Journal of Clinical Virology 51 (2011) 3843

Contents lists available at ScienceDirect

Journal of Clinical Virology

journal homepage: www.elsevier.com/locate/jcv

Analytical performance of Cervista HPV 16/18 genotyping test for cervical

cytology samples
Deborah A. Bartholomew a, , Ronald D. Luff b,1 , Neil B. Quigley c,2 , Michelle Curtis d,3 , Marilyn C. Olson d,4
a

Department of Obstetrics and Gynecology, The Ohio State University College of Medicine, 4053 West Dublin Granville Road, Dublin, OH 43017, USA
Director Anatomic Pathology for Clinical Trials, Quest Diagnostics, 1 Malcolm Avenue, Teterboro, NJ 07860, USA
VP, Geneuity CRS (a subsidiary of MPLN, Inc.), 250 East Broadway, Maryville, TN 37804, USA
d
Research and Development, Hologic Inc., 502 South Rosa Road, Madison, WI 53719, USA
b
c

a r t i c l e

i n f o

Article history:
Received 17 August 2010
Received in revised form 14 January 2011
Accepted 22 January 2011
Keywords:
HPV
Genotyping cervical cancer
Sensitivity
Specicity
Cross-reactivity

a b s t r a c t
Background: Human papillomavirus (HPV) types 16 and 18 are the 2 most frequent types associated with
cervical cancer. Identifying their presence or absence in cervical samples may assist in triaging women
for subsequent management. The Cervista HPV 16/18 genotyping test specically detects the presence
of HPV 16 and 18 in ThinPrep cervical specimens.
Objectives: The objective was to establish the analytical performance of the CERVISTA HPV 16/18 genotyping test.
Study design: These studies were performed in support of a regulatory submission to the US Food and Drug
Administration. Here we report the analytical sensitivity (limit of detection), accuracy compared to consensus L1 gene PCR/bi-directional sequencing, precision, reproducibility, and cross-reactivity (specicity)
of the genotyping test.
Results: Analytical sensitivity for detection of HPV 16 and 18 ranged between 625 and 1250
copies/reaction for both types. When compared to PCR/sequencing for women with atypical squamous
cells of undetermined signicance cytology, the positive percent agreement was 94.1% (95% condence
interval [CI], 89.896.7) and the negative percent agreement was 85.7% (95% CI, 82.488.4). The test
demonstrated high within-laboratory and inter-operator precision. Reproducibility within sites and
between 3 testing sites resulted in 100% agreement with expected results (150 positive, 90 negative
results). The genotyping test did not exhibit cross-reactivity to DNA from common low-risk HPV types
and other microorganisms found in the human female reproductive tract.
Conclusions: These analytical performance data support the use of CERVISTA HPV 16/18 genotyping test
for the detection and differentiation of HPV 16 and 18 in ThinPrep cervical cytology specimens.
2011 Elsevier B.V. All rights reserved.

1. Background

Abbreviations: HPV, human papillomavirus; HR, high-risk; ASC-US, abnormal

squamous cells of undetermined signicance; CIN, cervical intraepithelial neoplasia;
LSIL, low-grade squamous intraepithelial; HIST2H2BE, human histone 2, H2be; FOZ,
fold over zero; gDNA, genomic DNA; LoD, limit of detection; LoB, limit of blank;
CLSI, Clinical Laboratory Standards Institute; PCR, polymerase chain reaction; CI,
condence interval.
Corresponding author. Tel.: +1 614 293 9899/764 0172; fax: +1 614 889 6937.
E-mail addresses: Deborah.Bartholomew@osumc.edu (D.A. Bartholomew),
ronald.d.luff@QuestDiagnostics.com (R.D. Luff), nquigley@geneuity.com (N.B.
Quigley), michelle.curtis@hologic.com (M. Curtis), marilyn.olson@hologic.com
(M.C. Olson).
1
Tel.: +1 201 393 6007; fax: +1 201 462 4772.
2
Tel.: +1 865 273 1126; fax: +1 865 273 1123.
3
Tel.: +1 608 273 8933x8244; fax: +1 608 273 8718.
4
Tel.: +1 608 273 8933x8113.
1386-6532/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jcv.2011.01.016

There are more than 100 known types of human papillomavirus

(HPV), of which at least 13 appear to confer high-risk (HR) for cervical carcinogenesis: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59,
and 68.1,2 In most cases, HPV infection clears within 2 years by the
womans immune system, although some infections persist and can
lead to neoplastic progression.3,4 Both persistence of HPV infection and genotype of the infecting virus correlate with subsequent
diagnosis of cervical intraepithelial neoplasia (CIN),46 as well as
progression from CIN to cervical cancer.7 HPV 16 and 18 are responsible for approximately 70% of all cervical cancers worldwide; type
16 accounts for more than half of all cases and approximately 18%
of cases are associated with type 18.8 One large prospective cohort
study demonstrated that women with normal cytology who were
infected with HPV 16 or 18 had a higher 10-year cumulative incidence rate of CIN 3 than women with other HR HPV types or who
tested HPV-negative.9

D.A. Bartholomew et al. / Journal of Clinical Virology 51 (2011) 3843

39

Table 1
Organisms tested for cross-reactivity with the Cervista HPV 16/18 genotyping test.
Organisms added to PreservCyt solutiona

Puried DNA added to test reaction

Cloned DNA or PCR amplicons added to test reaction

Candida albicans
Corynebacterium pseudodiptheriticum
Enterococcus faecalis
Escherichia coli
Lactobacillus acidophilus
Proteus vulgaris
Staphylococcus aureus
Staphylococcus epideridis
Streptococcus mitis
Streptococcus pyogenes
Chlamydia trachomatis
Neisseria gonorrhoeae
Neisseria meningitides
Mycoplasma hominis

HSV, type 1
HSV, type 2
C. trachomatis
N. gonorrhoeae
N. meningitides
M. hominis

HPV types
1a, 6, 11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 53, 56,
58, 59, 66, 67, 68, and 70
HIST2H2BE
HIV type 1 (pol and env regions)

HPV = human papillomavirus; HSV = herpes simplex virus; PCR = polymerase chain reaction; HIST2H2BE = human histone 2, H2be; HIV = human immunodeciency virus.
a
These organisms were added to PreservCyt solution containing 100,000 Jurkat cells/mL.

Evidence suggests that testing for the presence of HR HPV

is an important component of cervical cancer screening programs when used in conjunction with cervical cytology for
preventing invasive cervical cancer.10 The American Society for
Colposcopy and Cervical Pathology advises that it is reasonable to
test for specic HPV types in HR HPV-positive/cytology-negative
women, and a positive test for HPV 16 or 18 should lead to
colposcopy.11 If HR HPV-positive, but negative for HPV 16 or
18, a conservative approach may postpone colposcopy until the
HR HPV infection is shown to be persistent in these patients,12
whereas a positive result for HPV 16 and/or 18 in women with
ASC-US or LSIL cytology may route these women directly to
colposcopy.

2. Objectives
The Cervista HPV 16/18 genotyping test (Hologic, Inc.; Marlborough, MA, USA) is approved by the US Food and Drug
Administration for adjunctive use with the Cervista HPV HR test
(Hologic, Inc.), in combination with cervical cytology screening, in
women 30 years and older. Here we report the analytical performance parameters of the CERVISTA HPV 16/18 genotyping test.

3. Study design
3.1. Samples
CERVISTA HPV 16/18 genotyping test was performed on cervical specimens collected in PreservCyt (Hologic, Inc.) solution, the
ThinPrep Pap Test (Hologic, Inc.) preservation system. A portion
of the cervical specimens was collected as part of a multicenter,
prospective clinical study. The study was conducted in the United
States, with participation from 89 sites across 22 states. All cervical samples (prospective collection and residual/remnant samples)
were collected under protocols reviewed and approved by institutional review boards from each participating site. Additional
samples included HPV-positive (SiHa and HeLa) and -negative
(Jurkat) cell cultures in PreservCyt solution and plasmids containing cloned HPV DNA. The genotyping test was analyzed against
cloned DNA from plasmids containing HPV types of low or undetermined risk (6, 11, 42, 43, 44, 53, 67, and 70) at 1 105 and 1 107
copies/reaction. For all experiments reported here, DNA was isolated from 2 mL using the GenndTM DNA Extraction kit (Hologic,
Inc.).13 Residual DNA from the clinical study samples extracted as
part of the CERVISTA HPV HR test was used for the CERVISTA HPV
16/18 genotyping test.

3.2. CERVISTA HPV 16/18 test

The CERVISTA HPV 16/18 genotyping test is a qualitative, in vitro
diagnostic test for the detection of DNA from HPV types 16 and 18.
The test uses the Invader chemistry (Hologic, Inc.), a signal amplication method for detecting specic nucleic acid sequences.14
As described previously, this method utilizes a primary reaction
that occurs on the targeted DNA sequence and a secondary reaction that produces a uorescent signal. Both types of reactions
rely on oligonucleotide hybridization, invasive structure formation,
and cleavage by the Cleavase enzyme (Hologic, Inc.).15 The genotyping test is designed to target multiple regions in the HPV 16
and 18 DNA genome including L1, E6, and E7, as well as human
histone 2, H2be (HIST2H2BE) DNA that serves as an internal control for detection of cellular DNA. A signal to noise value (sample
uorescence signal divided by the uorescence signal from a notarget control) is referred to as fold over zero (FOZ). A positive
result for HPV 16, HPV 18, or HPV 16 and 18 occurs when the FOZ
value is at or above an empirically derived cutoff value of 2.13. If
the FOZ values are below this cutoff, then the samples are considered negative. To demonstrate that the testing procedure has
been properly performed and sample genomic DNA (gDNA) was
present in sufcient quantity, the HIST2H2BE FOZ values (or average gDNA FOZ) must lie at or above an empirically derived cutoff
value of 1.5. Samples that generate average gDNA FOZ values below
1.5 are considered indeterminate in the absence of a positive HPV
signal.

3.3. Analytical sensitivity

The limit of detection (LoD) and limit of blank (LoB) were
determined according to the methods in the Clinical Laboratory
Standards Institute (CLSI) document, EP17-A.16 Nine characterized HPV 16- and 18-negative DNA samples isolated from cervical
specimens were tested in replicates of 8 (9 samples 8 replicates/sample = 72 data points) to determine the LoB. The LoB
represents the 95th percentile of HPV 16 and 18 FOZ values
obtained from a negative sample data set, when these FOZ values
are ranked from lowest to highest. Individual LoD were calculated for both HPV types using the LOB and the mean variance
of contrived positive samples. The LoD was computed as the HPV
copy number for which 5% of the FAM FOZ values fall at or below
the LoB. If exactly 5% was not observed, the LoD was described
as a range between the 2 closest copy number values. Cloned
HPV plasmid DNA, representing HPV 16 and 18, was tested at
concentrations of 625, 1,250, 2,500, and 5,000 copies per reaction, each in a background of 10, 100, 1,000 ng of Jurkat cell line

40

D.A. Bartholomew et al. / Journal of Clinical Virology 51 (2011) 3843

Table 2
Cross-reactivity assessment of the Cervista HPV 16/18 genotyping test with HR
HPV-cloned DNA samples.
Sample

HPV 16
HPV 16
HPV 18
HPV 18
HPV 31
HPV 31
HPV 31a
HPV 31a
HPV 31b
HPV 31b
HPV 31b
HPV 33
HPV 33
HPV 35
HPV 35
HPV 39
HPV 39
HPV 45
HPV 45
HPV 51
HPV 51
HPV 52
HPV 52
HPV 56
HPV 56
HPV 58
HPV 58
HPV 59
HPV 59
HPV 66
HPV 66
HPV 68
HPV 68

Copies/reaction

1 105
1 107
1 105
1 107
1 105
1 107
1 107
1 107
5 105
1 106
5 106
1 105
1 107
1 105
1 107
1 105
1 107
1 105
1 107
1 105
1 107
1 105
1 107
1 105
1 107
1 105
1 107
1 105
1 107
1 105
1 107
1 105
1 107

Table 3
Cross-reactivity assessment of the Cervista HPV16/18 genotyping test with cervical
and cell line samples.
Samplea

HPV 16

HPV 18

FOZ

FOZ

Assay result

12.09
12.65
0.97
0.99
1.02
2.06
2.33
1.06
1.13
1.60
2.00
0.89
0.93
0.93
0.92
0.96
0.96
0.82
0.90
0.92
0.92
0.92
0.95
0.94
0.96
0.93
0.91
0.92
0.92
0.94
0.92
0.95
0.96

1.00
0.99
15.43
16.70
0.99
0.96
1.01
1.02
0.98
1.02
1.00
0.91
0.90
0.92
0.92
0.96
0.95
0.96
1.02
0.95
0.93
0.93
0.94
0.94
0.97
0.99
0.93
0.91
0.92
0.92
0.91
0.94
0.99

Positive 16
Positive 16
Positive 18
Positive 18
Negative
Negative
Positive 16
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative

HR = high-risk; HPV = human papillomavirus; FOZ = fold over zero.

a
Repeat testing.
b
Additional dilutions prepared and tested.

gDNA per reaction. All positive samples were tested in replicates

of 8, resulting in 24 replicates per HPV plasmid copy number
used.
3.4. Cross-reactivity
As previously described,15 17 organisms commonly found in the
female anogenital tract, as well as several HPV types of high, low, or
undetermined risk (Table 1), were tested to assess potential crossreactivity. Additional tests for cross-reactivity utilized 36 residual
cervical DNA samples, determined by polymerase chain reaction
(PCR) and sequencing to contain various HPV types, as well as 4 cell
line samples: Jurkat (HPV-negative), SiHa (HPV 16-positive), Hela
(HPV 18-positive), and ACCURUN 370 (SeraCare Life Sciences),
consisting of a mixture of HPV 16-positive cells and HPV-negative
cells (Table 3).
3.5. Comparison with PCR/sequencing
The analytical performance of the HPV HR and 16/18 genotyping tests for 1,354 cervical specimens from women with ASC-US
cytology was measured against PCR/sequencing, as previously
described.15 In brief, residual DNA samples were amplied using
PGMY consensus primers for the HPV L1 gene.17 A portion of the
human beta-globin gene was amplied as an internal control in the
same reaction. Puried amplicons were used as sequencing templates in multiple sequencing reactions for HPV 16 and 18. The
sequencing data were analyzed using sequence alignment software.

S1
S2
S3
S4
S5
S6
S7
S8
S9
S10
S11
S12
S13
S14
S15
S16
S17
S18
S19
S20
S21
S22
S23
S24
S25
S26
S27
S28
S29
S30
S31
S32
S33
S34
S35
S36
HeLa-100 ng
HeLa-1000 ng
SiHa-100 ng
SiHa-1000 ng
Jurkat-100 ng
Jurkat-1000 ng
Accurun 370 control

Sequencing

HPV 16

HPV 18

Result

FOZ

FOZ

Cervista HPV
16/18 result

HPV 16
HPV 16
HPV 16
HPV 16
HPV 18
HPV 18
HPV 18
HPV 31
HPV 31
HPV 31
HPV 35
HPV 39
HPV 39
HPV 45
HPV 45
HPV 51
HPV 51
HPV 51
HPV 52
HPV 56
HPV 58
HPV 66
HPV 66
HPV 68
HPV 43
HPV 53
HPV 53
HPV 70
HPV 6
HPV 6
HPV 6
HPV 42
HPV 42
HPV 42
HPV 53
HPV 44
HPV 18
HPV 18
HPV 16
HPV 16
Negative
Negative
HPV 16

6.39
12.33
12.09
12.05
0.88
0.84
0.89
0.81
1.17
1.35
0.98
0.92
0.87
0.99
0.99
0.82
1.15
0.78
1.01
0.83
0.88
1.03
0.81
0.89
0.92
0.78
0.82
0.83
0.85
0.77
0.90
0.79
0.85
0.82
1.02
0.86
1.10
0.97
11.62
10.78
1.03
0.90
5.67

0.90
0.94
0.97
1.11
8.40
15.88
16.54
0.91
0.87
0.90
0.91
0.98
0.98
1.10
1.17
0.84
0.92
0.82
0.95
0.91
0.90
0.94
0.90
0.87
0.85
0.79
0.83
0.84
0.83
0.78
0.90
0.85
0.83
0.88
0.87
0.84
15.98
15.37
1.12
1.12
1.00
0.83
0.98

Positive 16
Positive 16
Positive 16
Positive 16
Positive 18
Positive 18
Positive 18
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Positive 18
Positive 18
Positive 16
Positive 16
Negative
Negative
Positive 16

HPV = human papillomavirus, FOZ = fold over zero.

a
Samples S1S36 are cervical cell DNA samples characterized by sequencing.

3.6. Precision and reproducibility

The within-laboratory precision of the HPV 16/18 genotyping
test was assessed according to the parameters set forth in CLSI EP5A2.18 The total number of measurements per sample was 84 (21
days 2 runs/day 2 replicates/run). Precision values were calculated for each target at each concentration tested.
Reproducibility of the HPV 16/18 genotyping test was assessed
at 3 external sites using 8 cell line samples (SiHa, HeLa, and Jurkat)
and 8 pooled HPV-positive and -negative cervical cytology specimens. The total number of measurements for each sample was 15
(3 sites 5 days 1 run/day). Two lots of the HPV 16/18 genotyping
kits and 3 lots of Gennd DNA Extraction Kits were used across
the 3 sites for the study.
4. Results
The LoD was 1.34 (0.10) FOZ for HPV 16 and 1.33 (0.07) FOZ
for HPV 18. The LoB values were 1.18 and 1.21 (FOZ) for HPV 16 and
18, respectively. As described in the CLSI guideline, the LoD for each

0
0
0
4
0
0
0
1
0
0
0
8

0
0
1
0
0

0
0
0
3

6
0
0
2
0

0
1
0
31

0
0
3
0
0

0
0
0
31

0
0
0
10

1
0
0
27
2

0
0
0
2

0
9
1
0
0

0
10
1
402

29
0
1
1
0

0
0
0
29

0
0
2
0
0
41
7
1
335
7

2
9
0
115

0
27
0
2
0

3
510
5
640

96
0
2
6
0
CERVISTA HPV HR negative

HPV 16 positive
HPV 16 & 18 negative
HPV 16 and/or 18 IND
Total

1
35
1
78

25
0
1
95
1
CERVISTA HPV HR positive
HPV 16 positive
7
HPV 18 positive
0
HPV 16 & 18 positive
0
HPV 16 & 18 negative
32
HPV 16 and/or 18 IND
2

HPV = human papillomavirus; PCR = polymerase chain reaction; ASC-US = atypical squamous cells of undetermined signicance; HR = high-risk; IND = indeterminate.
a
A PCR/sequencing HR IND result signies that there was evidence for the presence of HR HPV, but not substantial enough to meet the quality requirements (at least 2 sequencing reads from 2 independent amplicons) to
report the presence of a specic type.

205
43
13
503
12

6
565
7
1,354

41

0
0
1
3
0

Others
18 & others
16 & others

Multiple HR HPV Types

16, 18, & other

Others
18 & others
16 & others

2 HR HPV types

16 & 18
18

Others

One HR HPV type

16

HR negative
HR INDa

PCR sequencing
CERVISTA result

Table 4
Performance of Cervista HPV 16/18 genotyping test and PCR/sequencing results in women with ASC-US cytology.

target in relation to copy number is where 5% of the values fall at or

below the LoB. If exactly 5% does not occur, the LoD is described as
a range between the 2 closest copy number values. For HPV 16 and
18, 29% and 33%, respectively, of the FOZ values were below the LoB
at 625 copies, and 0% and 4% were below the LoB at 1,250 copies,
respectively. Therefore, the LoD was determined to fall within 625
to 1,250 copies/reaction. The positive cutoff for the test (FOZ value
of 2.13) was based on targeting a 5% positive rate in the negative for intraepithelial lesion or malignancy (NILM) population of a
multicenter clinical study.19 This was done by taking the 95th percentile of the maximum HPV 16 and 18 FOZ values for subjects 30
with NILM cytology. Based on this analysis, a FOZ value of 2.13 was
selected as the positive cutoff value for the test. This value would
correspond to approximately 2,500 copies per reaction.
The CERVISTA HPV 16/18 genotyping test did not exhibit any
cross-reactivity to the panel of puried viruses or bacteria, cloned
DNA from plasmids containing the HIST2H2BE sequence, or HPV
types of low or undetermined risk (6, 11, 42, 43, 44, 53, 67, and 70) at
1 105 and 1 107 copies/reaction (data not shown). When tested
against a HR HPV panel of 14 types of HPV cloned DNA at 1 105 and
1 107 copies/reaction, HPV 16 and 18 were positive as expected
(Table 2). The other HR types produced negative results, with the
exception of HPV 31 DNA, which yielded positive HPV 16 results at
1 107 copies/reaction in 1 out of the 3 samples tested. Titration
of the HPV 31 DNA to 5 106 copies/reaction eliminated crossreactivity (Table 2). When cervical and cell line samples (S1S36)
containing HPV were tested for cross-reactivity, only samples containing HPV 16 and 18 produced positive results (Table 3). Of the 3
cervical samples tested that contained HPV 31, all 3 were negative
for HPV 16 and 18.
Cervical specimens from women with ASC-US cytology collected as part of the multicenter, prospective clinical study were
tested with both the CERVISTA HPV HR and HPV 16/18 genotyping
tests, and these results were compared to PCR/sequencing results
(Table 4). There were 725 samples with HPV HR positive results and
valid HPV16/18 genotyping and PCR/sequencing results. The overall positive percent agreement between CERVISTA HPV 16 and/or
18 results versus PCR/sequencing was 94.1% (177/188: 95% condence interval [CI], 89.896.7) and the negative percent agreement
was 85.7% (460/537: 95% CI, 82.488.4). Of the 1,354 samples,
100 samples generated discrepant HPV 16/18 results between
the 2 genotyping methods. Twenty samples were CERVISTA HPV
16/18 negative and positive for HPV 16 or 18 by PCR/sequencing,
and 80 samples were CERVISTA HPV 16/18 positive and negative for HPV 16 or 18 by PCR/sequencing. There were 6 samples
that were CERVISTA HPV 16/18 positive but CERVISTA HPV HR
negative. This could be due to the HPV 16/18 genotyping test having slightly lower analytical sensitivity compared to the HPV HR
test.
High within-laboratory precision was demonstrated for both
HPV 16 and 18 (HPV 16, Table 1, Supplementary data; HPV 18,
Table 2, Supplementary data). The total within-laboratory coefcient of variation for the FOZ values ranged from 5% to 13% for HPV
16 and 6% to 26% for HPV 18. SiHa cell mixtures tested positive for
HPV 16 as expected; positivity ranged from 5% at low concentrations to 100% at high concentrations of SiHa cells (Table 5). HeLa
cell mixtures tested positive for HPV 18 as expected; positivity
ranged from 12% at low concentrations and 95% to 100% at higher
concentrations (Table 5). The interoperator results demonstrated
good precision overall when testing HPV 16- and 18-positive
DNA samples and/or cell lines between 3 operators (Table 5 and
Tables 1 and 2, Supplementary data). Among HPV 16 and 18 cloned
DNA samples tested, all samples tested positive at concentrations
of 5,000 and 20,000 copies/reaction, while at 1,250 copies/reaction,
HPV 16 and 18 cloned DNA samples did not produce any positive
results with the 2.13 FOZ cutoff (Table 5).

Total

D.A. Bartholomew et al. / Journal of Clinical Virology 51 (2011) 3843

42

D.A. Bartholomew et al. / Journal of Clinical Virology 51 (2011) 3843

Table 5
Between-operator precision for HPV 16- and/or 18-positive samples.
Sample

Copies/reactiona or cells/mLb

HPV 16

1,250a
5,000a
20,000a
1,250a
5,000a
20,000a
2,500 SiHa/97,500 Jurkatb
5,000 SiHa/95,000 Jurkatb
20,000 SiHa/80,000 Jurkatb
1,250 HeLa/98,750 Jurkatb
2,500 HeLa/97,500 Jurkatb
10,000 HeLa/90,000 Jurkatb
10,000b
20,000b
100,000b

84
84
84
84
84
84
84
84
84
84
84
84
84
84
84

HPV 18

SiHa/Jurkat

Hela/Jurkat

Jurkat

HPV 16 positive % (n)

HPV 18 positive % (n)

Operator 1

Operator 2

Operator 3

Total

Operator 1

Operator 2

Operator 3

Total

0 (0)
100 (28)
100 (28)
0 (0)
0 (0)
0 (0)
0 (0)
82 (23)
100 (28)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)

0 (0)
100 (28)
100 (28)
0 (0)
0 (0)
0 (0)
7 (2)
100 (28)
100 (28)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)

0 (0)
100 (28)
100 (28)
0 (0)
0 (0)
0 (0)
7 (2)
100 (28)
100 (28)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)

0 (0)
100 (84)
100 (84)
0 (0)
0 (0)
0 (0)
5 (4)
94 (79)
100 (84)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)

0 (0)
0 (0)
0 (0)
0 (0)
100 (28)
100 (28)
0 (0)
0 (0)
0 (0)
7 (2)
100 (28)
100 (28)
0 (0)
0 (0)
0 (0)

0 (0)
0 (0)
0 (0)
0 (0)
100 (28)
100 (28)
0 (0)
0 (0)
0 (0)
25 (7)
100 (28)
100 (28)
0 (0)
0 (0)
0 (0)

0 (0)
0 (0)
0 (0)
0 (0)
100 (28)
100 (28)
4 (1)
0 (0)
0 (0)
4 (1)
100 (28)
86 (24)
0 (0)
0 (0)
0 (0)

0 (0)
0 (0)
0 (0)
0 (0)
100 (84)
100 (84)
1 (1)
0 (0)
0 (0)
12 (10)
100 (84)
95 (80)
0 (0)
0 (0)
0 (0)

HPV = human papillomavirus.

a
HPV 16 or HPV 18 cloned DNA at the indicated concentration (copies/reaction) mixed with 100 ng of HPV-negative genomic DNA (Jurkat).
b
Genomic DNA isolated from HPV-positive cells (SiHa and HeLa) and/or HPV-negative cells (Jurkat) at the indicated concentration (cells/mL).

Reproducibility of the HPV 16/18 genotyping test was assessed

at 3 external sites. One hundred percent of the results obtained from
each sample were in agreement with the expected results at each
test site (150 positive, 90 negative). The percent agreement results
and mean FOZ values for the same samples tested at different sites
are presented in Table 6.
5. Discussion
The CERVISTA HPV 16/18 genotyping test demonstrated a
high degree of analytical sensitivity, analytical specicity, withinlaboratory precision, and between-laboratory reproducibility. The
specicity of the test for HPV 16 and 18 is critical when considering
the utility of the test in clinical practice. Importantly, this test did
not exhibit cross-reactivity to 22 different HPV types tested. HPV
16 cross-reactivity was observed with HPV 31 at high concentration (1 107 copies/reaction). HPV 31, which is found in less than
5% of invasive cervical cancers,8 is a member of the A9 clade and
phylogenetically related to HPV 16.20 Homologous regions between
these genotypes, as well as high copy number, likely contributed
to this cross-reactivity. When HPV 31-positive cervical samples

were tested in the current study, none of these samples generated

positive HPV 16 or 18 results.
When HPV 16/18 genotyping test results were compared to
PCR/sequencing results, the overall positive and negative percent
agreement was 94% and 85%, respectively, among women with
ASC-US cytology and CERVISTA HPV HR positive results. The discrepant results between the 2 genotyping methods could be due
to differences in analytical sensitivity and limitations with the
PCR/sequencing method. There were samples positive by CERVISTA
HPV 16/18 but negative for HPV 16 and/or 18 by PCR/sequencing.
The consensus PCR method used amplies multiple HPV types,
including both HR and low-risk types.17 Samples containing HPV
16 or 18 may appear to be negative when another type of HPV
out-competes the 16 or 18 type during the logarithmic amplication process. In addition, when HPV consensus primers targeted
against HPV L1 are used for PCR, integrated forms of the virus
may not be detected due to loss of portions of L1.21,22 Other
samples were negative by CERVISTA HPV 16/18 but positive for
HPV 16 and/or 18 by PCR/sequencing, which could be due to
the PCR method detecting lower HPV copy numbers in these
samples.

Table 6
Summary of Cervista HPV 16/18 genotyping test results between sites.
Sample type and concentration (cells/mL)

5,000 SiHa/95,000 Jurkat

10,000 SiHa/90,000 Jurkat
20,000 SiHa/80,000 Jurkat
2,500 HeLa/97,500 Jurkat
5,000 HeLa/95,000 Jurkat
10,000 HeLa/90,000 Jurkat
5,000 SiHa/2,500 HeLa/12,500 Jurkat
100,000 Jurkat
Pooled cervical sample
Pooled cervical sample
Pooled cervical sample
Pooled cervical sample
Pooled cervical sample
Pooled cervical sample
Pooled cervical sample
Pooled cervical sample

Expected result

Positive 16
Positive 16
Positive 16
Positive 18
Positive 18
Positive 18
Positive 16 and 18
Negative
Positive 16
Positive 16
Positive 18
Negative
Negative
Negative
Negative
Negative

Agreement with expected result, % (n) Total (n)a

HPV 16 FOZ
(across 3 sites)

HPV 18 FOZ
(across 3 sites)

Site 1

Site 2

Site 3

Mean

SD

Mean

SD

100 (5)
100 (5)
100 (5)
100 (5)
100 (5)
100 (5)
100 (5)
100 (5)
100 (5)
100 (5)
100 (5)
100 (5)
100 (5)
100 (5)
100 (5)
100 (5)

100 (5)
100 (5)
100 (5)
100 (5)
100 (5)
100 (5)
100 (5)
100 (5)
100 (5)
100 (5)
100 (5)
100 (5)
100 (5)
100 (5)
100 (5)
100 (5)

100 (5)
100 (5)
100 (5)
100 (5)
100 (5)
100 (5)
100 (5)
100 (5)
100 (5)
100 (5)
100 (5)
100 (5)
100 (5)
100 (5)
100 (5)
100 (5)

3.049
4.933
6.345
0.833
0.847
0.883
3.047
0.899
9.769
2.782
0.958
0.905
0.888
0.865
0.919
1.049

0.473
0.598
0.553
0.073
0.083
0.076
0.387
0.048
0.658
0.611
0.154
0.078
0.097
0.127
0.093
0.130

0.927
0.963
0.831
3.645
6.121
9.322
3.815
0.927
0.861
0.921
10.413
0.896
0.892
1.146
0.927
0.921

0.031
0.043
0.043
0.455
1.105
0.831
0.435
0.042
0.053
0.035
1.945
0.049
0.053
0.121
0.029
0.050

HPV = human papillomavirus; FOZ = fold over zero; SD = standard deviation.

a
The total number of measurements for each sample was 15 (3 sites 5 days 1 run per day).

15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15

D.A. Bartholomew et al. / Journal of Clinical Virology 51 (2011) 3843

The analytical performance demonstrated by this test validates

the results obtained with properly collected, stored, and puried
samples, but does not indicate whether or not a woman has, or will
develop, precancerous lesions or cervical cancer. The clinical performance of the CERVISTA HPV 16/18 genotyping test with regard
to detection of cervical disease is reported elsewhere.23 Due to the
prevalence and increased risk of developing cervical cancer with
HPV 16 and 18 infection,5,8 knowledge of the presence of these
types may be used to triage patients and guide management in
cervical cancer screening strategies.12
Acknowledgements
The authors would like to thank Stephen P. Day, LuAnne Chehak,
Angela Hudson, Joe King, Sarah Olson, and Tamara Sander (Hologic,
Inc.), Joellen Ledford (Molecular Pathology Laboratory Network),
Belinda Yen-Lieberman and Debra Kohn (Clinical Pathology, Cleveland Clinic Foundation), and Denise Quigley (Medical University
of South Carolina) for their technical contributions to this work.
The authors had full control over the direction and content of
the manuscript. Editorial support was sponsored by Hologic Inc.
and provided by Melanie Watson, PhD, at AlphaBioCom. Each
clinical trial site participated with the approval of the protocol by
their respective institutional review boards; all cervical samples
(prospective collection and residual/remnant samples) were
collected under protocols reviewed and approved by institutional
review boards.
Ethical approval: The authors Deborah Bartholomew, Ronald D.
Luff, Neil B. Quigley, Michelle Curtis and Marilyn C. Olson have no
ethical approval to declare.
Funding: The authors Deborah Bartholomew, Ronald D. Luff, Neil
B. Quigley have no funding to state. Michelle Curtis and Marilyn C.
Olson have funding to state: Hologic Inc.
Conict of interest: The authors Deborah Bartholomew and Neil
B. Quigley have no competing interest to declare. Ronald D. Luff has
competing interest: employee/shareholder at Quest Diagnostics;
Michelle Curtis and Marilyn C. Olson: employee and stockholder of
Hologic Inc.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.jcv.2011.01.016.
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