This case study describes a 7-year-old Thai boy who presented with a rash, fever, and seizures. He was initially diagnosed with epilepsy and treated with phenytoin, cef-3, and azithromycin. His condition worsened with the development of Steven Johnson Syndrome. Testing found he was positive for phenytoin on an ELISPOT test. The document discusses SJS pathogenesis, definitions, investigations including the ALDEN algorithm and ELISPOT test, and management of the patient's condition.

1. Theerapan Songnuy, M.D.
June 21, 2013

2.  Case study
 Definition
 Pathogenesis & Etiology
 Clinical Manifestation
 Investigations:
- ALDEN Algorithm
- In vitro: ELISpot test
 Conclusion

3.  A Thai boy, 7-y of age, from Samutsakorn province
 History :
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

4.  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

5.  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

6.  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

7.  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

8. 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

9.  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

10. 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 Syndrome
Etiology: Suspected Phenytoin, Cef -3 , or infection

11.  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

12.  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

13. 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)

14.  Epilepsy ( benign Rolandic)
- CT brain ( 10/1/2011) : normal
- EEG ( 27/5/2013) : normal
- If recurrent >>>> Keppra ( focal seizure , low risk
to allergy)

15.  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

16.  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 BSA
 High mortality rate 23%
Am J Dermatopathol 1997; 19: 127-132
J Am Acad Dermatol 2008; 58: 33-44

17.  Incidence 1.9 cases per million inhabitants/ y
 Annual incidence in HIV-positive population
approximately 1 case of TEN /1,000/y
 Factors 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

18.  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

19. Arch Dermatol 2003; 139: 33-36

20.  Clinical diagnosis
 Histological 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.

21. Neurology. 2011; 77: 2015-2033

22.  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

23.  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 BSA
 TEN : involves greater than 30% of BSA

24. Neurology. 2011; 77: 2015-2033

25. Neurology. 2011; 77: 2015-2033

26. Neurology. 2011; 77: 2015-2033

27. Neurology. 2011; 77: 2015-2033

28. Neurology. 2011; 77: 2015-2033

29. Neurology. 2011; 77: 2015-2033

30. Clinical Pharmacology & Therapeutics 2010; 1: 60-68

31.  Expert judgment
- Subjectivity
- Lack of standardization
- Poor reproducibility
 Probabilistic approaches
- Need to model probability distribution
- Impractical in routine practice
 Algorithm 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

32.  EuroSCAR, a case-control study of SJS/TEN
 Conducted in 6 countries; Austria, France, Germany,
 Israel, Italy, & the Netherlands
 Total 379 cases enrolled
 Between April 1997- December 2001
 Validated 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)

33.  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

34.  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
Clinical Pharmacology & Therapeutics 2010; 1: 60-68

35. Clinical Pharmacology & Therapeutics 2010; 1: 60-68

36.  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
Clinical Pharmacology & Therapeutics 2010; 1: 60-68

37.  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 )
Clinical Pharmacology & Therapeutics 2010; 1: 60-68

38. Clinical Pharmacology & Therapeutics 2010; 1: 60-68

39. Clinical Pharmacology & Therapeutics 2010; 1: 60-68

40. Clinical Pharmacology & Therapeutics 2010; 1: 60-68

41.  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

42.  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.

43.  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.

44.  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
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

45.  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
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

46. 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

47. 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

48.  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
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

50.  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

51.  12 pt were selected
 Offending drugs were defined ( typical medical history
such as onset, interval, clinical manifestation)
 Skin test & positive lymphocyte transformation test
 All 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 event
 16 drug-exposed donors who tolerated tested drug & had
negative LTT as a control group
Allergy 2010; 65: 376-384

52.  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

53.  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

54. - 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
-

55.  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)

56.  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

57. - 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

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70.  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

71. Thank You Very Much