Study population
Two different populations were enrolled in this study. The first population comprised 185 cases with cervical lesions, which contained detectable HPV DNA and underwent cervical cytopathological evaluation in the Department of Pathology of Guangdong Provincial People’s Hospital between November 2017 and March 2018. The second population comprised 290 cases with cervical lesions, which contained detectable HPV DNA and underwent cervical histopathological evaluation in the Department of Clinical Laboratory of the National Cancer Center/Cancer Hospital between March 2018 and October 2018. The inclusion criteria included an age between 21 and 80 years, the absence of pregnancy, an intact cervix, no history of cervical lesions, and no history of chemotherapy, radiotherapy or surgical treatment.
DNA extraction from cervical cells
In this study, for each patient, flushed the female genital tract with normal saline, probed a specimen collection brush into the cervix, turned the brush to take the cervical secretions after 5 seconds, and placed the brush in a 2 ml volume collection tube. The cervical specimens collection were performed by an experienced doctor as detailed for each test. And the cervical specimens were stored in a refrigerator (4°C) and analyzed within 14 days.
For the SureX HPV test, total cellular DNA was extracted from cervical specimens using the extraction work station Smart LabAssist-16/32 (Taiwan Advanced Nanotech Inc., Taiwan) according to the manufacturer's instructions. For Cobas 4800 HPV test, HPV DNA was extracted from cervical specimens using the automatic nucleic acid extractor cobas x480 DNA extractor (Roche Molecular Systems, Inc., USA). For the Venus HPV test, HPV DNA was extracted from cervical specimens using the automatic nucleic acid extractor Autrax workstation (Shanghai ZJ Bio-Tech Co., Shanghai, China). For the detection of 14 HR-HPV types, the three different method were used in accordance with the manufacturer's instructions. Negative and positive controls provided in the kits were included in each PCR test.
SureX HPV genotyping test
The SureX HPV test (Health Gene Technologies, Ningbo, China) utilized amplification of target HPV DNA by multiplex polymerase chain reaction (PCR) and capillary electrophoresis to detect and genotype 25 HPV types according to the length of specific amplification fragments. The HPV types were identified by the test including HPV 6, 11, 16, 18, 26, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 81, 82 and 83. For the SureX HPV test, specifically designed primers were targeted on early genes E6, E7 and E1 of HPV types, plasmid pcDNA 3.1(+) (pcDNA for short)and human β-globin locused to make sure the length of the amplified PCR products were at least 3 nucleotides difference. So, via PCR amplification of target DNA, 27 targets could be identified by capillary electrophoresis in a single analysis according to the length of PCR products. The measure of β-globin served as a quality control mechanism to confirm that a negative result was not due to inappropriate sample collection or failure of DNA extraction. The internal control pcDNA could monitor the PCR process and ensured that the testing procedure had been properly performed. Therefore, a validated specimen should show the specific peaks of pcDNA and β-globin. The peak height of pcDNA ought to be equal to or greater than 500 RFU (≥ 500). When the peak of β-globin was absent indicating insufficient cervical cells, we suggestd resampling should be conducted. For PCR amplification products (1µl) subjected to capillary electrophoresis in an ABI 3500 Dx/3500xL Dx genetic analyzer, the cutoff value for determining specimens of HPV positive was:i) Signal of a HPV type ≥ 300 RFU; or ii) Peak area ratio (the ratio of the peak area of a HPV type to the peak area of pcDNA)≥0.2.
Cobas 4800 HPV test
The cobas4800 HPV test (Roche Molecular Systems, Inc., USA) used primers to define a sequence of approximately 200 nucleotides within the L1 region of the HPV genome, which specifically detected 14 high-risk types (16, 18, 31, 33, 35, 39,45, 51, 52, 56, 58, 59, 66, and 68 ). The cobas4800 test mainly included two processes. Firstly, HPV DNA was extracted through automated sample preparation, and then the HPV and β-globulin target DNA sequences were amplified by PCR primers. The amplified target DNA sequences were combined with their corresponding fluorescent probes. There were 4 types of fluorescent probes for detection, namely HPV16, HPV18, β-globulin and 12 other high-risk HPVs. The probes were labeled with different fluorescent dyes, and the subtype of HPV in the sample were determined by real-time monitoring of fluorescent signals. In addition, β-globin was used as an internal control (IC) to ensure a sufficient sample quantity for HPV DNA detection. If the cycle threshold (Ct) cutoff value for HPV16 was ≤40.5, a sample was considered HPV-positive; if the Ct cutoff value was >40.5 and β-globin was effective, it was considered negative; otherwise, it was considered invalid. If the Ct cutoff value for HPV18 or any of the 12 other high-risk types was ≤40, a positive result was determined; and if the Ct cutoff value was >40 and β-globin was effective, a negative result was determined; otherwise, the result was considered invalid.
Venus HPV genotyping test
The Venus HPV genotyping test (Zhijiang Bio-Tech Co., Shanghai, China) was based on real-time fluorescence PCR technology and contained a specific ready-to-use system for the detection of 15 15 types of HR- HPV genotypes, including HPV 16, 18, 31,33,35,39,45,51,52,56,58,59,66, 68, 82. Detection of amplified HPV DNA fragments was performed in the fluorimetric channels FAM, HEX/VIC/JOE, TEXAS RED/Cal Red 610 and CY5 with the fluorescent quencher BHQ1. Human minibrain homolog (MNBH) was amplified as an internal control (IC) to indicate the presence of sufficient nucleic acid from the human MNBH gene. The Ct value was calculated. If the Ct value was ≤38.0, a sample was considered HPV-positive. If the Ct value of the IC was ≤ 32.0, and "undetermined" or "no CT" was displayed in the other channels, the sample was determined to be HPV-negative. If the Ct value was 38.0~40.0, the reaction was repeated. If the Ct value remained in this range and the amplification curve was a typical S-shape, the sample was considered HPV-positive; if the amplification curve was not a typical S-type, as the sample was considered HPV-negative.
Sequencing
PCR sequencing was performed for further confirmation of discordant results. Sequencing reactions was targeted on type-specific E6/E7 gene and performed using the ABI PRISM BigDye Terminator V3.0 kit (Applied Biosystems) and analyzed in an ABI 3730 genetic analyzer (Applied Biosystems) at Sangon Biotech Co. (Shanghai). DNA sequences were then compared with the sequences of known HPV types using the Basic Local Alignment Search Tool from the National Center for Biotechnology Information website (http://www.ncbi.nlm. nih.gov/BLAST).
Histological diagnosis
The cytopathological diagnosis was based on the nomenclature of the Bethesda system of cervical cytology. The histopathological diagnosis was classified according to the WHO histological criteria for cervical tumors and was used as the gold standard, with cervical intraepithelial neoplasia grade 2 (CIN2) and higher (CIN2+) considered positive. Cytological diagnosis was performed by the Department of Pathology, Guangdong Provincial People’s Hospital; pathological diagnosis, by the Cancer Hospital, Chinese Academy of Medical Sciences.
Statistical analysis
All statistical analyses were conducted using SPSS 23.0. The consistency checks were evaluated by the Kappa (k) values. Using CIN2+ as a reference, the sensitivity, specificity, and area under the receiver operating characteristic (ROC) curve (AUC) were calculated. All differences with P values of < 0.05 (two-tailed) were considered statistically significant.