Diagnosis of Tuberculosis

Diagnosis of TB by various methods

1. Diagnosis Of Tuberculosis By- Dr. Amita Yadav MBBS, MD (Patholog y)

2. Introduction- Tuberculosis is highly infectious chronic inflammatory disease caused by Mycobacterium tuberculosis characterised histologically by formation of caseating granuloma .

3. India accounts for 26% of the total global TB burden i.e. 2.0-2.5million new cases annually. In Out of all TB notified cases in India, 53% are smear positive cases and 28% are smear negative cases and 19% are extra pulmonary cases. Govt of India declared Tuberculosis a notifiable disease on 7th May 2012

4. Case definitions- A bacteriologically confirmed TB case is one from whom a biological specimen is positive by smear microscopy , culture or WRD ( WHO approved Rapid Diagnostics such as Xpert MTB/RIF).

5. A clinically diagnosed TB case is one who does not fulfil the criteria for bacteriological confirmation but has been diagnosed with active TB by a clinician . This definition includes cases diagnosed on the basis of X-ray abnormalities or suggestive histology and extrapulmonary cases without laboratory confirmation .

6. Classification based on history of previous TB treatment (patient registration group) New patients have never been treated for TB or have taken anti-TB drugs for less than 1 month . Previously treated patients have received 1 month or more of anti-TB drugs in the past. They are further classified by the outcome of their most recent course of treatment

7. Relapse patients have previously been treated for TB, were declared cured or treatment completed at the end of their most recent course of treatment, and are now diagnosed with a recurrent episode of TB (either a true relapse or a new episode of TB caused by reinfection ). Treatment after failure patients are those who have previously been treated for TB and whose treatment failed at the end of their most recent course of treatment. Treatment after loss to follow-up patients have previously been treated for TB and were declared lost to follow-up at the end their most recent course of treatment. (These were previously known as treatment after default patients .)

8. Classification based on drug Resistance Monoresistance : resistance to one first-line anti-TB drug only. Polydrug resistance: resistance to more than one first-line anti-TB drug (other than both isoniazid and rifampicin ). Multidrug resistance: resistance to at least both isoniazid and rifampicin . Extensive drug resistance: resistance to any fluoroquinolone and to at least one of three second-line injectable drugs ( capreomycin , kanamycin and amikacin ), in addition to multidrug resistance. Total Drug Resistance Or XXDR- Resistance to all anti TB drugs.

9. Mycobacterium species- M. tuberculosis complex- M. tuberculosis – most common M. bovis ( M. microti , M. africanum , M. canetti ) MOTT/NTM- Mycobacterium kansasii Mycobacterium fortuitum Mycobacterium avium-intracellulare Mycobacterium xenopi Mycobacterium chelatum M.leprae

10. CHARECTERISTICS OF M. TUBERCULOSIS Aerobic and non motile Straight or curved rods, beaded 0.2-0.5 µ in Diameter, 2-4 µ in Length Mycolic acid present in its cell wall, makes it acid fast It resists decolourization with 20% acid & alcohol It multiplies slowly, can remain dormant for decades ( Doubling time- 18-20 hours)

11. Sites Involved- Pulmonary TB (85% of all TB cases) Miliary TB is classified as PTB because there are lesions in the lungs. Extra-pulmonary sites Lymph node Genito -urinary tract Bones & Joints Meninges Intestine Skin Brain Larynx Lymph node Kidney Spine Lung Bone

12. Symptoms of Tuberculosis- 1.General Symptoms Fever, night sweats, weight loss Tiredness and Loss of appetite 2.Pulmonary Symptoms 3.Extrapulmonary Symptoms

13. Symptoms of Pulmonary TB- Persistent cough of two weeks or more, with or without expectoration.(Most Common) It may be accompanied by one or more of the following symptoms:- Chest pain, haemoptysis (coughing up of blood or blood stained sputum)), shortness of breath,

14. Symptoms of Extrapulmonary TB- Enlarged cervical lymph nodes with or without discharging sinuses ( TB Lymphadenitis ) Chest pain with or without dyspnoea (difficulty in breathing) in pleural TB Pain and swelling of the joints in bone tuberculosis (fever, backache, deformity in spine) Signs of raised intra-cranial tension like irritability, headache, vomiting, fever, stiffness of the neck and mental confusion in TB meningitis Painless haematuria (blood in urine) or sterile pyuria (pus in urine) in renal tuberculosis Infertility in genito -urinary TB.

15. Pathogenesis Of Tuberculosis-

16. Spectrum of Tuberculosis-

17. Primary Tuberculosis-

18. Secondary Tuberculosis-

19. Tuberculosis Lymph Node-

20. Tuberculosis Intestine-

21. Tuberculosis Skin (Lupus Vulgaris )-

22. Miliary Tuberculosis-

23. Histology-

24. Histology-

25. Cytology-

26. Diagnostic steps:- 1-History and clinical examination 2.Bacteriological Examination- a.Direct methods b.Indirect Methods

27. Other Investigations- Fine Needle Aspiration Cytology (FNAC) and direct smear examination Excision / Biopsy of specimen for histo -pathological examination Fluid for cytology, biochemical analysis and smear examination Urine for LAM antigen test. ADA level Radiology

28. Diagramatic Algorithm of Pulmonary Tuberculosis-

29. Blood Picture:- (nonspecific) Anemia due to either hemoptysis or chronic illness Relative Lymphocytosis High ESR

30. Methods of collection of sputum sample-

31. Conventional Diagnostic Methods:- Acid fast bacilli (AFB) smear microscopy and culture are still the“ gold standards ” for the diagnosis of active TB. Active tuberculosis (TB) is diagnosed by detecting Mycobacterium tuberculosis bacilli in specimens from the respiratory tract (pulmonary TB) or in specimens from other bodily sites ( extrapulmonary TB). -AFB smear microscopy and culture can also be used to monitor the effectiveness of treatment and can help to determine when a patient is less likely to be infectious.

32. Microscopy- Mycobacteria can be visually distinguished from other microorganisms by their thick lipid containing cell wall, which retains biochemical stains despite decolourisation by acid-containing reagents (known as ‘acid fastness’). Examination of two sputum specimens is adequate to identify the majority (95-98%) of smear-positive TB patients (WHO’s current policy on case-finding)

33. A case is defined as someone with one positive smear that is, at least 1 acid-fast bacillus in at least100 microscopic fields. In 2010, WHO confirmed the diagnostic accuracy of examining two consecutive smears on the same day to diagnose TB.

34. Microscopy- 1-Ziehl-Neelsen ( Ziehl-Neelsen is a hot acid-fast stain because the slide has to be heated during incubation with carbol fuchsin ) 2- Kinyoun (is a cold acid-fast staining procedure) 3-Fluorochrome procedure (primary staining is done with fluorescent dye) 4- AFB staining for colour blinds

35. Ziehl-Neelson Staining- Fix the smear with gentle heat. Cover the slide with filtered ZN carbol fuchsin . (8-10min) Heat the slide until steam rises but without boiling .

36. Wash D/W. Decolorize with 20% H 2 SO 4 or 3% Acid-alcohol. (3 min) Counterstain with methylene blue or malachite green. (3min)

37. Smear results reported as follows:

38. Fluorescence Microscopy- Fluorescence staining utilizes basically the same approach as Z-N staining, but carbol fuchsin is replaced by a fluorescent dye ( auramine -O, rhodamine , auramine-rhodamine , acridine orange etc), the acid for decolorisation is milder and the counter stain, though not essential, is useful to quench background fluorescence. Sensitivity- 52-97% Specificity-98%

39. 10% more sensitive than light microscopy (Sensitivity of light microscope- 32-94%) The most important advantage of the fluorescence technique is that slides can be examined at a lower magnification, thus allowing the examination of a much larger area per unit of time. In fluorescence microscopy, the same area that needs examination for 10 minutes with a light microscope can be examined in 2 minutes.

40. Principle- Mycobacteria retain the primary stain even after exposure to decolorizing with acid alcohol, hence the term “acid-fast”. A counter-stain is employed to highlight the stained organisms for easier recognition. Potassium permanganate is used as counter-stain and it helps prevent non-specific fluorescence.

41. With auramine staining, the bacilli appear as slender luminous rods, standing out clearly against a dark background. The identification of the mycobacteria with auramine O is due to the affinity of the mycolic acid in the cell walls for the fluorochromes .

42. Results reported as follows-

43. AFB Staining for colour blinds-

44. For color blind people (or in backgrounds where detecting red bacteria is difficult)  Victoria Blue  can be substituted for  carbol fuchsin   and  Picric acid  can be used as the counter stain instead of  methylene blue , and rest of Kinyoun technique can be used.

45. Recent Trend- A blue –green screen or filter is placed in front of high intensity illuminant ,so the bacilli appear black. If the counterstain is malachite green the background of cells almost disappears, so the bacilli are easily recognisable .

46. Advantage- Simple and inexpensive. Suitable for both peripheral level and higher level laboratories. Necessary for follow up of patients with susceptible TB.

47. Disadvantage- Direct sputum-smear microscopy is relatively insensitive: at least 5 000 to 10,000 bacilli per ml of sputum are required for a positive result. The sensitivity is further reduced in patients with extrapulmonary TB, children and in those who are coinfected with HIV.

48. Limitations- Cannot distinguish Mycobacterium tuberculosis complex from nontuberculous mycobacteria ; It cannot distinguish drug-susceptible strains from drug-resistant strains.

49. Testing for Latent TB infection- The lifetime risk of TB reactivation for a person with documented LTBI is estimated to be 5–10%, with the majority developing TB disease within the first five years after initial infection. A direct measurement tool for M. tuberculosis infection in humans is currently unavailable, hence, there is no gold standard for the diagnosis of LTBI. The tuberculin skin test (TST) and Interferongamma release assays (IGRAs) indirectly measureTB infection.

50. The tuberculin skin test (TST) and Interferon gamma release assays (IGRAs) indirectly measure TB infection by detecting memory T-cell response signifying the presence of host sensitization to M. tuberculosis antigens.

51. Tuberculin Skin Test- TST has been used to identify patients actively infected with TB, To measure the prevalence of infection in a community, and to select susceptible or high-risk patients for BCG vaccination . To detect persons at high risk for LTBI or at high risk to progress to disease

52. Tuberculin Skin Test Method- TST works by intradermally injecting 0.1 mL of 5 TU PPD on the forearm. ( Mantoux's method) On examination, after 48-72 hours, a positive reaction is indicated by erythema and induration of > 10 mm in size. Erythema (redness) alone is not taken as a positive reaction.

53. Interferon Gama Release Assay (IGRA )- QuantiFERON -TB Gold This in vitro test detects the release of interferon-gamma (IFN-γ) from lymphocytes of sensitized persons when their blood is incubated with peptide mixtures simulating two M. tuberculosis proteins called ESAT-6 and CFP-10 . The TB-specific antigens ESAT-6 and CFP- 10 that are only present in M. tuberculosis and are absent from all strains of M.bovis (BCG) and most environmental mycobacteria

54. The IFN-γ secreted by T-cells into the plasma is measured by ELISA to indicate the likelihood of TB infection .

55. T Spot TB Test- The T-SPOT. TB  technique uses isolated peripheral blood mononuclear cells to detect interferon γ released by stimulated T cells using the enzyme-linked immunospot assay. The cells are stimulated by the  Mycobacterium tuberculosis -specific antigens ESAT-6 and CFP-10, applied separately. The appearance of spots in the well indicates the presence of reactive T cells. These spots are then counted manually or with an automated plate reader. The result is interpreted as positive if there are at least 6 more spots in either or both of the ESAT-6 or CFP-10 panels than in the nil control panel; the spot count in the antigen panels must also be twice that of the nil control panel. The result is considered indeterminate if there are fewer than 20 spots in the positive control panel and fewer than 6 spots in the antigen panels.

56. Urinary LAM Antigen Test ( Chemogen ®) Detects LAM antigen by ELISA in the urine Lipoarabinomannan (LAM) is a complex glycolipid associated with cell wall of mycobacteria & is produced in substantial quantities by growing mycobacterium Rapid (2.5 hrs) Sensitivity: 80%

57. ADA level in tuberculosis- Adenosine deaminase  (also known as Adenosine aminhydrolase , or  ADA ) is an enzyme involved in  purine metabolism. There are 2  isoforms of ADA: ADA1 and ADA2. ADA2 is found predominantly in the human plasma and serum, and exists solely as a homodimer . In tubercular effusion we detect ADA2.

58. Tubercular pleural Effusion- In a journal of Lung India , an official publication of Indian chest society ,it has been concluded that  If 36 IU/L is taken as cut of limit the sensitivity and specificity of ADA for tuberculosis is 100 % and 77.7 %. More than 100 IU/L was exclusively seen in tubercular pleural effusion.

59. Tubercular Peritonitis- According to a study result published in Pubmed done on 2006  ADA levels showed high sensitivity (100%) and specificity (97%) using cut-off values from 36 to 40 IU/L. Optimal cut-off point was determined at 39 IU/L.

60. Tubercular Pericarditis - The median ADA level in the tuberculous group -71.7 U/L (range 10.3 to 303.6 U/L).

61. Tubercular Meningitis- In a research of Biochemistry Research Laboratory, Central India Institute of Medical Sciences , demonstrated that ADA activity in the CSF of TBM patients, using a cutoff value 11.39 U/L/min, can be useful for the early differential diagnosis of TBM.

62. Cobweb formation in CSF in Tubercular Meningitis--

63. Tubercular Meningitis-

64. Culture- Solid Media- Egg-based Media: Lowenstein-Jensen (LJ) Media Dorset Medium Serum containing Media: Loeffler’s Media Potato-based Media: Pawlowsky’s Media Blood containing Media: Tarshi’s Media Agar-based Media: Middlebrook 7H10 Middlebrook 7H11 Middlebrook Biplate (7H10/7H11 S Agar).

65. Solid Media- (Duration- 3-4 wks) 1- Löwenstein -Jensen (egg and also contain high concentrations of malachite green to overcome contamination with other bacteria) 2- Middlebrook 7H10 and 7H11 are ( agar-based media.) Middlebrook media have been shown to achieve slightly higher isolation yields than egg-based media, but are considerably more expensive.

66. L-J Media- Colonies are dry, rough, raised, irregular with wrinkled surface. They are creamy white initially, becoming yellowish or buff coloured on further incubation.

67. Culture result reported as follows: = no growth reported (1−9) = <10 colonies (report number of colonies) + = 10−100 colonies ++ = >100 colonies +++ = innumerable or confluent growth

68. Liquid Media- (duration- 10-14 days) BACTEC 12 B Medium BACTEC 460 TB BACTEC MGIT 960 EPS Culture System II Middlebrook 7H9 Broth SeptiChek AFB Dubo’s Medium Tween 80 ( Sorbitol Mono oleate )

69. BACTEC 460 ( rapid radiometric culture system ) Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C 14 – labelled palmitic acid & PANTA antibiotic mixture. P ---- Polymyxin B A ---- Amphotericin B N ---- Nalidixic acid T ---- Trimethoprim A ---- Azlocillin The antibiotic mixture inhibits the growth of contaminating bacteria.

70. Growing mycobacteria utilize the acid, releasing radioactive CO 2 which is measured as growth index (GI) in the BACTEC instrument. The daily increase in GI output is directly proportional to the rate & amount of growth in the medium.

71. Mycobacteria Growth Indicator Tube (MGIT)- Tube contains modified Middlebrook 7H9 broth base with oleic acid, albumin, dextrose, and catalase (OADC) enrichment & PANTA antibiotic mixture. Principle of the procedure- A fluorescent compound (which is sensitive to O 2 ) is embeded in silicone on the bottom of the tube. The actively respiring microorganisms consume the oxygen & allow the fluorescence to be observed using UV trans-illuminator lamp. (365nm)

72. MGIT (Mycobacterium Growth Indicator Tube)-

73. Sensitivity Specificity BACTEC 960/MGIT 81.5 % 99.6% BACTEC 460 85.8% 99.9% ,

74. Advantages- Culture and identification of M. tuberculosis provide a definitive diagnosis of TB as well as significantly increasing in the number of cases identified when compared with microscopy there is often an increase of 30–50%. Culture also provides the necessary isolates for conventional Drug Sensitivity Testing.

75. Disadvantage- Culture is more complex and expensive than microscopy; It also takes longer,requiring facilities for preparing media,processing specimens and encouraging the growth of organisms. Culture also requires specific laboratory equipment, technicians with additional skills, and appropriate biosafety conditions

76. Limitations- Specimens must be decontaminated before culture to prevent overgrowth by other microorganisms. To some extent, all decontamination methods are also harmful to mycobacteria ; therefore, culture is not100% sensitive.

77. Molecular Testing- Line Probe Assays Xpert MTB/ RIF

78. Line Probe Assays- Performing an LPA involves extracting DNA from M. tuberculosis isolates or directly from clinical specimens and using polymerase chain reaction.

79. (PCR) to amplify the resistance-determining region of the rpoB gene using biotinylated primers. Subsequently, labelled PCR products are hybridized with specific oligonucleotide probes immobilized on a strip. Colorimetric development of the captured and labelled hybrids enables the presence of M. tuberculosis complex to be detected as well as the presence of wildtype M. tuberculosis.

80. The post-hybridization reaction leads to the development of coloured bands on the strip at the site of probe binding, and it can be read by the laboratory technician.

81. It also detects mutations associated with drug resistance. If a mutation is present in one of the target regions, the amplicon will not hybridize with the relevant probe. Therefore, mutations are detected by a lack of binding to wild-type probes as well as by binding to specific probes for the most commonly occurring mutations.

82. Uses- Rapid diagnosis in smear negative samples 65 kDA protein encoding gene mpt64 gene Differentiate M. tb / NTM Species specific IS6110 Genetic markers for drug resistance Rifampicin – rpoB INH – codon 315 of katG

83. Advantages- Molecular LPAs enable rapid detection (in less than 48 hours) of resistance to rifampicin (alone or in combination with resistance to isoniazid ); LPAs are a high throughput technology, allowing up to 48 specimens to be processed simultaneously.

84. Disadvantages- LPAs do not eliminate the need for conventional culture and DST. Available LPAs are recommended for use only on smear-positive sputum specimens and isolates of M. tuberculosis. Current LPAs cannot replace phenotypic DST for second-line anti-TB agents.

85. There is incomplete cross-resistance among second line injectable agents. LPAs cannot identify resistance to specific second-line injectable agents; thus, they cannot be used to guide the choice of second-line agents included in individualized MDR-TB regimens.

86. Limitations- LPAs are suitable for use at the central or national reference laboratory level; they have the potential to be used at the regional level if the appropriate infrastructure can be ensured (three separate rooms are required). The sensitivity of LPAs to detect resistance to isoniazid is lower (approximately 85%) than that of culture methods.

87. Xpert MTB/ RIF assay- The Xpert MTB/RIF assay is an automated, cartridge-based nucleic acid amplification test (NAAT). The Xpert MTB/RIF assay is performed directly on sputum, processed sputum sediment and selected extrapulmonary specimens from adults and children.

88. Xpert MTB/RIF results reported as follows: T = MTB detected, rifampicin resistance not detected RR = MTB detected, rifampicin resistance detected TI = MTB detected, rifampicin resistance indeterminate N = MTB not detected; I = invalid / no result / error

89. Drug susceptibility testing- Absolute concentration method; Resistance ratio method; and Proportion method. Newer methods- 1.Microscopic observation drug-susceptibility (MODS) assay, 2.Colorimetric redox indicator (CRI) methods and 3.Nitrate reductase assay (NRA).

90. Absolute Concentration Method: This method uses a standardized inoculum grown on drug-free media and media containing graded concentrations of the drug(s) to be tested. Several concentrations of each drug are tested, and resistance is expressed in terms of the lowest concentration of the drug that inhibits growth; i.e., minimal inhibitory concentration (MIC). This method is greatly affected by inoculum size and the viability of the organisms.

91. The Resistance Ratio Method: It compares the resistance of unknown strains of tubercle bacilli with that of a standard laboratory strain. Parallel sets of media, containing two fold dilutions of the drug, are inoculated with a standard inoculum prepared from both the unknown and standard strains of tubercle bacilli. Resistance is expressed as the ratio of the MIC of the test strain divided by the MIC for the standard strain in the same set.

92. Proportion Method: It enables precise estimation of the proportion of mutants resistant to a given drug. Several 10-fold dilutions of inoculum are planted on to both control and drug –containing media; at least one dilution should yield isolated countable (50 -100) colonies.

93. When these numbers are corrected by multiplying by the dilution of inoculum used, the total number of viable colonies observed on the control medium, and the number of mutant colonies resistant to the drug concentrations tested may be determined. The proportion of bacilli resistant to a given drug is then determined by expressing the resistant portion as a percentage of the total population tested.

94. Microscopic observation drug-susceptibility (MODS) assay- MODS is a microcolony method that uses liquid culture. Drug-free and drug containing media are inoculated, and this is followed by microscopic examination of early growth. MODS is recommended as a direct or indirect test for rapid screening of patients suspected of having MDR-TB.

95. Colorimetric redox indicator (CRI)methods - CRI methods are indirect methods. A coloured indicator is added to liquid culture medium on a microtitre plate after M. tuberculosis strains have been exposed to anti-TB agents in vitro. Resistance is detected by a change in the colour of the indicator, which is proportional to the number of viable mycobacteria in the medium.

96. Nitrate reductase assay (NRA)- NRAs can be used as direct or indirect methods on solid culture. NRAs are based on the ability of M. tuberculosis to reduce nitrate, which is detected by a colour reaction

97. Radiology-

98. Miliary TB (Chest Radiograph)-

99. Summary-

100. Thank You.....