Tuberculosis (TB) is the leading infectious cause of death globally, and is responsible for more than 1.6 million deaths and about 10.6 million new cases annually . TB is caused by the Mycobacterium tuberculosis complex consisting of M. tuberculosis, M. africanum, M. bovis, M. canettii, M. microti, M. pinnipedii, M. orygis and M. caprae . As primarily a lung disease, TB is acquired through inhalation of droplet nuclei containing the causative agent . Extra-pulmonary spread and multi-organ involvement or disseminated disease occurs, particularly in immuno-compromised hosts . Early detection and treatment of active and latent TB are key factors in the control of the TB epidemic. Globally, it has been estimated that only 64% of TB cases are detected, indicating that 36% of the cases are not identified .
Confirmation of pulmonary TB infection should be performed in the laboratory using respiratory samples such as sputum (spontaneously expectorated or induced), broncho-alveolar lavage, nasal washings, or sampling gastric contents) .
Enhancing laboratory capacity and performance is crucial for TB control since laboratories play a pivotal role in the diagnosis of TB . Smear microscopy along with other tests are used for the diagnosis of TB in over 70% of laboratories in low and middle-income countries (LMICs), but smear microscopy has low sensitivity and can detect only about 60% of TB cases . In developed countries, the choice of diagnostic tests is guided by the need for accuracy and speed, as well as the clinical presentation of the patient. Smear microscopy, with its lower sensitivity, is not the primary method of TB diagnosis. It can still be used in some cases, such as for initial screening in resource-constrained settings, but the emphasis is on complementing it with more sensitive and specific tests like culture and molecular assays to ensure accurate and timely diagnosis. This approach is essential for effective TB management, reducing transmission, and addressing drug-resistant TB cases.
Additionally, about 25% of all TB cases involve infections outside of the lungs, and these presentations are often not diagnosed with smear microscopy . Culture is able to identify cases with low levels of Mycobacterium and provides additional benefits such as DST and identification of other Mycobacterium species . Due to the slow growth of Mycobacteria, results take 3–4 weeks or longer on Lowenstein Jensen (LJ) medium and about 2 weeks in liquid Mycobacteria Growth Indicator Tubes (MGIT)-960 medium, therefore faster and more accurate diagnostic tests are required to improve patient management .
Cutting-edge technologies in laboratory medicine have been developed for early detection of Mycobacterium and assessing drug susceptibility. These utilize liquid culture medium, nucleic acid amplification techniques (NAATs), DNA hybridization, mutation detection techniques, antibody, and antigen detection .
Advances in the diagnosis of pediatric tuberculosis
Approximately 1.2 million children were among the 10.6 million people worldwide who became sick with TB in 2021. Diagnosing and treating TB in children is challenging despite the fact that it is a preventable and treatable disease . Traditional samples such as sputum and gastric aspirates for TB testing are not very useful for diagnosing TB in infants as they are difficult to collect, making diagnosis of pediatric TB a significant challenge . The ease of stool collection from individuals makes it a significant advancement in the new technologies of TB diagnosis as it can be utilized for TB testing . The diagnosis of childhood TB has been significantly improved by an innovative stool sample processing kit . The development of the stool sample processing kit aimed to investigate the feasibility of utilizing an automated PCR test with stool samples as an alternative to culture techniques and smear microscopy in the diagnosis of TB in children . The kit is designed to process stool samples without requiring any specialized laboratory equipment or skilled personnel, and it works together with the Cepheid Expert MTB/RIF Ultra assay .
Improvement in smear microscopy for TB diagnosis
Microscopic examination of Ziehl-Neelsen (ZN) stained sputum smears has been in use for nearly 100 years . The light microscope smear microscopy method is not very effective for diagnosing TB, as it has low sensitivity , and it only has the ability to detect about 60–70% of TB cases . An alternative to light microscopy for TB diagnosis is fluorescence microscopy, which is reported to be more sensitive by 10% . This is because the fluorescent bacilli of M. tuberculosis can be observed at a lower magnification and a larger field of view than with light microscopy . On the other hand, fluorescence microscopy has some drawbacks compared to light microscopy. It requires a microscope with a mercury vapor lamp, which is more expensive, and the ultra violet lamp needs to be replaced frequently, about every 200–300 h . Additionally, the slides need to be read in a dark room, which can be an inconvenience .
Light Emitting Diode (LED) fluorescence microscopy is a recent technological improvement that uses an illumination system based on a light-emitting diode that has a much longer lifespan of 10,000 h compared to the traditional fluorescence microscope which uses a mercury vapor lamp . LED-based fluorescent microscopes are now accessible and reasonably priced . The effectiveness of these microscopes was evaluated by the WHO, and the findings indicated that their diagnostic accuracy is higher by approximately 10% compared to traditional smear microscopy.
Alternative culture-based methods for TB diagnosis
Isolation of M. tuberculosis on a culture medium has for a long time been regarded as the gold standard for the diagnosis of TB . Culture techniques also allow for the identification of specific isolates through either biochemical tests or molecular methods and DST . Solid egg-based media such as LJ and Stonebrink medium were commonly used for culture of TB until the early nineties .
Stonebrink medium is a specialized culture medium used for the isolation and growth of M. bovis. The LJ base contains ingredients such as agar, glycerol, malachite green and other nutrients. Alternatively, it contains bromothymol blue, a pH indicator that detects the production of acid by M. bovis colonies. The medium changes color as M. bovis metabolizes specific substrates. Stonebrink medium is primarily used in veterinary diagnostic laboratories and research settings to isolate and identify M. bovis from clinical specimens, particularly from bovine sources . However, a disadvantage of these solid media is the slow growth of the bacteria, which can take up to 8 weeks before the culture results are confirmed . The sensitivity of liquid culture is higher by up to 20% and the time required for detection is shorter (10–14 days compared to 2–4 weeks) than traditional solid media . Therefore, WHO recommends using both traditional solid media and liquid media for the primary isolation of Mycobacteria . To improve the accuracy of TB diagnosis, a liquid broth culture can be used. In this culture, Middlebrook 7H9 broth, which is supplemented with 10% OADC (oleic acid, albumin, dextrose, and catalase) and PANTA (an antibiotic mixture of polymyxin, amphotericin B, nalidixic acid, trimethoprim, and azlocillin) to prevent contamination from other microorganisms are added . Various commercial culture systems are currently available, including simple bottles and tubes such as MGIT (BD BACTECTM MGITTM), Septi-Chek Acid Fast Bacilli (BBL-Becton Dikinson Microbiology Systems), and MB Redox, as well as more advanced systems such as the semi-automated BACTEC 460TB and fully automated systems like BACTEC 9000 MB and BACTEC MGIT 960 (all from BD, USA), ESP Culture System II (Trek Diagnostics, USA), and MB/BacT ALERT 3D System (BioMérieux, NC) .
Lateral flow lipoarabinomannan
A quick and timely diagnosis is crucial in managing diseases, and point-of-care testing can provide immediate results. A test for TB that can be performed at the point-of-care detects a substance called lipoarabinomannan in urine. LAM is a component of the cell wall of Mycobacteria, which is released when cells break down and is excreted in urine after being processed by the kidneys . It is a potential biomarker of diagnosis of M. tuberculosis. Various brands of a test that detects LAM are available, such as Abbott Determine™ TB LAM Ag in the USA, which was approved by WHO in 2015. Another brand is the Fujifilm SILVAMP TB-LAM test, also known as the FujiLAM, which is a new type of lateral flow LAM test that has a higher sensitivity compared to the Alere Determine™ TB LAM Ag . The Urinary LAM is used for the diagnosis of TB among individuals co-infected with HIV and with lower CD4 counts and has demonstrated improved sensitivity. The test was however not been approved for use by WHO committee in 2019 due to its low sensitivity . but is currently a good contender as a point-of-care test (30).
Alere Determine™ TB LAM and Fujifilm SILVAMP TB-LAM tests are based on the detection of LAM in urine, there are differences in terms of manufacturer, reported sensitivity and specificity, ease of use, and the potential use as a stand-alone test . These differences may influence the choice of test in specific clinical and resource settings. It is therefore essential to consider the patient’s clinical condition and the available diagnostic options when deciding which test to use for TB diagnosis.
Serological tests for diagnosing pulmonary and extra-pulmonary TB.
Antigen detection has gained attention as a promising approach for TB diagnosis. Immuno-PCR (iPCR) and real-time immuno-PCR are techniques that combine immunoassay principles with the amplification power of PCR. Immuno-PCR (I-PCR) integrates the ease and adaptability of ELISA with the substantial amplification capability and heightened sensitivity of PCR, resulting in a notable enhancement in sensitivity when compared to a similar ELISA . These methods can enhance the sensitivity and specificity of detection . Recent research in this field has focused on identifying and validating TB-specific antigens for use in immuno-PCR. These assays aim to improve the accuracy of TB diagnosis, especially in cases of extrapulmonary TB where bacillary load is low.
Aptamers are single-stranded DNA or RNA molecules that can bind specifically to target molecules, including proteins and antigens. Aptamer-based assays have been explored for their potential in TB diagnosis . Aptamers can be designed to bind to TB-specific antigens and used in diagnostic tests. These assays offer the advantage of high specificity and sensitivity. The development of aptamer-based assays is an exciting area of research that may lead to more accurate and reliable TB diagnostics. More studies are required to develop immune response-based or serological tests that can provide accurate results .
Diagnosis of latent M.
tuberculosis infection
Individuals with latent TB infection have been infected with M. tuberculosis, but they do not exhibit symptoms of active TB disease . Until recently, the only way to detect M. tuberculosis infection was through the tuberculin skin test (TST), also known as the Mantoux test .TST involves intradermally injecting a small amount of a purified protein derivative (PPD) into the skin and waiting for an induration to develop at the injection site within 2 days, indicating exposure to M. tuberculosis . However, the test cannot distinguish between latent infection and active disease and its limitations have been widely publicized.TST can produce inaccurate results due to false positives, which may occur as a result of exposure to non-tuberculous Mycobacteria or the Bacillus Calmette-Guerin (BCG) vaccine.This is because the PPD used in the test contains similar antigens to those present in BCG and certain non-tuberculous Mycobacteria. Despite its limitations, skin testing is still the most commonly used approach for detecting TB infection .IFN-γ is an important factor in regulating cell-mediated immune responses against M. tuberculosis . This understanding has led to the development of alternative tests for detecting TB infection, known as IFN-γ-release assays (IGRAs). These are blood tests that measure the release of interferon (IFN)-gamma from T cells after they have been stimulated with TB-specific antigens .Two commercially available IGRAs are Quantiferon TB Gold and T-SPOT TB . However, since there is no standard reference test for detecting latent TB infections, it is difficult to determine the accuracy of these assays . The occurrence of false negative results in tests for latent TB infection can be attributed to various factors, including simultaneous anergy, recent TB infection, the presence of concurrent viral, bacterial or fungal infections, recent vaccination with live viruses, chronic renal failure, low protein states, diseases that affect lymphoid organs such as Hodgkin’s disease, lymphoma, chronic leukemia, and sarcoidosis, immunosuppressive drugs, very young age (<6 months) or elderly individuals, stress, incorrect antigen storage or handling, incorrect administration, and misinterpretation of test results . It is important to note that IGRAs are not capable of differentiating between latent TB infection and active TB disease, and therefore should not be used as a diagnostic tool for active TB.
DNA-based tools for the
diagnosis of TB
Numerous techniques have been documented for detecting M. tuberculosis using a PCR test. This involves using short sequences of DNA, known as oligonucleotide primers, to amplify a specific DNA fragment that is unique to this microorganism . The classic Nucleic Acid Amplification Tests can give results in 3–6 h . There are different types of tests available for detecting Mtb using PCR, including commercial kits and “in-house” tests . The in-house tests are based on a protocol that has been developed in a non-commercial laboratory . Each nucleic acid amplification test (NAAT) employs a distinct technique to amplify specific regions of nucleic acid in the genome of the M. tuberculosis complex There are various commercial NAATs available, and the U.S. Food and Drug Administration (FDA) has authorized the use of certain commercial NAATs for respiratory specimens exclusively . Several kits, including the Gene-Probe Amplified M. tuberculosis direct test (AMTD), the Roche Amplicor MTB test, the Cobas Amplicor test, the Abbott LCx test, and the BD-ProbeTec (SDA) test, are available for detecting M. tuberculosis.However, none of these methods have been authorized for direct detection of M. tuberculosis from extra-pulmonary specimens. While these technologies are rapid and highly specific, their performance characteristics may vary, and their sensitivity is still not as good as that of culture-based methods, especially for samples that are negative on smear microscopy .
Drug susceptibility testing
The occurrence and proliferation of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) are significant concerns in the medical and public health fields.
MDR-TB refers to tuberculosis that has developed resistance to at least two of the first line anti-TB medications, rifampicin and isoniazid . Extensively drug-resistant tuberculosis (XDR-TB) is a form of TB that is classified as both MDR/RR-TB and also exhibits resistance to at least one fluoroquinolone drug, as well as one or more Group A drugs. Group A drugs refer to a potent set of second-line medications used in the treatment of drug-resistant TB, including levofloxacin, moxifloxacin, bedaquiline, and linezolid.
Detecting drug resistance at an earlier stage is crucial as it shortens the period between the diagnosis of TB and the commencement of treatment, ultimately leading to better outcomes for patients and contributing to the control of resistant strains in the population by reducing their transmission . Conventional techniques for testing drug susceptibility are time-consuming . The standard proportion method, which is the most widely used approach and is carried out on Lowenstein-Jensen medium or Middlebrook agar, takes approximately 4 to 8 weeks to generate results. This method is considered an “indirect method” as it necessitates a sequential process that involves isolating Mycobacteria from the clinical specimen, identifying the M. tuberculosis complex, and then performing in vitro testing of the strain’s susceptibility to anti-TB medications.
Over the past 15 years, a range of culture- and molecular-based techniques have emerged, some of which are considered “direct methods” that use patient specimens and avoid the need to first isolate M. tuberculosis in a pure culture from clinical samples .Several low-cost methods have been suggested for the swift identification of drug resistance, particularly for use in low-income settings. These methods include the microscopic observation of drug susceptibility (MODS), thin layer agar (TLA), colorimetric redox indicator (CRI) techniques, and the NRA . These techniques are capable of providing susceptibility results within 1–2 weeks after inoculation. Both MODS and TLA testing involve directly inoculating drug-free and drug-containing media (liquid medium for MODS and solid medium for TLA with patient specimens . In these methods, the isolation of pure culture from clinical specimens is not required. Instead, cultures are examined under a microscope for early growth or micro-colonies. If growth is observed in drug-free media, it indicates a positive culture, while growth in both drug-free and drug-containing media suggests resistance. Colorimetric redox indicator (CRI) methods are considered indirect and therefore require a pure culture to be isolated from clinical specimens . These methods involve adding a colored indicator to a liquid culture medium in a microtiter plate after M. tuberculosis has been pre-incubated for several days with different antibiotics and concentrations of drugs in vitro. The color of the indicator is reduced based on the presence or absence of growth, and the degree of color change indicates the level of drug resistance . Detection of resistance using colorimetric redox indicator (CRI) methods is based on the change in the color of the indicator which is related to the number of viable Mycobacteria present in the medium after incubation with different antibiotics and drug concentration . Various growth indicators are utilized in these methods, including Alamar blue and Resazurin. The nitrate reductase assay, on the other hand, is a solid culture approach that utilizes LJ medium . It works by detecting the nitrite that is produced when M. tuberculosis reduces nitrate. A specific reagent called Griess reagent is added to the medium, which contains 1 mg/ml of potassium nitrate (KNO3). The NRA test can be used as a direct or indirect test. To perform resistance testing using the nitrate reductase assay, patient samples or a pure culture of M. tuberculosis is directly inoculated onto media containing antibiotics and media without antibiotics. The nitrate reductase assay detects the reduction of nitrate, which is indicated by a colored reaction .
The BACTEC MGIT 960 system is based on detecting reduced levels of oxygen in broth as a result of bacterial respiration . It is a commonly used automated liquid culture DST system for testing the susceptibility of first-line antibiotics and it is an indirect method which requires a positive culture of M. tuberculosis complex with inoculation into a liquid medium.
The author is a Microbiologist Certified infection control Auditor Kidney Hospital Srinagar [email protected]
