Pediatric Drug-resistant Tuberculosis

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

Proportional burden of world’s TB cases

http://www.worldmapper.org/display.php?selected=228#WHO/HTM/TB/2009.411

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

Serology

Antibodies secreted from circulating plasma B cells found in PBMCs

Accumulated antibodies in serum

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

Yes, induces false-positive ALS if TST placed within 2 months

Yes, variable effect

Concept Clinical specimen used

Affected by recent TST placement?

Table 1: Comparison of ALS to Serology ALS Assay

Serology

Antibodies secreted from circulating plasma B cells found in PBMCs

Accumulated antibodies in serum

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

Yes, induces false-positive ALS if TST placed within 2 months

Yes, variable effect

Concept

Clinical specimen used

Affected by recent TST placement?

Table 1: Comparison of ALS to Serology ALS Assay

Serology

Antibodies secreted from circulating plasma B cells found in PBMCs

Accumulated antibodies in serum

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

Yes, induces false-positive ALS if TST placed within 2 months

Yes, variable effect

Concept Clinical specimen used

Affected by recent TST placement?

Table 1: Comparison of ALS to Serology ALS Assay

Serology

Antibodies secreted from circulating plasma B cells found in PBMCs

Accumulated antibodies in serum

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

Yes, induces false-positive ALS if TST placed within 2 months

Yes, variable effect

Concept Clinical specimen used

Affected by recent TST placement?

Table 1: Comparison of ALS to Serology ALS Assay

Serology

Antibodies secreted from circulating plasma B cells found in PBMCs

Accumulated antibodies in serum

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

Yes, induces false-positive ALS if TST placed within 2 months

Yes, variable effect

Concept Clinical specimen used

Affected by recent TST placement?

Table 1: Comparison of ALS to Serology ALS Assay

Serology

Antibodies secreted from circulating plasma B cells found in PBMCs

Accumulated antibodies in serum

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

Yes, induces false-positive ALS if TST placed within 2 months

Yes, variable effect

Concept Clinical specimen used

Affected by recent TST placement?

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 unstimulated 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 culturepositive 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 2009 2. Thomas et al, Thorax Jan 2011

Demographic and clinical characteristics of patients. N=9 Drug-susceptible TB, (n=5)

Drug-resistant TB, (n=4)

2.5 [1.6–5]

10.5 [5–13]

Female gender (%)

2 (40%)

4 (100%)

Known TB contact

4 (80%)

4 (100%)

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%)

Median age in years [range]

Chest X-ray findings on presentation

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

Growth during the course of TB therapy

Change in BMI (median)

2.5

DS-TB M/DR-TB

2

1.5 1

0.5 0

After 2 months

After 6 months

ALS titers during the course of TB therapy ALS titers (in optical densities)

2.5

------ DS-TB, - - - DR-TB, - - - MDR-TB

2

1.5

1

0.5

0.35

0 0

2

4

6

8

Time (in months) 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 Prospective Cohort

Comparison of ALS as a diagnostic test Nested Case – Control to assess ALS as a biomarker

TB suspects (6mo-14yrs)

TB Cases

“Slow Responders”

“Normal Responders”

Non-TB Controls

Definitions Suspected 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 48 hrs  TST within preceding 8 weeks  BCG vaccine within preceding 8 weeks

Clinical Procedures Time

T= 0

T= 2 mo

T= 6mo

T= 12 mo

Procedure Interview for symptoms Anthropometrics Phlebotomy (ALS, drug levels) Sputum sample TB medications & adherence Inter-current illnesses

(TB cases only)

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



Feasibility  Large



sample size needed

Inclusion of immunocompromised children  Affect



of not having a diagnostic “gold standard”

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