, particularly macrophages, is vital for developing effective strategies to combat tuberculosis. This entry process is critical in establishing infection, and further insight into the interaction between M. tuberculosis virulence factors and host defenses is key to advancing vaccine and therapeutic development.

According to WHO data, approximately one‐third of the global population carries asymptomatic tuberculosis. The rise of multidrug‐resistant tuberculosis (MDR‐TB) in both developing and industrialized countries emphasizes the need for rapid diagnostic methods. While molecular techniques—such as DNA microarray, luciferase reporter assay, polymerase chain reaction (PCR), DNA and RNA probes, next‐generation sequencing, and whole‐genome sequencing—offer rapid, sensitive, and specific detection of M. tuberculosis , these methods are expensive and require technical expertise.

Author Contributions

N.M., J.S., M.E., A.B.B., M.V., and A.F.: design and concept of manuscript, data collection, and manuscript preparation, J.S., A.S., M.R., M.D., and A.F.: manuscript editing and manuscript review, all included authors read and approved the final manuscript.

Consent

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The authors confirm that the data supporting the findings of this study is available within the article.