This study was approved by the ethics committees of the Pontificia Universidad Católica
de Chile and the Servicio de Salud Metropilitano Sur-Oriente. All participants gave
written informed consent before entering the study. The detailed methods have been
described elsewhere 3]. Briefly, the study was conducted in a low to middle socio-economic area of Santiago,
Chile, within the setting of the national cervical cancer program. Three public primary
health centers and their referral hospital participated in the study with their regular
infrastructure, personnel and protocols; only HPV testing was newly implemented. We
invited women from the general population aged 25–64 who resided locally and were
not pregnant, hysterectomized or virgins. This population had not received HPV vaccination
(the HPV vaccine was incorporated into the public immunization program in 2014, targeting
girls age 9–10 years). A total of 8,265 women received both hrHPV DNA detection and
conventional Pap cytology; the 930 women who tested positive by either test underwent
colposcopy and directed biopsy, as did a control sample of 295 screen-negative women.
The current analysis is focused on the 776 hrHPV positive women with complete follow-up
data (88Â %) who were reflex tested for the presence of hrHPV 16 and 18 genotypes.
hrHPV DNA testing and HPV16/18 genotyping
Both tests were performed at the Molecular Biology Laboratory of the Pontificia Universidad
Católica de Chile University. Each sample was divided into two 500 ul aliquots. One
was tested for hrHPV using the Hybrid Capture 2 High Risk HPV DNA test (HC2, Qiagen,
Gaithersburg, USA), which detects viral DNA by nucleic acid hybridization and uses
a pooled probe set for 13 oncogenic HPV genotypes (HPV 16, 18, 31, 33, 35, 39, 45,
51, 52, 56, 58, 59, 68); samples were considered positive when the relative light
unit/cutoff was ?1.0. The second aliquot was stored at ?80° Celsius until genotyping,
1–2 years after collection. Genotyping was performed as a reflex test in HPV-positive
women, using the Digene HPV Genotyping PS test (Qiagen, Gaithersburg, USA), currently
available in Europe and Australia. This test is based on hybrid capture technology
with a working protocol similar to the HC2 test, but in which each sample is tested
against separate sets of type-specific probes allowing the individual detection of
HPV16, HPV18 and HPV45 20]; we only tested for HPV16 and HPV18. Reference cut-off values were determined per
manufacturer’s instructions; samples were considered positive when the relative light
unit/cutoff was ?1.3.
Conventional cytology
Cytologic evaluation was conducted at the area referral hospital, following regular
protocols, with results reported according to the 2011 Bethesda System. Cytotechnicians
and supervising pathologists were blind to the women’s HPV status.
Diagnostic confirmation
Colposcopy with directed biopsy was considered the gold standard for diagnostic confirmation.
These procedures were performed by specialists at the health centers’ referral hospital,
as part of their regular activities; according to standardized protocol, biopsies
were taken of all areas with acetowhite lesions or atypical vessels. Histological
examination of the biopsies was performed by hospital pathologists, with regular internal
quality control procedures in place; results were reported according to standard cervical
intraepithelial neoplasia (CIN) terminology. Women with negative colposcopies were
considered without lesions.
Analysis
Diagnostic accuracy of HPV16/18 genotyping for the detection of CIN2+ and CIN3+ among
hrHPV positive women was assessed calculating sensitivity, specificity, positive predictive
value (PPV) and negative predictive value (NPV), with 95Â % confidence intervals (CI);
crude estimates were obtained based on women with complete screening, triage and diagnostic
confirmation results.