Study design
The rate of positive test results and diagnostic performance of VIA/VILI, HR-HPV DNA detection and p16INK4a/Ki-67 dual-stain cytology was evaluated in a cross-sectional study in women living in Thika district, Kenya. Liquid-based cytology (LBC) samples were taken and sent to Heidelberg, Germany, where HR-HPV DNA testing and p16INK4a/Ki-67 immunostaining was performed. Additionally, Pap staining was performed from the LBC sample in Heidelberg. According to the study protocol, women with a positive VIA/VILI and/or HR-HPV and/or p16INK4a/Ki-67 test, and/or Pap cytology result were invited for colposcopy/biopsy to obtain a histopathologic diagnosis followed by treatment in cases were indicated. With the limitation that a sole positive HR-HPV DNA test only resulted in immediate colposcopy in women older than 30 years.
Ethical approval was obtained from the National Ethical Review Committee at the Kenya Medical Research Institute and women who agreed to participate signed an informed consent form.
Recruitment of women and cervical sampling
Women were recruited for the study in Thika District, Kenya in February 2010. The population is both rural and urban. Women who live in this region were invited to participate in the study through posters placed at markets places, churches, and health centers. Women were eligible for the study if they were (a) self-identified as residing in the region, (b) were ages 18 and above, (c) had an intact uterus, (d) did not report the use of vaginal medication for the previous two days, (e) did not report treatment for cervical disease for the previous 6 months, and (f) were not pregnant at the time of the study.
Cervical cells were collected by female nurses using the liquid-based cytology ThinPrep® Pap test Kit (Hologic, Bedford, USA) according to manufacturer’s instruction and stored in the PreservCyt solution vial (Hologic, Bedford, USA) at ambient temperature. All tests, Pap cytology, p16INK4a/Ki-67 cytology, and HR-HPV DNA detection were performed from the same vial at the Department of Applied Tumor Biology, Heidelberg, Germany. VIA/VILI was performed by the nurses after the cytology sample was taken and documented in a standard form.
Pap cytology
ThinPrep® slides were stained according to routine Pap staining laboratory protocols. All Pap cytology slides were read blinded to all other results at the Institute of Pathology, A2, Mannheim, Germany by an experienced cytotechnologist and gyneco-pathologist. The result was reported according to the Bethesda system.
p16INK4a/Ki-67 dual-stain cytology
A second ThinPrep® slide was prepared from all samples and stained manually with a mix of two monoclonal antibodies against p16INK4a and Ki-67, respectively using the CINtec PLUS kit (Roche mtm Laboratories, Heidelberg, Germany) according to the manufacturer’s instructions. The cervical cancer cell line C4-1 was used as positive control. The dual-staining results in brown visualization of p16INK4a protein and red visualization of Ki-67 protein. Samples were considered as p16INK4a/Ki-67 dual-stain positive when at least one cell was visible on the slide under a light microscope expressing both proteins, thus if at least one cell was found with a red nucleus (Ki-67) surrounded by brown cytoplasm (p16INK4a).
High-risk human papillomavirus detection
After processing for cytology, four mls of residual PreservCyt® solution was used for HR-HPV detection. The HR-HPV detection was carried out using the Hybrid capture 2® (hc2) test (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. A positive hc2 result was defined as RLU/Co ≥ 1.
Colposcopy, biopsy and histology
Colposcopy was performed at Thika District Hospital by an experienced gynecologist. Four to five punch biopsies were taken from the cervix of women with visible lesions. The tissue was placed in formalin and embedded in paraffin for hematoxylin & eosin staining. The slides were assessed by an experienced pathologist at Institute of Pathology, A2, Mannheim, Germany. A second section from all biopsies was stained immunohistochemically for p16INK4a (CINtec histology, mtm Laboratories, Heidelberg) and slides with a diffuse p16INK4a expression beginning in the basal and parabasal cell layers were considered as p16INK4a histology positive.
Data evaluation and disease endpoint
Results were computed as number of positive and negative test results in the entire study cohort, in different age groups and separated for cyologic and histologic diagnosis. Inter-assay agreements were assessed using Cohen's kappa statistics.
Women with negative VIA/VILI, HPV DNA, p16INK4a/Ki-67 and Pap cytology test were considered as disease negative. Women with a positive VIA/VILI and/or HR-HPV and/or p16INK4a/Ki-67 test, and/or Pap cytology result were invited for colposcopy/biopsy to obtain a histopathologic diagnosis (CIN2, CIN3+). Women who showed up for colposcopy but had a normal appearing cervix were not biopsied and were considered as disease negative. Since most women with positive screening test results did not show up for colposcopy as per protocol, a gold standard disease endpoint for the evaluation of the diagnostic performance of the tests was only available for a subset of study participants. Prevalence of positive test results and agreements of the different tests was assessed in the entire study population.