Nodal TB Diagnostics Modalities in children .pptx

Diagnostic Plan for Lymph Node Tb in Children

1. TB Lymphadenitis

2. 12-year girl at opd Painful s welling in right submandibular region for the last 10 -12 weeks (swelling increased initially but has remained of the same size for the past 6 weeks) H /O undocumented fever for 1m N o H/O cough, or appetite /weight loss. No other localizing symptoms Non contributary past medical and family history. Case

3. T hin built girl A single diffuse swelling 4x3 cm in the right submandibular region, ill-defined borders. S mooth surface, firm consistency, tender, non-fluctuant, noncompressible, mobile, and showed signs of matting No other lymph nodes palpable in the cervical, supraclavicular, or axillary areas and /or Hepatosplenomegaly. Pallor Throat- mild pharyngeal congestion, tonsils appear normal Other systems –normal O/E –

4. Differentials ? Reactive Typically, patients will have a history of a viral prodrome. The reactive lymph node(s) may be tender to palpation and have a unilateral or bilateral location. Most importantly, the lymphadenopathy typically resolves with resolution of the viral llness . Common viruses, including rhinovirus, adenovirus, influenza, parainfluenza, and respiratory syncytial virus, May induce a self-resolving and uncomplicated cervical lymphadenopathy. TB Unilateral Lymph node involvement beyond two weeks, invariably painless and without fever Always consider in prolonged fevers from community Contact history not always positive/reliable Mantoux/ IGRA are tests for latent TB IF TB strong clinical suspicion, FNA with cytology and Xpert MTB can help diagnose TB. Lymphoma Possible presentation FNAC will miss diagnosis if not representative sample. Best to do representative node biopsy in this case. At least get Chest Xray to see any mediastinal enlargement HIV Though uncommon, can be chronic HIV presentation Probe into details on parent’s health/ medical conditions and then, consider testing if strong suspicion. SLE No other diagnostic criteria like malar rash, photosensitivity, arthritis, serositis, oral ulcers etc. Remember, ANA can be positive in up to 15% of normal people

5. What Investigations will you order? Complete Hemogram , ESR ? RBS LFT/KFT HIV serology

6. Labs Hb 8.8 g/dl TC 4200 cells/cu.mm DC P74 L22 M4, with reactive lymphocytes Platelet Count: 4,70,000 cells/cu.mm P. smear : microcytic hypochromic RBCs, no abnormal cells. ESR 27 mm /hour AST 33 u/L ALT 43 U/L LFT : Normal EBV VCA IgM positive

7. Discuss Is this infectious mononucleosis or some other cause? How to proceed/ counsel parents?

8. What Should be the next plan of investigation? Radiology: • Chest X-ray • Ultrasonography • CT Scan • MRI/PET- CT (subject to clinical indication and availability) Sampling: Histo path and Microbial Dx

9. Labs Chest X ray : Normal Mantoux - 12mm / IGRA FNAC of cervical node : Reactive hyperplasia ANA- positive (++) USG Abdomen: Periportal Nodes with Hepatosplenomegaly USG of the neck

10. Discuss briefly IGRA/ Mantoux + , but FNAC is not suggestive - can not start ATT ANA + , but no distinguishing SLE features USG abdomen : HSM with peri-portal nodes as well- some disseminated process. Consider TB/ Lymphoma/ other chronic infections( HIV, Brucella, melioidosis) etc. Probe further into epidemiological history on animal contact, unpasteurized milk, soil/ water contact.

11. Tuberculin Skin Test (TST)/ Mantoux test [Ref: Elhassan et al.International Journal of Infectious Diseases 43 (2016) 25-29 Detjen et al Lancet Respir Med 2015;3:451–61 . ]. Screening test for LTBI diagnosis Limited value in the diagnosis of active tuberculosis False-negative reactions common in immunosuppressed patients False-positive reactions seen in non- tuberculous mycobacteria (NTM) and BCG vaccination. ADVANTAGES LIMITATIONS

12. Blood (1 ml) is collected into each of the three tubes, mixed with the reagents already in the tubes and incubated for 16 to 24 hours. Plasma is separated, and the IFN-g concentration in the plasma is determined using a sensitive ELISA.

13. Interferon Gamma Release Assay (IGRA) In vitro assays that measure T-cell release of IFN-γ in response to stimulation with the tuberculosis-specific antigens ESAT-6 and CFP-10 Two tests are commercially available QuantiFERON-TB Gold® T-SPOT.TB® (ELISpot) assay

14. Interferon Gamma Release Assay (IGRA) More specific than TST due to less cross-reactivity with BCG vaccination & NTM Limited value in the diagnosis of active tuberculosis including EPTB A positive IGRA may not necessarily indicate active TB. A negative IGRA result would not conclusively rule out active disease in an individual suspected to have TB ADVANTAGES LIMITATIONS

15. QFT-GIT T-Spot Initial Process Process whole blood within 16 hours Process peripheral blood mononuclear cells (PBMCs) within 8 hours, or if T-cell Xtend is used, within 30 hours M. Tuberculosis Antigen Single Mixture of synthetic peptides representing ESAT- 6, CFP – 10 and TB 7.7 Separate mixtures of synthetic peptides representing ESAT- 6 and CFT - 10 Measurement IFN-g Concentration Number of IFN – g producing cells (spots) Possible Results Positive, Negative, Indeterminate Positive, negative, borderline, invalid Difference in Currently Available IGRAs

16. TSPOT. TB QuantiFERON Gold In-Tube Sample Collection 1x tube heparinized blood Minimum blood vol. = 5ml 3x QFT blood tubes containing nil control, Mtb antigens (combined) or mitogen control 1ml blood per tube Total blood volume = 3ml Pre-incubation Sample preparation PBMC isolation, and addition of PBMCs to TSPOT. TB 96 well plate (250,000/well) Containing negative control, Panel A and B MTB Antigens (individually) or positive control MTB antigen ESAT 6, CFP 10 Inversion of tubes to ensure mixing of antigens/controls with blood Mtb antigens: ESAT-6, CFP-10, TB7.7 Incubation 16-20 hour incubation in TSPOT . TB in 96 well plate (37 C, 5% CO2) 16-24 hour incubation in QFT blood tubes (37 C) Post- incubation Sample preparation Removal of cells and antigens/controls by washing Centrifugation of QFT blood tubes for separation of plasma Assay Procedure ELISpot for enumeration of IFN – G producing cells

17. Characteristic Tuberculin Skin Test QuantiFERON TB T-Spot TB Time to result 48-72 hrs 24-36 h 36-48 h Complexity Low Moderate High TB antigen PPD-tuberculin(not specific to M. tb.) ESAT-6; CFP-10; TB-7.7 ESAT-6; CFP-10 Measurement Skin induration after in vivo stimulation ELISA-based measurement of IFN-y production by T-cells after in vitro stimulation ELISPOT-based measurement of IFN-y-producing T-cells (spots) after in vitro stimulation Minimum number of visit to complete testing 2 visits(within 48-72 h of placement) 1 visit Sample/ Method I D of 5 units of PPD-tuberculin Blood draw Reliability/ Variability of test result Limited variability with appropriate training Significant within-person variability Cross – reactivity with BCG vaccine Yes (particularly if vaccinated after infancy or repeatedly) No NTM cross reaction Many Few (M. kansasi, M. marinum,M. szulgai) Booster effect with repeated testing Yes No Booster effect after prior TST Yes Possible (but likely inconsequential if blood drawn < 3 days after TST Internal Controls No Yes Utility by Age Less reliable in children under 6-months of age Less reliable in children under 5-years of age children under Sensitivity with bacteriologically confirmed TB 75-85% 80-85% Sensitivity with bacteriologically confirmed TB 95-100% With BCG vaccination 49-65% 90-95% With BCG vaccination 89-100% Sensitivity in HIV- infected patients 45% Same as TST [36); T-SPOT.TB slightly less affected by immunosuppression than QFT

18. Radiological Diagnosis:

19. SMEAR MICROSCOPY Same-day results Low tech Inexpensive Widely available Can be used for treatment monitoring Useful mainly in Pulm TB Low sensitivity (50%) among all cases Not accurate for children, people with HIV Doesn’t give information on drug susceptibility Can’t distinguish MTB from other related bacteria (non-TB mycobacteria) ADVANTAGES LIMITATIONS

20. Tuberculous lymphadenitis H & E: hematoxylin and eosin; PAP: Papanicolaou

21. A Fine Needle Aspiration (FNA): FNA is a simple and inexpensive procedure FNA can be directly done from superficial nodes Deep nodes: USG/CT/EBUS guided aspiration can be done Material from FNA to be subjected to: ZN stain NAAT MGIT/L-J media Cyto-histopathology Limitations of FNA Limited sample amount Tissue morphology might be distorted in immunodeficiency i.e. HIV Tuberculous granuloma

22. Biopsy: When & What type Can be done where FNA is not diagnostic in the setting of strong clinical suspicion of TB or alternate diagnosis is suspected. Excisional biopsy is preferred over incisional biopsy excisional biopsy has highest diagnostic yield and incisional biopsy later may be associated with sinus tract formation . Mycobacterium tuberculosis can be cultured in 70-90% cases in excisional biopsy . Mediastinal lymph node biopsy is done with help of Bronchoscopy/mediastinoscopy/EBUS. Merits: Highest diagnostic yield Adequate sample can be sent for AFB, microscopy, culture, molecular test and immunohistochemistry marker for lymphoma/carcinomas can also be done. Limitations: Requires experience Local complications like infection, sinus tract formation , scarring

23. Excisional biopsy is the most invasive approach to diagnosis; however, it has the highest sensitivity and may produce a more rapid and favorable symptomatic response and has been recommended in cases involving multiple nodes. Complications of biopsy include postsurgical pain, wound infection, sinus formation and scar. Currently the combined Contrast Enhanced US-guided core biopsy with Xpert may be more helpful to improve the efficiency of early etiological diagnosis of TBLN being minimally invasive and does not warrant hospital stay.

24. Ultrasound is the first line investigation for cervical lymphadenopathy . Sonographic features that help to identify abnormal nodes include shape (round), absent hilum, intranodal necrosis, reticulation, calcification, matting, soft-tissue oedema, and peripheral vascularity. Tuberculous nodes - hypoechoic, round, without echogenic hilus and tend to show intranodal cystic necrosis, nodal matting, and adjacent soft-tissue oedema. On colour doppler, power Doppler, and 3D sonography, the vascular distribution of tuberculous nodes is varied and simulates benign and malignant nodes. Displacement of hilar vascularity is common in tuberculous nodes and is due to the high incidence of intranodal cystic necrosis, which displaces the vessels, in tuberculous nodes. Ultrasonography:

25. Ultrasound-guided core biopsy A n alternative diagnostic tool - increasingly being used to diagnose cervical masses. Logistic advantages over other procedures Performed under local anesthesia in outpatient clinics F ewer complications I ncreased patient satisfaction. H igh diagnostic accuracy - technically provides a larger tissue sample that retains its architecture and permits the use of a range of histochemical and immunohistochemical stains. Superior in yield compared to FNA and comparable to excisional biopsy , if local expertise is available. It can be further enhanced by the use of CE-EUS with the combined qualitative and quantitative analyses for lymphadenopathy to complement regular EUS and EUS-FNA

26. Diagnostic value of color Doppler ultrasound, elastography and contrast-enhanced multimodal ultrasound for detecting superficial tuberculous lymphadenitis

27. (A) Transaxial   18 F-FDG PET–CT demonstrating a right  supraclavicular lymph node  with an SUV max  of 5.7. (B) Coronal slice revealing multiple mediastinal lymph nodes with an SUV max  of 9.9 in the right lower paratracheal region. Hypometabolic areas noted in the nodes are suggestive of caseation/necrosis. PET- CT in a Case of tubercular LAP lymphadenopathy.

28. MICROBIOLOGICAL DIAGNOSIS SMEAR MIC R OS C O P Y ZN Stain Fluo r e s c en c e stain CULTURE T E C H NI Q U E S Solid Liquid M y c o b ac t e rium Growth Indicator Tube (MGIT) MOL E CULAR METHODS PCR Real Time PCR CB NAAT/ G e neX p e rt Line Probe Assay (LPA) TESTS FOR LTBI TST/ Mantoux test Interferon Gamma Release Assay (IGRA) O TH E R L i p oa r abi n om annan assay (LAM) Adenosine Deaminase (ADA)

29. 2. CULTURE TECHNIQUES SOLID (CONVENTIONAL) Egg based – Lowenstein- Jensen media Agar based – Middlebrook 7H10 or 7H11 media LIQUID (AUTOMATED) Mycobacterium Growth Indicator Tube (MGIT) BacT/Alert MGIT 960

30. 2. CULTURE TECHNIQUES contd… ADVANTAGES More sensitive than microscopy Avg time to growth detection – 4-6 weeks Useful for smear negative TB cases Drug susceptibility can be performed Useful for follow up SO L ID LIQUID ADVANTAGES 20% more sensitive than solid culture Avg time to growth detection – 10-14 days Same

31. AUTOMATED LIQUID CULTURES Considered the gold-standard for isolating mycobacteria. Increase the yield by 10-20% compared with solid media. Liquid media for culture and DST results in saving time. Prone to contamination . Requires stringent quality assurance systems. Expensive Requirement of well trained staff with extensive technical expertise. ADVANTAGES LIMITATIONS

32. Molecular Diagnosis From the fine needle aspirate Two cytological smears are prepared from each aspirate one to be air dried and the other spray fixed with commercial alcohol-based cytology fixative, and the needle and syringe are rinsed in either mycobacterial growth indicator tube (MGIT) medium for mycobacterial culture using the BACTEC MGIT 960 System (Becton Dickinson, Franklin Lakes, NJ) for a maximum of 8 weeks. or TB transport medium (0.7 mL sterile Middlebrook 7H9 medium) for Rapid diagnosis of M. tuberculosis complex Xpert MTB/RIF testing RIF sample preparation buffer is added to the TB transport bottle containing the aspirate (in a 2:1 ratio) and are processed for Xpert MTB/RIF testing according to the manufacturer’s instructions.

33. Xpert MTB/RIF from different samples SAMPLE SENSITIVITY SPECIFICITY SPUTUM 88% 99% LYMPH NODE ASPIRATE 85% 92.5% GASTRIC LAVAGE 83.8% 98.8% CSF 79.5% 98.6% PLEURAL FLUID 43.7% 98.1%

34. Ultra TM

35. TruNat TM

36. Method Direct Smear Culture CBNAAT TRU NAT CBNAAT ULTRA Limit of detection 10,000/ml 50 – 100/ml 131/ml 150 CFU/ml 16/ml Time to result 1 hour 9 – 12 day MGIT <2 Hours <2 Hours <2 Hours Cost Rs 100 - 200 Rs 800 - 1000 Rs 1500 - 2000 <1000 ???

37. A combination of cytology (positive for epithelioid cell granulomas, multinucleated giant cells, a granulomatous lesion with caseation and necrosis), and AFB identification in the specimen can help to diagnose TBL Imaging techniques such as CT or MRI can be used as an adjunct diagnostic tool for TBL . Imaging alone is insufficient to discriminate TBL from other necrotic lymphadenopathies. T-SPOT-TB has 91% sensitivity and 74% specificity for TBL diagnosis 

38. Sensitivity and specificity of various diagnostic tests commonly used for EP TB

39. Role of Tuberculin Skin Test? Role of TB PCR open system? Ancillary Aids

40. To Summarise….

41. Patients who should be investigated for LNTB Presumptive peripheral LNTB Enlarged LN (>2 cm across) in neck, axilla or groin. May have - fever, cough, weight loss Presumptive mediastinal LNTB Chest X-ray hilar widening, CT chest mediastinal LN in absence of active pulmonary TB May have - fever, cough, weight loss, tachypnea Presumptive abdominal LNTB Dull or colicky abdominal pain, weight loss, abdominal LN on USG, CT or MRI

42. Xpert MTB/ RIF assay MTB detectedR resistant MTB detected; R sensitive MTB not detected; In groups with high Risk of MDR- TB WHO recommended regimen for MDR-TB with H; Registration as RR-TB DST to at least H; Quinolones; SL injectable Modify MDR-TB treatment based on DST results; Update registration In groups with low risk of MDR-TB Repeat Xpert MTB/ RIF MTB detected; R sensitive MTB detected; R resistant WHO recommended Regimen fpr MTB-TB With H; Register as RR-TB DST to at least R; H; Quinolones; SL injectables Modify MTB-TB Treatment based on the DST results Update Registration In case of discordance on R result, refer sample for sequencing WHO recommended First line regimen; Registration as bacteriologically confirmed TB If TB still suspected Further investigation (CXR, repeat Xpert MTB/ RIF, Culture, etc

43. RECOMMENDED TESTS FOR PUL & EPTB CONDITION FREQUENCY SPECIMEN RECOMMENDED TESTS REMARKS Pulmonary TB 75% Sputum Smear microscopy Liquid culture CB NAAT In MDR suspected cases molecular method should be 1 st choice Tubercular lymphadenitis 35-40% Lymph node aspirate Smear microscopy Liquid culture (~70% positivity) CB NAAT Pleural TB 20% Pleural fluid Biochemical analysis ADA is useful screening test IGRA may be helpful Liquid culture (~80% positivity) CB NAAT has low sensitivity Smear microscopy lower sensitivity

44. RECOMMENDED TESTS FOR PUL & EPTB CONDITION FREQUENCY SPECIMEN RECOMMENDED TESTS REMARKS T ube r cular meningitis 5% CSF Biochemical analysis CBNAAT (~ 70% positivity) Culture (~ 80 % positivity) Negative result does not exclude diagnosis of TBM T ube r cular peritonitis 3.5% Ascitic fluid Biochemical analysis Culture (low sensitivity) Peritoneal biopsy (by laparoscopy) is often needed to establish the diagnosis Tubercular pe ri c a r d i tis Very low P er i c a r dial fluid Biochemical analysis Culture (~ 70% positivity) ADA IFN Y assay Disease of elderly with low TB prevalence Seen in HIV +ve patients

45. Superficial lymphadenopathy Sample Deep lymphadenopathy Sample Cytopathology • CBNAAT (GeneXpert MTB/RIF) • AFB • Culture(MGIT/LJ media) wherever possible Cytopathology • GeneXpert MTB/RIF • AFB • Culture (LJ media /MGIT) Bacteriologically Confirmed LNTB case Symptoms and signs of LNTB and has at least one of the following: • Positive microscopy for AFB • Positive culture of M tb • Positive validated PCR-based test (such as Xpert MTB/RIF) Clinically Diagnosed LNTB case A LNTB patient who has all of: • Negative microscopy, negative culture and PCR- based tests • Strongly suggestive radiological findings, histopathological findings, clinical course • No other diagnosis made to explain signs and symptoms Treat with drug sensitive ATT

46. Carry Home Message A detailed clinical examination and routine evaluation patients with deep lymphadenopathy, all peripheral lymph nodes must be assessed for the presence of lymphadenopathy and preferred for sampling. Chest radiograph to be performed in all patients to rule out pulmonary tuberculosis TB screening and diagnosis requires a combination of different methods and techniques to accurately test people There is no single TB test that can do it all Culture remains GOLD standard for the definitive diagnosis of TB in smear negative patients Culture techniques should be used for monitoring treatment response or follow up of case Molecular methods are promising with a very low turn around time Suspected MDR/XDR cases first diagnostic choice should be CBNAAT or LPA