Patient and Specimen Collection
This was a cross-sectional study with a minimum sample size of 30 participants. Seventy women were recruited from the Colposcopy Outpatient Clinic of Dr. Cipto Mangunkusumo Hospital, Jakarta, Indonesia, between June 2016 and June 2017. Respondents were recruited through consecutive sampling. All participants were sexually active, 20–67 years old, nonpregnant, and provided written informed consent prior to enrollment in the study. An obstetrician obtained a cervical sample and placed it in a Liqui-PREP liquid-based cytology (LBC) vial (LGM International Inc., Melbourne, FL, USA). The sample was subsequently divided into 5 mL portions for cytological examination and polymerase chain reaction (PCR). The samples were stored at a temperature of 4°C and were processed within 2 days of collection.

Liquid-Based Cytology Pap Smear
Liquid-based cytology Pap smears were performed based on the corporation’s protocols under the supervision of a pathologist.¹⁹ The cervical cytology was categorized using the 2014 Bethesda system: high-grade squamous intraepithelial lesions (HSIL); low-grade squamous intraepithelial lesions (LSIL); atypical squamous cells, cannot exclude HSIL (ASCH); atypical squamous cells of undetermined significance (ASCUS); and normal cytology or negative for intraepithelial lesion or malignancy (NILM).²⁰

DNA Extraction
A 1-mL aliquot of a 5-mL LBC sample was centrifuged (Sorvall Biofuge Primo R, Thermo-Fisher Scientific Inc., Waltham, MA, USA) for 30 min. at 13,000 rpm. The pellet was then resuspended in 200µL phosphate-buffered saline after removing the supernatant. DNA isolation was conducted with a High Pure PCR Template Preparation Kit (Roche, Mannheim, Germany), based on the corporation’s protocols, with 50µL of final elution volume.²¹ Purified DNA was kept at -20^oC for ≤1 week prior to being used for PCR.

DNA Positive Control
A positive control was obtained from the Laboratory of Virology and Molecular Biology DNA Collection, Microbiology Department, Medical Faculty, Universitas, Indonesia. The positive control was detected using PCR and confirmed via DNA sequencing.

PCR for Detecting HPV
A 266-bp fragment of human b-globin was amplified to validate the sample collection and/or the DNA extractions (Figure 1a) using specific primers (Forward/Reverse: 5′ -AAG AGC CAA GGA AAG GTA-3′ / 5′-AAC TTC ATC CAC GTT CAC-3′).²² The PCR was conducted in 10µL total volume:1x PCR buffer, 1.2mM MgCl₂, 0.2mM dNTP mix, 5x Q Solution, 0.25 µM primer mix, 1.25 U HotStar Taq DNA Polymerase (Qiagen, Hilden, Germany), and 2µL DNA sample. HPV was detected using consensus primers GP5+/GP6+: 5’-TTT GTT ACT GTG GTA GAT ACT AC-3’ / 5’-GAA AAA TAA ACT GTA AAT CAT ATT C-3’,²³ in a 20µL total volume:1x PCR buffer, 1.2mM MgCl₂, 0.2mM dNTP mix, 5x Q Solution, 0.25µM primer mix, 1.25 U HotStar Taq DNA Polymerase (Qiagen, Hilden, Germany), and 5 µL DNA isolate. Thermal cycler settings were 95°C for 15 min followed by 40 amplification cycles at the following conditions: 30s at 94°C, 90s at 49°C,45s at 72°C, and final elongation 10 min at 72°C.

PCR for Detecting U. urealyticum, U.parvum, C.trachomatis, and M.hominis
Conventional duplex PCR was used to identify U.urealyticum, U.parvum, C.trachomatis, and M.hominis. Duplex PCR was carried out twice using primers according to previous studies, with modifications.^24,25 The first duplex was performed to detect M.hominis (101 bp; Forward/Reverse: 5’-TTT GGT CAA GTC CTG CAA CGA-3’ / 5’-CCC CAC CTT CCT CCC AGT TA-3’ and C.trachomatis (71 bp; Forward/Reverse 5’-CAT GAA AAC TCG TTC CGA AAT AGAA-3 / 5’-TCA GAG CTT TAC CTA ACA ACG CATA-3’), in 20µLtotal volume:0.2mM dNTP mix, 1.2 mM MgCl₂, 1x PCR buffer, 5x Q Solution, 0.05 µM of M.hominis primer mix, 0.45µM of C.trachomatis forward primer, 0.15µM of C.trachomatis reverse primer, 1.25 U Taq polymerase (Qiagen, Hilden, Germany), and 4 µL DNA isolate. Thermal cycler conditions were set up for 15 min at 95°C followed by 40 amplification cycles at the following conditions: 30s at 94°C, 30s at 60°C, 30s at 72°C, and a final elongation of 7 min at 72^oC. The second duplex PCR was performed to detect U.urealyticum (127 bp; Forward/Reverse:  5’-GAT CAC ATT TTC ACT TGT TTG AAG TG-3’/ 5’-CAC GAG CAG ATT GCA TTA AGT CAG-3’)²⁶ and U.parvum (99 bp; Forward/Reverse: 5’-GAT CAC ATT TTC ACT TGT TTG AAG TG’3 / 5’-AAC GTC GT CCA TAA GCA ACT TTG-3’).²⁷ Duplex PCR was performed according to previous studies, with modifications.^26,27 A 20 µL PCR reaction was performed with the following reagents:1x PCR buffer, 0.2 mM dNTP mix, 1.2 mM MgCl₂, 5x Q Solution, 1.25 U HotStar Taq DNA Polymerase (Qiagen, Hilden, Germany), 0.3 µM of U.urealyticum primer mix, 0.4 µM of U.parvum primer mix, and 4 µL DNA isolate. Thermal cyclers were set up for 15 min at 95^oC followed by 40 amplification cycles at the following conditions: 30s at 94°C, 30s at 60°C, 30s at 72°C, and a final elongation of 7 min at 72°C. All PCR amplifications were performed using an Applied Biosystems GeneAmp PCR System 2700 (Thermo Fisher Scientific, Waltham, MA, USA). Polyacrylamide gel electrophoresis (9%) was performed to analyze the PCR products, and UV transillumination for DNA band documentation was performed using a GelDoc system (Bio-Rad, Hercules, CA, USA).

Statistical Analysis
Data were inputted into Microsoft Excel 2019 v16.0 (Microsoft, Redmond, WA, USA) and subsequently analyzed using IBM SPSS Statistics for Windows (version 20.0; IBM Corp., Armonk, NY, USA). A comprehensive descriptive analysis was conducted to generate summary statistics, including mean, median, standard deviation, and 95% confidence intervals, pertaining to baseline demographic and behavioral attributes. Data are presented as numbers and percentages with 95% confidence intervals. Pearson’s chi-square test was used to examine the associations between categorical variables, whereas Fisher’s exact test was implemented in instances where one or more cells exhibited an expected frequency ≤5. Statistical significance was determined by associations with P values <0.05.