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“ The renewals of the TB Laboratory Diagnosis ”

“ The renewals of the TB Laboratory Diagnosis ”. Nurhan Albayrak ( Clinical Microbiologist ) April 3, 2013 / Antalya. Outline. Flourecence microscopy Molecular tests for TB identification Moleculer tests for drug susceptibility testing. TB diagnosis. Bacteriological

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“ The renewals of the TB Laboratory Diagnosis ”

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  1. “Therenewals of the TB LaboratoryDiagnosis” Nurhan Albayrak (ClinicalMicrobiologist) April 3, 2013 / Antalya

  2. Outline • Flourecencemicroscopy • Moleculartestsfor TB identification • Moleculertestsfordrugsusceptibilitytesting

  3. TB diagnosis • Bacteriological • Smearmicroscopy • Culture (gold standart) • Moleculartests • Serologicaltests • Interferon gama releasingassay

  4. Smearmicroscopy • Because of themycobacterialcellwall lipit content • Lateculturepositivity (4-8 week) • Difficultytostainig • mikolikacidscarbonsaresomuch

  5. Smearmicroscopy • Advantages • Simple, cheap, fast (<1 hour) • Abundantbacilli in cavitarydisease • Disadvantages • Sensitivity 25-80% (5.000 bacil/ml) • Lesssensitivity in extrapulmonary TB and NTM • Needsexperience (10-15 slides/week) • Maximumcapacityforoneperson is 25 slides/daily • Deficiency • Didn’tdisriminateM. tuberculosiscomplexfrom NTM • Didn’tdisriminateviablebacteriafromthedeadone

  6. Renewal • LED (lightemittingdiode) • Comparedwith EZN staining (patientnumber is 2.355) • Sensitivity • 2 sputum LED %68,8-76,5, EZN %61,6-69,8 • 3 sputum LED %73,3-80,6, EZN %66,4-74,4 • Specivicity • 2 sputum LED %89,5-92,2, EZN %97,3-98,6 • 3 sputum LED %86,5-89,6, EZN %96,8-98,2 Cuevas LE, et al. PLoSMed. 2011

  7. Smearmicroscopy What’stheadvantages of LED? • Sensitivity↑ • Capacity↑ • Needtolabstaff↓ Is thereanyclinicaladvantage?

  8. Interpretation of theresults • Whatmeans AFB (+) results? • Acidfastbacillipositive • Mycobacteriapositive • M. tuberculosiscomplexpositive • M. tuberculosispositive • Whatmeans AFB (-) results? • Thisresultsdon’tmeansthat ‘thepatient is not TB’ • Becausethesensitivity is 25-80%

  9. Moleculartests • Fordetection • PCR • Real-time PCR • Microarray • Foridentification • For DST • Real-time PCR • Lineprobeassay • Sequencing

  10. Moleculartests • Based on theprinciple of amplification of theM. tuberculosis DNA • Advantages • Sensitivitychangedaccordingtothemethod (10-100%) • Fast • Disadvantages • Expensive • Deficiency • Didn’tdisriminateviablebacteriafromthedeadone • Didn’tvalidatedforextrapulmonary TB

  11. SensitivitySpecivicity • Metaanalysis with 45 investigation • Inpulmonary TB 9-100% sensitivity 25-100% specitivity Sarmiento OJ, et al. J ClinMicrobiol. 2009

  12. Renewal in moleculartests • XpertMtb/RIF assay • Detection of Mtband RIF resistance • Handlingfromsputum • Resulted in 2.5 hours

  13. Xpert • Metaanalysiswith 18 investigation, 10244 sample • Inpulmonary TB; sensitivity 90.4%, specivity98.4% • Detection limit; 131 CFU/ml • ForRIFresistance; sensitivity94.1%, specivity 97% Chang K, et al. J Infect. 2012

  14. WHO’srecommendationsforXpert (2011) • In MDR-TB suspectedcases • Means RIF resistance MDR? • RIF resistance is an indicatorfor MDR • Smearnegativecases • Sensitivity is 70%

  15. Drugsusceptibilitytesting • Conventional = phenotypicmethods • LJ / Agarproportion • Automatedliquidcultures • Molecular = genotypicmethods • Detectingthemutation • Real-time PCR • Lineprobeassay • Sequencing

  16. Advantages - disadvantages Molecularmethods Detectingmutaion DNA is enough High in mutation Fastfromsputum (1-7 days) Expensive Needexperience Needequipment BSL-2 Needverification Conventionalmethods Growthinhibition Needsviablebacteria Highfot HR Forsecondlinedrugs↓ Latefromculture (afterculture 4-35 days) Cheaperthanmoleculartests Simplethanmoleculartests Lessequipment BSL-3 Principle Sensitivity Turn-on time Technic Biosafety Deficiency

  17. Mutationregionsrelatedwithresistance %40-60 %25 %10 %95 %95 %60 %20 %60 %80-90 %90 Silva PEA, et al. J AntimicrobialChemother. 2011

  18. Lineprobeassay • AFB (-/+) sample; HR resistance • AFB (+) samle; EMB, FLQ and AG/CP resistance • Steps • DNA extraktion (45 min) • Amplification (2-3 h) • Hybridisatioon (2-3 h)

  19. Lineprobeassay (sL)

  20. Interpretation of theresults Real RFalse (+) False (-) • RIF resistance180 4 (2,2%)* 1 (0,5%) (rpoBmutation) • INH resistance391 2 (0,5%) 43 (10,9%) (katG / inhAmutation) • EMB resistance120 13 (10,8%) 23(19,1%) (embBmutation) • FLQ resistance13 0 1 (7,6%) (gyrAmutation) • AG/CP resistance12 1 (8,3%) 10 (83%) (rrsmutation) *Anymutationwithwildtypemissings

  21. Summary • Thenewmoleculartestsrecommended in identification of pulmonary TB cases • Usingmoleculartestsfor DST • Is fasterfor MDR detection • Recommendedforsomesituations • Don’tforgetthatthetestsneedtoverificationwithconvantionalmethods.

  22. Teşekkürler

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