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

Cavitary disease
- Actinomycosis, nocardiosis
- Staphylococcal disease
- Streptococcus pneumoniae
- Gram-negative pneumonia
- Anaerobic pneumonia
Miliary disease
- Mycoplasma pneumoniae
- Pneumocystis jirovecii
- Yeasts
  - Histoplasmosis
  - Coccidioides
  - Blastomyces
- Influenza virus
- Respiratory viruses
Constitutional disease
- Malnutrition
- HIV

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

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

Mycobacterium tuberculosis
Epidemiology
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

Organism
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)
Epidemiology
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

Organisms
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

Epidemiology

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

Diagnosis

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

Pneumonia – bacterial, viral
Malnutrition
HIV

Monitoring

See Monitoring tab

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.

Acid-Fast Bacillus (AFB) Culture and AFB Stain 0060152
Method: Stain/Culture/Identification/Susceptiblity

Recommended Use

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

Limitations

Mycobacteria are slow growing organisms; culture requires several weeks

Follow-up

DNA probes are available for M. tuberculosis complex (MTBC) 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

Recommended Use

Panel includes PCR testing to detect MTBC isolates and determine possible resistance to rifampin treatment

Test may be ordered for client-processed specimens; refer to specimen requirements

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

Recommended Use

Aid in the detection of latent disease among persons at increased risk for tuberculosis (TB)

Positive predictive value is less in low-risk populations

Specific for M. tuberculosis, not NTM or BCG vaccine

Test may be used in persons who have received BCG vaccine

Limitations

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

Recommended Use

Identify the species of MTBC 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

Limitations

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

Recommended Use

Comprehensive panel includes acid-fast bacillus culture and stain

Positive smears reflex to PCR amplification of MTBC species and rifampin resistance

Limitations

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

Recommended Use

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.

Recommended Use

Complete identification and susceptibility of clinically significant isolates of MTBC, M. kansasii, M. avium-intracellulare complex, M. fortuitum complex, M. abscessus complex, M. chelonae, M. immunogenum, and any isolate from a significant source

Limitations

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

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

Recommended Use

Order for susceptibility of clinically significant isolates of MTBC, M. kansasii, M. avium-intracellulare complex, M. fortuitum complex, M. abscessus complex, M. chelonae, M. immunogenum, and any isolate from a significant source

Antimicrobial Susceptibility, AFB/Mycobacterium tuberculosis Primary Panel 0060347
Method: Broth Macrodilution

Recommended Use

Order when phenotypic drug susceptibility testing is required for M. tuberculosis treatment

For genotypic (DNA sequencing) resistance testing, refer to TB drug resistance by sequencing test

Mycobacterium tuberculosis Drug Resistance by Sequencing 2011713
Method: Polymerase Chain Reaction/Sequencing

Recommended Use

Order for rapid identification of mutations associated with drug resistance for M. tuberculosis treatment

For phenotypic drug susceptibility testing, refer to the antimicrobial susceptibility, AFB/TB primary panel

Guidelines

Diagnostic Standards and Classification of Tuberculosis in Adults and Children. This official statement of the American Thoracic Society and the Centers for Disease Control and Prevention was adopted by the ATS Board of Directors, July 1999. Am J Respir Crit Care Med. 2000; 161(4 Pt 1): 1376-95. PubMed

Mazurek GH, Jereb J, Vernon A, LoBue P, Goldberg S, Castro K, IGRA Expert Committee, Centers for Disease Control and Prevention (CDC). Updated guidelines for using Interferon Gamma Release Assays to detect Mycobacterium tuberculosis infection - United States, 2010. MMWR Recomm Rep. 2010; 59(RR-5): 1-25. PubMed

General References

Balasingham SV, Davidsen T, Szpinda I, Frye SA, Tønjum T. Molecular diagnostics in tuberculosis: basis and implications for therapy. Mol Diagn Ther. 2009; 13(3): 137-51. PubMed

Cruz AT, Starke JR. Pediatric tuberculosis. Pediatr Rev. 2010; 31(1): 13-25; quiz 25-6. PubMed

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El-Sadr WM, Tsiouris SJ. HIV-associated tuberculosis: diagnostic and treatment challenges. Semin Respir Crit Care Med. 2008; 29(5): 525-31. PubMed

Glassroth J. Pulmonary disease due to nontuberculous mycobacteria. Chest. 2008; 133(1): 243-51. PubMed

Karussis D. The diagnosis of multiple sclerosis and the various related demyelinating syndromes: a critical review. J Autoimmun. 2014; 48-49: 134-42. PubMed

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Miranda C, Tomford W, Gordon SM. Interferon-gamma-release assays: Better than tuberculin skin testing? Cleve Clin J Med. 2010; 77(9): 606-11. PubMed

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Shingadia D. The diagnosis of tuberculosis. Pediatr Infect Dis J. 2012; 31(3): 302-5. PubMed

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Zar HJ, Connell TG, Nicol M. Diagnosis of pulmonary tuberculosis in children: new advances. Expert Rev Anti Infect Ther. 2010; 8(3): 277-88. PubMed

References from the ARUP Institute for Clinical and Experimental Pathology®

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