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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
Hot topic in the international research agenda for TB Drug development Diagnosis and diagnostic tests Epidemiology and public health Health services research Basic science research HIV MDR M. tuberculosis  vaccines Rylance et al, TLID 2010
The key role of the laboratory Care of TB patients starts with a QA diagnosis A robust network of Tb laboratories is required: Adaquate biosafety Modern diagnostics SOPs QAs Integrated laboratory network
Conventional TB diagnostic methods Slow and cumbersome Require sequential procedures for isolation, identification and in vitro sensitivity testing (DST) Require appropriate laboratory facilities and trained staff (AFB) Smear test: Insensitive, WHO and national guidelines recommend 2 to 3 smear tests to be performed by suspected patient Requires  skilled technician Time consuming: 1-2 hours Culture Highly sensitive Slow: 3 to 6 weeks Nucleic Acid Amplification methods: Highly specific Not optimal on clinical samples: Highly sensitive on smear positive samples (95-100%) Until now insufficient sensitivity on smear negative samples (60-75%) Very sensitive on culture isolates: For identification For Rif susceptibility testing
New Policy and Smear microscopy definition of a TB case New definition in 2007*: “ person with al least one smear-positive sample (1 AFB is sufficient) out of a total of two examined” 2 samples regardless the collection time *The definition/policy can be applied to countries performing microscopy under satisfactory quality assurance programmes
LED Fluorescence Microscopy Advantages :  increase in performance increase in lamp lifetime reduces initial, operating and maintenance costs No need for dark room
WHO recommendations on sputum smear microscopy (2010) ZN light microscopy performed on UNCONCENTRATED sputum is suitable for all laboratory service levels Concentration of sputum is NOT recommended in programmatic settings Fluorescence microscopy is recommended for increased sensitivity (add 10%) LED microscopy is recommended over conventional fluorescence
TB Culture* Advantages Definitive diagnosis of TB Increases case finding of 30-50% Early detection of cases Provide strains for DST and epidemiological studies Disadvantages Complex and expensive compared to microscopy Requires complex handling of specimens Skilled technicians Appropriate infrastructure and biosafety levels LIMITATIONS: need for decontamination and identification *coverage 500.000/1000000 Strip speciation tests needed for fast ID of Tbcomplex Molecular test for speciation of most common mycobacteria
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
Development of multi-drug resistant tuberculosis No single genetic alteration is responsible for the MDR phenotype (defined as resistance at least to INH and RMP). MDR develops by sequential acquisition and selection of mutations at different loci, usually because of inappropriate patient treatment.  Inappropriate treatment may lead to an increase of bacillary load and increase od spontaneous mutations while the disease progresses. Because MDR strains are the result of cumulative mutations, growth of  M tuberculosis  needs to be controlled in the host by concomitant treatment with more than one active drug.  Thus, TB treatment regimens that consist of  four  ACTIVE  drugs TBPANNET workpackage 6  midterm meeting
INH  = 1 in 10 6 RIF  = 1 in 10 8 EMB  = 1 in 10 6 Str  = 1 in 10 6 I + R  = 1 in 10 14 Frequenza delle mutazioni ai farmaci di prima linea
DST Phenotype based Genotype based TBPANNET workpackage 6  midterm meeting
Phenotypic DST TBPANNET workpackage 6  midterm meeting
WHO laboratory policies for culture and DST Automated liquid culture (2007) Use of liquid culture in the contest of comprehensive plan for strengthening lab capacity starting with national/central level Rapid speciation in combination with culture Second line DST (2008) to be conducted for selected drugs a central level Available at http://www.who.int/tb.dots/laboratory/policy/en/print.html
Standardization of procedures still difficult to set up The correct performance of DST requires the understanding of several steps such as: Origin of resistance and interpretative criteria Dosage and stability of the incorporated compounds Anti-mycobacterial activity of the incorporate drug Interpretation of results and data reporting TBPANNET workpackage 6  midterm meeting Drug Susceptibility Testing (DST): the technical challenge still persists
Determination of clinically significant proportion of resistant bacteria Clinically significant proportion In vitro growth of resistant cells in the presence of the critical concentration of the drug that is equal to or greater than 1% of the growth of the total population in the absence of the drug. The presence of 1% resistant cells represents a significant increase in resistance; laboratory assays are based on this assumption. Are molecular tests able to detect 1% mutant  subopulation?  TBPANNET workpackage 6  midterm meeting
Second line DST Recommended in appropriate structures for: Aminoglycosides, Fluoroquinolones, Polypeptides NOT recommended on routine for ethionamide, prothionamide, cycloserine,clofazimine, amox-clav, clarithromycin, linezolid)
TBPANNET workpackage 6  midterm meeting Cost-effectiveness Only available for selected drugs Only available for selected specimens Low sensitivity in AFB-negative and non-respiratory samples Genetic diversity may influence statistical parameters of molecular tests Based on single nucleotide mutations detection  Data on DST from specimens not suitable for culture Cross-resistance prediction Large number of specimens analyzed at the same time Standardization (automated systems) and TAT MOLECULAR DST ON  M. tuberculosis
Genes involved in drug-resistance for major anti-tubercular drugs TBPANNET workpackage 6  midterm meeting
Isoniazid resistance and  katG INH is a pro-drug that requires activation in INH-susceptible mycobacterial species.  the activation of INH results in a number of highly reactive compound that are capable of damaging the mycobacterial cell wall.  INH-resistant clinical isolates frequently loose their catalase-peroxidase activity (Middlebrook et al., 1954) Association of this enzyme with INH activation was proven when the mycobacterial catalase-peroxidase gene ( katG ) was cloned and sequenced. (Zhang et al., 1992)  The Ser315Thr mutation is found in 70-80% of high INH-resistant clinical isolates.  TBPANNET workpackage 6  midterm meeting Adapted from GLI training material
Isoniazid resistance and  inhA INH blocks the synthesis of cell-wall mycolic acids, the major components of the envelope of  M tuberculosis . One intracellular target of the drug is fatty-acid  enoyl-acyl carrier protein reductase (InhA) . (Basso et al., 1998) Mutations in the promoter region of  inhA  result in over-expression of the protein. The over-expressed enzyme may counter-balance the effect of INH and will result in a low-level resistance to the drug.  TBPANNET workpackage 6  midterm meeting
Isoniazid resistance and virulence The Ser315Thr mutation results in an enzyme without the ability to activate INH, but retains approximately 50% of its catalase-peroxidase activity. This altered catalase-peroxidase provides high-level resistance to INH, while retaining a level of oxidative protection against host antibacterial radicals.  Isolates that carry other, mutations in  katG  are exhibiting varying levels of INH-resistance and catalase-peroxidase activity. MTB Phagosome with viable MTB Lysosomes  with free oxigene radicals Phago-lysosomes with inactivated MTB TBPANNET workpackage 6  midterm meeting
Rifampin resistance RMP exhibits a significant early bactericidal effect on metabolically active  M tuberculosis , and excellent late sterilizing action on semidormant organisms undergoing short bursts of metabolic activity.  RMP inhibits mycobacterial transcription by targeting DNA-dependent RNA polymerase.  Frequency of spontaneous mutation to RMP is low, monoresistance is rare TBPANNET workpackage 6  midterm meeting
RIF resistance as surrogate marker for MDR TB RIF resistance as a mono resistance not very frequent (5-15% of them) 80-95% of RIF resistant strains are also resistant to INH Khue  et al.  A 10-year prospective surveillance of  Mycobacterium tuberculosis drug resistance in France 1995–2004 . Eur  Respiratory J. 2007 PAGE  |   TBPANNET workpackage 6  midterm meeting
The available commercial tests LiPA for Rif, INH LiPA for FQ IA  XPERT-TB/MDR TBPANNET workpackage 6  midterm meeting
Commercial Line Probe Assays Hain Lifescience Innogenetics INNO-LiPA-Rif.TB
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
Heteroresistance in  M. tuberculosis   Are molecular tests able to detect small fraction of mutated bacteria? The study showed that line probe assays underdiagnose drug resistance and may detect resistance only if >1% resistant bacteria are present (MTBDRplus) or if >10% resistant bacteria are present (InnoLipa Rif TB)
New generation of LiPA performs better in both Sm+ and Sm- samples Miotto al. JCM 2008 TBPANNET workpackage 6  midterm meeting
3 rd  generation of LiPA (MDR-Plus V2) will be available shortly Test is under validation on smear negative and scanty samples. Main advantages: all included in master mix Master mix stable at wide –range Clearer interpretation pattern TBPANNET workpackage 6  midterm meeting
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
Possible automation on LiPA   LiPAs require: Level II biosafety areas Skilled laboratory staff Amplicon Contamination control
Xpert MTB/RIF works on the  Gene Xpert System TB What else!
Boehme C, N Engl J Medicine, 2010 Assay procedure for MTB/RIF test
Moving to  “ district” : Dharamsala,  Delek Hospital
Proportion of TB cases detected  Time to detection Time to RFP resistance detection Advantages of fully automated real-time test
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
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
WHO recommendations (December 8 th , 2010) Strong recommendation  the new automated DNA test for TB should be used as the initial diagnostic test in individuals suspected of MDR-TB  or HIV/TB Conditional recommendation the new  automated DNA test may be used as a follow-on test to microscopy in settings where MDR-TB and or HIV is of lesser concern, especially in  smear-negative specimens (recognising major resource implications) Expected impact a three-fold increase in the diagnosis of  patients with MDR-TB a doubling in the number of TB/HIV cases diagnosed in areas with high rates of TB and HIV  (compared to microscopy diagnosis)
Currently recommended TB tests fit into the following tiered system:
 
Proportion of TB cases detected  time to detection Time to RFP resistance detection XPERT-TB/MDR TBPANNET workpackage 6  midterm meeting
Advantages and limits of Xpert MTB/RIF Simple to perform Minimal training Virtually cross contamination free Minimal biosafety requirement MTB and RIF resistance detection within 2 hours High sensitivity also in paucibacillary samples (HIV+) High performance in extrapulmonary samples Unknown the performance in children Few data in low-prevalence populations No data on INH res and other drugs
Genes involved in drug-resistance for major anti-tubercular drugs 40-80%
XDR molecular diagnosis: GenoType MTBDR sl  (Hain Lifescience)
 
Common problems in interpreting molecular results Discrepancies genotype/phenotype: rpoB  absence of WT8 (codo530-533) and absence of rpoB MUT3 inhA - 15 alone False negative due to duplication Double pattern Further research is needed to clarify the clinical  role of some mutation or mix infections
A common problem: the “double pattern” Hetero-resistance  =  equal representation of susceptible and resistant mutants of the same strain Mixed pattern  =  mutual presence of a resistant strain and a second, susceptible strain Not pure culture  Carry-over contamination
Common problems in interpreting molecular results: INH, E inhA - 15 alone: increased mic, needs to follow closely over time Ethambutol :  is therapy modified based on resistance data? Correlation of E 306 mut to phenotype and mic: Recent published and unpublished data have suggested correlation between increased mic and mutation TBPANNET workpackage 6  midterm meeting
TBPANNET workpackage 6  midterm meeting Codons analysed: PMA is a membrane impermeant that intercalates into both extracellular DNA and DNA in nonviable cells and is excluded from viable bacteria. Exposure to a light source renders PMA-DNA incapable of contributing to PCR. Method developed for microbial food pathogens and already tested on environmental samples. Propidium Monoazide TM  (PMA)
TBPANNET workpackage 6  midterm meeting PMA pretreatment of samples allows  selective amplification of DNA from live bacteria Miotto 2011 under revision ERJ
Molecular DST
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
Limitations How discriminatory are the 24 loci? Is the future in whole genome sequencing? Should we be including strains in clusters with missing loci? Patients could be included in more than one cluster 42244 42234 -22-4 422-4 422— Patients can switch clusters or drop out if loci are re-typed Investigating false clusters results in wasted resources and may indicate that the service is not effective How many missing loci are acceptable and does this provide an indication of the quality of the method? EQA offered by the ECDC to one “reference laboratory for each country. Results show high variability and little reproducibility and high need of training
Role of IGRAs and serological tests for active TB diagnosis What’s available What will be available
Challenges Identification of people with significant Mtb contact Calculation of the risk to develop active Tb Discrimination between active and latent Tb Test with the following properties High sensitivity High specificity Fast Easy (material, performance,…) Low cost
1.) Serological tests Not (yet) sufficient for Tb-diagnosis Lancet, 377:113-114, 2011:   „ WHO recommends against inaccurate tuberculosis tests “ !!! Pro Contra easy fast out of serum samples low cost low sensitivity low specificity
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
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
Accuracy of IGRA: sensitivity and specificity
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
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
Recent developments and perspectives Antigen different from the commercial RD1 peptides  (RD1 selected peptides, antigens of latency, Rv2628, HBHA) Use of extrasanguinous fluids   Broncheoalveolar fluid  Pleural effusions  Cerebrospinal fluid Intracellular cytokine staining combined with flow-cytometric analysis Characterisation of cytokine profiles and use of aTb-specific profile changes New skin tests using recombinant ESAT-6 and CFP-10 Analysis of alternative read-out parameters IP-10, MCP-2 TNF-  … ???
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
TBNET report Goletti et al, PLoS ONE 2008
IGRA at the site of TB disease: BAL vs blood From Jafari, AJRCCM 2009
IGRA at the site of TB disease: Pleural fluid vs blood Berlin, October 4th, 2008 From Losi et al, ERJ 2007 PLEURAL CELLS PBMC
Enumerate the proportion of IFN-γ, IL-2 and TNF-α single, di and tri-producing cells in response to ESAT6 and CFP-10 Single positive TNF-α producing CD4+ T-cells in response to CFP-10 discriminate active from latent TB with a sensitivity of 67% & specificity 80% Enumerating subpopulations using flow cytometry Harari A, Nature Med 2011
Predictive value of IGRA: HIV-negative  subjects Diel et al, AJRCCM 2010 in press
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
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
Efficacy of therapy based on IGRA results: NO DATA AVAILABLE
Trends for the future  IGRA on a chip Skannex.com
Conclusion Appropriate lab data guided TB therapy is often still a challenge New diagnostics and supporting policies are available, globally the next major step is translation of policy into practice.  The impact of new tests will depend: Availability of new generation of tests covering more targets on the extent of their introduction and capacity to interpret the results and translate results into clinical practice Use of the test for more than diagnosis alone TBPANNET workpackage 6  midterm meeting
Thank you! Acknowledgments

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  • 2. Hot topic in the international research agenda for TB Drug development Diagnosis and diagnostic tests Epidemiology and public health Health services research Basic science research HIV MDR M. tuberculosis vaccines Rylance et al, TLID 2010
  • 3. The key role of the laboratory Care of TB patients starts with a QA diagnosis A robust network of Tb laboratories is required: Adaquate biosafety Modern diagnostics SOPs QAs Integrated laboratory network
  • 4. Conventional TB diagnostic methods Slow and cumbersome Require sequential procedures for isolation, identification and in vitro sensitivity testing (DST) Require appropriate laboratory facilities and trained staff (AFB) Smear test: Insensitive, WHO and national guidelines recommend 2 to 3 smear tests to be performed by suspected patient Requires skilled technician Time consuming: 1-2 hours Culture Highly sensitive Slow: 3 to 6 weeks Nucleic Acid Amplification methods: Highly specific Not optimal on clinical samples: Highly sensitive on smear positive samples (95-100%) Until now insufficient sensitivity on smear negative samples (60-75%) Very sensitive on culture isolates: For identification For Rif susceptibility testing
  • 5. New Policy and Smear microscopy definition of a TB case New definition in 2007*: “ person with al least one smear-positive sample (1 AFB is sufficient) out of a total of two examined” 2 samples regardless the collection time *The definition/policy can be applied to countries performing microscopy under satisfactory quality assurance programmes
  • 6. LED Fluorescence Microscopy Advantages : increase in performance increase in lamp lifetime reduces initial, operating and maintenance costs No need for dark room
  • 7. WHO recommendations on sputum smear microscopy (2010) ZN light microscopy performed on UNCONCENTRATED sputum is suitable for all laboratory service levels Concentration of sputum is NOT recommended in programmatic settings Fluorescence microscopy is recommended for increased sensitivity (add 10%) LED microscopy is recommended over conventional fluorescence
  • 8. TB Culture* Advantages Definitive diagnosis of TB Increases case finding of 30-50% Early detection of cases Provide strains for DST and epidemiological studies Disadvantages Complex and expensive compared to microscopy Requires complex handling of specimens Skilled technicians Appropriate infrastructure and biosafety levels LIMITATIONS: need for decontamination and identification *coverage 500.000/1000000 Strip speciation tests needed for fast ID of Tbcomplex Molecular test for speciation of most common mycobacteria
  • 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
  • 10. Development of multi-drug resistant tuberculosis No single genetic alteration is responsible for the MDR phenotype (defined as resistance at least to INH and RMP). MDR develops by sequential acquisition and selection of mutations at different loci, usually because of inappropriate patient treatment. Inappropriate treatment may lead to an increase of bacillary load and increase od spontaneous mutations while the disease progresses. Because MDR strains are the result of cumulative mutations, growth of M tuberculosis needs to be controlled in the host by concomitant treatment with more than one active drug. Thus, TB treatment regimens that consist of four ACTIVE drugs TBPANNET workpackage 6 midterm meeting
  • 11. INH = 1 in 10 6 RIF = 1 in 10 8 EMB = 1 in 10 6 Str = 1 in 10 6 I + R = 1 in 10 14 Frequenza delle mutazioni ai farmaci di prima linea
  • 12. DST Phenotype based Genotype based TBPANNET workpackage 6 midterm meeting
  • 13. Phenotypic DST TBPANNET workpackage 6 midterm meeting
  • 14. WHO laboratory policies for culture and DST Automated liquid culture (2007) Use of liquid culture in the contest of comprehensive plan for strengthening lab capacity starting with national/central level Rapid speciation in combination with culture Second line DST (2008) to be conducted for selected drugs a central level Available at http://www.who.int/tb.dots/laboratory/policy/en/print.html
  • 15. Standardization of procedures still difficult to set up The correct performance of DST requires the understanding of several steps such as: Origin of resistance and interpretative criteria Dosage and stability of the incorporated compounds Anti-mycobacterial activity of the incorporate drug Interpretation of results and data reporting TBPANNET workpackage 6 midterm meeting Drug Susceptibility Testing (DST): the technical challenge still persists
  • 16. Determination of clinically significant proportion of resistant bacteria Clinically significant proportion In vitro growth of resistant cells in the presence of the critical concentration of the drug that is equal to or greater than 1% of the growth of the total population in the absence of the drug. The presence of 1% resistant cells represents a significant increase in resistance; laboratory assays are based on this assumption. Are molecular tests able to detect 1% mutant subopulation? TBPANNET workpackage 6 midterm meeting
  • 17. Second line DST Recommended in appropriate structures for: Aminoglycosides, Fluoroquinolones, Polypeptides NOT recommended on routine for ethionamide, prothionamide, cycloserine,clofazimine, amox-clav, clarithromycin, linezolid)
  • 18. TBPANNET workpackage 6 midterm meeting Cost-effectiveness Only available for selected drugs Only available for selected specimens Low sensitivity in AFB-negative and non-respiratory samples Genetic diversity may influence statistical parameters of molecular tests Based on single nucleotide mutations detection Data on DST from specimens not suitable for culture Cross-resistance prediction Large number of specimens analyzed at the same time Standardization (automated systems) and TAT MOLECULAR DST ON M. tuberculosis
  • 19. Genes involved in drug-resistance for major anti-tubercular drugs TBPANNET workpackage 6 midterm meeting
  • 20. Isoniazid resistance and katG INH is a pro-drug that requires activation in INH-susceptible mycobacterial species. the activation of INH results in a number of highly reactive compound that are capable of damaging the mycobacterial cell wall. INH-resistant clinical isolates frequently loose their catalase-peroxidase activity (Middlebrook et al., 1954) Association of this enzyme with INH activation was proven when the mycobacterial catalase-peroxidase gene ( katG ) was cloned and sequenced. (Zhang et al., 1992) The Ser315Thr mutation is found in 70-80% of high INH-resistant clinical isolates. TBPANNET workpackage 6 midterm meeting Adapted from GLI training material
  • 21. Isoniazid resistance and inhA INH blocks the synthesis of cell-wall mycolic acids, the major components of the envelope of M tuberculosis . One intracellular target of the drug is fatty-acid enoyl-acyl carrier protein reductase (InhA) . (Basso et al., 1998) Mutations in the promoter region of inhA result in over-expression of the protein. The over-expressed enzyme may counter-balance the effect of INH and will result in a low-level resistance to the drug. TBPANNET workpackage 6 midterm meeting
  • 22. Isoniazid resistance and virulence The Ser315Thr mutation results in an enzyme without the ability to activate INH, but retains approximately 50% of its catalase-peroxidase activity. This altered catalase-peroxidase provides high-level resistance to INH, while retaining a level of oxidative protection against host antibacterial radicals. Isolates that carry other, mutations in katG are exhibiting varying levels of INH-resistance and catalase-peroxidase activity. MTB Phagosome with viable MTB Lysosomes with free oxigene radicals Phago-lysosomes with inactivated MTB TBPANNET workpackage 6 midterm meeting
  • 23. Rifampin resistance RMP exhibits a significant early bactericidal effect on metabolically active M tuberculosis , and excellent late sterilizing action on semidormant organisms undergoing short bursts of metabolic activity. RMP inhibits mycobacterial transcription by targeting DNA-dependent RNA polymerase. Frequency of spontaneous mutation to RMP is low, monoresistance is rare TBPANNET workpackage 6 midterm meeting
  • 24. RIF resistance as surrogate marker for MDR TB RIF resistance as a mono resistance not very frequent (5-15% of them) 80-95% of RIF resistant strains are also resistant to INH Khue et al. A 10-year prospective surveillance of Mycobacterium tuberculosis drug resistance in France 1995–2004 . Eur Respiratory J. 2007 PAGE | TBPANNET workpackage 6 midterm meeting
  • 25. The available commercial tests LiPA for Rif, INH LiPA for FQ IA XPERT-TB/MDR 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
  • 28. Heteroresistance in M. tuberculosis Are molecular tests able to detect small fraction of mutated bacteria? The study showed that line probe assays underdiagnose drug resistance and may detect resistance only if >1% resistant bacteria are present (MTBDRplus) or if >10% resistant bacteria are present (InnoLipa Rif TB)
  • 29. New generation of LiPA performs better in both Sm+ and Sm- samples Miotto al. JCM 2008 TBPANNET workpackage 6 midterm meeting
  • 30. 3 rd generation of LiPA (MDR-Plus V2) will be available shortly Test is under validation on smear negative and scanty samples. Main advantages: all included in master mix Master mix stable at wide –range Clearer interpretation pattern 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
  • 39. WHO recommendations (December 8 th , 2010) Strong recommendation the new automated DNA test for TB should be used as the initial diagnostic test in individuals suspected of MDR-TB or HIV/TB Conditional recommendation the new automated DNA test may be used as a follow-on test to microscopy in settings where MDR-TB and or HIV is of lesser concern, especially in smear-negative specimens (recognising major resource implications) Expected impact a three-fold increase in the diagnosis of patients with MDR-TB a doubling in the number of TB/HIV cases diagnosed in areas with high rates of TB and HIV (compared to microscopy diagnosis)
  • 40. Currently recommended TB tests fit into the following tiered system:
  • 41.  
  • 42. Proportion of TB cases detected time to detection Time to RFP resistance detection XPERT-TB/MDR TBPANNET workpackage 6 midterm meeting
  • 43. Advantages and limits of Xpert MTB/RIF Simple to perform Minimal training Virtually cross contamination free Minimal biosafety requirement MTB and RIF resistance detection within 2 hours High sensitivity also in paucibacillary samples (HIV+) High performance in extrapulmonary samples Unknown the performance in children Few data in low-prevalence populations No data on INH res and other drugs
  • 44. Genes involved in drug-resistance for major anti-tubercular drugs 40-80%
  • 45. XDR molecular diagnosis: GenoType MTBDR sl (Hain Lifescience)
  • 46.  
  • 47. Common problems in interpreting molecular results Discrepancies genotype/phenotype: rpoB absence of WT8 (codo530-533) and absence of rpoB MUT3 inhA - 15 alone False negative due to duplication Double pattern Further research is needed to clarify the clinical role of some mutation or mix infections
  • 48. A common problem: the “double pattern” Hetero-resistance = equal representation of susceptible and resistant mutants of the same strain Mixed pattern = mutual presence of a resistant strain and a second, susceptible strain Not pure culture Carry-over contamination
  • 49. Common problems in interpreting molecular results: INH, E inhA - 15 alone: increased mic, needs to follow closely over time Ethambutol : is therapy modified based on resistance data? Correlation of E 306 mut to phenotype and mic: Recent published and unpublished data have suggested correlation between increased mic and mutation TBPANNET workpackage 6 midterm meeting
  • 50. TBPANNET workpackage 6 midterm meeting Codons analysed: PMA is a membrane impermeant that intercalates into both extracellular DNA and DNA in nonviable cells and is excluded from viable bacteria. Exposure to a light source renders PMA-DNA incapable of contributing to PCR. Method developed for microbial food pathogens and already tested on environmental samples. Propidium Monoazide TM (PMA)
  • 51. TBPANNET workpackage 6 midterm meeting PMA pretreatment of samples allows selective amplification of DNA from live bacteria Miotto 2011 under revision ERJ
  • 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
  • 54. Limitations How discriminatory are the 24 loci? Is the future in whole genome sequencing? Should we be including strains in clusters with missing loci? Patients could be included in more than one cluster 42244 42234 -22-4 422-4 422— Patients can switch clusters or drop out if loci are re-typed Investigating false clusters results in wasted resources and may indicate that the service is not effective How many missing loci are acceptable and does this provide an indication of the quality of the method? EQA offered by the ECDC to one “reference laboratory for each country. Results show high variability and little reproducibility and high need of training
  • 55. Role of IGRAs and serological tests for active TB diagnosis What’s available What will be available
  • 56. Challenges Identification of people with significant Mtb contact Calculation of the risk to develop active Tb Discrimination between active and latent Tb Test with the following properties High sensitivity High specificity Fast Easy (material, performance,…) Low cost
  • 57. 1.) Serological tests Not (yet) sufficient for Tb-diagnosis Lancet, 377:113-114, 2011: „ WHO recommends against inaccurate tuberculosis tests “ !!! Pro Contra easy fast out of serum samples low cost low sensitivity low specificity
  • 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
  • 63. Recent developments and perspectives Antigen different from the commercial RD1 peptides (RD1 selected peptides, antigens of latency, Rv2628, HBHA) Use of extrasanguinous fluids Broncheoalveolar fluid Pleural effusions Cerebrospinal fluid Intracellular cytokine staining combined with flow-cytometric analysis Characterisation of cytokine profiles and use of aTb-specific profile changes New skin tests using recombinant ESAT-6 and CFP-10 Analysis of alternative read-out parameters IP-10, MCP-2 TNF-  … ???
  • 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
  • 68. Enumerate the proportion of IFN-γ, IL-2 and TNF-α single, di and tri-producing cells in response to ESAT6 and CFP-10 Single positive TNF-α producing CD4+ T-cells in response to CFP-10 discriminate active from latent TB with a sensitivity of 67% & specificity 80% Enumerating subpopulations using flow cytometry Harari A, Nature Med 2011
  • 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
  • 74. Conclusion Appropriate lab data guided TB therapy is often still a challenge New diagnostics and supporting policies are available, globally the next major step is translation of policy into practice. The impact of new tests will depend: Availability of new generation of tests covering more targets on the extent of their introduction and capacity to interpret the results and translate results into clinical practice Use of the test for more than diagnosis alone TBPANNET workpackage 6 midterm meeting

Editor's Notes

  1. 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.
  2. 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.
  3. 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]
  4. 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.
  5. 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.
  6. 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.
  7. 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.
  8. 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
  9. 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.
  10. 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