Download as PDF, PPTX
More Related Content
Similar to TB Diagnosis_.pdf2345678987uytreert7890987654
TB Diagnosis_.pdf2345678987uytreert7890987654
- 1. TB Diagnosis Professor: MohammedWaheeb Al.Obaidi FACCP,FRCPE Consultant chest physician
- 2. Tuberculosis in history Devastatingeffect on society • 100 years ago one in five of the population was destined to die of tuberculosis. Families suffer psychologically, socially and economically. • TB is highly stigmatized, especially in women.
- 3. What is TB? •TB is an infectious disease that affects mainly the lungs (pulmonary TB, or PTB) but can also attack any part of the body (extrapulmonary TB, or EPTB)
- 4. What is TB? 4 TBis an infectious disease that affects mainly the lungs (pulmonary TB, or PTB) but can also attack any part of the body (extrapulmonary TB, or EPTB)
- 5. TB transmission (infection) Person-to-person via airbornetransmission in confined environment
- 6. Inhalation (1–5 μmØ) droplet infection 10–30% infection 90% LTBI 5–10% ACTIVE TB within 2 years 10% TB during lifetime 10% TB within 1 year if HIV+ 85% pulmonary TB 15% extrapulmonary TB No infection 33% pulmonary TB 33% extrapulmonary TB 33% both HIV− HIV+ Tuberculosisinfectionversus active disease 6
- 7. TB is causedby Mycobacterium tuberculosis and occasionally by other species belonging to the (M. tuberculosis complex: M. tuberculosis, M. africanum, M. bovis, M. bovis BCG, M. caprae, M. microti, M. canettii, and M. pinnipedii strains are responsible for the majority of mycobacterial infections worldwide. TB complex bacilli are also known as tubercle bacilli. A: ZN staining 1000x B: Fluorescent staining, 400x 7 Cause of TB A B
- 8. Central laboratory Intermediate laboratories Peripherallaboratories TB diagnostic services should be linked as a “laboratory network” 8 Adaptedto local situationand infrastructures
- 9. Diagnosis of TBby microscopy • Mycobacteria retain the primary stain even after exposure to decolorizing acid–alcohol, hence the term “acid-fast bacilli” (AFB). • Ziehl-Neelsen • Fluorescence • M. tuberculosis bacilli can be found singly, in clumps or in clusters. They are usually beaded and long bacilli. 9
- 10. Limitations of smearmicroscopy • Low sensitivity: 104–105 bacilli/ml. • Detects both dead and viable bacilli. • Does not distinguish tubercle bacilli from other mycobacteria. 10
- 11. Advantages of culture •Detects small numbers of bacilli (as few as 10 bacilli/ml, depending on the technique used). • Improves case detection: >25% over microscopy. • Provides definitive diagnosis of EPT. • Confirms diagnosis of TB in HIV+ patients. • Allows species identification. • Allows DST and drug resistance surveys. • Allows epidemiological studies. 11
- 12. Limitations of culture •High cost. • Slow growth of M. tuberculosis: delays results. • More sensitive to technical deficiencies. • Greater need for infrastructure: – qualified staff – equipment – additional safety measures. 12
- 13. CULTURE MEDIA • Thefollowing media are used for M. tuberculosis culture: • Egg-based solid media: • Löwenstein–Jensen(LJ) medium • Stonebrink medium • Ogawa medium • acid-buffered Ogawa medium. • Stonebrink medium has the same composition as Löwenstein–Jensen,except that glycerol is replaced by 0.5% sodium pyruvate.
- 14. Liquid media (formanual and automated use): • Herman-Kirchner liquid medium • Dubos oleic acid–albumin liquid medium • Middlebrook 7H9 liquid medium
- 15. • Each ofthese media has advantages and disadvantages: • solid media require longer (8–12 weeks) to give a positive culture, but they are less expensive than liquid media and allow the recognition of the morphology of colonies. • Liquid media are more expensive than solid media and more prone to contamination; they can easily be spilt (biosafety issue
- 16. Egg-based media • theyare easy to prepare; • the cost is low • they support good growth of tubercle bacilli; • they can be stored in a refrigeratorfor several weeks; • there is limited contamination during preparation; • malachite green suppresses the growth of non- mycobacterial organisms; • contamination may involve just a portion of the surface of the media, meaning the culture can be saved.
- 17. Automated broth systems 17 BACTECTM 460TB MGITTM 960 VersaTREKTM BacT/ALERTTM MP system Format SA CM CM CM Detection Radiometric Fluorimetric Manometric Colorimetric Capacity 60 960 384 240 Data management NO YES YES YES DST YES YES YES YES
- 18. • Radiometric method RadiolabelledCO2 detected by β-counter. • Fluorimetric method Fluorescence, inhibited by O2 in the medium, is released by oxygen consumption. • Colorimetric method CO2 production changes the colour of a sensor detected by a reflected light beam. • Pressometric method Pressure increase from gas production is detected by a pressure- detection device. Methodology of positive detection 18
- 19. •Liquid radiolabelled bottlesBACTECTM 12B for: –mycobacterial growth and detection; –DST. BACTECTM 460TB 19 First semiautomatic system. Still considered a “standard” for liquid culture and DST.
- 20. • Automatic systemfor mycobacteria detection • DST automatic (1999) • Non-radiometric • Fluorescence BBLTM MGIT™ • Non-invasive (no needles), totally automatic and computerized • High workload BACTECTM MGITTM 960 20
- 21. Fluorescence-labelled medium • Mycobacterialiquid medium. • Ruthenium salt, incorporated in a film on the bottom of the tube, registers oxygen variation. • Oxygen consumption by bacterial metabolism releases fluorescence. • Fluorescence is detected automatically by UV lamp. 21
- 22. Fluorescence technology Little orno fluorescence Positive culture F F FO2 FO2 FO2 FO2 FO2 F FO2 FO2 CO2 O2 O2 O2 O2 O2 O2 O2 O2 CO2 CO2 O2 F F F F FO2 FO2 F F F F CO2 O2 O2 O2 O2 O2 Strong fluorescence Sensor Broth Empty space Negative culture 22
- 23. •Automatic system •Non-radiometric •Colorimetric technology •Cultureof all material •Computerized data analysis BacT/ALERTTM MP 23
- 24. LES (liquid emulsionsensor) Single cell reading CO2 pH BacT/ALERTTM MP 24
- 25. • Automatic system •Non-radiometric • Pressometric technology • Culture from all kinds of samples • DST • Computerized data analysis VersaTREKTM 25
- 26. Detection technology VersaTREKTM monitorsthe pressure variation over time in the bottles, associated with O2 consumption and gas production (CO2, H2 and N2 – i.e. not just CO2). 26
- 27. The introductionof theDNA-probe technology has greatly improved the differentiation between mycobacteria species and the time needed for identification at species level has dramaticallydeclined. Rapid microbiologic diagnosis relied on microscopy until late in the eighties when direct nucleic acid amplification methods were introduced for this purpose. These methods have primarily been used for the detection of MTC and only scarcely for NTM. Molecular Diagnosis
- 28. Genes to distinguishall clinically important mycobacteria: (EXCEPT FOR THE MEMBERS OF THE MTC COMPLEX) 32 kDa protein gene 65 kDa heat shock protein gene (hsp65) 16S ribosomal RNA gene 23S ribosomal RNA gene 16S–23S ribosomal RNA internal transcribed spacer The 65 kDa protein gene can also be used for distinguishing clones of certain mycobacterial species (intra-species variation). Introduction
- 29. Identification by genomicsequencing Identification by PCR restriction analysis (PRA) Identification by commercial assays*: - Hybridization in liquid phase AccuProbe, bioMérieux - Hybridization in solid phase INNO LiPA Mycobacteria, Innogenetics GT-Mycobacterium and GT- MTBC, Hain Lifescience All molecularmethods requireDNA(or RNA) isolation * Commercial assays can be used to identify organisms from both solid and liquid culture. Methods – Overview
- 30. Molecular line probeassays for rapid screening of patients at risk of MDR-TB Policy statement by WHO and Partners June 27, 2008 Endorsement of the two commercialline probe assays for rifampicin resistance detection: Tests are CE marked and meet predefined performance targets in controlled evaluation studies Both tests are highly sensitive and specific for rifampicin resistancedetection from TB strains
- 31. 31 Advantages: - Easy toperform and easy read-out; cost- effective Disadvantages: - Limited number of probes that can be used; - Fails to distinguish insertions mutations Line Probe Assay (LiPA) Microarray Advantages: - High throughput for screening due to the high number of probes; - High automation(high standardization) Disadvantages: - Higher number of probes higher complexity in results interpretation; - Standardization requires reproducibility of data; - Cost-effectiveness? To be evaluated
- 32. 32 Commercial Line ProbeAssays HainLifescience Innogenetics INNO-LiPA-Rif.TB
- 33. 33 Sensitivity for INNOLiPA,MTBDR, and MTBDRplus based on frequency of targeted mutations INH RIF MDR Sensitivity: TruePositive/(True Positive+False Negative) Commercial molecular DST assays Miottoet al ESM,2008
- 34. The “Hain platform” •Targets the most common mutations conferring resistance to 1st and 2nd line drugs • Allows MDR and XDR definition • Test is simple to be performed But • Not automated • Some possibility of misinterpretation • Limited in the number of probes • P2 biosafety required • Possibility of cross contamination
- 35. Molecular DST: conclusions Althoughthese assays cannot replace conventional DST, the high sensitivity and specificity for RIF-R and INH-R can facilitate the early diagnosis and treatment of MDR-TB, particularly for patients with a history of prior TB treatment. Usefulness of molecular techniques in DST limited by the lacks of knowledge of all molecular mechanisms of resistance.
- 36. 36 If drug resistancesare suspected, molecular DST allows the early detection of mutations involved in drug resistances resulting in a better management of patients. Identification of mutations by molecular DST allows to predict cross-resistances among drugs (e.g. aminoglycosids). Large number of data on mutations are needed to develop commercial assays for fast detection of XDR strains Molecular DST: conclusions
- 37.