Study setting and design
This was a cross-sectional study of adult women attending the cervical cancer screening and gynecologic oncology unit of the department of obstetrics and gynecology, Jos Teaching Hospital in Jos, North Central Nigeria. Participants were enrolled between January 2020 and February 2022.
Study participants and data collection procedures
Screening and enrollment of eligible study participants were done at the cervical cancer screening and colposcopy clinic of the Department of Obstetrics and Gynecology of the Jos University Teaching Hospital, Jos, Nigeria. The source population were adult women who presented for cervical cancer screening, follow-up colposcopy for abnormal cytology report, or evaluation for suspected ICC. Women who were at least 30 years old, with no prior treatment for abnormal cervical precancer or invasive cancer and an intact uterus and cervix, and who were not pregnant were eligible for inclusion. Enrollment was stratified by cervical cytology. We enrolled women with a spectrum of negative intraepithelial neoplasia or malignancy (NILM), low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL), and women with confirmed invasive cervical cancer (ICC). After the principal investigator explained the study purpose and procedures and obtained written informed consent, each participant had a gynecological examination in the colposcopy room. A sterile dry metal speculum was gently inserted into the vagina to visualize the cervix and the vaginal fornices. Hybrid capture 2 Digene HPV collection brushes were used to obtain samples from cervixes, and cytobrushes were used to obtained samples for cytology from each patient. Subsequently, cervico-vaginal lavage samples were collected as described below. Details of cervical cytology reporting and colposcopic assessments have been described previously.[3]
Laboratory procedures for processing, preservation, and microbiome DNA extraction
Sample Collection and Processing
All sample collections were performed by the gynecological oncology team in the colposcopy assessment room. Cervical vaginal Lavage (CVL) sample was obtained by washing the upper vaginal walls and cervix of each of the female subjects with 10 mL of sterile Phosphate Buffer Saline applied and aspirated with a sterile disposable pipette and Drummond Pipet-Aid. The CVL sample was aseptically aspirated into 15mL sterile falcon tubes placed on ice, capped, and transported to the genomics laboratory for processing and storage within 1-4 hours of collection. The 10 mL of CVL sample in the falcon tube was vortexed and aliquoted into 2mL cryovials tubes. The aliquoted 2 mL CVL sample was centrifuged at 10,000 x g for 10 minutes and 1 mL of the resulting supernatant was pipetted and discarded while the pellet was resuspended in 1 mL of DNA/RNA shield and stored at -800C.
Extraction and Purification of DNA
The extraction and purification of the DNA from the CVL sample were performed using Quick-DNA™ Fecal/Soil Microbe Miniprep kit obtained from Zymo Research, USA, following manufacturer directions.
Briefly, the CVL was removed from -800C storage and allowed to thaw before it was resuspended by vortexing. 400 µl CVL was used for DNA extraction using the Zymo quick DNA fecal/soil microbe DNA Miniprep kit. Cat No: D6010, Lot No: 207380, 207953 &208889, according to the manufacturer’s protocol. The extracted DNA was quantified using a Qubit dsDNA HS Assay Kit with a Qubit 4 Fluorometer (Thermofisher, Scientific, USA), and stored at -80°C until use.
HPV DNA extraction
As described above the CVL samples were thawed, and 500 μl was pelleted by centrifugation at 3,000g for 10 minutes and re-suspended in 200 μl phosphate-buffered saline. DNA was extracted using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), following the manufacturer’s protocol. Each DNA extraction run contained positive and negative controls to monitor the extraction procedure and the extracted DNA was eluted in a final volume of 50μl. The extracted DNA was quantified using a Qubit 4 Fluorometer.
Detection of HR-HPV genotypes using the Real-time Polymerase chain reaction (qPCR)
For each DNA sample, HPV detection and genotyping were performed using AnyplexTM II HPV28 Detection kit (Seegene, Korea). Briefly, each PCR reaction was performed in a 20-μl reaction volume consisting of 5 μl aliquot of DNA added to AnyplexTM PCR Mix (15 μl aliquot each for mixtures A and B) on a CFX96 Real-time PCR system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer’s instructions. The thermal cycler conditions consisted of an initial incubation at 50°C for 4 min, denaturation at 95°C for 15 min, followed by 50 cycles of denaturation (30 sec at 95°C), annealing (1 min at 60°C), and elongation (30 sec at 72°C). Cyclic-Catcher Melting Temperature Analysis (CMTA) was performed after PCR cycles 30, 40, and 50. CMTA was performed by cooling the reaction mixture to 55°C, holding at 55°C for 30 sec, and heating from 55°C to 85°C (5 sec/0.5°C) with continuous fluorescent monitoring. The L1 gene of HPV DNA was the target of the assay, together with simultaneous targeting of a housekeeping gene (Human beta-globin) which was co-amplified as an internal control to monitor DNA purification efficiency, PCR inhibition, and cell adequacy. The results were exported and analyzed using the Seegene Viewer software provided by the manufacturer.[28]
Microbial community characterization
A two-stage PCR protocol was used on the extracted microbiome DNA to amplify the V3-V4 variable region of bacterial 16S rRNA genes, as described previously.[29] Briefly, gDNA was used as template for PCR amplification with the primers CS1_357wF[30] and CS2_806R[31] (ACACTGACGACATGGTTCTACACCTACGGGNGGCWGCAG and TACGGTAGCAGAGACTTGGTCTGGACTACNVGGGTWTCTAAT, respectively; linker sequences underlined). Reactions were performed in 10 µl volumes using repliQa HiFi ToughMix (QuantaBio). Cycling conditions were 2 min denaturation at 98°C, followed by 28 cycles of 98°C for 10 sec, 50°C for 1 sec, and 68°C for 1 sec. Subsequently, a second PCR amplification was performed, also in 10 microliter reactions in 96-well plates using repliQa HiFi ToughMix. Each well received a separate primer pair with a unique 10-base barcode, obtained from the Access Array Barcode Library for Illumina (Fluidigm, South San Francisco, CA; Item# 100-4876). One microliter of PCR product from the first stage amplification was used as template for the 2nd stage, without cleanup. Cycling conditions were 98°C for 2 minutes, followed by 8 cycles of 98°C for 10”, 60°C for 1” and 68°C for 1”. Libraries were then pooled and sequenced with a 15% phiX spike-in on an Illumina MiSeq sequencer employing V3 chemistry (2x300 base paired-end reads). Library preparation and sequencing were performed at the Genomics and Microbiome Core Facility (GMCF) at Rush University.
Quality control and taxonomic identification and classification into community state types (CSTs) was conducted by University of Maryland Institute for Genomic Science, as described previously.[32] [33]. CSTs were determined by the VALENCIA algorithm implemented by the University of Maryland Institute for Genomic Science, which uses a distance-based metric to classify each sample to a CST based on the similarity of the sample to the centroid of CSTs identified in a reference set. In brief, dominant taxa by CST are as follows: CST-I, L. crispatus; CST-II, L. gasseri; CST-III, L. iners; CST-IV, diverse; CST-V, L. jensenii.[33]
Data management
All data were entered in REDCap and exported as CSV file for coding and subsequent analysis on Stata version 14.1, College station, Texas, USA. The setting and use of REDCap for managing our research data has been reported in an earlier publication.[34]
Stratification of high-risk HPV status
The Seegene AnyplexTM II HPV28 Detection kit detects 28 human papillomavirus types [28].
High-risk HPV (HR-HPV) types include: HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 73, and 82. The low-risk types HPV (LR-HPV) include: HPV6, 11, 40, 42, 43, 44, 54, 61, and 70. The HPV status were categorized as follows: category 1 as HPV negative, category 2 as low-risk risk HPV, category 3 as single high-risk HPV (any of the HR-HPV), and category 4 as multiple high risk (2 or more of the HR-HPV types). For subsequent statistical analysis we recoded HPV types into category 1 as HPV negative; category 2 as low-risk HPV positive, and category 3 as any high-risk HPV positive (single or multiple).
Statistical analysis
We compared baseline socio-demographic, sexual risk characteristics, CSTs, HPV status, and HIV status between cervical pathology categories using the Pearson’s chi squared or Fisher’s exact tests for categorical variables and ANOVA for normally distributed continuous variables. To assess the association between vaginal microbiome CSTs with grades of cervical pathology, we considered the clinical relevance of the four cervical cytology categories. We combined NILM and LSIL as one group (normal or minor grades dysplasia) while HSIL and ICC were combined as the clinically important group that requires immediate evaluation and treatment. Observations with CST-II (n=4) were excluded (none was CST-V) from analyses as being too sparse for inference. We performed bivariable logistic regression for the primary exposure variable (CST) and other predictor variables using robust logistic regression to obtain unadjusted odd ratios, 95% confidence intervals (CI), and Wald p-values for associations between HSIL/ICC and NILM/LSIL. We used backward stepwise selection method to build final models checked by the Akaike’s Information Criteria for model selection, including age in years, HIV status, HPV status, CSTs, years of completed education, and total number of births (parity). We then modelled the analyses stratified by high-risk HPV versus negative for HPV (the numbers for low-risk HPV were too small for stratification in the model) to assess the possible effect of CST on cervical pathology probably mediated by high-risk HPV, a known epidemiologic factor in cervical carcinogenesis[2, 35].