tb pathogenesis and laboratory diagnosis .pptx

tb pathogenesis and laboratory diagnosis

1. Tuberculosis Dr Bhavika patel MBBS,MD Microbiology,DIPC Assistant Professor Department of microbiology GMERS MC, Valsad.

2. Learning objectives At the end of the session, the students will be able to understand: Classification of mycobacteria. Antigenic structure, pathogenesis, clinical manifestations, lab diagnosis, treatment, drug resistance and prophylaxis of TB.

3. Tuberculosis (TB)

4. Mycobacteria Tuberculosis Non sporing, Non-capsulated. Weakly Gram Positive. Strongly Acid Fast (due to mycolic acid in cell wall). Lipid Rich Cell Wall (confers resistance to disinfectants, detergents, common antibiotics and traditional stains). Long Generation time. Humans are the only natural reservoir. Person to person transmission, by infectious aerosols/droplets.

5. Antigenic Structure 1. Cell wall (insoluble) antigens: Peptidoglycan layer – maintains shape & rigidity Arabinogalactan layer - survival of M.tuberculosis within macrophages Mycolic acid layer - principal constituent, confers very low permeability, acid fastness and reduces the entry of most antibiotics Outermost layer - lipids, glycolipids & mycosides

6. Antigenic Structure (Cont..) 1. Cell wall (insoluble) antigens (Cont..): Proteins (e.g. porins , transport proteins) - found throughout various layers Plasma membrane - helps in attachment to host cell and is also a target antigen used for diagnosis

7. Antigenic Structure (Cont..) 2. Cytoplasmic (soluble) antigens: These include antigen 5, antigen 6, antigen 60 Used in serodiagnosis of tuberculosis.

8. Lin P et al. J Immunol 2010 Clearance Pulmonary TB Low grade TB “percolating“ Dormant infection Septic TB Miliary TB Extrapulmonary TB LTBI active TB Spectrum of M. tuberculosis infection

9. Tuberculosis or TB (short for tubercle bacillus) Is a common and often deadly infectious disease caused by mycobacterium usually mycobacterium tuberculosis. M.tuberculosis complex includes: 1.M.tuberculosis 2. M.bovis 3.Other member: M.africanum, M. microti , M. caprae, M. pinnipedii, M. canetti , M. suricattae, M. orygis , M.mungi

10. Pathogenesis

11. Modes of infection 1. Droplet infection Person to person by inhalation aerosols Mycobacterium tuberculosis (Pulmonary tuberculosis) 2.Ingestion of milk Infected cattle Mycobacterium bovis ( Intestinal tuberculosis) 3. Inoculation (in skin tuberculosis) 4.Transplacental route (rare route)

12. Pathogenesis - Risk Factors Sputum positive patients with Bacillary load at least 10 4 bacilli/mL Cavitary lesions in lung - more bacillary load Overcrowding in poorly ventilated rooms Low cell-mediated immunity – HIV Other comorbid conditions - Post-silicosis, postransplantation , hemodialysis , diabetes, IV drug abuse, smoking, etc. Age: Late adolescence and early adulthood Sex: women at 25–34 years, men in older ages

13. EVIDENCE OF INFECTION WITH M TUBERCULOSIS Chest x-ray / positive skin test

14. Host Immune Response - Cell-mediated Immune Response Macrophages present the mycobacterial antigens to T H (T helper) cells - T H 1and T H 2 subsets. TH1 cells release - IL-2 and IFN-γ - activate monocytes and macrophages Activation of T H 1 cells - development of two host responses: 1. A macrophage-activating response 2. Tissue-damaging response.

15. Host Immune Response - Cell-mediated Immune Response (Cont..) 1. A macrophage-activating response: IFN γ activates macrophages Tubercles: a favorable sign Hard tubercles : initially hard - central zone containing activated macrophages (epithelioid and giant cells) and a peripheral zone of lymphocytes and fibroblasts. Soft tubercles - central part undergoes caseous necrosis - Bacilli inhibited within this necrotic environment because of low oxygen tension and low pH - lesion heals and calcifies.

16. Host Immune Response - Cell-mediated Immune Response (Cont..) 2. Tissue-damaging response: Minority of cases, associated with risk factors -macrophage-activating response is weak & bacilli more virulent Mycobacterial growth inhibited only by an intensified delayed hypersensitivity reaction - lung tissue destruction

17. Host Immune Response - Cell-mediated Immune Response (Cont..) Spread of caseous necrosis 1. Direct draining into the airways - discharged with cough 2. Lymphatic spread - reseeding into same or opposite lung → disseminate to other organs. 3. Hematogenous spread to various organs

18. Host Immune Response - Cell-mediated Immune Response (Cont..) Humoral Immune Response: TH2 release - IL-4, IL-5 - activate B-cells - antibodies. M. tuberculosis being obligate intracellular organism , humoral immunity plays a minor role Anti-LAM antibodies play a role in preventing dissemination of tuberculosis in children.

19. Symptoms and Signs of Tuberculosis

20. Clinical Manifestations Tuberculosis (TB) is classified as: Pulmonary TB and Extrapulmonary TB

21. Comparison of primary and secondary pulmonary tuberculosis Features Primary PTB Post-primary (adult-type) /secondary PTB Results due to Initial exogenous infection with tubercle bacilli Exogenous reinfection Endogenous- reactivation of latent primary lesion Age group affected Children Adults Parts of lungs commonly affected Sub pleural lesion affecting, upper part of lower lobe and lower part of upper lobe Apical and posterior segments of the upper lobes (high oxygen tension) Lesions formed at the initial sites Fibrotic nodular lesions are formed ( Ghon focus) Hematogenous seedling in the apex of lungs called Simon’s focus Reactivated Simon focus with central caseation ( Assmann focus)

22. Comparison of primary and secondary pulmonary tuberculosis (Cont..) Features Primary PTB Post-primary (adult-type) /secondary PTB Lymph node Ghon focus with associated hilar lymphadenopathy is common (called as primary complex) Lymph node involvement is unusual Clinical feature It may be asymptomatic or may present with fever, productive cough (with or without hemoptysis ) and occasionally chest pain, night sweating, weight loss Lesions undergoing necrosis and tissue destruction, leading to cavity formation. Symptoms are similar, but more pronounced. Fate Lesions heal spontaneously. Primary complex becomes calcified (Ranke complex) Bronchogenic spread to the same or opposite lung form satellite lesions  caseating pneumonia Hematogenous spread to various parts of the body and granuloma formation. Rarely, heals spontaneously

23. Extrapulmonary Tuberculosis (EPTB) Tuberculous lymphadenitis – MC form (35% of all EPTB) Posterior cervical and supraclavicular lymph nodes - painless swelling in neck region without warmth or color change Pleural tuberculosis – (20%) - pleural effusion Tuberculosis of the upper airways - larynx, pharynx, and epiglottis

24. Extrapulmonary Tuberculosis (EPTB) (Cont..) Genitourinary tuberculosis: Renal tuberculosis Genital tuberculosis – Females: fallopian tubes & endometrium - infertility. Males: epididymis Skeletal tuberculosis - Weight-bearing joints (spine, hips & knees) Complications - collapse of vertebral bodies - kyphosis & paravertebral ‘cold’ abscess

25. Extrapulmonary Tuberculosis (EPTB) (Cont..) Tuberculosis of CNS – M/C in children, Tuberculous meningitis & tuberculoma are common forms Tuberculous pericarditis - direct extension from adjacent lymph nodes or following hematogenous spread

26. Extrapulmonary Tuberculosis (EPTB) (Cont..) Gastrointestinal tuberculosis - Terminal ileum and caecum Due to swallowing of sputum with direct seeding, hematogenous spread, or ingestion of cow’s milk contaminated with M. bovis Tuberculous skin lesions: Scrofuloderma – skin involvement by direct extension from underlying tuberculous lymphadenitis Lupus vulgaris : Apple jelly nodules are formed over face

27. Extrapulmonary Tuberculosis (EPTB) (Cont..) Miliary or disseminated tuberculosis : Hematogenous spread - yellowish 1–2 mm size granulomatous lesions resembling millet seeds in various organs. Post-TB aspergillosis: Chronic pulmonary aspergillosis - due to colonization of Aspergillus fumigatus in the residual TB cavities

28. Epidemiology Quarter of the current world population is infected asymptomatically with M. tuberculosis , of which 5–10% develop the clinical disease. World: 10 million new cases of TB occurred in 2018. Deaths due to TB - 12 Lakh in HIV-negative and 2.5 Lakh in HIV- coinfected people in 2018.

29. Epidemiology (Cont..) WHO regions: South-East Asia (44%), followed by Africa (24%) and the Western Pacific (18%) Countries: Eight countries accounted for two-third of the total TB burden, with India having the largest share. India: In 2018 - 27 Lakh cases occurred India - highest burden from Uttar Pradesh

30. Epidemiology (Cont..) WHO regions: South-East Asia (44%), followed by Africa (24%) and the Western Pacific (18%) Countries: Eight countries accounted for two-third of the total TB burden, with India having the largest share.

31. Laboratory diagnosis of Tuberculosis

32. Laboratory diagnosis of Tuberculosis Specimen collection In pulmonary TB: Sputum (2 specimens—spot and early morning), gastric aspirate (in children) In EPTB: Specimens vary depending on the site involved

33. Extrapulmonary specimens Sterile site specimens collected aseptically Optimum specimens CSF, pericardial fluid, synovial fluid and ascitic fluid Suboptimal specimens (organism load is less) Pleural fluid (20–50 mL is collected and centrifuged) Blood (indicated only for disseminated TB and co- infected with HIV) Specimens containing normal flora Swabs Considered suboptimal specimen. The only recommended swabs are: Laryngeal swabs: Collected early morning in empty stomach or Swab from discharging sinus Urine Three early morning specimens collected (500 mL/ specimen, centrifuged) on different days as TB bacilli in urine are shed intermittently Stool For disseminated TB in HIV infected patients and infants

34. Extrapulmonary specimens (Cont..) Specimens containing normal flora Other respiratory specimens Bronchial secretions (2–5 mL) Bronchoalveolar lavage (20–50 mL) Transbronchial and other biopsies (collected in sterile normal saline) Gastric lavage Recommended for children (tend to swallow sputum), or ICU patients (aspiration) Early morning lavage should be collected and processed early (<4 hours)

35. PRESUMPTIVE TB PT

36. Integrated DR-TB diagnosis and treatment algorithm 40 Rifampicin resistance detected 4 All TB patients All Presumptive TB 1 or Key Population 2 Non-responders Rifampicin resistance not detected 4   DS-TB regimen NAAT 3 FL-LPA 5 + SL-LPA 6 + LC DST 7 – Z, Bdq 8 , Cfz 8 , Mfx, Lzd, Dlm 8 No additional resistance detected 4   or H resistance detected 4 with KatG or InhA mutation (not both) & FQ resistance not detected 4   H resistance detected 4 with both KatG and InhA mutation or FQ resistance detected 4 Shorter oral Bedaquiline-containing MDR/RR-TB regimen 10 Longer oral M/XDR-TB regimen 11 FL-LPA 5 Additional resistance or intolerance or non-availability of any drug in use or emergence of exclusion criteria or return after LTFU or failure H mono/poly DR-TB regimen Modify H mono/poly DR-TB regimen as per replacement table H resistance detected 4 Non-responders Additional resistance or intolerance or non-availability of any drug in use or emergence of exclusion criteria or return after LTFU or failure Longer oral M/XDR-TB regimen, modified if needed as per replacement table Reflex testing for SL-LPA 6 + LC DST 7 – Mfx, Z, Lzd, Cfz 8 Other exclusion criteria 9 for shorter regimen PRESENT ABSENT After completing PTE, check on Nikshay or with C&DST lab, if LPA results are available NO YES YES Stop DS-TB Regimen FIRST SPECIMEN TESTED AT NAAT SITE SECOND SPECIMEN TESTED AT C-DST LAB Yes No

37. Integrated DR-TB diagnosis and treatment algorithm 41 Rifampicin resistance detected 4 All TB patients All Presumptive TB 1 or Key Population 2 Non-responders Rifampicin resistance not detected 4   DS-TB regimen NAAT 3 FL-LPA 5 + SL-LPA 6 + LC DST 7 – Z, Bdq 8 , Cfz 8 , Mfx, Lzd, Dlm 8 No additional resistance detected 4   or H resistance detected 4 with KatG or InhA mutation (not both) & FQ resistance not detected 4   H resistance detected 4 with both KatG and InhA mutation or FQ resistance detected 4 Shorter oral Bedaquiline-containing MDR/RR-TB regimen 10 Longer oral M/XDR-TB regimen 11 FL-LPA 5 Additional resistance or intolerance or non-availability of any drug in use or emergence of exclusion criteria or return after LTFU or failure H mono/poly DR-TB regimen Modify H mono/poly DR-TB regimen as per replacement table H resistance detected 4 Non-responders Additional resistance or intolerance or non-availability of any drug in use or emergence of exclusion criteria or return after LTFU or failure Longer oral M/XDR-TB regimen, modified if needed as per replacement table Reflex testing for SL-LPA 6 + LC DST 7 – Mfx, Z, Lzd, Cfz 8 Other exclusion criteria 9 for shorter regimen PRESENT ABSENT After completing PTE, check on Nikshay or with C&DST lab, if LPA results are available NO YES YES Stop DS-TB Regimen FIRST SPECIMEN TESTED AT NAAT SITE SECOND SPECIMEN TESTED AT C-DST LAB Yes No

38. Laboratory Diagnosis A SMEAR MICROSCOPY 1- Sputum smears stained by Z-N stain Two morning successive mucopurulent sputum samples are needed to diagnoise pulmonary TB . Advantage : - cheap – rapid - Easy to perform Disadvantages: - sputum ( need to contain 5000-10000 AFB/ ml.) - Young children, elderly & HIV infected persons may not produce cavities & sputum containing AFB.

39. Limitation of Microscopy for Tuberculosis. Repeated sample examinations. load on technical staff. Training and dedication of Microscopist. The load of bacilli must be more than 10,000 / 1 ml of sputum. Low in sensitivity < 50 % Repeated requests for samples Not dependable in pediatric age group.

40. Grading If the slide has Result Grading No. of fields to be examined More than 10 AFB /oil immersion field pos 3+ 20 1-10 AFB/ oil immersion field pos 2+ 50 10-99 AFB/100 oil immersion field pos 1+ 100 1-9 AFB/100 oil immersion field pos Scanty* 100 No AFB in 100 oil immersion field neg 100 *record actual number of bacilli seen in 100 fields

41. 2- Detecting AFB by fluorochrome stain using fluorescence microscopy: The smear may be stained by auramine-O dye. In this method the TB bacilli are stained yellow against dark background & easily visualized using florescent microscope . Advantages : - More sensitive - Rapid Disadvantages: - Hazards of dye toxicity - more expensive

42. Mycobacterium Tuberculosis Stained with Fluorescent Dye From Carl Zeiss microimaging GmbH ( FluoLED) Yellow bacilli with green background.

43. B.CBNAAT/True NAT Fully automated Cartridge based Nucleic Acid Amplification Test working on the principal of PCR Generates digital report in 2 Hours (Test Turn around Time*) Sensitivity is similar to Culture and highly specific for M.TB Complex (For Sputum Samples) Advantage – Detects Rifampicin Resistance additionally Extrapulmonary Samples – Gastric Lavage, BAL, CSF, Pericardial Fluid, Synovial Fluid, Pleural Fluid N.B., Samples should not contain blood, Formalin should not be added

44. C.Mycobacterial Culture Reasons to request mycobacterial culture: Patient previously on anti-TB treatment ( Relapse, Defaulter ) Still smear-positive after intensive phase of treatment or after finishing treatment Symptomatic and at high-risk of MDR-TB To test fluids potentially infected with M. tuberculosis Investigation of patients who develop active PTB during or after IPT. TB in health workers

45. 50 FLOW CHART CONTD. Screen by AFB smear & inoculate media (one liquid & one solid) Liquid Medium Solid Media MGIT BACTEC SEPTI-CHEK CMS Incubate At 37 ºC For 6 wks Incubate At 37ºC For 6 wks Incubate inverting At 37ºC For 8 wks Incubate At 37ºC For 6 wks Fluoresc- -ence detected Growth Index >10 Colonies or turbidity Growth detected Confirm by AFB smear Reinoculate on Solid media L J L J with RNA LJ with Pyruvic acid Incubate At 37ºC For 8 wks If growth Confirm on AFB smear

46. Solid media Cultures incubated at 35°c in the dark in an atmosphere of 5% to 10% co2 and high humidity Cultures are examined weekly for growth Most isolates appear between 3 and 6 weeks a few isolates appear after 7 or 8 weeks After 8 wks of incubation negative cultures are reported

47. Eight Week Growth of Mycobacterium tuberculosis on Lowenstein-Jensen Agar

48. Commonly used liquid media systems to culture and detect the growth of mycobacterium BACTEC 460 Mycobacteria growth indicator tube (MGIT) BACTEC MGIT 960 ( continuous growth monitoring systems)

49. Liquid media Use of liquid media system reduces the turn-around time for isolation of acid-fast bacilli to approximately 10 days compared with 17 days or longer in conventional methods Once growth is detected in the liquid media , an acid fast stain is performed to confirm the presence of acid-fast bacilli

50. Laboratory diagnosis of Tuberculosis (Cont..) 55 Culture identification Automated identification—by MALDI-TOF MPT 64 antigen detection—by ICT Different biochemical test – ex Nitrate reduction and niacin production are definitive for M.tb

51. DST (Drug Susceptibility Testing) 56 Phenotypic Methods Genotypic Methods

52. Methods for drug susceptibility testing Genotypic testing is much faster than phenotypic methods.

53. Choice of diagnostic technology DR diagnostic technology Choice NAAT/LPA First Liquid culture isolation and LPA DST Second Liquid culture isolation and liquid DST Third Solid LJ media- of up to 84 days, Liquid Culture (MGIT) up to 42 days, LPA up to 72 hours NAAT - 2 hours. Turn around time

54. Laboratory diagnosis of Tuberculosis (Cont..) Diagnosis of latent tuberculosis Tuberculin skin test (e.g. Mantoux test) Interferon gamma release assay (IGRA).

55. Laboratory diagnosis of Tuberculosis (Cont..) 60 Diagnosis of latent tuberculosis Tuberculin skin test (e.g. Mantoux test) Interferon gamma release assay (IGRA).

56. Mantoux Tuberculin Skin Test (TST) 0.1 ml of tuberculin purified protein derivative (-PPD) into inner surface of forearm intradermally. Read between 48-72 hrs. A positive tuberculin skin test result is supportive evidence in the diagnosis of TB in areas of low prevalence (or no vaccination); however, a negative tuberculin skin test result may occur in approximately one third of patients.

57. Cytokine Assays T- cell Interferon-Gamma Release Assay (IGRA) INF- y produced by T-lymphocytes , is capable of activating macrophages , increasing their bactericidal capacity against M tuberculosis and is involved in granuloma formation. Elevated concentrations of INF-y in TB is related to increased production at the disease site by effectors T cells. The sensitivity of an elevated level varies from 78 to 100% and specificity from 95 to 100%

58. IGRA is useful in targeted strategy for latent TB infection(LTBI) detection in low TB incidence settings More specific than Tuberculin Skin Test Can’t distinguish active from treated TB or LTBI. False positive results in - Hematologic malignancies Empyema.

59. Methods for detection of IFN-y Two new blood tests T-SPOT. TB [Oxford Immunec] – directly count the no of IFN-y secreting T cells. QuantiFERON-TB Gold [Cellestis Limited] – measures the concentration of IFNy secretion. Both tests based on detection of IFN-y in blood have been found to be more accurate than the tuberculin skin test in the diagnosis of latent TB infection. Future research should focus on the potential efficacy of quantification of specifically activated lymphocytes in body fluid and blood using IFN-Y release assay in the diagnosis of TB.

60. Comparison between TST and IGRA test A positive test result by either of the two methods available is not by itself a reliable indicator that the person will progress to TB disease as the possibility of false positive results cannot be ruled out. Conversely, a negative test result does not rule out TBI, given the possibility of a false-negative test result among at-risk groups, such as young children or among those recently infected.

61. Progress in TB Diagnosis Past Present Koch discovered tubercle bacillus 133 yrs back No major discovery Except TB Genome , IS6110, BACTEC 460 (liquid media) TB diagnosed by symptoms - prehistoric Still the same practice in many High Bruden Countries (HBCs) Tuberculin test - > 100yrs Still Commonly used Egg based media –almost 100yrs Still most commonly used AFB Smear for diagnosis – 133 yrs back Still the major diagnostic tool in many countries Radiological Diagnosis Still Important (X-ray, CT Scan)

62. Global and National TB Programmes The End TB Strategy (WHO) Revised National Tuberculosis Control Programme (RNTCP) National Strategic Plan, India (2020–2025) – Nikshay and 99DOTS

63. Vaccine Prophylaxis Against Tuberculosis - Bacillus Calmette-Guérin Vaccine (BCG) BCG strain: In India, WHO recommended Danish 1331 strain of BCG is used. Reconstitution of BCG: Available in lyophilized form, should be reconstituted before administration. Administration of BCG: 0.1 mL (0.1 mg TU) of BCG vaccine - administered above the insertion of left deltoid by intradermal route

64. Vaccine Prophylaxis Against Tuberculosis - Bacillus Calmette-Guérin Vaccine (BCG) (Cont..) Protection: Efficacy: Variable efficacy of 0–80% Duration of immunity - 15–20 years Protection to infants and young children against the development of complications - tuberculous meningitis and disseminated tuberculosis.

65. Vaccine Prophylaxis Against Tuberculosis - Bacillus Calmette-Guérin Vaccine (BCG) (Cont..) Complications following BCG: Most common complications - ulceration at the vaccination site and regional lymphadenitis Rarely - keloid or lupus lesion, osteomyelitis, non-fatal meningitis, progressivetuberculosis and disseminated BCG infection (“ BCGitis ”).

66. Vaccine Prophylaxis Against Tuberculosis - Bacillus Calmette-Guérin Vaccine (BCG) (Cont..) Indications of BCG Direct BCG: BCG is directly given to the newborn soon after birth. Indirect BCG: BCG is given after performing tuberculin skin test.

67. Vaccine Prophylaxis Against Tuberculosis - Bacillus Calmette-Guérin Vaccine (BCG) (Cont..) Contraindications to BCG include: HIV-positive child Child born to AFB positive mother Child with low immunity Generalized eczema Pregnancy.

68. Chemoprophylaxis Treatment of selected high-risk tuberculin reactors (i.e. people with latent tuberculosis) aims at preventing active disease. Isoniazid or ethambutol for six months - tried. Chemoprophylaxis has several shortcomings such as— (1) it is expensive, (2) risk of developing tuberculosis is minimal in tuberculin reactors, and (3) side effects of the drugs.

69. Chemoprophylaxis (Cont..) INH preventive therapy (IPT) - restricted to limited indications: Adults with HIV - unlikely to have active TB Children with HIV - no TB symptoms and unlikely to have active TB All children with HIV - successfully completed treatment for TB.