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Investigations in Tuberculosis and advances
- 1. Investigations in Mycobacterium Tuberculosisand Advances Dr. Nirish Vaidya Resident, First year Internal Medicine KUSMS, Kathmandu University Hospital
- 2. Mycobacterium tuberculosis- Characteristics •Gram positive •Obligateaerobe •Non-spore-forming •Non-motile rod •Mesophile •Slow generation time: 15-20 hours •May contribute to virulence •Lipid rich cell wall contains mycolic •Responsible for many of this bacterium’s characteristic properties 2
- 3. The Burden • Worldwide,9.6 million people are estimated to have fallen ill with TB in 2014 • In 2014, 6 million new cases of TB were reported to WHO, fewer than two-thirds (63%) of the 9.6 million people estimated to have fallen sick with the disease. This means that worldwide, 37% of new cases went undiagnosed or were not reported. • Of the 480 000 cases of multidrug-resistant TB (MDR-TB) estimated to have occurred in 2014, only about a quarter of these – 123 000 – were detected and reported • Delay in the diagnosis? • Delay in getting result? • To reduce this burden, detection and treatment gaps must be addressed, funding gaps closed and new tools developed • Laboratory methods play a crucial
- 4. TECHNIQUES 1. Detection ofMycobacterium 2. Molecular Methods 3. Identifications of Species 4. Immunological Methods
- 5. Sputum Microscopy • Carbolfuchsin • Fluorochrome stain such as auramine-rhodamine – Improves the sensitivity of Mtb detection – Recent advances in light-emitting diode (LED) technology have widened the applicability of fluorescent microscopy • Persons unable to cough up sputum – induce sputum – bronchoscopy – gastric aspiration The quantity of sputum (at least 5 mL)
- 6. Sputum smears stainedby Z-N stain Advantage: - cheap – rapid - Easy to perform - High predictive value > 90% -Specificity of 98% Disadvantages: - sputum ( need to contain 5000-10000 AFB/ ml.) Sensitivity: 40-70% - Young children, elderly & HIV infected persons may not produce cavities & sputum containing AFB. Solution: -Centrifused sample -Overnight Sedimentation
- 7. 7 New Policy andSmear microscopy definition of a TB case • New definition in 2007 (ISTC): “person with al one AFB is sufficient out of a total of two examined” • 2 samples regardless the collection time – Retrospective study • Nelson, SM, Deike, MA, Cartwright, CP. Value of examining multiple sputum specimens in the diagnosis of pulmonary tuberculosis. J Clin Microbiol 1998; 36:467. – overall, 92 percent of cases would have been detected with two specimens • Craft, DW, Jones, MC, Blanchet, CN, et al. Value of examining three acid-fact bacillus sputum smears for the removal of patients suspected of having tuberculosis from the "airborn precautions" category. J Clin Microbiol 2000; 38:4285. – a third sputum smear was of no additional value * 7
- 8. Interpretation of sputumstained by Z N Stain (WHO ) No No AFB/300 OIF Negative 1-9 No of AFB/100 OIF Mention the number 10-99 No of AFB/100 OIF 1+ 1-10 No ofAFB/50 OIF 2+ >10 No of AFB/ 20 OIF 3+
- 9. The smear maybe stained by auramine-O dye. TB bacilli are stained yellow against dark background & easily visualized using florescent microscope. Advantages: - More sensitive - Rapid Disadvantages: - Hazards of dye toxicity - More expensive LED Fluorescence Microscopy
- 10. Culture • GOLD STANDARD •Solid media: Lowenstein-Jensen – Brown, granular colonies "buff, rough and tough"). – Four weeks, due to the slow doubling • Liquid media: used in commercially available automated systems • Used to confirm diagnosis of TB
- 11. Cultures • Sensitivity: 80-85% •Specificity: 98% • Times needed: – Solid medium • 4-8 wks – Liquid medium • 2 wks Advantages: - More sensitive (need lower no. of bacilli 10-100 / ml) -Differentiate between TB complex & NTM using biochemical reactions - Sensitivity tests for antituberculous drugs Disadvantages: Slowly growing ( up to 8 weeks ) in solid medium
- 12. AFB smear vs.Cultures • AFB smear – Need 5000-10000/ml bacilli – Rapid diagnosis • Cultures – Need lower no.10-100/ml bacilli – More sensitive – Allows drug sensitivity test
- 13. Screening-Tuberculin test ( Mantouxtest) • Delayed HSR against tubercular protein • Inject intradermally 0.1 ml of 5TU PPD tuberculin • Read reaction 48-72 hours after injection • Measure only induration • Record reaction in mm 13
- 14. Classifying the tuberculinreaction • >5 mm is classified as positive in – HIV-positive persons – Recent contacts of TB case – Persons with fibrotic changes on CXR consistent with old healed TB – Patients with organ transplants and other immunosuppressed patients
- 15. Classifying the tuberculinreaction • >10 mm is classified as positive in – Recent arrivals from high-prevalence countries – Injection drug users – Residents and employees of high-risk settings – Mycobacteriology laboratory personnel – Persons with clinical conditions that place them at high risk – Children <4 years, or children and adolescents exposed to adults in high-risk categories
- 16. Classifying the tuberculinreaction • >15 mm is classified as positive in – Persons with no known risk factors for TB
- 17. Tuberculin Test: Interpretation •A positive test • The person’s body was infected with TB bacteria • Latent TB infection or TB disease??-Additional test needed • may develop reactivation type of T.B. • A negative tuberculin test • excludes infection in suspected persons • The person’s body did not react to the test, and that latent TB infection or TB disease is not likely. • are at risk of gaining new infection 17
- 18. False negative •Severe tuberculosisinfection (Miliary T.B.) - Hodgkin’s disease • Corticosteroid therapy - Malnutrition - AIDS • Children below 5 years of age with no exposure history False positive • Atypical mycobacterium • BCG vaccination – Usually <10 mm by adulthood 18 Recombinant Early Secretory antigenic target (ESAT)-6 instead of tuberculin have demonstrated safety and tolerability of such a test. May solve the problem but is on Phase I trial
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- 20. Septi-check AFB • Simultaneousdetection of Mycobacterium tuberculosis and non tuberculosis mycobacterial • Requires ~ 3 weeks of incubation • Specimens: Sputum, bronchoalveolar lavage, urine, stool, body fluids, biopsy tissues, wounds and skin • Advantage over convetions Mb isolation media -Small inocula -Short time
- 21. Microcolony detection onsolid media(MODS) • Uses simple light microscopy on plates with a thin layer of 7H11 agar medium • Identifies characteristics strings and tangles of M.tb • Rapid yield ~9-10 days • Low cost alternate for detecting drug resistance • Sensitivity 92% • High technical skills, biosafety cabins complex agar medium
- 22. Mycobacteria Growth Indicator Tube(MGIT) Tube contains modified Middlebrook 7H9 broth base All types of clinical specimens, pulmonary as well as extra-pulmonary ( except blood) could be cultured
- 23. BACTEC • Specimen isinoculated in 14C labeled 7H12 medium(MGIT) • Incubated • Mycobacterium multiply generating 14Co2, increase fluorescence in sensor of the bottle • The instrument scans the MGIT every 60 minutes for increased fluorescence. • An instrument-positive tube contains approximately 10^5 to 10^6 CFU/mL. • Can detect growth as early as 4 days and drug susceptibility in 21-28 days. • Cost effective
- 24. MB/Bac T • Non-radiometriccontinuous monitoring system that utilizes colorimetric sensor and reflected light to continuously monitor the Co2 concentration in culture medium • Good alternative to BACTEC – But takes slightly longer time ~15-20 days • The mean time to detection was 12.9 days by BACTEC MGIT960, and 15.0 days with BACTEC 460,compared with 27.0 days LJ Medium (Advances in the diagnosis of tuberculosis; C LANGE et al;Repirology)
- 25. ESP Blood culturesystem • Fully automated continuous monitoring culture system by evaluating gas consumption by microbes rather than production of CO2 as in BACTEC • Mean time of detection 11-16 days
- 26. • Detection andDiagnosis • – uncultivable or difficult to culture • – need for rapid answer • Prognosis and management • – need for quantitative information (viral load) • – susceptibility testing (drug resistance) without culture • • Molecular resistance testing Molecular Diagnostics Why? 26
- 27. Nucleic Acid Amplification(NAA) • Both detection and identification of M. tuberculosis through enzymatic amplification of bacterial DNA • Amplify M. tuberculosis-specific nucleic acid sequences using a nucleic acid probe. • Used mostly for confirmation of smear positive results • Quick results, diagnostic accuracy • Require as few as 1O bacilli from a given sample • Expensive
- 28. Nucleic Acid Amplification(NAA) • Specificity in the range of 98% to 99%. • Useful technology for rapid diagnosis of smear negative cases of active TB • Able to identify 50-60% of smear -ve culture +ve cases • Distinguish M.tb from NTM in smear +ve cases • Should not be used to replace sputum microscopy as an initial screen & culture remains the gold standard • Very high degree of quality control required
- 29. Polymerase Chain Reaction(PCR) • Allows sequences of DNA to be amplified in vitro • Detection is based on multiplication not of whole bacilli, as in culture, but of their genetic material, chromosomal DNA or ribosomal RNA, typical for MTB or MTB complex. • Most common target used for PCR is insertion sequence IS6110 which is specific for M. tb.
- 30. Advantages: - Rapid procedure( 3 – 4 hours) - High sensitivity (1-10 bacilli / ml sputum) Sensitivity ~95%, Specificity ~100% Disadvantage -High cost Polymerase Chain Reaction (PCR) 30
- 31. Xpert MTB/RIF • Realtime PCR assay to amplify a specific sequence of the rpo B gene • Xpert MTB/RIF detects M. tuberculosis as well as rifampicin resistance • Cartridge based Automated molecular test M.Tb and its resistance to Rifampin 31
- 33. Advantages with GeneXpert •Simultaneous detection of both MTB and rifampicin resistance, a marker for MDR strains • Unprecedented sensitivity for detecting MTB — even in smear negative, culture positive specimens • Results in two hours; requires no instrumentation other than the GeneXpert® System • On-demand results enable physicians to treat rapidly and effectively • 33
- 34. • Monoresistance toRifampicin ~5 % • Concurrent resistance with Isoniazide ~95% – Dx of MDR Tb with high level of accuracy • Xpert MTB/RIF has higher sensitivity for Sputum smear positive cases than Smear negative cases. Nontheless a valuable tool for smear negative cases • International standard for TB Care recommended Xpert MTB/RIF assay with Culture for Sputum Negative Suspected cases
- 35. Chromatography • Identification ofdifferent species • Used in reference labs for epidemiologic studies • Results within 2 hours • High cost
- 36. TB PNA FISH •Fluorescence in situ hybridization (FISH) using specific Peptide nucleic acid (PNA) probe allows differentiation between tuberculous and nontuberculous mycobacteria in AFB+ve cultures • Specificity and predictive values approaches 100%
- 37. • DNA amplificationtechnique • It is a variant of PCR, in which a pair of oligonucleotides are made to bind to one of the DNA target strands, so that they are adjacent to each other. A second pair of oligonucleotides is designed to hybridize to the same regions on the complementary DNA. Ligase Chain Reaction 37
- 38. • The actionof DNA polymerase and ligase in the presence of nucleotides results in the gap between adjacent primers being filled with appropriate nucleotides and ligation of primers • Mainly being used for respiratory sample • High overall specificity and sensitivity for smear +ve and –ve specimens. Ligase Chain Reaction 38
- 39. Loop-mediated isothermal amplification (LAMP) •The mixing of all reagent in a single tube, followed by an isothermal reaction during which the reaction mixture is held at 63°C 60-min incubation time. • It is a novel nucleic acid amplification method • 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) or on a solid medium (Ogawa’s medium). 39
- 40. Interferon-gamma release assays (IGRA) •Measure interferon (IFN-γ) released by sensitized T cells after stimulation by M. tuberculosis antigens. Measures immune reactivity to M.tb. • The test uses synthetic peptide antigens (ESAT-6,CFP-10) that simulate this protein to generate the immune response • No memory as in Mantoux, but tells us it’s a RECENT INFECTION
- 41. Available Interferon-gamma release assays(IGRA) tests Quantiferon Gold T-SPOT.TB Limitation is COST. NRs 4000/-
- 42. Advantages of IGRA •Quantitative reports – postive or negative • Result in single patient visit • Not affected by BCG status and NTB • Not affected by repeated IGRA
- 43. Disadvantages of IGRA •Does not tell Latent or active TB • COST • Results altered in immune compromised
- 44. Monocyte-amplified INF-γ release assays(MIGRAS) • Principle: – INF release leads to subsequent release of INF- responsive chemokines such as MIG and IP-10 – Measures this chemokines
- 45. Transcription mediated amplification (TMA) •Can identify the presence of genetic information unique to M. tuberculosis directly from pre processed clinical specimens • Sensitivity ~ culture • Sensitivity ~96% and Specificity 100% • Disadvantage – Positive results in both viable and dead bacilli
- 46. Other approaches • Detectionof anti-mycobacterium superoxide dismutase antibodies • MPB 64 patch test – Transdermal patch containing MPB 64, an antigen specific for M tuberculosis – Results obtained d after 3-4 days – Sensitivity 97.8% and Specificity 100% – Discriminated latent infection from active disease
- 47. FAST Plaque TB •Specific mycobacteriophages infect M.tb in specimen • Intracellular phages undergo replications with subsequent release of phages from host cells • Released phages are incubated along with other nonpathogenic organisms in agar plate – produces zone of clearing (plaques) – POSITIVE • Rapid <24 hours
- 48. FAST-Plaque-Response • Extention ofFAST-Plaque TB • Allows early detection of Rifampicin resistance
- 49. Adenosine Deaminase (ADA) •Surrogate marker in pleural, pericardial and peritoneal fluids and CSF • Sensitivity of 100%, Specificity 94.6% (study performed in India)
- 50. Detection of lipoarabinomanan •LAM is a cell lipopolysaccharide Ag of M tb • LAM-ELISA assay in urinary useful in HIV associated tuberculosis with low CD4 counts • Sensitivity 93%, Specificity 95% • Useless in Blood TB-ELISA
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