stevens johnson syndrome and laboratory diagnostic tools

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

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


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


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


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


ELISPOT Test


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


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

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

stevens johnson syndrome and laboratory diagnostic tools

Health & Medicine

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oral ulcer

oral care

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oral pain

oral mucositis