Mycobacterium tuberculosis - TB


Laboratory Testing

  • Mycobacterium tuberculosis

    Indications for Testing

    • Cough, fever, weight loss in at-risk patients

    Laboratory Testing

    • CDC TB testing and diagnosis recommendations
    • Concentrated acid-fast bacilli (AFB) smear
      • Traditional standard screening method (sputum)
        • Purified protein derivative (PPD) is old initial standard of diagnosis
      • Not as sensitive as culture – requires ~104 bacilli/mL sputum for detection
    • Culture
      • Culture provides definitive diagnosis
      • Susceptibility testing is mandatory
      • Most sensitive test for detection of mycobacteria – gold standard
      • Slow – 2-8 weeks
      • Should be performed on specimen from any site suspected for mycobacteria infection
    • Nucleic acid amplification testing
      • Less sensitive than culture
    • Latent TB – interferon alpha release assays (QuantiFERON®)
      • Not affected by previous vaccination with bacillus Calmette-Guérin (BCG)
      • Does not differentiate latent from active disease
    • Adenosine deaminase (ADA)
      • Adjunctive test – use in evaluating suspected extrapulmonary TB involving body cavities or cerebrospinal fluid
      • Reported sensitivity and specificity vary
        • Meta-analysis derived summary receiver operator curves from pleural fluid – sensitivity 92%; specificity 92%
      • Not specific for TB
      • AFB culture should always be performed concurrently on fluid

    Imaging Studies

    • Chest x-ray confirmation is required – may have been performed prior to TB testing
  • Nontuberculous (atypical) mycobacteria (NTM)

    Indications for Testing

    • Clinical setting suspicious for disease

    Laboratory Testing

    • Concentrated AFB smear
      • Traditional standard screening method (sputum)
      • Not as sensitive as culture – requires ~104 bacilli/mL sputum for detection
      • Not specific for atypical TB
    • Mycobacterial culture
      • Most sensitive test for detection of mycobacteria – gold standard
      • Slow – 2-8 weeks
      • Should be performed on specimen from any site suspected for mycobacteria infection
      • Positive culture from site of suspected disease required to make a diagnosis of disseminated disease
    • Nucleic acid amplified testing
      • Emerging recommended method for rapid, sensitive and specific detection of atypical TB
      • Less sensitive than culture

    Imaging Studies

    • Chest x-ray – cavities, frank parenchymal infiltrates (upper lobes more common), bronchiectasis, pulmonary nodules
      • Does not distinguish between TB and NTM
    • CT – may give better global picture of disease present

Differential Diagnosis


  • TB skin testing
    • Targeted tuberculin testing for latent TB infection for foreign-born persons entering the U.S. within the past 5 years
    • False positives in patients previously vaccinated with BCG or exposed to NTM
    • False negatives
      • Immunocompromised state
      • Severe illness
  • QuantiFERON Gold – if latent TB infection suspected
    • Should not be used in patients with active disease symptoms
    • Unaffected by previous BCG
    • May be used instead of PPD
    • Should not be used in children <5 years


  • Sputum culture
    • Follow up treatment with repeat cultures of sputum
    • 3-month culture may be predictive of relapse, if positive

Clinical Background

Mycobacterium tuberculosis (TB), as well as other nontuberculosis mycobacteria (NTM), can be infectious agents in humans.

  • Mycobacterium tuberculosis


    • Incidence and prevalence
      • ~3/100,000 reported in the U.S. (CDC, 2014)
      • 8-9 million new cases annually worldwide
        • 3-4 million cases are infectious pulmonary disease (smear positive)
    • Age – incidence increases with age; peak is 35-44 years in the U.S.
    • Sex
      • <25 years – M:F, equal
      • >25 years – M>F, 1.5-2:1
    • Ethnicity – in U.S., more frequent in African Americans and foreign-born individuals


    • Mycobacterium is a genus of the order Actinomycetales (Mycobacteriaceae family)
    • M. tuberculosis – rod-shaped aerobic bacterium with acid-fast staining properties
    • M. tuberculosis complex (MTB) – transmission via inhaled droplet nuclei
      • Includes M. tuberculosis, M. bovis, M. africanum, M. microti and others

    Risk Factors

    • Risk of infection (greatest risk)
      • Homeless, shelter dwelling
      • Incarceration
      • Alcoholism, intravenous drug abuse
      • Military (foreign deployment)
      • Foreign born
      • Immunocompromised (eg, HIV, transplant drugs, chemotherapy, chronic steroid treatment)
    • Risk of developing active disease once infected  
      • Comorbidity present (eg, HIV, immunosuppression, chronic renal failure, diabetes mellitus)
      • Recent tuberculosis infection (<1 year)
      • Malnutrition, alcoholism
      • Fibrotic lesions present on chest x-ray

    Clinical Presentation

    • Primary disease
      • Ranges from mild, self-limited illness to severe disseminated disease
    • Post-primary disease
      • Reactivation of latent infection
      • Moderate to severe; progressive and often fatal
    • Extrapulmonary TB
      • Gastrointestinal disease
      • Genitourinary
      • Lymphadenitis
      • Meningitis, central nervous system tuberculoma
      • Miliary
      • Pericarditis
      • Pleural disease
      • Skeletal (Pott disease)
    • TB in patients with HIV
      • Extrapulmonary disease is common
      • Atypical chest x-ray findings
      • Delays in diagnosis and treatment are common
      • Increased risk of latent disease reactivation or rapid progression of newly acquired infection
    Nontuberculous (atypical) mycobacteria (NTM)


    • Age – usually isolated
    • Transmission routes vary – cutaneous, inhalation, parenteral routes
      • Organisms are widely distributed in water and soil and by animals; person-to-person transmission has not been reported


    • Free-living organisms; more than 125 species of NTM
    • Nontuberculous organisms
      • M. avium complex, M. kansasii, M. fortuitum, M. abscessus, M. chelonae, M. marinum, M. haemophilum
    • Rare new organisms
      • M. nebraskense, M. parmense, M. saskatchewanense,  M. arupense, M. caprae, M. colombiense, M. florentium, M. montefiorense
    • Pathogenicity and clinical significance vary with species and host; asymptomatic infections are common

    Clinical Presentation

    • Cutaneous – M. fortuitum, M. abscessus, M. chelonae, M. marinum, M. haemophilum
      • Nodular or ulcerating chronic lesions that fail to respond to standard antimicrobial therapy
      • Lymphadenitis - M. avium complex, especially in children
    • Pulmonary – M. avium complex, M. kansasii, M. abscessus
      • Chronic cough usually present
      • Immunocompromised hosts
      • Increased in elderly and patients with underlying pulmonary disease
      • Bronchiectatic, nodular, or cavitary disease
    • Disseminated
      • Weight loss, fever, fatigue, lymphadenopathy, hepatosplenomegaly, gastrointestinal complaints
      • Usually immunocompromised patients (advanced HIV, transplant) 
    • Other (in-dwelling catheters, skeletal)


Clinical Background


  • High-incidence countries where children have contact with TB-positive adults – 30-40% risk for TB in child

Clinical Presentation

  • Much higher rate of progression to active disease
  • Cough typical in infants; fever common in adolescents
  • 25-35% have extrapulmonary presentation
    • 3 typical forms
      • Cervical adenitis
      • Miliary disease – most common in <2-3 years or immunocompromised
      • CNS disease – meningitis typically develops 3-6 months after primary infection; 50% are <2 years
    • Risk highest in infants, adolescents, immunocompromised children
  • Reactivation disease most common in adolescents
  • Congenital
    • Infants born to mothers who have disseminated disease
    • Failure to thrive in first 3 months (most common symptom)
    • Other – hepatomegaly, peripheral lymphadenopathy, respiratory distress


Indications for Testing

  • Clinical suspicion for disease

Laboratory Testing

  • Concentrated acid-fast bacilli smear
    • Not as sensitive as culture;  positive in 10-15%
    • Difficult to obtain sputum in children; may need to use early morning gastric aspiration
  • Culture
    • Yield positive in 20-30%
    • Best specimen is early morning gastric aspirate
    • Gastric lavage has better yield than pulmonary lavage – 3 samples best on sequential mornings
      • Requires hospitalization and fasting
    • Single hypertonic saline-induced sputum specimen may produce some bacteriologic yield as gastric aspirate
    • Highest yield in cavitary disease
  • Nuclear acid amplification test (NAAT)  – variable sensitivities may be good in children for rapid test if positive
  • QuantiFERON gold
    • Unable to distinguish between active and latent disease
    • Better than tuberculin skin test
    • Cannot be used in children <5 years (highest yield of indeterminate results)
  • Tuberculin skin test
    • Test of choice in children <5 years
    • Rarely positive in congenital disease

Imaging Studies

  • Chest x-ray
    • Enlarged perihilar and peritracheal nodes most common in children <5 years
    • Effusions unusual in children 3-5 years
    • Adult disease at age 8-10 years with apical segment involvement

Differential Diagnosis


  • See Monitoring tab

Indications for Laboratory Testing

  • Tests generally appear in the order most useful for common clinical situations
  • Click on number for test-specific information in the ARUP Laboratory Test Directory
Test Name and Number Recommended Use Limitations Follow Up
Acid-Fast Bacillus (AFB) Culture and AFB Stain 0060152
Method: Stain/Culture/Identification/Susceptiblity

Gold standard, most sensitive test for diagnosing mycobacteria

Specimen from any suspected site, including sputum, CSF, tissue, urine, and other body fluid or gastric aspirate

Susceptibility will be performed on organisms isolated from a sterile source and for isolates of Mycobacterium chelonae, M. abscesses, M. fortuitum complex, M. immunogenum, M. mucogenicum; susceptibility testing will be performed by request only on M. kansaii and M. marinum; susceptibility testing of M. gordonae is inappropriate

Mycobacteria are slow growing organisms; culture requires several weeks

DNA probes are available for MTB complex and M. avium-intracellulare complex as indicated

Other species require DNA sequencing or different molecular techniques for identification

For drug susceptibilities, refer to Antimicrobial Susceptibility - AFB Mycobacteria test

Mycobacterium tuberculosis Complex Detection and Rifampin Resistance by PCR 2010775
Method: Qualitative Polymerase Chain Reaction

Amplified test offers same day-results for infection and drug resistance

Specimen source required

QuantiFERON-TB Gold In-Tube 0051729
Method: Cell Culture/Semi-Quantitative Enzyme-Linked Immunosorbent Assay

Detect latent disease among persons at increased risk for TB

Specific for M. tuberculosis, not NTM or BCG vaccine

Do not use alone to diagnose or exclude TB or to assess possible latent disease; result interpretation requires a combination of epidemiological, historical, medical, and diagnostic findings

Negative result does not completely rule out TB infection; positive result does not differentiate active from latent TB

Mycobacterium tuberculosis Complex Speciation 0060771
Method: Qualitative Polymerase Chain Reaction

Identify the species of MTB complex to species level by comparing genomic deletion patterns; includes testing for M. tuberculosis, M. bovis, M. bovis BCG-vaccine strain, M. africanum, M. microti,  M. caprae

NTM may have indeterminate patterns of the genomic deletions used as targets in this assay

Changes in DNA sequence at the primer annealing sites may affect genomic deletion pattern determination and speciation

Acid-Fast Bacillus (AFB) Culture and AFB Stain with Reflex to Mycobacterium Tuberculosis Complex Detection and Rifampin Resistance by PCR 0060738
Method: Stain/Culture

Single-order test incorporating culture and stain with reflex to amplified MTB complex testing when smear positive

Available for respiratory specimens only

Low-level false-positive results can occur in specimens with high concentration of mycobacteria other than M. tuberculosis

Negative result does not exclude M. tuberculosis

Blood Culture, Acid-Fast Bacillus (AFB) 0060060
Method: Continuous Monitoring Blood Culture/Identification

Order for AFB culture of blood or bone marrow specimen

Acid-Fast Bacillus (AFB) Identification with Reflex to Susceptibility 0060997
Method: Identification/Susceptibility. Methods may include biochemical, mass spectrometry, nucleic acid probe, polymerase chain reaction, or sequencing.

Identify AFB isolated in pure culture with reflex to susceptibility testing, if indicated

Susceptibility testing may not be performed on all isolates (varies by species)

Antimicrobial Susceptibility, AFB/Mycobacteria 0060217
Method: Macrobroth Dilution/Microbroth Dilution


  • M. tuberculosis primary and secondary panel
  • M. avium-intracellulare complex c.
  • Laboratory should be notified when the presence of Mycobacterium genavense is suspected because this organism will not grow on media routinely used for mycobacterial isolation
  • Rapid-growing mycobacteria
  • Other slow-growing NTM
Additional Tests Available
Click the plus sign to expand the table of additional tests.
Test Name and NumberComments
Acid-Fast Bacillus (AFB) Stain Only 0060151
Method: Auramine O Stain

Monitor respiratory specimens on previously diagnosed patients

Should not be ordered without culture in previously undiagnosed patients

Acid-Fast Bacillus (AFB) Identification 0060999
Method: Identification. Methods may include biochemical, mass spectrometry, nucleic acid probe, polymerase chain reaction, or sequencing.

Identification of acid-fast organisms (AFB) isolated in pure culture

Blood Culture, AFB and Fungal 0060024
Method: Continuous Monitoring Blood Culture/Identification
Adenosine Deaminase, CSF 2006098
Method: Quantitative Spectrophotometry
Adenosine Deaminase, Peritoneal Fluid 2006101
Method: Quantitative Spectrophotometry
Adenosine Deaminase, Pleural Fluid 2006096
Method: Quantitative Spectrophotometry
Adenosine Deaminase, Pericardial Fluid 2009357
Method: Quantitative Spectrophotometry
Chylomicron Screen, Body Fluid 0098457
Method: Qualitative Electrophoresis