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Antibodies in Lymphocyte supernatant for the Diagnosis & Management of TB in children

Antibodies in Lymphocyte supernatant for the Diagnosis & Management of TB in children. Tania Thomas, MD, MPH. Outline. Principles of ALS Methodology Performance in adults and children Performance as a biomarker Proposed study. Tuberculosis: global estimates.

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Antibodies in Lymphocyte supernatant for the Diagnosis & Management of TB in children

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  1. Antibodies in Lymphocyte supernatant for the Diagnosis & Management of TB in children Tania Thomas, MD, MPH

  2. Outline • Principles of ALS • Methodology • Performance in adults and children • Performance as a biomarker • Proposed study

  3. Tuberculosis: global estimates Proportional burden of world’s TB cases http://www.worldmapper.org/display.php?selected=228#WHO/HTM/TB/2009.411

  4. Multi-drug Resistant TB • >500,000 cases annually • New TB cases > Previously treated TB cases

  5. Antibodies in lymphocyte supernatant (ALS) • Hypothesis: • Active TB results in continuous antigen stimulation, resulting in antibody producing cells in circulation. • Diagnostic principles: • Measures antibody secretion from in vivo activated plasma cells that migrate into peripheral circulation in response to active TB. • B cell assay • Not a serological assay

  6. Methods: PBMC harvest and culture • Phlebotomy: 3.5 - 10mL venous blood • Isolate and wash PBMCs • More cells = better responses, minimum of 5x106 cells/mL • Suspend PBMCs in tissue culture media and culture un-stimulated x 48-72hrs in CO2 incubator

  7. Methods: ELISA • Supernatants added to BCG-coated wells, incubated for 2 hours • Measure BCG-specific antibodies by ELISA • Positive controls: pooled sera from M. tb culture-positive patients. • Negative controls: conjugate and substrate alone • Pediatric positive test >0.35 OD • Calculated by taking average ALS titer from healthy control children +3 standard deviations

  8. Coating antigens Rehka et al, PLoSOne Jan 2011

  9. Performance in adults from Bangladesh • 49 TB cases, 35 ill controls & 35 healthy controls • ALS (>0.42) compared to smear microscopy: • Sensitivity: 92.5% • Specificity: 80% • PPV: 97% Raqib et al, JID 2003

  10. Performance in children from Bangladesh • 58 TB cases, 58 ill controls & 16 healthy controls • Compared to expert clinical diagnosis: • 92% were positive by ALS • 64-67% were positive by score cards • ALS assay performance: • Sensitivity: 91%; Specificity: 87% • PPV: 96%; NPV: 74% Raqib et al, CVI 2009

  11. p < 0.001 1

  12. Performance as a biomarker • Objectives: evaluate role of ALS as a test to monitor response to therapy (biomarker) • Compare differences in ALS titers between children with DS-TB and DR-TB • n=9, culture confirmed (15%) • 5 with drug-susceptible-TB (DS-TB) • 4 with any drug resistance • 2 with MDR TB (INH/RMP) • 1 with resistance to INH, SM • 1 with resistance to INH, SM, EMB 1. Raqib et al, CVI 2009 2. Thomas et al, Thorax Jan 2011

  13. Thomas et al, Thorax Jan 2011

  14. ------ DS-TB, - - - DR-TB, - - - MDR-TB DS-TB: ALS titers declined significantly after two months of first-line anti-TB treatment (p=0.016). Black dashed line represents the threshold value for a positive test, 0.35 OD. Thomas et al, Thorax Jan 2011

  15. Summary of ALS • Performs well as a diagnostic test among children with TB. • May be useful as a biomarker • In this cohort, a lack of significant decline over time was associated with drug-resistant TB • Validation studies are needed in larger cohorts of children.

  16. Proposed study

  17. Definitions

  18. Settings • Haydom Lutheran Hospital • ~400 beds • ~525 TB cases/yr • 12-15% among children <14yrs • Kilimanjaro Clinical Research Institute (KCRI)/ Biotechnology Laboratory (BL) • Mycobacteriology testing (culture, DST) • ELISA tests for ALS

  19. Population • Children aged 6 mo – 14 yrs • Presenting with signs/symptoms of TB • Pulmonary TB, miliary TB, TB lymphadenitis, TB meningitis, TB of the spine • Exclude those who have received: • TB treatment >48 hrs • TST within preceding 8 weeks • BCG vaccine within preceding 8 weeks

  20. Clinical Procedures

  21. Laboratory Procedures • Sputum: • #1: ZN microscopy at HLH • #2: send to KCRI/BL • concentrated AFB smear (Auramine staining) • liquid culture & first-line DST (using MGIT-960) • ALS: • HLH: • Phlebotomy and isolation of > 5 million PBMCs • Culture PBMCs in BCG-lined wells x48h • Freeze supernatants • KCRI/BL: • Measure IgG by ELISA

  22. Estimated Sample Size • N=330 to be enrolled over ~26 months • Yielding ~100 TB cases • ~20 children with “poor response” as measured by persistently elevated ALS titers.

  23. Potential problems • Misclassification bias • Difficulties of not having a diagnostic “gold standard” • Feasibility • Large sample size needed • Inclusion of immunocompromised children • Affect on performance of B-cell assay • Performance

  24. Thank you • UVA • Eric Houpt • Kristine Peterson • Bill Petri • Becca Dillingham • Yan Ge • Jean Gratz • Scott Heysell • Suzanne Stroup • Bangladesh • RubhanaRaqib • Dinesh Mondal • SayeraBanu • TahmeedAhmed • Tanzania • Gibson Kibiki • Stella Mpagama • Charles Mtabho • Sister Kimaro • Happy Kumburu • AtanasiaMaro • Norah Ndusilo • Sweden • Susanna Brighenti

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