Quantification of urinary LTE4, histamine and creatinine
Levels of LTE4 and histamine were measured in urine stored at -80oC, using leukotriene quantitative ELISA (Cayman Chemical, USA) and histamine quantitative ELISA (Abcam, UK) according to the manufacturer’s instructions. Assays were performed in urine diluted at 1:5 ratio for assessment of leukotriene levels and undiluted urine for used for measurement of histamine. Urinary creatinine levels were measured in all patients and healthy individuals in order to normalize the urinary LTE4 and histamine levels using the urinary creatinine colorimetric assay (Cayman Chemical, USA). These assays were carried out and interpreted according to the manufacturer’s instructions.
Determining Viral loads and the DENV serotypes
Viral RNA from all serum samples were extracted using QIAamp Viral RNA Mini Kit (Qiagen, USA) according to manufacturer’s instructions. Reverse transcription of the extracted viral RNA to cDNA was done using a high capacity reverse transcription kit (Applied biosystems, USA). To detect and quantify the DENV, a quantitative multiplex real time PCR was carried out as previously described (Fernando et al. , 2016), using the CDC real time oligonucleotide primers, and dual labeled probes for DENV 1-4 serotypes (Life technologies, USA). The reaction was performed for 20 seconds at 95°C for initial denaturation, followed by 40 cycles, 95°C at 3 seconds and 60°C at 30 seconds. The threshold cycle value (Ct) for each reaction was determined by manually setting the threshold limit. A multiplex method was optimized to quantify the four serotypes in a single reaction and viral quantification of unknown samples was performed using the standard curve.