Medical Ethics Approval
The study was approved by Universiti Malaysia Sarawak Medical Ethics Committee UNIMAS/NC-21. 02/03-02 Jld. 3 (17).
This is a cross-sectional study. The medical team consisting of gynecologists, general practitioners, nurses, a virologist, and volunteers traveled to Bario from the city of Kuching via the city of Miri for the two days cervical cancer screening program from 21st-22nd April 2019.
The careHPV system
The 3-piece careHPV system and a careHPV kit (Qiagen) was procured via Qiagen’s preferred distributor in Sarawak.
The careHPV system (Qiagen) together with the packaging weighs around 30kg and was brought to Bario by flight as checked-in baggage [Figure 1A]. A total of 90 careBrush and 90 careMedium, Repeater E3 (Eppendorf), 10-200ml pipet (Eppendorf), Combitips (Eppendorf), a box of 200ml long-reach tip (MBP), 6 plate seals (Qiagen), test tube rack (Nalgene), paper towel, a double socket Automatic Voltage Regulator (AVR), a 3-point socket adapter, and an autoclave bag were packed separately. The system was set up in one of the consultation rooms at the Bario Health Clinic No. 10 and the reliability of electricity supplied by the solar panels and diesel generator was evaluated [Figure 1B].
The purpose of the Cervical cancer outreach program was broadcasted via Radio Bario, a community-run broadcasting station that keeps the isolated highland community connected. The news was also conveyed during church services, by word of mouth, through community leaders and the Bario health clinic at least two weeks before the outreach.
Subjects and specimens collection
All women who have had at least one sexual encounter and presented to the cervical cancer screening program were offered the HPV test. Those ages <50-year-old were offered VIA.
Women who wished to know their HPV status and/or have their cervical examination done but do not wish to be part of the study were screened as well but data obtained from them were excluded in this manuscript.
Cervical sample collection, VIA, and HPV DNA test
Cervical swabs were clinician-collected using the careBrush (Qiagen) according to the manufacturer’s instructions and preserved in careMedium (Qiagen) at ambient temperature. The sample tubes were arranged and labeled corresponding to the coordinate of the 96-well plate to facilitate sample loading as well as to reduce pipetting error (Figure 3A). All of the samples were screened for hrHPV according to the manufacturer’s instruction by a certified operator (Figure 3B). The careHPV test detects the presence of 14 high-risk oncogenic HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 using full genome probes complementary to HPV DNA, specific DNA, specific antibodies, signal amplification and chemiluminescent detection. Positive results were defined as the relative light unit (RLU) equal to or greater than the cut-off value as measured by the luminometer.
Visual inspection via acetic acid (VIA) was performed for all participants aged <60 by two certified VIA providers from Universiti Malaysia Sarawak. Participants aged >60 years (peri- or post-menopausal) were not offered VIA as their columnar epithelium and transformation zone would have retreated back from the outer cervix into the endocervical canal (13), reducing the sensitivity and specificity of VIA.
hrHPV positive participants were recruited and a separate cervical swab was obtained using the Rover Cytobrush (BD) and preserved in Surepath Medium (BD) according to the manufacturer’s instruction. Two mL of the cell suspension was pelleted via centrifugation, Surepath medium removed by aspiration and the cell pellet was resuspended in 200 mL of phosphate-buffered saline pH7.4.
Viral nucleic acid was extracted using High Pure Nucleic Acid Extraction Kit (Roche) and eluted in 50ml of elution buffer supplied in the kit. Internal control was carried out against the human b-globin gene. Nested-PCR was carried out by using the MY09/MY11 as the outer primers and GP5+/GP6+ as the inner primers (14). Sanger sequencing service was outsourced to Apical Biotechnology (Kuala Lumpur, Malaysia) and the sequences obtained were compared with the sequences in the Genbank for HPV genotype identification. HeLa cell (HPV18 transformed cervical epithelial cells) and water were included as positive and negative controls respectively.