TABLE 2.

Test performance of the second-generation genomic-based assay for HPV16 DNA in HPV-OPC

Test characteristics	Analyte	Primer–probe region
		E7	HR E5L 2-4	BR E1-5
Limit of detection—Plasma (cp/mL)	pATCC HPV16	1.5	1.5	1.5
	SiHa	13	20	22
Limit of detection—Salivary rinse (cp/mL)	pATCC HPV16	3	2	3
	SiHa	23	19	12
Measuring range—Plasma (cp/mL)	pATCC HPV16	2.0-8.5 log10	2.0–8.5 log10	2.0–8.5 log10
	SiHa	2.0-5.0 log10	2.0-6.5 log10	2.0–6.5 log10
Measuring range—Salivary rinse(cp/mL)	pATCC HPV16	2.0–8.5 log10	2.0–8.5 log10	2.0–8.5 log10
SiHa	2.0–6.5 log10	2.0–6.5 log10	2.0–6.5 log10
Precision—Plasma (% CV)	pATCC HPV16
	2.0 E6 cp/mL	11%	28%	7%
	2.0 E4 cp/mL	8%	20%	13%
	2.0 E2 cp/mL	25%	24%	19%
	SiHa	5.0 E4 cp/mL, 10%	2.0 E5 cp/mL, 26%	2.0 E5 cp/mL, 24%
		1.0 E2 cp/mL, 11%	2.0 E3 cp/mL, 55%	2.0 E3 cp/mL, 23%
			2.0 E1 cp/mL, 65%	2.0 E1 cp/mL, 32%
Total precision—Salivary rinse (% CV)	pATCC HPV16
	8.4 E6 cp/mL	35%	39%	54%
	8.4 E4 cp/mL	50%	64%	50%
	8.4 E2 cp/mL	53%	57%	40%
	SiHa
	2.0 E5 cp/mL	31%	21%	24%
	2.0 E3 cp/mL	50%	33%	43%
	2.0 E1 cp/mL	62%	42%	65%
Accuracy—Plasma blind panel	Positive % agreement	100	100	100
	Negative % agreement	90	100	100
	Total % agreement	97	100	100
Accuracy—Salivary rinses from HNSCC cases	N = 28 salivary rinse DNAs tested	The Triple Test (E7 + HR E5L 2-4 + BR E1-5 results) detected HPV16 DNA in 27/28 (96%) of salivary rinses
Specificity	No positive results were obtained for any of the three primer/probe sets for HIV-1, EBV, Coxsackie A9, echovirus 11, adenovirus, influenza A virus, herpes simplex virus type 1, herpes simplex virus type 2, cytomegalovirus, BK virus, JC virus, human metapneumovirus, respiratory syncytial virus, human rhinovirus, HCV, HBV, parvovirus B19
Reference range	All positive results are reportable