A total of 235 married and sexually active women (mean age of 37.9 y; ages range 21 to 48 years) were recruited by the Department of cytopathology of Yonsei University College of Medicine for this study. Specimens collected from cervical liquid-based cytology medium were negative for intraepithelial lesions. Participants had no clinical STD symptoms that required a clinical visit, no history of STDs, and no abnormal Papanicolau tests (Pap smear) results. We also excluded patients with SIL or carcinoma, postmenopausal women, or those with previous operative or therapeutic history related to gynecologic disease. The study protocol was approved by the local institutional review board, and written informed consent was obtained from all participants.
Multiplex-PCR for STI
Pretreatment and DNA Extraction of cervicovaginal swab specimens
Cervix swab samples were collected from the posterior fornix and lavaged with 5 mL of sterile phosphate-buffered saline (PBS pH 7.4). PreservCyt specimens were tested by Seeplex® STI Master ACE Detection (Seegene, Seoul, Korea) or Seeplex® HPV4A ACE Screening (Seegene) within 1 month of collection. PreservCyt transport medium containing endocervical cells was vortexed vigorously, and 1-mL samples were transferred into 1.5-mL polypropylene tubes, each containing 1 mL of PreservCyt transport medium. The tubes were centrifuged at 13,000 × g for 15 min at 20°C. Supernatants were discarded, and each cellular pellet was suspended in 200 μL of PBS. DNA was purified from these samples using the QIAamp DNA Mini Kit according to the manufacturers' instructions (Qiagen, Hilden, Germany). The quality and quantity of purified DNA was measured by spectrophotometry.
Multiplex PCR
Four primer sets were tested by STI multiplex PCR: Seeplex® STI Master Panel 1, 2, 3, and HPV4A ACE Screening. Panel 1 comprised of Neisseria gonorrhoeae (NG), Mycoplasma hominis (MH), Chlamydia trachomatis (CT), Ureaplasma urealyticum (UU), and M. genitalium (MG). Panel 2 comprised Trichomonas vaginalis (TV), Gardenerella vaginalis (GV), Bacteroides fragilis (BF), Mobiluncus curtisii (MC), and M. mulieris (MM). Panel 3 comprised Candida glabrata (CG), C. tropicalis (CT1), C. parapsilosis (CP), Group β- streptococcus (GBS), and C. albicans (CA). Primers were designed such amplicon sizes differed sufficiently to be distinguished from each other; they ranged from 212 bp (UU) to 635 bp (CG) and the internal control was 981 bp. Optimized multiplex PCR was performed in 20 μL reactions containing DNA template, primer mixture (final concentration of each primer, 3 pmole), 2× Master mix (Seegene, Korea) and 30 μg/mL of 8-methoxypsoralen (MOP), which prevents contaminating DNAs from being amplified. PCR amplification was performed in an Applied Biosystem 9700 thermal cycler (Perkin-Elmer, Boston, MA, USA) with the following conditions: 94°C for 15 min, followed by 40 cycles of 94°C for 30 sec, 63°C for 1.5 min (or 60 °C for 1.5 min for HPV4A ACE Screening), and 72°C for 1.5 min; and final extension at 72 °C for 10 min. A DNA plasmid was added to the PCR reaction mixtures as an internal control to be co-amplified with the target DNAs collected from the clinical specimens. Sterile deionized water was included as a negative control in each batch of PCR reactions. Target organisms, target genes, accession numbers, and expected amplicon sizes are summarized in Additional File 1.
Multiplex-PCR-
HPV was detected with the Seeplex® HPV4A ACE screening kit for 14 HR-HPV types (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 67, 68, and 70) and 5 LR-HPV-types (6, 11, 42, 43 and 44). In addition individual genotyping of HPV-16 and HPV-18 was performed with different multiplex primer mixtures.
Hybrid capture 2 (HC2)
HC2 HPV test (Digene, Gaithersburg, MD) was performed according to the manufacturer's instructions to detect HPV, starting from 4 mL of liquid cytology specimens. The HC2-13 HR probe cocktail detects HPV types-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. Relative light unit/cut-off ratio between 1 and 2.5 relative light units was used as the threshold for a positive result.
HPV Microarray
We used an HPV genotyping DNA microarray (Biocore Ltd, Korea, Seoul) with multiple oligonucleotide probes of L1 sequence of 26 types of HPV (HR: 16,18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 69, 73 and 53; LR: 70, 6, 11, 32, 34, 40, 42, 43, 44, 54, 55, 57, 61 and 62). L1 consensus PCR products were hybridized to the probes on the microarray, and HPV genotypes were identified with a fluorescence scanner (GenPix 4000B, Axon Instruments Inc, CA) with a 532 nm laser for excitation of Cy3.
DNA sequencing and cloning
Discrepant results among the HC2, DNA microarray, and Seeplex® HPV4A ACE screening kit results were resolved by sequencing with the PGMY09/PGMY11 primer sets. PCR reactions (20 μL) were performed with 2 μL template DNA, 1 μM of each primer, and 2x Master mix (Seegene, Korea). PCR amplification was carried out with an Applied Biosystem 9700 thermal cycler (Perkin-Elmer) with the following parameters: 94°C for 15 min followed by 40 cycles of 94°C for 30 sec, 55°C for 1 min, and 72°C for 1 min, and a final extension at 72°C for 10 min. The entire volume of each PCR reaction was run on a 2% agarose/1× Tris-acetate-EDTA gel containing ethidium bromide. The 465-bp bands amplified by the PGMY09/PGMY11 primers were excised and transferred to 1.5-mL tubes. DNA was extracted with the QiaexII gel extraction system (Qiagen, Valencia, CA) and eluted with 20 μL Tris-EDTA buffer. The products were ligated into the pGEM-T Easy vector (Promega, Madison, WI), transformed into chemically-competent JM109 cells, and plated onto two Luria-Bertani (LB) plates containing ampicillin, isopropyl-D-thiogalactopyranoside, and 5-bromo-4-chloro-3-indolyl-D-galactopyranoside. DNA was extracted from the white colonies with the Qiaprep Spin kit (Qiagen) and sequenced with the ABI Big Dye Terminator v3.1 cycle sequencing kit and an ABI 377 sequencer (Applied Biosystems, Foster City, CA). Sequences were trimmed to exclude the amplification primer and vector sequences, and the resulting fragments were analyzed by BLAST http://www.ncbi.nlm.nih.gov/blast. The top hit for each sequence was listed as the genotype.
Statistical Analysis
The agreement rate of kappa value with respect to a quantitative measure of agreement between assay methods was calculated. The sensitivity of the HPV multiplex PCR, HC2 or DNA microarray relative to DNA direct sequencing is the proportion of positive samples in each assay among sequencing positive samples, and the specificity is the proportion of negative samples in each assay among those negative samples in direct sequencing. The positive predictive value is the proportion of positive samples in DNA direct sequencing, among those positive samples in each assay, and the negative predictive value is the proportion of negative samples in sequencing among those negative in each assay.