Laboratory and clinical aspects of human papillomavirus testing

Figures & data

Figure 1.  Key targets of human papillomavirus genome for detection.

Table 1.  Parameters for assessing the clinical performance of HPV tests in cervical cancer screening.

Terminology	Characteristics to measure
Sensitivity^Citation1	The proportion (expressed as percent) of patients with a certain disease (e.g. CIN III or invasive cervical cancer) who have a POSITIVE HPV test. E.g., a sensitivity of 95% for CIN III means that 5 of 100 CIN III cases will be NEGATIVE (false-negative) and will be missed by this test. Sensitivity is affected mainly by the test itself and to some extent by the nature of samples submitted for testing.
Specificity^Citation2	The proportion of subjects (expressed as percent) without a certain disease (e.g. CIN II or higher severity) who have a NEGATIVE HPV test. E.g., a specificity of 70% means that if 100 women without CIN II or higher severity participate in the screening, 30 will be POSITIVE (false-positive) by the test. Specificity is affected mainly by the test itself and to some extent by the nature of samples submitted for testing.
Positive predictive value	The chance (expressed as percent) that a POSITIVE HPV test result indicates the presence of a certain disease (e.g. invasive cervical cancer). In the context of cervical screening, this predictive value can be further divided into predictive value for the current situation or in the next 5 or 10 years. The positive predictive value of a test depends mainly on the prevalence of the disease in the target population. Cancer screening tests, including HPV, are bound to have low positive predictive value when applied to the general population.
Negative predictive value	The chance (expressed as percent) that a NEGATIVE HPV test result excludes the presence of a certain disease (e.g. CIN II or higher severity).Under the context of cervical screening, this predictive value can be further divided into predictive value for current situation or in the next 5 or 10 years. The negative predictive value of a test depends mainly on the prevalence of the disease in the target population.

¹The definition presented here refers to “clinical” sensitivity. From the laboratory performance point of view, the concept of analytical sensitivity is often used, which means the lowest amount of analyte required in the specimen to generate a positive result, (also referred to as detection limit, e.g. 100 copies of viral DNA). For the HPV test, increasing the analytical sensitivity may not always increase the clinical sensitivity, but usually specificity is lost.

²From the laboratory performance point of view, “specificity” means cross-reaction with similar targets. This is more relevant for assays intended for HPV typing. e.g. an assay with poor specificity may misidentify an HPV 58–positive sample as HPV 33.

Table 2.  Commercially available HPV tests.

Molecular target	Type differentiation	Test name	Principle	Manufacturer
HPV DNA (full genome)	High-risk types as a group, not type-differentiating	Hybrid Capture 2 HPV DNA Test	Hybridization	QIAGEN, Gaithersburg
		CareHPV Test	hybridization	QIAGEN, Gaithersburg
HPV DNA (L1 ORF)	High-risk types as a group, not type-differentiating	Amplicor HPV Test	PCR	Roche, Branchburg
		Cervista HPV HR Test^Citation1	Hybridization (Invader)^Citation2	Hologic, Madison
HPV DNA (L1 ORF)	Differentiate 13 or more high-risk types	CLART	Reverse line-blot Hybridization on PCR products	Genomica, Coslada
		INNO-LiPa HPV Genotyping	Reverse line-blot Hybridization on PCR products	Innogenetics, Gent
		Linear Array HPV Genotyping Test	Reverse line-blot Hybridization on PCR products	Roche, Branchburg
		Digene HPV Genotyping RH Test (RUO)	Reverse line-blot Hybridization on PCR products	Digene, Hilden
HPV DNA (E1 ORF)	Differentiate 13 or more high-risk types	Infiniti HPV-HR QUAD Assay	Microarray on PCR products	Autogenomics, Carlsbad
		PapilloCheck	Microarray on PCR products	Greiner Bio-one, Frickenhausen
HPV DNA (L1 ORF)	Limited type differentiation – HPV 16/18	Cervista HPV 16/18 Test^Citation1	Hybridization	Hologic, Madison
		CORBAS 4800 HPV Test	Real-time PCR	Roche, Pleasanton
		Real Time High Risk (HR) HPV Test	Real-time PCR	Abbott, Des Plaines
HPV E6/E7 mRNA	14 high-risk types as a group, not type-differentiating	APTIMA HPV Assay	TMA	GenProbe, San Diego
HPV E6/E7 mRNA	Limited type differentiation – HPV 16/18/31/33/45	NucliSENS EasyQ HPV^Citation3	NASBA	BioMerieux, Marcy-l’Étoile
		PreTect HPV-Proofer^Citation3	NASBA	Norchip, Klokkarstua

NASBA = nucleic acid sequence-based amplification; TMA = transcription-mediated amplification.

¹Also targets E6 and E7.

²The “Invader” reaction involves two simultaneous isothermal reactions. A primary reaction is based on hybridization with two sequence-specific oligos to the same target, creating a single-nucleotide overlap. The overlap together with its 5′ flap will be cleaved. In the secondary reaction, the cleaved flap combines with a fluorescence resonance energy transfer (FRET) probe that generates a fluorescent signal. As a result, each released 5′ flap from the primary reaction cycles on and off the FRET probes, enabling the secondary reaction to further amplify the target-specific signal to 1–10 million-fold.

³Same technology marketed under different brand names in different countries.

Figure 2.  Natural history and management of cervical HPV infection.

Table 3.  Commonly used primers targeting the L1 region of HPV genome.

Primer name	Primer sequences	Remarks	References
MY09/11	Forward (5′-GCMCAGGGWCATAAYAATGG-3′) Reverse (5′-CGTCCMARRGGAWACTGATC-3′)	One of the commonly used first generation primers.Target a ~450-bp fragment which is usually too long for formalin-fixed tissues.The amplified product allows type identification by restriction fragment length polymorphisms, hybridization with type-specific probes or by sequencing.	143
PGMY09/11	PGMY11-A (5′-GCA CAG GGA CAT AAC AAT GG-3′)PGMY11-B (5′-GCG CAG GGC CAC AAT AAT GG-3′)PGMY11-C (5′-GCA CAG GGA CAT AAT AAT GG-3′)PGMY11-D (5′-GCC CAG GGC CAC AAC AAT GG-3′)PGMY11-E (5′-GCT CAG GGT TTA AAC AAT GG-3′)PGMY09-F (5′-CGT CCC AAA GGA AAC TGA TC-3′)PGMY09-G (5′-CGA CCT AAA GGA AAC TGA TC-3′)PGMY09-H (5′-CGT CCA AAA GGA AAC TGA TC-3′)PGMY09-I (5′-GCC AAG GGG AAA CTG ATC-3′)PGMY09-J (5′-CGT CCC AAA GGA TAC TGA TC-3′)PGMY09-K (5′-CGT CCA AGG GGA TAC TGA TC-3′)PGMY09-L (5′-CGA CCT AAA GGG AAT TGA TC-3′)PGMY09-M (5′-CGA CCT AGT GGA AAT TGA TC-3′)PGMY09-N (5′-CGA CCA AGG GGA TAT TGA TC-3′)PGMY09-P (5′-GCC CAA CGG AAA CTG ATC-3′)PGMY09-Q (5′-CGA CCC AAG GGA AAC TGG TC-3′)PGMY09-R (5′-CGT CCT AAA GGA AAC TGG TC-3′)	An improved version of MY09/MY11 targeting the same 450-bp fragment.Use in the Linear Array HPV Genotyping Test (Roche, Branchburg), where the HPV types are identified by hybridization with type-specific probes.	144
GP5+/6+	Forward (5′−TTT GTT ACT GTG GTA GAT ACT AC-3′) Reverse (5′−GAA AAA TAA ACT GTA AAT CAT ATT C-3′)	Targets a ~150-bp fragment suitable for formalin-fixed tissues.Commonly used in large-scale epidemiological studies.Reported to have sub-optimal sensitivity for a variant of HPV 52 commonly found in East Asia due to mutation at the primer binding site.	145,153
SPF	Forward primer (5′−GCI CAG GGI CAT AAC AAT GG-3′)Two reverse primers:(5′−GTI GTA TCI ACA ACA GTA ACA AA-3′) and (5′−GTI GTA TCI ACA TCA GTA ACA AA-3′)	Targets a short (~65-bp) fragment.Good for samples with fragmented DNA such as formalin-fixed tissues.Modified versions include SPF6 and SPF10 with the addition of more primers.SPF10 primers are used in the INNO-LiPa HPV Genotyping assay, but sequences not disclosed.	146
L1F/L1R	Forward (5′−CGT AAA CGT TTT CCC TAT TTT TTT-3′) Reverse (5′−TAC CCT AAA GAC CCT ATA CTG-3′)	Targets a ~255-bp fragment, a length that may not be retained in formalin-fixed tissues.	147

Figure 3.  Improved positive predictive value of HPV test by refining the target population. Figure shows the age-specific prevalence of high-risk HPV infection and age-specific distribution of cervical cancer cases in Hong Kong¹⁹⁶. Restricting HPV test to women aged ≥ 35 years avoids the age-related infection peak, and covers women with higher incidence of cervical cancer. By setting the testing population to women aged ≥ 35 years (in case of Hong Kong as used in this example) will therefore improve the positive predictive value of HPV testing for cervical cancer.

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