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Yasuyuki Fujita, MD,a Naoya Yoshioka, MS,a Riichiro Abe, MD, PhD,a Junko Murata, MD, PhD,a Daichi Hoshina, MD,a Hirokatsu Mae, MS,b and Hiroshi Shimizu, MD, PhDa Sapporo, Japan
Background: Life-threatening adverse drug reactions such as Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) sometimes start with clinical features of ordinary drug-induced skin reactions (ODSRs) and it may be difﬁcult to make a correct diagnosis before severe mucocutaneous erosions occur. We have reported that serum granulysin levels are elevated (cut off: 10 ng/mL) in patients with SJS/TEN before generalized blisters form. Objective: We sought to develop a rapid detection system for elevated serum granulysin to predict the progression from ODSRs. Methods: Serum samples from 5 patients with SJS/TEN at 2 to 4 days before mucocutaneous erosions formed were analyzed. Sera from 24 patients with ODSRs and 31 healthy volunteers were also investigated as control subjects. We developed a rapid immunochromatographic assay for the detection of high levels of serum granulysin using two different antigranulysin monoclonal antibodies. Results: The immunochromatographic test showed positive results for 4 of 5 patients with SJS/TEN but only one patient of 24 with ODSRs. The results correlated closely with those of enzyme-linked immunosorbent assays. Limitations: The validation of the long-time stability in this test strip has not been investigated. Conclusion: This novel test enables the prediction of SJS/TEN occurrence in patients even when only features of ODSRs are noted clinically. ( J Am Acad Dermatol 2011;65:65-8.) Key words: adverse drug eruption; diagnostic test; granulysin; Stevens-Johnson syndrome; toxic epidermal necrolysis.
tevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are life-threatening adverse drug reactions characterized by blister formation and widespread skin detachment.1 In the
Abbreviations used: ODSRs: sFasL: SJS: TEN: ordinary drug-induced skin reactions soluble Fas ligand Stevens-Johnson syndrome toxic epidermal necrolysis
From the Department of Dermatology, Hokkaido University Graduate School of Medicine,a and Sapporo Immuno Diagnostic Laboratory.b The first two authors contributed equally to this article. Funding sources: None. Conflicts of interest: None declared. Accepted for publication April 26, 2010. Reprint requests: Riichiro Abe, MD, PhD, Department of Dermatology, Hokkaido University Graduate School of Medicine, N15 W7, Kita-ku, Sapporo 060-8638, Japan. E-mail: aberi@med. hokudai.ac.jp. Published online April 20, 2011. 0190-9622/$36.00 ª 2010 by the American Academy of Dermatology, Inc. doi:10.1016/j.jaad.2010.04.042
early stage, SJS/TEN presents clinically as edematous papules or erythema multiformeelike target rashes, which are very similar to those of ordinary druginduced skin reactions (ODSRs). Such a clinical course makes it difficult to reach a diagnosis of SJS/TEN in the early stage, and this results in high mortality. There is an urgent need for a method to distinguish between early-stage SJS/TEN and ODSRs. The method should be as fast as possible, because SJS/TEN usually occurs within a few days. Furthermore, the technique should be as clinically 65
a control line was created by the mucocutaneous erosions appear.2.3 It would be immobilization of antimouse IgG. The plates were then 30% involvement. R&D Systems. MBL. The samples and standards Patients (recombinant granulysin. granulysin protein to the immunochromatographic test strips. The plates were then washed and serum granulysin to diagnose and predict the early blocked with phosphate-buffered saline containing stage of SJS/TEN. we papules will develop severe drug 96-well flat-bottomed plates developed a rapid immunoeruptions. we can predict whether munosorbent assay as previthan those of sFasL. We levels are increased in patients who later found that both serum grandevelop Stevens-Johnson syndrome or Enzyme-linked ulysin and sFasL are higher toxic epidermal necrolysis. we The plates were incubated with tetramethylbenzidine obtained serum samples from 35 patients with substrate (Sigma. taneous or ocular lesion ﬁrst eroded or ulcerated Switzerland) for 30 minutes at room temperature. Another diagnostic markers. and it may be diagnostic device. SJS refers to cases with mucosal erosions and MN) were incubated for 2 hours at room temperature. Nagoya. on these observations. revealing a visidifficult to distinguish life-threatening Chung et al4 recently reble result line. Among several candidates for brane area of the test device in a dry state.1 mg/mL of biotinysurface area.3 Of these. Thoiry. and the procedures were approved by RESULTS the Ethical Committee of the Hokkaido University We ﬁrst applied diluted recombinant human Graduate School of Medicine. were coated with 5 mg/mL chromatographic assay for of RB1 antibody and stored the detection of high-level overnight at 48C. MBL) was (sFasL) and found that it is elevated in the sera of immobilized on a nitrocellulose membrane to form a patients with SJS/TEN in the early stage. a murine brought the same results (Fig 1. but sFasL serum via RB1 and comigrated uplevels are too low (cut off: ward until the granulysin was Drug reactions sometimes start with 100 pg/mL) for use in a rapid sandwiched with the immoedematous papules. epidermal detachment of less than 10% of the body Then they were reacted with 0. we examined soluble Fas ligand granulysin monoclonal antibody (RC8.2 mg/mL of horseradish-peroxidaseSJS/TEN was deﬁned as the day when the mucocuconjugated streptavidin (Roche Diagnostics. Serum samples from patients with Technologies. Likewise. Informed consent was obtained from all patients. Disease onset in patients with treated with 0. to conﬁrm the threshold and reliability of the assay. before result line. ODSRs (n = 24) and healthy volunteers (n = 31) were also analyzed. A). We recently found that serum granulysin of patients with SJS/TEN. Berthold listed in Table I. sandwich-enzyme-linked imhigher (cut off: 10 ng/mL) With this test. highly expressed in blisters within 15 minutes. (day 1). such as using immunochromato(RB1. The patient information is using a microplate reader (Mithras LB940. The entire test drug reactions from ordinary drug ported that granulysin is procedure was completed reactions early in their course. we investigated 5 patients whose room temperature. bilized RC8. The optical density was measured at 450 nm SJS/TEN (day e2 to e4). The granulysin in very useful to be able to the serum sample speciﬁcally predict the occurrence of bound to the microparticles CAPSULE SUMMARY SJS/TEN. Japan. and 3 repeated investigations In the immunochromatographic test.3 From multiple Japanese institutions. Basel.66 Fujita et al J AM ACAD DERMATOL JULY 2011 simple as possible. Japan) was conjugated with graphic test strips that are available for the detection microparticles and then placed on the glass memof inﬂuenza infections. monoclonal antibody speciﬁc to human granulysin d d d . MO) for 30 minutes at SJS/TEN. Approximately 10 ng/mL of sample Immunochromatographic assay yielded a result line.6.1% Tween-20 (washing buffer) and blocked with 10% fetal bovine serum in washing buffer at room METHODS temperature for 2 hours. immunosorbent assay in patients with early-stage The granulysin concentraSJS/TEN than in patients We report a novel tions of the serum samples with ODSRs. Minneapolis. and TEN refers to those with more than lated RC8 antibody for 1 hour. France). 0. Sapporo. and then 1 mol/L sulfuric acid was sera had been collected before the diagnosis of added.5 Serum levels immunochromatographic assay to were measured with a of granulysin are 100 times detect high levels of serum granulysin. Based patients with nonspecific edematous ously described.7 In brief. St Louis.
TEN. y Sex Diagnosis Affected skin area Causative drug Serum granulysin (d) 1 2 3 4 5 17 66 27 80 25 M F F M F SJS TEN SJS SJS SJS 20% 70% \10% 5% Only mucosal lesions Carbamazepine Imatinib Unknown Phenytoin Unknown 52. This pattern is similar to that . are seen.35 6 9. Although 20% of the cases could be missed. Normal human serum as negative control (1. we should use the mathematical tool called SCORTEN that has been developed. apoptosis of target cells in a mechanism involving caspases and other pathways.91 ng/mL. The only sample with a negative result had granulysin at the normal level of 2.1 ng/mL. 6. a member of the saposin-like protein family of lipid-binding proteins.5 ng/mL). Very recently. Immunochromatographic test strip detects elevated granulysin. Normal human serum as negative control (3. and it induces Fig 1. exhibits potent cytotoxicity against a broad panel of microbial targets. careful daily and hourly monitoring of the patient for a few days should take place. Based on this observation. Age. NUMBER 1 Fujita et al 67 Table I. transplanted cells. Approximately 10 ng/mL of granulysin is considered a positive result. A. Positive results are shown as a band (indicated by the arrow). Four of 5 SJS/TEN samples showed positive results (Fig 1. and serum granulysin has decreased to 5. serum granulysin was 52. bacteria. including tumor cells. Patient information Patient No. 2. 1. one in 24 ODSRs samples and none of 31 healthy volunteers showed positive bands in this immunochromatographic assay. The results of the immunochromatographic test correlated closely with early diagnosis for SJS/TEN (P = 1. Recombinant human granulysin as positive control. we then applied serum samples to detect the elevated granulysin levels. DISCUSSION We succeeded in developing a rapid immunochromatographic test for the detection of high-level serum granulysin that puts our previous ﬁndings to practical use. to assess the severity of illness and to predict mortality. and parasites. male.J AM ACAD DERMATOL VOLUME 65. Female. Stevens-Johnson syndrome. It would not be necessary for every morbilliform drug eruption. The test showed a sensitivity of 80% and a specificity of 95. M. in order to look for necrotic keratinocytes. average 6 SEM). it would be a useful adjunct in diagnosing SJS/TEN. 1 to 5. Furthermore. We suggest that the test be applied when clinical ﬁndings hinting at SJS/TEN.02 3 10À3. Serum taken from patient 1 with early Stevens-Johnson syndrome (SJS )/toxic epidermal necrolysis (TEN ) 3 days before blister formation. toxic epidermal necrolysis. two biopsies should be done as soon as SJS/TEN are suspected.8 If the results of either method are negative. for hematoxylin-eosin processing and immediate frozen sections. No bands are observed. damaging negatively charged cell membranes.4 ng/mL). we showed that granulysin levels of sera from patients with SJS/TEN are significantly elevated before the development of skin detachment or mucosal lesions. such as target lesions. Seven days after blister formation in same patient with SJS/TEN. Conversely. fungi. Diluted recombinant granulysin is applied.10 Granulysin plays important roles in host defense against pathogens. Detection of serum granulysin by immunochromatographic assay.2 42.7 (e3) (e3) (e4) (e2) (e2) F.9 Granulysin.7 ng/mL. 3. 4. All the positive samples had elevated granulysin as detected by enzyme-linked immunosorbent assay analysis (30. analyzed by Fisher exact probability test).8% for SJS/TEN versus ODSRs.11 Chung et al4 reported that granulysin was identified as the most highly expressed cytotoxic molecule in blisters of patients with SJS/TEN. B. which is another sensitive test.9 2. B). Although patient showed only edematous erythema and papules without mucosal manifestations. However.7 ng/mL.2 12.5 The elevated serum granulysin levels decrease rapidly within 5 days after disease onset.1 14. SJS.
Yoshioka N. Mudgil AV. Medication use and the risk of Stevens-Johnson syndrome or toxic epidermal necrolysis. et al. Yang JY. Rosmarin DM. Shimizu T. 3. Shimizu H. Majima T. 8. J Am Acad Dermatol 2007. Murata J. Ann Intern Med 2009.151:514-5. Chung WH. Huang SP. Shimizu H. et al. Nakamura H. 10. Kelekar A. Watanabe H. SCORTEN: a severity-of-illness score for toxic epidermal necrolysis. Kelly JP. Anderson T. Nat Med 2008. . Takamori Y. Hung SI. Serum granulysin level as a novel prognostic marker in patients with gastric carcinoma. Kaspar AA. Granulysin in human serum as a marker of cell-mediated immunity. Bastuji-Garin S. Abe R.68 Fujita et al J AM ACAD DERMATOL JULY 2011 observed with sFasL. Wei CY. Shimizu H. Nagasawa M. Su SC. J Gastroenterol Hepatol 2007.3 When granulysin levels for patients with SJS/TEN in the early stage were compared with those levels for patients with ODSRs and healthy control subjects. Rzany B. Abe R.162:1515-20. Okada S. Poulain FR. Fouchard N. 9. J Invest Dermatol 2000.14:1343-50. Kasahara Y. Tsujimoto H. J Allergy Clin Immunol 2008. Bertocchi M. Wolkenstein P. Drouvalakis KA.333:1600-7. 4. Fujita Y. Revuz J. Roujeau JC. 5. Increased soluble Fas ligand levels in patients with Stevens-Johnson syndrome and toxic epidermal necrolysis preceding skin detachment.115:149-53. Krensky AM. 2. 7. Saigusa S. Curr Opin Immunol 2003. Roujeau JC. Kumar J.22:1322-7.56:181-200. Granulysin as a marker for early diagnosis of the Stevens-Johnson syndrome.5 This novel test enables the early diagnosis of SJS/TEN in patients with cutaneous adverse drug reactions that are otherwise indistinguishable from ODSRs. REFERENCES 1. Abe R. Ichikura T. Naldi L. Suzuki K. et al. et al. Clayberger C. 6. Takano S. Sugasawa H. Ogawa K. Granulysin is a key mediator for disseminated keratinocyte death in Stevens-Johnson syndrome and toxic epidermal necrolysis.167:350-6. Kawarabayashi N.33:1925-33. Murata J.122:992-1000. Eur J Immunol 2003. Toxic epidermal necrolysis and Stevens-Johnson syndrome are induced by soluble Fas ligand. 11. et al.15:560-5. Pereira FA. N Engl J Med 1995. Stern RS. the differences were statistically significant. Toxic epidermal necrolysis. Shibaki A. Am J Pathol 2003. Granulysin. J Immunol 2001. A distinct pathway of cell-mediated apoptosis initiated by granulysin.