ANTIBODIES IN LYMPHOCYTE SUPERNATANT FOR THE DIAGNOSIS & MANAGEMENT OF TB IN CHILDRENTania 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 SerologyALS Assay Serology

Concept Antibodies secreted from circulating

plasma B cells found in PBMCs

Accumulated antibodies in serum

Clinical specimen used PBMCs SerumPositive 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 SerologyALS Assay Serology

Concept Antibodies secreted from circulating plasma B cells found in PBMCs

Accumulated antibodies in serum

Clinical specimen used PBMCs SerumPositive 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 SerologyALS Assay Serology

Concept Antibodies secreted from circulating

plasma B cells found in PBMCs

Accumulated antibodies in serum

Clinical specimen used PBMCs SerumPositive 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 SerologyALS Assay Serology

Concept Antibodies secreted from circulating plasma B cells found in PBMCs

Accumulated antibodies in serum

Clinical specimen used PBMCs SerumPositive 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 SerologyALS Assay Serology

Concept Antibodies secreted from circulating plasma B cells found in PBMCs

Accumulated antibodies in serum

Clinical specimen used PBMCs SerumPositive 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 SerologyALS Assay Serology

Concept Antibodies secreted from circulating plasma B cells found in PBMCs

Accumulated antibodies in serum

Clinical specimen used PBMCs SerumPositive 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 symptomsAnthropometricsPhlebotomy (ALS, drug levels)

(TB cases only)

Sputum sampleTB medications & adherenceInter-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