Study design and population

A retrospective cross-sectional study design was employed. Sample collection and pathological analysis were carried out at Pathologist Without Borders, Accra, Ghana, a private anatomical pathology diagnostics company that reports averagely, 12,000 cases a year. Archived formalin fixed paraffin embedded (FFPE) tissue blocks of cervical lesions and nasopharyngeal lesions diagnosed between January 2019 and December 2021 were retrieved from the tissue bank. In all, 403 FFPE tissues blocks of cervical and nasopharyngeal biopsies were retrieved.

Ethical approval

Ethical approval to conduct the study was obtained from the Ethics Committee of Basic and Applied Sciences (ECBAS), University of Ghana, with identification number ECBAS 045/21-22.

Processing of biopsies

The selected FFPE tissue blocks were sectioned using a Leica microtome RM2235 and Leica microtome blade (Biosystems, USA). Ten (10) µm thick sections of seven series were taken from each FFPE tissue using a new blade to avoid cross-contamination.

The sections were transferred into Eppendorf tubes with the blade that was used to section that block. Two sections were fixed on slide and stained with hematoxylin and eosin, and examined under a microscope by a Board-Certified Pathologist to confirm the presence or absence of cancer. Confirmed cervical (51) and nasopharyngeal (48) cancer biopsies, and cervical and nasopharyngeal non-cancer tissues were selected as controls.

Detection of HPV in cervical and nasopharyngeal FFPE tissuesDNA and RNA extraction

Genomic DNA and total RNA were extracted from the cervical and nasopharyngeal cancer tissues using the Quick-DNA/RNA™ FFPE Kits from Zymo-Research (Cambridge, UK). Briefly, five series of 10 µm sections from each FFPE tissue block were deparaffinized using a deparaffinization solution followed by digestion with proteinase K. The lysate was incubated at 55 °C for 4 h and subsequently at 94 °C for 20 min to de-crosslink the sample, and then purified using Zymo-Spin™ column technology according to the manufacturer’s instruction. The flow-through was collected for RNA extraction. The DNA was then eluted in 50 µl of DNA/RNase free water and stored at − 20 °C until use. For the RNA purification, 1 volume of ethanol (95–100%) was added to the flow-through (1:1) and mixed thoroughly. The flow-through was then transferred into a new Zymo-Spin™ IICR column in a collection tube and centrifuged. The RNA remained in the spin column and was eluted in 50 µl of DNA/RNase free water after several steps of purification, according to the manufacturer’s instruction. NanoDrop™ (ThermoFischer, USA) was used to determine the concentration and purity of the DNA and RNA extracted. Extracted DNA and RNA from HPV-infected Hela cells were used as positive controls for the PCRs.

HPV detection

Nested-PCR assay was performed using the primers and protocol previously described with some modifications [24]. The first round PCR reaction for detection was carried out in a total volume of 25 µL containing template DNA (2 µL), 12.5 µL enhanced fidelity polymerase OneTaq® Quick-Load® 2X Master Mix with standard buffer (New England Biolabs, USA), 0.2 µM each of MY09/11 primers; forward: 5′-CGT CCM ARR GGA WAC TGA TC-3′ and reverse: 5′-GCM CAG GGW CAT AAY AAT GG-3′ and topped up with deionized water. Amplification was performed in a Techne Prime thermal cycler (Cole Parmer, USA) with the following conditions: 94 °C for 30 s, 49.5 °C for 1 min, and 68 °C for 1 min for a total of 35 amplification cycles. The first cycle was preceded by a 30 s denaturation step at 94 °C. The last cycle was followed by an additional 5 min elongation step at 68 °C. The second round PCR was carried out using 2 µL of the MY09/11 primer PCR product as a template and GP5+ /6+ primer with sequences; forward: 5′-CGT CCM ARR GGA WAC TGA TC-3′ and reverse: 5′-GCM CAG GGW CAT AAY AAT GG-3′.

The same reagents, concentrations and amplification condition were used as for the first round. The MY09/11 primer set amplified approximately a 450 bp fragment within the HPV L1 structural gene while the GP5+ /6+ primer amplified approximately a 140 bp fragment within the 450 bp fragment. The pairing of MY09/11 and GP5+ /6+ primers further amplified a 190 bp fragment from the 450 bp fragment [24].

HPV18 LCR amplification

A nested-PCR assay was performed according to the protocol previously described by Awua et al., with some modifications [25].

Primers used for the PCR were as follows; HPV18LCR primer for first round PCR; forward: 5′-GTGTTTGTGGTATGGGTGTT-3′ and reverse: 5′-GTATAGTATGTGCTGCCCAA-3′ and HPV18LCR2 second round PCR; forward: 5′-CGGTTGCATAAACTATGTAT-3′ and reverse: 5′-TCGGTTGCCTTTGGCTTATG-3′.

Briefly, the first round PCR reaction was carried out in a total volume of 25 µL containing template DNA (2 µL), 12.5 µL enhanced fidelity polymerase, OneTaq® Quick-Load® 2X Master Mix with standard buffer (New England Biolabs, USA), 0.2 µM each of primer set and topped up with deionized water.

Amplification was performed in a Techne Prime thermal cycler (Cole Parmer, USA) with the following conditions: 95 °C for 1 min, 57.1 °C for 1 min, 30 secs; and 68 °C for 1 min, 30 secs for a total of 35 amplification cycles. The first cycle was preceded by a 5 min denaturation step at 95 °C. The last cycle was followed by an additional 5 min elongation step at 72 °C. The second round PCR was carried out using 2 µL of the first round PCR product as template. The same reagents, concentrations and amplification condition were used as described above.

Genotyping of HPVs by Sanger sequencing

HPV L1 and LCR amplicons from the GP5 + /6 + and HPV18LCRR2 primers were selected for Sanger sequencing after the PCR products have been separated in 2% ethidium bromide-stained agarose gels. The representative samples for sequencing were a single band with molecular weights of 140 bp (GP5 + /6 +) for the L1 gene and 375 bp (HPV18LCRR2) for the LCR. Sample tubes containing the selected amplicons were clearly labelled and shipped for sequencing with instructions.

HPV L1 and LCR sequences analysis

Following the sequencing of HPV L1 gene and LCR, the L1 sequences obtained were aligned with HPV18 L1 reference sequences; DQ059013 (Mauritius), EF202155 (African), MH028425 (Italy), EF202147 (European), AY262282 (Asia), and MN689568 (Iran) retrieved from NCBI GenBank Database (https://www.ncbi.nlm.nih.gov/). All retrieved sequences were trimmed along with the sample sequences. Multiple sequence alignment of L1 genes and LCRs nucleotide sequences were separately performed using ClustalX, version 2.0 (European Bioinformatics Institute) [26]. Similarity and percent identity matrix of L1 sequences were analyzed using, BioEdit (version 5.0.9) software programme [27]. Phylogenetic trees using ClustalX Neighbor-Joining (NJ) algorithm, were generated separately for L1 and LCR sequences. The reliability was assessed by the calculation of bootstrap with 1000 replicates.

RT-PCR for HPV18 E6 and E7 expression

Ten (10) blocks each of HPV 18 positive cervical and nasopharyngeal cancer tissues, and 10 blocks each of HPV 18 positive cervical and nasopharyngeal non-cancer tissues (controls) were selected to determine E6/E7 mRNA expression. Briefly, 2 μl of total RNA was added to the master mix containing; OneTaq One-Step reaction mix, OneTaq One-Step enzyme mix (25X) (OneTaq® One-Step RT-PCR Kit, New England Biolabs, USA), HPV E6, forward primer: 5′-GTATGGAACAACATTAGAACAGCAA-3′; reverse primer: 5′-GTGGCTTTTGACAGTTAATACACC-3′ and E7, forward primer: 5′-GCATGGAGATACACCTACATTG-3′;

reverse primer: 5’-TGGTTTCTGAGAACAGATGG-3’ in a total volume of 10 µl. Amplification was done using QuantStudio 3 system (ThermoFischer, USA), according to the dye method. The following amplification conditions were used; 55 °C for 10 min, 95 °C for 1 min, 95 °C for 15 s, 60 °C for 1 min, 68 °C for 1 min, and 68 °C for 5 min according to the protocol of OneTaq® One-Step RT-PCR Kit. All reagent reconstitutions were carried out on ice. GAPDH was used as an internal control and the master mix without template RNA was also used as a negative control.

Statistical analysis

Statistical analysis was performed with GraphPad prism software version 9.0. Data normality was checked by Shapiro–Wilk test. Normally distributed data was presented as mean and standard deviation whereas non-normally distributed data was depicted as median with interquartile range. Statistical comparisons among and between more than two groups were determined by One-way analysis of variance (ANOVA) or Kruskal–Wallis test. Differences between two groups was analyzed by the two-tailed unpaired t-test or Wilcoxon-sum signed rank test. Associations between categorical variables was determined using Chi square (χ2) or Fisher’s exact test. Sequencing data for groups was analyzed using bioinformatic approaches. The fold change in HPV E6 or E7 expression was calculated using the 2−ΔΔct. method. p-value < 0.05 was considered statistically significant.