DIAGNOSIS OF TUBERCULOSIS, NEWER TECHNIQUES, TST.pptx

TB Diagnosis

1. Diagnosis of Tuberculosis, Tuberculin skin test, New techniques Dr. Ajaypal Singh Junior Resident Pulmonary medicine Gmc patiala

2. Definition of presumptive TB Presumptive pulmonary TB : Refers to person with any of the symptoms and signs suggestive of TB including: Persistence Fever > 2 Weeks , Weight loss Night sweats 4) Cough for > 2 weeks Any pulmonary abnormality in chest radiograph Presumptive Extra pulmonary TB – Refer to as organ specific symptoms and signs like Swellings of lymph node, Pain and Swelling Joint, Neck stiffness, Disorientation etc

3. METHODS OF DIAGNOSIS OF PULMONARY TUBERCULOSIS 1)DIRECT METHODS: Detects mycobacteria and its products 2)INDIRECT METHODS : Antigen and Antibody Detection 3)RADIO-DIAGNOSIS : CXR,CT AND MRI

4. DIRECT METHODS 1)Direct Microscopy -ZN stain , Flourence microscopy. 2)Culture -Traditional, Rapid methods.  3) molecular methods – CBNAAT ,LPA

5. INDIRECT METHODS 1) Antibody detection : Banned by GOI TB STAT-PAK ELISA Insta test TB 2) Antigen detection : Banned by GOI TB MPB 64 patch test. Quantiferon -GOLD test. 3) Biochemical Assays : ADA

6. Antibody Detection Test : Banned by GOI in june 2012 A60 is the antigen used for both pulmonary and extra pulmonary , adult and childhood TB. sensitivity of these test has ranged between 30 to 100 per cent. presence of antibody does not indicate current disease or past infection . presence of antigen may be a better indicator of the disease than the antibody However, antigen quantity in circulation is usually very limited and masked by the antibody and hence difficult to detect

7. Antigen Detection Test 1) Lipoarabinomannan Urine Test : Lipoarabinomannan is a component of the TB bacterial cell wall. The test exists in ELISA and simplified “tube” format . 2 ) Flow-through Filter Tests : detection of Mycobacterium tuberculosis in sputum or body fluids with a polyclonal antibody, using a flow-through device.

8. Sputum smear microscopy Use of microscopy in diagnosis of TB is of paramount importance, as culture takes a long time before the results are ready. 10,000 AFB /ml required. -Sensitivity = 50% The tubercle bacilli are Gram positive but they do not take the stain readily . Mycobacteria retain the primary stain even after decolourization with acid alcohol ; hence the term as “acid-fast ”.

9. A counter-stain is employed to highlight the stained organisms for easier recognition . In the carbol-fuchsin [ Ziehl-Neelsen ] , acid-fast organisms appear red against a blue background . Acid fastness is based on the integrity of the cell wall. Cell wall of Myc. Tuberculosis is made up of MYCOLIC ACID which connect outer Arabinoglactan and inner peptydoglycan . These form a dense pallisade , arranged in rope-like structure, which gives the cell wall its thickness and is largely responsible for acid fastness

10. Fluorescent Staining Ziehl-Neelsen staining is a time consuming process for staining as well as examination. F luorescence microscopy is recommended where > 50 smears are examined per day, and if electricity is continuously available . Fluorescence staining utilizes basically the same approach as Z-N staining, but carbol fuchsin is replaced by a fluorescent dye .

11. Grading for FM 1 length =40 fields Negative Zero AFB/1 length scanty 1 – 19 AFB / 1 length 1+ 20 -199 AFB /1 length 2+ 5 – 50 AFB /1 field 3+ > 50 AFB/1 field

12. Value of Smear Examination in Extra-pulmonary Specimens B enefit of microscopy in these specimens is limited because of their pauci bacillary nature and it is, therefore, recommended that the extra-pulmonary specimens be referred for culture and other molecular techniques. Various extra pulmonary specimen includes : 1 ) Gastric Washings 2) Laryngeal Swabs 3 ) Pus and Thick Aspirates 4) Pleural and Pericardial Fluid 5 ) Cerebrospinal Fluid 6) Urine 7) A scitic fluid 8) lymph node FNAC 9) Blood

13. ISOLATION OF MYCOBACTERIA BY CULTURE Provide definitive diagnosis by establishing the viability and identity of the organisms D istinguish between different mycobacterial species as well as to perform drug susceptibility tests . M. tuberculosis proliferates extremely slowly [generation time 18 to 24 hrs ]. D etect as few as 10 to 100 bacilli per ml of sputum . M. C used medium is L-J medium. It contains eggs, asparagine, glycerol and some mineral acids Cultural Characters : G rowth appears in about two weeks but may be delayed up to six to eight weeks. Optimum temperature for growth is 37 °C. Increased [CO2] tension [5% to 10%] enhances growth

14. Culture Media

15. Culture Colony Characteristics tubercle bacilli give rise to discrete, raised, irregular, dry and wrinkled colonies which may be white in colour in begning to buff colour . Virulent strains form long serpentine cords in the liquid media while avirulent strains grow in a more dispersed fashion . The clinical specimen after concentration, is inoculated onto two bottles of L-J medium and incubated at 37 ° C. Cultures are examined initially after 7 days to rule out the presence of rapid growing mycobacteria.

16. N egative result is given, if no growth appears after eight weeks. Z-N stained smear made from the same is examined and routine biochemical tests put up . All cultures should be examined 3 – 4 days after inoculation to detect gross contaminants. Thereafter cultures are examined weekly . With doubtful cultures, the acid-fastness should be confirmed by Z-N staining. Diagnosis for sputum positive material is obtained within a week while for negative results incubation has to continue for six weeks. culture system has also been used for drug susceptibility testing for the first-line drugs . DST is set up after identification tests Takes 7-9 weeks for DST.

17. Rapid Culture Methods BACTEC radiometric system : measure radioactive CO2 liberated during decarboxylation of 14C labelled substrates. BACTEC 12B vial is inoculated, mycobacteria , utilize the 14C labelled substrate [ palmitic acid] and release 14CO2. BACTEC instrument measures quantitatively the radioactivity in terms of numbers on a scale ranging from 0 to 999, designated as the growth index [GI ]. The daily increase in GI is directly proportional to the rate and amount of growth in the medium. By adding inhibition agents inhibition of metabolism is indicated by reduced production of 14CO2, which in turn is indicated by decrease in GI. This basic principle is utilized in isolation of mycobacteria and drug susceptibility testing in this method. Not used due radioactive hazards.

18. Rapid Liquid Tuberculosis Culture A lso known as Mycobacteria Growth Indicator Tube [MGIT ]. culture is done using manual or automated systems in which tubes contain enriched Middlebrook 7H9 broth and an oxygen sensitive fluorescent sensor embedded in silicone on the bottom of the tube. The presence of oxygen dissolved in the broth quenches emissions from the compound . As the actively growing and respiring mycobacteria consume the dissolved oxygen, the sensor glows indicating mycobacterial growth . The fully automated version can incubate up to 960 samples.

19. Higher recovery rates especially in smear negative specimens. DST – 4-14 days Negative –42 days Can be made to read out negative by 28 days Manual version also available. MPT64 protein detection-based immunochomatographic test is applied for confirmation of myco . Tuberculosis. If culture became positive and myco . Tuberculosis detected on MPT64 then result given as culture positive and MTB detectected . If culture became positive and MTB not detected on MPT64 then result given as culture positive and MTB not detected .

20. MOLECULAR DIAGNOSIS OF TUBERCULOSIS Rapid and sensitive tools for the diagnosis of tuberculosis are needed, due to the increased incidence of tuberculosis epidemics and the length of time required by classical diagnostic tests, especially among human immunodeficiency virus (HIV)-infected patients. In this context, the recent advances in cloning and characterization of M. tuberculosis genes has allowed the application of basic molecular biology techniques to the examination of clinical samples, such as sputum and bronchoalveolar lavage (BAL), for the molecular diagnosis of tuberculous infection .

21. Polymerase Chain Reaction Sequencing The most widely used method for defining genetic resistance for drug sensitivity testing . commonly used for characterising mutations in the rpoB gene in rifampicin resistant strains and to detect mutations responsible for other antituberculosis drugs. This process is repeated sequentially 25-40 times, thereby creating millions of copies of target sequence. The amplified sequence can then be detected by agarose gel electrophoresis

22. Ingredients of Polymerase Chain Reaction Primers: µM 0.1-0.5 Deoxy-nucleotides triphosphate (dNTPs): µM 200-250nucleotides Co-factors: Cations: MgCl 2 mM 1.5-6 Buffer pH 8.3-8.8 DNA polymerase: 0.5-2.5 U Target DNA:  1 µg

23. Three steps of PCR: Denaturation, annealing and extension

24. Role of PCR in pulmonary TB Useful technology for rapid diagnosis of smear – ve cases of active TB. Able to identify 50-60% of smear - ve cases; this would reduce the need for more invasive approaches to smear - ve cases Distinguish M.tb from NTM in smear + ve cases as IS6110 sequence is not found in NTM. Should not be used to replace sputum microscopy. Sensitivity, specificity, & for PCR is 83.5%, 99% & respectively.

25. Disadvantages Very high degree of quality control required. Variation from lab to lab remain significant. In pts. on ATT, PCR should not be used as an indicator of infectivity as this assay remains + ve for a greater time than do cultures High false + ve results in patients previously treated with ATT in contacts of sputum + ve active cases.As it can detect dead bacteria. High Cost

26. Geno Type Assays Two Geno Type assays :For TB diagnosis [ Geno Type Mycobacteria Assay ] CBNAAT For detection of rifampicin and isoniazid resistance [ Geno Type MTB DR Assay ].LPA

27. Xpert MTB/RIF assay (CBNAAT-cartridge based NAA test A utomated PCR diagnostic test that can detect presence of M.tb & resistance to RIFampicin (by detecting mutation in 81bp region) It is automatic , fast & sensitive Accurate diagnosis is obtained in 1hr 45 mins by adding a reagent to the sputum sample & 15mins later its pippeted into a cartridge that is inserted into the diagnostic instrument. Sensitivity for PTB in adults= 88% ( 98% for smear positive TB , 68% for smear negative TB).

28. CBNAA- Limitations They are able to detect nucleic acids from both living and dead organisms so in pts on ATT, CBNAAT should not be used as an indicator of infectivity as this assay remains positive for a greater time, than do cultures NAA should always be performed in conjunction with microscopy and culture . Used only for diagnosis and not for follow ups.

29. LPA(Line Probe Assay) It is a genotypic method use PCR and reverse hybridization with specific oligonucleotide probes fixed to nitrocellulose strips in parallel lines. Resistance targets are rpoB for RIF Kat G and inhA for INH. higher sensitivity for H resistance Both H&R Sensitivity for R is >95%, H is 70-80 % Specificity for R & H is 98% Results within 48 hours

30. Differences types Hain GenotType MTBDRplus It targets 23s rRNA gene space region. It detects both R and H resistance InnoLiPA Rif It targets 16s - 23s rRNA gene space region. It detects only R resistance

31. CONCLUSION: LPA is applicable on sputum+ve specimens only. Gene Xpert can’t monitor progress of treatment. HENCE PHENOTYPIC CULTURE REMAINS THE GOLD STANDARD

32. Interferon-gamma release assays for latent tuberculosis infection Banned by GOI in 2012 markers of Myc. TB infection. Indicate a cellular immune response to M. tuberculosis. C annot distinguish between latent infection and active TB disease. should not be used for diagnosis of active TB . A positive result not necessarily indicate active TB a negative IGRA result may not rule out active TB.

33. Blood Assays for M. tuberculosis 1) QuantiFERON ® -TB Gold In-Tube (Banned by GOI) Measures Interferon-gamma (IFN-y) 2) T-SPOT. TB Measures peripheral blood mononuclear cells that produce IFN- γ

34. Loop-mediated isothermal amplification[ LAMP ] LAMP is used for detection of M.tb complex, M.avium , and M.intracellulare directly from sputum specimens as well as for detection of culture isolates grown in a liquid medium (MGIT ). Simple procedure, starting with the mixing of all reagents in a single tube, followed by an isothermal reaction during which the reaction mixture.

35. ADVANTAGES: Due to its easy operation without sophisticated equipment, it will be simple enough to use in: Small-scale hospitals, Primary care facilities Clinical laboratorie in developing countries. Difficulties : Sample preparation Nucleic acid extraction Cross-contamination

36. Microscopic-Observation Drug-Susceptibility Assay[MODS] facilitates the detection of Mycobacterium tuberculosis, directly from the sputum. This innovative technique utilizes a liquid medium which facilitates faster growth of the TB bacillus and thereby aids in the early microscopic visualization of characteristic cord formation. incorporation of drugs allows rapid and direct drug-susceptibility testing concomitantly with the detection of bacterial growth .

37. IMMUNOLOGICAL MAREKER Adenosine deaminase : enzyme catalyzing the deamination reaction from adenosine to inosine . 2 isoforms of ADA . ADA-1 - many tissues including red blood cells . ADA-2 - only in macrophages and monocytes. ADA acts in proliferation and differentiation of lymphocyte, especially T lymphocyte . If the pleural fluid ADA level is berween 40 and 70 U/L and the patient has a lymphocyte-to-neutrophil ratio of more than 0.75, a presumptive diagnosis of TB can be made. If the patient's pleural fluid ADA level is below 40, the diagnosis of TB is unlikely.

38. Interferon- γ : produced by the CD4 lymphocytes from patients with tuberculous pleuritis cutoff level of 3.7 IU/mL yielded a sensitivity of 0.98 and a specificity of 0.98 measurement of IFN-γ levels in pleural effusions is also likely to be a useful tool for diagnosis of TB pleurisy .

39. Tuberculin Skin Test T uberculin skin test [TST] is mainly used to detect infection with tubercle bacilli. T est is based on the fact that infection with Mycobacterium tuberculosis produces sensitivity to certain components, which are contained in culture extracts called tuberculins . H owever , has a limited value for the diagnosis of TB disease among adults living in areas where TB is highly endemic The interpretation of TST is complicated by cross-sensitivity to tuberculin induced by infection with environmental mycobacteria or by bacille Calmette -Guerin [BCG] vaccination

40. HISTORICAL BACKGROUND Sir Robert Koch : produced a filtrate prepared from heat sterilized concentrated broth cultures of human tubercle bacilli, in the late 19th century. This product named as old tuberculin [OT ]. OT was gradually replaced by a low dose, intradermal test using a purified protein derivative [ PPD] that was well tolerated. Clement Von Pirquet observed in 1907 that a tiny scratch with a little quantity of tuberculin resulted in a local reaction at the test site. Moro in 1908 announced his patch test, where tuberculin was incorporated into an ointment that was smeared onto the skin, with a piece of gauze over it

41. Charles Mantoux developed the intradermal test to be administered by injection as a measured volume [ Mantoux test ]. Heaf test which used a simple instrument, that caused six spring loaded needles to pierce the skin with a drop of undiluted OT. Tine test was developed as a disposable multiple puncture test where the tuberculin was introduced into the skin by puncture with four tines coated with dried tuberculin. most of these tests have become obsolete and only the Mantoux technique which allowed quantitative measurement has stood the test of time and is now the standard method of administration of the TST.

42. IMMUNOLOGICAL BASIS OF TUBERCULIN TEST I ndividuals infected with Mycobacterium tuberculosis respond with delayed type hypersensitivity [DTH] to the TST. T he sensitization is induced by natural mycobacterial infection or by vaccination with BCG, a live attenuated mycobacterial strain derived from Mycobacterium bovis . Clinically, it is a manifestation of previous infection with tubercle bacilli or a variety of nontuberculous mycobacteria [NTM ]. I njection of the tuberculin antigen leads to migration and proliferation of the sensitized T-cell lymphocytes to the test site. These T-cells release cytokines and chemokines , which further attract other lymphocytes and monocytes. These reactions along with increased permeability of the local blood capillaries lead to an induration at the test site

43. SKIN CHANGES IN TUBERCULIN SKIN TEST characteristic features : include a delayed course reaching a peak more than 24 hours after injection and an induration with occasional vesiculation and necrosis.. DTH reaction peaks at 48 to 96 hours with an area of erythmatous induration, which resolves in a week’s time. size of this induration is, thus, maximal between 48 to 96 hours after the test Product and Dosage : Standard test employs a single batch tuberculin, i.e., PPD RT-23 However, 2 TU of PPD RT-23 is now recommended as the standard dose.

44. Administration of Test given on the mid-volar aspect of the forearm since this area is usually hair free. The skin is lightly stretched and the needle point is inserted with its bevel facing upward into the superficial layers of the skin and 0.1 ml of tuberculin is injected slowly. hold the syringe only by the barrel and not to touch the plunger before the needlepoint has been satisfactorily inserted into the skin. A satisfactory test should raise a flat pale pea-sized wheal with clear pits of hair follicles and a well demarcated border and without leakage of tuberculin.

45. Reading of the Test R esult is read between 48 to 96 hours after the test in good day light. keeping the forearm flexed, by carefully palpating the site of injection using one finger induration may be easily recognized as a firm well circumscribed density. S mall indurations may be missed if not sought carefully with a light touch. M arked with the ballpoint pen and the maximum transverse diameter is measured in ‘ millimetres ’ [mm] with a transparent ruler. test result should never be recorded as ‘positive’ or ‘negative’ and must always be recorded in ‘mm of size’. Record should also be made of vesicles, bullae, lymphangitis , ulceration and necrosis at the test site. B

46. False positive reaction Infection with non tubercular mycobacterium. Previous BCG vaccination Incorrect method of TST Incorrect interpretation of reaction Incorrect bottle of antigen used False negative reaction Cutaneaus anergy (inability to react to antigen due to weak immune respone ) Recent TB infection Very old TB reaction Very young age(<6 months) Recent live virus vaccination Overwheling TB disease Incorrect method of TST Incorrect interpretation

47. New techqnies URINE-BASED SCREENING [stamp trial] Rapid urine-based Screening for Tuberculosis to reduce AIDS-related Mortality . February 2015 to January 2018. The overarching aim of this work is to determine whether use of a fundamentally new approach to rapid diagnostic screening for HIV-associated  tuberculosis  ..

48. Trunat tb test The TrueNat TB test is a new molecular test that can diagnosis TB in one hour as well as testing for resistance to the drug rifampicin. This  test for TB  uses a sputum sample taken from each patient. Only about 0.5 ml of the sample is required compared with about 1 ml needed for the American  GeneXpert  machine

49. Urease activity of  M. tuberculosis M. tuberculosis  urease is a likely bacterial virulence factor, which may subvert the host acidification of the phagosome microenvironment . It may used as suportive diagnostic technique.

50. Diaskintest Diaskintest is an innovative skin test for the mass screening of  tuberculosis. Diaskintest belongs to the IGRA tests, which are the most promising line in the diagnostics of infectious diseases. Diaskintest is highly specific, and does not show false-positive reactions in BCG-vaccinated persons. Diaskintest allows the identification of only a group of individuals with active tuberculosis infection or those at high risk of TB, minimizing the rate of false- positiveresults .