Tuberculosis (TB) is a highly communicable disease caused by the bacterium Mycobacterium tuberculosis, significantly impacting global health and remaining one of the leading causes of death worldwide. The disease primarily affects the lungs but can also target other parts of the body, such as the kidneys, spine, and brain. TB spreads through the air when an infected person coughs, sneezes, or talks, releasing tiny droplets containing the bacteria. The COVID-19 pandemic has severely disrupted TB services globally, leading to an 18% decline in newly diagnosed cases from 7.1 million in 2019 to 5.8 million in 2020, setting TB control efforts back to 2012 levels. Sixteen countries accounted for 93% of this reduction, with India, Indonesia, and the Philippines being the hardest hit due to diverted health resources and reduced attention and funding for TB programs.^1-3

In response, the Indian National Strategic Plan for TB Elimination (2017-2025) aims to eliminate TB in India by 2025 and achieve zero catastrophic costs for families affected by TB by 2020. This ambitious goal involves reducing TB incidence to less than one case per million population per year and addressing direct medical expenses and indirect costs like lost income due to illness to prevent TB from driving families into poverty. Breaking the chain of transmission is essential for TB elimination, necessitating early detection, prompt and effective treatment, and ensuring treatment completion to prevent disease spread. Accurate and efficient diagnosis is crucial, enabling timely identification of TB cases and reducing the risk of transmission. However, diagnostic delays and health system failures pose significant barriers to effective TB control.^4,5

These challenges stem from limited access to healthcare facilities, lack of awareness, inadequate diagnostic tools, insufficient laboratory infrastructure, lack of trained personnel, and delays in reporting and follow-up, resulting in missed or late diagnoses with severe consequences for TB patients, including prolonged illness, increased transmission, and higher mortality rates. Addressing these challenges requires strengthening health systems, improving diagnostic capacity, and ensuring timely access to accurate and effective diagnostic services for all individuals with TB symptoms.^6-8

Microbiological Diagnosis^9-15

Microscopy: Various microscopy methods are employed to detect TB, each with its advantages and specific applications.

• ZN Stain: The Ziehl-Neelsen (ZN) stain is a traditional and widely used method for TB detection. It involves staining the bacteria with carbol fuchsin, decolorizing with acid-alcohol, and counterstaining with methylene blue. Mycobacterium tuberculosis retains the red color of carbol fuchsin, distinguishing it from other bacteria. This method is cost-effective and straightforward but requires skilled technicians to interpret the results accurately.

• Fluorescent Microscopy with Light-Emitting Diodes (LED): This technique uses fluorochrome stains, such as auramine O, which bind to mycobacteria. The stained bacteria fluoresce under LED illumination, enhancing visibility against the background. Fluorescent microscopy is more sensitive than ZN staining and allows for faster examination of specimens, increasing diagnostic efficiency.

• Front-Loaded Microscopy: This method involves collecting and examining two specimens on the same day a patient presents to the clinic. If the initial smears are negative, the patient is asked to return with a morning specimen the next day. This approach reduces the need for multiple clinic visits, accelerating the diagnostic process and improving patient compliance.

• Sodium Hypochlorite (Bleach) Microscopy: Adding a standardized sodium hypochlorite solution to sputum samples, followed by overnight sedimentation, enhances TB detection by approximately 15%. The bleach helps to concentrate the mycobacteria, making them more visible under the microscope. This method is particularly useful in resource-limited settings where more advanced diagnostic tools may not be available.

• Vital Fluorescent Staining: This technique uses fluorescein diacetate (FDA) to stain only living, cultivable bacteria, providing real-time information on bacterial viability. Unlike most fluorescent stains, FDA stains only living bacteria, which can guide antimicrobial therapy decisions before culture reports are available. This method is beneficial for monitoring treatment response and assessing bacterial load during therapy.

• Newer Microscopic Technologies: Innovations like TBDx and CellScope represent significant advancements in TB microscopy.

  • TBDx: This automated system integrates robotic loading of stained slides and high-resolution digital image analysis to provide rapid results. With a capacity of 200 slides, TBDx reduces the burden on laboratory technicians and enhances diagnostic accuracy.

  • CellScope: A portable digital fluorescence microscope (FM) that provides enlarged, digitalized images for review. CellScope is particularly useful in field settings and remote areas where traditional laboratory infrastructure may be lacking.

Culturing Mycobacterium tuberculosis remains the gold standard for TB diagnosis due to its high sensitivity and ability to provide drug susceptibility testing.

• Microcolony Detection on Solid Media: Early detection of microcolonies on solid media can significantly reduce diagnostic time. This method involves incubating specimens on solid agar and monitoring for the growth of microcolonies, which can be detected sooner than visible colonies, thereby accelerating the diagnostic process.

• Septi-Check AFB Method: This technique combines solid and liquid media to enhance TB detection. It uses a biphasic medium that supports the growth of mycobacteria, allowing for faster and more reliable detection compared to traditional methods.

• BACTEC Systems: Automated culture systems like BACTEC MGIT 960, ESP II, VersaTREK, and BACTEC MYCO/F LYTIC offer faster and more accurate TB diagnosis by automating the detection and drug susceptibility testing processes.

  • BACTEC MGIT 960: This fully automated system is designed for detecting mycobacterial growth and conducting drug susceptibility testing. It utilizes a Middlebrook 7H9 broth base, enriched with OADC (oleic acid, albumin, dextrose, and catalase) and PANTA antibiotics to inhibit contaminant growth. The system detects fluorescence emitted by the mycobacteria, indicating positive growth. It can handle a high volume of samples and provides results in a matter of days rather than weeks.

  • Other Systems: The ESP II culture system, VersaTREK, and BACTEC MYCO/F LYTIC are additional automated systems that facilitate rapid TB detection and susceptibility testing. These systems differ in their mechanisms of detecting bacterial growth but share the common goal of reducing diagnostic turnaround time and increasing diagnostic accuracy.

     

Advancements in microbiological diagnosis, including microscopy and culture techniques, have significantly improved the detection and management of TB. These methods offer varying degrees of sensitivity, specificity, and practicality, allowing for tailored diagnostic approaches in different healthcare settings.

PCR (Polymerase Chain Reaction)^20-25

PCR (Polymerase Chain Reaction) is a powerful technique used to amplify small segments of DNA, enabling the detection of Mycobacterium tuberculosis with high sensitivity and specificity. Various forms of PCR are employed in TB diagnostics, each offering unique advantages:

• Nested PCR: This method involves two successive rounds of PCR amplification, which significantly increases the sensitivity of TB detection. The first round amplifies a larger fragment of DNA, and a second round amplifies a smaller, internal fragment. This technique reduces the likelihood of false-negative results by ensuring that even low levels of bacterial DNA are detectable.

• Real-Time PCR (Xpert MTB/RIF, Cepheid): The Xpert MTB/RIF assay revolutionizes TB diagnostics by providing rapid, accurate detection of TB bacteria and rifampin resistance within two hours. It utilizes a fully automated, cartridge-based system that integrates sample preparation, DNA amplification, and detection in a single-use disposable cartridge, minimizing contamination risks. The Xpert Ultra, an improved version of this assay, has a lower detection limit of 16 bacilli/ml, making it more sensitive and capable of detecting TB in paucibacillary cases where bacterial load is low.

   

Line Probe Assays (LPA)^26-28

Line Probe Assays (LPA) are DNA strip-based tests designed to identify the genetic mutations associated with drug resistance in Mycobacterium tuberculosis. These assays offer a rapid and reliable means to determine the drug resistance profile of TB strains, which is crucial for effective treatment planning:

• Principle: LPA detects specific mutations in the TB genome that confer resistance to first-line and second-line anti-TB drugs. The process involves extracting DNA from TB isolates or directly from clinical specimens, amplifying target regions using multiplex PCR, and hybridizing the PCR products to probes immobilized on a strip. The pattern of probe binding indicates the presence of resistance-associated mutations.

• Commercial Kits: Two main commercial kits are used for LPA:

  • GenoType: This kit identifies mutations in genes associated with resistance to rifampicin, isoniazid, and second-line drugs.

  • INNO-LiPA: Similar to GenoType, it detects mutations conferring resistance to multiple drugs, providing a comprehensive resistance profile.

• Systems:

  • TwinCubator: A manual system for performing LPAs. It involves manually handling the hybridization and development steps, requiring careful attention to detail and consistency.

  • GT-Blot: An automated system that streamlines the hybridization process and development of colored bands on the test strip, enhancing reproducibility and reducing the potential for human error.

     

TrueNat^29-31

TrueNat is an innovative molecular diagnostic tool designed for the rapid detection of TB and rifampicin resistance, particularly suited for use in resource-limited settings:

• TrueNat System: This portable, battery-operated device integrates DNA extraction, amplification, and detection into a single platform. It comprises two main components:

   

  • Trueprep AUTO v2: This device automates the extraction and purification of DNA from sputum samples, ensuring consistent and reliable results.

  • Truelab Real-Time micro PCR Analyzer: This device performs real-time PCR amplification and detection, providing semi-quantitative results that indicate the presence of TB bacteria and rifampicin resistance.

     

• WHO Recommendation: The World Health Organization (WHO) endorses the use of TrueNat MTB and MTB Plus as initial diagnostic tests for TB, recommending them over traditional smear microscopy or culture due to their rapid turnaround time and higher sensitivity.

• Detection Limits: TrueNat MTB has a detection limit of 100 colony-forming units (CFU)/ml, while TrueNat MTB Plus can detect as low as 30 CFU/ml, making it highly effective in identifying TB cases with low bacterial load.

   

TB-LAMP (Loop-Mediated Isothermal Amplification)^31-33

TB-LAMP is a manual, molecular assay that detects TB bacteria rapidly, providing a valuable diagnostic tool in settings with limited laboratory infrastructure:

• Procedure: The TB-LAMP test involves three main steps:

  • Sample Preparation: The PURE (ultra-rapid extraction) method is used to prepare the sample. This involves adding specific buffers to liquefy and lyse the sputum sample, followed by rapid DNA extraction.

  • LAMP Reaction: The prepared sample is then subjected to a LAMP reaction, which amplifies the DNA at a constant temperature using a set of specially designed primers. The reaction produces a large amount of DNA quickly, making it visible to the naked eye or under a simple fluorescence detector.

  • Detection: The results are obtained within an hour, with the amplified DNA forming a visible precipitate or fluorescence signal that indicates the presence of Mycobacterium tuberculosis. This method does not require sophisticated equipment, making it ideal for field use and low-resource settings.

     

Molecular methods for TB diagnosis, including PCR, LPAs, TrueNat, and TB-LAMP, represent significant advancements in the rapid, accurate detection of TB and drug resistance. These technologies enhance the ability to diagnose TB promptly, initiate appropriate treatment, and reduce transmission, thereby improving patient outcomes and supporting global TB control efforts.

Interferon-Gamma Release Assays (IGRAs)^34-36

Interferon-Gamma Release Assays (IGRAs) are advanced immunological tests used to detect latent tuberculosis infection (LTBI) by measuring the immune response to TB antigens. These tests have become increasingly important in TB control and management due to their specificity and convenience.

• Principle: IGRAs operate on the principle of measuring the release of interferon-gamma (IFN-γ) from T-cells in response to specific TB antigens. When blood samples from an individual are mixed with these antigens, T-cells that have been sensitized by previous TB exposure release IFN-γ. The amount of IFN-γ produced is then quantified using an enzyme-linked immunosorbent assay (ELISA) or enzyme-linked immunospot (ELISPOT) technique. Unlike the traditional tuberculin skin test (TST), IGRAs do not cross-react with the Bacille Calmette-Guérin (BCG) vaccine or most non-tuberculous mycobacteria, making them more specific for Mycobacterium tuberculosis infection.

• Applications: IGRAs are widely used as a substitute for the TST in various clinical scenarios:

  • Contact Investigations: In cases of exposure to a person with active TB, IGRAs help identify individuals who have been infected and might benefit from preventive therapy.

  • Pregnancy: Pregnant women at risk for TB can be safely tested with IGRAs, as these tests do not involve injecting substances into the skin, unlike the TST.

  • Health Care Workers: Regular screening of health care workers, especially those in high-risk settings, is crucial. IGRAs provide a reliable method for serial testing, helping to identify LTBI and prevent the progression to active TB.

     

Lateral Flow Urine Lipoarabinomannan Assay (LF-LAM)^37-40

Lateral Flow Urine Lipoarabinomannan Assay (LF-LAM) is a rapid diagnostic test used to detect active TB, particularly in individuals with advanced immunosuppression, such as those with HIV/AIDS. This test is valuable in specific clinical situations where conventional diagnostic methods may be less effective.

• Principle: The LF-LAM assay detects the presence of lipoarabinomannan (LAM), a glycolipid component of the mycobacterial cell wall, in urine samples. LAM is released from metabolically active or degenerating TB bacteria and can be detected in the urine of individuals with active TB. The test employs a lateral flow format, similar to pregnancy tests, where the urine sample is applied to a test strip that contains antibodies specific to LAM. The binding of LAM to these antibodies produces a visible band on the test strip, indicating a positive result.

• Procedure: The LF-LAM test is straightforward and can be performed manually at the point of care:

  • Sample Collection: A urine sample is collected from the patient.

  • Application: A specified volume of urine (usually 60 μL) is applied to the test strip.

  • Incubation: The test strip is allowed to incubate at room temperature for approximately 25 minutes.

  • Interpretation: The intensity of any visible band on the test strip is compared against a manufacturer-supplied reference card, which includes bands of varying intensities (grades 1 to 4) to facilitate interpretation.

     

• Use Case: While LF-LAM is not recommended as a routine screening test for TB due to its lower sensitivity compared to other diagnostic methods, it is particularly useful in certain scenarios:

  • HIV/AIDS Patients: In individuals with advanced HIV infection, where TB diagnosis is challenging due to atypical presentations and low bacillary load, LF-LAM provides a rapid and non-invasive diagnostic tool.

  • Resource-Limited Settings: The simplicity and minimal equipment requirements of the LF-LAM test make it suitable for use in settings with limited laboratory infrastructure, where more sophisticated diagnostic tools may not be available.

     

The diagnosis of latent TB infection (LTBI) and active TB has been significantly advanced by the development of Interferon-Gamma Release Assays (IGRAs) and Lateral Flow Urine Lipoarabinomannan Assay (LF-LAM). IGRAs offer a specific, convenient alternative to the TST for detecting LTBI, particularly useful in contact investigations, during pregnancy, and for health care workers. LF-LAM provides a rapid, point-of-care diagnostic option for active TB, especially valuable in HIV/AIDS patients and resource-limited settings. Together, these innovative diagnostic tools enhance the ability to accurately detect and manage TB, contributing to global efforts to control and eventually eliminate this devastating disease.

The advancements in tuberculosis (TB) diagnostics, including microscopy, culture techniques, and molecular methods, have significantly enhanced the accuracy, speed, and reliability of TB detection and drug resistance profiling. Innovations such as the BACTEC MGIT 960 system, Real-Time PCR (Xpert MTB/RIF), Line Probe Assays (LPA), TrueNat, and TB-LAMP have revolutionized TB diagnostics, enabling rapid and efficient diagnosis even in resource-limited settings. Interferon-Gamma Release Assays (IGRAs) and the Lateral Flow Urine Lipoarabinomannan Assay (LF-LAM) have further improved the diagnosis of latent TB infection (LTBI) and active TB, particularly in high-risk populations. These advancements are crucial for timely detection, effective treatment, and management of TB, contributing significantly to global TB control efforts and the ambitious goal of eliminating TB. Continued investment in these diagnostic technologies and robust health system strengthening are essential to sustain progress and achieve TB elimination.

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