Table 6. Evaluation of the three optimised RT-qPCR assays using patient samples with Central Nervous System (CNS) infections.

Patients with RT-qPCR ‘positive’ test results.

Number	Age	Sex	Serology Results (PU)*			JEV RT-qPCR Results (Cq)**						Pan-flavivirus nested RT-qPCR		Sequencing Results
			Admission Serum JEV IgM	Convalescent Serum JEV IgM	CSF JEV IgM	Serum			CSF
						Pyke	NS2A	NS3	Pyke	NS2A	NS3	CSF	Serum
1	17	F	$\mathrm{Positive}\left(15.3\right)$	$\mathrm{Positive}\left(56.5\right)$	$\mathrm{Positive}\left(32.8\right)$	ND	ND	ND	Negative	$\mathrm{Positive}\left(36\right)$	$\mathrm{Positive}\left(37\right)$	Negative	Negative	JEV sequence identified◆
2	42	F	Negative (8.6)	ND	Negative (2.9)	$\mathrm{Positive}\left(31\right)$	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Non-specific amplification
3	60	M	Negative (3.9)	ND	Negative (3.5)	Negative	Negative	Negative	$\mathrm{Positive}\left(31\right)$	Negative	Negative	Negative	Negative	Non-specific amplification
4	60	M	Negative (3.6)	ND	Negative (3.6)	$\mathrm{Positive}\left(40\right)$	Negative	Negative	Negative	Negative	Negative	ND	ND	Non-specific amplification
5	58	F	$\mathrm{Positive}\left(15.9\right)$	ND	Negative (3.4)	Negative	Negative	Negative	$\mathrm{Positive}\left(34\right)$	Negative	Negative	ND	ND	Non-specific amplification
6	-	-	$\mathrm{Positive}\left(54.6\right)$	ND	$\mathrm{Positive}\left(80.8\right)$	Negative	Negative	Negative	$\mathrm{Positive}\left(40\right)$	Negative	Negative	Negative	Negative	Non-specific amplification
7	-	M	Negative (2.5)	ND	Negative (2.6)	Negative	Negative	Negative	$\mathrm{Positive}\left(32\right)$	Negative	ND	ND	ND	Non-specific amplification
8	12	M	Negative (6.8)	ND	$\mathrm{Positive}\left(57.2\right)$	Negative	Negative	Negative	$\mathrm{Positive}\left(39\right)$	$\mathrm{Positive}\left(35\right)$	Negative	$\mathrm{Positive}$	ND	JEV sequence identified◆
9	26	M	Negative (5.8)	ND	Negative (3.3)	Negative	Negative	-	$\mathrm{Positive}\left(36\right)$	Negative	Negative	ND	ND	Non-specific amplification
10	27	M	Negative (4.6)	ND	Negative (3.6)	$\mathrm{Positive}\left(28\right)$	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Non-specific amplification
11	33	M	Negative (3.2)	ND	Negative (2.8)	$\mathrm{Positive}\left(40\right)$	Negative	Negative	Negative	Negative	Negative	ND	ND	Non-specific amplification
12			Negative (6.4)	ND	Negative (4.9)	Negative	Negative	Negative	$\mathrm{Positive}\left(36\right)$	Negative	Negative	ND	ND	Non-specific amplification
13	-	-	$\mathrm{Positive}\left(27.8\right)$	ND	$\mathrm{Positive}\left(29.9\right)$	Negative	Negative	Negative	$\mathrm{Positive}\left(32\right)$	Negative	Negative	Negative	Negative	Non-specific amplification

* JEV-Dengue IgM Combo ELISA kit (Panbio ELISA), Inverness Medical Innovations, Brisbane, Australia (formerly Panbio Ltd.). PU = Panbio Unit, calculated from OD according to manufacturer’s instruction. JEV IgM Positive, Negative or Equivocal cut-offs calculated according to the manufacturer’s instructions.

** Pyke, NS2A and NS3 JEV RT-qPCR assays were reported as negative if the Cq>40 or not detected.

^◆ JEV sequence identified as part of a previous study, for Patient 1 by sequencing following cell culture, and for Patient 2 by sequencing of the hemi-nested qPCR product.

ND Not done.