Comparison of the GenoFlow human papillomavirus (HPV) test and the Linear Array assay for HPV screening in an Asian population.
ABSTRACT High-risk human papillomavirus (HR-HPV) DNA detection in cervical cytology samples is useful for primary screening of cervical cancer and for triage of patients with equivocal cytological findings. The GenoFlow HPV array test (GF assay; Diagcor Bioscience Inc., Hong Kong) was recently developed to detect 33 HPV genotypes by a "flowthrough" hybridization technology. In this study, we assessed the analytical sensitivity and reproducibility of the GF assay and compared its genotyping results with those of the Linear Array (LA) assay (Roche Molecular Diagnostics, Indianapolis, IN), using 400 archived liquid-based cytology samples representing the full range of cytology findings. Genotyping findings of the GF and LA assays were concordant or compatible for 93.44% of tested samples, with a good (κ = 0.797) to very good (κ = 0.812) strength of agreement for assay-common and oncogenic HPV types, respectively. The two assays showed good (κ = 0.635) agreement in detecting infections with multiple HPV genotypes. The lowest detection limits of the GF assay for HPV16 and HPV18 were 25 copies and 20 copies, respectively. Repeat testing of 60 samples by use of two different lots of the GF assay revealed no discordant results, suggesting good reproducibility of the assay. Both assays achieved approximately 80% and 100% sensitivity for identifying cases of atypical squamous cells of undetermined significance (ASC-US) and low-grade squamous intraepithelial lesions (LSIL) with subsequent detection of LSIL+ and high-grade squamous intraepithelial lesions or higher (HSIL+) in 2 years, respectively. Among ASC-US samples, the GF assay achieved the highest specificity (23.08%) for indicating subsequent identification of HSIL compared with the LA (19.23%) and Hybrid Capture 2 (HC2) (8.97%) assays. The GF and LA assays showed significant discrepancy in detecting HPV genotypes 11, 26, 39, 52, and 66. More sensitive detection of HPV52 by GF assay offers an advantage in regions where HPV52 is more prevalent. The sensitivity of the GF assay for detecting patients with HSIL+ was noninferior to that of the LA assay.
- SourceAvailable from: ufsc.br[show abstract] [hide abstract]
ABSTRACT: More than ever, clinicians need regularly updated reviews given the continuously increasing amount of new information regarding innovative cervical cancer prevention methods. A summary is given from recently published meta-analyses on three possible clinical applications of human papillomavirus (HPV)-DNA testing: triage of women with equivocal or low-grade cytological abnormalities; prediction of the therapeutic outcome after treatment of cervical intraepithelial neoplasia (CIN) lesions, and last not but not least, primary screening for cervical cancer and pre-cancer. Consistent evidence is available indicating that HPV-triage with the Hybrid Capture-2 assay (HC2) is more accurate (significantly higher sensitivity, similar specificity) than repeat cytology to triage women with equivocal Pap smear results. When triaging women with low-grade squamous intraepithelial lesions (LSIL), a reflex HC2 test does not show a significantly higher sensitivity, but a significantly lower specificity compared to a repeat Pap smear. After treatment of cervical lesions, HPV testing easily detects (with higher sensitivity and not lower specificity) residual or recurrent CIN than follow-up cytology. Primary screening with HC2 generally detects 23% (95% confidence interval, CI: 13-23%) more CIN-2, CIN-3, or cancer compared to cytology at cut-off atypical squamous cells of undetermined significance (ASCUS) or LSIL, but is 6% (95% CI: 4-8%) less specific. By combined HPV and cytology screening, a further 4% (95% CI: 3-5%) more CIN-3 lesions can be identified but at the expense of a 7% (95% CI: 5-9%) loss in specificity, in comparison with isolated HC2 screening. Sufficient evidence exists to recommend HPV testing in triage of women with atypical cytology and in surveillance after treatment of CIN lesions. In the United States, recently reviewed knowledge has resulted in the approval of combined cytology and HC2 primary screening in women older than 30 years. However, in Europe, cytology-based screening still remains the standard screening method. The European screening policy will be reviewed based on the longitudinal results of randomised population trials which are currently underway.Vaccine 09/2006; 24 Suppl 3:S3/78-89. · 3.49 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Links between human papillomaviruses (HPVs) and cervical cancer were first suspected almost 30 years ago. DNA of specific HPV types has since been found in almost all cervical cancer biopsies. HPV oncogenes that are expressed in these cells are involved in their transformation and immortalization, and are required for the progression towards malignancy. Epidemiological studies have underlined that HPVs are the main aetiological factor for cervical cancer. But how has this knowledge been translated into the clinic to allow the prevention, screening and treatment of cervical cancer?Nature reviews. Cancer 06/2002; 2(5):342-50. · 35.00 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Type-specific detection of human papillomavirus (HPV) is increasingly important for monitoring temporal and age-specific changes in type-specific prevalence in support of HPV vaccination efforts. The impact of sampling, extraction and assay characteristics on HPV results is increasingly recognized. Inter-assay comparability studies have been performed, but the robustness of type-specific results has neither been emphasized nor has the degree of intra-assay reproducibility been addressed. Here we describe the general and type-specific reproducibility of the linear array HPV genotyping test (Roche Molecular Diagnostics, Indianapolis, IN). Extracts of 276 cervical samples from two ongoing epidemiologic HPV studies were retested while blinded to prior results. The testing involved five different reagent lots and three technologists. Concordance for HPV detection (sample positive versus negative for any of the 37 types) was high (98.2%, kappa=0.959). Type-specific concordance for individual HPV types was also high (99.4%, kappa=0.915), and most samples (83.0%) showed complete concordance for all types. Type-specific reproducibility of the linear array HPV genotyping test is good but not perfect. The results suggest that type-specific performance should be included in the evaluation of HPV typing formats.Journal of Clinical Virology 09/2008; 42(4):412-4. · 3.29 Impact Factor
Comparison of the GenoFlow Human Papillomavirus (HPV) Test and
the Linear Array Assay for HPV Screening in an Asian Population
Oscar Gee-Wan Wong,aC. K. Lo,aJoanne N. K. Chow,aObe K. L. Tsun,aElaine Szeto,aStephanie S. Liu,bHextan Y. S. Ngan,band
Annie N. Y. Cheunga
Departments of Pathologyaand Obstetrics & Gynaecology,bQueen Mary Hospital, The University of Hong Kong, Hong Kong Special Administrative Region, China
?200 HPV types, around 15 anogenital types are associated with
HR-HPV DNA testing has been proposed as a tool for primary
with the equivocal cytology finding of atypical squamous cells of
undetermined significance (ASC-US) (23). For these purposes,
HR-HPV cocktail tests such as the Hybrid Capture 2 (HC2) test
However, it has been demonstrated that specific identification of
HPV16 and HPV18 can highlight patients at the greatest risk of
developing cervical intraepithelial neoplasia 3 or above (CIN3?)
(6, 15, 28). HPV genotyping tests such as the Linear Array (LA)
test (Roche) may hence provide more specific information.
clinical performance by using established tests as benchmarks
(20). The HC2 test (Qiagen, Gaithersburg, MD [previously Di-
gene]) is the first U.S. FDA-approved HR-HPV DNA test. It is a
signal-amplified cocktail assay that detects 13 common HR-HPV
types (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68). It
has been validated extensively in many large studies (3, 19). The
The GenoFlow human papillomavirus array test (GF test) is a
new HPV assay, developed by Diagcor Bioscience Inc., that is ca-
pable of genotyping 33 types of HPV (27). The GF test is a reverse
uman papillomavirus (HPV) is an established essential etio-
logical factor for cervical cancer (31). Among the estimated
on a membrane by a rapid flowthrough hybridization process. In
well as some HPV variants.
results of the GF and LA tests, as well as their clinical sensitivity
and specificity, for a selected cohort of patients with different cy-
ibility of the GF test were also evaluated.
MATERIALS AND METHODS
vical cytology samples were retrieved from the archive of the Cervical
Cytology Laboratory, Queen Mary Hospital. The specimens were ran-
epithelial lesions (HSIL) (ASC-H) (40 samples), atypical glandular cells
Received 2 October 2011 Returned for modification 15 November 2011
Accepted 3 February 2012
Published ahead of print 15 February 2012
Address correspondence to Annie N. Y. Cheung, firstname.lastname@example.org.
Supplemental material for this article may be found at http://jcm.asm.org/.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
0095-1137/12/$12.00Journal of Clinical Microbiologyp. 1691–1697 jcm.asm.org
(AGC) (30 samples), low-grade squamous intraepithelial lesions
(LSIL) (100 samples), HSIL (60 samples), and squamous cell carci-
noma (SCC) (30 samples). All of the ASC-US samples had been tested
for HR-HPV by the Digene HC2 HPV test. The follow-up cytology/
colposcopic biopsy findings obtained within 2 years were retrieved.
The use of archival human cytology specimens for research purposes
was approved by the local institutional ethics committee (HKU/HA
HKW IRB no. UW-10-301).
use of a Qiagen blood minikit according to the “Blood and Body Fluid
Spin Protocol” of the kit instructions. DNA concentration was deter-
mined by spectrophotometry, but the same volume (5 ?l) of extracted
the manufacturer’s instructions.
HPV genotyping by GF and LA assays. DNA samples were tested for
HPV by the GF and LA assays according to instructions of the manufac-
turers. Briefly, for the GF assay, DNA extracted from a sample by use of a
QIAamp blood minikit (Qiagen) was amplified by use of a biotin-labeled
5 ?l of DNA template, 19.25 ?l of the master mixture provided, and 0.75
?l of DNA Taq polymerase (5 U/?l) in a Perkin-Elmer GeneAmp PCR
system 9700 apparatus (Applied Biosystems). The reaction mixture was
denaturation at 95°C, 30 s of annealing at 55°C, and 30 s of elongation at
72°C, followed by a final extension for 5 min at 72°C. The amplified
products were subsequently denatured and then hybridized to probes
After a stringent wash, hybridized DNA was detected with streptavi-
din-alkaline phosphatase followed by colorimetric development using
nitroblue tetrazolium–5-bromo-4-chloro-3-indolylphosphate (NBT-
BCIP). The results were interpreted by direct visualization.
The LA test uses a biotinylated PGMY09/11 primer set to amplify a
450-bp region of the L1 gene and is capable of detecting 37 HPV geno-
types, including 15 HR types. DNA was amplified by PCR in a Perkin-
Elmer GeneAmp PCR system 9700 apparatus (Applied Biosystems). The
denatured PCR product was then hybridized to an array strip containing
immobilized oligonucleotide probes. The results were interpreted by us-
ing the reference guide and reading the matching individual types down
the length of the strip.
Interpretation of genotyping results. The results of GF tests were
interpreted according to the manufacturer’s instructions. A valid HPV-
positive result must include visible signals at the “universal,” “HC” (hy-
bridization control), and “AC” (amplification control) probe spots. A
valid negative result must include signals at the HC and AC probe spots.
An HPV of unknown genotype was present when only the universal, HC,
and AC probe spots were positive. Some HPV types share the same probe
spots and cannot be distinguished by GF assay, e.g., HPV66 and HPV68,
HPV54 and HPV55, and HPV40 and HPV61. Both genotypes were inter-
The results of LA tests were read according to the manufacturer’s
reading the matching individual types down the length of the strip. A
result is valid only when at least one of the globin (low and high) signal
bands is visible. The LA assay offers no separate detection for HPV52.
Instead, a signal band for HPV types 33, 35, 52, and 58 and three separate
bands for HPV33, HPV35, and HPV58 are present. HPV52 positivity is
established only if the HPV33/35/52/58 signal is present and the HPV33,
HPV35, and HPV58 signals are absent.
Assessment of analytical sensitivity and reproducibility. Plasmids
containing the full-length genomes of HPV16 and HPV18 were used to
determine the analytical sensitivity of the GF assay (18). The plasmids
1 ? 101, and 5 copies/?l. Each dilution also contained 32 ng/?l genomic
DNA of C33A, an HPV-negative cervical cancer cell line. The smallest
amount of input HPV DNA tested was 5 copies. Each dilution was tested
three times with the GF assay, and the lowest copy number that could be
detected every time defined the analytical sensitivity of the assay.
the reproducibility of the assay. Among the 60 samples, there were 20
ASC-US, 20 LSIL, and 20 HSIL. The samples were tested with a different
lot of the GF assay.
test. Cases showing discordant results and discrepancy in terms of HR-
samples were amplified by both PGMY09/11 and MY09/11 primer sets in
separate PCRs. The absence of an amplification product suggested that
there was no detectable HPV DNA. PCR products were sequenced, and
resultant sequences were matched with known HPV sequences. In addi-
identifies (types) HPV16 and HPV18 and at the same time indicates the
presence or absence of the rest of the high-risk types (types 31, 33, 35, 39,
45, 51, 52, 56, 58, 59, 66, and 68).
Data analysis and statistics. A kappa value of 0 indicates no agree-
ment better than chance, and a kappa value of 1 indicates perfect agree-
?0.81 indicate a poor, fair, moderate, good, and very good strength of
agreement, respectively (2). The nonparametric McNemar test was used
to analyze the complementarities of the detection methods and to deter-
mine if the results obtained by the two methods were significantly differ-
values of ?0.05 were considered statistically significant.
dant or compatible. To compare the HPV genotyping results of
the GF and LA assays, we performed two independent HPV tests
on 400 liquid-based cervical cytological samples, using both as-
says. The two assays are capable of (Table 1) detecting different
but largely overlapping sets of HPV genotypes (Table 1). In this
report, “assay-common HPV genotypes” refers to those HPV ge-
notypes detectable by both assays. The 400 samples were selected
to encompass various cytological diagnoses, including normal,
samples yielded either HPV-negative or HPV-positive results,
whereas the amplification control signal could not be visualized
for four samples tested by the GF assay, meaning that there was
either insufficient DNA to be amplified or a PCR inhibitor was
present in the extracted DNA. Therefore, comparable results of
the GF and LA assays were obtained for a total of 396 samples.
In comparing results from the GF and LA assays, “concordant”
exactly the same or that both assays yielded HPV-negative results. A
common HPV genotype in the results of the GF and LA assays. To-
As shown in Table 2, the GF and LA assays gave rise to either
concordant or compatible results for 93.44% (52.78% plus
40.66%) of the samples. Remarkably, for serious lesions (HSIL
and SCC), the results of the GF and LA assays were highly consis-
tent, giving concordant results for more than 70% of the samples,
with no discordant results (Table 2). This difference reached sta-
tistical significance (chi-square test; P ? 0.0001).
GF and LA assays highly agree on samples positive for
HR-HPV. We transformed the HPV genotyping results into HR-
Wong et al.
jcm.asm.orgJournal of Clinical Microbiology
HPV-positive and HR-HPV-negative results by using the follow-
ing criteria: if a sample was positive for at least 1 of the 15 HR-
agreement for HR-HPV-positive samples, reaching an absolute
agreement of 93.69%. Cohen’s ? value was 0.812, meaning that
the two assays showed very good agreement (Table 3). When
the criterion was changed to assay-common genotypes, absolute
agreement was 95.45%, and Cohen’s ? value was 0.797. When
only HSIL and SCC cases were considered, Cohen’s ? values were
and oncogenic HPV types, respectively (see Table S1 in the sup-
Interassay agreement of individual HPV genotypes. When
individual HPV genotypes were examined, the GF and LA assays
agreed for most genotypes (Table 4). Most importantly, the abso-
lute interassay agreement values for HPV types 16 and 18 were
96.72% and 99.49%, respectively (Table 4). Cohen’s ? values for
these two HPV vaccine-covered oncogenic HPV types were 0.897
and 0.966, respectively (Table 4). However, we also noted a statisti-
two assays (Table 4). Notably, the GF and LA assays differed signifi-
cantly in detecting the HR-HPVs HPV39 and HPV52 (P ? 0.0044
warts (Table 4). Notably, every genotype detectable by the GF assay
was detected at least once within our cohort. The rarest type was
HPV72, which was detected in only one sample. The frequency of
detection of each HPV type assayed by the GF assay is presented in
Single- and multiple-infection detection of GF and LA as-
says. One of the advantages of line blot or dot blot assays such as
TABLE 1 HPV genotypes recognized by GF and LA assays
GF assay LA assay
Carcinogenic (group 1) 16
Probably carcinogenic (group 2A) 68b
Possibly carcinogenic (group 2B) 26b
Not classifiable by carcinogenicity
aAccording to reference 4.
bHPV66 and HPV68, HPV54 and HPV55, HPV40 and HPV61, HPV57 and HPV71,
and HPV84 and HPV26 share probe spots on the GF membrane.
cThe GF assay provides a universal probe for detection of HPV types outside the panel.
According to the manufacturer, types 54 (new subtype), 74 (new subtype), CP8304
(81), 87, and 89 have been detected successfully in clinical samples.
TABLE 2 Concordant/compatible HPV detection (assay-common
genotypes) in relation to cytological classification between GF and LA
No. (%) of samples with the indicated result
between GF and LA assays
Total no. of
Total210 (52.78)161 (40.66)25 (6.57)396
TABLE 3 HPV positivity agreement of GF and LA assaysa
GF assay result
No. of samples with LA assay result
Total 34749396 31185396
aThe absolute agreement levels between assays for assay-common genotypes and
oncogenic genotypes were 95.45% and 93.69%, respectively. Cohen’s ? values for assay-
common genotypes and oncogenic genotypes were 0.797 and 0.812, respectively. P
values determined by the McNemar test for assay-common genotypes and oncogenic
genotypes were 0.4795 and 1.0000, respectively.
bAssay-common genotypes are HPV genotypes detectable by both assays.
cOncogenic genotypes are types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73,
Evaluation of GenoFlow HPV Test
May 2012 Volume 50 Number 5 jcm.asm.org 1693
the GF and LA assays is their ability to detect multiple infections.
We categorized the genotyping results of the GF and LA assays
types present (? ? 0.635; P ? 0.01 by chi-square test) (Table 5).
Notably, more multiple-infection cases were identified by the GF
assay (190 cases) than by the LA assay (154 cases).
Discrepancy analysis of HR-HPV discordant cases. Among
the 396 cases compared, 25 cases showed discordant GF and LA
assay results. In other words, for 25 cases, the results from the GF
cases, 13 disagreed on their detection of HR-HPV. These cases
cocktail HR-HPV test capable of indicating the presence of
HPV16, HPV18, or non-HPV16/18 HR-HPV or the absence of
HR-HPV. We could not detect any HPV by the PCR method in 9
(Table 6). Among the 4 PCR-positive cases, 3 agreed with the LA
assay (one of the genotypes suggested by the test) and 1 agreed
with the GF assay. Taken together, the results show that 5 cases
matched by PCR and LA assay, 6 cases matched by PCR and GF
(Table 6). In 8 cases, the results of the Cobas 4800 system agreed
with the LA assay, and in 5 cases the Cobas 4800 system results
agreed with the GF assay (Table 6).
Analytical sensitivity and interlot reproducibility of GF as-
say. To assess the analytical sensitivity of the GF assay, serial dilu-
tions of two plasmids, harboring the full genomes of HPV16 and
HPV18, were prepared (10,000 copies/?l to 5 copies/?l). Each
dilution also contained the same amount (32 ng/?l) of genomic
25 copies and 20 copies, respectively.
To assess the interlot reproducibility of the GF assay, 60 sam-
ples were selected for repeat testing with a different lot of the test.
Of these 60 samples, 49 (81.67%) yielded exactly the same HPV
failed to be amplified in one of the tests and hence could not be
compared (Table 7).
Sensitivity and specificity of GF and LA assays for triage of
tology follow-up data were available for 119 of the ASC-US cases,
and all of the ASC-US samples had been tested for HR-HPV by
HC2 assay. To compare our results with those of the HC2 test,
identify cases that would progress to LSIL/CIN1?, their sensitiv-
ities were 81.82% and 87.88%, respectively, which are lower than
that of the HC2 test (96.97%).
All three tests could identify all cases that would progress to
assays achieved higher specificities than that of the HC2 test
(23.08% for GF assay, 19.23% for LA assay, and 8.97% for HC2
ASC-H and AGC are rare but nonetheless significant cytology
findings (14, 22). In total, there were 138 cases of ASC-US,
ASC-H, or AGC with available follow-up data. We calculated the
sensitivity and specificity of using the GF and LA assays for pre-
dicting disease on follow-up. When ASC-H and AGC cases were
included, the sensitivity and specificity of detecting cases that
tively, for the GF assay and 80.56% and 40%, respectively, for the
LA assay. The sensitivity and specificity of detecting cases that
would progress to HSIL/CIN2? were 84.00% and 25.45%, re-
spectively, for the GF assay and 88.00% and 26.55%, respectively,
for the LA assay.
In summary, we found that the GF assay and LA assay per-
formed very similarly for triage of cases with equivocal cytology
Sensitivity of GF assay is noninferior to that of LA assay for
detecting HSIL?. A test of noninferiority was performed using
data from the HSIL and SCC cases (20). To assess the noninferi-
ority of the sensitivity (i.e., relative sensitivity of no lower than
TABLE 5 Single and multiple HPV infections identified by GF and LA
GF assay result
No. of samples with LA assay result
Single HPV type
Multiple HPV types
Total 191154 51396
aCohen’s ? value for the number of HPV types detected was 0.635.
TABLE 4 Interassay agreements for individual HPV (assay-common)
genotypes detected by GF and LA assays
No. of samples positive
aDetermined by the McNemar test. Values in bold show significant results.
bHPV84 and HPV26 share the same probe spot in the GF assay.
Wong et al.
jcm.asm.orgJournal of Clinical Microbiology
90%), the GF and LA test results were tabulated and the noninfe-
riority score T was calculated (Table 8). The null hypothesis was
rejected (T ? 3.24893; P ? 0.00058), and hence the sensitivity of
test score for the noninferiority of specificity calculated based on
cases with diagnoses lower than HSIL, however, did not reach
statistical significance (T ? 0.91698; P ? 0.17958), and hence the
null hypothesis (i.e., the relative specificity was lower than 98%)
was not rejected.
The Diagcor GF HPV test provides a faster alternative to the LA
us to test similar numbers of samples (?25) by the GF assay is
approximately 25% shorter than that required for the LA assay.
This is partly attributable to the adaption of flowthrough hybrid-
ization technology, which significantly shortened the hybridiza-
tion step of the GF assay (27). However, before it is adopted for
clinical or research use, the performance of the GF assay needs to
be characterized. The GF test has been compared to the LA assay
only in a study with a limited number of samples (27). Our study
provides a more comprehensive comparison of the GF and LA
assays. Generally, we found the results from the two tests to be
detecting the presence of HR-HPV without detailing the individ-
Therefore, when used for triage of ASC-US cases, the two tests
achieved similar sensitivities and specificities. At the individual
HPV type level, the two tests agreed for most (13/15 types)
HR-HPV types, being significantly different only in detecting
HVP39 and HPV52 (Table 4).
ful tools for epidemiological research (1, 9, 13, 16). In addition,
identification of specific oncogenic HPV types may bear implica-
tions for the management of HPV-positive women. In a recent
study involving more than 40,000 patients, detection of HPV16,
HPV18, or both had a better sensitivity and similar positive pre-
dictive value (PPV) for detection of CIN3 or worse than for
previous study also found that detection of HPV16 and HPV18
improved the sensitivity of identifying HPV-positive ASC-US
TABLE 6 Discrepancy analysis for cases showing discordant results and discrepant HR-HPV statuses in the two tests
Genotype(s) detected bya:
PCR and sequencingb
Cobas 4800 HPV test
LA assay GF assayResult Matchc
16, 42, 62
Non-type 16/18 HR-HPV
Non-type 16/18 HR-HPV
Non-type 16/18 HR-HPV
Non-type 16/18 HR-HPV
18, non-type16/18 HR-HPV
aUnderlining indicates HR-HPV types.
bPCR was done with both PGMY09/11 and MY09/11 primer sets.
cXX, sequencing result does not match either GF or LA assay result.
TABLE 7 Samples yielding different results between two different lots of
Genotype(s) detected by lot:
66/68, 81, 84/26
16, 18, 56
52, 58, 11
18, 31, 51, 84/26
33, 52, 11
51, 52, 11
16, 18, 56, 70
52, 58, 11, 57/71
aNA, not available.
TABLE 8 Comparison of GF and LA test results by test of noninferiority
Case type and GF test
HSIL and SCC casesa
No. of cases with LA test result
Normal, ASC-US, ASC-H,
AGC, and LSIL casesb
a? ? 0.90, T ? 3.24893, and P ? 0.00058.
b? ? 0.98, T ? 0.91698, and P ? 0.17958.
Evaluation of GenoFlow HPV Test
May 2012 Volume 50 Number 5 jcm.asm.org 1695
cases that will progress to HSIL or worse in an Asian screening
population (28). It remains to be tested whether detection of any
other HPV types could similarly highlight patients at particular
risk of disease progression.
It is particularly reassuring that the absolute interassay (GF
assay versus LA assay) agreement levels for HPV types 16 and 18
for these two HPV vaccine-covered oncogenic HPV types were
ment than when assay-common (? ? 0.797) or oncogenic (? ?
0.812) HPV types were compared as a group.
assay because no amplification control (AC) signal was present in
them was actually positive for HPV43/44. One of the failed sam-
amplification of the ?-globin control. However, the four samples
needs to be optimized.
We evaluated the interlot reproducibility of the GF assay by
imately 80% (49/60 samples) of the samples yielded exactly the
similar to that previously reported for the LA assay (83%) (24).
GF assay is adequate. Among the 11 cases which yielded differ-
ent results when tested with two different lots of GF tests, most
of the differences involved low-risk HPV types (Table 7). How-
ever, in a few cases (4/11 cases), HR-HPV was detected in only
one of the two GF tests involving different lots of kits. Such
interlot variability, though it may have clinical implications, is
difficult to avoid.
between the LA and GF tests. Unlike all other HPV types, there is
while these types also have their own separate probe bands. Ac-
on the HPV52/33/35/58 probe band, HPV52 positivity can be
HPV33/35/58 probe bands. In other words, the LA test cannot
detect HPV52 in samples with multiple infections with HPV52
and HPV33/35/58. The GF test is free of this limitation, and in-
deed, significantly more HPV52-positive cases were detected by
GF assay than by LA assay (Table 4). Such a difference may have
HPV52 is more prevalent than in Western countries. In the last
only HPV16 and HPV18 (7). We also found that HPV52 was
the Guangzhou region of China in a recent study (17). Moreover,
HPV52 prevalence exceeded that of HPV16 in patients with normal
be 2.7% in studies carried out mostly in South America and Europe
On the other hand, we noticed that several pairs of HPV types
could not be distinguished unequivocally by the GF test due to
probe spot sharing. Most of the types in question were low-risk
HPV types to be established as carcinogenic by epidemiological
data (21). Recently, HPV66 was classified as carcinogenic (8).
Since the carcinogenicity of HPV types may differ by an order of
magnitude, we think that it is more desirable to develop separate
probe spots for these two HPV types.
This study was supported by research funds from the Diagcor Bioscience
1. Alsbeih G, et al. 2011. Prevalence and genotypes’ distribution of human
papillomavirus in invasive cervical cancer in Saudi Arabia. Gynecol. On-
2. Altman DG. 1991. Practical statistics for medical research. Chapman and
Hall, London, United Kingdom.
3. Arbyn M, et al. 2006. Chapter 9. Clinical applications of HPV testing: a
summary of meta-analyses. Vaccine 24(Suppl 3):S78–S89.
4. Bouvard V, et al. 2009. A review of human carcinogens. B. Biological
agents. Lancet Oncol. 10:321–322.
5. Castle PE, et al. 2009. Evaluation of a prototype real-time PCR assay for
carcinogenic human papillomavirus (HPV) detection and simultaneous
HPV genotype 16 (HPV16) and HPV18 genotyping. J. Clin. Microbiol.
6. Castle PE, et al. 2011. Performance of carcinogenic human papillomavi-
rus (HPV) testing and HPV16 or HPV18 genotyping for cervical cancer
study. Lancet Oncol. 12:880–890.
7. Chan PK, et al. 2009. Distribution of human papillomavirus types in
decades. Int. J. Cancer 125:1671–1677.
8. Cogliano V, et al. 2005. Carcinogenicity of human papillomaviruses.
Lancet Oncol. 6:204.
9. De Francesco MA, et al. 2010. Prevaccination distribution of human
papillomavirus types in Italian women with high-risk lesions and cervical
neoplasia. Intervirology 53:417–425.
10. Gravitt PE, et al. 2000. Improved amplification of genital human papil-
lomaviruses. J. Clin. Microbiol. 38:357–361.
11. Gravitt PE, Peyton CL, Apple RJ, Wheeler CM. 1998. Genotyping of 27
human papillomavirus types by using L1 consensus PCR products by a
single-hybridization, reverse line blot detection method. J. Clin. Micro-
12. Halfon P, et al. 2010. Comparison of the clinical performance of carci-
assay. J. Clin. Virol. 47:38–42.
13. Howell-Jones R, et al. 2010. Multi-site study of HPV type-specific prev-
alence in women with cervical cancer, intraepithelial neoplasia and nor-
mal cytology, in England. Br. J. Cancer 103:209–216.
14. Iavazzo C, et al. 2008. The histological outcome of glandular dyskaryo-
sis—AGUS—reported in Papanicolaou smears. J. BUON 13:97–100.
15. Khan MJ, et al. 2005. The elevated 10-year risk of cervical precancer and
possible utility of type-specific HPV testing in clinical practice. J. Natl.
Cancer Inst. 97:1072–1079.
16. Kreimer AR, et al. 2011. The epidemiology of oral HPV infection among
a multinational sample of healthy men. Cancer Epidemiol. Biomarkers
17. Liu SS, et al. 2011. Prevalence and risk factors of human papillomavirus
PLoS One 6:e19244.
18. Liu SS, Leung RC, Chan KK, Cheung AN, Ngan HY. 2009. Evaluation
of a newly developed GenoArray human papillomavirus (HPV) genotyp-
ing assay by comparison with Roche Linear Array HPV genotyping assay.
J. Clin. Microbiol. 48:756–764.
Wong et al.
jcm.asm.orgJournal of Clinical Microbiology
19. Mayrand MH, et al. 2007. Human papillomavirus DNA versus Papani-
20. Meijer CJ, et al. 2009. Guidelines for human papillomavirus DNA test
older. Int. J. Cancer 124:516–520.
21. Munoz N, et al. 2003. Epidemiologic classification of human papillo-
mavirus types associated with cervical cancer. N. Engl. J. Med. 348:
22. Sherman ME, Castle PE, Solomon D. 2006. Cervical cytology of
thelial lesion (ASC-H): characteristics and histologic outcomes. Can-
23. Sherman ME, Schiffman M, Cox JT. 2002. Effects of age and human papil-
loma viral load on colposcopy triage: data from the randomized Atypical
24. Steinau M, Swan DC, Unger ER. 2008. Type-specific reproducibility of
the Roche linear array HPV genotyping test. J. Clin. Virol. 42:412–414.
25. Takehara K, et al. 2011. Human papillomavirus types 52 and 58 are
prevalent in uterine cervical squamous lesions from Japanese women.
Pathol. Res. Int. 2011:246936.
26. van Hamont D, van Ham MA, Bakkers JM, Massuger LF, Melchers WJ.
genotyping test and the Roche linear array HPV genotyping test. J. Clin.
27. Wong FK, Ching JC, Chow JK. 2010. Comparison of the DiagCor Geno-
Flow human papillomavirus array test and Roche linear array HPV geno-
typing test. Open Virol. J. 4:169–174.
28. Wong OG, Lo CK, Szeto E, Cheung AN. 2011. Efficacy of Abbott
RealTime high risk HPV test in evaluation of atypical squamous cells of
undetermined significance from an Asian screening population. J. Clin.
29. Wright TC, Jr. 2007. Cervical cancer screening in the 21st century: is it
time to retire the PAP smear? Clin. Obstet. Gynecol. 50:313–323.
for the triage of women with high-risk HPV? cytology-negative results.
Am. J. Clin. Pathol. 136:578–586.
31. zur Hausen H. 2002. Papillomaviruses and cancer: from basic studies to
clinical application. Nat. Rev. Cancer 2:342–350.
Evaluation of GenoFlow HPV Test
May 2012 Volume 50 Number 5jcm.asm.org 1697