Smear microscopy is simple, inexpensive and requires minimum training. It has multiple advantages like high specificity, high reliability with low inter-reader variation, can be used for diagnosis as well as follow up (for monitoring progress and defining cure), quick same day turnaround time, feasible at peripheral health institutions and also the results (grading) correlate with infectivity in pulmonary TB. NTP will replace smear microscopy with WHO recommended molecular diagnostics as the initial diagnostic test for all presumptive TB cases in a phased manner. Until the full replacement, the microscopy (preferably LED) will continue to be used for diagnosis.
4.3 Tools for diagnosis of TB
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Chest X-Ray, being a very sensitive tool, is ideal for the purpose of screening. However, any abnormality in chest radiograph should be further evaluated for TB including microbiological confirmation. For the diagnosis in the absence of microbiological confirmation, X-ray can be used as a tool for supportive evidence. Though (1) the inter and intra observer variability is high in X ray, (2) no X ray shadow is specific for TB and (3) 10-15% of culture positive cases may remain undiagnosed (under reading) when x-ray alone is used as a diagnostic tool, careful clinical assessment and supportive X-ray findings can be used to diagnose TB and such cases will be considered as clinically diagnosed TB. It is also useful for diagnosing extra pulmonary TB like pleural effusion, pericardial effusion, mediastinal adenopathy and miliary TB. A qualified physician should decide on the diagnosis of TB based on X-ray findings.
This test, using standard Tuberculin at a standard dose, is used for diagnosing Latent Tuberculosis Infection (LTBI) as per the national guidelines. (Refer to TPT section). Tuberculin skin test only express the presence of infection. It does not confirm TB disease. This test will be used for certain group of people before placing them on TB preventing therapy. This is also used as an adjunct tool in the diagnosis of TB among children (unable to produce sample). It has to be kept in mind that previous exposure to non-TB Mycobacteria (NTM) may yield false-positive test result. Conversely, the tuberculin skin test result may give false negative results under certain circumstances, for e.g., when the patient is immune compromised (co infected with HIV or on immune suppressive therapy), suffering from severe malnutrition or miliary TB etc.
Culture is still considered as the gold standard, detecting a higher proportion of patients among presumptive TB cases. A properly performed culture can detect low numbers of TB bacilli, even to the tune of less than 100 bacilli per ml. Culture, though a highly sensitive and specific method for TB diagnosis, requires 2-8 weeks to yield results and hence does not help in early diagnosis. However, culture is widely used for follow up of patients on drug resistant TB treatment to detect early recurrence.
The Liquid culture system: Mycobacteria Growth Indicator Tube (MGIT) is an automated culture system that detects the growth of mycobacteria faster than a solid culture. The liquid culture results are usually available within a maximum of 14 days. DST results are available 14-26 days after the cultures turn positive. However, the liquid culture needs a TB Containment Lab, which is operationally challenging to create as well as maintain.
The phenotypic DST using solid media and Liquid media (MGIT) is used for first line and second line as well as newer drugs (after standardization).
Cartridge Based Nucleic Acid Amplification (CB-NAAT) and Line Probe Assay (LPA) are the two molecular diagnostic tools introduced in Bangladesh in 2012. Currently, the GeneXpert, which is a CB-NAAT test, is the most widely used molecular diagnostic tool to detect TB and drug resistant tuberculosis. Also NTP in planning to introduce WHO recommended new molecular tools e.g. TrueNat and Xpert MTB\XDR etc.
Xpert MTB/RIF (GeneXpert): GeneXpert, which is a CB-NAAT test, detects DNA sequences, specific for Mycobacterium tuberculosis complex and rifampicin resistance by polymerase chain reaction. It concentrates Mycobacterium tuberculosis bacilli from sputum samples, isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR. The process identifies clinically relevant, rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons. Results are obtained from unprocessed sputum samples in 105 minutes. The Xpert MTB/RIF assay is suitable for deployment at all levels of the health facilities, although certain operational requirements need to be ensured, such as uninterrupted power supply, temperature-controlled settings and well-trained laboratory staff.
Line Probe Assay (LPA): Polymerase Chain Reaction (PCR) based technologies using various modifications are used for detecting the presence of putative resistance genes (rpoB for rifampicin, katG and inhA for Isoniazid, gyrA and gyrB for Fluoroquinolones, rrs and eis for second line injectables etc.). The most widely evaluated and used assays are Line Probe Assays (LPA) which are based on in-situ hybridization on nitrocellulose strips of specific genetic targets for resistance genes. The first line LPA (FL-LPA) can detect resistance to rifampicin and isoniazid while the second line LPA (SL-LPA) can do so for Fluroquinolones and second line injectable drugs (FQs and the SLIDs) within1-2 days. However, the process needs to be conducted in moderate risk level TB laboratory with at least 3 clean rooms, which, is operationally difficult to create and maintain. Further, only smear positive samples can be subjected to LPA. Smear negative samples need to be inoculated on culture (Solid / Liquid) and the growth subjected to DST on LPA.
These are special tests for confirmation of extra-pulmonary TB and should be performed by the concerned specialists. NTP will establish at least one center at district level equipped with FNAC, histopathology, GeneXpert to diagnose EP TB .