Performance of Commercial Reverse Line Blot Assays for Human

Papillomavirus Genotyping
Martin Steinau,a Juanita M. Onyekwuluje,a Mariela Z. Scarbrough,a Elizabeth R. Unger,a Joakim Dillner,b and Tiequn Zhouc
WHO Human Papillomavirus Laboratory Network Global Reference Laboratory, Chronic Viral Diseases Branch, Division of High-Consequence Pathogens, and National
Center for Emerging and Zoonotic Infectious Diseases Pathology, Centers for Disease Control and Prevention, Atlanta, Georgia, USAa; WHO Human Papillomavirus
Laboratory Network Global Reference Laboratory, Region Skne, Malm, and Departments of Laboratory Medicine, Medical Epidemiology, and Biostatistics, Karolinska
Institute, Stockholm, Swedenb; and Quality, Safety and Standards Team, Immunizations, Vaccines and Biologicals Department, World Health Organization, Geneva,
Switzerlandc

The performance of three line blot assays (LBAs), the Linear Array HPV genotyping assay (LA) (Roche Diagnostics), INNO-LiPA
HPV Genotyping Extra (LiPA) (Innogenetics), and the reverse hybridization assay (RH) (Qiagen), was evaluated using quantitated whole genomic human papillomavirus (HPV) plasmids (types 6, 11, 16, 18, 31, 33, 35, 39, 51, 52, 56, 58, 59, and 68b) as well
as epidemiologic samples. In a plasmid titration series, LiPA and RH did not detect 50 international units (IU) of HPV type 18
(HPV18) in the presence of 5 104 IU or more of HPV16. HPV DNA (1 to 6 types) in the plasmid challenges at 50 IU or genome
equivalents (GE) were identified with an accuracy of 99.9% by LA, 97.3% by LiPA, and 95.4% by RH, with positive reproducibility of 99.8% (kappa 0.992), 88.2% (kappa 0.928), and 88.1% (kappa 0.926), respectively. Two instances of mistyping occurred with LiPA. Of the 120 epidemiologic samples, 76 were positive for high-risk types by LA, 90 by LiPA, and 69 by RH, with a
positive reproducibility of 87.3% (kappa 0.925), 83.9% (kappa 0.899), and 90.2% (kappa 0.942), respectively. Although
the assays had good concordance in the clinical samples, the greater accuracy and specificity in the plasmid panel suggest that LA
has an advantage for internationally comparable genotyping studies.

he introduction of human papillomavirus (HPV) vaccines has

highlighted the need for standardized accurate HPV genotyping assays to ensure comparability of results in laboratories worldwide, both before and after vaccine introduction. A large variety of
assays, using both in-house methods and commercial kits, are
available for HPV detection and typing (6), and more can be anticipated. International proficiency testing organized by the WHO
HPV LabNet has documented significant differences in sensitivity,
specificity, and reproducibility between different laboratories using a variety of testing platforms (2). Variation in performance for
laboratories using the same assay implicates laboratory practice;
however, the results also indicated differences between assays. The
WHO HPV LabNet has further recognized the need for HPV typing assays that can easily be standardized, require minimal equipment for assay performance and interpretation, and can be used
in a variety of laboratory settings. Following its meeting on the
standardization of HPV assays and the role of the WHO HPV
LabNet in supporting vaccine introduction (WHO, Geneva, Switzerland, 23 to 25 January 2008 [http://www.who.int/biologicals
/publications/meetings/areas/vaccines/human_papillomavirus
/HPV%20Jan%20meeting%20report_20080909%20_Clean
_.pdf]), it was agreed that commercial assay kits should be evaluated in collaborative studies for proficiency.
Reverse line blot assays (LBAs), based on consensus amplification of conserved regions of HPV followed by hybridization to
type-specific probes on line blot strips, have been widely used.
Available LBAs differ in the primer set (1, 3, 4) used in the amplification phase and in the number and sequences of the detection
probes. Beyond thermocyclers for target amplifications, only temperature control water baths and visual inspection are required to
carry out these assays. Therefore, the LBA platforms have the potential to meet the needs of the WHO for high-performance genotyping that does not involve expensive equipment.

0095-1137/12/$12.00

Journal of Clinical Microbiology

p. 1539 1544

Studies have been conducted previously to compare and evaluate the performance of these HPV typing assays. Typically, these
were restricted to DNA extracts from patient-collected anogenital
specimens, which cannot be validated independently and limit the
analysis to relative comparisons and assessments of type prevalence found by the individual assays (5, 7, 10). The use of plasmid
standards offers clear advantages for more objective evaluations
beyond that level. Cloned, full-length HPV genomic DNA
(gDNA) provides complete control over genotype identity as well
as copy number input and eliminates the need for a gold standard
test.
We selected three commercial LBAs using different primer sets
and subjected them to use with a number of different test samples.
These included plasmid standards as well as patient extracts from
different sources. The objective was a comparison of these kits
performance, with the potential needs of the WHO in mind. Specifically, assay sensitivity, specificity, and reproducibility were assessed.
MATERIALS AND METHODS
Study design. HPV plasmids were used to prepare test samples to assess
the assays performance parameters. Human genomic DNA was added as
carrier diluent to a final concentration of 1 ng/l in each plasmid preparation to simulate the situation in actual samples. For the evaluation of

Received 2 December 2011 Returned for modification 3 January 2012

Accepted 14 February 2012
Published ahead of print 22 February 2012
Address correspondence to Martin Steinau, MSteinau@cdc.gov.
Copyright 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JCM.06576-11

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TABLE 1 HPV types in the plasmid test samples

HPV type(s) ina:
HPV types (copy no.) for
plasmid titration

Individual
plasmid
Plasmid mix

HPV18 (5E01), HPV16 (5E00)

HPV18 (5E01), HPV16 (5E01)
HPV18 (5E01), HPV16 (5E02)
HPV18 (5E01), HPV16 (5E03)
HPV18 (5E01), HPV16 (5E04)
HPV18 (5E01), HPV16 (5E05)
HPV18 (5E01), HPV16 (5E06)
No HPV
Water blank

HPV6
HPV11
HPV16
HPV18
HPV31
HPV33
HPV35
HPV39
HPV45
HPV51
HPV52
HPV56
HPV58
HPV59
HPV68b
No HPV

HPV16, HPV68b
HPV18, HPV59
HPV6, HPV56
HPV35, HPV11
HPV39, HPV52
HPV31, HPV51
HPV33, HPV58
HPV45, HPV16, HPV52
HPV18, HPV39, HPV59
HPV6, HPV35, HPV51
HPV33, HPV18, HPV56
HPV58, HPV16, HPV11, HPV45
HPV6, HPV59, HPV52, HPV39
HPV31, HPV33, HPV35,
HPV58, HPV56
HPV18, HPV31, HPV11,
HPV45, HPV68b
HPV52, HPV39, HPV45,
HPV68b, HPV51
HPV6, HPV31, HPV35, HPV16,
HPV56, HPV59
HPV18, HPV33, HPV39,
HPV51, HPV58, HPV68b

a
All plasmids in the individual and mixed samples were prepared to represent the
equivalent of 50 IU or GE per test sample, the sensitivity suggested by the WHO HPV
LabNet.

competition between HPV types with unequal copy numbers, a titration

series containing a constant 50 international units (IU) of HPV type 18
(HPV18) DNA and different numbers of HPV16 in 10-fold increments
from 5 100 to 5 106 IU was prepared. To evaluate detection of HPV at
the desired level of sensitivity, i.e., 50 IU or genome equivalents (GE) per
assay, the final concentration of each type was adjusted so that 50 IU (or
GE) would be assayed. Samples containing multiple types (from 2 to 6 of
the 15 types in different combinations) were prepared in order to evaluate
the impact of multiple types on assay performance (Table 1). Two additional control samples containing no template DNA and one with only
human genomic DNA (Roche Diagnostics) were also prepared. Since
sample source and collection medium can influence the performance of
molecular assays, particularly those based on PCR technology, we included extracts of cervical cells in specimen transport medium (STM) and
archived formalin-fixed paraffin-embedded (FFPE) tissue in the test
panel. These samples are described below. In particular, FFPE extracts are
known to be challenging for assays with large amplicons, and the LBAs
could perform differently.
The final testing panel included 66 plasmid challenge samples (7 titration, 15 individual, and 18 multiple, each in duplicate), 60 epidemiologic
samples, and 2 negative controls. Six replicate aliquots of the testing panel
were prepared to provide duplicate testing on each of the three LBA plat-

forms. All DNA samples were randomly coded by a member of the laboratory not involved in testing, disguising the origin of the sample, expected HPV status, and types. Two technologists independently tested a
panel on each of the platforms and interpreted the results. Prior to initiating testing, each technologist successfully completed proficiency testing
on each assay platform, correctly identifying one to five HPV types in 10
unknown samples with at least 80% accuracy.
Each assay used a 10-l DNA aliquot per PCR. Results for all possible
HPV types were entered into MS Access database tables. A sample was
termed HPV positive when at least one type was detected, HPV negative if
none of the 15 types but the genomic control was positive, and inadequate
if neither HPV nor the control was detected. Testing was completed over
a period of 2 weeks. The order in which each technologist used each
platform varied.
Plasmid samples. Full-length clones of HPV types 6, 11, 16, 18, 31, 33,
35, 39, 45, 51, 52, 56, 58, 59, and 68 in plasmid vectors (100 ng in TrisEDTA [TE] buffer) were provided by the WHO HPV Global Reference
Laboratory (Region Skne, Malm, Sweden). Each plasmid was diluted to
5 104 genome equivalents per ml in 200 g/ml yeast tRNA solution
(Invitrogen, Carlsbad, CA). The number of genomic copies for HPV16
and -18 had been previously standardized by WHO and was measured in
international units (IU) accordingly. Copy numbers of other types were
calculated from the molecular mass and concentration as genome equivalents (GE). Human genomic DNA used as background for HPV plasmid
samples was obtained from Roche Diagnostics, (Indianapolis, IN) and
diluted to 10 ng/l in 0.1 mM TE.
Epidemiological samples. Residual DNA extracts were retrieved from
anonymous archived epidemiologic samples from studies of HPV, either
from populations with a high HPV prevalence or from HPV-associated
cancers. These were randomly selected without knowledge of prior HPV
results to include 30 DNA extracts from cervical cells in Digene STM
(Qiagen, Valencia, CA) and 30 from archived FFPE cancer tissues.
HPV genotyping. The selection of kits to be evaluated was based on
the following criteria: (i) the ability to detect and individually identify all
or the majority of the 13 high-risk HPV types 16, 18, 31, 33, 35, 39, 51, 52,
56, 58, 59, and 68b and the two low-risk types 6 and 11; (ii) no requirement for expensive instrumentation to perform and interpret the assay;
and (iii) commercial availability in the United States. Meeting most of
these criteria, the Linear Array HPV genotyping assay (LA) (Roche Diagnostics, Indianapolis, IN), INNO-LiPA HPV Genotyping Extra (Innogenetics, Ghent, Belgium), and the reverse hybridization probe assay (RH)
(Qiagen, Valencia, CA) were chosen. All three tests are based on general
HPV amplification via a low-stringency PCR amplification of the L1 region and subsequent detection via reverse line blot assay with type-specific
probes. Technical details are compared in Table 2.
The LA uses PGMY 09/11 consensus primers and identifies a total of
37 individual types. The test was performed according to the manufacturers specification, except that 40 l DNase-free water was added with 10 l
template DNA to fill to the required volume. The hybridization and washing steps of the reverse line blot assay were done automatically with Beeblot instruments (Bee Robotics, Caernarton Gwynedd, United Kingdom).
HPV52 is detected only by an XR probe which cross-hybridizes with

TABLE 2 Technical specification of commercial LBAsa

Parameter

LA

LiPA

RH

Primer set
HPV amplicon size (bp)
PCR vol (l)
Hybridization temp (C)
Detection signal
Genomic control
Control amplicon size (bp)

PGMY 09/11
450
100
53
Horseradish peroxidase
-Globin
268

SPF10
65
50
49
Alkaline phosphatase
HLA-DPB1
280

GP5/6
150
50
50
Alkaline phosphatase
-Globin
258

LA, linear array HPV genotyping test; LiPA, line probe assay; RH, reverse hybridization.

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Journal of Clinical Microbiology

Performance of HPV Line Blot Assays

TABLE 3 Impact of increasing amounts of HPV16 on detection of 50 IU HPV18 and 10 ng genomic control DNA
Resultb
LA

LiPA

RH

Test

HPV16 input
(IU)a

HPV16

HPV18

-Glob

HPV16

HPV18

Human DNA

HPV16

HPV18

Genomic
DNA

First
Second

5E00
5E00

First
Second

5E01
5E01

First
Second

5E02
5E02

First
Second

5E03
5E03

First
Second

5E04
5E04

FP

First
Second

5E05
5E05

First
Second

5E06
5E06

First
Second

0
0

a
b

The input also included 50 IU of HPV18 and 10 ng of genomic control DNA.

, detected; FP, false-positive result (HPV39 detected instead of HPV18). -Glob, -globin.

HPV33, -35, and -58. In the presence of any of these three types, HPV52
cannot be identified unequivocally.
LiPA applies SPF10 primers and includes probes to detect 28 HPV
types. The assay was performed in accordance with the manufacturers
protocol using an AutoBlot 3000H (MedTec, Buffalo, IL) for hybridization to the genotyping strips. Some types are defined by a single positive
probe on the genotyping strip (i.e., HPV6, -11, and -16), but others are
interpreted as a combination of two to four probes (i.e., HPV18, -33, and
-58). While the manufacturer provides interpretation of type detection,
including possible types, only those that were detected unequivocally
were included in the analysis.
The RH was not officially available in the United States at the time that
this study was conducted, and the kits were kindly provided by Qiagen.
The test utilizes the GP5/6 primers and line probes to distinguish 18
high-risk types; it does not detect HPV6 and -11. PCR amplification and
line blot detection were carried out as specified by Qiagen. Hybridization
and washing of HPV genotyping strips were achieved manually using a
Gemini Twin shaking water bath (SciGene, Sunnyvale, CA) and an aspiration system with 8-needle Stream Splitter (Art Robbins Instrument,
Sunnyvale, CA).
A water blank and SiHa DNA were included as negative and positive
controls, respectively, in every run with each assay to monitor validity.
The results from these controls were not included in the analysis.
Analysis. The 15 HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56,
58, 59, and 68 were considered for the analysis. Some calculations (positive samples, accuracy, and reproducibility) for RH results were restricted
to the 13 high-risk types as indicated. The two technologists results for
each sample and platform were counted as independent events. For plasmid samples, accuracy was calculated as the percentage of correctly detected types (true positive and true negative) from the total number of
types assessed in all samples (total types assessed 15 types per assay 66
samples 2 assays 990, or 858 if restricted to 13 high-risk types).

May 2012 Volume 50 Number 5

Results from STM and FFPE extracts were compared descriptively.

Positive reproducibility (PR) was calculated as the percentage of HPV
types identified in both replicates among the total number of types detected in either of the duplicate samples. Unweighted Cohens kappa coefficients were calculated with SPSS 15.0 for Windows for all types assessed (positive and negative) in the relevant sample set.

RESULTS

Titration of HPV16 against 50 IU HPV18. The results for detection of HPV16, HPV18, and the genomic control at all input concentrations of HPV16 are listed in Table 3. When HPV16 is present at below 5 104 IU, all three targets are detected by all assays.
While LA results were robust to increasing amounts of HPV16,
LiPA and RH failed to detect 50 IU HPV18 and at the highest input
of HPV16 failed to detect the genomic control. The negative control containing only human gDNA gave expected results with all
assays, as did the no-template control (data not shown).
Detection of HPV plasmid standards. Table 4 lists false-positive, false-negative, and inadequate results obtained by LBA
among plasmid samples. Both technologists results are reported,
so the number of results is double the number of samples. In the
single HPV plasmid challenges, the accuracy of LA was 29/30
(96.7%), that of LiPA was 27/30 (90.0%), and that of RH was
23/30 (76.7%). (RH detected 23/26 [88.5%] if HPV6 and -11 were
excluded.) The challenges with multiple HPV plasmids included a
total of 61 HPVs. Of these, LA identified all 122 types correctly
(100%), LiPA 100/122 (82.0%), and RH 83/122 (68.0%). If HPV6
and -11 were excluded, 83/108 (76.9%) types were correctly found
by RH.
The accuracy for all 15 types in the 66 plasmid samples was

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TABLE 4 Incorrect line blot assay results in HPV plasmid challenges

HPV type(s) not detected witha:
No. of types
(no. of samples) LA
LiPA
RH
1 (30)
2 (14)
3 (8)
4 (4)
5 (6)

6,c 6,c 11,c 11,c 52, 68, 68

6,c 6,c 11,c 11,c 56, 52
52, 52, 68, 68, 59, 6,c 6,c 39
11,c 11,c 6,c 6,c 39, 52, 59
58, 58, 39, 39, 52, 52, 11,c
11,c 68, 68, 68, 68
39,b 39,b 58, 68, 59, 59 68, 68, 6,c 6,c 59, 59

59 (IA) 39 (IA), 58 (FP 52), 59

58, 59, 59
39,b 39,b 68, 59
58 (FP 52), 59, 59
39, 39, 68, 68, 68,b 68b

6 (4)
a

Each entry reflects failure to detect a type by one of the technologists. IA, inadequate;
FP, false positive and falsely detected type.
b
Detected as possible types.
c
Probe not included in assay.

FIG 2 Venn diagram illustrating instances of type-specific concordance and

99.9, 97.3, and 95.4% for LA, LiPA, and RH, respectively. Restricted to the 13 high-risk types, RHs accuracy was 96.7%.
Epidemiologic samples. Results for STM and FFPE samples
are combined, as differences between the two sample types were
negligible for all LBAs. From the 120 test results derived from each
assay, the genomic control probe was positive in 116, 90, and 87
tests by LA, LiPA, and RH, respectively. However, results were
adequate in 118, 118, and 111 tests by LA, LiPA, and RH, respectively, because some samples were positive for HPV but negative
for the genomic control.
One or more HPV types were detected in 76, 90, and 69 samples by LA, LiPA, and RH, respectively. Complete type-specific
agreement among all three LBAs was noted for 81 samples
(67.5%). Of these, 42 were HPV positive and 39 HPV negative. An
additional 24 samples (20%) had at least one type detected concurrently by all assays, and one sample was HPV positive in all
assays but without type agreement.
The overall detection of individual types was very similar for
the three LBAs, with the exception of HPV52, which was detected
only by LiPA in 9 cases (Fig. 1). LA detected a total of 111 types
(108 without HPV6 and -11) in 76 samples, LiPA detected 121
types (111 without HPV6 and -11) in 90 samples, and RH found

discordance between assays (restricted to 13 high-risk types, i.e., types 16, 18,
31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68). Numbers in parentheses indicate
the total number of HPV types detected by each LBA.

97 types in 69 samples. Instances of type concordances and discordances between the assays are illustrated in Fig. 2.
Reproducibility. All test samples with the exception of the
seven titration samples were included in the reproducibility assessment. In the 33 test samples prepared with single or multiple
plasmids, expected results were obtained for a total of 76 instances
of HPV type detection (67 without HPV6 and -11). Differences in
reproducibility between STM and FFPE samples were not significant, and they are combined in Table 5.
DISCUSSION

Results from the plasmid standards revealed differences between

the HPV typing assays. The titration of HPV16 in different
amounts against constant HPV18 template numbers highlighted
the robustness of the assays to competing types with different
concentrations. Copy numbers of more than 5 103 or a 100fold-larger template amount of HPV16 suppressed amplification
of HPV18 in LiPA and RH PCRs. These observations are in line

FIG 1 Type-specific detection by the three LBAs in the 120 epidemiologic samples. White bars indicate results found by LA, gray bars those found by LiPA, and
black bars those found by RH.

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Journal of Clinical Microbiology

Performance of HPV Line Blot Assays

TABLE 5 Positive reproducibility of line blot assays for plasmid

standards and extracts from epidemiologic samples
Value for:
Sample (n)

Parameter

LA

LiPA

RHa

Plasmid DNA (33)

No. of types to be detected

No. of discrepant types
No. of samples affected
Positive reproducibility (%)
Kappa
No. of positive types (by
either test)
No. of discrepant types
No. of samples affected
Positive reproducibility (%)
Kappa

76
1
1
98.7
0.992
63

76
9
6
88.2
0.928
66

67
8
8
88.1
0.926
51

8
6
87.3
0.925

11
9
83.3
0.899

5
5
90.2
0.942

Epidemiologic (60)

For RH analysis, types 6 and 11 were omitted and the total number of types adjusted.

with assessments by van Doorn et al. (9), who found that 100
copies of HPV18 were outcompeted by a 1,000-fold-higher concentration of HPV16. Only the LA was able to detect both types
even at 100,000-times-higher HPV16 concentrations. The larger
reaction volume (100 l in LA versus 50 l in the others) might be
responsible for the superior tolerance to competition. The extreme differences in copy number are unlikely to occur in actual
biologic samples; however, pushing the technical limits of the assays allowed for evaluation of robustness to a variety of challenges.
All three LBAs generally could detect 50 IU or GE of the single
high-risk HPV types. Only HPV68b was not detected by either RH
duplicate, which is not surprising since the limit of detection
(LOD) is stated as 105 viral copies in the RH detection kit handbook. The sporadic lack of reproducibility may indicate that this
input amount is in the range of the lowest LOD for at least some
types (Table 3).
Significant deficiencies were seen in samples that included
more than one type. LiPA failed to detect 18% of the types included in the 36 multiplasmid challenges. The 22 missed types
consisted exclusively of HPV39, -58, -59, and -69. In some instances, types 39 and 68b were identified as possible types due to
the LiPAs multiprobe set (Table 4). Nevertheless, both HPV39
and -68b were also missed in other samples that were free of this
ambiguity, suggesting unequal amplification efficiency or competition. Disregarding HPV6 and -11, the RH failed to detect 20.5%
of the types in this subset. Besides HPV68b, types 52, 59, and 39
were missed in several instances. A critical limitation of the RH
might result from the large discrepancy in detection sensitivities
for different HPV types. According to the handbook, LODs differ
25,000-fold, ranging from four copies for HPV16 to 100,000 copies for HPV53 and -68.
Reproducibility of results for the plasmid challenges was greatest for the LA but was generally good in all assays. Among the
results derived from the patient samples, the RH had the highest
positive reproducibility (90.2%), but the denominator was also
lowered to 51 types since HPV6 and -11 are not detected by this
test. It was rather surprising that no significant differences were
found between STM and FFPE extracts, as fractured and poorquality DNA from archived tissues should favor shorter amplicon
lengths as targeted by the SPF primers (8). The sample size may
not have been sufficient to allow differences in assay performance
to be identified. Generally, results from the patient samples were

May 2012 Volume 50 Number 5

comparable between the assays, with at least partial agreement in

87.5%. Performance differences found with the plasmid samples
might reflect rather extreme situations which are not relevant for
the majority of real clinical specimens.
False-positive results were a particular concern. Two instances
were observed among LiPA results in plasmid samples. In both
cases, the algorithm for identifying the expected type (HPV18 or
-58) requires that more than one probe hybridizes with the amplicon. As only one of the required probes hybridized, another
type (HPV39 or -52) was falsely indicated. It is likely that individual LiPA probes differ in their affinity to the same amplicon and
generate an incomplete band pattern at small target amounts,
which would consequently lead to attributing the result to an incorrect type. Detection of HPV52 by LiPA might be particularly
vulnerable to this problem. In the LiPA, the HPV52 amplicon
hybridizes to a single probe. However, that probe also hybridizes
to amplicons of five additional HPV types that are distinguished
by combinations with other probes. In this regard, it is noteworthy
that HPV52 was detected in 9 of the 15 types found exclusively by
LiPA among the patient samples (Fig. 1), and two of three patient
samples that were exclusively HPV positive by LiPA had only type
52. It seems likely that at least some of these cases are false positives.
Conversely, some HPV52 may have been missed by the LA, as
it has a similar shortcoming and detects HPV52 only through the
cross-binding XR probe. While this ambiguity occurred in 28
cases among the LA results, only two of these samples tested positive for HPV52 by LiPA.
Every laboratory test is also influenced by the accuracy of human handling. The samples prepared from plasmid DNA yielded
one inadequate result each by LA and LiPA, and the (single) HPV
types not detected in these results were also counted as missed.
Although the tests were performed with utmost care in a clinically
certified laboratory, operator errors cannot be ruled out as a cause
and may have lowered the real performances of the tests. However, this possible distortion should be minimal.
Analysis for this comparison study was restricted to the most
relevant high-risk types as well as HPV6 and -11. It should be
considered, however, that LA detected 50, LiPA 16, and RH 4
additional HPV types in the 60 patient samples. Depending on the
number of types covered by each assay, the scope of HPV detection will always be limited and does not directly allow an HPVnegative interpretation.
For the majority of samples, HPV typing results will be very
similar and comparable for the three LBAs evaluated. In difficult
situations such as multiple infections, low copy numbers, or large
difference in viral copies, LA has an advantage. Some limitations
of LiPA are due to its multiple-probe detection system. RH is
disadvantaged by low sensitivity for some types, particularly
HPV53, -68, and -82. LA performed nearly perfectly and is hampered only by the cross-reacting XR probe for HPV52 detection.
ACKNOWLEDGMENTS
This work was supported by the WHO via a project funded by the Bill and
Melinda Gates Foundation.
The findings and conclusions in this report are those of the authors
and do not necessarily represent the views of the supporting agencies.

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