Citation: Macios, A.; Nowakowski,
A. False Negative Results in Cervical
Cancer Screening—Risks, Reasons
and Implications for Clinical Practice
and Public Health. Diagnostics 2022,
12, 1508. https://doi.org/10.3390/
Academic Editor: Paula Soares
Received: 4 May 2022
Accepted: 13 June 2022
Published: 20 June 2022
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False Negative Results in Cervical Cancer Screening—Risks,
Reasons and Implications for Clinical Practice and
Anna Macios 1, 2, * and Andrzej Nowakowski 2
1Doctoral School of Translational Medicine, Centre of Postgraduate Medical Education,
Marymoncka Street 99/103, 01-813 Warsaw, Poland
2Department of Cancer Prevention, The Maria Sklodowska-Curie National Research Institute of Oncology,
Roentgen Street 5, 02-781 Warsaw, Poland
*Correspondence: firstname.lastname@example.org; Tel./Fax: +48-22-546-30-67
False negative (FN) results in cervical cancer (CC) screening pose serious risks to women.
We present a comprehensive literature review on the risks and reasons of obtaining the FN results of
primary CC screening tests and triage methods and discuss their clinical and public health impact and
implications. Misinterpretation or true lack of abnormalities on a slide are the reasons of FN results
in cytology and p16/Ki-67 dual-staining. For high-risk human papillomavirus (HPV) molecular tests,
those include: truly non-HPV-associated tumors, lesions driven by low-risk HPV types, and clearance
of HPV genetic material before sampling. Imprecise disease threshold deﬁnition lead to FN results in
visual inspection with acetic acid. Lesions with a discrete colposcopic appearance are a source of FN
in colposcopic procedures. For FAM19A4 and hsa-miR124-2 genes methylation, those may originate
from borderline methylation levels. Histological misinterpretation, sampling, and laboratory errors
also play a role in all types of CC screening, as well as reproducibility issue, especially in methods
based on human-eye evaluation. Primary HPV-based screening combined with high quality-assured
immunocytochemical and molecular triage methods seem to be an optimal approach. Colposcopy
with histological evaluation remains the gold standard for diagnosis but requires quality protocols
and assurance measures.
Keywords: cervical cancer; screening; false negative results
The fundamental objective of screening is to distinguish healthy persons from those
in an early, asymptomatic phase of certain diseases by the use of available, validated,
inexpensive, non-invasive, and widely accepted screening tests [
]. As no ideally accurate
screening test exists, planning of screening procedures and algorithms is a matter of
balancing potential beneﬁts and harms [
]. Both false negative (FN) and false positive (FP)
results, i.e., negative results in persons with the disease and positive results in healthy
persons, respectively, should be maximally limited to minimize the risk of progression of
the disease that escaped detection, unfavorable prognosis, and delayed treatment, as well
as overdiagnosis, overtreatment, high costs, and needless anxiety .
The aim of this work is to discuss the risks and reasons of FN results of cervical cancer
(CC) screening tests and triage methods, as well as their implications on screening process.
1.1. Cervical Cancer Neoplasia
A great majority of cervical neoplasia is driven by infections with oncogenic types
of human papillomavirus (HPV) [
], and the development of only a small fraction of
neoplastic lesions is considered HPV-independent [
]. In most cases, HPV infections
acquired mainly by sexual contacts are self-limiting and regress spontaneously; however,
Diagnostics 2022,12, 1508. https://doi.org/10.3390/diagnostics12061508 https://www.mdpi.com/journal/diagnostics
Diagnostics 2022,12, 1508 2 of 24
a fraction of them progress to cancer precursor lesions, termed cervical intraepithelial
neoplasia (CIN), and rarely to invasive cancer [
]. At each stage of CIN, lesions may
regress spontaneously, but the probability of regression decreases when lesion severity
]. Currently, mild CIN (CIN1) is considered a benign cellular manifestation
of productive HPV infection with high regression potential, which, in most cases, requires
only surveillance [
]. Moderate CIN (CIN2) and severe CIN (CIN3), which is the direct
precursor of cancer, require treatment due to their higher risk of progression [
or excisional treatment of CIN2/CIN3 is used in non-pregnant women to avoid progression
to cancer [
]. The period between HPV infection and cancer invasion takes at least
in a great majority of cases [
]. The natural history of rare, HPV-unrelated
cervical neoplasia is not well-established yet.
1.2. Cervical Cancer Screening
Due to a long period of precancerous lesions development and the possibility of
their accurate identiﬁcation and effective treatment, CC screening has been introduced in
many countries [
]. Screening procedures aim at detecting CIN2 and CIN3 lesions in
asymptomatic women and providing them appropriate treatment and follow-up. Therefore,
screening may reduce CC incidence and, hence, mortality [
]. However, high coverage
of the target population and high quality of screening, triage, treatment, and follow-up
procedures, as well as the appropriate organization of the screening processes need to be
assured to achieve these goals .
Exfoliative cervical cytology remains the basic test for CC screening in developed coun-
tries. However, as the discovery of the role HPV in the aetiology of CC emerged [
], the pos-
sibility of applying new methods based on HPV detection in screening
HPV-based molecular testing was proven to be approximately 1.5 times more sensitive
than cytology-based screening in the detection of CIN2+ and CIN3+ [
]. Low- and middle-
income countries (LMIC) rely on cheaper methods, such as visual inspection with acetic
acid (VIA), which is recommended by the World Health Organization (WHO) in cases
when no other option is available or affordable [
]. Depending on the screening test,
different triage methods of screen-positive women are incorporated, including colposcopy
with colposcopically-targeted or random biopsies, a mixture of other screening tests, or
immediate treatment [
]. Other methods, such as dual staining (DS) for the simultaneous
expression of p16 and Ki-67 proteins in cells or detecting hypermethylation of the FAM19A4
and miR124-2 genes, are still being extensively studied and gradually implemented into
opportunistic screening programs [
]. Nevertheless, none of CC screening and triage
tests and methods have perfect accuracy. Limited speciﬁcity results in FP results. Less than
perfect sensitivity poses a risk of more dangerous FN results and consequences, which are
studied and discussed in this paper (Table 1).
Diagnostics 2022,12, 1508 3 of 24
The basic parameters of screening tests and triage methods, with potential reasons of false negative results occurrence. Sensitivity and speciﬁcity data
were retrieved from comprehensive meta-analyses. Pooled rates were reported with range of parameter analyzed in brackets. For dual staining, 95% CI from
meta-analyses were given in brackets. Cytology parameters were reported for atypical squamous cell of undetermined signiﬁcance benchmark; * for punch biopsies
taken in CIN1+ women; ** considering studies with excisional treatment following punch biopsies only; *** for women with low-grade cytological abnormalities
(atypical squamous cells of undetermined signiﬁcance, low-grade squamous intraepithelial lesions).
Sensitivity (%) Speciﬁcity (%) Potential Reasons for False Negative
CIN2+ CIN3+ CIN2+ CIN3+
Pap test 66 (34–96) 70 (39–85) 96 (86–99) 97 (85–99) (1) misclassiﬁcation;
(2) sampling errors;
(3) true lack of abnormal cells;
(4) subjectivity of evaluation.
Liquid-based cytology 76 (52–94) 76 (52–98) 92 (77–97) 92 (73–97)
High-risk HPV test 93 (61–100) 97 (81–100) 89 (64–95) 89 (69–94)
(1) true lack of HPV DNA in sample, due to non-HPV associated histological
type of lesion;
(2) histological misclassiﬁcation of endometrial cancer as cervical cancer;
(3) cancer developed from HPV type not detectable by screening tests
(probably carcinogenic, low-risk types);
(4) clearance of HPV infection before sampling;
(5) sampling and laboratory errors.
Visual inspection with
acetic acid 79 (65–91) 83 (58–95) 85 (74–95) 84 (74–94) (1) low reproducibility, observer-dependence;
(2) inaccurate deﬁnition of disease threshold.
Colposcopy with or without
91 (56–100) *
81 ** 91 (75–100) * 25 (0–79) *
63 ** 18 (0–65) *
(1) low reproducibility, observer-dependence;
(2) discrepancies between lesion severity in terms of histology and
(3) histological misclassiﬁcation of biopsies taken during colposcopy.
p16/Ki-67 dual staining *** 84 (77–89) 88 (58–98) 77 (70–82) 72 (67–76) (1) slide misinterpretation.
Methylation of FAM19A4 and
miR124-2 genes 68 79 78 77
(1) truly low risk of profession of precancerous lesion to cancer resulting in a
negative methylation result interpreted as FN;
(2) true lack of abnormal cells in a sample;
(3) borderline methylation level.
Diagnostics 2022,12, 1508 4 of 24
2. CC Screening and Triage Tests and Methods, Their Performance, and Reasons for
2.1. Screening Tests
For decades, the Pap smear remained a basic screening test for the secondary preven-
tion of CC in the more afﬂuent countries and has been proven to be very effective in the
reduction of CC incidence [
] and mortality [
]. Originally, cytology was performed as
a conventional smear (Pap test). Over time, liquid-based cytology (LBC) was developed
to reduce the fraction of slides unsatisfactory for evaluation obtained while using the Pap
test. LBC utilizes a liquid medium to preserve cells, which are subsequently transferred
to a slide in a thin layer [
]. The rate of slides that were inadequate for evaluation due
to overlapping cells, obscuring blood, or inﬂammation was reduced by the use of LBC,
compared with conventional cytology; however, no overall impact on limiting unsatisfac-
tory evaluation rates was observed [
]. Studies comparing the sensitivity of conventional
cytology and LBC provide conﬂicting results [
] regarding whether LBC is more [
equally sensitive [33–35], compared to Pap test.
Published studies reported speciﬁcity of cytology at 86–100% [
]. However, its limited
] and sensitivity, ranging around 30–87% [
], cause a serious risk of
missing precancerous lesions. The issue of interval cancers—invasive cancers diagnosed
after normal result of screening test within the screening interval [
]—is vital in screening
programs that are based on a Pap smear [
]. “European guidelines of quality assurance
in CC screening” recommended auditing all CC cases and their screening history, with
special attention paid to those with negative cytology results prior to cancer diagnosis; all
FN slides should be seeded among randomly selected screening slides and rescreened [
The audit is, indeed, performed in many countries, with a heterogenous methodology
Among the reasons for FN cytological results are: (1) misclassiﬁcation of an abnormal
slide as normal, which results in no referral for further diagnostic examinations; (2) sam-
pling errors; (3) true lack of abnormal cells on a slide due to lesion location—submucosal
or deep in the endocervical canal; and (4) rater-dependence, with low reproducibility.
Misclassiﬁcation of Abnormal Slide as a Normal One
Interpretation of a slide as normal, despite the presence of abnormal cells, was pointed
out as a reason for FN diagnoses in over 50% of slides in many studies. In a pooled analysis
by DeMay, 655 no intraepithelial lesions or malignancy (NILM) slides were evaluated,
and 340 of them were reclassiﬁed as abnormal (51.9%) [
], which was in line with re-
sults obtained by Kenter et al. (53.3%) [
], Bulk et al. (61.1%) [
], and, more recently,
Komerska et al. (54.2%) 
. Other researchers, however, reported the rate of misinterpre-
tation as low as 4.6% to 15% [
]. Emphasis on certiﬁcation, training, and proﬁciency
tests for laboratory staff may limit the rate of misclassiﬁcation .
Small number of abnormal cells on the slide was shown to impede proper evalua-
]. In a study by Sherman et al., 50% of re-evaluated false-negative slides had
<100 cells with features of squamous intraepithelial lesions [
]. In a similar analysis, 34 out
of 43 FN smears (79%) contained <100 abnormal cells, compared to 5/45 in true positive
cases (11%) .
Sampling Errors—Misclassification of Unsatisfactory for Evaluation Slides as Normal Ones
Sampling issues are a common reason for FN cytology results. In studies on the
reevaluation of NILM slides preceding cancer diagnosis, a fraction of slides were subse-
quently rated as unsatisfactory for evaluation, according to the Bethesda system [
with inﬂammation and obscuring blood hampering the proper diagnosis [
the scale of this phenomenon is not clearly established. In the study by Ejersbo et al.,
81% of all negative cytologies preceding CC diagnosis were recognized as sampling er-
rors after a review [
]. According to Sherman et al., among 18 patients with an overall
Diagnostics 2022,12, 1508 5 of 24
123 multiple normal slides preceding CIN3+ diagnosis, 7 had at least 2 slides reclassiﬁed
as unsatisfactory for evaluation (38.9%) [
]. In an audit of FN in Poland, only 3 amongst
48 re-evaluated normal slides preceding CC diagnosis were reclassiﬁed by all three experts
as unsatisfactory for evaluation (6.3%) .
True Lack of Abnormal Cells on the Slide
Some FN slides truly do not contain abnormal cells. This may be related to the ab-
sence of CC precursor lesion on the cervix at the time of sampling and extremely short
time of cancer development and invasion [
], as well as the nature of the lesion and
its location deep in endocervical canal or under the mucosa, which impede proper sam-
]. The risk factors of rapid onset of CC are not well-investigated; it was shown
Hildesheim et al.
that they are non-speciﬁc and similar to those conditioning a typical
development of the disease .
Histology of the Lesion
Adenocarcinoma (ADC), a histological subtype of CC, was identiﬁed signiﬁcantly
more frequently than squamous cell carcinoma (SCC) among women with FN cytological
results, compared to those with screen-detected cancer, due to their common location in the
endocervical canal and more difﬁcult sampling [
]. Based on the audit of over four
thousand CC cases diagnosed in Sweden in 2002–2011, Wang et al. concluded that women
with normal screening results had an 89% lower risk of SCC and only 60% lower risk of
ADC diagnosis [
]. The risk of obtaining a FN result, compared to a true positive cytology
report, was shown to be over three times higher in women subsequently diagnosed with
ADC, compared to SCC, and almost two times higher in those with diagnosis of carcinoma
other than squamous and glandular type [
]. It also coincides with the trend of the raising
rate of ADCs detection in countries with CC screening implemented; since this type of
carcinoma is more difﬁcult to diagnose in screening, the treatment of precancerous lesions
is suspended, and they may develop to the fully invasive form of disease .
Overall, the lower sensitivity of cytological screening for the detection of ADC than
SCC is a complex issue and may result from a true lack of abnormal glandular cells on the
slide due to: (1) the location of the lesions and less efﬁcient sampling [
], (2) difﬁculties
in the proper identiﬁcation of these cells on the slide and abnormal features [
(3) the possibly faster development and higher rate of progression of ADC precursors [
Data on the performance of cervical cytology in screening for cases of histology, other than
SCC and ADC, are scarce [61,68,69].
Cytology is an examination with limited reproducibility. As shown by Stoler et al. in
the ALTS study (ASC-US/LSIL Triage Study) on almost ﬁve thousand cytological slides, the
kappa coefﬁcient was only moderate (
= 0.46, 95% CI 0.44–0.48) for an atypical squamous
cell of undetermined signiﬁcance (ASC-US) and low-grade squamous intraepithelial lesion
(LSIL) cytologies [
]. Sørbye et al. found similar agreement on a set of 100 slides, with
uniform distribution of NILM, ASC-US, LSIL, atypical squamous cells—cannot exclude
high-grade intraepithelial lesions (ASC-H) and high-grade intraepithelial lesions (HSIL)
results, with the least agreement in low-grade abnormalities [
]. In the ATHENA study,
four laboratories in the United States evaluated over forty-six thousand LBCs; despite
the similar background population, the differences between them in the rates of abnor-
mal results were substantial (3.8–9.9%) [
]. All these results conﬁrm the subjectivity of
cytological evaluation, which may also impact the rates of FN results.
2.1.2. High Risk HPV Tests
Depending on the oncogenic potential of speciﬁc HPV genotypes, the International
Agency for Cancer Research (IARC) classiﬁed 12 HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52,
56, 58, and 59), as associated with a high risk (HR) of CC development. Eight types (26, 53,
Diagnostics 2022,12, 1508 6 of 24
66, 67, 68, 70, 73, and 82) are considered probably carcinogenic due to the more common
occurrence in CC cases than in cohorts with normal cytology [
]. The oncogenic potential
of the subsequent nine types (6, 11, 34, 40, 42, 43, 53, 54, and 73) is classiﬁed as low .
As a primary prevention against HPV infection, prophylactic HPV vaccines are cur-
rently available, and many countries worldwide have decided to implement HPV vaccina-
tion into national immunization programs [
]. The bivalent vaccine against the HPV-16
and -18 types is being gradually replaced with quadrivalent (against HPV types 6, 11, 16,
and 18) and nine-valent (against HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58) vaccines.
All of them were shown to be safe and effective [
], with the highest efﬁcacy in the
prevention of precancerous lesions reaching 100%, when vaccinating girls without previous
contact with the virus [79,80].
Only two likely carcinogenic genotypes (66 and 68), according to IARC classiﬁcation,
are included in the standard HPV tests used in the primary screening. Due to the rare
occurrence of remaining genotypes among CC cases (26, 53, 67, 70, 73, and 82), a possible
slight increase in sensitivity at the expense of a high decrease in speciﬁcity, extending the
spectrum of HPV types included in validated screening tests, is not recommended [
According to the meta-analysis by Koliopoulos et al., the sensitivity of HR HPV tests
for the detection of CIN2+ lesions in group of women aged 30 or more was 93.9%, compared
to 72.2% for cervical cytology (both LBC and Pap). HPV tests were shown to be statistically
signiﬁcantly more sensitive for CIN2+ detection than both conventional cytology and LBC,
with relative sensitivities of 1.52 and 1.18, respectively. Speciﬁcity was signiﬁcantly lower
for HPV testing (with relative speciﬁcities of 0.94 for Pap test and 0.96 for LBC). Similar
results were obtained for CIN3+ detection .
On account of the increasing number of HPV tests on the market without proper
validation, some criteria ensuring safety and high-quality performance were urgently
]. In 2009, Meijer et al. published a list of requirements to be fulﬁlled by the
producer, in order to introduce the test for primary HPV screening, which included: (1) a
sensitivity of at least 90% of sensitivity of the Hybrid Capture 2 test for CIN2+ detection
30 years old; (2) a speciﬁcity of at least 98% of speciﬁcity of the Hybrid
Capture 2 test for CIN2+ detection in women
30 years; and (3) the intra-laboratory
reproducibility and inter-laboratory agreement, with a lower conﬁdence bound of at least
]. According to recent study by Poljak et al., 254 distinct HPV tests and over 425 test
variants exist on the market; however, over 90% of them were not validated in accordance
with Meijer’s criteria .
The use of HPV tests for detecting HR HPV types in screening signiﬁcantly reduced
the problem of FN results, compared to cytological-based screening, due to the signiﬁcant
increase in sensitivity [
]. Despite the high sensitivity, the number of women at risk of FN
results in HPV testing increased as the method spread around the world, and this should
be carefully monitored [
]. The reasons for a truly negative HPV test result preceding CC
diagnosis include the following: (1) the true lack of HPV DNA related to the non-HPV-
associated nature of cancer; and (2) the histological misclassiﬁcation of endometrial cancer
as of cervical origin. FN results may potentially follow from: (1) cancer development from
infection with low-risk oncogenic types; (2) clearance of HPV infection before sampling
leading to the conclusion of HPV-unrelated disease; and (3) sampling and laboratory errors.
A. Reasons of Negative HPV Test Results in CC Cases
True lack of HPV DNA in the sample
The homogenous group of SCC, which accounts for about 75–85% of all CC cases
], is highly associated with HPV infection [
]. The second type, ADC, is
rarer, with many subtypes distinguished and some of them being extremely rare [
overall rate of HPV-negative (HPV(
)) ADCs ranges between 15% and 48% and strongly
depends on the subtype [
]. A low viral load in all ADC types additionally impedes
the capability of its detection; as a consequence, the diagnosis is stated later, and the
detected tumors are larger .
Diagnostics 2022,12, 1508 7 of 24
According to the International Endocervical Adenocarcinoma Criteria and Classi-
ﬁcation, two groups of ADCs were extracted, depending on the HPV infection status:
(1) non-HPV-associated (endometrioid, gastric type, serous, clear cell, and mesonephric
carcinoma) and (2) HPV-associated (usual, villoglandular, mucinous (not otherwise speci-
ﬁed), mucinous intestinal, mucinous signet ring, and invasive stratiﬁed mucin-producing
]. Detailed information on HPV-dependent and -independent CC types is
presented in Table 2, based on the current WHO Classiﬁcation of Tumors .
HPV-dependency in cervical cancer types, according to the WHO Classiﬁcation of Tumors;
NOS—not otherwise speciﬁed.
HPV-Dependance Precursor Lesions Invasive Lesions
Squamous intraepithelial lesions:
Squamous cell carcinoma:
Adenocarcinoma in situ adenocarcinoma, usual type
Adenocarcinoma, mucinous type:
mucinous NOS adenocarcinoma
signet-ring cell adenocarcinoma
Adenocarcinoma, mucinous type:
stratiﬁed mucin-producing carcinoma
adenosquamous and mucoepidermoid
neuroendocrine tumors, NOS
small cell neuroendocrine carcinoma
large cell neuroendocrine carcinoma
carcinoma admixed with neuroendocrine
Mesonephric remnants adenocarcinoma, mesonephric type
Atypical lobular glandular
dysplasia adenocarcinoma, gastric type
squamous cell carcinoma, HPV-independent
adenocarcinoma, clear cell type
Genetic tumor syndromes:
Diagnostics 2022,12, 1508 8 of 24
Table 2. Cont.
HPV-Dependance Precursor Lesions Invasive Lesions
haematopoetic proliferations and neoplasia
solitary ﬁbrous tumor, NOS
NTRK (neurotrophic tyrosine receptor
kinase)-rearranged spindle cell neoplasm
alveolar soft part sarcoma
In general, HPV(
) CC cases were shown to be associated with higher age, worse
prognosis, diagnosis of ADC, and higher risk of relapse and distant metastases [
Histological misclassiﬁcations seem to explain a part of the HPV(
) CCs phenomenon.
Endometrial cancer, which is not, or barely, related to HPV infection [
], may be misclassi-
ﬁed as being of cervical origin. This erroneous classiﬁcation may also partially explain the
higher age of HPV(
) CC cases, compared to HPV-positive (HPV(+)) ones, since the risk of
endometrial cancer is the highest in women around 70 years old [100,101], which is about
10 years later than the peak of CC incidence.
In the study by the Cancer Genome Atlas Research Network, 8 out of 178 primary CC
cases were reclassiﬁed as endometrial-like (5%); a total of 7 of them were HPV(
a study of 371 biopsy-proven CCs, 21 of 31 HPV(
) cases were pointed out as being of non-
cervical origin (68%) [
]. Lower rates of misclassiﬁed CCs were reported by Pirog et al. in
the retest of 760 ADC cases collected worldwide [
]: 49 of 731 cases (6.7%) were reclassiﬁed
by the board of four pathologists: 23 of them (3.1%) were classiﬁed as not, or doubtfully,
being of cervical origin, with 26 (3.6%) classiﬁed as non-epithelial neoplasias. Indeed, inter-
and intra-rater agreement in the histological classiﬁcation of non-SCC depends on the
type of carcinoma and is rather low, with a kappa coefﬁcient (measuring the agreement
beyond the chance) of 0.44, indicating moderate agreement, according to Landis and Koch
]. However, another study showed low reproducibility in HPV-associated
ADCs and higher reproducibility for non-HPV-associated ADCs .
B. Reasons of Potential FN HPV Test Results in CC Cases
Cervical Cancer Developed from Infection with Low-Risk Oncogenic Types
Published studies conﬁrmed the relationship between the HPV types classiﬁed by
WHO as probably, or possibly, carcinogenic (which are not being detected by primary
HPV screening tests) and the development of a fraction of CC cases. Those cases are
usually single-infected, which makes them impossible to diagnose with contemporary HPV
In the ATHENA trial, with over 46 thousands women involved, 55 HPV(
cases were found [
]. After re-evaluation of the LBC samples with more sensitive tests,
22 cases were reclassiﬁed as high-risk HPV(+) (40.0%), and the next 10 were reclassiﬁed
as low-risk HPV(+) (18.2%) with HPV-73 or HPV-82 detected. Halec et al. showed the
transcriptional activity of HPV types 26, 53, 66, 67, 68, 70, 73, and 82 in cases with CC and
single HPV infection of these types [
]. The HPV-73 type was also conﬁrmed as oncogenic
by Amaro-Filho et al. [
]. Of 544 CIN3+ cases with a single infection in Japan [
HPV types listed by Halec et al. were found in 28 cases, with single infection being only
5.1%. Another study of 136 CC cases showed the reclassiﬁcation of 6 out of 14 (42.9%)
) women as HPV(+), with HPV-11, 16, 18, 45, and 68 detected [
a study of the Latin USA population, single HPV-90 prevalence was unexpectedly high,
Diagnostics 2022,12, 1508 9 of 24
with 9.4% of infected women having additional cytological abnormalities. Most women
who were diagnosed with HPV-90 (96.8%) had a single infection [
]. In the study of
1739 patients with SCC, 14 women (0.8%) were infected with one of the WHO-probable
carcinogenic HPV types, and all of them were reported as single-infected .
Hit and Run Theory
A hit and run theory may bring another explanation for HPV(
) CCs phenomenon.
The hypothesis is broad and related not only to CC. It states that some types of cancer need
the viral infection to initiate the oncogenesis; however, as the mutations are accumulated,
the need for oncogenic factor presence no longer exists, and the initial infection clears [
Subsequent cancer is, indeed, viral-driven, but the test performed after clearance is unable
to detect absent viral DNA.
According to the work by Tjalma et al. on over 6000 CIN2+ women, comparing
HPV prevalence in high-grade CIN and invasive CC, the rate of HPV(
) high-grade CIN
cases was lower than the rate of HPV(
) invasive CCs (2% vs. 8%, respectively) [
Differences were even sharper in a study by Coutleéet al.: 11.5% of invasive CCs were
), whilst only 0.2% of CIN2 and CIN3 women no detected HPV DNA [
observation stayed in line with the hit and run hypothesis, as the longer time needed for
invasion may also lead to HPV infection clearance. However, this hypothesis clearly needs
further investigation. Studies should be planned, in order to prove the absence of HPV
DNA in invasive cancer cases, as well as the presence of HPV DNA and expression of genes
involved in carcinogenesis in the precursor lesions sampled prior to cancer diagnosis.
Sampling Errors, Laboratory Errors
Aside from the test-speciﬁc factors, other common mistakes may be responsible for FN
results. Sampling errors may result in a lack of the necessary amount of HPV DNA material;
a similar problem is vital in cytological screening [
]. As self-sampling methods for HPV
detection emerged, the question was raised regarding whether the rate of unsatisfactory for
evaluation self-collected samples will be acceptable. Recently published studies suggested
that proper test selection for self-sampling results in a sensitivity comparable with clinician-
sampling; therefore, sampling errors will be of less importance .
Another classical reason for obtaining false-negative results is laboratory error, fol-
lowing inappropriate laboratory procedures, poorly trained staff, no validation or quality
assurance of procedures, and the use of screening tests with no analytical and clinical
2.1.3. Visual Inspection with Acetic Acid
In settings where ﬁnancial resources and the number of well-trained health workers
are limited, the WHO recommends using VIA with the immediate treatment of suspected
treatable lesions as a screening method. VIA involves the application of a 3% to 5% solution
of acetic acid on the cervix and observation of the acetowhite lesions using the naked
eye after at least 1 min since application. If necessary and appropriate, treatment with
cryotherapy or loop electrosurgical excision procedure (LEEP) should be provided. This
approach is aimed at ensuring a high negative predictive value, reducing the loss to
follow-up, and treating all women in need using scant ﬁnances .
The sensitivity of VIA screening for CIN2+ detection ranged widely between 42% and
92% in 26 studies, where all subjects underwent conﬁrmatory testing, with a pooled sensi-
tivity of 80% [
]. Similar results were shown in a meta-analysis by Arbyn et al. [
results, however, may be overestimated, due to some methodological issues [
a fraction of studies on VIA effectiveness for symptomatic women were included [
Secondly, colposcopically-targeted biopsy was most often used as a gold standard, and
this method is correlated with VIA results, since both of them rely on a visual evalua-
]. Thirdly, in many settings, women were screened for the ﬁrst time in their
lives, which may result in the easier detection of more advanced lesions [120,121].
Diagnostics 2022,12, 1508 10 of 24
The phenomenon of FN VIA results is not widely discussed in the literature, due
to the lack of long-term follow-up of women in developing countries. Besides, the gold
standard of colposcopy with biopsy is not always performed in all screened women; in
many studies, only VIA(+) participants underwent conﬁrmatory testing [
]. The potential
causes of false-negative VIA results may include: small size, endocervical and submucosal
location of the lesions, and various reactions of the abnormal epithelium on acetic acid
application; however, these have not been well-investigated in the literature on the subject.
Low reproducibility and observer-dependence are the next reasons.
VIA, as an unquantiﬁable method based on the human eye evaluation only, is strongly
observer-dependent and has low reproducibility. The results of the study in rural Nigeria
showed that, depending on the provider, 0–21% of women were suspected to have cancer,
and 0–25% of women were assessed as VIA(+), despite the homogenous background
]. In the study carried out in Andhra Pradesh, India, women were examined
by six gynecologists, and their positivity rate varied between 4% and 31% [
plays an important role in ensuring the optimal performance of VIA-based screening [
In a study on over 140 thousand of women in India, retraining resulted in a drop in
positivity rates from 17% to 10%. Additional training entailed an increase in the agreement
of both cytological and histological evaluations, as well .
Additionally, the deﬁnition of the disease threshold may impact the accuracy of VIA,
as it is not strict. As stated in guidelines by JHPIEGO, abnormalities looking similar
to “raised and thickened white plaques or acetowhite epithelium, usually near the SCJ
(squamocolumnar junction)” are of clinical signiﬁcance when using VIA for screening, as
well as “cauliﬂower-like growth or ulcer; fungating mass”, which should be considered a
cancer suspicion [
]. Cryotherapy is recommended if the whole SCJ is visible, the lesion
is fully visible, and it does not exceed 75% of the ectocervix [
]. All of these criteria are
ambiguous, and the impact of human subjective evaluation cannot be completely excluded,
regardless of the degree of detail in the description. The need for the appropriate and
regular training of health workers providing VIA is, therefore, essential. Additionally,
the use of artiﬁcial intelligence is currently discussed as a helpful tool for distinguishing
clinically signiﬁcant lesions from others .
2.2. Triage Methods
2.2.1. Colposcopy with or without Colposcopically-Directed Biopsy
Colposcopy is one of the most commonly used triage methods to screen positive
women in cytology and HR HPV-based screening [
]. Colposcopy protocols included the
comprehensive evaluation of the cervix, with the use of the magniﬁed appearance after the
application of an acetic acid solution and Lugol’s iodine, identiﬁcation and initial grading
of abnormalities, collection of tissue material for histological examination, and excisional
procedures, if necessary [127,128].
Despite the numerous attempts to standardize the procedure for the proper training
and certiﬁcation of colposcopists, the accuracy of the procedure is far from perfect, and
FN results are common. According to the meta-analysis by Mitchell et al., the pooled
rate of positive cone biopsies, widely accepted as a gold standard, among colposcopically-
directed negative biopsies in women with cytological abnormalities reached approximately
]. In a prospective study of follow-up women with cytological abnormalities and
negative colposcopy, 9% were subsequently diagnosed with CIN2+ [
]. A follow-up of
participants of ALTS study with adequate baseline colposcopy revealed 30% of them were
diagnosed with CIN2+ over the 2 years since their baseline test .
The rate of CIN2/CIN3 detected by colposcopy with targeted biopsy reached 56% in
2112 women with cytological abnormalities [
]. On the other hand, according to meta-
analysis by Underwood et al., over 91% of CIN2+ women were correctly identiﬁed, but
the authors admitted that high sensitivity values probably followed from veriﬁcation bias.
When restricted to the studies with immediate excisional treatment following colposcopy,
sensitivity dropped down to 81.4% for CIN2+ detection [
]. Underestimation of diagnosis
Diagnostics 2022,12, 1508 11 of 24
in colposcopically-directed biopsy is especially common in women subsequently diagnosed
with microinvasive CC .
Likewise in cytology, colposcopic examination, as a method based on the evaluation by
human eye, is subjective and observer-dependent. Poor agreement between colposcopists
was reported in the literature [
] with lowest agreement for evaluating low-grade
]. Low reproducibility (with kappa coefﬁcient of about 0.2) for critical
issues impacting the ﬁnal result—sharpness of margins, atypical vessels, or lesion presence—
was also demonstrated [
]. In a study by Pretorius et al. 7 physicians achieved
sensitivity of colposcopy with colposcopically-targeted biopsy between 28.6% and 92.9%
for CIN3+ detection [
]. The size of the lesion and its severity was shown to have an
impact on accuracy of the colposcopic impression—it appeared more precise when more
quadrants of cervix were affected by the lesion .
Sensitivity of colposcopy is believed to depend on colposcopists’ experience; however,
many studies contradict this statement [
]. Gage et al. reported similar sensitivity
for nurse practitioners, general gynecologists, gynecologic oncology fellows and gyneco-
logic oncologists [
]. Bekkers et al. demonstrated higher sensitivity for junior compared
to senior colposcopy fellows but at the expense of lower positive predictive value which is
a logical effect of less self-conﬁdence resulting in tendency of upgrading diagnoses and
taking more biopsies by less experienced colposcopists [
]. Colposcopy performance
improvement may be achieved, however, by providing standardized training to colposcopy
]. For the last decade, efforts have been made for developing standardized
colposcopic terminology by, i.e., the International Federation for Cervical Pathology and
Colposcopy (IFCPC) [
] and American Society for Colposcopy and Cervical Pathology
]. Still, further work is needed to disseminate the proposed nomenclature
and train medical staff.
Discrepancies between Actual Lesion Severity and Colposcopic Image
The issue of colposcopically-targeted and random biopsies taken during colposcopy
was widely discussed in the literature. Since the severity of lesion did not always cor-
respond with the most visually advanced area of the colposcopic appearance, random
biopsies were suggested, in order to elevate colposcopy sensitivity [
]. The study by
Pretorius et al. reported that 37% of CIN2+ cases were recognized by random biopsy and
not by colposcopically-directed biopsy [
]. Huh et al. analyzed the results of 2796 women
who participated in ATHENA study, with random biopsies performed during colposcopy,
due to no visible lesions on the cervix: 20.9% and 18.9% of biopsies were subsequently
diagnosed as CIN2+ and CIN3+, respectively [
]. In the study by Gage et al., taking two
or more biopsies during one procedure was found to signiﬁcantly improve the sensitivity
of colposcopy [
], which was conﬁrmed by others [
]. Endocervical curettage was
shown to improve sensitivity of colposcopy performance, with the yield of 2% to 15% for
CIN2+ detection [149–151].
A part of the FN colposcopies phenomenon may also occur due to histological mis-
classiﬁcations. According to a study by Ismail et al. based on 100 colposcopic biopsies, as
evaluated by 8 experienced colposcopists, the overall agreement was moderate but highly
dependent on lesion severity, ranging from a kappa of 0.175 for CIN1 and CIN2 to 0.832 for
invasive cancer [
]. In a similar study by McCluggage et al., the average of overall agree-
ment between pathologists was poor, with a kappa of around 0.3 [
]. In a histological
review in Poland, only 205 out of 368 histological samples that were primarily assessed as
CIN2+ were subsequently conﬁrmed in an expert diagnosis (55.7%) [
]. However, the
discrepancies resulting in normal- and high-grade result were rather rare and, based on the
mentioned studies, occurred in <1% of paired evaluations.
Diagnostics 2022,12, 1508 12 of 24
2.2.2. P16/Ki-67 Dual Staining
DS for the simultaneous detection of p16 and Ki-67 proteins within the same cell be-
came a widely investigated triage procedure, due to its high sensitivity, relatively high speci-
ﬁcity, and possibility to perform on the residual sample after the HPV test and LBC [
In the DS procedure, at ﬁrst, the cellular material is stained to visualize cells containing
both proteins. Next, the slide is prepared for microscopic evaluation. The threshold for
positivity is predominantly set at 1 dually-stained cell.
DS was shown to have a higher sensitivity in detecting precancer lesions and provide
longer negative reassurance in HPV(+) women than negative cytology, with 5 years relative
risk of CIN2+ detection of 0.7 [
]. In published studies, the sensitivity for CIN3+
detection was stable and achieved about 92% [
]. The sensitivity for CIN2+
detection seemed to not depend on the examined population, either. For women with
ASC-US/LSIL, the sensitivity was 88% [
]; for HPV(+) women, it was 86% [
for the HPV(+) cohort with abnormal cytology, it was 85% [
]. In a meta-analysis by
Peeters et al., 84% sensitivity and 77% speciﬁcity were reported for CIN2+ detection, with
similar values for CIN3+ detection in the ASC-US+ women (88% and 72% for sensitivity
and speciﬁcity, respectively) .
Published evidence pointed out difﬁculties in slide interpretation as a reason of im-
proper DS result. As the threshold for positivity is usually set at one dual-stained cell on
the slide, the correct exclusion of the positivity of each visible cell is crucial. Cells presented
in groups on the slide, or with a scant cytoplasm and weak p16 staining, seemed to be
challenging for readers [
], as well as, on the contrary, those with extremely strong
background staining, which hampered appropriate evaluation of nucleus staining [
A low number of dually stained cells, sometimes with only one visible, may occur and
lead to oversight [
]. The level of subjectivity in evaluation may be decreased, and
the total agreement can be increased by training [
]. Nevertheless, the reported
reproducibility is good to excellent for interobserver agreement, with kappa values ranging
between 0.65 and 0.91 [162,163,165].
The issue of factors inﬂuencing overall positivity of a DS slide is by far better in-
vestigated in the literature than the matter of strict FN DS reports. It was shown by
Benevolo et al.
that the longer time between sampling and immunostaining or between
immunostaining and evaluation of the slide, the higher probability of its positive interpreta-
tion. Other factors like referral cytology result, presence of CIN2+ or HPV mRNA detection
were also naturally shown as associated with slide positivity [
]. To our best knowledge
no other reasons of possible FN reports for DS were discussed in published reports.
2.2.3. Methylation of FAM19A4 and hsa-miR124-2 Genes
The test measuring methylation levels of speciﬁc genes involved in the process of car-
cinogenesis was shown as a promising method of precancerous lesions detection in HPV(+)
women. Many potential biomarkers have been veriﬁed to ﬁnd appropriate ones [
According to the data published in 2020, four methylation tests of different genes are
currently commercially available on the European market for cervical cancer detection:
two of them to be performed on cervical smear: GynTect
(based on genes: ASTN1, DLX1,
ITGA4, RXFP3, SOX17, and ZNF671) and QIAsure Methylation test kit (FAM19A4 and
miR124-2 genes) and two on cervical scrapes: PAX1 DNA detection kit (PAX1 gene) and
ZNF582 DNA detection kit (ZNF582 gene) [
]. The performance of the assay detecting
the hypermethylation of FAM19A4 and hsa-miR124-2 was demonstrated as promising:
at ﬁrst in research on cell cultures [
] and afterwards in humans [
] and at
present this test seems to be the best validated one.
In published studies virtually all CC cases were properly identiﬁed by methylation
tests based on these genes. In a cross-sectional study of 519 CC cases from 25 countries
in 5 continents, 510 (98.3%) had positive methylation test result and—worth noting—the
Diagnostics 2022,12, 1508 13 of 24
set of samples consisted of both cervical scrapes and cervical tissues and covered SCCs
and ADCs as well as adenosquamous cell carcinomas and other rare CC histotypes [
) cases were correctly detected by the methylation test (18/19, 94.7%).
The results of smaller studies performed on both cervical scrapes and clinician-taken
], as well as on self-samples [
], stayed in line with the
results of Vink et al., pooling all results of 208 out of 213 CC cases, which were detected
by the methylation test (97.7%), including 31/31 for self-samples (100%) and 177/182 for
other samples (97.3%). The sensitivity for detecting CIN2+ and CIN3+ lesions, however,
was lower and ranged between 56.1% to 80.3% for CIN2+ lesions and between 69.6% to
94.7% for CIN3+ lesions [175,178,180].
Lower Risk of Progression
The negative result of methylation test in women with CIN2 or CIN3 may indicate
low risk of progression to cancer in these persons. De Strooper et al. used the length of
preceding HPV infection in women with precancerous lesions as a proxy for advancement
of the lesion and risk of progression to cancer. In his study all CIN2+ lesions preceded
by HPV infection lasting >5 years were detected by the methylation test (51/51) and
only 8/19 (42%) of early CIN2/CIN3 lesions (with <5 years since prior HPV infection)
were detected [
]. Similar results were published by Bierkens et al. for CADM1 and
MAL genes; levels of methylation of those genes in women with CIN2/CIN3 were higher
for cases with
5 years of preceding HPV infection compared to those with <5 years of
preceding HPV infection .
This explanation of lower performance of methylation test in less advanced lesion
is in line with observation that CIN2/CIN3 lesions were more commonly methylation
negative in women below 30 years old [
]. Indeed, the regression rate in younger
women is higher and is probably caused by a shorter time of associated HPV-infection [
However, the lower sensitivity in detecting CIN2/CIN3 lesions in young women may be
clinically beneﬁcial by applying surveillance instead of treatment in women with lower
risk of cancer progression.
Borderline Methylation Level
Samples with methylation level near the cutoff value seem to be less reproducible.
Bonde et al. observed that 80 samples among 983 selected for both intra- and inter-
laboratory agreement analysis had discordant results (8.1%), with 77 of them being close
to the methylation positivity threshold (96% of all samples with discrepant results). Addi-
tionally, the only case of CC undetected by methylation test in this study had borderline
methylation level . This phenomenon may be responsible for both FN and FP results
and indicate the need of establishing cutoff properly.
3. Prospects in Cervical Cancer Screening
In order to address still unmet needs in CC screening some novel methods have been
proposed. HPV testing on ﬁrst-void urine samples seems to be an acceptable method for
reaching underscreened women, with sensitivity and speciﬁcity comparable with respective
indicators of HPV test performed on clinician-taken samples [
]. HR HPV digital
droplet polymerase chain reaction test, based on the fractionation of a sample into droplets
and subsequent detection and quantiﬁcation of HPV DNA in each of them, may improve
the sensitivity of HPV detection in CIN lesions and early CC tumors [
promising, yet more research is needed for safe implementation these methods into real-life
3.1. Implications for Screening
To balance sensitivity and speciﬁcity of screening process both indicators of the test
as well as interval between screens should be taken into consideration. More frequent
testing increases sensitivity and reduces FN rate at the expense of decreasing speciﬁcity and
Diagnostics 2022,12, 1508 14 of 24
rising number of FPs. Risks of both CC and precancer occurrence after a negative screening
test result were investigated in studies with large sample size and long-term follow-up
to assess negative predictive value of the test and establish acceptable intervals [
Nevertheless, in some countries, the opportunistic screening without any restrictions on
the interval competes with the organized program. Opportunistic screening is, however,
not recommended, as it leads to a high smear consumption in only a part of population,
limits the accessibility to screening for some women, and may result in poor quality of
offered procedures and low cost-effectiveness [
]. Only organized screening programs
with intervals set according to the risk of FN and FP results enable limiting these adverse
3.1.1. Screening Tests
No screening or triage test ideally distinguishing healthy and diseased people exists,
neither for CC screening nor for any other type of screening. Sensitivity of 100% is beyond
the reach of current possibilities and both policymakers and screening organizers should be
aware of it. Women must be informed that participation in screening signiﬁcantly reduces
the risk of both incidence and mortality of CC but it does not eliminate it completely.
Emphasis should be placed on the use of certiﬁed and validated screening tests, only to
ensure highest possible quality. Additionally, quality assurance procedures and quality
control should be in place.
It is impossible to avoid FN results in cervical cancer screening but due to their serious
consequence efforts should be undertaken to limit their burden. The whole set of reasons
of FN results may be divided into broadly understood errors and test deﬁciencies. To
minimize human factor extensive and regular training should be introduced, however, it
does not always guarantee satisfactory effects and may be difﬁcult to run in some settings.
According to the “WHO guidelines for screening and treatment of cervical pre-cancer
lesions for cervical cancer prevention”, HPV testing is considered superior to both cytology-
based and VIA screen-and-treat approach by means of overall effectiveness of screening
]. HPV-based CC screening is less subjective than cytology or VIA, may be
automated, run on a large scale of self-collected material, or as a part of point-of-care
infrastructure and “see-and-treat” protocols. Its main advantage is the minimization of
FN result rate. While comparing the cytology currently being replaced with the HPV
test, in the sense of FN results rate for CIN2+ detection, the latter seem better, with about
7% of potential false HPV(
) results, compared to 13–70% for the Pap test [
]; a similar
comparison can be made for VIA as well, with the rates ranging between 8% and 58% [
Although HPV-based screening is very sensitive, false-negative results may occasion-
ally occur [
]. As supporters of co-testing state, adding cytology to the primary HPV
testing increases the screening sensitivity and gives the opportunity to detect HPV(
subtypes of ADC, which are invisible for HPV testing. However, ADCs cause considerable
difﬁculties for cytological detection and even co-testing may be insufﬁcient for detecting
]. The study on over 1.2 million co-tested women showed that 3.5% of precancers
and 5.9% of cancers were initially HPV(−) and cytology-positive . However, bearing
in mind the low rate of CC incidence, the yield of using co-test, compared to the HPV test
alone is approximately only ﬁve additional cases detected per million screened. In the light
of hit and run hypothesis at least some of precancer lesions should be successfully detected
by the HPV test before infection clears and HPV(
) CC develops. Keeping in mind also
high costs of cytology performance [
] and maintenance of cytological infrastructure, the
use of primary HPV testing alone seem more reasonable than co-testing.
3.1.2. Triage Methods
A relatively low speciﬁcity of HPV-based screening, however, leads to an excessive
number of referrals for colposcopy, colposcopies performed, and treatment procedures
executed. Accurate triage methods are, therefore, crucial for avoiding both overdiagnosis
Diagnostics 2022,12, 1508 15 of 24
and overtreatment. Some actions were taken to support proper decision when using
subjective screening and triage tests; this may both limit the number of FN and FP results.
Colposcopy still serves as a gold standard in many countries with screening programs
implemented worldwide. A few decades ago, cervicography, based on taking pictures of
the cervix, which were subsequently evaluated by trained colposcopists, was proposed as
a useful tool [
]. More recently, attempts were made to digitize cervigrams, in order to
facilitate their storage and usage [
]. The combination of deep-learning algorithms on col-
lected cervical images and the medical history of patients allowed us to develop automated
visual evaluation (AVE) application that supports providers in proper decision-making,
regarding patients’ management, especially in LMIC. The results seemed promising [
However, further research is needed for its proper validation before its implementation into
real-life screening [
]. Additionally, appropriate nomenclature and colposcopic protocols
were developed as a response to the need for the uniﬁcation of standards [
the further need for their dissemination and implementation in screening programs is vital.
DS obtained approval by the U.S. Food and Drug Administration with the indication
for HPV(+) women with HPV genotypes other than 16 and 18, in order to determine the
need for colposcopy referral, as well as for HPV(+) women with HPV 16 or 18 detected, to
guide their management [
]. There have been some endeavors to automize and objectify
DS assessment. Wentzensen et al. showed that DS using artiﬁcial intelligence (AI) and
deep-learning protocols had a lower positivity rate, equal sensitivity, and higher speciﬁcity,
compared to both manually evaluated cytology and DS [
]. However, these methods
need further validation.
Among the investigated triage procedures, the methylation of FAM19A4 and miR124-2
genes provided promising results, in terms of high sensitivity for cancer detection, and
allowed for the use of self-sampled vaginocervical material. However, the rate of FN
methylation results is higher for less severe CIN2 and CIN3, which are the main targets
of CC screening. According to the aforementioned studies, the methylation test correctly
identiﬁes cases with long-lasting HPV infection; however, further studies are required, in
order to assess the potential for the progression, regression, and persistence of HPV(+)
methylation-negative HSIL cases.
To conclude, FN results are unavoidable in screening, and this phenomenon can only
be minimized by careful quality assurance. Human error can be reduced, but not excluded,
by both training and use of AI methods. In light of the published studies and despite
some inevitable adverse effects, HPV-based screening seem to be the most efﬁcient, in
regard to FN rates. The triage methods of HPV(+) women are still being investigated.
Both DS and methylation of FAM19A4 and miR124-2 genes seem promising for the future;
however, colposcopy with appropriate protocols and quality measures incorporated will
probably still serve as a reliable triage examination, until further studies will be performed
to elaborate on the most optimal protocol for proper risk stratiﬁcation and management of
A.M. performed the literature search and drafted manuscript. A.N. designed
the study and critically revised the manuscript. All authors approved the ﬁnal version of the
manuscript to be submitted and agreed to be accountable for all aspects of the work. All authors
have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
No new data was created or analyzed in this study. Data sharing is not
applicable to this article.
Conﬂicts of Interest: Authors declare no potential conﬂict of interests relevant to this article.
Diagnostics 2022,12, 1508 16 of 24
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