Laboratory Tests and Ancillaries
Sputum Smear Microscopy
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Sputum smear microscopy is recommended for all individuals highly suspected of pulmonary tuberculosis and those with chest X-rays suggestive of PTB. Two sputum specimens are submitted for evaluation, amounting to at least 3 mL (ideal volume: 5-10 mL). The patient must submit at least one early-morning sputum sample, which usually has the highest yield. Two sputum samples submitted on the same day are comparable to two consecutive days-sputum specimens. These samples have similar sensitivity and specificity rates.
Pulmonary tuberculosis cases without sputum smear results are classified as “smear not done." Sputum smear microscopy is also used in monitoring the treatment response of patients. Fluorescence microscopy is preferred over conventional microscopy because of its higher sensitivity and better time efficiency. Individuals suspected to have PTB who are either negative for AFB smear or unable to provide appropriate sputum samples are recommended to still submit proper sputum specimens via induction, rather than having to obtain them through flexible bronchoscopy, which is recommended for individuals suspected to have pulmonary tuberculosis who are incapable of providing induced sputum specimens. If there is a need for immediate diagnosis, transbronchial biopsy (TBB) may also be done for patients who are incapable of providing induced sputum specimens.
Sputum samples of post-bronchoscopy PTB-suspected patients should be sent for culture and AFB smear. Patients under miliary TB consideration, with negative AFB smear, with no other available specimens to submit for laboratory diagnosis, and unable to provide induced sputum are recommended to have specimens obtained by means of flexible bronchoscopy. Samples should include TBB and/or bronchial brushings. An AFB smear is indicated for specimens from sites that are being considered for EPTB.
Smear-Positive Pulmonary Tuberculosis (PTB)
Smear-positive PTB is considered smear-positive if ≥1 sputum smear specimens at the start of treatment in countries with well-functioning external quality assurance (EQA) systems are positive for AFB. In countries with no functional EQA, a smear-positive PTB is considered when two or more initial sputum smear examinations are positive for AFB, or one sputum smear examination is positive for AFB with abnormalities consistent with active PTB in a chest X-ray, or one sputum smear is positive for AFB with a culture positive for M tuberculosis.
Smear-Negative Pulmonary Tuberculosis (PTB)
Smear-negative PTB is considered in patients with a negative sputum smear but with a positive culture for M tuberculosis or when at least two sputum samples at the start of treatment in countries with a functional EQA system are negative for AFB. To confirm the diagnosis of TB, sputum culture for M tuberculosis is advised in patients who live in areas with HIV prevalence of >1% in pregnant women or ≥5% in TB patients that have smear-negative sputum. This is also considered in patients who are being treated by a clinician with a full course of an anti-TB regimen with abnormalities in a chest X-ray consistent with active PTB; the patient is either positive for HIV or, if with negative or unknown HIV status, lives in an area of low HIV prevalence or has no improvement in response to a course of broad-spectrum antibiotics (excluding anti-TB drugs, fluoroquinolones, and aminoglycosides).
Rapid Molecular Tests
Nucleic acid amplification tests (NAATs) are now recommended as the initial diagnostic test for TB in patients with signs and symptoms or screened positive for tuberculosis due to better detection of M tuberculosis if available.
Rapid molecular tests are divided into low-complexity automated nucleic acid amplification test (LC-aNAAT) (Xpert® MTB/RIF and Xpert MTB/RIF Ultra assays and Truenat® MTB Plus and MTB-RIF Dx assays); low-complexity manual nucleic acid amplification test (LC-mNAAT) (Loopamp™ MTBC Detection Kit [TB LAMP]); and moderate-complexity automated NAATs (MC-aNAAT) (Abbott RealTime® MTB and Abbott RealTime MTB RIF/INH, BD MAX™ MDR-TB, cobas® MTB and cobas MTB-RIF/INH, FluoroType® MTB, and FluoroType MTBDR). LC-aNAATs and MC-aNAATs are used as initial tests for the diagnosis of TB with drug-resistance detection, while LC-mNAATs are used to diagnose TB infection without detection of drug resistance. LC-aNAATs or LC-mNAATs should be used on respiratory samples as initial diagnostic tests for tuberculosis rather than smear microscopy or culture in patients with signs and symptoms of tuberculosis or those who screened positive. Screening tests include chest X-ray with or without computer-aided detection, C-reactive protein (CRP) in people living with HIV, and rapid molecular diagnostic tests for tuberculosis.
For patients with bacteriologically confirmed TB, biological specimens are positive by smear microscopy, culture, or tests such as Xpert MTB/RIF; LC-mNAATs should be used on respiratory samples as initial tests for detection of resistance to Rifampicin, rather than culture-based DST. For patients with signs and symptoms of TB meningitis, LC-mNAATs on cerebrospinal fluid should be used for the initial diagnosis of TB meningitis, rather than smear microscopy or culture. For patients with signs and symptoms of EPTB, LC-mNAATs on lymph node tissue aspirate, pleural tissue, pleural fluid, synovial fluid, peritoneal fluid, or pericardial fluid should be used for the initial diagnosis of tuberculosis rather than smear microscopy or culture. For patients with HIV who have signs or symptoms of tuberculosis, screen positive for TB, are seriously ill, or have advanced HIV disease, concurrent testing using LC-aNAATs on respiratory samples and LF-LAM should be used as the initial diagnostic strategy for diagnosing TB, rather than LC-aNAATs on respiratory samples alone.
An initial pulmonary sample should undergo NAAT. In patients who are AFB smear-positive and with negative NAAT, TB disease is improbable. In patients who are AFB smear-negative and with a moderate to high index of having TB disease, a positive NAAT enables the assumption of TB disease; however, a negative NAAT does not disregard TB disease. In patients who are unlikely to have TB disease, NAAT is not recommended since the yield of results that are false-positive is very common.
Xpert MTB/RIF
Xpert MTB/RIF is a highly sensitive and specific cartridge-based fully automated NAAT for the detection of M tuberculosis and Rifampicin resistance. It is 68% sensitive and 99% specific when compared to sputum culture; 88% sensitive and 98% specific when compared with smear microscopy; 94% pooled sensitivity and 98% pooled specificity for Rifampicin resistance; 84.9% sensitive for EPTB (eg lymph nodes, lung aspirates); and 79.5% sensitive for cerebrospinal fluid analysis (CSF) for TB meningitis.
A single automated polymerase chain reaction test (molecular test) using the GeneXpert platform can detect M tuberculosis bacteria and Rifampicin resistance within 2 hours of starting the test. This is recommended as an initial diagnostic tool for tuberculosis and Rifampicin resistance detection in adults and children with PTB signs and symptoms. In children, sputum, gastric aspirate, nasopharyngeal aspirate, and stool may be used. Xpert MTB/RIF is recommended for CSF examination of patients suspected to have TB meningitis rather than smear microscopy or culture. This is also recommended for detection of Rifampicin resistance in adults and children with signs and symptoms of EPTB rather than culture or DST. Xpert MTB/RIF is recommended for repeat testing of children with signs and symptoms of pulmonary tuberculosis in high prevalence settings using sputum, gastric fluid, nasopharyngeal aspirate, and stool specimens. This is also recommended as a confirmatory test for suspected TB patients with negative sputum smear microscopy results.
Xpert MTB/RIF may also be used as an initial diagnostic tool for non-sputum specimens (lymph node aspirate/biopsy, pleural fluid, peritoneal fluid, pericardial fluid, synovial fluid, or urine) from adult and pediatric patients with signs and symptoms of EPTB rather than smear microscopy or culture; disseminated TB in HIV-positive adults and children using blood as a specimen; and in patients with chest X-ray findings pertinent to pulmonary tuberculosis.
Xpert MTB/RIF Ultra (Xpert Ultra)
Xpert MTB/RIF Ultra uses a similar platform as the Xpert MTB/RIF and was developed to overcome the limitations in sensitivity for TB diagnosis of Xpert MTB/RIF. This is more sensitive compared to Xpert MTB/RIF in detecting M tuberculosis in smear-negative culture-positive specimens, extrapulmonary specimens, specimens from HIV-infected persons, and pediatric specimens. This is recommended as an initial diagnostic tool for TB and Rifampicin resistance in adults and children with PTB signs and symptoms. In children, nasopharyngeal aspirate may be used.
Xpert MTB/RIF Ultra is recommended for CSF examination of patients suspected to have TB meningitis. This is recommended for detection of Rifampicin resistance in adults and children with signs and symptoms of EPTB. This may be used in the following: Initial diagnostic test in adults and children for the corresponding form of EPTB using lymph node aspirate or biopsy; repeat testing of children with signs and symptoms of PTB in both low- and high-prevalence settings using sputum and nasopharyngeal aspirate specimens; as a replacement for culture as the initial test in patients with signs and symptoms of pulmonary tuberculosis or chest X-ray with lung abnormalities or both.
It is not recommended to perform repeated testing with Xpert Ultra in adults with signs and symptoms of PTB who initially had Xpert Ultra trace results. Trace results will require follow-up, which includes reassessing clinical symptoms and information on the prior history of tuberculosis.
Hologic Amplified Mycobacterium Tuberculosis Direct (MTD) Test
An MTD test is a nucleic acid probe test for the identification of M tuberculosis complex rRNA from sputum, bronchial, or tracheal specimens. A negative result does not mean that M tuberculosis complex is not present since the procedure may be influenced by different factors (eg handling of samples, laboratory errors).
Interferon-Gamma Release Assay (IGRA)
IGRA is a diagnostic blood test used to identify a patient’s immune reactivity to M tuberculosis. This may be considered for the initial diagnosis of children suspected to have tuberculosis. Example tests are T-SPOT.TB (T-Spot), TB-IGRA, QuantiFERON-TB Gold Plus (QFT-Plus), STANDARD E TB-Feron ELISA, and LIAISON QFT-Plus CLIA (Diasorin). IGRA along with tuberculin testing should not be used in low- and middle-income countries for the diagnosis of pulmonary or extrapulmonary TB or for the initial diagnostic work-up of adults (including people living with HIV) with suspected active TB due to suboptimal sensitivity and specificity.
In determining the presence of latent TB infection (LTBI), IGRA is preferred over tuberculin skin test (TST) in patients ≥5 years old who are likely infected with M tuberculosis, those with low-intermediate risk of disease progression, patients highly suspected of LTBI, and those previously vaccinated with BCG or who may not follow up for TST reading. Tuberculin skin testing may be performed instead if IGRA is unavailable or not preferred by the patient. One may perform IGRA instead of TST in patients ≥5 years of age at high risk for M tuberculosis, those at low to intermediate risk for disease progression, and if an LTBI test is warranted. For patients at high risk of disease progression, IGRA may be performed if initial TST is negative. Consider the second test result as confirmatory of M tuberculosis infection if positive. There is no need for M tuberculosis examination for individuals with low risk of having the disease, except in certain instances (eg as instituted by law), wherein patients ≥5 years old should undergo IGRA preferably (or TST as an alternative), with those whose results revealed infection should undergo another test (TST or IGRA, whichever is not used initially) for confirmation.
Loop-mediated Isothermal Amplification (TB-LAMP)
TB-LAMP is based on a loop-mediated isothermal amplification (LAMP) reaction, which requires <1 hour to perform and can be read with the naked eye under ultraviolet (UV) light. This is a low-complexity manual NAAT that may be used as a replacement test for sputum-smear microscopy. An example test is Loopamp™ MTBC Detection Kit (TB LAMP). Initial test without drug resistance detection for the diagnosis of pulmonary tuberculosis in adults with signs and symptoms and a follow-up test for sputum-smear negative results in adults with signs and symptoms consistent with pulmonary tuberculosis.
Skin Tests
Tuberculin Skin Test (TST, Mantoux Method)
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A tuberculin skin test is recommended for the initial diagnosis of children suspected to have tuberculosis but with no history of contact with persons with active TB disease. This is not recommended as an initial diagnostic tool for adults. This may be used to screen for LTBI and is also used as a screening examination for pediatric patients in contact with persons with active PTB. A tuberculin skin test is considered positive in the following instances: Induration of ≥5 mm in immunocompromised patients with diagnostic evaluation indicating current or previous TB, individuals who are in close contact with people with pulmonary tuberculosis, and those who are receiving immunosuppressive agents; induration of ≥10 mm for those at increased risk for LTBI and those with medical conditions that predispose them to progress from LTBI to TB disease; and induration of ≥15 mm for all other individuals. A positive skin test is not conclusive of an active TB disease and other confirmatory tests should be performed.
Mycobacterium tuberculosis Antigen-based Skin Tests (TBSTs)
Mycobacterium tuberculosis antigen-based skin tests are used for the indirect detection of TB infection through injection of MTB-specific antigens to elicit a localized skin reaction, which is more specific than TST. Example tests are Diaskintest®, Siiltibcy™, and C-TST.
Culture
A culture is used for definitive diagnosis of tuberculosis in patients with negative sputum smears, especially in areas with high HIV prevalence. Blood and urine culture should also be performed in HIV-infected patients with a CD4 count of <100 cells/mm3. This is also advised in patients in whom drug resistance is likely considered. Specimens should undergo culture for M tuberculosis in both liquid and solid media. A liquid system detects mycobacteria more and increases the case yield by 10% and provides DST results within 10 days as compared to solid media. This is also performed on specimens from patients in whom EPTB is suspected. Positive results can be used for documentation of EPTB and assist in patient evaluation, but a negative result should not rule out the possibility of EPTB. A culture isolate indicative of mycobacterial growth should be sent to a regional laboratory for genotyping.
Lateral Flow Urine Lipoarabinomannan Assay (LF-LAM)
Lateral Flow Urine Lipoarabinomannan Assay involves detection of lipoarabinomannan (LAM) antigen in urine. An example test is Determine™ TB LAM Ag. This may be used as an initial test for TB diagnosis without drug resistance detection. This has shown improved sensitivity for the diagnosis of tuberculosis in persons coinfected with HIV. LF-LAM is strongly recommended by the WHO to diagnose active TB in HIV-positive individuals in inpatient settings with the following: Signs and symptoms consistent with pulmonary and/or EPTB; seriously ill (respiratory rate >30/minute, temperature >39°C, heart rate >120/minute, and unable to walk unaided) or advanced HIV disease (CD4 cell count <200 cells/mm3 or WHO clinical stage 3 or 4); or CD4 cell count <200 cells/mm3 irrespective of signs and symptoms of tuberculosis. LF-LAM may be used in outpatient settings to support the diagnosis of active TB in HIV-positive individuals with signs and symptoms of pulmonary and/or EPTB or who are seriously ill and have a CD4 cell count of <100 cells/mm3 irrespective of signs and symptoms of TB.
LF-LAM is not recommended for use in outpatient settings to support the diagnosis of active TB in HIV-positive individuals in the following conditions: Without assessment for symptoms of TB; without symptoms of TB and unknown CD4 cell count or without symptoms of TB and CD4 cell count ≥200 cells/mm3; and without symptoms of TB and with CD4 cell count of 100-200 cells/mm³.
Drug Susceptibility Testing (DST)
Drug susceptibility testing is ideally done to all patients at the start of the treatment to identify and provide the most appropriate therapy. This should be done at or prior to the start of treatment in the following: New patients who had active TB after contact with a documented MDR-TB patient; all previously treated patients, especially patients with sputum smear-positive after 3 months of therapy, treatment failure, patients lost to follow-up, and those with disease relapse after treatment; patients living with HIV; HIV-infected TB patients with CD4 counts <200 cells/mm3; and all new patients in countries with >3% MDR-TB level in new patients.
Drug susceptibility testing should be performed for at least Isoniazid and Rifampicin. Susceptibility testing for fluoroquinolones is recommended by the WHO before the initiation of shorter, all-oral Bedaquiline-containing MDR-TB regimens. In patients who are either positive for AFB smear or hologic amplified MTD, a rapid molecular DST for Rifampin, with or without the inclusion of Isoniazid, is recommended if any of the following is present: History of TB treatment; birthplace or place of residency for 1 year in a country wherein the average prevalence of TB is ≥20/100,000 or that of MDR-TB is ≥2%; exposure to MDR-TB patients; and HIV disease.
A culture-based DST is the gold standard in the detection of drug resistance, and the rapid molecular DST is only for a selected few, such as those enumerated above. Rapid molecular DST (LC-aNAATs and MC-aNAATs) is recommended by the WHO as the initial test to detect drug resistance prior to the initiation of appropriate treatment for all patients, including new patients and patients with a previous history of therapy for tuberculosis. Conventional DST provides results within a week for liquid media or a month for solid media; therefore, patients should be given an empirical regimen while awaiting the results. A shorter waiting time for DST results may stop the continued spread of MDR, prevent an increase of resistance, and avoid an increased risk of a patient defaulting from treatment due to adverse effects of unnecessary empirical drugs.
High Complexity Reverse Hybridization-Based NAATs for Detection of Pyrazinamide Resistance
This may be used on M tuberculosis isolates for the detection of Pyrazinamide resistance rather than culture-based phenotypic DST. Pyrazinamide resistance is strongly associated with MDR/RR-TB.
Line-Probe Assay
Line-probe assay is a rapid molecular-based DST that provides results for Isoniazid or Rifampicin within 1-2 days, which can be used in deciding the treatment appropriate for the patient. Example tests include GenoType® MTBDRplus v1 and v2, GenoType MTBDRsl, Genoscholar™ NTM+MDRTB II, and Genoscholar PZA-TB II. This is a confirmatory diagnostic tool for sputum-smear-positive patients and culture-positive TB isolates. This is not to be used as an initial test for TB detection; it should be used together with culture and drug susceptibility testing. The GenoType M tuberculosis drug-resistant second-line assay (MTBDRsl), or GenoType MTBDRsl line probe assay, may be used to detect resistance to fluoroquinolones and second-line injectable medications in patients diagnosed with MDR-TB or RR-TB.
Low-Complexity Automated NAATs (Xpert MTB/XDR)
New class of diagnostics intended for use as a reflex test in specimens determined to be M tuberculosis-positive. This can be used to complement existing DSTs that only test for Rifampicin. This can rapidly detect resistance (under 90 minutes) to Isoniazid, fluoroquinolones, Ethionamide and Amikacin.
Targeted Next-Generation Sequencing
A targeted next-generation sequencing uses amplification of selected genes with next-generation sequencing technology to detect resistance to different drugs with a single test. Example tests are Deeplex® Myc-TB, AmPORE-TB®, and TBseq®. This can also identify specific mutations associated with resistance. This may be used on respiratory samples of patients with bacteriologically confirmed PTB disease to diagnose resistance to Rifampicin, Isoniazid, fluoroquinolones, Pyrazinamide and Ethambutol.
Phenotypic Drug Susceptibility Testing for Anti-TB Drugs
This is a test for all fluoroquinolones, and injectable aminoglycosides are recommended for RR-TB and MDR-TB patients.
Tests For Extrapulmonary Tuberculosis
Cell counts and chemistries are performed on samples from EPTB-infected regions (eg abdominal, pleural, joint fluids, etc). Measurement of adenosine deaminase (ADA) may be considered for patients suspected to have TB meningitis, pleural, peritoneal, or pericardial TB. Measurement of interferon-γ (IFN-γ) may be used for patients suspected of pleural or peritoneal TB. Histologic examination may also be considered, together with AFB smear microscopy, mycobacterial cultures, and NAAT.
Imaging
Chest X-ray
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A chest X-ray is a sensitive but non-specific test to detect tuberculosis (98% sensitive, 75% specific for any abnormality suggestive of TB). This cannot establish the diagnosis of TB alone but may be used for screening along with C-reactive protein (CRP) in people living with HIV and rapid molecular diagnostic tests for tuberculosis. A chest X-ray is used to evaluate patients with negative sputum smears and/or negative Xpert MTB/RIF and identify other possible diagnoses.