1. Update in the Diagnosis of Tuberculosis in Children Ana M. Alvarez, MD, FPIDS Associate Professor Division of Pediatric Infectious Diseases and Immunology University of Florida College of Medicine/Jacksonville

2. Objectives At the end of this session, participants will be able to: Discuss the appropriate work up for pediatric patients with suspected tuberculosis Review the tests currently available to diagnose TB infection Review molecular testing in TB diagnostics

3. Work up for Children with Suspected TB History and physical exam Tuberculin skin testing (TST) or IGRAs Chest x-rays Microscopy and molecular testing

4. Importance of Contact Investigation How are pediatric cases discovered? Active – Contact investigation: 25-80% – Screening of high risk groups: 3-35% Passive – Symptomatic children: 15-45%

5. Pediatric Tuberculosis Clinical Presentations Pulmonary TB 75-80% of presentations in children Primary: Primary focus with hilar adenopathy with or without focal infiltrates, usually mild to moderate symptoms Chronic/re-activation: upper lobes cavitation (adult-type) Extra-pulmonary TB Almost any organ: lymph nodes, ears/mastoids, bones, GI, etc. Meningitis Diagnosis usually requires a HIGH index of suspicion LP should be performed in ALL <12 mo old patients with suspected TB disease. Low threshold to do it in 12-23 mo old patients Disseminated/Miliary Results from hematogenous spread, affecting two or more organs

6. Testing for M. tuberculosis Infection Two testing methods available for the detection of M. tuberculosis infection in the United States: Mantoux tuberculin skin test (TST, PPD) Interferon-gamma release assays (IGRA) These tests DO NOT exclude TB infection and they DO NOT distinguish LTBI from TB disease Decisions about medical and public health management should include other information, and not rely only on TST or IGRA results.

7. Diagnosis of TB Infection: Tuberculin Skin Test (TST)

8. Mantoux Tuberculin Skin Test: Interpreting the Reaction Interpretation of TST reaction depends on size of induration and person’s risk factors for TB

9. Mantoux Tuberculin Skin Test: Interpreting the Reaction Induration of > 5 mm is considered positive for: People living with HIV Recent close contacts of people with infectious TB People with chest x-ray findings suggestive of previous TB disease People with organ transplants Other immunosuppressed patients (e.g.TNF-α antagonists and high dose steroids for extended duration)

10. Mantoux Tuberculin Skin Test: Interpreting the Reaction Induration of > 10 mm is considered a positive reaction for: People who inject drugs People who live or work in high-risk congregate settings People with certain medical conditions that increase risk for TB: diabetes, chronic renal failure, silicosis, malnutrition, certain malignancies, gastrectomy Children younger than 4 years old

11. Mantoux Tuberculin Skin Test: Interpreting the Reaction Induration of > 15 mm is considered a positive reaction for people who have no known risk factors for TB

12. Mantoux Tuberculin Skin Test: False-Positive Reaction Factors that can cause people to have a positive reaction even if they do not have TB infection: Infection with nontuberculous mycobacteria BCG vaccination Administration of incorrect antigen Incorrect measuring or interpretation of TST reaction

13. Mantoux Tuberculin Skin Test: BCG Vaccine People who have been vaccinated with BCG may have a false-positive TST Children vaccinated at < 2 months of age: 40% are TST negative at 1 year of age >95% TST negative at 5 years of age. Persons vaccinated at > 2 months of age: >95% TST negative after 7 years. Multiple doses of BCG or repeat doses at older ages can cause persistence of TST positive reaction There is no reliable way to distinguish between reaction caused by TB infection or by BCG vaccine Individuals should always be further evaluated if they have a positive TST

14. Mantoux Tuberculin Skin Test False-Negative Reaction Factors that can cause false-negative reactions: Anergy Recent TB infection (within past 8 – 10 weeks It can take 2 – 8 weeks after TB infection for body’s immune system to react to tuberculin Younger than 4 months of age Recent live-virus (e.g., measles or smallpox) vaccination Incorrect method of giving the TST Incorrect measuring or interpretation of TST reaction

15. INTERFERON-GAMMA RELEASE ASSAYS (IGRA)

16. Interferon Gamma Release Assays IGRAs use purified antigens from MTB to stimulate peripheral- blood lymphocytes to produce gamma interferon Three IGRAs approved by the U.S. FDA and are commercially available in the U.S.: QuantiFERON®-TB Gold test (QFT-G); QuantiFERON®-TB Gold In-Tube test (QFT-GIT); T-SPOT®.TB test (T-Spot) Note: Latest guidelines: CDC guidelines for QFT-GIT and T- SPOT published June 2010 www.cdc.gov/mmwr Vol. 59, No. RR-5www.cdc.gov/mmwr

17. IGRA: General Points IGRAs are highly specific (~95%) Substantially more specific than the PPD Distinguish reactions due to BCG and most non-tuberculous mycobacteria (except M. kansasii, M. marinum, M. szulgai, M. flavescens) IGRAs have moderate to high sensitivity vs. PPD QFT being as sensitive as PPD (70-80%) in immunocompetent T-SPOT TB is more sensitive (~90%) than QFT and PPD in the immunocompromised

18. IGRA: Advantages Requires a single patient visit to conduct the test Controlled laboratory test Results can be available within 24 hours No booster responses with subsequent tests More specific than TST Use of IGRA may increase acceptance of LTBI tx

19. IGRA: Disadvantages Errors in collecting or transporting blood specimens can decrease their accuracy Limited data on the use of IGRAs for: Children younger than 5 years of age Persons recently exposed to M. tuberculosis Immunocompromised persons Serial testing Tests may be expensive

20. Current Recommendations for the Use of TST and IGRAs in Children TST is preferred, IGRA acceptable Children < 5y of age* IGRA preferred, TST acceptable Children ≥ 5 y of age who have received BCG* Children ≥ 5 y of age who are unlikely to return for TST reading TST and IGRA should be considered when The initial and repeat IGRA are indeterminate The initial test (TST or IGRA) is negative and: Clinical suspicion for TB is moderate to high Risk of progression and poor outcome is high The initial TST is positive and: >5y of age with history of BCG, healthy without known TB exposure AND/OR additional evidence needed to increase compliance Nontuberculous mycobacterial disease is suspected AAP, Red Book 2015 *Some experts would use IGRA in children ≥3 y of age

21. Copyright © 2015 American Academy of Pediatrics. All rights reserved. Guidance on Strategy for Use of TST and IGRA by age and BCG-Immunization Status

22. Evaluation of a Positive TST or IGRA LTBI versus Disease Review for symptoms of tuberculosis Physical exam for signs of TB Chest X-Ray – careful interpretation by expert If all of the above are normal, the diagnosis is LTBI If any of the above is abnormal, consider work up for TB disease

23. Chest Radiograph in Children with TB

29. Importance of Source-case Investigations TB in children <5 years of age (especially <2) typically indicates recent transmission. Public health measure Identify the individual who infected the child, who is probably transmitting TB to others Diagnostic measure Isolating the organism from the source case provides likely drug susceptibility of the child’s organism Yield is higher than yield of diagnostic microbiology in children

30. When should we culture children? Cultures should be obtained in young children when: Source case isolate is not available Known or suspected MDR TB The child is immunocompromised Cases of extrapulmonary TB

31. Specimen Collection: which is the best sample in children? Gastric aspirates Obtain with NG tube upon awakening the child and before ambulation or feeding Problems Invasive Expensive: need for overnight stay One study suggested it can be done as outpatient, but results not duplicated Low yield: <50% in children; <75% in infants Induced sputum Hypertonic saline neb to irritate airway and produce cough Inpatient or outpatient Problems Risk of bronchospasm – need to give bronchodilators Limited experience in very young children Most centers are not familiar or comfortable with the procedure in children

32. Specimen collection: which is the best sample in children? For young children (less than 5 y?) Gastric aspirates* For older children without a productive cough Induced sputum* For older children and adolescents with productive cough Spontaneous sputum* Culture pleural fluid, CSF, urine, other body fluids and biopsy specimens * Send samples collected on 3 separate days

33. AFB Staining Ziehl-Neelsen stain Kinyoun method Auramine-rhodamine stain (fluorochrome)

34. Direct Detection Using Nucleic Acid Amplification Test (NAAT) NAAT rapidly identify a specimen via DNA amplification Can provide results within 24-48 hours Benefits may include Earlier lab confirmation of TB disease Earlier respiratory isolation and treatment initiation Improved patient outcomes Interruption of transmission A single negative NAAT does not exclude TB

35. Nucleic Acid Amplification Test (NAAT) Gen-Probe AMPLIFIED Mycobacterium Tuberculosis Direct (MTD) Test Roche AMPLICOR MTB Test Gen-Probe MTD-2 (modified version) is faster and more sensitive – can be used on smear negative samples

36. Nucleic Acid Amplification Tests (NAAT) If NAAT and AFB smears are positive: Patients are presumed to have TB and should begin treatment If NAAT is positive and AFB smears are negative: Patients are presumed to have TB and should begin treatment If NAAT is negative and AFB smears are positive: Patients may have nontuberculous mycobacteria infection (NTM)

37. AFB Culture Remains gold standard for confirming diagnosis of TB Results within 1 to 6 weeks when liquid media used, as long as 10 weeks using solid media Conduct drug-susceptibility testing on initial isolate

38. Molecular Detection of Drug Resistance Several molecular assays can detect mutations that confer resistance Molecular detection should be used for patients with high risk for MDR TB Conventional drug susceptibility testing should be done in conjunction with molecular tests

39. NAATs for detection of drug resistance MYCOResist (PCR-Sequencing) Detects mutations in katG gene indicating resistance to INH and rpoB gene indicating resistance to rifampin. GenoType MTBDRplus (Hain Lifescience, Nehren, Germany) Solid-phase Hybridization Technique Simultaneous molecular genetic identification of M. tuberculosis complex and its resistance to rifampin and isoniazid Xpert® MTB/RIF (Cepheid, Sunnyvale, CA, USA)

40. DNA amplification test Simultaneous detection of both MTB and rifampin resistance (rpoB gene) Highly sensitive in both smear positive and smear negative samples High degree of specificity Results in less than 2 hours

41. Xpert® MTB/RIF in Children Meta-analysis published in March 2015* 15 studies in children <16 y/o: 420 + cultures Compared with culture Sensitivity: 62% - sputum/induced sputum 66% - gastric aspirates Specificity: 98% - sputum/induced sputum and gastric aspirates *The Lancet Respiratory Medicine 2015; 3:451-61

42. In summary… In spite of advances in TB laboratory diagnosis, neither TST, IGRAs, molecular testing (Xpert MTB/Rif) or cultures definitely rule out TB. More research studies are crucial. Currently nothing replaces good clinical judgment to decide when to start anti-TB treatment in children with presentations suspicious for TB.

43. Thank you! Gracias! Shukran! شكرًا