Study design and participants
A cross sectional study that uses a non-probabilistic sample was conducted. Patients attending dermatology clinics at Farwania Health District Area were recruited in the study consecutively from November 2016 to May 2017. Immunocompetent patients with previous clinical diagnosis of external anogenital warts scheduled for cryotherapy or laser treatment were asked to sign the consent form after a verbal explanation from the dermatologist soliciting their participation. At the time of sampling, wart specimens were collected in universal transporting medium (HealthLink Inc., Copan Italia S.p.A., Italy), and stored at − 80 °C. Ethical approval was obtained from the Health Sciences Center Ethical Committee (Number: VDR/EC/2310) and Ministry of Health.
Demographic and clinical information were taken from each patient, including: age (years), gender (male, female), nationality (Kuwaiti, non-Kuwaiti), number of warts (one, two to four, and at least five), duration of the presence of warts (< one month, one to six months, seven to 12 months, and more than 12 months), size of each wart (in centimeter) (< one, one, between one and two, more than two), partners has warts (yes, no), and if warts were subject to prior treatment (yes, no). None of the participants received HPV vaccination, because HPV vaccination has not yet been implemented in Kuwait, although its implementation is currently being discussed at the Ministry of Health.
DNA extraction and controls
Received tissue samples were stored at − 80 °C. Due to the large number of samples received, all warts per patient were minced together and were subjected to one DNA isolation. Tissue was minced with sterile scissor and forceps on a Petri dish and about 25-mg of tissue was weighed. The tissue was washed in phosphate buffered saline (PBS) and transferred into 1.5-ml Eppendorf tubes. Genomic DNA was extracted from tissue samples using a QIAmp DNA Mini kit (Qiagen, USA) according to the manufacturer’s instructions.
HPV type 2, HPV type 1 and HPV type 16 vectors (American Type Culture Collection, Manassas, VA, USA) were used as controls in PCR experiments.
Real time PCR
The real-time PCR assay was carried out to screen for HPV DNA in the genital warts. Five microlitres (5-μl) of the extracted DNA was combined with 12.5-μl of 2X Syber Green master mix (Applied Biosystems, Foster City, CA, USA) containing ROX as a passive reference, and 10 pmol of forward and reverse primers (10-uM, described below). All sets of the primers were custom synthesised by Thermo Fisher Scientific, Waltham, Massachusetts, USA. The mixture was made up to 25-μl volume with nuclease free water (Ambion, Austin, TX, USA). In order to reduce the number of false positive or negative results, samples were analyzed in duplicate on a 96 optical well reaction plate (Applied Biosystems, Foster City, CA, USA). Positive and negative controls were included in each amplification batch. In order to quantify the HPV in the samples, a five to 10 fold serial dilution of the known positive control DNA was performed alongside the samples. Real-time PCR amplification was carried out in ABI 7500 real-time PCR (Applied Biosystems, Foster City, CA, USA).
The real-time PCR assay was carried out on three different plates. The first plate was used to determine the integrity of the target DNA by β-globin PCR assay, amplifying a target of 268- bp fragment, as described previously by Lum and Le Marchand in 1998 [
20]. The nucleotide sequence of β-globin primers is as follows: β-globin forward PCR primer: 5′–TGGGTTTCTGATAGGCACTGACT-3′. β-globin reverse PCR primer: 5′-AACAGCATCAGGAGTGGACAGAT-3′.The PCR amplification was initiated at 95 °C for ten minutes and completed by 45 amplification cycles (denaturation at 95 °C for 15 s, annealing at 55 °C for 45 s and extension at 65 °C for one minute).
The second plate was used to screen for the presence of HPV infection using MY11/GP6 primers from the HPV L1 ORF [
6]. The nucleotide sequences of MY11/GP6 primers [
6] are as follows: MY11 forward primer: 5′- GCM CAG GGW CAC AAY AAT GG -3′ and GP6 reverse primer: 5′- GAAAAATAAACTGTAAATCATATTC-3′. The expected size of amplified fragment is 185-bp. The PCR amplification was initiated at 95 °C for ten minutes and completed by 45 amplification cycles (denaturation at 95 °C for 15 s, annealing at 45 °C for 45 s and extension at 65 °C for one minute).
The third plate was used to screen for the presence of HPV infection using HVP2/B5 primers from the HPV L1 ORF [
18]. The nucleotide sequences of HVP2/B5 primers are as follows: HVP2 forward primer: 5′- TCN MGN GGN CAN CCN YTN GG -3′; B5 reverse primer: 5′- AYN CCR TTR TTR TGN CCY TG -3′. The expected size of the amplified fragment is 650-bp. The PCR amplification was initiated at 95 °C for ten minutes and completed by 45 amplification cycles (denaturation at 95 °C for 15 s, annealing at 50 °C for 45 s and extension at 65 °C for one minute).
Fluorescence spectra were recorded during the elongation phase of each PCR cycle. Sequence Detection Software (SDS v1.7) of ABI 7500 real-time PCR was used to generate the amplification curve for each reaction. A dissociation curve was generated after each reaction to differentiate between specific and non-specific amplicons. On the basis of the amplification curve, all samples with HPV amplification starting at any cycle and up to cycle number 40 (with a cut-off line of 0.2) were selected for the analysis. Only samples with a dissociation curve between 70 °C and 80 °C and with a derivative value between 0.100–0.500 were considered HPV DNA positive.
Sanger sequencing
Samples showing positive amplification for the presence of HPV by MY11/GP6 were subjected to conventional PCR assay. The expected HPV genotype spectrum to be detected from mucosal warts using MY11/GP6 includes, HR HPV genotypes: 9, 16, 18, 26, 31, 33, 35, 38, 39, 45, 51, 52, 55, 56, 58, 59 and 68, LR HPV genotypes: 6, 11, 40, 42, 53, 54, 57, 66 and 81 [
16].
HPV DNA in warts was amplified by nested PCR prior to sequencing, using the AmpliTaq Gold Master Mix (Applied Biosystems, Foster City, CA, USA) HVP2/B5 primers were used in the first PCR, and CN1F/CN1R, CN2F/CN2R, CN3F/CN3R and C4F/C4R primers [
18] in the second PCR. The first PCR amplification was initiated at 95 °C for ten minutes and completed by 35 amplification cycles (denaturation at 95 °C for 15 s, annealing at 50 °C for 45 s and extension at 68 °C for one minute). The second PCR amplification was carried out using 3 μl of the first PCR product, and the same cycling conditions. The expected broad-spectrum HPV genotypes to be detected from cutaneous warts using HVP2/B5 and CN1F/CN1R, CN2F/CN2R, CN3F/CN3R and C4F/C4R primers includes HPV types from genera alpha (HPV 2, 3, 7, 10, 27, 28, 29, 40, 43, 57, 77, 91 and 94), gamma (HPV 4, 65, 95, 48, 50, 60 and 88), mu (HPV 1 and 63), and nu (HPV 41) [
12].
The PCR products were purified using a PCR purification kit (NucleoSpin Extract II PCR Purification Kit, Macherey-Nagel GmbH & Co.KG, Düren, Germany) as per the manufacturer’s instructions. They were then subjected to Sanger sequencing reaction using BigDye terminator v3.1 cycle sequencing mix (Applied Biosystems, Foster City, CA, USA), and the nested PCR primers described above. Post sequencing PCR purifications were performed to remove unbound fluorescent dye deoxy terminators using BigDye XTerminator™ Purification kit (Applied Biosystems, Foster City, CA, USA). The samples were denatured for 2 min at 95 °C and immediately chilled on ice and loaded on an ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Samples were electrophoresed on a 50-cm capillary array using POP 6 polymer (Applied Biosystems, Foster City, CA, USA) as a separation medium.
The samples were analyzed using Sequencing Analysis software v 3.7 (Applied Biosystems, Foster City, CA, USA). The HPV sequence alignment was performed with sequences presented in the GenBank database using BLASTn software (
https://blast.ncbi.nlm.nih.gov/) and the Los Alamos Data National Laboratory Theoretical Biology and Biophysics HPV database (
https://pave.niaid.nih.gov/).
When obtained sequences were of poor quality due to low yield of PCR product, the PCR products were cloned into the pGEM-T Easy Vector (Promega, Madison, WI) following purification with the Wizard SV Gel and PCR Clean-Up System kit (Promega), and the sequencing of inserts was performed using M13 forward and reverse primers.
Statistical analysis
Data of the clinical diagnosis, demographic data and virological analysis were tabulated and analyzed using SPSS statistical software ‘Statistical Package for Social Sciences, SPSS version 25.0’ (IBM Corp, Armonk, NY, USA). Descriptive statistics: frequencies/percentages, measures of center and dispersion were calculated. For testing equality of means, the two-sample t-test was used for all continuous variables when normality was held, otherwise Mann Whitney U test was used. For comparing means of three groups, either analysis of variance or Kruskal-Wallis test was used depending on the assumptions being satisfied. Pearson chi square test for independence or Fisher exact test were used to test associations between two categorical variables when the assumptions were satisfied. To model a binary outcome with a set of covariates, logistic regression was implemented, and crude and adjusted odds ratios and their 95% confidence intervals were estimated. All tests were two tailed and a P value less than 5% was considered statistically significant.