Pediatric Drug-resistant Tuberculosis
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Transcript 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 <14yrs
Kilimanjaro Clinical Research Institute (KCRI)/
Biotechnology Laboratory (BL)
Mycobacteriology
ELISA
tests for ALS
testing (culture, DST)
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
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