Garcinia mangostana L. has earned significant interest as a plant base compound with anticancer properties. Despite α-mangostin’s superior properties as an anticancer agent, its applications are limited due to its poor solubility and physicochemical stability, rapid systemic clearance, and low cellular uptake. Our review aimed to summarize and discuss the nanoparticle formulations of α-mangostin for cancer drug delivery systems from published papers recorded in Scopus, PubMed, and Google Scholar. We investigated various types of α-mangostin nanoformulations to improve its anticancer efficacy by improving bioavailability, cellular uptake, and localization to specific areas These nanoformulations include nanofibers, lipid carrier nanostructures, solid lipid nanoparticles, polymeric nanoparticles, nanomicelles, liposomes, and gold nanoparticles. Notably, polymeric nanoparticles and nanomicelles can increase the accumulation of α-mangostin into tumors and inhibit tumor growth in vivo. In addition, polymeric nanoparticles with the addition of target ligands can increase the cellular uptake of α-mangostin. In conclusion, nanoformulations of α-mangostin are a promising tool to enhance the cellular uptake, accumulation in cancer cells, and the efficacy of α-mangostin as a candidate for anticancer drugs.Natural compounds are emerging as effective agents for the treatment of malignant diseases. The active constituent of α-mangostin from the pericarp of  Cancer is the second leading cause of death globally and was responsible for an estimated 9.6 million worldwide deaths in 2018 ,4. CurreGarcinia mangostana Linn known as the queen of fruits. It has revealed several anticancer properties  is 103.76 ± 7.45 nm; α-mangostin nanoparticles coated with CREKA peptide [CRE-NP(α-M)] is 106.93 ± 3.69 nm with a zeta potential of −31.77 ± 2.12 mV, respectively. The negative charge generated by the nanoparticles renders an optimal possibility to develop the EPR effect when examined in vivo. The nanoparticles effectively reduced the production of secreted mass extracellular matrix both in vitro (NIH3T3 cells) (5.8-fold) and in vivo by blocking the TGF-β signaling pathway and significantly inhibiting the tumor growth (<70%), inducing apoptosis and necrosis on an orthotopic mice model. Biodegradable nanocarriers (PEG-PLA) demonstrate controlled and continuous release behavior. Additionally, the use of PEG can avoid the elimination of nanoparticles by the reticuloendothelial system, which further increases the circulation time in the body. Moreover, CREKA peptide as a target ligand can bind to the fibroin–fibronectin complex that is overexpressed in the tumor mass extracellular matrix, thereby increasing the accumulation in the tumor region. From the results achieved, we can infer that this formula could be amplified as an efficient method to enhance the therapeutic effects .Samprasit et al. formulated α-mangostin-loaded chitosan/alginate (CS/ALG) nanoparticles cross-linked with genipin (GP), used the ionotropic gelation method, and evaluated antitumor activity against the colorectal cancer cells (HT-29) . ChitosaThe viability of the cells treated with varying concentrations of blank GP nanoparticles showed no substantial decrease in cell viability. As a result, blank GP nanoparticles have little effect on cell viability and protected drug carrier. The cytotoxicity of the α-mangostin-loaded GP nanoparticles was dose-dependent. Cell viability was lower at concentrations of 200 and 300 μg/mL of α-mangostin-loaded GP nanoparticles compared to the control. These findings are associated with α-mangostin’s cytotoxicity, confirming that α-mangostin-loaded nanoparticles and α-mangostin have antitumor action against colorectal adenocarcinoma cells. It concluded that chitosan/alginate nanoparticles cross-linked with genipin could be promising candidates for a controlled release drug delivery system of α-mangostin to the large intestine .50 of α-mangostin, α-mangostin-chitosan nanoparticle, and α-mangostin-chitosan-kappa carrageenan was 8.2, 6.7, and 4.7 g/mL, respectively [Wathoni et al. developed an enteric-coated nanoparticle of α-mangostin-loaded chitosan-kappa carrageenan. Kappa carrageenan is a natural polymer used as an encapsulator that will protect nanoparticles from gastric acid because of its low sensitivity to pH and ionic strength. This formula yields an average particle size of 200–400 nm, high entrapment efficiency, and increased solubility. In vitro release testing showed that the nanoparticles produced an initial burst release for pH 1.2 and 7.4, respectively, followed by a slow and sustained release. Cytotoxicity in MCF-7 cells showed an increase in nanoparticle activity compared to free α-mangostin. The ICectively .d(+)-glucopyranose. As there are numerous hydroxyl groups linked to the top and bottom of the molecule, it is water-soluble, but the inside of the cyclic structure is hydrophobic and may trap hydrophobic molecules; this configuration is known as the “molecular pocket”. The number of glucose units determines the cavity size, and the cavity diameters of α-, β-, and γ-cyclodextrin are 5.7, 7.8, and 9.5 Å, respectively [Cyclodextrins are non-reducing oligosaccharides of starch-modified products with a ring-shaped chemical structure and are formed through the cyclization process by CGTase activity (cyclodextrin glycosyltransferase). Suitable reaction conditions will produce three main groups of cyclodextrin: α-, β-, and γ-cyclodextrins consisting of 6, 7, and 8 units of  linked ectively ,114,115.ectively .50 than γCDNP/α-mangostin. It is relevant to the cavity size of CDs; γCDNP/α-mangostin have a larger cavity than βCDNP/α-mangostin. Impressively, in vivo anticancer efficacy showed changes in the tumor volume and tumor growth ratio after i.v. (10 mg/kg) of α-mangostin and βCDNP/α-mangostin into BALB/c mice bearing CT26WT tumors. α-mangostin showed tumor volume and tumor growth ratio gradually increased over 22 days and reached 1145 ± 194 mm3 in tumor volume and tumor growth, indicating an increase of ~10-fold. While, the βCDNP/α-mangostin showed appreciable suppression of tumor growth. The tumor volume was 501 ± 372 mm3 and the tumor growth ratio was ~4 [Cyclodextrin-based nanoparticles (CDNP) encapsulating α-mangostin were developed by a polyaddition reaction using epichlorohydrin and examined cytotoxicity in CT26WT cancer cells. The cyclodextrin-containing hyperbranched polymer was produced by a poly-addition reaction between cyclodextrin and epichlorohydrin (ECH) that capably integrates numerous cyclodextrins into the polymer using a relatively easy synthesis technique. The results showed that βCDNP showed a higher loading ratio of α-mangostin. The cell viability results after 24 and 48 h of incubation indicated the CDNP themselves are inert, while in the case of CDNP α-mangostin complexes, the cell viability is different depending on the type of CD. The αCDNP/α-mangostin and γCDNP/α-mangostin showed high cytotoxicity for cells after 24 h. However, cell viability decreased significantly for βCDNP/α-mangostin after 48 h. It indicates that the α-mangostin in the αCDNP and γCDNP systems can be easily released from the polymer network structure so that almost no cells survived after 24 h of incubation. In contrast, because βCDNP can strongly resist α-mangostin release, the βCDNP system does not show any toxicity after 24 h. However, due to the presence of other candidates of the guest molecule for βCD, such as cholesterol in the cell membrane, the α-mangostin in βCDNP was probably gradually exchanged by the guest molecules, resulting in releasing α-mangostin and inducing higher toxicity after 48 h . Continuo was ~4 . The inco was ~4 . Consequo was ~4 .50 values than α-mangostin . Furthermore, fluorescence microscopy revealed that βCDNP/α-mangostin is carried into cancer cells and changes the shape of the cell’s nucleus. These findings suggest that βCDNP/α-mangostin improves bioavailability and anticancer efficacy [In another study, M.P. Nguyen Thi et al. synthesized ßCDNP/α-mangostin with particles size of <50 nm and zeta potential of −38 mV. Cytotoxicity against lung cancer cells A549 demonstrated that βCDNP/α-mangostin exhibited lower ICefficacy .Nanomicelles are colloidal nano-sized structures with a hydrophobic core and a hydrophilic shell . Nanomic50 of 7.7 ± 0.1 and 8.9 ± 0.2 μg/mL, respectively. Cytotoxicity results show intracellular release of drug payload, which occurred due to the degradative effect of lysosomal enzymes and also show the interaction of PVP and α-mangostin does not affect α-mangostin’s cytotoxicity [In a study from A.F.A. Aisha et al., α-mangostin was prepared in PVP by solvent evaporation method and the intracellular delivery through endocytosis that may enhance the antitumor efficacy of α-mangostin was examined. The cellular uptake assay on human colon tumor cell line 116 (HCT16) showed high permeability of α-mangostin through the cytoplasmic membrane around the treated cells whereas nanomicelles showed fluorescence in the form of spherical particles. These results suggest that cellular uptake from nanomicelles can be mediated through endocytosis. The cytotoxic effect of α-mangostin and nanomicelles exhibited a significant cytotoxic effect in a dose-dependent manner with ICtoxicity .3) compared to the free α-mangostin (0.7 g and 910 mm3). The α-mangostin nanomicelles held smaller tumors than α-mangostin. In conclusion, a strategy was presented in which the α-mangostin delivery system in nanomicelles comprehensively increases the anti-tumor activity of α-mangostin in vivo [Another study of nanomicelles nanoformulation was used to improve the α-mangostin anti-melanoma effect. The α-mangostin/MPEG-PCL has a core structure with PCL (hydrophobic) which absorbs α-mangostin and MPEG (hydrophilic) as shells. The molecular modeling examined the interaction between the αM and MPEG-PCL as carriers which were observed by comparing the interactions in the aquatic environment and the tumor environment. The results showed that the interaction between α-mangostin and MPEG-PCL in an aqueous environment tended to persist with a spherical particle shape, whereas in the tumor environment, MPEG-PCL did not appear to be able to maintain their spherical particles so that it was easier for αM to exit the nanoparticle system. In in vitro cytotoxic testing, α-mangostin nanomicelles possessed a stronger inhibitory effect compared to the α-mangostin on A375 and B16 melanoma cells and exhibited low cytotoxicity for non-tumor cell lines . The growth inhibitory effect of the α-mangostin and α-mangostin nanomicelles shows inhibition on melanoma cell colonies. Surprisingly, no colony formation was observed in A375 and B16 cells treated by α-mangostin nanomicelles. However, several colony formations under the same condition were formed in the α-mangostin group. These results suggest that α-mangostin nanomicelles have a more intense restraining effect on the colony formation rate in melanoma cells compared to α-mangostin. Moreover, the improved apoptotic activity of α-mangostin nanomicelles was determined compared with α-mangostin. It is because the nanomicelles had a better cellular uptake behavior than the α-mangostin. Furthermore, in vivo anticancer activity of the α-mangostin nanomicelles was observed using A375 cells injected subcutaneously into the female BALB/c athymic mice. The α-mangostin nanomicelles possessed a better effect on tumor growth inhibition  and indicated by a decrease in tumor weight (0.62 g for nanomicelles and 1.14 g for α-mangostin), inhibited tumor growth, decreased proliferation, suppression of angiogenesis, and an increase in the apoptosis index that was two times higher than that of α-mangostin. This increase in activity is due to the particle size of nanomicelles at the nanoscale, being able to pass through the inter-endothelial junctions of a tumor by passive diffusion, and the use of PEG to increase the stability of nanoparticles in blood circulation and high retention in the tumor region .Liposomes are membrane vesicles composed of amphiphilic lipids that surround the water nucleus. Liposomes form spontaneously as lipid molecules are dispersed in a liquid medium, resulting in nanometer to micrometer size. Liposomes are formed by a phospholipid sheath composed of one or more lipid bilayers with hydrophilic head groups and hydrophobic tail groups ,121. Thi50 was 2–4 times greater than α-mangostin. It is due to the disparity in the cellular absorption process, where α-mangostin can reach cells through passive diffusion, while α-mangostin liposomes can enter cells through adsorption, endocytosis, or the fusion mechanisms, then accompanied by an α-mangostin release from its carriers. Although free α-mangostin showed relatively higher toxicity than α-mangostin liposomes, the use of liposomes as nanocarriers can still be used as a promising α-mangostin delivery system for anticancer therapy to avoid direct toxicity of α-mangostin to normal cells (drug targeting) [R. Benjakul et al. developed liposomes nanoformulation and evaluated their cytotoxic effect and mechanism inducing cell death in various human carcinomas , human colon carcinoma (HT-29), human breast carcinoma (MCF-7), and human colon carcinoma (Caco-2) cells). Liposome was prepared with phosphatidylcholine and cholesterol using the reverse-phase evaporation method. The particle size, polydispersity index, and zeta potential were 113.98 ± 2.95 nm, 0.13 ± 0.01, and −25.6 ± 0.07 mV, respectively. The cytotoxic effect trends of α-mangostin liposomes on human carcinoma cell lines revealed that α-mangostin liposomes were less toxic than α-mangostin. In all four measured cells, the ICrgeting) .50 value of 2.4 times between α-mangostin and α-mangostin liposomes . Furthermore, the cell viability significantly reduced after 48 and 96 h in the α-mangostin liposome group compared with the α-mangostin. Due to the α-mangostin liposome group, α-mangostin is released slowly and the cells could absorb it gradually so that it kills fewer cells than free α-mangostin within 24 h and length of the period (48 and 96 h). It is consistent with the release of in vitro liposome that exhibits a sustained drug release profile. As for α-mangostin, cell viability varied with the concentration during the first 24 h. The results of this study indicate that liposomes exhibited a more effective cytotoxic effect against Hep-G2 cells as compared with free α-mangostin [In another study from M.P. Nguyen Thi et al., α-mangostin liposomes were formulated with dioleoylphosphatidylcholine and cholesterol, and a cytotoxicity test was conducted on human hepatocellular carcinoma (Hep-G2) cells. There was a significant decrease in the ICangostin .Gold nanoparticles are excellent carrier molecules for cancer drug delivery. They can be synthesized in a variety of sizes and surface characteristics, which make them promising candidates as drug delivery vehicles. Multifunctional gold nanoparticles are now widely used in cancer therapy because of their inertness and biocompatibility. The surface of the gold nanoparticles can be easily modified to provide a controlled release strategy using internal or external stimuli ,125,126.Recently, nanoformulations have been taken into consideration as a drug carrier as they have improved the pharmacokinetic properties of the drug to increase its efficiency and reduce side effects. In cancer treatment, targeted treatment only targeted the cancer cells to be killed and less harm to the normal cells is increasingly desirable. Nanotechnology has improved cancer therapy in many ways, such as selective recognition of cancer cells, targeted drug delivery, and overcoming the limitations of α-mangostin.It is generally known that nanoparticles with smaller sizes and modified surface properties can reach or penetrate certain areas that normally cannot be passed through. Surface properties, particularly the surface charge of the particles, can also influence the distribution of the nanoparticles and possibly lead to higher toxicity in both cancer and non-cancer cells. Therefore, future research can perform surface functionalization of α-mangostin nanoformulations with specific antibodies, proteins, nucleic acids, and small molecules that can actively attach to cancer cells. Currently, CREKA peptide and thioaptamer are ligands that use α-mangostin nanoformulations. The use of these two ligands showed to increase the intracellular delivery and the efficacy of α-mangostin. There are many types of target ligands  that can develop in nanoformulations of α-mangostin to increase the anticancer effectiveness of α-mangostin.Polymeric nanoformulation (PEG-PLA-CREKA), β-cyclodextrin nanoparticles, liposomes, gold nanoparticles, and nanomicelles (MPEG-PCL and MPEG-PLA) have particle size characteristics >150 nm, which are suitable for entry into cancer cells and increased cellular uptake of α-mangostin nanoparticles. Cytotoxicity testing in vitro also showed increased activity in the tested cancer cell lines, except for in β-cyclodextrin nanoparticles, there was a decrease in cytotoxicity in the CT26WT cell line and liposomes (phosphatidylcholine and cholesterol) in Calu-3, HT-29, MCF-7, and Caco-2 cell lines. It is due to differences in the cellular mechanism for uptake of free α-mangostin, which can pass into cells by passive transport (diffusion) mechanism, the β-cyclodextrin and liposome nanoformulations can be taken up into cells by adsorption and endocytosis and related to the α-mangostin release from the carrier. MPEG-PLA and MPEG-PCL nanomicelles were proved to improve the bioavailability of α-mangostin in mice which showed significantly reduced α-mangostin elimination. In addition, the in vivo anticancer activity showed better inhibition of tumor growth than α-mangosin.Polymeric, fibroin, solid lipid nanoparticles are very promising to be developed due to biodegradable components, high-loading capacity (up to 17%), and easy preparation, but these formulas produce large particle sizes up to 400 nm. The development of nanoformulations for α-mangostin such as enteric coating nanoparticles (chitosan-kappa carrageenan) and mucoadhesive (NLC-CS) is an alternative for oral delivery of α-mangostin, by modifying the release stimulated by pH and the surface charge interactions of the nanoparticles that can target the release at specific areas of the gastrointestinal tract. Even though in vitro studies have been shown to increase cytotoxicity activity through the activity of inhibiting proliferation and increasing apoptosis against cancer cells, further studies regarding anticancer activity in vivo are needed. Although recent reports on α-mangostin nanoformulation to date appear promising, most of the published data come from in vitro and in vivo  trials aIn recent years, various nanoformulations of α-mangostin have been developed to improve bioavailability and effectiveness in cancer treatment. α-mangostin exhibits excellent anticancer properties but its poor solubility, rapid elimination, and poor pharmacokinetic properties hinder its usability as a potent drug against cancer. Many techniques have been used to address this issue, one of which is the development of nanosized delivery vehicles. For α-mangostin formulated as nanoparticles, this development is very welcome because they are not only able to improve the dispersion of α-mangostin in aqueous solution, but also provide other advantages not obtained from conventional delivery techniques. These advantages include changeable particle size as well as modifiable surface characteristics. For example, the conjugation of α-mangostin nanoparticles with targeting ligands such as peptides and aptamers can provide specific targeting to cancer cells. With the nanoformulation, the researchers can improve the bioavailability and therapeutic properties of α-mangostin nanoparticles to achieve high therapeutic efficacy. Moreover, the α-mangostin nanoformulation appears to have good affinity and selectivity against cancer cells while imparting negligible toxicity to normal cells.


HPV cervical cancer screening (CCS) must use validated HPV tests based on the molecular detection of either viral mRNA (Aptima HPV Assay—AHPV) or DNA. AHPV has demonstrated the same cross-sectional and longitudinal sensitivity for the detection of HSIL/CIN2+ lesions but with greater specificity than HPV-DNA tests. The study aimed to estimate the total costs of a CCS with a primary HPV test based on the detection of mRNA compared to DNA in women aged 35–65 years for the National Health System.A decision-tree-based model to estimate the cost of the CCS until the first colposcopy was designed based on Spanish CCS guidelines. The total cost  for CCS with AHPV or DNA tests (HC2 and Cobas) was calculated, including HPV test, liquid-based cytology (LBC) and colposcopy, for a population of 7,263,529 women aged 35–65 years (assuming 70% coverage). Clinical inputs derived from a literature review were validated by a multidisciplinary expert panel. Data from head-to-head studies between different HPV tests were selected.The use of AHPV showed reduction of 290,541 (− 35%) and 355,913 (− 40%) LBC compared to HC2 or Cobas, respectively. Furthermore, AHPV avoided 151,699 (− 47%) colposcopies versus HC2 and 151,165 (− 47%) versus Cobas. The total cost of CCS was € 282,747,877 with AHPV, € 322,587,588 with HC2 and € 324,614,490 with Cobas. Therefore, AHPV savings € − 39,839,711 versus HC2 and € − 41,866,613 versus Cobas.Assuming that 70% of women from 35 to 65 years attend the CCS programme, the cost of screening up to the first colposcopy using AHPV would provide cost savings of up to € 41.9 million versus DNA tests in Spain.The online version contains supplementary material available at 10.1186/s12905-021-01310-8. Approximately 85% of these deaths occur in developing countries  and two HPV DNA tests [Hybrid Capture 2 high-risk HPV DNA test  and Cobas 4800 HPV test ].The Hybrid Capture 2 (HC2) high-risk HPV DNA test is considered the gold standard of HPV assays, as its performance was validated in many randomized controlled trials. HC2 collectively detects 13 high-risk (hr) HPV genotypes . Aptima human papillomavirus (AHPV) is an in vitro nucleic acid amplification test for the qualitative detection of E6/E7 viral transcript mRNA from 14 h HPV types in cervical smear samples. The hr HPV types detected by the assay are 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. Likewise, the Cobas 4800 test detects the same 14 h HPV genotypes as the AHPV assay. However, while the Roche assay detects hr HPV DNA, the Aptima assay detects hr HPV oncogenic mRNA expression and is designed to be more specific in identifying clinically significant hr HPV infections that are likely to lead to high-grade squamous dysplasia and neoplasia.In the model, two different scenarios were compared: scenario 1, AHPV versus HC2; and scenario 2, AHPV versus Cobas 4800. Although some of these HPV tests give, in addition to the overall result for all hr HPV types including (positive/negative) provide the partial result for HPV 16 and HPV 18, this partial result was not considered in this analysis.A literature review was conducted in Medline through PubMed to identify scientific publications regarding the clinical evidence available in this field and to extract the probabilities required for the model. Details of the search strategy applied for the literature review is shown in Additional file An initial selection of 1408 localized references was performed by reading the title and abstract. Subsequently, 80 studies were considered relevant for this analysis and were selected and reviewed in full text. The probabilities of the different clinical data included in the analysis were obtained from those clinical studies considered the most relevants. Among all publications, the studies providing data from direct comparisons between the selected HPV tests were preferred and prioritized as sources for inputs.In this sense, the prevalence of HPV for base case was obtained from a transversal head-to-head study about HPV tests in a screening population . This stRegarding the proportion of women with abnormal LBC after a positive HPV test result, several studies were identified –28. ThesFor the alternative case, the prevalence of women with abnormal LBC was 6.5% .Other tThe analysis was carried out from the perspective of the Spanish National Health System (NHS); therefore, only direct health care costs were included, comprising HPV and diagnostic test costs . Figure Unitary costs were obtained from the available scientific literature or regional public information , 29, 30 Table Several sensitivity analyses (SA) were performed to incorporate the uncertainty into the analysis and to observe the effect of these modifications on the results. The following parameters were varied individually: 1) One-way SA was carried out considering a possible reduction in the women from 35 to 65 years of age who would attend their screening appointment, assuming that 36.3% of these women attended the CCS in private practice  and thatAssuming that 70% of women aged 35–65 years would attend the CCS, when AHPV was used instead of HC2 or Cobas 4800 as the primary test, there was a reduction of 290,541 (35% decrease) and 355,913 LBC samples (40% decrease), respectively. Furthermore, the use of AHPV avoided 151,699 (47% reduction) colposcopies compared to HC2 and 151,165 (47% reduction) compared to Cobas 4800 , the use of AHPV provided a cost-saving of € − 2,537,896 versus HC2 and € − 26,669,032 versus Cobas 4800.Based on the decrease in primary LBC, colposcopy and biopsy costs, when the upper value of the cost reported in the literature was applied, using AHPV would save up to € − 49,733,682 compared to HC2 and € − 52,257,582 compared to Cobas 4800  reported a 21% reduction in colposcopy referrals in the 12 months following the change from HC2 to AHPV in women with an ASC-US diagnosis . In our The present model has some limitations. First, the parameters used in the analysis have been extracted from different sources. However, all parameters are based on official sources or on publications with a high level of clinical evidence, and values were validated by a multidisciplinary expert panel. The potential uncertainty associated with some of the parameters was tested in a univariate SAs. Second, the influence of the additional procedure costs performed in women with a positive HPV test result  was also tested in a SA. As observed, none of these two SAs changed the conclusions of the base case results.Another possible limitation could be related to the use of clinical data extracted from studies conducted in other countries, as no robust head-to-head comparative studies have been conducted in Spain. The literature review provided a wide variety of reports; however, a limited number of studies with direct comparisons between mRNA and DNA tests were found. Among all available studies, two of them , 25 evalThis study used an analytic model to assess the health cost after a CCS programme implementation (from the beginning of the implementation to the end of the first colposcopy) with a HPV mRNA test (AHPV) compared to an HPV DNA test (HC2 or Cobas 4800) in women aged 35–65 years in Spain. Initially, the decision tree model was developed and designed considering the entire time horizon for a complete CCS programme based on current recommendations from national and European guidelines  and was Despite the limitations described above, the results of the SA confirmed that the uncertainty associated with the parameters used in this analysis did not represent a significant deviation from the results obtained in the base case, showing that AHPV is the least costly option, reducing testing in all the scenarios evaluated.In conclusion, assuming that 70% of women from 35 to 65 years attend the population-based CCS programme in Spain, the cost of screening up to completion of the first colposcopy using AHPV could generate health cost savings up to 39.8–41.9 million euros when compared to DNA testing.Additional file 1. Appendix 1: Search strategy in PubMed database for LBC. This appendix shows the search strategy carried out in the PubMed database to obtain LBC-related transition probability data to feed the model. Appendix 2: Search strategy in PubMed database for HPV test. This appendix shows the search strategy carried out in the PubMed database to obtain VPH test-related transition probability data to feed the model.


This study was designed to investigate the genetic evidence for repurposing of GLP1R (glucagon‐like peptide‐1 receptor) agonists to prevent heart failure (HF) and whether the potential benefit exceeds the benefit conferred by more general glycemic control.P=1.69×10−4), and higher left ventricular ejection fraction . The magnitude of these benefits exceeded those expected from improved glycemic control more generally. The results were similar in sensitivity analyses, and we did not find evidence to suggest that these associations were mediated by reduced coronary artery disease risk.We applied 2‐sample Mendelian randomization of genetically proxied GLP1R agonism on HF as the main outcome and left ventricular ejection fraction as the secondary outcome. The associations were compared with those of general glycemic control on the same outcomes. Genetic associations were obtained from genome‐wide association study summary statistics of type 2 diabetes mellitus (228 499 cases and 1 178 783 controls), glycated hemoglobin (n=344 182), HF , and left ventricular ejection fraction (n=16 923). Genetic proxies for GLP1R agonism associated with reduced risk of HF (odds ratio per 1 mmol/mol decrease in glycated hemoglobin 0.75; 95% CI, 0.64–0.87; This genetic evidence supports the repurposing of GLP1R agonists for preventing HF. Patients with type 2 diabetes mellitus are at increased risk of developing heart failure and evidence from randomized controlled trials supports that GLP1R (glucagon‐like peptide‐1 receptor) agonists reduce this risk.All data used in this work are publicly available and anonymized. All contributing studies received appropriate ethical approval and patient consent.GLP1R) encoding proteins corresponding to drug targets.The Mendelian randomization (MR) approach uses genetic variants as proxies to investigate the causal effect of an exposure on an outcome.P<5×10−8) and uncorrelated (r2<0.1) variants in the GLP1R gene (genomic position on build GRCh37/hg19: chromosome 6:39 016 574–39 055 519) that associated with type 2 diabetes mellitus liability in the largest published genome‐wide association study meta‐analysis ,P<0.05) associations with glycated hemoglobin in the UK Biobank (n=344 182).GLP1R (or vice versa).We identified genetic proxies for the effect of GLP1R agonism as genome‐wide significant  and 2 intronic variants . This estimate was similar in MR analysis only using the missense variant rs10305420 . Analyses excluding this variant provided similar evidence of effect, suggesting that this variant did not solely drive the estimates . Consistent with previous reports,P=7.75×10−11). This estimate was smaller in magnitude than the estimate obtained for genetically proxied GLP1R agonism . There was no evidence of an association between genetically proxied glycemic control more generally and LVEF . This estimate was smaller in magnitude than that obtained for genetically proxied GLP1R agonism (Pdifference=0.03). Corresponding scatter plots for all analyses are provided in Figures Genetically proxied GLP1R agonism associated with a higher LVEF  and overall glycemic control  by type 2 diabetes mellitus liability showed similar evidence for a reduction in heart failure risk. There was no significant heterogeneity in the MR estimates generated by the different variants when considering either heart failure or LVEF as outcomes  and improved glycemic control  with heart failure risk, and directionally concordant but nonsignificant associations of genetically proxied GLP1R agonism with LVEF .Analyses weighting the genetic proxies for GLP1R agonism . We did not select this variant for inclusion in our analysis as its association with type 2 diabetes mellitus achieved only a nominal level of statistical significance (P=0.001), and not the more stringent genome‐wide level of statistical significance achieved by the variants in our investigation. In contrast, meta‐analyses of clinical trials have supported a nominally significant (P=0.043 before adjustment for multiple comparisons) beneficial effect of GLP1R agonism on myocardial infarction.A similar genetic approach was previously used to support a protective effect of GLP1R agonism on coronary artery disease riskGLP1R (rs10305420) and gene expression data to strengthen the validity of our findings, further experimental work is necessary to determine the mechanism by which these variants influence GLP1R expression or function. In particular, the directionally concordant association of the rs2268647 intronic variant on gene expression in the pancreas, but discordant effect on GLP1R expression in myocardial tissue warrants further exploration. The MR estimates reflect the consequence of a lifelong genetic perturbation of GLP1R signaling and cannot be extrapolated to predict the magnitude of effect from shorter, discrete pharmacological interventions. The limited number of genetic variants available to instrument GLP1R agonism precluded more extensive sensitivity analyses for horizontal pleiotropy. In particular, modeling an intercept term (as in the MR‐Egger regression approach) can in some scenarios mitigate bias from unbalanced horizontal pleiotropy but was not appropriate in our analysis of GLP1R agonism because of the availability of only 3 genetic proxies. We used summary‐level genetic associations with heart failure and LVEF and therefore could not perform stratified analyses, such as by sex or diabetes mellitus status. Finally, these genetic data were predominantly gathered from individuals of European ancestry and these results may therefore not generalize to other ethnic groups.The key strength of our work is the use of randomly allocated genetic proxies to study the effects of GLP1R agonism. The genetic proxies used in these analyses were further validated by their associations with glycated hemoglobin, which reduces risk of bias due to winner's curse and permits the contextualization of the MR estimates on the glycated hemoglobin scale. The genetic associations with LVEF were adjusted for body mass index, which allowed standardization for body size. A study limitation is the absence of available large‐scale genetic summary data for heart failure subtypes. Although we used a missense variant in In conclusion, we identified genetic proxies for the effects of GLP1R agonism, and applied these proxies in MR analyses to generate evidence supporting a protective effect on risk of heart failure. Further investigation of GLP1R agonist repurposing to prevent heart failure in the context of clinical trials is warranted.D.G. was supported by the Wellcome Trust 4i Programme (203928/Z/16/Z) and British Heart Foundation Centre of Research Excellence (RE/18/4/34215) at Imperial College, and a National Institute for Health Research Clinical Lectureship (CL‐2020‐16‐001) at St. George's, University of London. S.B. is supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (204623/Z/16/Z). B.F.V. was supported by the National Institutes of Health (DK101478) and a Linda Pechenik Montague Investigator award. P.S.T. and K.‐M.C. are supported by the VA Cooperative Studies Program with funding from the VA award I01‐BX003362. S.M.D. was supported by the Department of Veterans Affairs Office of Research and Development (IK2‐CX001780). Million Veterans Program research data are funded by Office of Research and Development, Veterans Health Administration and supported by award no. MVP000. MVP‐based articles do not represent the views of the VA, the US Food and Drug Administration, or the US Government. This work was supported by funding from the National Institute for Health Research . The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care. The funding sources had no role in the design, acquisition of data, analysis, interpretation or write up of this study.D.G. is employed part time by Novo Nordisk and has received consultancy fees from Policy Wisdom. J.M.M.H. is an employee of Novo Nordisk. S.M.D. has received grants from the U.S. Department of Veterans Affairs, Calico Labs, and Renalytix AI plc outside the submitted work. The remaining authors have no disclosures to report.Tables S1–S6Figures S1–S5Click here for additional data file.


Co-infection of High Risk Human Papillomavirus (HR-HPV) and HIV is thought to favour initiation of intraepithelial squamous cell lesion and subsequent progression to cervical carcinoma.Evaluation of cytological physiognomies in relation to possible age influence and the genotype distribution of human papillomaviruses among HPV/HIV co-infected and HPV monoinfected women in Kisii, Kenya.The case-control study enrolled 42 HPV/HIV co-infected and 42 HPV monoinfected women. Cervical swabs were collected in ThinPrep vials for HPV tying and cytological analysis. HPV subtypes were assayed by Xpert® HPV system (GXHPV-CE-10).Mono-infected women aged 30–39 years had the highest proportion of low grade squamous intraepithelial lesion (LSIL) at 14 (16.67%) while the co-infected aged 50–59 years had the highest proportion of high grade squamous intraepithelial lesion (HSIL) at 9 (10.71%). HPV-16 genotype was the most predominant and it increased with age rise. Older coinfected and mono-infected women (>40 years) had HSIL and LSIL as the most predominant cytological grade respectively.The predominance of HPV-16 and HPV-18/45 genotypes in the study setting is a consideration that would benefit targeted prophylactic vaccination programs. HPV testing and cervical cancer screening for young and older women on a regular basis ought to be reinforced. Immunosuppression due to Human Immunodeficiency Virus (HIV) is associated with greater prevalence and broader range of the high risk human papilloma virus (HR-HPV) genotypes in women with cervical cancer Infection with high risk HPV types is closely associated with intraepithelial neoplasia progressively resulting in cervical carcinoma In the case-control study conducted at Kisii Teaching and Referral Hospital (KTRH) located in Kisii County Kenya, participants enrollment inclusion and exclusion criteria was essentially carried out as previously described Briefly, in the one stop sample collection, consented women were recruited into the study. HIV testing was then done according Ministry of Health, National AIDS & STI Control Program (NASCOP) 2018 national testing guidelines using Determine® rapid test kit , and the positive results confirmed by Uni-Gold® . There after the participants underwent a preliminary visual inspection with acetic acid (VIA) prior to cervical samples collection with a cyto-broom. Te Pap smears slides were prepared in duplicate for Papanicolaou (Pap) staining. Three independent pathologists read the slides. The cells were classified according to the revised standardized Bethesda classificationDescriptive statistics was applied for cervical lesions and HPV characterization and the significance of their difference tested by chi-square statistics. Significance level was set at P < 0.05.The enrolment of participants was consecutive and purposive which recruited 1854 consented participants. The women first underwent Visual Inspection with 5% Acetic Acid (VIA). There were 410 (22.22%) VIA positive women while 1435 (77.7%) were VIA negative. All the 410 VIA positive samples qualified for cytological analysis and HPV testing. The 121 VIA/HIV positive women had cervical cell exfoliated and smears prepared for cytological evaluation. A portion of the cervical sample was preserved for HPV characterization. Out of the 121 samples, 4 (3.3%) samples/slides were classified as unsatisfactory for cytology while 117 (96.69%) were satisfactory. The satisfactory samples had 79 (67.52%) women with normal cytology and 38 (32.47%) with abnormal cytology. There were 289 VIA positive/HIV negative women. These 289 women had cervical cell samples exfoliated and smears prepared for cytological evaluation. Out of 289 Pap smear samples, 8 (2.76%) were classified as unsatisfactory for cytology while 281 (97.23%) were satisfactory for cytology. The satisfactory samples had 241 (85.76%) women with normal cytology while 40 (14.23%) had abnormal cytology. All the unsatisfactory samples from VIA/HIV positive 4 (3.3%) and those of VIA positive/HIV negative 8 (2.76) were also HPV negative. Abnormal cytology had 38 (32.47%) VIA/HIV positive and 40 (14.23%) VIA positive/HIV negative women who were HPV positive. Only 4 (5.06%) with normal cytology from the VIA/HIV positive and 2 (0.83%) of VIA positive/HIV negative were HPV positive. In total 42 (34.71%) of HIV positive were HPV positive (cases) while 42(14.53%) of HIV negative were HPV positive (controls). The summary of the recruitment process is in Among the women recruited, age categories between HPV/HIV co-infected women (cases) and those in HPV monoinfected women (controls) group were analysed. The highest proportion of cases were in the upper age limit (50 -59 years), with 14 (33.33%) cases. Controls in this age limit were only 5 (11.90%). The highest proportion of controls was in the age category 30 – 39 years where there were 10 (23.81%) cases compared to 15 (35.71%) controls in this age category. Frequencies of the cases and controls in different age categories are represented in The distribution of women required to recruit the adequate sample size of cases (121) compared to that of controls (289), with controls requiring more than twice the population of cases is summarized in Overall mean age for the participants was 37.79 ± 10.21 years. 42 HPV/HIV co-infected women (cases) had mean age of 40.36 ± 11.318 years while 42 HPV mono-infected women (controls) had mean age of 35.21 ± 9.495 years.Prevalence of cytological grades in the study population The overall cytological categories in both co-infection (HPV/HIV) and mono-infections (HPV) combined were classified according to the revised standardized Bethesda classificationThe categorization of cytological grades was done according to the ages of women and the trends presented in The prevalence of HPV types was done in the four age brackets namely; >29, 30–39, 40–49 and 50–59 years . In the The most prevalent HR-HPV subtype was HPV 16 which was consistent with other settings HPV characterization in the current study population showed that HPV type 16 had the highest prevalence of 26 (20.8%) followed by HPV types combination  with 19 (22.6%). HR-HPV type 16 had a higher prevalence of 4 (4.8%) in all ages compared to HPV type (18/45) which had 2 (2.4%) in this population. A similar dimension was observed in the study conducted in the Central Kenya at Thika where the prevalence of HPV type 16 was 4.6% The presented findings had some limitations. The current study was not within reach to evaluate socio-demographic determinants where differences in sexual behaviour and population genetics in a geographical area or ethnicity Low grade lesions were predominant in women aged < 29 with high proportions of HSIL in coinfected and LSIL in monoinfected women aged >40 years and <40 years respectively. Targeted prophylactic vaccines should incorporate HPV-16 and HPV-18/45, due to predominant infection rates in the women in south western Kenya while HPV testing and cervical cancer screening for young and older on a regular basis ought to be reinforced. There was a higher prevalence of HRHPV types in high grade lesions in co-infections which were double that of mono-infections.


Clearance of anal HPV infection was more frequently observed in the bacteriotherapy group (p = 0.067). Conclusion. Being an interim analysis, we limit ourselves to report the preliminary results of the current study. We refer the conclusions relating to the possible effectiveness of the intervention to the analysis of the definitive data.Background. Anal HPV infection, anal dysplasia and, ultimately, anal cancer are particularly common in HIV-infected men who have sex with men. Treatment of anal dysplasia, aiming to prevent evolution to squamous cell carcinoma of the anus, is currently limited to direct ablation and/or application of topical therapy. The aim of the present study is to investigate the effect of oral bacteriotherapy  on anal HPV infection and HPV-related dysplasia of the anal canal in HIV-infected men who have sex with men. Methods. In this randomized, placebo-controlled, quadruple-blinded trial (NCT04099433), HIV-positive men who have sex with men with anal HPV infection and HPV-related dysplasia were randomized to receive oral bacteriotherapy or placebo for 6 months. Anal HPV test, anal cytology and high resolution anoscopy with biopsies of anal lesions were performed at baseline and at the end of the study. Safety and tolerability of oral bacteriotherapy were also evaluated. Interim analysis results were presented. Results. 20 participants concluded the study procedures to date. No serious adverse events were reported. In respect to participants randomized to placebo, individuals in the experimental arm showed higher rate of anal dysplasia regression ( Squamous cell carcinoma of the anus (SCCA) is almost invariably associated with Human Papilloma Virus (HPV) infection in the anal canal and, similarly to cervical cancer, it is typically preceded by the onset of local dysplasia, namely: low grade and high grade squamous intraepithelial lesions  ,2.Although HPV infection, as well as LSIL and HSIL, can regress spontaneously, HIV infected individuals show high rates of HPV persistence and high incidence of dysplasia ,4,5,6. NModulation of microbial flora with the use of probiotics, either through oral or local administration, promoted the clearance of genital HPV infection and dysplasia among the HIV-negative female population in prospective not-controlled clinical trials ,10,11,12Herein we report the interim analysis results of a randomized controlled trial (RCT) investigating the effect of oral bacteriotherapy on anal HPV infection and HPV-related dysplasia in highly active anti-retroviral therapy (HAART)-treated HIV+ MSM.The present randomized, placebo controlled, quadruple blind study , was designed to investigate the effect of oral bacteriotherapy on anal HPV infection and HPV-related dysplasia in HAART treated HIV+ MSM. Two study arms were compared: an arm formerly called “experimental group” in which oral bacteriotherapy was administered, and an arm called “control group” whose participants took a placebo. Randomization was independently carried out on a 1:1 basis by an external society. Study design is reported in Primary outcomes were: (i) change from baseline in the number of HPV positive anal swabs, (ii) change from baseline in the number of dysplastic lesions.The rate of adverse events throughout the study was evaluated as secondary outcome.Participants were enrolled at the HIV Outpatient Clinic of Policlinico Umberto I Hospital of “Sapienza” University of Rome, Italy.Subjects were considered eligible for the study if: (i) >18 years old; (ii) on stable HAART since at least 12 months with undetectable HIV RNA in plasma (lower limit of detection 37 copies/mL); (iii) positive for anal HPV infection with at least 1 histology proven area of anal dysplasia of any grade.Exclusion criteria were: (i) refusal to provide written informed consent; (ii) known diagnosis of inflammatory bowel disease; (iii) use of antibiotics and/or probiotics within the 3 months prior to screening for enrollment in the study. To avoid the risk of bias, HPV vaccinated subjects were not enrolled and initiation of vaccination course during the study was not allowed. Similarly, the use of over-the-counter probiotics represented a reason for withdrawal from the study.Demographic and clinical data were collected at baseline (T0) and after 6 months (T1) on electronic CRF. At each study visit (T0 and T1) participants underwent: (1) anal HPV DNA detection and genotyping; (2) anal cytology; (3) high resolution anoscopy (HRA) with biopsies of areas suspicious for dysplasia. A self-reported questionnaire investigating the presence of gastrointestinal symptoms  was administered to all participants at T0 and T1.HPV DNA extraction was performed using QIAamp Blood and Tissue kit . HPV identification has been performed through PCR using a pair of specific primers: MY09 and MY11, which allowed the amplification of a 450 bp fragment of the late L1 region of HPV. PCR products corresponding to proper fragments were purified with QIAquick PCR purification kit. DNA sequencing was performed with an automatic DNA sequencer , according to the manufacturer specifications . Sequence similarity was determined by BLAST and ClustalW programs. Samples were classified as “multiple infection” when L1 and E6/7 sequencing gave discordant results .Clearance of anal HPV infection was defined as: (a) negative swab at the end of the study in participants with positive swab at baseline, (b) positive swab at the end of the study that shows a different genotype from baseline .Cytology specimens were collected over a period of 15–20 s, smeared onto a glass slide and fixed with alcohol within 15 s from brush withdrawal from the anus. The slides were stained using Papanicolau stain; cytology results were provided following the LAST recommendations .HRA were conducted at T0 and T1 by the same provider. HRA technique and areas of suspected dysplasia were defined following the 2016 IANS International Guidelines for Practice Standards in the Detection of Anal Cancer Precursors . BiopsieT1 HRA was planned within 30 days from the end of intake of the study product but due to the SARS-CoV2 pandemic it was delayed by an additional 30 days in 6 individuals .Streptococcus thermophilus DSM24731®, Lactobacillus plantarum DSM24730®, Bifidobacterium breve DSM24732®, Lactobacillus paracasei DSM24733®, Lactobacillus delbrueckii subsp. bulgaricus DSM24734®, Lactobacillus acidophilus DSM 24735®, Bifidobacterium longum DSM24736®, Bifidobacterium infantis DSM24737®, (trade name: Vivomixx® in EU and Visbiome® in USA). Probiotics were administered at the concentration of 1800 billion bacteria/day . The placebo was composed of maltose and silicon dioxide. Sachet envelopes and content were indistinguishable between probiotics and placebo. Since probiotics needed refrigeration, storage at +4 °C was required for both compounds. All participants were told to ingest the study product at least 30 min before or 2 h after introducing food.Participants were randomly assigned to receive a 6-month course of oral supplementation with daily probiotics or placebo. The probiotic compound was composed as follows: 2) was used to test group differences of proportions.Since literature data on the topic of the study are scarce and since this is a pilot study, the number of participants was arbitrarily set at 40 subjects. The interim analysis of 50% of the overall population to be enrolled in the trial and the final analysis with 100% of sample size enrolled were planned. Statistical analysis was conducted with Statistical Package for Social Science software, version 25 . Unless otherwise stated, data were presented as median and interquartile range  for continuous variables, and as proportions, simple frequencies (n) and percentages for categorical variables. The Kolmogorov–Smirnov test and histograms were applied to assess the normal distribution of data. The presence of statistically significant differences between groups was assessed using Student t-test or Mann–Whitney test for normally and non-normally distributed variables, respectively. The Chi-square test  with 95% confidence intervals (95% CIs) were calculated for all associations. A two-sided The study was independently approved by the Committee of the Public Health and Infectious Diseases Department of “Sapienza” University of Rome and by the local Ethics Committee . Written informed consent was obtained from each participant prior to the enrollment in the trial. The study protocol was registered in the ClinicalTrials.gov register (Identifier number: NCT04099433).Probiotic compound and placebo were donated by the probiotic manufacturing company without any obligation. The probiotic manufacturing company did not take part in data analysis nor did it obtain access to study data or results prior to their publication.Since the trial is still ongoing, here we report the interim analysis (as per protocol) of the results of the first 20 participants that concluded the protocol.Among 20 subjects that have concluded the study protocol to date, 9 have been randomized to daily intake of oral probiotics and 11 to placebo. Baseline characteristics of the study population are reported in The concordance between anal biopsies and diagnosis of dysplasia was 100%. Total number of dysplasia observed at baseline was 17 in the experimental arm and 19 in the control arm. In each group, five subjects showed more than one lesion.All participants reported unprotected receptive anal intercourse during the study. 44.5% of participants in the experimental arm reported sexual contacts exclusively with the usual partner and 55.5% with multiple sexual partners. In the control arm, 36% of participants had receptive anal intercourses with the usual partner and 64% with multiple sexual partners.At the end of the study, individuals in the experimental group showed significant improvements in the considered outcomes as compared to the control group, as shown below.Nutritional and metabolic status of patients were also evaluated, as part of the routine follow-up for HIV+ individuals. No differences in blood pressure, body weight, BMI and lipid profile (cholesterol and triglycerides) were observed between groups at T1 (nor at baseline).Main characteristics of the study population at T1 are reported in p = 0.067).At T1, 67% of subjects in the experimental arm and 18% in the control group showed clearance of baseline anal HPV  showed clearance of baseline HPV infection but acquired a new genotype. In the control arm: two participants cleared baseline HPV infection but showed acquisition of a new genotype at T1.In the experimental arm: two participants with cytologic LSIL at baseline showed absence of abnormalities at T1. In the control arm: one participant with normal cytology at enrollment showed onset of LSIL at T1 and one participant with cytologic LSIL at baseline showed HSIL at T1 (HSIL was subsequently confirmed by histology).Among the population of the experimental arm: total number of dysplasia decreased from 17 to 12, the median number of lesions decreased from one with IQR 1–2 to one with IQR 0–1 and the number of participants with multiple dysplasia decreased from five to two. Persistence of dysplasia with unchanged HRA characteristics was observed in 55% of subjects, while persistence with worsening of HRA appearance was observed in 11% of participants.Among the population of the control arm: total number of dysplasia increased from 19 to 30, the median number of lesions increased from two with IQR 1–3) to two with IQR 2–4 and the presence of multiple dysplasia increased from five to nine participants. Persistence of dysplasia with unmodified characteristics at HRA was observed in 54% of participants, while persistence with worsening of HRA appearance was observed in 73% of participants.The regression of at least one dysplastic lesion was observed in 88% of subjects in the experimental arm versus 9% in the control arm. At the end of the study a normal HRA with absence of abnormal area was observed in 33% of the experimental group population and 0% of the control group.Onset of new dysplasia was observed in 22% of participants in the experimental arm (LSIL in both cases) and 73% of participants in the control arm, 37.5% of whom showed histology-proven HSIL. The number of SIL on HRA examination is shown in At baseline, condyloma of the anal canal were observed in two participants in the experimental arm and one in the control arm. At T1, while regression was observed in all cases, onset of new condylomas was observed in one subject in the control arm.p = 0.028) as well as an increased likelihood of regression of pre-existent dysplasia . Moreover, the intake of probiotic compound was protective against the onset of new lesions  and protective against worsening of persistent dysplasia .Multivariate linear regression confirmed the observed results. In the study population, being assigned to probiotic intake was associated with an increased likelihood of HPV clearance , lower rate of onset of new anal dysplasia (p = 0.023) and lower rates of worsening of persistent lesions (p = 0.004), with respect to participants randomized to placebo. Clearance of anal HPV infection was more frequently observed in the bacteriotherapy group (p = 0.067), however it did not reach statistical significance. No adverse effects or safety concerns were reported in the experimental group.In synthesis in this study, 20 participants concluded the protocol procedures to date. Individuals in the experimental arm showed a higher rate of anal dysplasia regression (p = 0.067) despite the trend observed .Major limitations of this interim analysis of the study were the small sample size of participants enrolled, short study interval and the weak p-values in the clearance of baseline anal HPV (Since this is an interim analysis, and considering its limits, we limit ourselves to reporting the preliminary results of the current study, without drawing conclusions. We refer the conclusions relating to the possible effectiveness of the intervention with this specific probiotic formulation to the analysis of the definitive data.The findings of the interim analysis are in line with the evidence coming from similar studies that have been undertaken in female patients with HPV-related cervical lesions ,25.Although seemingly encouraging and worthy of disclosure, these results are nevertheless too preliminary and therefore potentially biased to draw ultimate conclusions.


Human Papillomavirus (HPV) primary cervical screening was implemented across England during 2019, where cervical cell samples are first tested for HPV and cytology is used to triage HPV-positive results. Around 8.5% of women who attend test HPV-positive with normal cytology . We aimed to explore women’s information needs and suggestions for improvements to result communication following an HPV+/normal result, among those with higher and lower levels of education. In‐depth interviews were conducted with 30 women aged 24–63 who had tested HPV+/normal at routine screening. Secondary qualitative data, not previously reported, were analysed using Framework Analysis to compare themes between those with education lower-than-degree-level vs. degree-level-or-higher (n = 15 in each group). Regardless of education level, women had unanswered questions about their result meaning and the HPV primary screening protocol. Expectations of cervical screening did not always match the service provided, especially regarding content of letters and mode of result delivery. Women with lower education were less clear about the meaning of normal cytology and its link to HPV; and had difficulty sourcing information after their result. Pragmatic suggestions were made for preferences in content, wording, format, and delivery of information in patient communications. Overall, our findings point to areas which can be used by policymakers and healthcare professionals to inform content and communication of results, as HPV primary screening continues to be implemented and refined worldwide. Future research should use these suggestions to develop patient materials and then test them to assess content engagement and information recall. Around Concerns have been expressed regarding how accurately women will interpret the meaning of an HPV+/normal result, partly due to lack of immediate follow-up or routine clinical contact. Some women may be learning about the link between HPV, its sexually transmitted nature, and cervical cancer for the first time . A recenAlthough existing literature has identified common themes following receipt of an HPV-positive result , little The aim of this secondary qualitative analysis was to explore women’s information needs and their suggestions for improvements to result communication following an HPV+/normal result at routine HPV primary screening in England. We also compared themes between women with lower than degree level vs. degree level or higher education to assess whether information needs differed based on educational attainment.2We performed secondary thematic analysis using interview data from a primary qualitative study exploring reasons for anxiety in women testing HPV+/normal . The dat. degree-level-or-higher education. Education categorisation was chosen for consistency with previous HPV research , Confidentiality Advisory Group (18/CAG/0118), and Cervical Screening Research Advisory Committee (ODR1819_005).3Interviews were conducted with 30 women: 15 with lower-than-degree education (LE) and 15 with degree-or-higher education (HE). Women attended the interview on average 35.5 days after their HPV+/normal result (range: 22–76 days). Women’s experiences, information needs, and suggestions covered five themes: (i) receiving the result by letter; (ii) content and structure of the result letter; (iii) information seeking after the result; (iv) questions about result meaning; and (v) HPV primary screening and public awareness. Overall, information needs were relatively consistent across high and low education groups, but nuanced differences were identified and are highlighted where applicable. 3.1“I only recognised that there was another test or there’s another result for cell something, erm, when I was doing the survey for this [study]… I probably didn’t pay attention to it because it just said ‘normal’.” Most women were unaware that they could test HPV-positive at cervical screening until after they received their result, except a minority who had been informed at their screening appointment. Several reported confusion regarding the information in their result letter and could only accurately recall the HPV-positive aspect. Women with lower education particularly struggled to recall their normal cytology result.good news”, whereas “if it was bad news I’d hear on a phone call or something” . Others expected to receive a follow-up call or appointment from a healthcare professional (HCP):“It might have been a lot better if the doctor… someone from the practice phones, could phone me and say, “We’ve received this, and how do you feel about it?” Regardless of education level, for some, there was a prior expectation that they would only receive a mailed letter if their result was negative and “3.2polite” and “well written”. Others thought it was “blunt” and “not very humanised”. Many felt the letter was “informative to a point” but there was not enough information to understand the result. Several expressed that more information would have helped put their “mind at rest”. A few referred to an analogy they had read which helped them to understand HPV clearance by the immune system and reassured them that HPV+/normal was low risk:I think explaining it with an analogy like it’s… you know, it’s like a cold, your immune system can clear it, that… I thought that was quite clear. Women had mixed views about the wording of the result letter. Some thought it was ““The [same] standard letter is not appropriate and should be changed to standard letter one, standard letter two, standard letter three … and what action needs to be taken.” Nearly all women wanted their letter to feel more personalised. Notably, women who had received a 2nd or 3rd consecutive HPV+/normal result after 12-month recall expressed a need for information in their letter covering the future screening protocol and treatment procedures:good news”  before the “bad news” (i.e. HPV-positive result). Some suggested the letter should be restructured to draw more attention to the HPV and cytology result separately. Pragmatic suggestions included highlighting results in bold; splitting results under subheadings for ‘cytology’ and ‘HPV’; and displaying key information as bullet points. A few suggested visual aids or signposting to an online video.Regarding letter structure, some said they would prefer to get the “3.3minefield” . Women with lower education expressed difficulty sourcing and interpreting online HPV content. Often this was due to uncertainty about what information to trust and through reading conflicting information.“I don’t always like Googling things because there’s so much out there that how do you - how do you know what - what’s the right information and what’s not” Nearly all women searched online for information immediately after reading their letter. In some instances, online information was viewed as helpful. However, many found it unhelpful comparing the process to searching through a ““the emotional side… It was informative in a way that I could take what knowledge I could from it but not, it wasn’t so like black and white” .In contrast, women with higher education reported higher satisfaction and discussed the importance of supportive information sources. Some particularly liked the Jo’s Cervical Cancer Trust website (UK charity), describing it as on “I kinda felt that she [nurse] didn’t really have any more information than what was already out there anyway… I guess I was just hoping to get a bit more reassurance really” Some women visited a HCP after receiving their result for reassurance and clarification. Interactions with HCPs were reported as positive overall. However, some women with lower education reported less satisfaction from clinical interactions.waste their [HCP’s] time” . Alternative suggestions, considered to be acceptable, included visiting a sexual health clinic or calling a helpline. A preference for additional information placed alongside the result was expressed by most women, such as in a leaflet. The majority said information should be given to them rather than “the onus” being on them to seek it out. Women expressed interest in receiving a link to “a trusted website” . Few reported reading the cervical screening information leaflet which was posted alongside their screening invitation letter.Some reported not visiting their GP so as not to “3.4Nearly all women had unanswered questions relating to their HPV+/normal result. Topics suggested for inclusion in letters primarily centred on HPV-positivity and its link to cervical cancer. Information which women thought would help their interpretation included whether they had a high-risk HPV type, their short and long-term cancer risk, and cancer survival statistics. Several wanted information about sexual transmission, how they could test positive for HPV in a long-lasting relationship, and potential implications for their partners and risk of re-infection. Women with lower education expressed greater uncertainty about whether HPV was sexually transmitted and wanted this confirmed; as well as information on whether they should inform (ex)partners. A few younger women requested information on whether HPV could affect fertility.HPV but [their] cervix was clear” . Some thought HPV and cytology results were “completely separate” .Some wanted information about the normal cytology aspect of their HPV+/normal result, but this was a minor theme. Women with lower education expressed less clarity regarding how HPV and cytology results were related to one another; and were confused about how they could have “3.5“I think what would have helped as well, the fact that if they [sample taker] had mentioned that this is a new test.” “I think if it was explained why you have to wait twelve months, it would be a lot better. A lot of things can happen in twelve months” Few women had heard of HPV primary screening at the time of the interview. Of those that had, most found out after their result and wanted the switch in methods made explicit earlier in the screening process. Regardless of whether women formally knew about HPV primary screening, nearly all commented on 12-month early recall to screening and wanted information to explain the rationale for the change in interval. Many expressed the need for better communication and information provision before or during their screen. Some wanted their screening nurse to explain HPV testing and its implications, to reassure and prepare them for a potential HPV-positive result.Minor themes included the lack of public awareness campaigns about HPV primary screening and the need for better education, particularly related to sexual health and the benefits of cancer screening.9Our findings provide insights into the information needs of women receiving HPV+/normal results at routine HPV primary screening. We identified themes that build on, and contextualise, current literature on HPV primary screening knowledge and women’s experiences of receiving HPV-positive results. Regardless of education level, most women reported poor knowledge of HPV and HPV testing and had several unanswered questions about the meaning of their HPV+/normal result. Expectations of the cervical screening programme did not always match with the service provided, especially regarding content covered in letters and mode of result delivery. Pragmatic suggestions for improvements to routine information can be used to enhance patient communications and inform patient aspects of clinician training, as HPV primary screening continues to be refined and implemented worldwide.Consistent with previous research on abnormal cervical screening results, most women struggled to interpret the meaning of the new HPV+/normal result . They ex, differences in cancer risk and the need for colposcopy after three consecutive results made clear.Key pieces of information reported as helpful, partly in this study and from other work internationally, includes that HPV is common; it can remain dormant for a long time; only long-lasting infection leads to cervical cancer; there are minimal effects on partners; and HPV is usually cleared by the immune system without needing treatment . Novel tIn terms of letter format and structure, women suggested that results could be listed in bold font with information split under separate headings, clearly distinguishing between different aspects . Some also wanted the “good news” before the “bad news”, i.e., their normal cytology result first. These suggestions could be cost-effectively implemented in patient communications for most routine screening programmes. However, it is possible that ordering normal cytology information first may mean that some women overlook the HPV-positive aspect, if they do not read the full letter. Hence, HPV+/normal letter restructuring should be formally tested through research to assess impact on information recall and content engagement.Most women went online or visited a GP to find information about testing HPV+/normal after their result. Searching online was largely reported as confusing and unhelpful, especially by women with lower-than-degree-level education. Other studies have found that women with lower education have lower HPV knowledge and difficulty interpreting key aspects of screening . This emVisual aids or links to online videos were discussed and have been suggested as a potential solution to help women interpret complex information, particularly statistical information relating to cancer risk . A systeAs HPV primary screening is implemented worldwide, our findings point to areas which can be used by policymakers and healthcare professionals to improve content and communication of HPV+/normal results. Pragmatic suggestions for letter restructuring paired with adoption of evidence-based content could help women process and better understand an HPV+/normal result. Our results were relatively homogenous by level of education; therefore, further quantitative research would be needed to ascertain whether differing educational materials would be needed for each group. As health systems move towards more digital approaches, this may provide opportunities for personalised screening services rather than a one-size-fits-all national programme. Since our study took place, the NHSCSP in England has incorporated a new frequently asked questions (FAQ) section as part of their HPV+/normal result letters, which should begin to address some of the concerns raised by women. Similar policy could be adopted in other countries. Topics and suggestions highlighted in this study can be used to cost-effectively develop, enhance, or refine FAQs and leaflet content in addition to improving letter wording. Targeted efforts to deliver healthcare professional training on HPV primary screening and communication skills may also facilitate provision of information to women at screening.9.1To our knowledge, this is the first qualitative study to explore information needs in women receiving routine HPV+/normal results at HPV primary screening. Ecological validity was ensured through recruitment linked to routine clinical management at HPV primary screening. Our relatively diverse and well‐characterised sample allowed us to draw thematic comparisons between low- and high-education groups, which may be important for identifying inequalities in burden. However, our sample did not represent women without formal qualifications, who may represent a distinct group in terms of health literacy and need. Due to the relatively small numbers, we were unable to explore intersections between other important sociodemographic factors, such as ethnicity or age. Recall bias could be a limitation given interviews were carried out on average 35.5 days after result. Lastly, the primary qualitative study used for our secondary analysis purposively sampled women based on varying anxiety scores, which could be considered both a strength and limitation. Nonetheless, it is important that our secondary findings are interpreted in this context.10Women across all levels of education had unanswered questions about their HPV-positive with normal cytology result and the HPV primary screening protocol. Given that HPV primary screening is still a novel programme that continues to be piloted and implemented internationally, this research provides timely suggestions for service improvement whilst advancing the academic literature. Future research should use the suggestions outlined in this paper to develop and test patient communications with women from a range of sociodemographic backgrounds; and assess content engagement and information recall.10.13039/501100000272National Institute for Health Research (NIHR) as part of a fellowship awarded to Emily McBride (DRF-2017-10-105); the views expressed in this paper are not necessarily those of the NHS, the NIHR, or the Department of Health and Social Care. Jo Waller was funded by 10.13039/501100000289Cancer Research UK (C7492/A17219).This study was funded by the Emily McBride conceived the study, managed the project, and conducted the participant interviews. Sophie Mulcahy Symmons and Emily McBride analysed the data and drafted the manuscript. All authors contributed to, and approved, the final version of the manuscript.The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dr Emily McBride and Dr Jo Waller sat on the NHS Cervical Screening Programme, HPV Primary Screening Pilot Steering Committee from 2016 to 2019.


Validation was conducted using 1,300 consecutive cervical samples from women attending an organized population-based cervical screening program enriched with 300 cytologically abnormal samples. Overall high-risk HPV test concordance was assessed by kappa values; the concordance for HPV-16 and HPV-18 was assessed for Alinity m, RealTime, and cobas, and the Linear Array (Roche) was used for more detailed genotyping concordance. In the total study population, the relative sensitivity and specificity for cervical intraepithelial neoplasia grade 2 or worse (CIN2+) and CIN3+ of Alinity m compared to HC2 was 1.02  and 1.03 , respectively. The relative specificity for nondiseased subjects (≤CIN1) was 1.01  . Alinity m showed noninferior clinical accuracy among women 30 years or older when cobas or RealTime was used as a comparator. HPV genotype-specific concordance between Alinity m and the three comparator tests showed excellent agreement, with kappa values ranging from 0.82 to 1.00. In conclusion, Alinity m fulfills the international accuracy requirements for use in cervical cancer screening and shows excellent HPV genotype-specific concordance with three clinically validated HPV tests.Only clinically validated human papillomavirus (HPV) tests should be used in cervical cancer screening. VALGENT provides a framework to validate new HPV tests. In the VALGENT-3 study, the clinical accuracy of the recently launched Abbott Alinity m HR HPV assay (Alinity m) to detect cervical precancerous lesions was assessed against the standard comparator test  and against two previously validated alternative comparator tests (Abbott RealTi The strong etiological association recognized between persistent high-risk human papillomavirus (hrHPV) infection and the development of cervical cancer , 2 has r7–Alphapapillomaviruses are grouped into low-risk and high-risk HPV types. The International Agency for Cancer (IARC) considers 12 HPV genotypes  carcinogenic due to an etiological link with developing cervical cancer and with precursor lesions (group I carcinogens), with HPV types 16 and 18 being the most potent carcinogenic agents, accounting for approximately 70% of all cervical cancers . Following the VALGENT protocol , 300 samEthical approval was obtained for this study from the Slovenian Medical Ethics Committee (consent no. 83/11/09 and 109/08/12).me High Risk HPV test , and cobas 4800 HPV test . All tests were performed according to the manufacturer’s instructions.All 1,600 samples were tested with Alinity m, Hybrid Capture 2 HPV DNA test , RealTiAlinity m is a fully automated hrHPV test launched in 2019 that targets the conserved L1 region of HPV DNA of 14 hrHPV types. hrHPVs are detected with genotype-specific probes in five distinct channels: HPV-16, HPV-18, HPV-45, group A , and group B  (HC2 (Qiagen), launched in 1998, is a semiquantitative test and one of two recommended standard comparator tests in the international validation guidelines. HC2 detects 13 genotypes of hrHPV  was used in this study as a standard comparator test against which the clinical performance of Alinity m was evaluated. Sample testing with HC2  was carrme (Abbott), launched in 2008, is an automated multiplex real-time PCR test that targets the L1 region of 14 different hrHPV genotypes and has been clinically validated in several previous studies , launched in 2011, is a multiplex real-time PCR test that is fully automated and targets the L1 region of 14 different hrHPV genotypes with the same HPV genotyping capability as RealTi studies , 28–31. Linear Array (Roche) is a test for HPV that has full genotyping capacity, and it can discriminate between 37 low- and high-risk HPV genotypes that are frequently used for epidemiological and virological studies . This stAccording to the criteria of the Slovenian program for cervical cancer screening, women are given an immediate colposcopy referral relying on an atypical squamous cell threshold that cannot exclude high-grade lesions or worse (ASC-H) or according to the study protocol if they were positive for HPV-16/18, irrespective of cytology findings. Specimens are taken via punch biopsy during colposcopy from any region suspected of cervical intraepithelial neoplasia (CIN), and certified pathologists with more than 20 years of gynecological pathology experience examine the samples .The diseased group  included women with histologically confirmed cervical intraepithelial neoplasia grade 2 or worse (CIN2+) and grade 3 or worse (CIN3+). Clinical specificity estimates were calculated for the control or nondiseased group, which included patients who had received two negative cytology results in a row upon enrollment and also at the following screening 12 to 48 months later.me were included as additional comparator tests because they have clinical accuracy similar to that of HC2 and genotyping capacity similar to that of Alinity m. CIN2+ and CIN3+ clinical sensitivity and ≤CIN1 clinical specificity were calculated for Alinity m, HC2, RealTime, and cobas. We assessed clinical performance for the complete study population, regardless of age, and in patients 30 and older. We used the McNemar (McN) test to compare differences between paired proportions. The noninferior accuracy of Alinity m versus the comparator tests was assessed with the matched noninferior statistic pnon-inferiority (pni) proposed by Tang et al. (In addition to the standard comparator (HC2), cobas and RealTig et al. , acceptime  values and McNemar statistics were used to assess genotype-specific concordance for the HPV genotypes common to Alinity m and Linear Array, cobas, and RealTime . The κ vme .The statistical significance level was set at 0.05. Statistical analyses were carried out with STATA version 14 .n = 19) or had incomplete follow-up (n = 236). Thus, 1,212 women with two subsequent negative cytology results within at least 3 years  and 126 patients with CIN2+  were used for assessment of clinical accuracy. Of 126 patients with CIN2+, 45 had CIN2 and 81 had CIN3+, as shown in Of the 1,600 samples that were tested with Alinity m, seven samples (0.4%) were excluded from further analyses due to negative β-globin results. me are shown in Within the total study population, 124/126 CIN2+ and 81/81 CIN3+ cases showed positivity with Alinity m, which corresponds to sensitivity for CIN2+ and CIN3+ of 98.4%  and 95.1 , respectively see . Out of pni = 0.0001, pni = 0.0006, and pni < 0.0001), respectively, for the entire study population. Stratifying the analyses for women 30 years or older yielded similar results .In the entire study population, Alinity m had a somewhat higher sensitivity than HC2: 1.02  for CIN2+ and 1.03  for CIN3+. The specificity for ≤CIN1 of Alinity m was marginally higher than that of HC2 . The sensitivity for CIN2+ and CIN3+ and specificity for ≤CIN1 of Alinity m was noninferior to that of HC2  and 1.02 , respectively. The relative specificity for ≤CIN1 of Alinity m was 0.99  compared to RealTime and 1.00  compared with cobas (Tables S1 and S2).The relative sensitivity for CIN2+ of Alinity m versus RealTime, and Alinity m versus Linear Array, whereby HPV-16, HPV-18, and HPV-16/18 results include both single or multiple infections. The concordance for HPV-45 between Alinity m and Linear Array was 99.7% (κ = 0.81). When genotype detection by Alinity m versus cobas and Alinity m versus RealTime was stratified by clinical setting (screening and enriched populations), excellent agreement was observed again   and, overall, 14 hrHPV genotypes (Table S3).Tables 2 to Because international and national evidence-based recommendations continue to propose replacing cytology with HPV testing as a primary tool for screening for cervical cancer, validating novel HPV tests based on international criteria using established comparative frameworks (such as VALGENT) is deemed vital for global high-quality cervical cancer screening efforts and the cervical cancer 2030 elimination goals proposed by the World Health Organization .In addition to detecting 14 hrHPV genotypes, the Alinity m assay provides distinct information on the three most carcinogenic HPV genotypes  and separates the four subsequently most carcinogenic genotypes into group A , covered by the nonavalent HPV vaccine, from seven other less carcinogenic HPV genotypes . This study showed Alinity m’s clinical sensitivity and specificity for detecting CIN2+ in patients 30 years or older to be 99.0%  and 100.0% , respectively. Compared with the standard comparator test, HC2, in this study, Alinity m demonstrated noninferior CIN2+ sensitivity and specificity in both women 30 years and older as well as in the entire study population. The results of this study confirm those generated in the other Alinity m validation study, in which the clinical accuracy of Alinity m was assessed on 3,145 women 30 years or older and clinical sensitivity for CIN2+ and specificity for ≤CIN1 were 100.0% and 92.4%, respectively . Therefome, cobas, and Linear Array. Compared to these three clinically validated HPV assays, noninferior accuracy of Alinity m for CIN2+ and CIN3+ was observed. A previous validation study showed noninferior clinical sensitivity and specificity of Alinity m compared to HC2 and performance of Alinity m comparable to that of HC2, cobas, and RealTime in terms of 3-year negative predictive value  (me [0.04]) compared to those that had a normal baseline cytology (0.65) (In addition to the standard comparator assay (HC2), this study provides comparison data for Alinity m’s performance against three other clinically validated HPV assays: RealTi result) . In party (0.65) . In addiy (0.65) . Anotherme, and Linear Array. The research findings that have accumulated in the past years, coupled with recent U.S. FDA approval of the BD Onclarity HPV  assay for extended genotyping, suggest that extended genotyping will play a significant role in clinical practice in the future, in particular for populations with high vaccine coverage for HPV. Because Alinity m showed excellent performance in every clinical and analytical evaluation published to date and, in addition to aggregate information for 14 hrHPV genotypes, provides separate information for all hrHPV genotypes covered by the bivalent, quadrivalent, and nonavalent HPV vaccines, it can be deemed an important tool in possible new management algorithms for hrHPV risk-based screening for primary cervical cancer. Unfortunately, performance of hrHPV assays is the main focus of the current international validation guidelines  in a separate study to assess different triage options in HPV-based primary cancer screening settings, including partial and extended genotyping. Thus far, only a single pooled analysis nested in the VALGENT framework on the accuracy of HPV-16/18 genotyping to triage women with LSIL was performed. It clearly showed that the presence of HPV-16/18 justifies immediate referral to colposcopy, and that women with LSIL carrying other HPV types cannot be returned to routine screening and require further active monitoring , 40. WitIn conclusion, on the basis of the results of this evaluation study and previous ones (