5 μl of SuperScript III RT/Platinum Taq Mix (SuperScript III Platinum SYBR Green One-Step Quantitative RT-PCR

Kit, Invitrogen, Carlsbad, CA, USA). The amplification conditions consisted of reverse transcription at 50 °C for 30 min, 95 °C for 5 min for Taq inhibitor inactivation, followed by 45 cycles of 95 °C for 10 s, 54 °C for 30 s and 72 °C for 30 s. Melting curve analysis was used to confirm the specificity of the amplicons. Positive (extracted RNA from control strains of DENV1-4) and negative controls were included in each PCR run and the run only accepted if all controls gave appropriate results. The serotype of dengue virus was sought from the acute plasma specimen in all patients with serologically confirmed dengue infection using a nested RT-PCR assay,12 modified by the Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok, Thailand.19 The Panbio Dengue Early ELISA (cat. no. E-DEN01P, lot. no. 08140; Panbio, Brisbane, Queensland, Buparlisib datasheet Australia) was used to detect NS-1 antigen

in the acute plasma specimens only, following the manufacturer’s instructions. A positive specimen was defined as having >11 Panbio units; <9 Panbio units was defined as negative; and 9–11 Panbio units was equivocal and the specimen retested to confirm the result. Panbio units were calculated by first determining the assay cut-off value: the lot specific Ribociclib calibration factor was multiplied by the average absorbance result of the kit calibrator Buspirone HCl (internal control, run in triplicate). Subsequently, an index value was calculated for each patient specimen by dividing the specimen absorbance result by the cut-off value. Finally the Panbio units were determined by multiplying the index value by 10. We used the dengue IgM (Panbio: cat. no. E-DEN01 M, lot. no. 08316) and IgG (Panbio: cat. no. E-DEN02G, lot. no. 09080) antibody capture ELISAs to detect IgM and IgG antibodies in both the acute and convalescent plasma specimens, following

the manufacturer’s instructions. A positive specimen was defined as having >11 Panbio units for IgM and >22 for IgG antibodies; <9 and <18 Panbio units was defined as negative for IgM and IgG antibodies, respectively; 9–11 Panbio units was equivocal for IgM and 18–22 Panbio units was equivocal for IgG antibodies and the specimen retested to confirm the result. Panbio units were calculated as described above. Dengue infection was classified using the above described commercial serological assays as ‘confirmed’ acute dengue infection based on WHO dengue diagnostic criteria as defined in Table 1. ‘Confirmed’ acute dengue cases were those that demonstrated an IgM or IgG antibody sero-conversion based on paired serum collections.16 Patients with static IgM positivity (i.e., positive in both acute and convalescent specimens, but with no rise in Panbio units) were considered to have evidence of recent dengue infection. All statistical analyses were performed by using STATA/SE for Macintosh, version 10.