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.