PPT Cirillo " Diagnostic laboratory news"
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PPT Cirillo " Diagnostic laboratory news" Symposium on TB, 14 October, IV Session (physicians & surgeons), Monza, Italy.
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PPT Cirillo " Diagnostic laboratory news"
- 1. Le novità diagnostiche di laboratorio Daniela Maria Cirillo WHO Collaborating Centre for integrated laboratory strengthening on TB San Raffaele Scientific Institute Milano 14/15 ottobre 2011
- 9. Sensitivity and Specificity of NAA tests in clinical specimens FEMS Immunol Med Microbiol ]] (2009) 1–9 Low clinical relevance, not recommended to replace conventional test Amplicor 97 > 95 40 - 73 >95 27 – 98 > 95 AMTB 92-100 > 95 40 - 93 > 95 93 >95 BD Probe Tec 90-100 92 33 - 100 83 – 97 76 >90 Real-time PCR 78-95 100 78 100 80 100 LAMP 97.7 99 48.8 99 ND ND Smear-positive Smear-negative pulmonary pulmonary Extrapulmonary Sensitivity Specificity Sensitivity Specificity Sensitivity Specificity
- 13. Phenotypic DST TBPANNET workpackage 6 midterm meeting
- 19. Genes involved in drug-resistance for major anti-tubercular drugs TBPANNET workpackage 6 midterm meeting
- 26. Commercial Line Probe Assays Hain Lifescience Innogenetics INNO-LiPA-Rif.TB
- 27. Comparison GenoType® MTBDRplus and INNO-LiPA Rif.TB GenoType ® MTBDR INNO-LiPA Rif.TB Company Hain Lifescience Innogenetics M. tuberculosis detection Yes Yes Detection of RMP Resistance in M. tb Complex Yes Yes Detection INH Resistance in M. tb Complex Yes No Strip Assay Yes Yes PCR based Yes Yes From liquid or solid culture Yes Yes Direct assay Yes Yes (modified version) TBC Detection: 23S-rRNA/16S-rRNA Yes Yes RMP-Resistance: rpoB gene Yes Yes INH-Resistance: katG gene/inhA gene Yes No Universal control Yes No rpoB unicontrol Yes No kat G/inHA u nicontrol Yes No
- 29. New generation of LiPA performs better in both Sm+ and Sm- samples Miotto al. JCM 2008 TBPANNET workpackage 6 midterm meeting
- 31. Molecular line probe assays for rapid screening of patients at risk of MDR-TB Policy statement by WHO and Partners June 27, 2008 Endorsement of the two commercial line probe assays for rifampicin resistance detection TBPANNET workpackage 6 midterm meeting
- 32. Possible automation on LiPA LiPAs require: Level II biosafety areas Skilled laboratory staff Amplicon Contamination control
- 33. Xpert MTB/RIF works on the Gene Xpert System TB What else!
- 34. Boehme C, N Engl J Medicine, 2010 Assay procedure for MTB/RIF test
- 35. Moving to “ district” : Dharamsala, Delek Hospital
- 36. Proportion of TB cases detected Time to detection Time to RFP resistance detection Advantages of fully automated real-time test
- 37. Performance of the Xpert MTB/RIF assay for single assay *Xpert MTB/RIF sensitivity > Amplicor or Probe TEC sensitivity High-prevalence population SENSITIVITY (%) SPECIFICITY (%) Boehme et al , 2010, N Eng J Med 98.2 99.2 SMEAR + CULTURE + Total patients: 1730 72.5 SMEAR - CULTURE + 96.6 98.1 RIF detection SENSITIVITY (%) SPECIFICITY (%) Helb et al , 2010, J Clin Microbiol 100 100 SMEAR + CULTURE + Total patients: 107 Vietnamese, 64 Ugandan 71.7 SMEAR - CULTURE + 100 98.2 RIF detection
- 38. Performance of the Xpert MTB/RIF assay Extra-pulmonary specimens Pulmonary specimens in low-prevalence population *Stool, urine and gastric fluid sensititvity > tissue sensitivity SENSITIVITY (%) SPECIFICITY (%) Marlowe et al , 2011 J Clin Microbiol 98 100 SMEAR + CULTURE + Total: 217 72 SMEAR - CULTURE + nd nd RIF detection SENSITIVITY (%) SPECIFICITY (%) Hillemann et al , 2011 J Clin Microbiol 77.3* 98.2 ALL CULTURE + Total: 521 nd nd RIF detection
- 40. Currently recommended TB tests fit into the following tiered system:
- 42. Proportion of TB cases detected time to detection Time to RFP resistance detection XPERT-TB/MDR TBPANNET workpackage 6 midterm meeting
- 44. Genes involved in drug-resistance for major anti-tubercular drugs 40-80%
- 45. XDR molecular diagnosis: GenoType MTBDR sl (Hain Lifescience)
- 51. TBPANNET workpackage 6 midterm meeting PMA pretreatment of samples allows selective amplification of DNA from live bacteria Miotto 2011 under revision ERJ
- 52. Molecular DST
- 53. Typing: the gold standard Mycobacterial Interspersed Repetitive Units -Variable Number of Tandem Repeats (MIRU-VNTR) Based on tandem repeats in mini-satellite regions of the genome. The original 15 loci consisted of 5 exact tandem repeats (ETR A-E) followed by 10 MIRUs. Described by Supply et al ., 2006. VNTR-424 VNTR-1955 VNTR-2163b VNTR-2347 VNTR-2401 VNTR-3171 VNTR-3690 VNTR-4052 VNTR-4156 VNTR locus MIRU2 MIRU4 (ETR-D) MIRU10 MIRU16 MIRU20 MIRU23 MIRU24 MIRU26 MIRU27 MIRU31 (ETR-E) MIRU39 MIRU40 ETR-A ETR-B ETR-C
- 58. 2.) T-cell based tests APC T cell antigens/ peptides cytokine induction cytokine induction cytokine induction ELISA ELISPOT assay T.SPOT. TB QuantiFERON TB gold IGRA IFN- release assay PPD (= tuberculin) ESAT-6, CFP-10, Tb7.7 ESAT-6, CFP-10 Skin test
- 59. Immunology based Tb-tests Serological tests Antibodies against Mtb-specific antigens Skin tests Local immune reaction (PPD-specific T cells) Interferon gamma release assays (IGRAs) Mtb-specific T cells (PPD, ESAT-6, CFP-10) APC T cell antigens/ peptides
- 60. Accuracy of IGRA: sensitivity and specificity
- 61. Summary of pooled values from the metanalysis performed by Pai et al, and by Sester and Sotgiu et al Test Sensitivity for active TB Specificity for active TB Percentage TST Pai et al, 2008 77 95/79 Sester et Sotgiu et al, 2010 65 75 QFT-IT Pai et al, 2008 70 96 Sester et Sotgiu et al, 2010 80 79 T-SPOT. TB Pai et al, 2008 90 93 Sester et Sotgiu et al, 2010 81 59
- 62. IGRA in HIV+ with active TB Source Patient number Test Sensitivity (indeterminate results included) Sensitivity (indeterminate results excluded) Seshandri et al, 2008 13 QFT 23 33 Vincenti et al, 2007 13 QFT ND 85 Raby et al, 2008 59 QFT-IT 63 76 Aabye et al, 2009 68 QFT-IT 65 83 Leidl, ERJ 2009 31 QFT-IT 68 68 Kabeer et al, 2009 105 QFT-IT 65 78 Goletti et al, 2010 28 QFT-IT 61 73 Vincenti et al, 2007 13 T SPOT TB ND 85 Markova, 2009 13 T SPOT TB 62 89 Jiang, 2009 32 T SPOT TB 66 66 Leidl, ERJ 2009 19 T SPOT TB 89 100
- 64. Sensitivity, specificity and diagnostic odds ratio of the different assays for the immune diagnosis of TB Goletti et al, CMI 2006 Assay Sensitivity % Specificity % Diagnostic odds ratio RD1 ELISPOT assays Selected peptides 70 91 22 Intact proteins 83 56 6 RD1 commercially available assays T-SPOT TB 91 59 15 QFT Gold 83 59 7
- 65. TBNET report Goletti et al, PLoS ONE 2008
- 66. IGRA at the site of TB disease: BAL vs blood From Jafari, AJRCCM 2009
- 67. IGRA at the site of TB disease: Pleural fluid vs blood Berlin, October 4th, 2008 From Losi et al, ERJ 2007 PLEURAL CELLS PBMC
- 69. Predictive value of IGRA: HIV-negative subjects Diel et al, AJRCCM 2010 in press
- 70. Rates of disease progression in IGRA+ve vs TST Country Test Incidence of active TB in IGRA+ groups Comment Gambia [Hill et al. 2008] ELISPOT (in-house) 9/1000 person-yr High burden Colombia [del Corral et al. 2009] In-house CFP-10 assay 7/1000 person-yr High Burden Senegal [Lienhardt et al. 2010] ELISPOT (in-house- 32 SFC cut-point) 9/1000 person-yr High burden Turkey [Bakir et al. 2008] ELISPOT (in house similar to T-SPOT TB ) 21/1000 person-yr Intermediate Germany [Diel et al. 2010] QFT-IT 73/ 1000 person-yr Low burden
- 71. Multifunctional T cells correlate with active TB disease From Caccamo, EIJ 2010, modified IFN- γ , IL-2, TNF- α IFN- γ , IL-2 IFN- γ
- 72. Efficacy of therapy based on IGRA results: NO DATA AVAILABLE
- 73. Trends for the future IGRA on a chip Skannex.com
Editor's Notes
- Je vous parle d ’ un nouveau test pour le diagnostic de la tuberculose. Pourquoi? Qu ’ est-ce que c ’ est la TB? Et pourquoi a t-on besoin d ’ un nouveau test? Pour répondre a ces questions j ’ ai préparé une liste d ’ objectifs.
- A recent meta-analysis assessed the diagnostic accuracy of the commercial NAA tests described above (Ling et al., 2008). Sensitivity (not specificity) of different tests (except real time PCR) depends on samples smear results - positive or negative. It is interesting that NAA tests’ sensitivity of extrapulmonary samples varies much between diffrent tests. Real time PCR shows equal sensitivity for all kind of samples (pulmonary smear positive and negative and extrapulmonary). Specificity of all indicated tests for all kind of samples is nearly the same and quite high. Overall, a pooled sensitivity of 85% (range 36–100%) and specificity of 97%(range 54–100%) was reported. Both values showed significant heterogeneity, which could not be explained even after analysing the results of each test separately. The major conclusion was that the accuracy of these NAATs with respiratory samples was highly variable, with sensitivity giving lower values than specificity. For these reasons, they probably still do not have enough clinical relevance and could not be recommended to replace the conventional tests for diagnosing pulmonary tuberculosis.
- ISTC Training Modules 2008 In any given population of TB bacilli, naturally occurring resistant organisms occur at the rates as shown. [Review above] The greater the burden of disease, the larger the bacillary population and the greater the risk for harboring drug-resistant mutants and acquiring drug resistance. [Note: Year drug was discovered is listed under each drug.] [Image Credit: CDC/Dr. Ray Butler; Janice Carr. Illustration Credit: National Institute of Allergy and Infectious Diseases (NIAID). Illustrator: Krista Townsend]
- In this slide, MTB/RIF assay procedure is described. In brief, a sputum sample is treated for 15 minutes with sodium hydroxide and isopropanol-containing reagents, which decrease MTB viability by at least 6 logs. The sample is transferred into the cartridge preloaded with PCR buffers. The cartridge is then loaded into the instrument and an automatic process completes the sample processing, DNA extraction and real-time PCR, thus eliminating the risk of cross-contamination. The test turnaround time is less than 2 h, similar to smear microscopy and many time faster than culture methods.
- Up to now, the few papers that report the MTB/RIF performance, show for pulmonary specimens, a sensitivity of more than 98% in smear- and cultures positive samples, and of almost 72% in smear negative culture positive samples. The overall specificity is 99-100%. In addition, MTB/RIF correctly detected rifampicine resistance in more than 96% patients, with a high specificity.
- Similar sensitivity and specificity percentages are observed when the Xpert MTB RIF performance is evaluated in low MDR- prevalence population. A good performance, even if we see lower values, is observed in extrapulmonary samples and this indication may imply the isolation of TB strains from other sample in particular patients like children.
- Finally, here I reported the last WHO recommendations. WHO strongly recommends Xpert MTB/RIF should be used as the initial diagnostic test in individuals suspected of MDR-TB or HIV/TB and it may be used as a follow-on test especially in smear-negative specimens. The expected impact of Xpert MTB/RIF implementation would be a three-fold increase in the diagnosis of patients with MDR-TB and TB/HIV cases and an increase of treated TB at least may reduce mortality, secondary resistance and ongoing transmission.
- So, summarizing the Xpert MTB RIF characteristics: It is simple to perform It can be performed by low skilled technicians in absence of biosafety infrastructure and this (next slide) will make Xpert MTB/RIF test suitable for district and sub-district levels. It shows an high sensitivity also in paucibacillary samples simplifying the diagnosis of smear negative TB as HIV+ High performance in extrapulmonary samples and The advantage of MTB and RIF resistance detection within 2 hours However we have no data on Xpert MTB/RIF performance in children and few data in low-prevalence populations It requires uninterrupted and stable electronic power supplies and yearly calibration Size for storage issues
- Highlight the high variation between the different commercial kits (e.g. low sensitivity of QFT on extrasanguin but the high sensivity for same sample using T-spot). compare values to that of TST.
- Sensitivity of our in house assays based on selected peptides was lower compared to the other assays; however the specificity was much higher and more importantly the diagnostic odds ratio was significantly higher compared to the other tests