A 43-year-old man was admitted at the emergency room of the Sardjito General Hospital with a chief complaint of rash on the overall body. The ethnicity was Javanese, based on the ethnicity of parents and both grandparents. Vital sign examination showed an elevated blood pressure of 170/100 mmHg, heart rate of 80 beats/min, respiratory rate of 20/min and normal temperature.
Three days prior to the admission, he complained of fever, headache, muscle pain, and took over-the-counter medicine. Two days before the admission, rashes occurred on the face in addition to red eyes and mouth ulcers. These symptoms got worse on the day before the admission when the rash spread to the overall body. Physical examination showed erythema on the overall body (more than 30%), eye discharge, and mouth ulcers (Figure 1). Laboratory examination showed an increased leukocyte count with increased percentage of neutrophil, decreased erythrocyte counts, and markedly elevated liver enzymes and renal function tests (Table 1).
The diagnosis of SJS was established based on the skin and mucosal involvement and positive Nicolsky's sign. One month before the admission, the patient had started allopurinol and antihypertensive medication. The patient had a history of hypertension in the last year before the complaints started. The patient was administered methylprednisolone of 31.25 mg/24 h. Allopurinol was discontinued, but antihypertensive medications were continued. Dressing with NaCl 0.9% was given for 15 min twice a day. At discharge, the patient did not show either new erythematous patches or vesiculobullous lesions.
Three ml peripheral blood was sampled from both patients by using an EDTA-containing vacutainer from each patient. Blood samples were transported to the Molecular Biology Laboratory of the Faculty of Medicine, Public Health and Nursing Universitas Gadjah Mada. Buffycoat was isolated from blood samples by 1500 RPM cold centrifugation for 10 min. Further, DNA was extracted from 100 ul of buffycoat by using a QIAamp(R) DNA minikit (Qiagen, Switzerland) by following the instruction manual. The quality of extracted DNA was analyzed through DNA electrophoresis with the objective to observe single genomic DNA as well as 260/280 nm absorbance ratio of >=1.75 by using a spectrophotometer (DeNovix DS-11). The quantity of DNA was obtained through multiplication of 50 ug/ml x 260 nm spectrophotometer read-out x dilution factor. Further, purified DNA was stored in a -20 C freezer until used. Purified DNA was transported in cold chain to the School of Medicine and Health Sciences, Atma Jaya Catholic University of Indonesia, for further downstream genomic analysis.
 HLA-B*58:01 was determined by using the ExProbe  HLA B*58:01 Typing Kit (TBG Biotechnology Corp., Taipei, Taiwan) following its instruction manual. The detection system was based on the use of a real-time quantitative polymerase chain reaction (qPCR) technique containing primer mixes and SYBR green dyes. The presence of amplification is detected by activation of fluorescence and a positive indication of the existence of allele specific sequence within the genomic DNA. An internal control primer pair that amplifies a conserved region of the housekeeping gene, cystic fibrosis gene, was included in the PCR reaction mix, and the PCR product of the internal control primer pair serves as an indication of the integrity of the PCR reaction. A negative control was also included in the kit, which should provide a negative result after the completion of the PCR.
For each PCR reaction, a mix of 4.5 ul primer mix, 10.5 ul qPCR buffer mix, and 3 ul of template DNA was made, containing either positive or negative controls or purified DNA at concentrations ranging from 5 to 40 ng/mul. The thermal cycling was run using the BioRad CFX96 Real Time Detection System programmed as follows: one cycle of heating at 95 C for 3 min, 36 cycles of denaturation at 93 C for 30 s, annealing at 62 C for 45 s, and elongation at 72 C for 40 s. An additional step done for data collection included recording Melt Curve between 65 C and 95 C for 15 s, at increments of 0.5 C. Upon run completion, the melt peak threshold was set at 40 -dRFU/dT to determine melt temperature when peaks were present.
Indication of the HLA-B*58:01 allele was observed as the presence of peaks in the melt curve plot profile of the internal control and target allele region at 73.5 C-79 C and 88.5 C-90 C, respectively, or a single peak at 88.5 C-90 C. Whereas the absence of HLA-B*58:01 is shown by a presence of peak at 73.5 C-79 C denoting internal control only, but not the target allele region. HLA-B*58:01 typing of both samples showed positive results (Figures 2, 3).
Ethical approval was obtained from the Medical Research Ethics Committee at Universitas Gadjah Mada no KE/FK/1150/EC. Written consent was obtained from each patient after receiving adequate information about the study.