Pediatric Drug-resistant Tuberculosis

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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
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

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






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
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