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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
http://www.worldmapper.org/display.php?selected=228#WHO/HTM/TB/2009.411
Proportional burden of world’s TB cases
Multi-drug Resistant TB
>500,000 cases annually New TB cases >
Previously treated TB cases
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
Table 1: Comparison of ALS to Serology
ALS Assay SerologyConcept Antibodies secreted
from circulating plasma B cells found
in PBMCs
Accumulated antibodies in serum
Clinical specimen used PBMCs Serum
Positive response in LTBI or prior TB disease?
No Yes
Positive response in children, HIV/TB co-infection or EPTB?
Yes Inconsistent
Positive response to prior BCG vaccination?
No Yes
Ability to monitor treatment response?
Yes Inconsistent
Affected by recent TST placement?
Yes, induces false-positive ALS if TST
placed within 2 months
Yes, variable effect
Table 1: Comparison of ALS to Serology
ALS Assay SerologyConcept Antibodies secreted
from circulating plasma B cells found in PBMCs
Accumulated antibodies in serum
Clinical specimen used PBMCs Serum
Positive response in LTBI or prior TB disease?
No Possibly
Positive response in children, HIV/TB co-infection or EPTB?
Yes Inconsistent
Positive response to prior BCG vaccination?
No Yes
Ability to monitor treatment response?
Yes Inconsistent
Affected by recent TST placement?
Yes, induces false-positive ALS if TST
placed within 2 months
Yes, variable effect
Table 1: Comparison of ALS to Serology
ALS Assay SerologyConcept Antibodies secreted
from circulating plasma B cells found
in PBMCs
Accumulated antibodies in serum
Clinical specimen used PBMCs Serum
Positive response in LTBI or prior TB disease?
No Possibly
Positive response in children, HIV/TB co-infection or EPTB?
Yes Inconsistent
Positive response to prior BCG vaccination?
No Yes
Ability to monitor treatment response?
Yes Inconsistent
Affected by recent TST placement?
Yes, induces false-positive ALS if TST
placed within 2 months
Yes, variable effect
Table 1: Comparison of ALS to Serology
ALS Assay SerologyConcept Antibodies secreted
from circulating plasma B cells found in PBMCs
Accumulated antibodies in serum
Clinical specimen used PBMCs Serum
Positive response in LTBI or prior TB disease?
No Possibly
Positive response in children, HIV/TB co-infection or EPTB?
Yes Inconsistent
Positive response to prior BCG vaccination?
No Yes
Ability to monitor treatment response?
Yes Inconsistent
Affected by recent TST placement?
Yes, induces false-positive ALS if TST
placed within 2 months
Yes, variable effect
Table 1: Comparison of ALS to Serology
ALS Assay SerologyConcept Antibodies secreted
from circulating plasma B cells found in PBMCs
Accumulated antibodies in serum
Clinical specimen used PBMCs Serum
Positive response in LTBI or prior TB disease?
No Possibly
Positive response in children, HIV/TB co-infection or EPTB?
Yes Inconsistent
Positive response to prior BCG vaccination?
No Yes
Ability to monitor treatment response?
Yes Inconsistent
Affected by recent TST placement?
Yes, induces false-positive ALS if TST
placed within 2 months
Yes, variable effect
Table 1: Comparison of ALS to Serology
ALS Assay SerologyConcept Antibodies secreted
from circulating plasma B cells found in PBMCs
Accumulated antibodies in serum
Clinical specimen used PBMCs Serum
Positive response in LTBI or prior TB disease?
No Possibly
Positive response in children, HIV/TB co-infection or EPTB?
Yes Inconsistent
Positive response to prior BCG vaccination?
No Yes
Ability to monitor treatment response?
Yes Inconsistent
Affected by recent TST placement?
Yes, induces false-positive ALS if TST
placed within 2 months
Yes, variable effect
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
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
Coating antigens
Rehka et al, PLoSOne Jan 2011
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
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
p < 0.001
1
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 20092. Thomas et al, Thorax Jan 2011
Demographic and clinical characteristics of patients. N=9 Drug-susceptible TB,
(n=5)Drug-resistant TB,
(n=4)
Median age in years [range]
2.5 [1.6–5] 10.5 [5–13]
Female gender (%) 2 (40%) 4 (100%)
Known TB contact 4 (80%) 4 (100%)
Chest X-ray findings on presentation
Hilar LAD only: 3 (60%)
LAD & infiltrates: 2 (40%)
Hilar LAD only: 1 (25%)
LAD & infiltrates: 3 (75%)
Baseline ALS titer, median [range]
1.42 [0.41–2.07] 0.62 [0.38–1.53]
Resolution of fever by 2 months
3 (75%)* 0 (0%)
Resolution of cough by 2 months
4 (80%) 1 (25%)
TB: tuberculosis, LAD: lymphadenopathy, ALS: antibody in lymphocyte supernatant, measured in optical densities. BMI: body mass index for age and gender.
* of the 4 children with drug-susceptible TB who presented with fevers.
Thomas et al, Thorax Jan 2011
------ 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
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.
Proposed study
DefinitionsSuspected TB: > 2 of the following symptoms :
• chronic cough (>2 weeks),• fevers or night sweats,• loss of weight, or failure to gain weight,• painless superficial lymphadenopathy
Possible TB: “suspected TB” and > 1 of the following:
• TB contact• No alternative definitive diagnosis established
Probable TB: “suspected TB” with favorable response to TB treatment and >2 of the following:
• TB contact• TST >10mm induration (or >5mm if HIV+ or sev. malnourished)• Radiological evidence consistent with TB disease• Failure to respond to broad-spectrum antibiotics• Symptoms of meningitis associated with pleocytosis (>20 WBC) and lymphocytic predominance (>50%)
Definite TB: “suspected TB” and 1 of the following:
• >1 specimen positive for AFB on microscopy• >1 culture positive for M. tuberculosis
“Slow” responder: > 2 of the following at the 2-month follow up visit:
• No improvement in each of the TB symptoms at presentation;
• Inappropriate weight gain or presence of weight loss;
• No improvement/worsening of TB findings on Xray• Persistently positive sputum smear
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
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
Clinical Procedures
TimeProcedure
T= 0 T= 2 mo
T= 6mo T= 12 mo
Interview for symptoms
Anthropometrics
Phlebotomy (ALS, drug levels)
(TB cases only)
Sputum sample
TB medications & adherence
Inter-current illnesses
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
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.
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
Thank you UVA
Eric Houpt Kristine Peterson Bill Petri Becca Dillingham Yan Ge Jean Gratz Scott Heysell Suzanne Stroup
Bangladesh Rubhana Raqib Dinesh Mondal Sayera Banu Tahmeed Ahmed
Tanzania Gibson Kibiki Stella Mpagama Charles Mtabho Sister Kimaro Happy Kumburu Atanasia Maro Norah Ndusilo
Sweden Susanna Brighenti