TUBERCOLOSI:UNA	  MALATTIA	  SOCIALE	        21-­‐22	  SETTEMBRE	  2012	        Ia	  tappa:	  da	  Kock	  a	  Xpert	      ...
Outline	  •    Urgent	  need	  for	  an	  improved	  diagnosis	  •    Microscopy	  •    Culture	  and	  DST	  needs	  for	...
Urgent	  need	  for	  new	  diagnosGcs	  •  TB	  case	  detec/on	  gaps:	  	      –  cases	  undiagnosed	          •  Inac...
Microscopy	  :	  a	  century	  old	  procedure	           Rapid test          Inexpensive n   Does not allow species iden...
TB	  Culture	  	  Advantages                             Disadvantages•  Definitive diagnosis of TB          •  Complex an...
Culture:	  solid/	  liquid	  	  solid                                 liquid•  Low cost for reagents, not         •    Com...
M. tuberculosis identification	  Morphology/         Molecular	                   Immuno-­‐	  Biochemical	        tests	  ...
DST	  •  Definitive diagnosis of DRTB                           3	  main	  methods	        Absolute	                      ...
DST:	  Liquid/solid	  mtedia	  comparison	   Liquid	  media	  compared	   o	  solid	  media	              Advantages	  com...
SENSITIVITY	  OF	  NEW	  DIAGNOSTICS	                                                                     Fluorescent/    ...
Is	  new	  real-­‐Gme	  technology	  improving	  the	  sensiGvity	  of	           molecular	  tests?	  
New	  generaGon	  of	  tests	  does	  not	            improve	  sensiGvity	  Tortoli	  et	  al	  2012	  JCM	  Methods	  Am...
Are	  molecular	  tests	  tools	  for	  difficult	             to	  diagnose	  cases?	   •  TB	  in	  Children	   •  Extrapu...
Xpert	  TB/RIF:	  Performance	  in	  children	  
Extrapulmonary	  Tuberculosis:	         Xpert	  TB/(Rif)	                            Tortoli	  et	  al	  ERJ	  2012	  
Diagnosis	  in	  difficult	  cases	  •  Children	  diagnosis	  is	  not	  microbiologically	     confirmed	  in	  40-­‐60%	  ...
Unsolved	  Problems	  of	  Molecular	    Tests	  for	  Direct	  Diagnosis	  of	               Tuberculosis	  	  •  Subop/m...
Conventional DST: technical challenges•    Adequate	  infrastructures	  and	  biosefety	  levels	  •    MGIT	  DST:	  the	...
MECHANISMS	  OF	  DRUG	  RESISTANCE	  IN	           M.	  tuberculosis	                Zhang	  Y	  and	  Yew	  W,	  Int	  J...
Commercial	  Molecular	  tests	  for	  e	  Tdopted	  in	  WHO	  Global	  plan	  (2006-­‐2015):development	  and	  roll	  o...
LPA	  performance	  i(n	  isolates	  and	  clinical	              LPAs:	  performances	   *based	  on	  2°	  generaGon)	  ...
TAT	  to	  Rif	  –R	  detecGon	  and	  reporGng	                                       RIF-­‐R	  detecGon	                ...
What	  can	  be	  achieved	  or	  parGally	  achieve	     with	  the	  current	  molecular	  tools	    •  Diagnosis	  of	 ...
DR	  tesGng:	  Rifampicin	                         Drobniewski	  et	  al	  
DR	  tesGng:	  Rifampicin	                                                 Bactericidal	  an/bio/c	  that	                ...
ISONIAZID	                                                       ISONIAZID	                                          INH:	...
PPV	  and	  NPV	  for	  Rif	  resistance	  at	  different	  prevalence	  of	  Rif	  resistance	                            ...
Discrepancies	  with	  MGIT	  DST	  •  In vitro growth of Rif sensitive strains from   samples identified as Rif res or MD...
Heteroresistance	  Real	  heteroresistance	                   False	  heteroresistance	  •  Co-­‐presence	  of	  mutated	 ...
Role	  of	  point	  mutaGons	  in	  predicGng	   clinical	  outcome:	  embB	  Codon	  306	  •  Low	  sensi/vity	  of	  emb...
Commercial	  tests	  for	  Fluoroquinolones	  and	  injectables	  •  Commercial	  LPA	  tests	  	  for	  detec/on	  of	   ...
Molecular	  test	  performances	  should	  be	      evaluated	  in	  the	  epidemiological	  contests	  •  PPV	  and	  NPV...
Role	  of	  Molecular	  typing	                     •    To	  iden/fy	  epidemiological	  links	  between	  TB	           ...
Needs	  for	  berer	  tools	  Among	  3037	  pa/ents	  with	  new	  cases	  of	  tuberculosis	  and	  892	  with	  previou...
Lab-­‐on	  Chip	  for	  molecular	  diagnosGcs	  PCR:•  Ultra-Fast PCR•  Asymmetric Cy-5 PCR strategyMicroarray:•  Orienta...
Open	  Issues	  •  How	  to	  monitor	  the	  response	  to	  therapy?	       –  Sputum	  smear	  is	  s/ll	  guiding	  de...
New	  diagnosGcs?	  Nanopore	  USB	  sequencing	  
AcknowledgmentsEmanuele	  Borroni	  Andrea	  M.	  Cabibbe	  Irene	  Festoso	  Paola	  Mantegani	  Paolo	  Mioro	  Luca	  N...
PPT Cirillo "I Stage: from Koch to Xpert"
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PPT Cirillo "I Stage: from Koch to Xpert"

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PPT Cirillo "I Stage: from Koch to Xpert"

  1. 1. TUBERCOLOSI:UNA  MALATTIA  SOCIALE   21-­‐22  SETTEMBRE  2012   Ia  tappa:  da  Kock  a  Xpert   D  Cirillo   San  Raffaele  Scien/fic  Ins/tute   Milan  Italy  
  2. 2. Outline  •  Urgent  need  for  an  improved  diagnosis  •  Microscopy  •  Culture  and  DST  needs  for  standards  and  EQAs  •  Molecular  diagnosis  is  a  reality  •  What  can  be  achieved  with  current  molecular   tools  •  What  cannot  be  achieved  with  the  current   molecular  tools  •  Global  prospec/ve    
  3. 3. Urgent  need  for  new  diagnosGcs  •  TB  case  detec/on  gaps:     –  cases  undiagnosed   •  Inaccessible  facili/es   •  Not  self  repor/ng,  not  returning     •  Wrong  diagnosis   –  Cases  diagnosed  in  private  care  and  not  reported  •  Infec/on  control  (Stop  TB/MDR-­‐TB   transmission)  •  Guide  treatment  •  Monitoring  treatment    
  4. 4. Microscopy  :  a  century  old  procedure   Rapid test Inexpensive n  Does not allow species identification n Not applicable to all samplesn  Specificity for Mycobacterium spp:>95% Sensitivity: 25-65% (90 % of higlyn infectious cases) Fluorescence Ziehl-Neelsen staining Positive Predictive Value for TBn  1st AFB smear 80-82 %depends on epidemiological situation 2nd AFB smear 10-14 % LED  microscopy  recommended   over  light    and  fluorescent   3rd AFB smear 5-8 % microscopy  
  5. 5. TB  Culture    Advantages Disadvantages•  Definitive diagnosis of TB •  Complex and expensive compared to microscopy•  Increases case finding of •  Requires complex handling of 30-50% specimens•  Early detection of cases •  Skilled technicians•  Provide strains for DST •  Appropriate infrastructure and biosafety levels and epidemiological studies LIMITATIONS: need for decontamination and identification*coverage 500.000/1000000
  6. 6. Culture:  solid/  liquid    solid liquid•  Low cost for reagents, not •  Complex and expensive can be automated automated (MGIT) •  Highest infrastructure and•  Culture level infrastructure biosafety levels•  Low contamination rate •  Case finding increased 10% over•  Long time to positivity solid•  Colony morphology •  Diagnostic delay reduced to days•  ID required •  ID required •  DST only for selected drugs•  DST only for selected drugs Strip speciation tests for fast ID of Tbcomplex Molecular test for speciation of most common mycobacteria
  7. 7. M. tuberculosis identification  Morphology/ Molecular   Immuno-­‐  Biochemical   tests   cromatographic   tests   LPAs   test     Probes  on  liquid   phase   Sequencing   EQA  and  standardiza/on   Spoligotyping   Enzyme   7 restric/on  
  8. 8. DST  •  Definitive diagnosis of DRTB 3  main  methods   Absolute   Propor/on   concentra/on   Resistant  Ra/o   methods   method   method    
  9. 9. DST:  Liquid/solid  mtedia  comparison   Liquid  media  compared   o  solid  media   Advantages  compared  to  solid  media:   •   more  rapid   •   high  quality  of  media   •   fully  automated  system   •   tes/ng  of  1st,  2nd  ,  and  new  drugs  (Linezolid)   •   safety:  plas/c  tubes   •   pyrazinamide  sensi/vity  test     Break  points  for  2nd  line  drugs   Disadvantages:   recently  revised   •   expensive   S/ll  poor  correla/on  with  clinical   •   higher  contamina/on  rate   outcome,  some  tes/ng  not  fully   •   dependency  on  a  company   reliable   •   no  DST  for  Cycloserine        
  10. 10. SENSITIVITY  OF  NEW  DIAGNOSTICS   Fluorescent/ LEDmicroscopy   Immunochrom.f LAMP-­‐TB     5,000-­‐10,000/ml   or  specia/on   100-­‐1000/ml   1,000,000/ml   Solid  Culture   Line-­‐Probe  Assay     1000-­‐10,000/ml   100-­‐1000/ml   Automated   NAAT    Liquid  Culture   100-­‐150/ml   10-­‐100/ml   0 1 2 3 4 5 6 Log  cfu/ml  
  11. 11. Is  new  real-­‐Gme  technology  improving  the  sensiGvity  of   molecular  tests?  
  12. 12. New  generaGon  of  tests  does  not   improve  sensiGvity  Tortoli  et  al  2012  JCM  Methods  Amplicor  n  on  NAs  amples  Taqman  vs   based  oon  15000  s detec/on  have  intrinsic  limita/ons:  •  Absence  on  specificity  ubop/mal  specimen  selec/on,  quan/ty,  Improvement  i f  target:  s quality,..  Decrease  in  invalid  results  Improvement  in  post  test  probability  •  Subop/mal  sample  prepara/on      
  13. 13. Are  molecular  tests  tools  for  difficult   to  diagnose  cases?   •  TB  in  Children   •  Extrapulmonary  Tuberculosis  
  14. 14. Xpert  TB/RIF:  Performance  in  children  
  15. 15. Extrapulmonary  Tuberculosis:   Xpert  TB/(Rif)   Tortoli  et  al  ERJ  2012  
  16. 16. Diagnosis  in  difficult  cases  •  Children  diagnosis  is  not  microbiologically   confirmed  in  40-­‐60%  of  cases,  current   molecular  tools  s/ll  subop/mal.  A  host/ pathogens  biomarkers  approach  is  probably   needed  •  Extrapulmonary  TB:  the  performance  of   molecular  tools  varies  and  should  be   considered  separately  for  each  specific   specimen  type.    
  17. 17. Unsolved  Problems  of  Molecular   Tests  for  Direct  Diagnosis  of   Tuberculosis    •  Subop/mal  sensi/vity/specificity  versus  a  gold   standard:   –  Compared  to  culture  in  liquid  media   –  Compared  to  a  combined  standard  based  on  the   “inten/on  to  treat”/  response  to  treatment     Decreased   confidence  in  the   test  
  18. 18. Conventional DST: technical challenges•  Adequate  infrastructures  and  biosefety  levels  •  MGIT  DST:  the  gold  standard  •  MDRTB  :  3-­‐6  weeks;  XDRTB  :  6-­‐9  weeks  •  Reproducibility  and  accuracy  of  results  are  drugs  dependent:   –  Rifampicin,  isoniazid  :  good  results   –  Second-­‐line:  fluoroquinolones  and  injectables   CorrelaGon  of  sensiGvity  test   results  and  clinical  outcome  is   difficult  to  evaluate  and  we  have   very  limited  or  no  evidence    for   Pyr,E,  and  2nd  line  drugs  other   than  INJ  and  FQs  on  MDR  cases     In  addi/on:     Capital  cost  of  facili/es   Cost  of  maintenance   Cost  of  staff         Van  Deun  A.  et  al  2011.  IJTLD  15(1):116-­‐124  
  19. 19. MECHANISMS  OF  DRUG  RESISTANCE  IN   M.  tuberculosis   Zhang  Y  and  Yew  W,  Int  J  Tuberc  Lung  Dis  2009  
  20. 20. Commercial  Molecular  tests  for  e  Tdopted  in  WHO  Global  plan  (2006-­‐2015):development  and  roll  out  of  new  technologies  to  b a B/MDR  TB  detecGon  endorsed  by  WHO   resources-­‐limited  senngs   GenoType  MTBDRplus,  InnoLiPA  Rif.TB   • Reverse  hybridiza/on,  colorimetric  reac/on   • Results  in  6-­‐7  h   •   some  flexibility  (n  probes/strip:  30-­‐40)   •   Technical  exper/se:  some   • Biosafety  lev  2   Xpert  MTB/RIF     • Integrated/automated  qPCR   • Results  in  2h   • Closed  system  (limited  number  of  probes:   <10)   •   Technical  exper/se:  none  
  21. 21. LPA  performance  i(n  isolates  and  clinical   LPAs:  performances   *based  on  2°  generaGon)     Rifampicin   samples   Isoniazid   Inno-­‐LiPA  Rif.TB   GenoType  MTBDRplus*   GenoType  MTBDRplus  Hot-­‐spot  rpoB  gene   cod.  315  katG  gene   nt  -­‐8,-­‐15,-­‐16  inhA  gene   Morgan  M  et  al  2005.  BMC  Infect  Dis  5:62   Ling  DI  et  al  2008.  Eur  Respir  J  32:1165-­‐1174   Ling  DI  et  al  2008.  Eur  Respir  J  32:1165-­‐1174  Sensi.vity  95-­‐98%   Sensi.vity  95-­‐99%   Sensi.vity  82-­‐93%  Specificity  98-­‐100%   Specificity  97-­‐100%   Specificity  95-­‐100%     Decontaminated  clinical  specimens  (AFB-­‐posi?ve)   Dec.  clin.  spec.  (AFB-­‐pos)   Sensi.vity  95-­‐99%   Sensi.vity  72-­‐92%   Specificity  97-­‐99%   Specificity  96-­‐99%  
  22. 22. TAT  to  Rif  –R  detecGon  and  reporGng   RIF-­‐R  detecGon   Time  to  report  to  treatment  center  Boehme  CC  et  al  2011.  Lancet  377(9776):1495-­‐505  Xpert  MTB/RIF:  0-­‐1  d   Xpert  MTB/RIF:  0-­‐1  d          (Microscopy:  1-­‐2  d)  LPA:  10-­‐26  d*   LPA:  27-­‐53  d*  Culture  DST:  30-­‐124  d**   Culture  DST:  38-­‐102  d**  (culture:  42-­‐62  d)   Some  results  not  reported/lost  *  test  on  AFB-­‐pos  clinical  specimen  +  test  on  clinical  isolate  for  AFB-­‐neg  cases  **  DST  performed  by  MGIT  +  DST  performed  on  LJ  
  23. 23. What  can  be  achieved  or  parGally  achieve   with  the  current  molecular  tools   •  Diagnosis  of  TB,  rifampicin  resistant  TB,  MDR-­‐TB  in   smear  posi/ve/nega/ve  samples   •  Iden/fica/on  of  up  to  80%  fluoroquinolones  and  up   to  40-­‐80%  of  injectable  resistant  cases  among  MDR-­‐ TB  cases   •  Improvement  of  diagnosis  of  TB  in  extrapulmonary   samples   •  Support  to  diagnosis  of  TB  in  children   •  Diagnosis  of  NTMs  infec/on  
  24. 24. DR  tesGng:  Rifampicin   Drobniewski  et  al  
  25. 25. DR  tesGng:  Rifampicin   Bactericidal  an/bio/c  that   inhibits  the  bacterial  DNA-­‐ dependent  RNA  polymerase.     Target:  β-­‐subunit  of  the  RNA   polymerase  (encoded  by  rpoB),   blocking  elonga/on  of  the  RNA   chain.   codon  526/  531  high  level   resistance  to  rifampicin,   MutaGons  in  a  “hot-­‐spot”  region  of  81  bp  of   rifabu/n  e  rifapen/n   rpoB  gene  (Rifampin  resistance-­‐determining    516  e  522  high  PPV  for   region)  →  RIF  resistance  (>  95%)   rifabu/n  res        
  26. 26. ISONIAZID   ISONIAZID   INH:  targeGng  mycolic  acid  biosysthesis   MutaGons  in  KatG  gene  prevent  INH   acGvaGon  (cod.  315,  60-­‐90%)       MutaGons  in  the  direct  target  inhA   (inhA  belongs  to  the  family  of  short-­‐chain   dehydrogenases/reductases.  It  is  essenGal  in  MTB)     MutaGons  in  the  promoter  of  inhA   gene  leading  to  drug  tritraGon  (direct   target  over-­‐producGon)     Only  Kat  G  315  and  inhA     are  included  in  LPA    Ratan  A  et  al.  EID  1998  
  27. 27. PPV  and  NPV  for  Rif  resistance  at  different  prevalence  of  Rif  resistance   WHO/HTM/TB/2011.2    
  28. 28. Discrepancies  with  MGIT  DST  •  In vitro growth of Rif sensitive strains from samples identified as Rif res or MDR by LPA•  Few cases from strains tested by LPA•  rpoB absence of WT8 (codo530-533) and absence of rpoB MUT3•  Strains bearing the mutations grow slowly in liquid culture and are identified as Rif sensitive•  inhA mut strains sensitive to INH by MGIT DST Discordant  reports  are  confusing  for  pa/ents     Management  if  not  appropriately  explained    
  29. 29. Heteroresistance  Real  heteroresistance   False  heteroresistance  •  Co-­‐presence  of  mutated   •  Laboratory  cross   and  wild  type  popula/on   contamina/on  due  to  •  Two  different  strains     insufficient  control  of   amplicons   LPAs  can  detect  heteroresistance  from   clinical  strains:  clinical  role?  
  30. 30. Role  of  point  mutaGons  in  predicGng   clinical  outcome:  embB  Codon  306  •  Low  sensi/vity  of  embB306  locus  for   predic/ng  Eth  Res  and  MDR-­‐TB  (35.5%),  High   specificity  for  iden/fying  MDR-­‐TB  (92.6%;   87/94).  The  posi/ve  predic/ve  value  (77.4%;   24/31)  and  the  nega/ve  predic/ve  value   (66.4%;  87/131)  of  this  locus  are  moderate.   Xin  Shen,  AAC2007  •  Plinke  et  al  AAC  2006,  Mioxo  et  al  ERJ  2012:   higher  PPV  for  ETH  resistance    
  31. 31. Commercial  tests  for  Fluoroquinolones  and  injectables  •  Commercial  LPA  tests    for  detec/on  of   resistance  to  second  line  drugs  show  a  high   PPV  and  a  low  NPV  due  to  the  limited  number   of  muta/ons  included  in  the  tests  •  PPV  and  NPV  may  vary  with  the  genotypic   background  of  the  strains  and/or  with  the   prevalence  of  specific  genotypes  in  the  target   popula/on   Mioxo  et  al.  ERJ  2012  
  32. 32. Molecular  test  performances  should  be   evaluated  in  the  epidemiological  contests  •  PPV  and  NPV  may  vary  in  different  genotypic   backgrounds  •  High  prevalence  of  specific  clones  bearing   selected  muta/ons  not  included  in  current   diagnos/c  assays  may  modify  PPV  and  NPV  
  33. 33. Role  of  Molecular  typing   •  To  iden/fy  epidemiological  links  between  TB   pa/ents  to  detect  and  control  outbreaks   early  and  rapidly   •  Rule  out  suspected  outbreaks  and  confirm   transmission  has  NOT  occurred   •  To  iden/fy  incorrect  TB  diagnosis  based  on   false-­‐posi/ve  cultures  and  thus  avoid   unnecessary  inves/ga/on  and  treatment   •  To  dis/nguish  exogenous  re-­‐infec/on  from   endogenous  reac/va/on  in  pa/ents  with  a   past  history  of  TB   •  Discover  unusual  transmission  senngs  and   transmission  between  different  regions   •  Monitor  the  size  of  clusters  and  thus  monitor   progress  towards  TB  elimina/on   •  Vaccine  and  DR  detec/on  implica/ons  
  34. 34. Needs  for  berer  tools  Among  3037  pa/ents  with  new  cases  of  tuberculosis  and  892  with  previously  treated  cases,  5.7%  (95%  confidence  interval  [CI],  4.5  to  7.0)  and  25.6%  (95%  CI,  21.5  to  29.8),  had  mul/drug-­‐resistant  (MDR)  tuberculosis.  Among  all  pa/ents  with  tuberculosis,  approximately  1  of  4  had  disease  that  was  resistant  to  isoniazid,  rifampin,  or  both,  and  1  of  10  had  MDR  tuberculosis.    Approximately  8%  of  the  pa/ents  with  MDR  tuberculosis  had  extensively  drug-­‐resistant  (XDR)  tuberculosis  
  35. 35. Lab-­‐on  Chip  for  molecular  diagnosGcs  PCR:•  Ultra-Fast PCR•  Asymmetric Cy-5 PCR strategyMicroarray:•  Orientation probes•  Hybridization Control probes•  Hybridization Negative Controls probes Current Lay out: ID of MTBC, relevant NTMs MDR phenotype   Lab-on-chip architecture 2 PCR reactors of 12.5 uL volume each (Total 25 ul) 1 Hybridization chamber of 30 uL All the reaction modules A 126 spots DNA microarray are fluidically integrated 2 in-let ports compatible with standard micro-pipettor tips Integrated Heaters and Sensors
  36. 36. Open  Issues  •  How  to  monitor  the  response  to  therapy?   –  Sputum  smear  is  s/ll  guiding  decisions  on  admission  and  discharge   –  Sputum  culture  is  s/ll  the  only  reliable  monitoring  tool  for  MDR   pa/ents  •  Pa/ents  with  H  monoresistance  may  go  undetected,  in  R  res   H  should  be  le{  un/l  proven  R?  •  Contact  treatment  in  absence  of  H  sensi/vity  data  •  Discrepancy  between  phenotypic  and  genotypic  results  •  Are  all  the  muta/ons  in  rpoB  equally  contribu/ng  to   resistance?  •  Muta/ons  to  key  second  line  drugs  and  cross  resistance  •  How  to  report  molecular  data  in  order  to  provide  clinical   guidance?  
  37. 37. New  diagnosGcs?  Nanopore  USB  sequencing  
  38. 38. AcknowledgmentsEmanuele  Borroni  Andrea  M.  Cabibbe  Irene  Festoso  Paola  Mantegani  Paolo  Mioro  Luca  Norbis  Enrico  Tortoli  Ilaria  Valente  Diego  Zallocco    Emerging  Bacterial  Pathogens  Unit  WHO  Suprana/onal  Reference  Laboratory  for  TB  Control  San  Raffaele  Scien/fic  Ins/tute  Milano,  ITALY  Fulvio  Salvo  and  Delek  Hospital  Staff  AISPO    Alberto  Mareelli  Alberto  Roggi  and  SRL/  NTP  Burkina  faso    Ins/tute  of  Infec/ous  and  Tropical  Diseases  University  of  Brescia  Brescia,  ITALY   Thanks for your attention! Lanfranco  Farorini   GB  Migliori   Is/tuto  Superiore  di  Sanità   FSM,Tradate   Roma,  ITALY  

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