Background: Recent data have shown that the human papillomavirus (HPV) vaccine could impact on a decrease in high-grade cervical intraepithelial lesions (HSIL) in women who have undergone surgical treatment. This study aimed to evaluate the efficacy of human papilloma virus (HPV) vaccination against persistent/recurrent disease in patients undergoing conization for high-grade squamous intraepithelial lesion/cervical intraepithelial neoplasia-grade 2-3 (HSIL/CIN 2-3). Methods: From January 2009 to March 2019, 563 patients with HSIL/CIN 2-3 underwent conization. The population was divided into two groups according to vaccination status: vaccinated-group (V-Group) and non-vaccinated-group (NV-Group). Bivalent or quadrivalent vaccines were administered indiscriminately. A follow-up was scheduled every 6–12 months according to clinical guidelines. The mean follow-up was 29.6 vs. 36.5 months in the V-group and NV-group, respectively. Results: 277 (49.2%) women were vaccinated, while 286 (50.8%) were not. Overall, persistent/recurrent HSIL/CIN 2-3 was presented by 12/277 (4.3%) women in the V-Group and 28/286 (9.8%) in the NV-Group . Vaccination was associated with a 57% reduction in HSIL persistence/recurrence after treatment. When no disease was present in the first 6-month follow-up visit, persistence/recurrence rates were very low in both groups: 1.1% in the V-Group vs. 1.5% in the NV-Group (p > 0.05). The factor associated with a high-risk of HSIL persistence/recurrence was the presentation of a positive co-test in the first control after treatment (p < 0.001). Conclusions: Our results corroborate the benefit of HPV vaccination in woman treated for HSIL/CIN 2-3, showing a reduction of persistent/recurrent HSIL/CIN 2-3. Persistent High Risk-Human Papillomavirus (HR-HPV) infections are strongly associated with the development of High-grade Squamous Intraepithelial Lesion/Cervical Intraepithelial Neoplasia (HSIL/CIN) 2-3 ,2. ProphPatients who have received excisional therapy (conization) for HSIL/CIN 2-3 have a 0.4–19% risk of lesion recurrence . In addiIn April 2015, the Community of Madrid pioneered an HPV vaccination strategy in patients under 45 years of age with a diagnosis of HSIL/CIN 2-3 who had undergone conization in the previous 3 years. The Gynaecology Oncology Department of the San Carlos Clinical Hospital was among the first centers to benefit from initiating prophylactic vaccination against HPV in treated women. In the last 5 years, different Spanish regions have incorporated free prophylactic vaccination against HPV for women treated for HSIL/CIN 2-3. Since October 2018, the Ministry of Health has supported the Adult Vaccination Calendar for risk groups  (against genotypes 16/18) or quadrivalent vaccine [4v-HPV]  was administered indiscriminately, depending on the funding policies prevailing at that time. Patients vaccinated with at least one dose of any of the available vaccines were considered as cases. The 2v vaccine had a vaccination schedule of 0–1–6 months, while the 4v was administered at 0–2–6 months.The vaccination status at conization (defined by the date of administration of the first dose close to the treatment date), as well as the dates of administration of each dose, were collected from the patients’ records.The study was carried out according to the Guidelines to Good Clinical Practice of the International Conference on Harmonization and the Declaration of Helsinki. Enrolled patients signed an informed consent during follow-up visits. The study was carried out according to the clinical protocols of the hospital, without any additional procedures. The study was approved by the Ethics Committee of our institution (Number 19/320-E).Quantitative variables are expressed with their mean and standard deviation (SD). Variables showing an asymmetric distribution were expressed with the median and interquartile range (IQR). The association of qualitative variables between the two study groups was compared using the χ2 or Fisher exact test. Quantitative variables were compared by the Student’s T or the Mann-Whitney U test according to the distribution.p value less than 0.05 was considered as significant. The data were analyzed with IBM-SPSS software 21.0.The Kaplan-Meier method was used to estimate the distribution of persistence/recurrence in the study groups. A univariate Cox regression analysis was used to find the variables associated with HSIL/CIN 2-3 persistence/recurrence. For all tests, a From January 2009 to January 2019, 563 patients were evaluable and included in the study See .Positive margins in the cone specimen were found in 131 women (23.4%): 72 involved only endocervical margins, 42 only exocervical, and 17 involving both margins. Of these 131 women, 29 underwent a second conization because of involvement of the endocervical margin, and only one woman continued to show positive margins in the re-conization specimen, being considered as a positive margin for the follow-up. Finally, 103 (18.3%) women were considered as having positive margins during the follow-up period.Overall, 277 (49.2%) were vaccinated and 286 (50.8%) were not. The mean age was 36.91 years (SD 8.15). Of the 277 vaccinated women, 255 (92.1%) received the three doses, 16 (5.8%) received two doses, and 6 (2.2%) received only one dose.Among the vaccinated women, 67 (25.1%) received the first dose prior to conization: 12 (17.9%) received the first dose within 30 days before the intervention, 42 (62.7%) between 1 to 9 months, and 13 (19.4%) more than 9 months before conization.Two hundred women were vaccinated after treatment: 14 (7%) received the first dose the first month after conization, 135 (67.8%) at between one and six months, and 51 (25.12%) were vaccinated six months after treatment. Ten women were vaccinated during follow-up, but the dates were not reported.The mean time from treatment to the first visit was 7.1 months (SD 8). The status at this control is shown in p < 0.001) for the non-vaccinated women.The mean follow-up was 33.1 months . For the vaccinated women, the mean follow-up was 29.6 months , being 36.5  (p = 0.814). At the end of follow-up, 55 of 563 (9.8%) women had persistent/recurrent HPV 16–18 infection. Persistent/recurrent infection was found in 28 of 286 (9.7%) women in the non-vaccinated group and in 27/277 (9.74%) women in the vaccinated group (p < 0.05). No differences were found when comparing HSIL/CIN 2-3 between the two groups considering only patients with no disease at the 6-month follow-up visit.p = 0.529.Considering only HSIL/CIN 2-3 recurrence associated with the vaccine genotype (16/18), a lower non-significant recurrence rate was found in vaccinated women compared to non-vaccinated women: 8/277 (2.9%) vs. 11/286 (3.8%); p < 0.001). No differences were found in relation to recurrent HPV-16/18 infection between the vaccinated and non-vaccinated groups (4/184 (2.2%) vs. 3/195 (1.5%), respectively).However, the two vaccinated women with HSIL/CIN 2-3 recurrence showed another genotype not included in the HPV vaccines: both women received the first dose of the vaccine one month after conization. The recurrence appeared 3 years later carrying an HPV 58 in the first woman and 2 years later carrying an HPV 33 in the second woman. On the other hand, when no disease was detected at the 6-month follow-up visit, the risk of recurrent HPV 16/18 infection decreased from 48/184 (26.1%) to 7/379 (1.8%) (p = 0.014), and the factor associated with a high risk of persistent/recurrent was the presence of a positive co-test at 6 months after conization (p < 0.001).To our knowledge, this is the largest Spanish study to evaluate the effect of the HPV vaccine in women treated for HSIL/CIN 2-3 in clinical practice with the same follow-up protocol. The results of our study support the use of HPV vaccination to reduce the risk of developing persistent/recurrent HSIL/CIN 2-3 after surgical treatment.Several studies ,9,11 havp = 0.814), according to the findings of del Pino et al.  in the vaccinated vs. 6/286 [2.1%] in the non-vaccinated group). This could be explained, first of all, because the vaccine does not have a therapeutic but a prophylactic effect, and, in the second place, due to the fact that antibody titres have a maximum peak 7 months after the administration of the first dose ,23 and mThe strength of our study includes the large size of the cohort and the long follow-up period following the same protocol. In addition, our study showed low rates of positive margins and positive co-tests at the 6-month follow-up visit, according to the quality standards of the Colposcopy Unit. Correct patient treatment could justify the low percentage of persistent/recurrent lesions regardless of vaccination status. On the other hand, some limitations must be taken into account: women were vaccinated at different time points, not all women received the full vaccination schedule, and, due to the retrospective nature of the study, the vaccination type (2v-HPV or 4v-HPV) could not be analyzed. In addition, we cannot be sure that no women returned to the screening program, presented late recurrence, and were evaluated another hospital. Finally, another limitation is that the mean length follow-up was shorter in the vaccinated group because very few women were vaccinated before 2015.In conclusion, this study corroborates the benefit of HPV vaccination in women treated for HSIL/CIN 2-3, showing that HPV vaccination is associated with a reduction in the risk of persistent/recurrent HSIL/CIN 2-3 after conization and that the factor associated with a high risk of persistent/recurrent HSIL/CIN 2-3 is to have abnormal pap-smear results and/or a positive HR-HPV genotype at the first control after treatment.


More effective treatments are needed for human papilloma virus (HPV)-induced cancers despite HPV virus vaccination. The oncogenic HPV protein targets are currently undruggable and intracellular and therefore there are no antibodies to these targets. Here we report the discovery of TCR mimic monoclonal antibodies (TCRm mAb) specific for the HPV E7 protein p11-19, YMLDLQPET, when presented on the cell surface in the context of HLA-A*02:01 by use of human phage display libraries. One of the mAbs, 3F8, was able to specifically mediate T cell- redirected cytotoxicity, in a bispecific T cell engager (BiTE) form. While further studies are required to assess the therapeutic potential of this approach, the study provided the proof of concept that TCRm mAb could be a therapeutic strategy for HPV-induced human cancers. High risk HPV causes malignant transformation following persistent infection and is the cause of virtually all cases of cervical cancer and represents nearly 30% of all infection-related cancers . The virHPV type 16 E6 and E7 proteins are consistently expressed in HPV-associated cancers and are thus ideal targets for vaccine design. Therefore, therapeutic vaccines have mostly focused on E6 and E7 as target antigens to induce HPV-specific T cell responses. Various formats of vaccines including naked DNA, short and overlapping long peptides, fusion constructs with Toll-like receptor agonists, have been studied. However, significant clinical benefit over historical controls remains to be observed . PrimingWe hypothesize that a mAb that mimics TCR recognition of a HPV-derived epitopes presented by HLA class I molecules could be an effective immunotherapeutic approach targeting HPV-induced malignancies. The advantages of mAb therapy are well known and include their high target specificity, high efficacy, limited side effects, prolonged half-life, availability, and infrequent dosing. These features make a mAb therapy particularly useful and practical in undeveloped countries. In addition to its inherent properties, mAb can be engineered into many formats to enhance its potency and not patient-specific as adoptive T cell therapy.We selected a well-defined HPV-E7-derived CD8 T cell epitope , as the target for the discovery of a TCRm mAb. This epitope has been reproducibly detected in most cervical cancer biopsies and HPV positive cancer cell lines in the context of HLA-A*02:01 molecule by mass spectrometry –14. The After written and witnessed informed consent on Memorial Sloan-Kettering Cancer Center [MSKCC] Institutional Review Board–approved protocols, peripheral blood mononuclear cells [PBMCs] from HLA-typed healthy donors were obtained by Ficoll density centrifugation. Tumor cell lines used in this study were obtained from American Tissue Culture Collection [ATCC]. mAbs against human HLA-A2 (clone BB7.2) conjugated to fluorescein isothiocyanate [FITC] or allophycocyanin (APC), and its isotype control mouse IgG2b/FITC or APC were purchased from Biolegend. Goat F(ab′)2 anti-hIgG conjugated with phycoerythrin [PE] or FITC, mouse anti-human CD3 mAb, and 6x-His Tag mAb/FITC were purchased from Invitrogen. APC conjugation kit-lighting link (ab201817) was purchased from Abcam and was used to label 3F8, 2A5 and 1B1 mAbs according to manufacturer’s instruction. Human isotype control hIgG1 antibody was purchased from Bingo Biotech [catalog number ET901].All peptides were purchased and synthesized by Genemed Synthesis Inc . Peptides were >95% pure . The pepFor cell surface staining, cells were incubated with appropriate mAbs for 30 min on ice, washed, and incubated with secondary antibody reagents when necessary. Flow cytometry data were collected on a LSR Fortessa (BD Biosciences) and analyzed with FlowJoV10.6.1 software.E. coli and subsequent in vitro refolding and assembly in the presence of high concentrations of HPV-E7p11, WT1-RMF or HPV-AAAA peptide.The method used follows directly the original established protocol –17. BrieA proprietary naïve, semi-synthetic human single chain fragment variable (scFv) phage display library  was usedThe full-length IgG for 1B1, 2A5 and 3F8 was constructed by cloning variable heavy into human IgG1 constant domain and variable light into human kappa light chain backbone. The gene was cloned into pcDNA3.4 vector for expression in mammalian cells. For BiTE construction, 3F8 scFv in vL-vH was linked via G4S linker to an anti-CD3 L2K scFv  in vH-vL6/mL with >95% viability at linear growth. Enhancers were added on the following day and culture was harvested on day 5. Media was cleared by centrifugation and filtering through a 0.22μm membrane. Cleared supernatants were processed for protein purification using either MabSelect SuRe [IgG] or His-trap excel resin (BiTE) .Full length human IgG1 and 3F8 BiTE were expressed in Expi293F cells  as per manufacturer guidelines. Briefly, cells were transfected at 3x10The specificities of the fully human IgG1 mAbs for the HPV-E7/A2 complex were determined by staining T2 cells pulsed with or without HPV-E7p11-19, WT1-RMF or HPV-E7AAAA peptides, after directly conjugating mAbs to APC. Binding of the BiTE was determined by staining cells with the 3F8 BiTE, followed by anti-His/PE secondary antibody. The fluorescence intensity was measured by flow cytometry. The same method was used to determine the binding of the mAbs to peptide un-pulsed tumor cell lines with endogenous expression.51Cr-release assay. In brief, target cells were labeled with Na251CrO4 [50 uCi/million cells] for one hour, washed and incubated with effector cells for 5 hours. The supernatant fluid was collected and radioactivity was measured in a gamma counter. Percentage specific lysis was determined from the following formula: 100 × [/(maximum release − spontaneous release)]. Maximum release was determined by lysis of radiolabeled targets in 2.5% Triton X-100.The 3F8-BiTE or its control BiTE at indicated concentrations were incubated with target cells and PBMCs at different effector: target [E/T] ratios for 5 hours. The cytotoxicity was measured by standard Binding affinities of candidate antibodies in solution were determined using surface plasmon resonance (SPR) MASS-2 instrument [Bruker]. Biotinylated HLA-E7 peptide complex ligand (46 kDa) was immobilized on Biotin-Tag Capture Sensor (Bruker). Candidate antibodies (150 kDa) serially diluted in running buffer  starting at 1μM concentration were injected over the chip at 25°C. The complex was allowed to associate and dissociate for 180 and 300s, respectively. The data were processed using steady state 1:1 interaction model with double referencing.g at 4°C, and supernatant fluids were isolated. For immunopurification of HLA class I ligands, 0.5 mg of W6/32 antibody (BioXCell) were bound to 40 mg CN-Br activated sepharose and incubated with the protein lysate overnight. HLA complexes and binding peptides were eluted five times using 1% TFA. Peptides and HLA complexes were separated using C18 columns . C18 columns were preconditioned with 80% ACN (MilliporeSigma) in 0.1% TFA and equilibrated with 2 washes of 0.1% TFA. Samples were loaded, washed again twice with 0.1% TFA, and eluted in 300 μL of 30%, 40%, and 50% acetonitrile in 0.1% TFA. All three fractions were pooled, dried down using vacuum centrifugation and stored at -80°C until further processing. HLA ligands were isolated by solid-phase extractions using in-house C18 minicolumns. Samples were analyzed by high-resolution/high-accuracy liquid chromatography-tandem mass spectrometry (LC-MS/MS]) . MS and MS/MS were operated at resolutions of 60,000 and 30,000, respectively. Only charge states 1, 2, and 3 were allowed. The isolation window was chosen as 1.6 thomson, and collision energy was set at 30%. For MS/MS, maximum injection time was 100 ms with an automatic gain control of 50,000. MS data were processed using Byonic software  through a custom-built computer server equipped with 4 Intel Xeon E5-4620 8-core CPUs operating at 2.2 GHz and 512 GB physical memory (Exxact Corporation). Protein FDR was disabled to allow complete assessment of potential peptide identifications. Oxidization of methionine; phosphorylation of serine, threonine, and tyrosine; as well as N-terminal acetylation were set as variable modifications for all samples. Samples were searched against a database comprising UniProt human reviewed proteins supplemented with the HPV proteome as well as common contaminants. For mirror plots ion intensities were exported and re-plotted with Graphpad prism.Cell lines were cultured until 10M to 30 million cells were available and then harvested. Harvested cells were pelleted and washed 3 times in ice-cold sterile PBS  Immunoprecipitation, HLA ligand separation and LC-MS/MS were performed as previously described . BrieflyTo select phage clones that mimic TCR recognition, e.g. recognizing amino acids in the middle of the peptide/HLA complex, we performed the following screening strategies: first, counter screening against a peptide/MHC complex with a peptide with a non-similar sequence to HPV using peptide RMFPNAPYL (from the WT1 protein) that binds to HLA-A2 with high affinity. WT1-RMF/HLA-A2 complex to remove all clones that bound to the HLA-A2 molecule. Second: positive screening for HPV-E7p11 (YMLDLQPET) /HLA-A2 complex to broadly select clones that bound to the desired HPV complex. Third: screening against a mutant peptide E7-AAAA [YMAAAAPET]/HLA-A2 complex to remove phage clones that bind to either end of the peptide. In addition, specificity to middle amino acids LDLQ should reduce binding to many potential human proteomic off-target peptides. Positive clones specific for the HPV E7p11/HLA-A2 complex were selected by 3–4 rounds of panning, confirmed by ELISA and live cell binding to T2 cells pulsed with HPV-E7p11 or other control peptides. Three out of total 19 phage clones were selected and engineered into full length human IgG1 format for further characterization.The specificity of full length human IgG1 mAbs was evaluated using T2 cells, pulsed with or without HPV-E7p11, HPV-AAA or control peptide RMF, and tested for their binding capacity. All three hIgG mAbs, named 1B1, 2A5 and 3F8, bound to T2 pulsed with HPV-E7p11, but not to T2 cells alone, or pulsed with irrelevant RMF peptide. When T2 cells were pulsed with HPV-AAAA peptide, the 1B1 and 2A5 mAbs retained binding at a lower level, but 3F8 completely lost binding . These dThe binding positions of the mAbs were further investigated by pulsing T2 cells with HPV-E7p11 analog peptides that were substituted with alanine at each amino acid position . 1B1 andTo further assess the specificity of the mAbs, a panel of HLA-A2-positive or negative cell lines  that areThe low affinity of the mAb 3F8 suggested it might not have useful functional activity in an IgG format, especially for ultra-low density antigens such as HPV E7 PV HOpeptide/MHC complexes; therefore, we proceeded to engineer 3F8 to a bispecific T cell engager (BiTE) format to enhance its potency. Binding specificity of 3F8 BiTE for HPV-E7p11 was tested using T2 cells. There was no binding to either T2 alone or T2 when pulsed with RMF peptide, but the 3F8 BiTE bound well to T2 pulsed with the HPV target peptide . For theAlthough BiTE recognition is monovalent for both the target and the T cell, this level of binding should be sufficient to elicit functional cytotoxicity. We next assessed the efficacy and potency of 3F8 BiTE-directed T cell cytotoxicity in 5 hr 51Cr- release assays against T2 cells pulsed with HPV or control RMF peptide. Compare to control BiTE, 3F8 BiTE showed cytotoxicity against T2 pulsed with HPV E7-p11-19, down to levels below 10ng/ml. These data demonstrated that 3F8, while a low affinity mAb, was able to kill the target with specificity.To test if 3F8 mAb was able to recognize the naturally processed HPV-E7p11-20 epitope presented by HLA- A*02:01 molecules, multiple tumor cell lines that are HLA-A*02:01 and HPV-E7 positive derived from human cervical cancer were tested for the binding of the 3F8 hIgG1 and cytotoxicity of the 3F8-BiTE . The BiTThe development of TCRm mAbs is emerging as a powerful strategy to effectively target intracellular tumor antigens and expands the therapeutic opportunities for currently undruggable oncogenic and tumor-associated proteins. Virus-derived tumor antigens such as from HPV remain an attractive target because of their lack of normal tissue expression. The challenge for T cell-based therapies including TCRm mAbs remains the identification of suitable epitopes to ensure on-target specificity. While various HPV16-derived peptides have been tested as vaccines, the HPV-E7p11-19 was an epitope that was reproducibly detected by mass spectrometry analysis of HPV 16-E7-expressing cervical cancer cell lines and biopsies from patients with cervical cancer –14. TherHere, we show the discovery of the first TCRm mAb specific for the HPV16-E7p11 epitope when presented by HLA-A2 on the cell surface. Our initial characterization has demonstrated the specificity and the functional activity of a mAb clone, 3F8. Alanine scanning showed a broad range of amino acids from positions 3–8 that were important for the recognition by the mAb 3F8. This predicts a low risk for broad cross-reactivity to similar off-target amino acid sequences that may be in the proteome. A BiTE form of 3F8 was also able to kill the T2 cells pulsed with the target peptide, but not irrelevant peptide, despite the low affinity of the mAb. This could be due to an ability of the BiTE engaging a serial killing of the target cells once T cells are activated . However


To establish the sensitivity and specificity of a human papillomavirus (HPV) and tumor marker DNA/mRNA assay for detecting cervical cancer that is transferrable to a Lab-on-a-chip platform and determine its diagnostic benefit in early stage disease when used in conjunction with high-resolution endovaginal magnetic resonance imaging (MRI).Forty-one patients  had DNA and RNA extracted from cervical cytology swab samples. HPV16, HPV18, hTERT, TERC/GAPDH and MYC/GAPDH concentration was established using a loop mediated isothermal amplification (LAMP) assay. Thresholds for tumor marker detection for Group1 were set from Group2 analysis . Group 1 participants underwent endovaginal MRI. Sensitivity and specificity for cancer detection by LAMP and MRI individually and combined was documented by comparison to pathology.Sensitivity and specificity for cancer detection was 68.8% and 77.8% if any tumor marker was positive regardless of HPV status (scenario1), and 93.8% and 55.8% if tumor marker or HPV were positive (scenario 2). Adding endovaginal MRI improved specificity to 88.9% in scenario 1 (sensitivity 68.8%) and to 77.8%% in scenario2 (sensitivity 93.8%).Specificity for cervical cancer detection using a LAMP assay is superior with tumor markers; low sensitivity is improved by HPV detection. Accuracy for early stage cervical cancer detection is optimal using a spatially multiplexed tumor marker/HPV LAMP assay together with endovaginal MRI. Defining the presence and extent of any residual disease crucially determines subsequent surgical management . As wome3 because of confounding appearances from scarring and fibrosis after CKC biopsy or LLETZ  . The lator LLETZ . In thesOf the 14 high-risk HPV types carcinogenic to humans, HPV-16 is consistently the most prevalent, detected in 60% of cases of cervical cancer . HPV-16 0C and have been used for the detection of infectious diseases such as malaria, dengue fever, bacterial and viral infections, notably SARS-CoV-2  methods  incorporRS-CoV-2 –20. The RS-CoV-2 , 22 but RS-CoV-2 , 24, TERRS-CoV-2 –28 for eMolecular Diagnostics Using a novel Lab-on-a-chip and MRI for detecting cervical cancer, MODULAR, NCT03380741) was conducted in accordance with the Declaration of Helsinki (1964), and local research ethics committee and Health Research Authority (HRA) approval. Patients were studied in 2 groups: 1) new diagnosis of cervical cancer 2) non-cancer HPV negative controls. Written informed consent was obtained from each patient. In this hypothesis testing pilot study, where a biomarker could be positive or negative, and assuming the prevalence of a biomarker positive value (tumor DNA or HPV 16 or 18 DNA) among cancer patients is ~75% in women aged 30-39 years  referred for MRI to a tertiary oncology centre prior to being considered for curative surgery were invited to participate, so they formed a consecutive series of cases. Women with neuroendocrine tumors or unusual histological subtypes, or those unable to have MRI because of ferromagnetic implants or claustrophobia were excluded. A control group taken from women attending a separate local colposcopy clinic for follow up of either conservatively managed or previously treated cervical dysplasia, who were judged to have a normal cervix on colposcopic examination was recruited to establish threshold values for tumor marker positivity and confirm validity of a negative HPV result. As part of the routine management of the patients attending the colposcopy clinic for follow up, tests for both HPV 16 and 18 DNA  and HPV E6/7 mRNA  was obtained through The Doctors Laboratory (TDL). GenoID is a PCR based assay for the HPV L1 gene, followed by an ELISA based 96 well hybridisation assay to a cocktail of probes for the type-specific detection of high-risk HPV from 20 HPV phage types . PreTectA cervical swab was taken either at an out-patient visit or prior to an examination under anesthesia (EUA) in all study subjects with cancer. In those patients with a normal cervix at colposcopy, the sample was taken as part of the colposcopic examination. Following insertion of a speculum, the cervix was swabbed with a standard cervical smear brush and the exfoliated cells deposited in PreservCyt transport medium. A separate, sequential cervical swab sample was examined conventionally to assess cytology and HPV DNA and RNA typing as per standard institutional clinical practice. Cytological sampling was adequate in all cases, so that inadequate sampling did not lead to withdrawal from the study in any instance.The exfoliated cells were pelleted and PreservCyt solution discarded. The remaining pellet was washed with phosphate buffered saline (PBS) solution and pelleted again. The PBS solution was discarded. DNA and RNA were extracted from the pellet using Qiagen AllPrep kit  according to the manufacturer’s instructions. Total DNA and RNA yield was determined using a NanoDrop ND-2000 spectrophotometer . Only those samples which yielded both DNA and RNA were selected for analysis.A LAMP assay that is transferrable to a lab-on-a-chip was utilised using a conventional qPCR platform for readout. LAMP methods initially designed using 4 primers targeting 6 regions  and wher4 (100 mM stock), 0.28 μL of dNTPs (25 mM stock), 0.30 μL of BSA (20 mg/mL stock), 0.13 μL of NaOH (200 mM stock), 0.80 μL of Betaine (5M stock), 0.13 μL of Syto9 Green (20 μM stock), 0.02 μL of Bst 2.0 DNA polymerase , 0.13 μL of avian myelobastosis virus , 0.05 μL of Rnase inhibitor , 1 μL of extracted RNA or DNA and 0.50 μL of 10 × LAMP primer mixture , and enough nuclease-free water (ThermoFisher Scientific) to bring the volume to 5 μL. In experiments targeting DNA, AMV and Rnase inhibitor were replaced by water in the reaction mix. This LAMP recipe has been previously published , 0.30 μL of MgSOublished , 19. Reavia the relative fold gene expression2^-(ddCt) method: a mean delta Ct (threshold cycle) was calculated from the patients in the control group, and used to calculate the relative fold change in the cancer patients. Any detection of HPV 16 or 18 DNA or RNA was considered positive. The results of the reference GenoID and Norchip tests were not available to the reader of the LAMP assay at the time of interpretation.An hTERT mRNA result was considered positive if detection occurred within 30 min. Both the TERC DNA relative copy number and MYC mRNA expression relative to GAPDH DNA and mRNA respectively were calculated via a 4 mm diameter tube .The intramuscular administration of Hyoscine butyl bromide (Buscopan) 20 mg decreased artefacts from bowel peristalsis.All scans were performed on a 3.0 Tesla Philips Achieva  with a dedicated in-house developed 37 mm ring-design solenoidal receiver coil that has been previously described , 32, 33.T2-W images were obtained in three planes orthogonal to the cervix together with matched coronal Zonal Oblique Multislice (ZOOM) diffusion-weighted images (DWI). Sequence details are given in Following definitive surgical excision, formalin fixed tissue specimens were sectioned at three to four millimeter intervals, embedded in paraffin and 2-3 micron sections mounted on glass slides. Hematoxylin and eosin (H&E) stained sections were reviewed by a specialist gynecological-oncology histopathologist and the presence or absence of residual tumor was recorded.Statistical analysis used commercially-available software GraphPad Prism for Windows,  and utilized primarily sensitivity and specificity analyses with 95% confidence limits (Wilson/Brown method) for comparison of the LAMP assay with the gold standard (qPCR or histology), the endovaginal MRI with histology and a combination of LAMP assay and endovaginal MRI with histology. Accuracy as defined by [(true positive) + (true negative)]/[(true positive) + (true negative) +  + ] were calculated. These analyses represent the clinical performance of the tests. As we did not perform repeat experiments due to low amount of starting material it was not possible to estimate the precision (degree to which the measurements were repeatable under the same conditions) of the experiments.Between August 2018 and June 2019, 27 patients with newly diagnosed Stage 1 cervical cancer  and 14 non-cancer HPV negative  controls (Group 2) were prospectively recruited. Mean age and BMI were 34.7 years (range 25-51 years) and 23.7 (range 16.9-35.5) respectively. In Group 1, initial diagnosis was made with a LLETZ in 20 patients  and with punch biopsy in 7. Seventeen patients had squamous cell carcinoma (SCC), 10 had adenocarcinoma, with grade of disease distributed between well, moderate and poor differentiated in 5, 14 and 4 cases respectively (4 ungraded on histology). Lymphovascular space invasion was present in 7 cases, absent in 17 cases and not mentioned in 3. In Group 1, 26 of 27 patients had high-resolution MRI (I declined) and cervical swabs for LAMP assay analysis; patients in Group 2 had cervical swabs for LAMP assay analysis only.In Group 1, 23 underwent primary surgery within 4 weeks of diagnosis . On final histology, 12 of these patients had residual tumor. 2 further patients had radical trachelectomy after neoadjuvant chemotherapy and 2 had chemoradiotherapy following adverse findings at examination under anesthesia. These 4 patients were considered positive for tumor as swabs and diagnostic biopsies confirming tumor presence had been taken prior to treatment. Overall, 25 of 27 patients in Group 1 had endovaginal MRI, available histology and sufficient DNA/mRNA extracted on swabbing for inclusion in the analysis. DNA/mRNA extraction was sufficient for analysis in all patients from Group 2 Figure 2In Group 1, following DNA/RNA extraction the mean yield of DNA and RNA was 31.79ng/µL and 57.37ng/µL respectively. Nucleic acid purity (assessed by ratio of light absorbance at 260nm and 280nm) gave a mean of 1.62 for DNA  and 2.01 for RNA  indicating some contamination in the DNA samples, but that pure RNA was successfully extracted. The extracted nucleic acid yield was insufficient in 1 patient (negative on histology), so they were excluded from further analysis. In Group 2 mean yield of DNA and RNA was 43.28ng/µL and 56.07ng/µL respectively with a mean 260/280 ratio of 1.28 and 1.98 respectively, suggesting contaminants remained in the DNA samples but that RNA extraction was successful.In Group 1, 15 of 25 patients were positive for HPV 16 or 18 DNA and/or RNA on the gold standard DNA (GenoID) or E6/7 mRNA (Norchip) tests. Eleven were Type 16 and 4 were Type 18. Nine patients were negative for these HPV types, and in 1 case the results of the test were missing. Of these, 14 were positive on LAMP assay and 10 were negative, time to positive of clinical samples and the limits of detection for the synthetic HPV16 and 18 primers used are given in In the non-cancer controls (Group 2), hTERT was positive at the outer limit of detection in 7 cases with a mean Ct of 42.9 minutes. Therefore, this was used as a Ct threshold for a positive result for the presence of cervical cancer. The positivity of TERC and MYC on LAMP assay was assessed by relative expression to GAPDH. Based on the 2^-(ddCt) for TERC/GAPDH and MYC/GAPDH from the controls in Group 2, the threshold of cancer detection for these markers was set at 3.12 and 0.155 respectively. Limits of detection and time to positive for the synthetic primers designed for hTERT, TERC, MYC, GAPDH and HPV mRNA and of clinical samples are given in The hTERT LAMP assay results agreed with the PCR test for the presence or absence of cancer in 31 of 39 samples . The TERC/GAPDH DNA copy number PCR test was only successfully recorded in 26 of 39 samples; of these the standard deviation of the Ct for GAPDH was >0.25 in 14 cases and the standard deviation of the Ct for TERC was >0.25 in a further 4 cases, making the TERC/GAPDH replicable in only 8 cases. Similarly, with the MYC PCR assay a result was recorded in 25 of 39 samples; of these the standard deviation of the Ct for GAPDH was >0.25 in 10 cases and the standard deviation of the Ct for MYC was >0.25 in a further 10 cases, making the MYC/GAPDH replicable in only 5 cases. Comparison of LAMP assay with PCR was therefore not possible for TERC and MYC due to variability of the PCR results.Sensitivity and specificity for cancer detection was documented by comparison to the gold-standard of pathology. At the threshold set for hTERT, there was a sensitivity of 31.3% and specificity of 77.8%, accuracy 48% for tumor detection. Relative TERC/GAPDH DNA copy number was successfully recorded in 24 cases on LAMP assay and achieved a sensitivity of 40% and specificity of 100%, accuracy 62.5% at the threshold relative expression of 3.12. MYC/GAPDH relative expression was recorded in all 25 patients on LAMP assay and achieved a sensitivity of 43.8% and a specificity of 100%, accuracy 64% at a threshold relative expression of 0.155.To evaluate the performance of the combined markers within the LAMP assay in detecting residual disease two scenarios were applied. In the first, tumor was considered present if any tumor marker  was present with or without HPV positivity and tumor absent if all tumor markers were absent regardless of the presence or not of HPV. Using these criteria, gave the LAMP a sensitivity of 68.8% , specificity of 77.8% , positive predictive value of 84.6% and a negative predictive value of 77.8%, accuracy 72%. In the second scenario, tumor was considered present if any tumor marker or HPV was present, and tumor was considered absent if all tumor markers and HPV were absent. Using these criteria, the LAMP had a sensitivity of 93.8% , specificity of 55.8% , positive predictive value of 78.9% and a negative predictive value of 83.3%, accuracy 80% Table 3.Of the 25 patients scanned, 20 had tumor present on MRI and 15 of these were confirmed at histology . In the 5 patients who were negative for tumor on MRI, 4 cases had no residual disease on histology and 1 was positive for tumor Figure 4If patients in Group 1 were considered positive only if they were positive on MRI and LAMP tumor markers (scenario 1), sensitivity was 68.8% but specificity improved to 88.9%, accuracy 76%; for scenario 2 sensitivity was 93.8%specificity 87.5% and accuracy 88% Table 4.versus 0.4%; RR 4.0 and 95% CI 3.3–4.8)  . A metan3.3–4.8)  and was 3.3–4.8)  and a Co3.3–4.8) . The comValidation of the methodology against conventional PCR showed largely equivalent results for both the DNA and RNA tests in the HPV and hTERT primers. The PCR primers for MYC and TERC/GAPDH performed poorly in the experiments, perhaps indicating alternative PCR primers should be used as their technical sensitivity was below expectation in the clinical samples. Similarly, validation of the HPV-16 and the tumor markers against standard PCR was also equivalent. The pH-LAMP HPV Type 18 DNA marker, as developed by Luo et al. , did notApproaches combining HPV DNA testing with cytology have been previously tried  to optimThe three pH-LAMP tests  each had poor sensitivity but excellent specificity for predicting the presence of residual tumor. The need for three pH-LAMP nucleic tests is warranted to cover a range of possible scenarios; comparison between these showed that all 3 were positive in 1 case, 2 were positive in 6 cases and 1 was positive in 7 cases. The poor sensitivity of the tumor marker tests is counteracted by having the HPV markers included as part of the assay, as their sensitivity for detecting cancer was high. Unfortunately, the HPV 18 DNA LAMP assay designed by Luo et al.  in synthOther markers could be considered for inclusion on spatially multiplexed chip technology in future. Because the expression of HPV viral E7 leads to an increase of cyclin-dependent kinase inhibitor p16 (p16INK4a), p16 would be a possible candidate. However, as p16 overexpression, fundamentally is a marker of HPV infection, it was not selected for the current study. It provides a similar sensitivity and specificity profile to the HPV markers. A meta-analysis of seventeen studies showed a pooled sensitivity and specificity of p16INK4a to detect CIN2 or worse in patients with squamous intraepithelial lesions was 83.8% and 65.7% respectively . DNA metNucleic acid based tests have yet to be evaluated at a population screening level: the change to HPV DNA primary screening has only recently been adopted , especiain vitro diagnostic test that generates results in 30 minutes with analytical sensitivity comparable to the RT-PCR assays. However, it is limited to COVID-19 hence does not allow multi-pathogen detection, and the sensitivity is expected to be reduced due to the all-in-one test kit approach when compared to the full sample extraction methodology. We optimised our isothermal methods to enable the compatibility to our microchip technology as an alternative to fluorescence and time-consuming incubation. This approach has been shown to hold significant potential for the development of a cheap, portable and quantitative diagnostic tool . Cancer Research UK and the Engineering and Physical Sciences Research Council also supported the Cancer Imaging Centre at the Institute of Cancer Research and Royal Marsden Hospital in association with the Medical Research Council and Department of Health C1060/A10334, C1060/A16464. We also acknowledge National Institute for Health Research funding to the Biomedical Research Centre at Royal Marsden Hospital/Institute of Cancer Research and to the Clinical Research Facility in Imaging.The views expressed in this publication are those of the author(s) and not necessarily those of the National Health Service, the National Institute for Health Research or the Department of Health.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Dependence on the internet/social media as the source of HPV knowledge was associated with a significantly lower intention to get HPV vaccination (p = 0.002). The coverage of the HPV vaccination among female university students in health schools in Jordan appeared extremely low; however, three-fourths of the students who had heard of HPV were willing to receive the HPV vaccination if provided freely. Complacency appeared as a major factor for HPV vaccine rejection. Increasing the levels of knowledge and awareness of HPV infection and its association with cervical cancer through reliable sources is recommended. This can be helpful for the individual benefit of the students besides the potentially positive role they can play in community education. Countering vaccine conspiracy beliefs with proper education and awareness programs can be helpful to appraise the role of HPV vaccines in cancer prevention.Cervical cancer is a leading cause of morbidity and mortality in women worldwide. The availability of prophylactic vaccines for high-risk types of human papillomavirus (HPV) infection represents an important advancement in the prevention of cervical cancer. In Jordan, the availability of the HPV vaccination is restricted to individuals who are willing to pay. The aim of the current study was to evaluate the willingness and attitude of female university students in health schools/faculties in Jordan to get HPV vaccination and their knowledge about the virus. A self-administered online questionnaire was distributed in October 2021, which comprised 27 items to evaluate HPV knowledge, history of HPV vaccination, intentions to get the HPV vaccine, and the reason(s) behind vaccine refusal for those who rejected vaccination. The study sample comprised 836 participants: medical students (39.7%), pharmacy students (26.0%), dental students (21.2%), and nursing students (13.2%). Only 524 participants had heard of HPV prior to the study (62.7%), of which 48.7% knew about the availability of HPV vaccines. The lowest level of HPV knowledge was observed among nursing students. Only 19/524 students reported a history of HPV vaccination (3.6%). The overall willingness to receive HPV vaccination if provided freely was 75.0%, while only 16.0% were willing to pay for the vaccine. The most common reason for HPV vaccine rejection was the perceived low risk to get HPV infection. Significantly higher intentions to get HPV vaccination were found among older participants and medical students. The embrace of vaccine conspiracy beliefs was associated with a significantly less willingness to get the HPV vaccination ( Cervical cancer is considered a leading cause of morbidity and mortality among females worldwide . SpecifiHuman papillomavirus (HPV) infection is among the most common sexually transmitted infections (STIs) worldwide ,6. It haThe vast majority of cervical cancer cases can be linked to infection by high-risk HPV types , as evidenced through the detection of the virus DNA in almost all cervical cancers ,13. AddiThe huge burden of HPV-related cancers worldwide, with an absence of curative antiviral therapies necessitates a focus on the continuous development of therapeutic and preventive approaches to tackle such a public health issue . ScreeniVaccination against high-risk HPV types represents the main preventive measure against HPV-related cancers . CompareVaccination against HPV is recommended in routine immunization programs starting at age 11 or 12 years, through to age 26 years . The vacTherapeutic use of the HPV vaccination can also be considered, which entails targeting the clearance of infections that have already been established . NeverthScarce data on HPV infections and cervical cancer are available from Jordan. The latest report issued by the HPV Information Centre in October 2021 reported an annual cervical cancer diagnosis of 115 women with 71 mortalities from the disease in the country . During Despite the previous evidence of the low prevalence of STIs in Jordan, updated studies are needed to assess the recent trends of such infections in the country . The mosA recent study that evaluated the prevalence of abnormal cervical smears in women at a specialized cancer center in the country (King Hussein Cancer Center) argued against the introduction of population-based HPV testing and vaccination . This arThe previous survey studies addressing knowledge, attitude, and practices towards HPV-associated cancers have shown variable defects and gaps in Jordan, mainly regarding the awareness of HPV vaccine availability. Two recent studies among medical and dental students at the University of Jordan showed that a considerable number of students were not aware that HPV vaccines are available ,41. AddiTherefore, this study aimed to evaluate the overall intention of female university students in four health schools/faculties  to get vaccinated against HPV. In addition, the study sought to assess the overall level of HPV knowledge and its association with HPV vaccine acceptance. Finally, we aimed to assess the potential effect of embracing vaccine conspiracy beliefs on HPV vaccine acceptance.A self-administered questionnaire was developed based on a review of relevant previous studies addressing HPV knowledge and attitudes towards HPV vaccination, especially among female college students in various settings ,46,47,48The face validity of each item was checked by the first and the senior authors before the pilot test was conducted. The instrument was validated in a pilot test with six female respondents, which were not included in final analysis. There was a subsequent minor revision of the questionnaire following the pilot test.The survey was distributed online using a snowball sampling approach starting with the contacts of the authors . The survey link was also shared on different social media platforms (Facebook and Twitter) and the instant messaging application WhatsApp. Survey distribution took place in October 2021, with no incentives for participation.The study was approved by the Department of Pathology, Microbiology and Forensic Medicine (meeting 02/2021/2022) and by the Scientific Research Committee at the School of Medicine/University of Jordan (reference number: 5083/2021/67). Informed consent was ensured by the presence of an introductory section of the survey used in this study, with a mandatory question asking for agreement from the respondent to participate in the study. All collected data were treated with full confidentiality.The calculation of the minimum required sample size was conducted using the CheckMarket online sample size calculator . AssuminThe survey comprised five sections as follows: the first introductory section included a short summary of the study aims with a mandatory item indicating the agreement to participate in this study. The second section comprised six items assessing demographic characteristics of the participants . The third section comprised an introductory item to assess whether the participants had heard of HPV before the survey, with a request not to complete the survey if the answer was no. This was followed by eight items (correct/yes vs. incorrect/no) assessing HPV knowledge, with a request to answer based on the participant’s prior knowledge as follows: (1) HPV infections are rare; (2) HPV is sexually transmitted; (3) Genital warts are caused by HPV; (4) HPV can cause cervical cancer; (5) Men cannot get HPV; (6) Individuals can be infected by HPV for years without knowing; (7) HPV can be cured by antibiotics; and (8) Are you aware of the availability of HPV vaccines?The fourth section comprised five questions as follows: (1) Have you received the HPV vaccination?; (2) Given that HPV vaccines are safe and effective to protect from infection by the most common HPV types associated with cervical cancer, are you willing to get the HPV vaccine is it is provided for free?; (3) If you answered the previous question with “no”, what is/are the main reason(s) for your refusal to get the HPV vaccine? (multiple selections were enabled for the following responses: (a) I consider these vaccines unsafe; (b) I consider these vaccines ineffective; (c) I do not consider myself at risk of HPV infection; (d) I do not consider HPV as a common infection in Jordan; (e) For me, it is inconvenient to be vaccinated; and (f) Visiting the doctor makes me feel uncomfortable, which keeps me from being vaccinated); (4) Are you willing to pay to get the HPV vaccination?; and (5) What was your main source of knowledge about HPV? (multiple selections were enabled for the following responses: (a) School courses; (b) University courses; (c) Television/radio/newspaper; (d) Family/friend communication; (e) Internet/social media outlets; and (f) Healthcare providers).The final section comprised seven items of the previously validated vaccine conspiracy beliefs scale (VCBS) . These iThe main outcome measure in this study was the willingness to receive HPV vaccines, with responses dichotomized as yes vs. no. The knowledge of HPV was assessed among the participants who had heard of HPV prior to this study, with the aforementioned eight items. Each correct/yes response was scored as one, while the incorrect/no responses were scored as zero, yielding a minimum score of zero and a maximum HPV knowledge score of eight. For the VCBS, a response with “strongly disagree” was given the minimum score of 1, and the maximum score of 7 was given to the “strongly agree” response with a subsequent, direct relationship between the VCBS and the general embrace of vaccine conspiracies. The internal consistency of the VCBS was shown by Cronbach’s alpha value of 0.901.2). For continuous variables (HPV knowledge score and VCBS), analysis with categorical variables were assessed using the Mann–Whitney U test (M–W) and the Kruskal–Wallis (K–W) test. The association between vaccine conspiracy beliefs and the willingness to receive the HPV vaccination was assessed using multinomial logistic regression. The p value of 0.050 was assumed as the statistical significance cut-off.Analysis was done through the International Business Machines (IBM) Statistical Package for the Social Sciences (SPSS) for Windows, Version 22.0. . Descriptive statistics (mean and standard deviation (SD)) were used to describe the scale variables (VCBS and HPV knowledge score). The association between categorical variables were evaluated using the chi-squared test . Divided per school/faculty, the highest level of HPV prior knowledge was observed among medical students (75.00%), followed by dental students (63.84%), pharmacy students (57.60%), while the lowest level was seen among nursing students  and among medical students (mean: 6.61), compared to dental students (mean: 6.06), pharmacy students (mean: 6.04), and nursing students . Additionally, HPV knowledge was higher among students with a higher household income (>1000 JOD) compared to those with a lower income .Based on the 8-item HPV knowledge score, the study participants who had heard of HPV displayed a mean score of 6.30 (SD = 1.33). Knowledge was higher among Jordanian students , only 19 students reported a previous history of HPV vaccination (3.63%). These participants were medical students (n = 10), dental students (n = 6), two pharmacy students, and a single nursing student. The highest percentage of previous history of HPV vaccination was observed among Palestinian students , followed by Kuwaiti students , and Jordanian students .Among the study participants with previous knowledge of HPV prior to this survey . However, the willingness to pay for HPV vaccination was reported only among 84 students (16.03%).n = 194, 77.91%), followed by pharmacy students , dental students , and nursing students . However, this difference lacked a statistical significance . The acceptance of the HPV vaccination was higher among students with a higher monthly household income; however, this difference lacked statistical significance .The highest level of HPV vaccine acceptance was observed among medical students .The students that reported a willingness to receive the HPV vaccination displayed a significantly higher HPV knowledge score compared to those who reported unwillingness to get HPV vaccines , the most commonly cited reasons for vaccine rejection were those denoting complacency. Given that selection of multiple items was allowed, the most common reason for refusal of HPV vaccination was “I do not consider myself at risk of HPV infection” , followed by “I do not consider HPV as a common infection in Jordan” , and “For me, it is inconvenient to be vaccinated” .The mean VCBS for the participants who had heard of HPV prior to this study was 20.21 (SD = 8.40). A significantly higher VCBS was noticed among the students who rejected the HPV vaccination compared to those who were willing to receive it , while the p value for HPV knowledge was 0.813.The correlation between HPV vaccine acceptance with vaccine conspiracy beliefs and HPV knowledge was evaluated using multinomial logistic regression. The HPV knowledge score was divided into two categories based on the overall mean (>6.00 vs. ≤6.00), and the VCBS was also divided into two categories (≤20.00 vs. >20.00). The covariates used included: age (<20 years vs. ≥20 years); nationality (Jordanian vs. non-Jordanian); school/faculty ; and monthly income (≤1000 JOD vs. >1000 JOD). Higher VCBS was correlated with a statistically significant higher likelihood of HPV vaccine rejection , followed by internet/social media outlets , and healthcare providers . The dependence on the following sources of knowledge regarding HPV were associated with more willingness to receive the HPV vaccination: university courses (271/341 (79.47%) vs. 122/183 (66.67%), p = 0.001, χ2 test); and healthcare providers (91/109 (83.49%) vs. 302/415 (72.77%), p = 0.021, χ2 test). On the contrary, dependence on the internet/social media outlets was associated with less willingness to get the HPV vaccination (166/242 (68.60%) vs. 227/282 (80.50%), p = 0.002, χ2 test, The majority of students with prior knowledge on HPV pointed to university courses as their source of knowledge regarding the virus, with multiple selections being allowed  among the study participants who heard of HPV prior to participation. Nevertheless, the willingness to receive HPV vaccines if provided freely was reported among three-fourths of respondents who knew about HPV. To put this result into a broader perspective, a recent study among young women in Saudi Arabia showed a slightly lower level of HPV vaccine acceptance (64.3%) . ConsistCultural and religious factors can play a major role in attitudes towards vaccination . In a maOn the other hand, the intentions to get HPV vaccination were extremely variable in countries with a majority of Muslims in South East Asian countries. A recent study by Khatiwada et al. showed that 95.8% of university students in Indonesia stated that they would receive the HPV vaccination . AnotherA massive drop in the acceptance of HPV vaccination was noticed in the willingness to pay for the vaccine, which was also reported by a study among health students in Malaysia . In MalaIn relation to HPV knowledge, a striking finding was that a majority of the participants were unaware of the availability of HPV vaccines. A study among female college students in Lebanon showed a higher level of knowledge regarding the availability of such vaccines; however, it also showed poor to moderate knowledge in relation to the number of doses and price of these vaccines .Among those who rejected the HPV vaccination in this study, complacency emerged as the most common contributing factor. This appears conceivable considering the previous evidence of a low incidence of cervical cancer and HPV infections in the country ,34. CompA reluctance to receive HPV vaccines has also been reported in various different studies. A study that involved female college students aged 18–26 in Michigan showed that 31.3% of the study sample did not intend to get the HPV vaccination, with concerns of safety and side effects as the most frequently cited reasons for vaccine refusal . Low HPVOne novel result of this study was the finding of a significant correlation between the embrace of general vaccine conspiracy beliefs and HPV vaccine rejection among female students in Arab countries. Previous studies have linked vaccine conspiracy beliefs with COVID-19 vaccine hesitancy, and this effect appears to extend beyond the recently approved vaccines targeting the control of the pandemic ,72,73,74An interesting result of this study indicated that the dependence on healthcare providers and university courses were associated with significantly higher levels of vaccine acceptance; thus, highlighting the role of proper education and encouragement in cancer prevention. This pattern was also reported in a recent study from India, which praised the role of healthcare providers and classroom instructions in acceptance of the HPV vaccination . FurtherAnother important finding in this study was the correlation between the dependence on the internet/social media platforms with a lower intention to get HPV vaccines as opposed to the reliance on healthcare providers or university courses for HPV information. A previous study by Massey et al. reported on the possibility of the spread of misinformation/disinformation by anti-vaccination proponents using media channels such as Instagram and YouTube ,78. ThusLimitations of this study included the use of a convenience sampling approach, with the possibility of selection bias. Thus, the generalizability of the results should be approached with caution. In addition, survey studies entail the possibility of careless or multiple responses which cannot be ruled out. Sampling error that is related to the decision of the student to participate in the survey based on the previous knowledge of the survey subject should be considered as well. Thus, the genuine level of knowledge among students can be lower than the level reported in this study. The study participants  are expected to have a higher level of knowledge compared to the general population, which was shown in recent studies that investigated COVID-19 knowledge ,81. ThusSeveral gaps in HPV knowledge were identified in this study. The willingness of female students in health schools/faculties in Jordan to get HPV vaccination appeared acceptable if the vaccine was provided for free. The major reason behind HPV vaccine refusal was related to a low perception of being at risk of HPV infection. Rejection of the HPV vaccination was associated with the embrace of general vaccine conspiracy beliefs and reliance on the internet/social media as a source of knowledge regarding HPV. Thus, dismantling such beliefs and rigorous fact-checking such sources can be valuable to tackle hesitancy in HPV vaccine uptake. Special attention should be paid to incorporate refined educational courses for nursing students considering their low level of knowledge about the virus and its vaccination. The improvements in HPV knowledge among university students can also be beneficial at the population level, considering the potential role of such a group in community education.


Women living with HIV (WLHIV) are prone to harbor several high-risk human papillomavirus (HR-HPV) genotypes and to develop cervical cancerous lesions. Data on HPV prevalence in these women are needed to inform immunization programs, especially in Asia where few data are available. We conducted a systematic review and meta-analysis to estimate the prevalence of HPV and HR-HPV cervical infection in WLHIV in Asia and identify possible sources of heterogeneity for HR-HPV carriage. Pooled prevalence and its 95% confidence interval (95CI) were estimated using the inverse-variance weighting method. Linear regression weighted on study size was used to identify sources of heterogeneity. Among 7834 WLHIV (40 studies), the prevalence of HPV infection was 42.6% , and 34.6%  harbored HR-HPV genotypes, with significant heterogeneity across countries. In India, Thailand, and China, HPV-16 was the most frequent genotype (10.3%), followed by HPV-52 (5.4%), HPV-58 (5.0%), HPV-18 (4.1%), and HPV-33 (3.3%). In these women, most of whom were receiving antiretroviral therapy, we did not identify determinants of heterogeneity for HR-HPV infection. Our results underline the need for immunization programs based on nonavalent or new generation vaccines to prevent cervical cancer in WLHIV in Asia. Human papillomavirus (HPV) infection is the main determinant of cervical cancer in women . Nearly According to the World Health Organization (WHO), Asia has the highest incidence rate of cervical cancer after sub-Saharan Africa. Each year, 300,000 new cervical cancers are diagnosed among Asian women, and 150,000 die from cervical cancer . DependiHuman immunodeficiency virus (HIV) infection is also a strong determinant of cervical cancer . The proOver the last two decades, the scaling-up of antiretroviral therapy (ART) has dramatically increased the life expectancy of WLHIV ,20,21, lA global meta-analysis was carried out by Clifford et al. to estimate the worldwide prevalence of HPV and HR-HPV in WLHIV, but data on Asia were limited .In our meta-analysis, we estimate the prevalence of HPV and HR-HPV cervical infection in WLHIV in Asia and identify possible sources of heterogeneity for HR-HPV carriage.This systematic review was designed according to the Preferred Reporting Items for Systematics Reviews and Meta-Analyses (PRISMA) statement ,24.We focused our systematic review on the Asian continent, including countries of the Middle East (Asia–Africa boundary). Source material was obtained from peer-reviewed published literature, limited to English and French, using the following key search terms and connectors: (human immunodeficiency virus or AIDS) AND (HPV or human papillomavirus) AND  AND . The search was conducted in the Medline database using either MeSH headings or free-text terms. The literature was last searched in October 2020, targeting the last 30 years.Titles and abstracts of the articles were assessed. Articles were excluded if (1) the language was not English or French; (2) the study did not take place in Asia; (3) subjects were not women; (4) subjects were women not living with HIV; (5) the localization of HPV infection was not the cervix; and (6) HPV prevalence  was not provided. Meta-analyses and literature reviews were also excluded. When abstracts were unavailable or failed to provide sufficient information, full articles were retrieved to assess eligibility criteria. Furthermore, to avoid duplicates, if data subsets originating from a single study were published in more than one article with redundant information, only the publication with the largest sample size was included in the meta-analysis.For each study, the following information was extracted:-Study settings: study identification ; location  where the study was performed.-Sociodemographic data of WLHIV: number of WLHIV enrolled; mean age of WLHIV; and percentage of WLHIV who: use condoms consistently , have at least a primary education, smoke, are sex workers, and have had at least one pregnancy.-Data related to HPV infection: number of WLHIV tested for HPV infection; HPV test used; number of HPV genotypes tested; percentage of WLHIV with HPV infection and multiple HPV infection, and mean number of genotypes for those with multiple infection; percentage of WLHIV with any HR-HPV infection; percentage of WLHIV positive for each of the 13 HR-HPV genotypes, according to WHO’s classification [-Data related to HIV infection: mean CD4 cell-count nadir and when tested for HPV; mean HIV-RNA viral load and percentage of WLHIV with detectable viral load when tested for HPV; percentage of WLHIV receiving antiretroviral therapy, and mean duration since treatment initiation.The extracted data were cross-checked by two independent reviewers (FV and TD). A third expert was consulted if needed (SL). When mean was not provided, we used the median and the interquartile range to compute the mean and the standard deviation .Within each study, we estimated the prevalence of HPV infection as the number of WLHIV harboring any HPV genotype, divided by the total number of women who underwent HPV testing. The prevalence of HR-HPV infection overall and by oncogenic genotype was calculated as the number of WLHIV harboring any of the 13 oncogenic genotypes, divided by the total number of women who underwent HPV testing. The 13 oncogenic genotypes  were considered based on the WHO classification [Q test. We used a linear regression weighted on study size to estimate the strength of association between HR-HPV infection and potentially confounding, random effects: mean age; mean CD4 at time of HPV testing; use of antiretroviral therapy (yes/no); undetectable viral load (yes/no); consistent condom use (yes/no); educational level (at least primary school vs. no school); smoking (yes/no); sex workers (yes/no); parity history. Associations are reported using exponential of β coefficients (Exp(β)) with their 95CI.In the meta-analysis, we estimated the pooled prevalence and its 95% confidence interval (95CI) using the inverse-variance weighting method for HPV infection, HR-HPV genotypes combined, and for each HR-HPV genotype. The analysis of pooled prevalence of HR-HPV was restricted to countries with at least three studies and more than 100 WLHIV, in which women were tested for the 13 HR-HPV genotypes. We tabulated the prevalence of HPV and HR-HPV infection across countries using Forest plots. We compared the pooled prevalence within countries using the chi-squared test. We used a random-effects model to reflect the differential scaling of antiretroviral therapy programs throughout Asia . HeterogA sensitivity analysis was performed to estimate pooled prevalence of HR-HPV genotypes, including all studies providing prevalence for genus alpha genotypes: genus alpha-5 group , alpha-6 group , alpha-7 group , alpha-9 group  and alpha-11 group .p value of ≤0.05 was considered statistically significant. Analyses were conducted with the R software environment (version 3.6.1) using the Meta package.All tests were two-tailed, and a Systematic review identified 312 records. After screening titles and abstracts, we excluded 216 articles, including five duplicates. Of the remaining 96, 40 were eligible and were included in the meta-analysis. Among these 40 studies (7834 WLHIV), 15 were from India, 9 from Thailand, 6 from China, 2 from Cambodia, Taiwan, and Iran; only one was available from South Korea, Vietnam, Oman, and Israel.3 (14 studies).p < 0.01). The overall prevalence of multiple HPV infection was 15.8% .p < 0.01).Among 6925 women living in countries where at least three studies had been carried out, the estimates of prevalence ranged from 38.7%  in India (15 studies) to 39.3%  in Thailand (nine studies), and 42.0%  in China (six studies) (p < 0.01). Except for China, significant heterogeneity was observed in India and Thailand (p < 0.01). Results were similar in the sensitivity analysis including all studies providing prevalence for genus alpha genotypes (enotypes .3), no factor was significantly associated with higher prevalence of HR-HPV infection. Our meta-analysis reveals that 42.6% of the WLHIV in Asia were infected with HPV, and 34.6% harbored oncogenic HPV genotypes. In India, Thailand, and China, countries for which information on HR-HPV genotypes was available, HPV-16 was the most frequent genotype (10.3%), followed by HPV-52 (5.4%), HPV-58 (5.0%), HPV-18 (4.1%), and HPV-33 (3.3%). In these women, most of whom were receiving antiretroviral therapy and with a good immunological and virological response, we did not identify any determinant of heterogeneity for HR-HPV infection.In a previous global meta-analysis, the prevalence of HPV among WLHIV was estimated at 33% across nine studies in Asia, varying by cytological finding from 25% in those with normal findings, to 76% in those with atypical squamous cells of undetermined significance or higher grade lesions (ASCUS+) . The difCompared to previous meta-analyses describing HPV prevalence among HIV negative women, our results show that WLHIV in Asia have a 1.4 (IC95 1.3–1.4) to 6.7 (IC95 6.6–6.8) increased risk of being infected with HPV, depending on population characteristics ,29,30,31In our meta-analysis restricted to India, Thailand, and China, HR-HPV prevalence was consistent at around 34.6%, and the distribution of genotypes was similar to that previously reported, with the highest rate for HPV-16 and HPV-52 infections ,33.3), suggesting that HIV treatment reduces the risk of HR-HPV infection [We could not confirm the role of known factors associated with a higher prevalence of HR-HPV, such as age and educational level, or of factors related to HIV infection . However, most women (62.6%) had an undetectable viral load and a good immunological status  HPV varies according to the test used. Incoming technologies for HPV DNA testing, including circulating HPV DNA (ct-DNA), could improve the prevalence estimations and risk stratification .A recent study demonstrated an increased risk of cervical cancer among WLHIV, compared to uninfected women , underliIn Asia, the scaling-up of antiretroviral therapy programs seems to be effective for protecting WLHIV from oncogenic HPV infection. HPV-16 was the most frequent genotype, but closely followed by HPV-52, -58, -18, and -33, which underlines the need for immunization programs based on nonavalent or new generation vaccines to fight cervical cancer in these women .


Compared to urban Veterans, rural Veterans are more likely to be older (55-74), not employed, have less education, more service-related disabilities and unmet healthcare needs. Interviews with a national sample of community-based outpatient clinic providers described highly-rural Veterans who are “off the grid.” These Veterans, by choice and/or circumstance, do not have access to reliable internet, associated devices or knowledge/skills. Providers described the difficulties of connecting with these Veterans even by phone. The healthcare shift to virtual telehealth modalities in response to COVID-19 highlights the digital divide as a social determinant of health. For “off-the-grid” Veterans, past experiences and present-day circumstances converge to perpetuate and exacerbate inequalities in accessing healthcare. Their situation underscores that telehealth is not a panacea for increasing access to care and confronts us with the moral imperative to reach those with whom it may be most difficult to connect to span social, geographic and digital divides.


To assess the knowledge about cervical cancer and HPV infection and the awareness towards and perceived barriers of HPV vaccination amid medical students in Jordan.The present study is a cross-sectional survey that was conducted for a period of three months in the College of Medicine at six different universities in Jordan. Third-year to sixth-year students from these medical colleges in Jordan were invited to participate in the study.There were 504 students that took part in the study with 42.3% being males and 57.7% females. The mean knowledge score of students in our survey was 21.4 ± 4.4 out of 34, which was categorized as a moderate level of knowledge regarding cervical cancer and HPV. Only 40.5% knew about the availability of the HPV vaccine in Jordan, and 65.9% accepted the idea that it is necessary to introduce the HPV vaccine for school girls in Jordan.This study highlights that there is inadequate knowledge about cervical cancer and its screening among medical students in Jordan. Despite the limited awareness about the HPV vaccine among the study’s participants, there is a favorable opinion towards the introduction of the vaccine for school girls in Jordan. The data provide a benchmark on the level of knowledge about cervical cancer and awareness about HPV, which can be used to formulate an effective awareness program. Cervical cancer (CC) is the fourth most common cancer in females with 270,000 women dying from the disease annually; 90% of whom live in developing countries. In North Africa and the Middle East, CC affects 19,500 women per year, leading to 9,930 deaths annually. By 2035, these numbers are expected to double in this region unless effective public health interventions are introduced .CC, caused by human papillomavirus (HPV), is the major cause of years of life lost to cancer in the developing world. Since it affects women in their most productive years, CC has a disturbing effect on the well-being of families . In JordAmongst all known risk factors, persistent infection from high-risk HPV plays a considerable role in the pathogenesis of CC. The available screening tests for cervical cancer include conventional and liquid-based cytologic tests  while the most common test to detect HPV is to use a polymerase chain reaction (PCR) assay . Almost Successful achievements in basic and clinical research have expanded the possibilities of CC prevention by introducing HPV testing as part of the screening technology and, most importantly, by the production of efficacious prophylactic HPV vaccines . It is eThe first vaccine against HPV for the primary prevention of CC was licensed in 2006 by the Food and Drug Administration (FDA) of the United States of America (USA). Currently, two prophylactic vaccines against HPV are registered: quadrivalent Gardasil  and bivalent Cervarix . Both vaccines have good profiles for efficacy in preventing HPV infection and are well tolerated. Published studies have recognized a high efficacy of over 92% against precancerous lesions among women . HoweverSeveral studies, mostly from developed countries, have demonstrated that the knowledge about HPV infection and the acceptability of HPV vaccines amongst health care professionals and the general public vary from low to high . RecommePhysicians can play an imperative role in circulating knowledge about CC and the available preventive vaccines. Therefore, the awareness of physicians and medical students will greatly impact the success of CC prevention. The awareness programs should focus on family physicians and gynecologists depending on the known immunizer in each country. Targeted community education that responds to concerns about the HPV vaccine has proven to be effective . It is tThe objectives of this study were to evaluate the knowledge about CC and HPV infection and awareness towards and perceived barriers of HPV vaccination among medical students in Jordan who have the essential role as health care providers to raise community awareness and to modify population behavior. This is the first time in Jordan that the assessment of their knowledge about CC and HPV is being done.The present study is a cross sectional survey that was designed to investigate the knowledge about CC and awareness about the HPV vaccine among medical students in Jordan.The protocol of the study was approved by the Scientific Research Committee at Yarmouk University. The study was conducted after obtaining ethical approval from the Institutional Review Board (IRB) of Jordan University of Science and Technology (JUST) and King Abdulla University Hospital (KAUH), Irbid, Jordan (13/128/2019).The questionnaire was distributed utilizing an electronic format, through Google Forms. The link to the survey was shared with medical students in six universities in Jordan. The questionnaire was prefaced by a page explaining the nature and objectives of the study and the voluntary nature of participation with a consent statement if they would like to take part in the study. Participants who completed the questionnaire gave electronic informed consent by declaring their acceptance to fill out the questionnaire. The questionnaire was terminated automatically if participants declined to take part. This procedure was approved by the IRB committee.The participants were assured that the outcomes of the research would not be used for routine appraisal of the participants. The individuals were requested to complete the questionnaire without textbooks or consulting materials.The study was conducted for a period of three months in the College of Medicine at six different universities in Jordan. Third-year to sixth-year medical students from these medical colleges in Jordan were invited to participate in the study.The study questionnaire was developed by authors after an extensive review of literature. A pre-validated questionnaire, consisting of items modified from questionnaires in other studies  was usedThe final version of the questionnaire consisted of three parts. The first one was about the demographic information of participants which included age, gender, year of study, and average monthly income. The second part assessed the knowledge of participants about CC and HPV. Finally, the third part assessed the participants’ awareness and acceptance of HPV vaccination.The knowledge of the students about CC was evaluated using 14 multiple choice questions with 34 statements related to disease etiology, risk factors, clinical features, and screening recommendations according to the World Health Organization (WHO). Each answer was scored as incorrect or correct. The respondent was given a zero point for each wrong answer and one point for each correct answer.The total knowledge score was calculated for each participant out of 34. Participants were categorized to have poor, moderate, and good knowledge if their score was 0–17, 18–25, and 26–34, respectively.Regarding respondents’ awareness about HPV vaccination, this part consisted of seven questions. Each question was scored out of two points. A poor awareness level was allocated to the medical students with a maximum 39% of awareness mean score (0–5 points), an average awareness to those with at a 40–69% of awareness mean (6-9 points), and a good awareness level to people with over 70% of awareness mean (10-14 points).The last three questions in the questionnaire assessed the acceptance of HPV vaccination among medical students in Jordan, the perceived barriers of HPV vaccination, and their source of information about it.α), values ≥ 0.70 were considered adequate. All p-values were two sided and any p-value of less than 0.05 was considered statistically significant.Data was analyzed using SPSS software version 24. Descriptive data was expressed as frequencies and percentages. Chi square was used to analyze significant differences between categorical variables. Student’s t-tests were used to compare the means between two groups. Analysis of Variance (ANOVA) was used to compare the means between three or more groups. Analysis of Variance (ANOVA) was used to compare the means between three or more groups. The questionnaire’s internal consistency and reliability was assessed by measuring Cronbach’s alpha coefficient , Jordan University of Science and Technology (25.0%), University of Jordan (16.1%), Hashemite University (13.5%), Mutah University (6.3%), and Albalqa University (5.8%). About 42.3% were males and the mean age was 22.3 ± 1.6 years. n = 413, 81.9%) and that HPV is responsible for a wide array of diseases including CC .Knowledge was assessed using 14 questions with a total of 34 points related to disease diagnosis, risk factors, symptoms, and relation to HPV. In regards to knowledge about CC, the mean knowledge score for students was 21.4 ± 4.4. Concerning its epidemiology, 60.7% of students answered correctly that CC is a leading cause of gynecological cancer.Student knowledge about the clinical features of CC and percentages of correct answers were 35.5% for no symptoms and 80.6% for bleeding of the vagina. The percentages of students who were aware that fever, itching, and swelling of the cervix were not among the clinical features of the disease were 87.5%, 70.0%, and 54.85% respectively. The mean percentage knowledge score of this part was 59.7% ± 16.5%.Concerning knowledge about CC screening and vaccine, 26.4% of participants knew correctly that women aged 45–60 years should screen according to WHO once every 5 years. On the other hand, 76.6% were aware that there is a vaccine that protects from CC. The mean percentage knowledge score of this part was 55.2% ± 25.9%.Most students (90.3%) knew that HPV is responsible for a wide array of diseases including CC. Most of them (88.9%) were aware that HPV is transmitted sexually. About two thirds of students were aware that HPV subtypes 6 and 11 are commonly associated with genital warts (65.1%) and HPV subtypes 16 and 18 are commonly associated with CC (70.4%). The mean percentage knowledge score of this domain was 67.9% ± 21.5%.t-test and analysis of variance and there was a significant difference between the mean knowledge score for male students 20.6 ± 4.7 compared to female students 22.0 ± 4.0 . The knowledge score was significantly associated with the year of study with the highest mean score among students in the sixth year with 23.6 ± 3.5  and the least mean score among third-year students 18.4 ± 4.2. There was no significant association between family income, place of living, or nationality and knowledge score .The association of socioeconomic factors with knowledge score was assessed using Regarding student’s awareness about the HPV vaccine, only 40.5% knew about the availability of the HPV vaccine in Jordan, 71.4% were aware that the HPV vaccine should be given at an age between 11 and 29, and about half (54.0%) knew that it could be given to boys also. There were 21.0% that were familiar that girls or women do not need to be screened for HPV before getting vaccinated and that CC protection provided by the HPV vaccine is 70%. Less than one third of the students (30.8%) were aware that the HPV vaccine cannot be given to a woman already having HPV infection while 19.0% of students knew that three doses are required for protection in women. The mean awareness score of students about the HPV vaccine was 5.7 ± 2.8 with a range 0-13 which is classified as average awareness .p-value < 0.001). On the other hand, the awareness score was not associated with all other demographic data  as shown in The awareness score about the HPV vaccine was significantly associated with the year level of study as there was a significant difference between the mean awareness score for students in the sixth year with 6.3 ± 2.8 . On the other hand, the acceptance of introducing the vaccine was not associated with all other demographic data nor with the knowledge score or awareness score .There were 332 students (65.9%) that accepted the idea that it is essential to introduce the HPV vaccine for schoolgirls in Jordan. The association of demographic data with the acceptance of introducing the HPV vaccine was assessed using chi-square analysis, and it was higher among students in the sixth year at 71.1% (Among the obstacles that prevent the receipt of the vaccine or advice about taking HPV vaccination were high cost (53.8%) and inadequate information about the vaccine (62.5%) as shown in In the current study, general CC and HPV knowledge was moderate which was similar to the result of a study done with medical, dental, and nursing students in South India . Most ofIn our study, most medical students were able to recognize that infection and risky sexual practices are common risk factors for CC; these results were similar to other studies . HoweverLess than half of students correctly reported PCR as a test used for the detection of HPV infection and knew the appropriate frequency of CC screening in women . This showed an inadequacy of knowledge which is important as a preventive measure for CC. In addition, the knowledge score was significantly associated with gender and year of study and this was consistent with other studies where the score was higher among female students  and highThe HPV vaccine offers a major breakthrough to limit the global burden of CC . Many stLess than one third of students were aware that the HPV vaccine cannot be given to a woman already having the HPV infection, and 19% of students knew that the appropriate number of doses of the HPV vaccine is three with an overall awareness score of 5.7 ± 2.8. This indicates an inadequate awareness about the HPV vaccine which was similar to a result from a study of university students in India .In the current study, about two thirds of students (65.9%) thought that it is important to introduce the vaccine in school girls in Jordan which indicates a favorable acceptance of using the vaccine in Jordan. In the present study, more than half of the students (62.5%) reported inadequate information about CC and the HPV vaccine as an obstacle preventing the receipt of the vaccine or advice about taking HPV vaccination. Our results were similar to a study on medical students in India . TherefoThe high response rate and the inclusion of all medical colleges in Jordan enhance the generalizability of our results. However, this is a cross sectional study and therefore causal relationships between variables cannot be established. We could also not detect the response rate as the questionnaire was distributed electronically. In addition, a systematic sampling method must be used to identify respondents from each level, but a convenient sampling method was used in the current research to reach the determined sample number. Our study is considered a starting point for future studies on this sensitive topic exploring the attitudes and barriers to vaccination among women in Jordan.This study highlights insufficient knowledge about CC and its screening among medical students in Jordan. Despite the limited awareness about the HPV vaccine among the study’s participants, there is a favorable opinion towards the introduction of the vaccine in school girls in Jordan. More emphasis should be placed on the medical curriculum taught in undergraduate education. Suitable educational campaigns should be stratified at hospitals along with workshops and seminars which highlight the importance of CC screening in women and increase the awareness about HPV among physicians. Medical students who are the future health care providers can educate their patients, address their sensitive cultural concerns, and later increase the health seeking behavior in women in Jordan especially if they are properly aware of CC and hence its burden reduced.10.7717/peerj.11611/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.11611/supp-2Supplemental Information 2Click here for additional data file.


The VA Voluntary Service has developed and implemented a new social prescription program called Compassionate Contact Corps which was created during the COVID-19 pandemic when in-home volunteers could no longer enter Veterans’ homes. The program targets Veterans who are lonely, socially isolated or seeking additional social connection. Volunteers and Veterans are matched based on common interests. Trained volunteers provide support by making periodic phone calls. Program referrals are made from VA providers in several clinical programs (e.g. Home-based Primary Care). To date, CCC has been implemented in more than 80 sites in the VA, with 310 volunteers, 5,320 visits, and 4,757 hours spend with Veterans.