Title: TITLE: Does full HPV genotyping perform similarly well in clinician-collected cervical
samples and self-collected vaginal samples when using the CLART HPV4S assay?
Version: 4 Date: 25 Dec 2022
Reviewer:  Sveinung Wergeland Sørbye
Reviewer's report:
Solnæs et al. have included 212 women aged 30-59 years with cytological ASC-US diagnosis
and analyzed clinician-collected and self-collected vaginal samples with the CLART HPV4S
assay to compared the two types of samples with respect to HPV detection and genotype
agreement.  They found the same HPV prevalence in self-collected vaginal samples compared
to clinician-collected cervical samples 19.3% (41/212) vs 18.4% (39/212).  HPV was detected
with both sampling methods in 13.2% (28/212) women. The detection rate of CIN2+ was 6.6%
(14/212). The CLART HPV4S assay detected approximately the same number of CIN2+ cases
in the self-collected vaginal samples compared to the clinician-collected cervical samples (13
vs 11 cases). The same genotypes were detected in 75% (21/28) of the HPV-positive paired
samples.  In conclusion, the CLART HPV4S assay performed similarly in self-collected vaginal
samples and in clinician-collected cervical samples with respect to both HPV detection and
genotype agreement.
The claims are properly placed in the context of the previous literature. The experimental data
support the claims. The manuscript is written clearly enough that most of it is understandable to
non-specialists. The authors have provided adequate proof for their claims, without overselling
them. The authors have treated the previous literature fairly. The paper offers enough details of
methodology so that the experiments could be reproduced.
Comments
HPV self-sampling has been widely supported by the scientific community following a strong
body of literature on the subject. Self-sampling is important in cervical cancer screening as it
has been shown to improve participation. It is well documented that HPV-testing has proven
superior to cytology with regards to sensitivity in detection of CIN and cancer. The value of
self-collected samples is reliant on the quality of the molecular testing performed, as well as the
patients’ preference in sampling procedure and compliance to follow up on positive test results.
Self-collection of cervical samples is reported to be highly acceptable and preferred by most
women, being a promising approach to enhance women’s participation in regular screening for
cervical cancer prevention. It offers significant benefits over conventional sampling in terms
of cost, coverage and convenience for patients. Self-sampling reaches high-risk groups who
currently have limited access to national health system screening for personal and practical
reasons. Numerous studies comparing self-collected and clinician-collected samples for HPV
detection show good concordance when clinically validated PCR-based methods are used. In a
meta-analysis by Arbyn et al., self-sampled HPV tests based on PCR for the detection of CIN2+
were shown not to have statistically different sensitivity or specificity compared with clinician-
sampled tests.
Usually, when comparing two different sampling methods for HPV-testing, both samples are
taken the same day. In this study, the clinician-collected cervical samples and self-collected
vaginal samples was taken at different times with a median 43 days between the paired samples.
When most HPV-infections are transient, and women may acquire HPV every time they have
sex, this will make the results less reliable.
Usually, when comparing two different sampling methods for HPV-testing, all women with at
least one positive HPV-test, should have colposcopy and biopsy. In this study, only women with
a positive Roche Cobas 4800 clinician-collected cervical samples had histology. This will make
the results less reliable.
If I understand the results correctly, 46 women had a positive Cobas 4800 with 14 CIN2+ (PPV
30.4%), 39 women had a positive CLART in clinician-collected samples detecting 11 CIN2+
(PPV 28.2%) and 41 women had a positive CLART in self-collected samples detecting 13
CIN2+ (PPV 31.7%). The results are similar using different HPV-tests and sampling methods.
Line 35, "The study found a slightly higher but non-significant HPV prevalence" => "The study
found the same HPV prevalence"
(The HPV prevalence is not non-significant, but the difference between the HPV prevalence is
not different. If a difference is not statistically significantly different, it is no difference.)
Line 36, "The CLART HPV4S assay detected a few more CIN2+ cases" => "The CLART
HPV4S assay detected approximately the same number of CIN2+ cases"
Line 69-71, "Accordingly, a European cohort study conducted in a referral population has
demonstrated that the risk of CIN2 and CIN grade 3 and cancer (#CIN3) is genotype specific
with large variations"
Yes. But the purpose of the screening program is not to find as many CIN3+ as possible, but to
prevent cervical cancer. The most important is the genotype distribution in cervical cancer, not
in CIN3+. The three most important genotypes are HPV 16, 18 and 45. The next types are 33,
31, 58 and 52 (Arbyn 2014, Tjalma 2012, Tjalma 2013).
In Sweden, women with HPV type 16, 18 and 45 are followed-up closely, also when normal
cytology (Livmoderhalscancerprevention - nationellt vårdprogram, page 91).
https://cancercentrum.se/globalassets/vara-uppdrag/prevention-tidig-upptackt/gynekologisk-
cellprovskontroll/vardprogram/nationellt-vardprogram-cervixcancerprevention.pdf
Lline 145-147, "To account for the low number of HPV positives, the genotypes were
hierarchically subdivided into five groups based on the oncogenicity for cervical (pre-)cancer
(6): 1) HPV16/18, 2) HPV31/33/58, 3) HPV45/51/52, 4) HPV35/39/68, and 5) HPV56/59/66."
HPV type 45 should be classified as highly oncogenic (group 1), not middle oncogenic (group 3).
Line 185, "The HPV prevalence (any type) detected in the self-samples was not significantly
higher than the prevalence in the clinician-collected samples" =>  "The HPV prevalence (any
type) detected in the self-samples was similar to the prevalence in the clinician-collected
samples"
(When a difference is not statistically different, there is no difference. Actually, one concern
has been that the sensitivity of self-samples are lower than clinician-collected samples, but
most studies find at least the same sensitivity in the self-samples versus the clinician-collected
samples)
Line 207-208, "Using self-sampling resulted in slightly more HPV16/18 cases than when
clinician-collected samples were used" => "Using self-sampling resulted in approximately the
same number of HPV16/18 cases than when clinician-collected samples were used"
Line 208-209, "However, more HPV31/33/58 cases were found when clinician-collected
samples were used" => "Approximately the same number of HPV31/33/58 cases were found
when clinician-collected samples were used"
Line 216, "The self-samples seemed to detect a few more CIN2+ cases than clinician-collected
sample" => "The self-samples detected approximately the same number of CIN2+ cases as
clinician-collected sample"
Line 220-221, delete "Self-samples detected a few more HPV16/18 cases than clinician-
collected samples, whereas clinician-collected samples found a few more HPV31/33/58 cases
than self-sample"
Line 286-287, "One could imagine two outcomes from a HPV-positive self-sample: 1)
HPV16,18,31,33,58 positive women could be directly referred for colposcopy" => "One could
imagine two outcomes from an HPV-positive self-sample: 1) HPV16,18,31,33,45,52,58 positive
women could be directly referred for colposcopy"
Line 289-291, "It may also be possible, that the best risk stratification strategy among HPV-
positive self-samplers could be a combination of genotyping and DNA methylation profiling"
add "or a 7-type HPV mRNA test" (Aranda Flores 2021)
Table 1, you have calculated the sensitivity and specificity of self-samples using the HPV
positivity rate in clinician-collected samples as the gold standard. I would recommend using
histologically conformed CIN2+ as the gold standard, both for self-samples and for clinician-
collected samples.
Table 2, In my experience HPV type 45 should be grouped with HPV 16/18, not with HPV 51/52
References
Arbyn, M., Tommasino, M., Depuydt, C. and Dillner, J. (2014), Are 20 human papillomavirus
types causing cervical cancer?. J. Pathol., 234: 431-435. https://doi.org/10.1002/path.4424
Tjalma WA, Depuydt CE. Don't forget HPV-45 in cervical cancer screening. Am J Clin Pathol.
2012 Jan;137(1):161-2; author reply 162-3. doi: 10.1309/AJCPYB6C4HIMLZIX. PMID:
22180491.
Tjalma WA, Fiander A, Reich O, Powell N, Nowakowski AM, Kirschner B, Koiss R, O'Leary
J, Joura EA, Rosenlund M, Colau B, Schledermann D, Kukk K, Damaskou V, Repanti M,
Vladareanu R, Kolomiets L, Savicheva A, Shipitsyna E, Ordi J, Molijn A, Quint W, Raillard A,
Rosillon D, De Souza SC, Jenkins D, Holl K; HERACLES/SCALE Study Group. Differences
in human papillomavirus type distribution in high-grade cervical intraepithelial neoplasia and
invasive cervical cancer in Europe. Int J Cancer. 2013 Feb 15;132(4):854-67. doi: 10.1002/
ijc.27713. Epub 2012 Jul 24. PMID: 22752992.
Aranda Flores, C.E., Gomez Gutierrez, G., Ortiz Leon, J.M. et al. Self-collected versus clinician-
collected cervical samples for the detection of HPV infections by 14-type DNA and 7-type
mRNA tests. BMC Infect Dis 21, 504 (2021). https://doi.org/10.1186/s12879-021-06189-2