Theerapan Songnuy, M.D.June 21, 2013
Diagnostic Tools for Steven Johnson Syndrome/Toxic
Epidermal Necrolysis
Case studyDefinitionPathogenesis & EtiologyClinical ManifestationInvestigations: - ALDEN Algorithm - In vitro: ELISpot test Conclusion
Outlines
A Thai boy, 7-y of age, from Samutsakorn provinceHistory :
CC: Referred with generalized rash & oral lesion 4 d PTA
PI : 2 wk PTA he developed twitching at one side of oral angle & progressed to generalized tonic-clonic seizure. Unconsciousness was observed for 10 min before carried to private hospital
At the hospital, high fever was detected, no stiff neck, others were unremarkable
Case Study
Initial work up: CBC : Hb 12.6 Hct 40.6 WBC 10760 N 29 L 64
Eo 5 plt 419,000 Blood glucose : 107 Blood Chem : Na 141 K 3.9 Cl 100 HCO2 24 Ca 7.1 UA no cell H/C no growth Dengue NS1, IgG, IgM neg Influenza neg
Case Study
Imp: Epilepsy, Fever cause ? Hypo-calcemia
Managements: 1. Phenytoin loading , then 5 MKD oral 2. Calcium gluconate 10 ml iv drip* 2
doses 3. Cef-3 2 gm iv OD * 3 d 4. Zithromax ( 250) 2 tabs oral OD * 5 d 5. Doxycyclin 1 tab oral BID* 1 d
Case Study
Progress Note:
12 d PTA ( one day after admission) - He developed MP rash on trunk , face, with itchy , no oral ulcer, no eye
lesion - CBC: Hb 11.7 Hct 37 WBC 3490 N 46 L 34 plt 329,000 - Imp: viral exanthem
9 d PTA - He continued fever & progressive rash with itchy , no oral or eye
involvement - Imp: viral exanthem VS drug allergy - Management: Hydroxycine, calamine lotion
Case Study
4 d PTA - Progressive erythematous rash with cracked –dry
lips, conjunctival injection - Phenytoin was discontinued
At home, he had persistent fever & rash spreading on
chest wall
Today, he got high fever with oral pain, decreased intake then came to KCMH
Case Study
PH : - The first child of family, pre-term 30 wk - He had experience of seizure without fever 2 y
ago then was admitted & took diazepam iv . At the hospital, he had fever without other signs. He was intubated for 2 d & discharged without anti-epileptic drug - Vaccine: complete - G& D: normal - No family history of epilepsy
Case Study
Physical Examination:
GA : A Thai boy, good consciousness Bw 39 kg Ht 129 cm Ideal Bw 27 kg Wt for Ht 144 % VS : BT 40.5 PR 134/min RR 32 BP 130/80
Skin : Erythematous MP rash on trunk, palm, sole totally confined to 15 % of BSA
HEENT: Bilateral conjunctival injection & muco-purulent discharge, no corneal lesion, mild sunken eye ball, no puffy eyelids, red-cracked lips, oral mucositis
Case Study
Physical Examination:
RS : Equal breath sound, no adventitious sound CVS: Normal S1 S2 , no murmur
Abd : Soft , not tender, liver & spleen can’t be palpated Genitalia: No ulcer seen, not tender
Ext: No joints swelling, no edema, not tender Neuro: E4M6V5, others were unremarkable
Imp: Steven Johnson SyndromeEtiology: Suspected Phenytoin, Cef -3 , or infection
Case Study
Investigations
CBC: Hb 11.5 Hct 37 WBC 4360 N60 L23 Eo 5 (218)
plt 393,000 Blood chemistry: BUN 9 Cr 0.42 TB 0.25 DB 0.14 AST 43 ALT 52 ALP
113 alb 3.5 Ca 8.4 Po4 4.6 Mg 1.04 Na 135 K 5 Cl 98 HCO3 22
Case Study
Managements :
1. Hydrocortisone 5 mg/kg/dose iv q 6 hr * 5 d then prednisone 2 MKD * 2 d, 1 MKD * 3 d, 0.5 MKD * 3 d, 0.25 MKD * 2 d >>> off 2. Clindamycin 30 MKD iv devided q 8 hr* 7 d then oral * 3 d ( oral infection)
3. Levofloxacin ( 500) 1 od * 7 d 4. Oral care : wet dressing, xylocain oral viscous, NSS
5. Eye care : Vislube ed, Cravit ed, Maxitrol eo, fluoromethalone ed 6. Antihistamine: CPM 7 mg iv q 6 hr , cetirizine 1 od
Case Study
Progress Note: Clinical gradually improved after admission
Further investigations:
- HSV IgM negative, IgG positive
- EBV IgM negative, IgG negative
- Mycoplasma IgM negative, IgG negative
- PCR for mycoplasma negative
- HLA*B 1502 Negative
- ELISPOT for Phenytoin, Cef-3, Azithromycin ( pending)
Case Study
Epilepsy ( benign Rolandic) - CT brain ( 10/1/2011) : normal - EEG ( 27/5/2013) : normal - If recurrent >>>> Keppra ( focal seizure ,
low risk to allergy)
Case Study
F/U 4 wk after onset 1. Epilepsy
2. SJS 2.1 Eye involvement - Improve but sub-conjunctival fibrosis (right) - On FML ED, Xanalin ED, Vislube ED
2.2 ELISPOT to Phenytoin positive to Cef-3 & Azithromycin : negative - Peeling both hands, crusted upper lip - 20% urea cream apply
Case Study
Definition : - The process of epidermal necrolysis resulting
extensive blisters & detachment of skin & mucous membrane
- SJS & TEN are two forms of epidermal necrolysis ,
differing to the amount of skin detachment relative
to BSAHigh mortality rate 23%
Am J Dermatopathol 1997; 19: 127-132
J Am Acad Dermatol 2008; 58: 33-44
Stevens Johnson Syndrome/Toxic Epidermal Necrolysis
Incidence 1.9 cases per million inhabitants/ yAnnual incidence in HIV-positive population approximately 1 case of TEN /1,000/yFactors affecting SJS/TEN incidence : - Regional differences in drug prescription - Genetic background - Coexistence of cancer - Concomitant radiotherapy
Lancet 1999;353:2190-2194
Drug Saf 2005; 28: 917-924
N Engl J Med 1995;333:1600-1607
J Eur Acad Dermatol Venereol 2006; 20: 588-590
Epidemiology
Keratinocyte apoptosis followed by necrosis CD 8 T cells, cytolytic molecules FasL & granulysin
Etiology: - Drugs : sulfonamide , aminopenicillins, cephalosporin, quinolones, carbamazepine, phenytoin, phenobarbital, NSAIDs, allopurinol, corticosteroids
- Genetic susceptibility: - Carbamazepine associated with HLA B* 1502 - Allopurinol associated with HLA B* 5801
N Engl J Med 1995;333:1600-1607
Nature 2004; 428: 486
Pharmacogenomics 2008;9: 1617-1622
Pathogenesis
Clinical Manifestation
Arch Dermatol 2003; 139: 33-36
Clinical diagnosisHistological work up : - immediate cryosection or formalin-fixed
section revealing necrotic epidermis all layers
Differential Diagnosis - Autoimmune blistering diseases ; linear IgA
dermatosis acute generalized exanthematous pustulosis, staphylococcal scalded skin syndrome etc.
Diagnosis
Neurology. 2011; 77: 2015-2033
A retrospective study, over a 6-yr period
To study epidemiology & clinical of pt with SJS, TEN,
SJS-TEN overlap, & DRESS caused by anti-epileptic drugs
To analyzed subsequent alternative anti-epileptic drug used for pt after their SCAR episodes
Aims
Data were collected from Jan 2003-Dec 2009 Two clinical branches of Chang Gung Memorial
Hospital
SJS/TEN referred to the collection of SJS, SJS-TEN overlap, and TEN
SJS : widespread small macules or blisters with skin detachment of less than 10% of BSA
SJS-TEN: involves 10 %- 29% of BSATEN : involves greater than 30% of BSA
Methods
Neurology. 2011; 77: 2015-2033
Neurology. 2011; 77: 2015-2033
Neurology. 2011; 77: 2015-2033
Neurology. 2011; 77: 2015-2033
Neurology. 2011; 77: 2015-2033
Neurology. 2011; 77: 2015-2033
Clinical Pharmacology & Therapeutics 2010; 1: 60-68
Expert judgment - Subjectivity - Lack of standardization - Poor reproducibilityProbabilistic approaches - Need to model probability distribution - Impractical in routine practiceAlgorithm method - Based on decision trees or successive evaluation of criteria - Intra- and inter-evaluator agreements are usually high - Results depend on the weight given to each criterion
Assessment of causality of adverse events from drug use
EuroSCAR, a case-control study of SJS/TENConducted in 6 countries; Austria, France, Germany, Israel, Italy, & the Netherlands
Total 379 cases enrolledBetween April 1997- December 2001Validated by an expert committee ( blinded to details of
drug exposures) on medical histories, medical records, clinical photo, & biopsies
Medical information was collected by trained interviewers Global drug risks quantified with multivariate RRs
restricted to recent initiation of the drug ( within 8 wk)
Origin of SJS/TEN cases used in algorithm evaluation
To create an algorithm that can be used by clinicians & not capable of discriminating between the effect of various drugs which patient exposed
ALDEN Algorithm: - First step : algorithm elaborated by a group of experts, based on knowledge of the results of the SCAR study
- Second step : algorithm assessment of drug was carried out on all cases in the EuroSCAR study The results were compared with those provided by case-control analysis of the same cases
Aims
Reproducibility of the algorithm - Comparing score for 101 drugs by two
investigators - K concordance : 0.73 : individual score of each medication 0.71 : classification of medication in
causality group 0.71 : determination of the drug with the
highest score for each patient
Results
Clinical Pharmacology & Therapeutics 2010; 1: 60-68
Clinical Pharmacology & Therapeutics 2010; 1: 60-68
Range from -12 to + 10 1. Very probable : score > 6 2. Probable : score 4-5 3. Possible : score 2-3 4. Unlikely : score 0-1 5. Very unlikely : score < 0
Score of ALDEN Algorithm
Clinical Pharmacology & Therapeutics 2010; 1: 60-68
By Algorithm score: - One drug classified in probable or very probable (69 pt ) - Two drug classified in probable or very probable ( 3 pt) - No drug classified in probable or very probable ( 28 pt)
By French pharmaco-vigilance method - One drug classified in possible or probable ( 23 pt) - Two drug classified in possible or probable ( 17 pt) - No drug classified in possible or probable ( 60 pt)
Outcomes from causality assessment differ significantly between two method ( p < o.oo1, X2-test )
Comparison of ALDEN with the French pharmaco-vigilance method
Clinical Pharmacology & Therapeutics 2010; 1: 60-68
Clinical Pharmacology & Therapeutics 2010; 1: 60-68
Clinical Pharmacology & Therapeutics 2010; 1: 60-68
Clinical Pharmacology & Therapeutics 2010; 1: 60-68
Very strong correlation between two method ( r= 0.90, p< 0.001 at 95% CI 0.74-0.97)
Limitations: - created by experts who were aware of the results of case-control analysis & looking for a good correlation - Due to more than half of cases are related to a limited number of “high-risk” drug, there was a rather high a priori probability of observing an agreement - This algorithm is more specific to SJS/TEN, may be
not appropriate to apply for other adverse events
Conclusion
ELISPOT Test
- In 1983 , new technique for enumeration of Ab-secreting cells - Built on the same solid-phase immuno enzymatic principles as ELISA - Ag was immobilized to a solid support to bind Ab released by cultured splenocytes
Sedgwick JD & Holt PG. A solid-phase immunoenzymatic technique for the enumeration of specific antibody-secreting cells. J Immunol Methods. 1983; 57: 301-309.
Investigation (Vitro)
Later, 1983, Czerkinski & colleagues named “ Enzyme-Linked Immunospot” ( ELISPOT) - Modified by using coated –Ab to a solid phase - Waiting for capture Ag ( cytokines) secreted
by cultured cell - More popular - Some researcher called “ reversed ELISPOT”
Czerkinski CC, Nilsson LA, Nygren H, Ouchterlony O & Tarkowski A. A solid-phase enzyme-linked immunospot ( ELISPOT) assay for enumeration of specific antibody-secreting cells. J Immunol Methods. 1983; 65: 109-121.
ELISPOT Test
Fields of application
- Two hundred times more sensitive than ELISA in
detecting secreted cytokines - Cytokines ; IFN-gamma, TNF-alpha, IL-2, IL-4
etc. from peripheral blood lymphocytes - Used for vaccine development, AIDS, cancer,
infectious, autoimmune, allergy & transplantation researches
ELISPOT Test
Kalyuzhny A E. Chemistry and Biology of the ELISPOT Assay. From Methods in Molecular Biology . Vol 3.2: Handbook of ELISPOT: Methods and Protocols . Edited by : A.E. Kalyuzhny O Humana Press Inc., Totowa, NJ
Immunochemical principles of ELISPOT Assay
- Sandwich principle as ELISA - Two differences - ELISA measures real concentration of
cytokine but ELISPOT detects secreting cells - ELISA analyses cell-free media but ELISPOT combines immunoassay &
bioassay
ELISPOT Test
Kalyuzhny A E. Chemistry and Biology of the ELISPOT Assay. From Methods in Molecular Biology . Vol 3.2: Handbook of ELISPOT: Methods and Protocols . Edited by : A.E. Kalyuzhny O Humana Press Inc., Totowa, NJ
Kalyuzhny A E. Chemistry and Biology of the ELISPOT Assay. From Methods in Molecular Biology . Vol 3.2: Handbook of ELISPOT: Methods and Protocols . Edited by : A.E. Kalyuzhny O Humana Press Inc., Totowa, NJ
ELISPOT Test
Kalyuzhny A E. Chemistry and Biology of the ELISPOT Assay. From Methods in Molecular Biology . Vol 3.2: Handbook of ELISPOT: Methods and Protocols . Edited by : A.E. Kalyuzhny O Humana Press Inc., Totowa, NJ
Performance of the test depends on quality of : 1. Antibodies ( both capture & detection) 2. Enzyme conjugate 3. Chromo-genic substrates 4. Membrane-backed plates
- Secretion activity of cells determined by the number of - Spots on the plate - Spot should have strong staining intensity, well-defined
edge - Spot should have a small diameter to avoid merging
Nuts & Bolts of ELISPOT Assay
Kalyuzhny A E. Chemistry and Biology of the ELISPOT Assay. From Methods in Molecular Biology . Vol 3.2: Handbook of ELISPOT: Methods and Protocols . Edited by : A.E. Kalyuzhny O Humana Press Inc., Totowa, NJ
To evaluate whether drug-reacting cytotoxic cells can be detected in the peripheral blood of patients in remission
To find out this method might be helpful in drug allergy diagnosis
Aims
12 pt were selected Offending drugs were defined ( typical medical history such as onset, interval, clinical manifestation) Skin test & positive lymphocyte transformation testAll drugs were used in nontoxic concentration
Patients also were tested with tolerated drugs in some case, if no additional drug exposure was known ,
we used as “ nonculprit” drug ( was shown to induce strong granzyme B production or CD 107a expression in other allergic individuals)
All pt were clinical remission 5 mon-15 y after acute event16 drug-exposed donors who tolerated tested drug & had negative LTT as a control group
Methods
Allergy 2010; 65: 376-384
Cell preparation & culture medium - Peripheral blood mononuclear cell were isolated by density gradient centrifugation & frozen in 90% fetal calf serum ( Oxoid, Pratteln, Switzerland) plus 10% dimethyl sulfoxide
- After thawing, cells were cultured or preincubated overnight in 24-well plate ( 5* 106 cell/well) with medium alone or with 1 ng/ml of IL-7/IL-15 mixture ( PeproTech EC Ltd, London,UK)
- Culture medium consisted of RPMI-1640 supplement with 10% pooled, heat-inactivated human AB serum, 25 Mm Hepes buffer, 2 M L-glutamine, 100 U/ml penicillin & 25 ug/ml transferrin Allergy 2010; 65: 376-384
Method
Enzyme-Linked immuno-spot assay - Ninety-six-well filtration plates were coated with capture anti-human GzB mAb solution - Then washed & blocked according to manufacturer’s protocol
- Freshly thawed PBMCs ( 8*105 cells/well) were incubated with culture media ( negative control) or culprit drug & tolerated drug for 20,48 or 72 h at 37 C degree in a 5% CO2 incubator
- Cells were plated into 96-well U-bottomed tissue culture plates & transferred into GzB-mAb-coated plates for the last 20 h of stimulation
Method
- For experiment with IL-7/IL-15 pre-incubation
- Freshly thawed PBMCs were incubated overnight in 24-well plate ( 5* 106 cells/well) with 1 ng/ml of IL-7 & IL-15, followed by four washing steps to remove residual cytokines
- Cells were counted & incubated with antigens in 96-well U-bottomed tissue culture plates ( 5*105
cells/well) for 2 d at 37 C degree in 5% CO2 incubator
- For last 20 h, cells were placed to the GzB mAb-coated paltes. - ELISPOT plates were developed as to manufacturer’s instruction-
Method
ELISPOT procedure’s instruction
- Plates were washed with PBS/Tween 20 0.1% - Then biotinylated anti-GzB mAb was added for 90 min at 37 C degree
- After washing for 3 times, streptavidin-alkaline phosphatase was added for 1 h at 37 C degree - Spots were visualized with nitroblue tetrazolium/5-bromo-4-
chloro-3-indolylphosphate p- toluidine salt
- Then analyzed using a Bioreader 3000 CL/PRO ( BIO-SYS GmbH, Karben, Germany)
Method
CD 107a assay & flow cytometry
- Amount of 1* 10 6 per well PBMCs were cultured in 96-well
U-bottomed tissue culture plates at 37 C in a 5% CO2 incubator with indicated drug concentration
- Incubation with CM alone or nonculprit drug ( negative control) - PBMCs from healthy donors were cultured with different
drugs
- Monensin 6 ug/ml and 3 ul of anti-CD107a fluorescein were added to each well for the last 5 h of incubation
Method
- Following stimulation, PBMCs were taken to a 96-well V-bottom plate, wased once in ice-cold cell wash
- Then surface-stained for 25 min at 4 C in the dark
- Directly conjugated with antibodies: anti CD3 allophycocyanin ( APC), anti CD4 phycoeythrin,
anti CD 8 peridinin chlorophyll protein complex, & anti CD 56
- After two washing, cells were resuspended in Cell Wash
& analyzed using a FACSCantoTM FlowCytometer
Method
Results
Allergy 2010; 65: 376-384.
Allergy 2010; 65: 376-384
Allergy 2010; 65: 376-384
Allergy 2010; 65: 376-384
Allergy 2010; 65: 376-384
Allergy 2010; 65: 376-384
Allergy 2010; 65: 376-384
Allergy 2010; 65: 376-384
Allergy 2010; 65: 376-384
Allergy 2010; 65: 376-384
Allergy 2010; 65: 376-384
Drug-specific cytotoxic mechanism is detected in peripheral blood with various forms of delayed
DHRs
GranzymeB ELISPOT is used as supplemental tool in vitro diagnosis of drug allergies
Short exposure to IL-7, IL-15 enhances drug specific response in Granzyme ELISPOT , help to identify the
offending drug in allergic pt with weak proliferative response
Conclusion
Thank You Very Much