Diversity of high risk human papilloma viruses in women treated with antiretroviral and in healthy controls and discordance with cervical dysplasia in the South of Benin

Population study

The investigation was carried out within 1 year period in two groups of adult women: one group with HIV1 infection and under ARV therapy in the National University Hospital (CNHU-HKM), designated as CH group (n = 86); and one control group without HIV infection and attending the hospital Mènontin for gynecologic routine checkup and designated as ME group (n = 86). Women in group CH with HIV1 infection are under stringent anti-retroviral treatment at CNHU-HKM while women in group ME without HIV infection are not receiving any treatment. The CH group is under free stringent ARV treatment program offered by the Ministry of Health in BENIN.

The inclusion criteria are women of 20 to 60 years old (average 38 years) and sexually active. The exclusion criteria are women in menstruation or within third month post-partum. Signed Informed consent was obtained from all women before the collection of cervical uterine smears (CUS). Sample collections were carried out in the gynecological service under the supervision of a gynecologist and nurses. The Cervical Uterine junction was visualized with colposcopy before CUS collection [11, 12]. This study was approved by the Research Ethic Committee of the Institute of Biomedical Science and Applications (CER-ISBA), and by the Ministry of Health in Republic of BENIN. HIV status was determined in the referral hospital (CNHU-HKM) with a rapid HIV test (Determine^(R), Abbott Diagnostics) or Genie2^(R) (Bio-Rad, Marnes-La-Coquette, France) test as previously reported [13].

Cell collection and processing

To collect CUS for HPV and lamin A/C analyses, a disposable sterile speculum was introduced in the vagina along with a disposable cytobrush to reach the cervical-uterine junction where cells were collected by rotating the cytobrush clockwise twice. The brush was placed in a 50 ml collection tube containing 5 ml of sterile ice cold phosphate buffer saline (PBS) to collect cells. Cells were kept on ice and delivered within an hour to the laboratory (Unit of Biochemistry and Molecular Biology UBBM, Cotonou, BENIN) for processing. All collection tubes were centrifuged to gather cell pellets which were washed once with ice cold PBS before splitting them in several Eppendorf tubes (i) for the extraction of genomic DNA to genotype HR-HPV; (ii) for the analysis of lamin A/C proteins by western blot. The procedures followed were in accordance with the ethical standards of our institutional committee on human experimentation and in accordance with the Helsinki Declaration as previously reported [12].

Reagents

HPV multiplex primers, Agarose powder, Ethylenediaminetetraacetic acid (EDTA), 2- deoxyribonucleic acid (DNA) ladder and ethidium bromide were from Sigma-Aldrich (France). Tris-Base, glycine, sodium dodecyl sulfate, bis-acrylamide and nitrocellulose membrane, the protein ladder and the peroxidase conjugated secondary antibodies anti-rabbit and anti-mouse were purchased from Bio-Rad Inc (USA). Sodium chloride (NaCl), potassium chloride (KCl), Tween-20, protease inhibitor phenyl-methyl-sulfonyl fluoride (PMSF), 2-mercaptoethanol, methanol, glycerol, 1,4-Dithiothreitol (DTT), sodium fluoride (NaF), sodium azide (NaN₃), Tris-Hydrochloride (tris-HCl) and sodium dodecyl sulfate (SDS, NaC₁₂H₂₅SO₄) were from Sigma-Aldrich (USA). The primary antibody against lamin A/C was purchased from Transduction Lab (USA). Antibody against ß-tubulin was from Santa Cruz Biotechnology (CA, USA). The chemo-luminescence reagent “Super Signal West Dura Extended Duration Substrate” made by PIERCE was from Thermo Scientific (Rockford, IL USA) and was used on Western blot membranes for protein revelation after exposure to X-ray films [12].

DNA extraction

DNA was extracted from cell pellets (i) by the phenol-chloroform method after cell membrane disruption with lysis buffer containing proteinase K (20 mg/ml) and RNAse as previously reported [12]. Phenol was added to the cell lysate (V/V) and centrifuged at 10,000 rpm at 4 °C for 10 min to collect the upper aqueous phase into a new Eppendorf tube followed by the addition of chloroform (V/V) and another centrifugation (10,000 rpm at 4 °C for 10 min). The supernatant was collected in a new Eppendorf tube and ice cold ethanol (96 %) was added at -20 °C for 4 h to precipitate DNA. The DNA pellet was recovered after centrifugation (12,000 rpm at 4 °C for 10 min), was washed with ice cold ethanol (70 %) and then collected after centrifugation at 12,000 rpm at 4 °C for 5 min. The DNA pellet was air dried at 55 °C for 20 min, and solubilized in tris-EDTA (TE) buffer. The concentration of DNA was measured with a spectrophotometer (260 nm). Soluble DNA extract is stored in freezer until needed for genotyping of HPV by Polymerase chain reaction “PCR” [12].

PCR and genotyping of HPV

The quality of DNA was assessed by amplifying ß-actin gene before setting up the PCR with HPV multiplex primers for genotyping. A nested multiplex PCR (NMPCR) assay that combines degenerate E6/E7 consensus primers and type specific primers was done as previously reported [12]. For each PCR reaction, positive and negative controls were used. A first consensus PCR was performed with human sequences (GPEG from SIGMA-Aldrich) to confirm the presence of HPV DNA. After the first PCR the amplified product was used again for a second amplification with the set of multiplex primers (nested PCR) to determine the genotype of HPV as previously described [12]. The multiplex primers used can detect the high grade oncogenic HPV and the low grade oncogenic HPV. The genotypes were divided in 4 groups according to the set of primers. The set 1 of primers is able to detect HPV16; HPV 18; HPV 31; HPV 59; HPV 45 and yield PCR products of 457; 322; 263; 215 and 151 base pairs (bp), respectively. The set 2 of primers is able to detect HPV 33; HPV 6/11; HPV 58; HPV 52; HPV 56; and yield PCR products of 398; 333; 274; 229 and 181 bp respectively. The set 3 of primers is able to detect HPV35; HPV 42; HPV 43; HPV 44 with PCR products of 358; 277; 219 and163 bp respectively. The set 4 of primers is able to detect HPV68; HPV 39; HPV 51; HPV 66; yielding PCR products of 333; 280; 223 and 172 bp respectively. The PCR products were run through 1.5 % agarose gel to separate amplicons according to their size. Amplified bands were detected by ethidium bromide staining under UV light. The HPV genotype was determined according to the molecular size of the band as specified previously [12].

Cell lysates and lamin A/C analysis

Cell pellets (ii) were put in lysis buffer A [50 mM tris-HCl (pH 7.9), 150 mM NaCl, 0.1 mM EDTA, 1 % NP-40, 0.5 mM DTT, 0.5 mM PMSF, 30 mM NaF and 0.5 % protease inhibitor cocktail] and kept on ice for 30 min with vigorous agitation every 5 min as previously described [12]. Samples were boiled in SDS sample buffer for 5 min and store in freezer until analysis. Before western blotting protein samples were boiled again, loaded on 7.5 % SDS-polyacrylamide gels and run at 100 volts for 2 h in tris-glycine buffer, followed by a transfer to nitrocellulose membranes with transfer buffer containing tris-glycine and 20 % methanol. The membranes were blocked with 5 % milk in 1X Tris-buffered-saline (TBS) containing 0.1 % Tween-20 (TBST) for 30 min at room temperature before incubation in primary antibody rabbit-anti-lamin A/C at room temperature. The blots were washed 4 times for 10 min with TBST and incubated with HRP-conjugated secondary antibody anti-rabbit. The blots were washed 4 times for 15 min with TBST and incubated for 3 min in Super Signal West Dura Extended Duration Substrate before exposure to x-ray film and a film developer for the detection lamin A/C or of ß-tubulin as a loading control [12].

Statistical analyses

This study is a transversal case control study between HIV infected group (CH) and non HIV group (ME). The Fisher test (F-test) was used on Excel software to compare the prevalence of HPV infection between groups CH and ME. The CUS with HPV are given the value of 0 if there are not infected with HR-HPV, the value of 1 if they are infected with one type of oncogenic HPV, the value of 2 if they are co-infected with two types of HR-HPV, the value of 3 if they are co-infected with three types of HR-HPV, the value of 4 when are co-infected with 4 types of HR-HPV and the value of 5 when they are co-infected with five types of HR- HPV; the difference between group CH and ME is considered significant when p <0.05.

The non-parametric Mann-Whitney test was used to compare lamin A/C profile between both groups. Lamin A/C profiles were given the value of 3 when normal expression was observed, the value of 2 for weak expression of lamin A/C and the value of 0 for total absence of lamin A/C. The difference between group CH and ME groups is considered significant when p <0.05.

Video colposcopy

Women uterine cervix was examined with a video Colposcopy (SONY) by a gynecologist. Staining with dilute acetic acid followed by Lugol’s iodine solution was done to reveal the cervical lesions if present. The lesions of the cervix appeared white when stained with acetic acid (visual inspection with acetic acid, VIAA+) and yellow when stained with Lugol’s iodine solution (visual inspection with Lugol’s solution, VILI) as previously reported [11, 12].

Ethical considerations

The protocol of this study including objectives, participants, sample collection, analysis, and reporting of data was approved by the Ethics Committee of the Institute of Biomedical Sciences and Applications (CER-ISBA) and by the Ministry of Health in Republic of BENIN. The objectives of the study were explained to all participants before inclusion. Signed informed consent was freely obtained from all participants before sample collections for the study [12].