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 ELISA (IgG) May be used in conjunction with PPD or QuantiFERON® testing to diagnose active versus latent disease Positive QuantiFERON® with positive ELISA diagnoses progression to 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
  • When combined with IgG ELISA – may help differentiate latent from unsuspected active disease
    • Positive QuantiFERON^® and positive IgG indicate progression to active disease
  • 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 43.6/100,000 reported in the U.S. (CDC 2010) 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

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. This statement was endorsed by the Council of the Infectious Disease Society of America, September 1999. Am J Respir Crit Care Med. 2000; 161 (4 Pt 1) :1376-1395.PubMed
• Jereb JA. Progressing toward tuberculosis elimination in low-incidence areas of the United States. Recommendations of the Advisory Council for the Elimination of Tuberculosis. MMWR Recomm Rep. 2002; 51 (RR-5) :1-14.PubMed
• Mazurek M, Jereb J, Vernon A, LoBue P, Goldberg S, Castro K. 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

• Balasingham SV, Davidsen T, Szpinda I, Frye SA, Tonjum T. Molecular diagnostics in tuberculosis: basis and implications for therapy. Mol Diagn Ther. 2009; 13 (3) :137-151.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) :525-531.PubMed
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• Miranda C, Tomford JW, Gordon SM. Interferon-gamma-release assays: Better than tuberculin skin testing?. Cleve Clin J Med. 2010; 77 (9) :606-611.PubMed
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ARUP Institute for Clinical and Experimental Pathology®

• Aldous WK, Pounder JI, Cloud JL, Woods GL. Comparison of six methods of extracting Mycobacterium tuberculosis DNA from processed sputum for testing by quantitative real-time PCR. J Clin Microbiol. 2005; 43 (5) :2471-2473.PubMed
• Anderson BL, Welch RJ, Litwin CM. Assessment of three commercially available serologic assays for detection of antibodies to Mycobacterium tuberculosis and identification of active tuberculosis. Clin Vaccine Immunol. 2008; 15 (11) :1644-1649.PubMed
• Cloud JL, Carroll KC, Cohen S, Anderson CM, Woods GL. Interpretive criteria for use of AccuProbe for identification of Mycobacterium avium complex directly from 7H9 broth cultures. J Clin Microbiol. 2005; 43 (7) :3474-3478.PubMed
• Cloud JL, Hoggan K, Belousov E, Cohen S, Brown-Elliott BA, Mann L, Wilson R, Aldous W, Wallace RJ Jr, Woods GL. Use of the MGB Eclipse system and SmartCycler PCR for differentiation of Mycobacterium chelonae and M. abscessus. J Clin Microbiol. 2005; 43 (8) :4205-4207.PubMed
• Cloud JL, Meyer JJ, Pounder JI, Jost KC Jr, Sweeney A, Carroll KC, Woods GL. Mycobacterium arupense sp. nov., a non-chromogenic bacterium isolated from clinical specimens. Int J Syst Evol Microbiol. 2006; 56 (Pt 6) :1413-1418.PubMed
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• Cooksey RC, de Waard JH, Yakrus MA, Toney SR, Da Mata O, Nowicki S, Sohner K, Koch E, Petti CA, Morey RE, Srinivasan A. Mycobacterium cosmeticum, Ohio and Venezuela. Emerg Infect Dis. 2007; 13 (8) :1267-1269.PubMed
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• Lu J, Grenache DG. Development of a rapid, microplate-based kinetic assay for measuring adenosine deaminase activity in body fluids. Clin Chim Acta. 2012; 413 (19-20) :1637-1640.PubMed
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Actinomyces and Nocardia Species
Adrenal Insufficiency
Human Immunodeficiency Virus
Mold-Associated Infections
Sarcoidosis
Yeast-Associated Infections