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WHO recommended tests of tuberculosis

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WHO recommended tests of tuberculosis

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WHO recommended tests of tuberculosis

  1. 1. WHO RECOMMENDED TESTS FOR TB -DR. PRAPULLA CHANDRA
  2. 2. INTRODUCTION • Tuberculosis (TB) remains one of the world’s deadliest communicable diseases. • In 2013, an estimated 9 million people developed TB and 1.5 million died from the disease. • Of the estimated 9 million people, india accounts for 24% 0f the cases.
  3. 3. • In 2013, about 64% of the estimated 9 million people who developed TB were notified as newly diagnosed cases. That is, the remaining 3 million cases were either not diagnosed, or diagnosed but not reported to national TB programmes (NTPs). • Early diagnosis of TB and initiating optimal treatment would not only enable cure of an individual patient but also will curb the transmission of infection and disease to others in the community.
  4. 4. TESTS ENDORSED BY WHO • MICROSCOPY -light and LED microscopy -same day diagnosis CULTURE BASED TESTS -commercial liquid culture systems and rapid speciation -non commercial culture and DST (MODS, NRA, CRI) MOLECULAR TESTS -line probe assays -Xpert MTB/RIF
  5. 5. WHO RECOMMENDATIONS ON NEW TECHNOLOGIES 2006-2010 Year Policy Recommended 2007 Commercial Liquid Cultures (Automated and Manual) and DST Rapid Speciation –using immuno-chromatographic assays 2008 Line Probe Assays (LPA) 2009 LED based FM and Dual Mode Systems including low load settings Non-Commercial Methods for Culture & DST (MODS, CRI, NRA etc) 2010 Automated Cartridge Based Nucleic acid technologies – Xpert
  6. 6. DIRECT METHODS • Direct microscopy ( ZN, KINYOUN, FLUOROCHROME) • Culture ( traditional, rapid methods) • Detection of DNA/RNA of mycobacterial origin (PCR, LCR, LAMP, NAA, FAST PLAQUE)
  7. 7. DIRECT MICROSCOPIC EXAMINATION • Hallmark of staining is Ziehl-neelsen staining slides • Easiest & quickest diagnostic test • Limited sensitivity (46-78%) but specificity is almost 100% • Centrifugation & fluorochrome staining (auramine O) with UV microscopy markedly increase the sensitivity & a large no. of slides can be examined in a much shorter time.
  8. 8. CONCENTRATION METHODS FOR SMEARS • Autoclaving • 1% sodium hypochlorite • Sputum treated with 2-4% NAOH • Urine, c.s.f, pleural fluids centrifused and deposit
  9. 9. Decontamination • Eliminate normal flora from the non-sterile samples (micobacteria is acid and alkaline resistant) • Homogenization to release the bacteria from the sample and allow access to the nutrient present in the media N-acetyl-cysteine: homogenization NaOH: decontaminant Neutralization by phosphate buffer Transportation: 1% cetyl pyridium chloride Homogenization Sample mixing Phosphate buffer Centrifugation Pellet
  10. 10. • Sputum Concentration and decontamination methods are not recommended by WHO as there is no sufficient evidence.
  11. 11. Z-N staining method Primary stain: Carbol-fuschin solution 0.3% • Decolourising agent: 3% alcohol, or 20% Sulphuric acid • Counterstain: Methylene blue 0.3% • Observe under microscope
  12. 12. Microscopy- precautions • To use sterile containers/collection pots • Sterile reagents, slides and equipment • Do not use tap water for staining(saprophytic mycobacteria) • New slides only(old slides may harbour fungal spores in scratches)
  13. 13. Reporting on AFB Microscopy Number of bacilli seen Result reported None per 100 oil immersion fields Negative 1-9 per 100 oil immersion fields Scanty, report exact number 10-99 per 100 oil immersion fields 1+ 1-10 per oil immersion field 2+ > 10 per oil immersion field 3+
  14. 14. FLUORESCENT MICROSCOPY Fluorescent dye (Auramine O and Rhodamine B) • Good for labs with high workload. • Auramine O- Bright yellow • Rhodamine B- Yellow orange.
  15. 15. Reporting in flouroscent technique No of bacilli per HPF GRADE <6 per field 1+ 6-100 per field 2+ >100 per field 3+
  16. 16. LED FLUORESCENCE MICROSCOPY (light emitting diode fluorescent microscopy)
  17. 17. LED FLUORESCENCE MICROSCOPY • Increase the sensitivity of smear microscopy and improve the efficiency of facilities. • Much larger area of the smear to be seen, • More rapid examination of the specimen (up to four times faster) and making it easier to count bacilli. • ADVANTAGES: • More sustainable , • User-friendly than the quartz-halogen lamps or high-pressure mercury vapour lamps typically used in FM
  18. 18. COMPARISON -overall sensitivity is 93% and specificity is 99% -6% more sensitive than Z-N microscopy & 5% more sensitive than conventional flouroscent microscopy RECOMMENDATIONS • conventional fluorescence microscopy be replaced by LED microscopy with auramine staining in all settings where fluorescence microscopy is currently used. • LED microscopy be phased in as an alternative to conventional Ziehl-Nelsen light microscopy. • the switch to LED microscopy be made according to a carefully phased implementation plan.
  19. 19. SAME DAY DIAGNOSIS • Same-day diagnosis ('spot-spot') versus the conventional strategy ('spot- morning'), with two specimens and direct Ziehl-Neelsen microscopy -only 2.8% less sensitive and similar specificity •Same-day diagnosis ('spot-spot morning') versus the conventional strategy ('spot-morning-spot') with three specimens and direct Ziehl-Neelsen microscopy -3% more sensitive and similar specificity
  20. 20. WHO RECOMMENDATIONS • countries that have implemented the current WHO policy for two- specimen case-finding consider switching to same-day diagnosis, especially in settings where patients are likely to default from the diagnostic pathway • countries that are still using the three-specimen case-finding strategy consider a gradual change to same-day diagnosis, once WHO- recommended external microscopy quality assurance systems are in place and good-quality microscopy results have been documented
  21. 21. CULTURE METHODS
  22. 22. Less common media • Powlowski potato media • Tarshish blood media • Dubos media • Sulas media • Soutons media
  23. 23. Conditions for better growth • Temp b’n 25-40c. Ideally 37c • 5-10% co2 enhances the growth • Ph 6.4-7 • Addition of low percentage of glycerol to the medium enhances the growth of human strains • Minimum time for growth to appear 2 weeks and can take upto 6-8 weeks
  24. 24. Colony charecteristics • M.TB: these are raised rough ,discrete, dry, wrinkled , creamy white • M.bovis: smooth , moist, white, break up more readily when touched
  25. 25. Rapid culture techniques • BACTEC system • MB/BAC T system • Mycobact growth indicator tube (MGIT) • Septi chek AFB method • ESP culture system • Microscopic observation drug sensitivity(MODS)
  26. 26. • BACTEC -Radiometric method -growth is ascertained by liberation of 14CO2 as metabolised by mycobacteria -average time: 8days -can also be used for drug susceptibility testing DRAWBACKS: -high cost -disposal of radiactive waste
  27. 27. MB/BacT SYSTEM • Non radiometric continuous monitoring system • Based on calorimetric detection of co2 • Slightly longer time than BACTEC (14 days) • Prone to contamination
  28. 28. MGIT +ve tubes MGIT 960 instrument
  29. 29. MGIT 320 System and Manual Reader • Holds 320 tubes for an annual capacity of approx 2700 specimens/year • Optimal use of valuable lab space • Flexible configuration as bench top or stand- mounted • Manual UV Reader as back up for MGIT 960/320 • few specimens (in low load labs) • Power problems • MGIT tubes to be incubated in regular incubators
  30. 30. Liquid Culture Systems MGIT 960 STRENGTH WEAKNESS Automated system – growth & detection (except Blood) Capacity to incubate & monitor 960 tubes every 60 minutes for Increase in fluorescence Can handle 8000 cultures/year System alerts when tubes become +ve No radioactive material used BSL III/negative Pressure Environment Continuous Electricity Reagent costs and AMC High Contamination rates Training in handling Liquid culture Speciation if+ve* Cold chain transport for sputUM
  31. 31. MODS – MICROSCOPIC OBSERVATION DRUG SUSCEPTIBILITY • Microscopic colonies (micro-colonies) of M. tuberculosis are observed in the culture media using an inverted microscope Uses tissue culture plate - the wells coated with different drugs in different concentration are used - presence of growth with INH /RIF /SM/EMB can be detected. • Time taken 7 to 14 days
  32. 32. MODS – MICROSCOPIC OBSERVATION DRUG SUSCEPTIBILITY • difficulty distinguishing between the micro-colonies of TB and some nontuberculous mycobacteria (NTM). • requires experienced personnel. • , biosafety of laboratory workers must be taken into consideration.
  33. 33. STRIP SPECIATION • Detect a TB-specific antigen (MPB64) from positive liquid or solid cultures to confirm the presence of organisms belonging to M.tuberculosis complex. • Speciation is necessary to differentiate M.tuberculosis complex and other mycobacteria grown in cultures. • ADVANTAGES: • Results in 15 min • Sensitivity & specificity of 98.6% and 97.9% • no additional equipment or consumables are needed to perform the test and can detect TB even when mixed with nontuberculous mycobacteria.,
  34. 34. STRIP SPECIATION
  35. 35. Rapid Speciation of Positive Cultures • Immunochromatographic tests - Rapid strip test that detects a TB-specific antigen (MPB 64) from culture • Bacterial growth derived from solid or liquid culture can be used • Outputs: • Control band • Test band: • Present = M.tb complex • Absent = not M.tb complex • Increased yield of NTMs with liquid cultures necessitates prompt identification of mycobacterial growth as TB vs NTM
  36. 36. COLORIMETRIC REDOX INDICATORS • Based on the reduction of an indicator solution added to a liquid culture medium after TB organisms have been exposed to different antibiotics. • Isoniazid and rifampicin resistance is detected by a change in colour of the indicator. • ADVANTAGES: • Highly sensitive (about 95%) and specific for the detection of MDR-TB • Faster than conventional solid or liquid culture DST methods • Results between 7 and 14 days after culturing. • do not require sophisticated equipment
  37. 37. LIQUID CULTURE SYSTEM • liquid culture medium, enriched with oxygen • As bacteria grows in the culture, the oxygen is utilized, causing it to be fluorescent when placed under UV light. • ADVANTAGES: • Faster • High sensitivity &specificity nearly 100% • Diagnosis in 7-14 days • Both automated and manual systems perform well in detection of isoniazid and rifampicin susceptibility. • Not as effective for ethambutol and streptomycin
  38. 38. • Measures nitrate reduction to indicate resistance to isoniazid and rifampicin. • Based on the property of TB to reduce nitrate to nitrite& color change of the culture media. ADVANTAGES NRA (NITRATE REDUCTION ASSAY)less expensive to than liquid culture techniques for DST • Results are earlier than by eye examination of colonies in solid culture • the specificity and sensitivity of NRA were comparable to traditional solid culture methods for DST of isoniazid and rifampicin • . NRA does not need sophisticated equipment, is not complex to perform • Disadvantages • The culture is killed by the mix reagent used to develop the assay, requiring that multiple cultures be prepared if comparative testing will be performed. Only fresh cultures must be used (<14 days). NITRATE REDUCTION ASSAY
  39. 39. Detection of mycobacteria directly from clinical samples • Genotypic methods {NAA} • PCR (polymerase chain reaction) • LAMP (loop mediated isothermal amplification) • TMA (transcription mediated amplification) • Ligase chain reaction • AMPLICOR assay • MTD test (mycobacterium tuberculosis direct test) • BD probe Tec MTB test • Phenotypic methods • FAST plaque TB method
  40. 40. LIMITATIONS OF NAA • No drug susceptibility information • Detects nucleic acid from both dead and living organisms • May be falsely positive in persons having recent infection and undergone treatment.
  41. 41. • Line-probe assays are designed to identify M. tuberculosis complex and simultaneously detect mutations associated with drug resistance • They are a family of novel DNA strip-based tests that use PCR and reverse hybridization methods for the rapid detection of mutations associated with drug resistance • Line-probe assay has high sensitivity and specificity when culture isolates are used. • The majority of studies had sensitivity of 95% or greater, and nearly all were 100% specific Line Probe Assays
  42. 42. Two commercial assays available Genotype MTBDRplus (Hain LPA), INNO-LipA Rif.TB Validated for use directly from smear-positive sputum (MTBDRplus) or from TB cultures Manual and automated systems Twincubator – 10 /run GT Blot – 48/run rpoB for rifampicin resistance (InnoLiPA) rpoB for rifampicin, katG and inhA for isoniazid resistance (MTBDRplus) Commercial Assays
  43. 43. Results of Hain MTBDRplus •97% of smear-positive specimens gave results within 1–2 days (24-48 hrs) •Good sensitivity and specificity -Rifampin: sensitivity: 98%; specificity: 99% -Isoniazid: sensitivity: 89%, specificity: 99%
  44. 44. Same technology as Hains MDRplus Target genes identified are: • FQ – gyrA (oflox, Moxi) • Aminoglycosides and Polypeptides –rrs (Kana, Amik, Vio, Capreo) • EMB- embB (Ethambutol) LPA for diagnosis of XDR TB
  45. 45. Use of LPA under the program Integrated national plan for LPAs with MDR-TB management and lab capacity strengthening Use of LPAs on smear-positive sputum and from cultures if smear negative (insufficient evidence for direct testing in smear negatives) LPAs do not replace conventional culture + DST Commercial assays recommended Lab infrastructure, procedures and biosafety
  46. 46. GENEXPERT® MTB/RIF 1. The new, rapid and fully automated Xpert® 2. MTB/RIF test is cartridge-based automated DNA amplification test 3. Highly sensitive for confirmation of both smear positive and smear negative samples. 4. The Xpert® MTB/RIF assay uses 3 specific primers and 5 unique molecular probes to ensure a high degree of specificity 5. Assay targets the rpoB gene, which is critical for identifying mutations associated with rifampicin resistance
  47. 47. GENEXPERT® MTB/RIF 1. Advantages: • highly accurate results in less than 2 hours. • Simultaneous detection of both MTB and rifampicin resistance, • up to 95% of rifampicin resistance strains are INH resistance
  48. 48. Xpert MTB/RIF assay & GeneXpert instrument • diagnostic molecular test that uses modern technology: • Extraction and purification DNA/RNA & • Amplification & • Multiplex detection (automated)
  49. 49. Xpert MTB/RIF assay & GeneXpert instrument
  50. 50. Xpert MTB/RIF assay & GeneXpert instrument • Closed system – no contamination risk • Controls – Positive control included in test kit • Reagents – All reagents in self-contains kit, kit contains a pipette to transfer liquid sputum from container to cartridge • Storage/stability (including reagents) – Maximum shelf-life of 14 months
  51. 51. Xpert MTB/RIF assay & GeneXpert instrument • Reagents stable at temperature 2-280 • Instrumentations – Requires annual maintenance • Power requirement – Low power requirement compared with other PCR system • Training – Approximately 1 day of training required
  52. 52. Sensitivity for AFB+/culture+ 98.2% • Sensitivity for AFB-/culture+ 72.5% • Specificity 99.2% Rifampicin resistance detection Sensitivity – 98% • Specificity – 99% Xpert MTB/RIF assay & GeneXpert instrument Sensitivity and specificity
  53. 53. WHO RECOMMENDATIONS ON XPERT MTB/RIF • SHOULD BE USED: -as the initial diagnostic test in adults and children presumed to have MDR-TB or HIV-associated TB -as the initial diagnostic test in testing cerebrospinal fluid specimens from patients presumed to have TB meningitis
  54. 54. • MAY BE USED: -as the initial diagnostic test in adults and children presumed to have TB -as a follow-on test to microscopy in adults presumed to have TB but not at risk of MDR-TB or HIV-associated TB, especially in further testing of smear-negative specimens -as a replacement test for usual practice (including conventional microscopy, culture, and/or histopathology) -for testing of specific non-respiratory specimens (lymph nodes and other tissues) from patients presumed to have extrapulmonary TB
  55. 55. Drug Susceptibility Testing 7 - 10 days 3 - 4 weeksSolid Media Löwenstein-Jensen (Middlebrook) Liquid Media BACTEC 460 TB MGIT Molecular based Methods InnoLipa GenoTypeMTBDR Xpert MTB ‚home made‘- methods Hours – 1day
  56. 56. Indirect tests • ANTIBODY DETECTION • TB stat PAK • ELISA • INSTA TB TEST • ANTIGEN DETECTION • TB MPB 64 PATCH TEST
  57. 57. INTERFERON GAMMA RELEASE ASSAY • much less likely than the tuberculin skin tests (TST) to be confounded by exposure to environmental mycobacteria or by prior BCG vaccination. Does not boost responses • do not require a second clinical contact to evaluate the test result, • better sensitivity for HIV-infected people and patients with extrapulmonary TB • Disadvantages • lower sensitivity than the TST for past infection. Assays do not discriminate between active and latent TB infection. • moderately complex and requires standard ELISA equipment • . Blood samples have to be incubated within 16 hours of being collected, which may require the use of portable incubators • Requires that blood be drawn from the patient with a needle, which can lead to other infections. • application of such tests in disease-endemic, developing countries is the subject of extensive investigation.
  58. 58. INTERFERON GAMMA RELEASE ASSAY
  59. 59. There is insufficient data and low quality evidence on the performance of IGRAs in low- and middle-income countries, typically those with a high TB and/or HIV burden • IGRAs and the tuberculin skin test (TST) cannot accurately predict the risk of infected individuals developing active TB disease • Neither IGRAs nor the TST should be used for the diagnosis of active TB disease
  60. 60. WHO RECOMMENDATIONS RECOMMENDED NOT TO USE : • Commercial TB serodiagnostic tests. • Interferon-gamma release assays for detection of active TB (all settings).
  61. 61. Sensitivity (cfu/ml) of pulmonary TB diagnostics Solid culture
  62. 62. Phage based tests
  63. 63. • Can also be used on urine samples • Results available in 1 day • As it is cheap can be used in resource poor countries
  64. 64. TESTS BEING REVIEWED BY WHO IN 2015 • TB LAMP (loop mediated isothermal amplication) • URINARY LAM (lipoarabinomannan) • MOLECULAR DST METHODS- hains MTBDR sl tests
  65. 65. TECHNOLOGIES IN DEVELOPMENT • VOLATILE ORGANIC COMPOUNDS -breathlink -prototype breath analyser device MOLECULAR TECHNOLOGIES -alere Q -B SMART -genedrive MTB/RIF ID -LATE PCR -genepert XDR catridge -trueArray MDRTB -INFITINIMTB assay -fluorotype MTB/ fluorotype MTB RNA
  66. 66. • CULTURE BASED TECHNOLOGIES -BNP middlebrook -TREK sensititre MYCOTB mic plate OTHER TECHOLOGIES -TB rapid screen -TBDx
  67. 67. OVERVIEW OF WHO RECOMMENDATIONS
  68. 68. • LED microscopy: For use at all laboratory levels as replacement of conventional fluorochrome and light microscopy. • Commercial liquid culture and DST systems: For use at central/regional reference laboratory level, as current reference standard. • Rapid speciation strip technology: For use with conventional culture and DST at central/regional reference laboratory level, to identify Mycobacterium tuberculosis. • Commercial molecular line probe assays for 1st-line anti-TB drugs: For use at central/regional reference laboratory level for rapid detection of rifampicin (alone or with isoniazid) resistance. Suitable for use on smearpositive specimens or culture isolates.
  69. 69. • Selected non-commercial DST methods: MODS(microscopic observation of drug susceptibility), NRA(nitrate reductase assay), CRI(calorimetric redox indicator): For conditional use at central/reference laboratory level for detection of rifampicin resistance only. MODS and NRA suitable for use on smear-positive specimens or culture isolates, CRI suitable for use on culture isolates only. • Automated real-time nucleic acid amplification - Xpert MTB/RIF system: For rapid detection of pulmonary and extrapulmonary TB and rifampicin resistance in both adults and children at decentralised laboratory and health care centres.
  70. 70. NOT RECOMMENDED DUE TO CURRENT INSUFFICIENT EVIDENCE • Sputum concentration and decontamination methods. • Phage-plaque technology for rapid rifampicin resistance. • Thin-layer agar methods for rapid culture and DST. • Interferon-gamma release assays as replacement for the tuberculin skin test for detection of latent TB in low- and middle-income (typically high TB and/or HIV) settings. • Molecular line probe assays for 2nd-line anti-TB drugs. • Loop-mediated isothermal amplification test kit for TB.
  71. 71. RECOMMENDED NOT TO USE : • Commercial TB serodiagnostic tests. • Interferon-gamma release assays for detection of active TB (all settings).
  72. 72. THANK YOU

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