Head and neck cancer (HNC) usually arises from squamous cells of the upper aerodigestive tract that line the mucosal surface in the head and neck region. In India, HNC is common in males, and it is the sixth most common cancer globally. Conventionally, HNC attributes to the use of alcohol or chewing tobacco. Over the past four decades, portions of human papillomavirus (HPV)-positive HNC are increasing at an alarming rate. Identification based on the etiological factors and molecular signatures demonstrates that these neoplastic lesions belong to a distinct category that differs in pathological characteristics and therapeutic response. Slow development in HNC therapeutics has resulted in a low 5-year survival rate in the last two decades. Interestingly, HPV-positive HNC has shown better outcomes following conservative treatments and immunotherapies. This raises demand to have a pre-therapy assessment of HPV status to decide the treatment strategy. Moreover, there is no HPV-specific treatment for HPV-positive HNC patients. Accumulating evidence suggests that phytochemicals are promising leads against HNC and show potential as adjuvants to chemoradiotherapy in HNC. However, only a few of these phytochemicals target HPV. The aim of the present article was to collate data on various leading phytochemicals that have shown promising results in the prevention and treatment of HNC in general and HPV-driven HNC. The review explores the possibility of using these leads against HPV-positive tumors as some of the signaling pathways are common. The review also addresses various challenges in the field that prevent their use in clinical settings. Head and neck cancer (HNC) constitutes a large group of cancers arising in different anatomical sites of the head and neck (HN) region, comprising the lip and oral cavity, larynx, nasopharynx, hypopharynx, oropharynx, nasal cavity, paranasal sinuses, and salivary glands. Over 90% of these neoplastic tissues are squamous cell carcinomas (SCCs). According to WHO estimates for 2019, HNC was one of the leading forms of cancer with 931,931 new cases, representing 4.9% of all cancer cases . Lip andTobacco use, excessive alcohol consumption, and lately, infection of human papillomavirus (HPV) are the established risk factors for HNC . The risMost patients with HNC seek clinical intervention at advanced stages of the disease . This trRecently, there has been a renewed interest in phytochemicals and herbal derivatives with therapeutic correlates from traditional medicine in the treatment and prevention of HNC due to their safety, availability, efficacy, and low cost. A number of studies carried out to investigate screening of phytochemicals using different HNC cell lines, animal models, and clinical evaluation in patients showed potent anticancer activities in a small set of phytochemicals. However, very limited number of studies addressed the impact of these herbal derivatives on HPV infection and HPV-positive HNC. In this article, we have systematically reviewed the existing data on various phytochemicals demonstrating chemotherapeutic and chemopreventive activities against HNC with a special emphasis on phytochemicals/herbal derivatives that showed anticancer effects against HPV-positive HNC. Further, major deficiencies and actionable leads in this field have been highlighted.HNC is a group of neoplastic diseases that can be broadly classified based on their anatomical site, histological origin, and etiological factors .Oral SCC (OSCC) arises from mucosal areas of the lips, front 2/3rd of the tongue, gums, internal lining of cheeks and lips, floor of mouth below the tongue, hard palate, and the area behind the wisdom teeth , and conThe pharynx is a channel located in the region of the neck midline. The pharynx is majorly classified into three regions: the nasopharynx , oropharynx , and hypopharynx (behind the opening of larynx) . GloballThe nasal cavity is the upper most part of the respiratory tract. The nasal cavity is surrounded by four types of paranasal sinuses: frontal sinuses, sphenoid sinuses, paired maxillary sinuses, and ethmoid sinuses. Paranasal sinus malignancies are rare, accounting for less than 3–5% of the total HNC . The nasThe internal space of the larynx is a pyramid shaped about 5 cm long, connecting the pharynx to the trachea and is a part of the respiratory system. According to Exocrine glands and salivary glands function to secrete saliva in the oral cavity. Three type of salivary glands are present: parotid gland (situated front of both ears), submandibular gland (posterior of the mandible), and sublingual gland  . In the In the oral cavity, the mucosa is of masticatory, specialized, and mobile type. It covers around 25% of the oral cavity. In order to understand mechanical forces caused by mastication, it is covered by specialized, orthokeratinized, stratified squamous epithelium. Depending on the anatomic site, over 60% of the mucosa in the oral cavity is lined by the stratified squamous epithelium. The upper surface of the tongue is lined by specialized mucosa, with numerous lingual papillae .Histologically, the tumors of the HN region are classified as carcinoma, adenocarcinoma, lymphoma, and blastoma depending upon the tissue from where they are originating . For insCarcinomas mostly spread in the regions of the larynx, nasopharynx, and least in maxillofacial bones and oral cavity regions, whereas predominant anatomical sites for lymphomas were lymph nodes, followed by the maxillofacial bones. In contrast, sarcomas occurred most frequently in the maxillofacial bones, face/scalp, and the nose area . The disIn the oral cavity, leukoplakia (white plaque) and its variants, erythroplakia (fiery red patch) and submucous fibrosis , are three conditions that are highly associated with the development of oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC). Malignant transformation rates of leukoplakia range from 8.9 to 17.5 percent . The bucDysplasia can be categorized as mild , moderate , and severe . This classification of dysplasia by the WHO is referred to as the gold standard for histological diagnosis of oral potentially malignant disorders (OPMDs). The WHO defines OPMDs as “clinical presentation that carry a risk of cancer development in the oral cavity, whether in a clinically definable precursor lesion or in clinically normal mucosa” . Epithelin situ at high-risk sites unless a biopsy proves otherwise  has classified carcinogens in groups, group 1: tobacco smoking, secondhand smoking, and smokeless tobacco for HNCs, which are sufficient for evident carcinogenicity in human -1-(3-pyridyl)-1-butanone, as they are established carcinogens. In HNC of HNSCC type, the latter one is more associated with increasing the risk of cancer development . Tar is Studies have shown that development of HNC is strongly related with dose-dependent tobacco smoking but can also occur with low daily usage . MoreoveAlcohol-associated HNC: HNC is also associated with alcohol abuse. Studies suggest that alcohol consumption and cigarette smoking are differentially associated with the risk of HNSCC subtypes , radium, glue, formaldehyde as well as solvent fumes used in furniture and shoe production, and asbestos are the main risk factors for sinonasal carcinomas. Hypopharygeal and laryngeal carcinoma are associated with the use of coal for heating or cooking  is closely linked to that of Epstein–Barr virus (EBV) infection. This neoplasm is an uncommon disease with very low prevalence in most countries , which is responsible for resistance to EGFR inhibitors . HPV-posBased on transcription milieu, HNC showed constitutively active nuclear factor-κB (NF-κB) irrespective of their HPV status. However, a detailed molecular dissection of the constitutively active NF-κB complex showed the presence of p50:p65 heterodimer in HPV-positive tumors, whereas homodimer of p50:p50 was found in HPV-negative tumors . SimilarSTAT3, another transcription factor that is linked with carcinogenic outcome, was strongly associated with HPV-negative HNC and was characteristically low in HPV-positive tumors . SOX2 amTreatment of HNC requires a multi-modality approach depending on the stage and site of the tumor . Early tRadiation therapy (RT) is often performed as an adjunct to surgery or in concurrence with chemotherapy . Wendt eDespite a clear prognostic advantage and better response to therapy, therapeutic management for HPV-positive HNC is almost the same as that of any HPV-negative HNC. Considering the younger age of the patients, there have been efforts to reduce the long-term toxicity of anticancer treatment without risking the survival benefits . ReductiDespite aggressive treatment and organ preservation with current clinically administered curative therapies, the overall 5-year survival is less than 50% . With exLimitations of these therapies have prompted clinical and translational research for better chemotherapeutics with less treatment-associated toxicities. Many studies are focusing on biologically active compounds from herbal origin to develop chemotherapeutic agents with fewer side effects and higher efficacy . Many ofin vitro and in vivo experimental systems  comprising c-Rel, RelA (p65), RelB, NF-κB1 (p50 and p105), and NF-κB2 (p52), which plays important roles in immunity, inflammation, cell proliferation, survival, and differentiation . Many baSTAT3, a central transcription factor and known oncogene, works downstream of EGFR, and TGFα signaling also plays a key carcinogenic role in HNC . GuggulsIn 90% HNC, the PI3K/AKT/mTOR pathway is upregulated . WheneveMagnolia plant, reduced the level of Bcl-xL protein, while Bax expression in xenograft HNC tumors increased. It also reduces the expression of mTOR and its downstream p70S6K  among 40 patients with advanced SCCs. They achieved a response rate of 16% with isotretinoin, which included a complete response, a partial response, and a minor response. In the methotrexate-treated group, however, the response rate was 5%. The median survival rate from the start of treatment was also lower in the methotrexate group (4 months) than that in the isotretinoin group (4.5 months)  . Another months) . There aCancer chemoprevention refers to the use of agents to retard the progression of carcinogenesis, reverse, or inhibit it. The aim of chemoprevention is to lower the risk of developing invasive or clinically significant disease. Chemopreventive phytochemicals thus seek to occasion a chemopreventive response when the primary tumors have not reached a critical size, or seek to block and reverse development of a diagnosed premalignant tumor, or prevent metastasis and growth of metastatic tumors . AngiogeA battery of phytochemicals reportedly possess cell invasion, migration, angiogenesis, and metastasis inhibitory activities . These pIn vitro studies conducted with epigallocatechin-3-gallate, berberine, gypenosides, phenethyl isothiocyanate, resveratrol, tricetin, nobiletin, evodiamine, salvianolic acid A, gallic acid, pimosylvin, and extracts of Eclipta prostrata, Physalis angulata, Selaginella tamariscina, Leucaena leucocephala, Duchesnea indica, rasberries (Rubus idaeus), and Galium verum downregulated the expression of MMPs  and 1 placebo arm, with a clinical response in 50% of patients in the three combined arms and a 58% clinical response rate in the two combined higher dose arms , an active component of turmeric and a perennial herb, has been shown to suppress the expression of HPV oncogenes mediated by downregulation and reduced transactivation of AP-1 and NF-kB superfamily members, representing a novel mechanism regulating HPV-induced oral carcinogenesis . Its effinhibitedSTAT3 and promoted cell death. Similar effects were also observed in in vivo and clinical study including female C57BL/6 mice  and 10 human subjects, respectively. The pilot clinical trial demonstrated consistent bioavailability of sulforaphane, promising sustainable chronic administration. Although it is a cost-effective and natural product, further studies planned with encapsulated broccoli extract are required to enhance the ease of acceptability and dispensing. Also, HPV-specific chemopreventive effects are yet to be determined (Sulforaphane, an isothiocyanate, derived from broccoli sprout extracts; treatment of HPV-negative HNC cell lines–UM-SCC-22A, UM-SSC-1, and CAL33–and HPV-positive cell line SSC090 led to dose- and time-dependent stimulation of NRF2 signaling for carcinogen detoxication. It also dephosphorylated termined .via cell cycle arrest and induction of apoptosis (in vivo conditions and clinical trials along with direct correlation with HPV activity.6-Gingerol, a β-hydroxy ketone, derived from ginger rhizome, inhibited tumor cell proliferation and induces cellular toxicity, cell cycle arrest, apoptosis, and caspase 3/7 activation, as observed in KB and SCC4 cells. Also, the caspase-3–dependent proapoptotic activity was stimulated. It also inhibited cell cycle progression arresting the cells in G2 and M phases. Hence, 6-gingerol can be considered as a safe and potent chemotherapeutic/chemopreventive compound acting poptosis . Furtherin vivo as observed in CAL27 and HeLa-derived mouse xenografts. It induces the activation of TRPA1 and apoptosis via the mitochondria-dependent pathway. It promoted mitochondrial dysfunction via reducing mitochondrial membrane potential significantly (Thymol, a monoterpene derivative phenol, is a TRPA1 agonist found in thyme and oregano. It inhibited cellular proliferation and exhibited long-lasting cytotoxic effects as observed in CAL27, SSC4, and SSC9 cell lines. It also inhibited tumor growth ficantly . Its HPVvia preventing malignant transformation of cells, regulating cell signal transduction pathways, increasing apoptosis, and modulating cell cycle (via caspase-3 activation and inhibits DNA alkylation. With low toxicity and various beneficial bioactivities, apigenin can be considered as a potential chemopreventive agent against cancers, particularly, in smokers with HR-HPV coinfection (Apigenin, a flavonoid, found abundantly in flowers of plants, vegetables, and fruits, exerts anticarcinogenic effects ll cycle . It inhinfection .Hence, most of the phytochemicals mentioned above showed anticancer activity in HPV-positive cells, where only a limited studies focused on HPV-specific effects. Thus, considerable attention should be paid to analyze the correlation between anti-HNC and anti-HPV activity of the phytochemicals as a chemopreventive and chemotherapeutic measure to prevent HPV-HNC.Despite their encouraging pharmacological activities, there are bottlenecks in the translation of phytochemical-based therapies applicable in clinical settings.Low bioavailability: Many phytochemicals suffer from having poor aqueous solubility and low retention in blood circulation. Pharmacological concentration of these phytochemicals in blood and tumor tissues is low because of poor absorption, high rate of metabolism, chemical degradation, and speedy clearance. It has been reported that serum levels of curcumin were quite low, reaching a maximum of 0.06 ± 0.01 μg/ml after oral administration of 500 mg/kg in rats ( in rats . Ravindr in rats . A pilot in rats . Chen et in rats . Similar in rats , whereas in rats .Obstacles associated with the use of phytochemicals for treating and preventing cancer can be overcome with advances in the field of nanotechnology. A 10-fold dose advantage was achieved without any loss of effectiveness by encapsulating ECGC in polylactic acid–polyethylene glycol nanoparticles . IncreasToxicity: Although phytochemicals may show toxicity when administered in high doses, they exhibit less adverse effects than conventional therapies. In a clinical trial with 50 oral leucoplakia patients, significant toxicity, severe enough to cause withdrawal of 6 patients, was observed with the use of isoretinoin (retinoin . Additioretinoin . MoreovePharmaceutical industry challenges: Pharma-research into phytochemicals and herbal derivatives has experienced a slow decline during the recent times (nt times . This caPoor independent agents: While phytochemicals may not be efficient as standalone chemotherapeutic agents, many groups have established their efficacy as adjuvants to traditional therapies. A study demonstrated the benefits of combining sulforaphane with cisplatin and 5-fluorouracil (in vivo. In an Italian study conducted among 23 patients undergoing treatment with 5-fluorouracil and cisplatin, prolonged responses were reported with the use of retinol palmitate in chemotherapy intervals. Toxicity levels were acceptable, and treatment did not interfere with the quality of life (rouracil . Sulfora of life . A study of life .Preclinical efficacy vs. clinical response: The cause for discrepancy in effectiveness of phytochemical agents in preclinical and human clinical trials has been conjectured to arise because of differences in dosage, metabolic differences, bioavailability, differences in circulating tissue levels of chemopreventive agents in humans and animals, exposure conditions to damaged tissue vs normal tissue, follow-up time, and the assessed ends. Second, high doses are often administered to animals in contrast to low doses admisible to humans in clinical trials. Although animal models have significantly helped in the identification of carcinogens, and chemopreventive and chemotherapeutic agents, they are not available for every HNC organ site. Furthermore, existing models cannot mimic human exposure complexities of carcinogens, metabolic competence, turnover of cells, and their repair capacity.Phytochemicals show immense potential in the field of HNC chemotherapy and chemoprevention agents. In this evolving landscape, the success of employability of phytochemicals depends on our ability to decipher their molecular mechanics. Using phytochemicals in combination with another or in conjunction with existing chemotherapeutic practices or an alternate therapy is an area worth exploring.in vivo in laboratory conditions and can also be screened by using bioinformatic tools. There is a strong requirement to develop HPV-based concurrent therapies so that HNC can be treated more effectively. There are many associated challenges with the use of natural compounds. In pharmacological doses, the adverse effects of these natural compounds such as increased toxicity and low bioavailability are amplified. For chemoprevention to be feasible in treating premalignant lesions, the compound must be well tolerated and have long-lasting benefit. Moreover, the various signaling pathways contributing to HNC tumorigenesis mandate the use of compounds with multiple molecular targets. It is noteworthy that molecular targets of many such phytochemicals in active HNC are now well known (We have also observed that there has not been much phytochemical-related research on HPV-induced HNC. However, numerous phytochemicals that are effective against HPV-induced cervical cancer have been reported in the literature . In todall known . It is a


Background: Routine human papillomavirus (HPV) immunization in Belgium is currently regionally managed, with school-aged girls receiving the 9-valent HPV (9vHPV) vaccine in Flanders and Wallonia-Brussels with a national catch-up program for females only. This study will assess whether expanding these programs to gender-neutral vaccination (GNV) with the 9vHPV vaccine is a cost-effective strategy in Belgium.Methods: A validated HPV-type transmission dynamic model estimated the potential health and economic impact of regional vaccination programs, comparing GNV versus female-only vaccination (FOV) with the 9vHPV vaccine in individuals aged 11–12 years in Flanders, GNV with the 9vHPV vaccine versus FOV with the 2-valent HPV (2vHPV) vaccine in individuals aged 12–13 years in Wallonia-Brussels, and national catch-up GNV versus FOV with the 9vHPV vaccine for those aged 12–18 years. Vaccination coverage rates of 90, 50, and 50% in both males and females were used in the base cases for the three programs, respectively, and sensitivity analyses were conducted. All costs are from the third-party payer perspective, and outcome measures were reported over a 100-year time horizon.Results: GNV with the 9vHPV vaccine was projected to decrease the cumulative incidence of HPV 6/11/16/18/31/33/45/52/58-related diseases relative to FOV in both Flanders and Wallonia-Brussels. Further reductions were also projected for catch-up GNV with the 9vHPV vaccine, including reductions of 6.8%  for cervical cancer, 7.1% (386 cases) and 18.8%  for head and neck cancer in females and males, respectively, and 30.3%  and 44.6%  for genital warts in females and males, respectively. As a result, a GNV strategy would lead to reductions in HPV-related deaths. Both regional and national catch-up GNV strategies were projected to reduce cumulative HPV-related disease costs and were estimated to be cost-effective compared with FOV with incremental cost-effectiveness ratios of €8,062, €4,179, and €6,127 per quality-adjusted life-years in the three programs, respectively. Sensitivity analyses were consistent with the base cases.Conclusions: A GNV strategy with the 9vHPV vaccine can reduce the burden of HPV-related disease and is cost-effective compared with FOV for both regional vaccination programs and the national catch-up program in Belgium. In 2007, the Superior Health Council of Belgium announced recommendations for human papillomavirus (HPV) vaccination for females aged 10 to 13 years and catch-up vaccination for females aged 14 to 26 years in addition to screening for the prevention of cervical cancer . HPV is The importance of HPV vaccination is highlighted by the World Health Organization’s global strategy to achieve 90-70-90 targets  by 2030 in an attempt to eliminate cervical cancer . As a reVaccination in Belgium is made available through one of three options: reimbursement by the National Institute for Health and Disability Insurance, free of charge through regional community vaccination programs, or full price at pharmacies . HoweverTwo HPV vaccines are currently available as part of the Belgium national reimbursement program: the 2-valent HPV  and the 9-valent HPV  vaccines . The 9vHIn Flanders, in 2010, the 4-valent HPV  vaccine was made available for free in a 3-dose schedule through a regional vaccination program for females in the first year of secondary school (11–12 years old) . In 2014In 2011, the 2vHPV vaccine was made available in the Wallonia-Brussels region through a free school-based program to females in the second year of secondary school  . VaccinaBoth Flanders and the Wallonia-Brussels regions share similar age-standardized incidence rates for cervical cancer . However, the Flanders region has a higher mortality rate from cervical cancer  . In BelgThere is a strong rationale for immunizing females and males against HPV. Although a declining trend in the incidence/prevalence of cervical cancer and other HPV-related diseases has been observed in females with the introduction of HPV vaccinations for girls in many countries, the burden of HPV-related diseases in men remains high, particularly HPV-related anal and oral cancers . AdditioBeginning in September 2019, both the Flanders and the Wallonia-Brussels regions in Belgium have switched to a gender-neutral HPV vaccination (GNV) program , joiningA previously validated and published HPV-type transmission dynamic model simulating the natural history of HPV infections and estimating the cost associated with HPV-related diseases in women has been extended to include all HPV-related diseases in both sexes . HPV infThe age-structured mathematical model  accountsModel input variables of the epidemiological model are divided into demographics, sexual behavior, disease and treatment patterns, and screening. A detailed summary of these parameter groups is provided in Population figures and annual all-cause mortality rates for the general population by sex and age were retrieved from local Belgium-specific data . An overData from France were utilized for sexual behavior variables with the exceptions mentioned below . The annThe degree of sexual mixing among members of different age cohorts and sexual activity groups  were extracted from United States-specific data,  and was Model input variables to simulate the natural history of the disease as well as disease and treatment patterns utilized local Belgium-specific data when available or estimated through model calibration .Belgium-specific data on the incidence of HPV-related diseases were retrieved from previously published data and the Belgian Cancer Registry . Female Annual mortality rates for each HPV-related cancer, stratified by age and stage , are summarized in in situ {CIS}], vaginal cancer [including the precancerous lesions VaIN 1/2/3], vulvar cancer [including the precancerous lesions VIN 1/2/3], anal cancer, genital warts, and head and neck cancer for females; and anal cancer, genital warts, penile cancer, head and neck cancer for males) recognizing their symptoms and seeking treatment, and the proportions who were treated were estimated through the model calibration. Similarly, the proportions of females and males who were treated for genital warts were estimated through the model calibration.The proportions of the populations with HPV-related diseases , and the proportion of females receiving gynecological cancer screening tests at least once every 3 years (53.70%) were calculated based on data from the Belgian Healthcare Knowledge Center (KCE) and a 2007 expert Belgium survey . CervicaThe prophylactic efficacy or the degree of protection offered by the vaccine was based on clinical trial data . The durThe inputs for the economic model included vaccine strategy, vaccine properties, cost parameters, and health-related quality of life.The vaccination schedule was assessed according to the product label, i.e., 2-dose schedule for children under 15 years and 3-dose schedule for children between 15 and 18 years old. Our base-case analyses were conducted according to three different scenarios: 1) in the Flanders region, strategy of GNV vs. FOV with 9vHPV vaccine administered as two doses in ages 11 to 12 years old; 2) in the Wallonia-Brussels region, strategy of GNV with the 9vHPV vaccine vs. FOV with the 2vHPV vaccine, both administered as two doses in ages 12 to 13 years old; and 3) in the national catch-up program, strategy of GNV vs FOV with 9vHPV vaccine administered as two doses in ages 12 to 14 and 3 doses in ages 15 to 18 years old.GNV versus FOV with the 9vHPV vaccine in children ages 11 to 12 years was assessed at a 90% vaccine coverage rate (VCR) in Flanders, reflecting the high coverage in this region . GNV witCost parameters used in the model included cost per episode of care, cost of vaccination, and cost of screening and diagnostic tests; costs were reported in 2017 euros (€). A detailed summary of all costs used in the model is provided in The base model adopted the public prices of vaccines in Belgium .The estimated costs per episode of care of each HPV-related disease, defined as the cost of management from diagnosis to resolution of the case, are summarized in For cervical and vaginal cancers, a 2016 report by the Belgian National Institute for Health and Disability Insurance (INAMI) was used to extract the cost associated with screening (PAP smear) and related office visits, whereas colposcopy and biopsy costs were retrieved from a 2015 update on HPV primary screening by the KCE . Costs fAll costs are from the perspective of the third-party payer .Discount rates of 1.5% for quality-adjusted life years (QALYs) and 3% for costs were applied to the model. These discount rates were in accordance with the Belgian KCE Guidelines for pharmaco-economic evaluation .The health utility values for the Belgian general population and for those with HPV-related diseases were derived from several sources. Health utilities for the Belgian general population by age and sex were based on data from the United Kingdom  and are The model was used to estimate the following health outcome parameters: cumulative incidence of HPV-related diseases associated with HPV types targeted by the 9vHPV vaccine (6/11/16/18/31/33/45/52/58); number of prevented cases of HPV-related diseases (expressed as the cumulative reduction in HPV 6/11/16/18/31/33/45/52/58-related incident cases); number of prevented HPV-related deaths (expressed as the cumulative reduction in HPV 6/11/16/18/31/33/45/52/58-related incident cases). The model was also used to estimate the following economic outcome parameters: cumulative HPV-related disease health care costs; QALYs of the model population; and the incremental cost-effectiveness ratios (ICERs), which are calculated with the quotient: Incremental vaccination costs/Incremental QALYs. Model calculations were performed using the mathematical software package Mathematica®, Version 10.4 .Outcome measures were reported over a time horizon of 100 years because this was an appropriate time frame from which the system approached steady state and most benefits and costs of vaccination could be realized.Validation of the original model has been previously addressed . ConsultThe incidence rates for genital warts; CIN 1/2/3; and cervical, vaginal, vulvar, anal, penile, and head and neck cancers were calibrated.All HPV-related diseases were considered, in accordance with the Belgian Superior Health Council recommendations and other health technology assessment agencies considering HPV vaccination . Data weDeterministic one-way sensitivity analyses were conducted to assess the sensitivity of ICER values to variables that have been shown to be impactful to cost effectiveness. One variable is the VCR, which were assessed at different levels  . The lowProjected onto the population of Flanders, 9vHPV vaccination in both males and females demonstrated greater cumulative reductions in HPV 6/11/16/18/31/33/45/52/58-related diseases relative to FOV over a 100-year period . GNV witAs a result of reductions in the incidence of HPV-related diseases, the model predicts cumulative reduction in HPV 6/11/16/18/31/33/45/52/58-related deaths over 100 years , with thUpon evaluating the economic impact of the associated projected reductions in incidence associated with GNV relative to FOV with the 9vHPV vaccine, the Flanders region is expected to see a 5.9% reduction  in cumulative HPV 6/11/16/18/31/33/45/52/58-related disease costs ; 54.1 anGNV with the 9v HPV vaccine is a cost-effective strategy versus FOV with the 9vHPV vaccine in the Flanders region, with an ICER of €8,062 per QALY. Sensitivity analyses projected that decreasing the VCR improves ICER, with a 50% VCR leading to an ICER of €4,042 per QALY, whereas boosting the VCR to 95% increases the ICER to €8,854 per QALY . DecreasIn the Wallonia-Brussels region, GNV with the 9vHPV was also estimated to provide greater cumulative reductions in HPV6/11/16/18/31/33/45/52/58-related disease compared with FOV with the 2vHPV vaccine . The modThe model also projected the greatest number of averted deaths would be for head and neck cancer , cervical cancer (525), anal cancer , and penile cancer (29) .In the Wallonia-Brussels region, GNV with the 9vHPV vaccine was estimated to result in a 13.4%  reduction in HPV6/11/16/18/31/33/45/52/58-related disease costs after 100 years compared with FOV with the 2vHPV vaccine ; 23.4 anGNV with the 9v HPV vaccine is a cost-effective strategy versus FOV with the 2vHPV vaccine, with an ICER of €4,179 per QALY, well below previously estimated ICERs for FOV conducted in 2007  . SensitiCatch-up HPV vaccination with the 9vHPV vaccine through the Belgian national program in both males and females demonstrated greater cumulative reductions in HPV 6/11/16/18/31/33/45/52/58-related diseases relative to FOV with the 9vHPV vaccine over a 100-year period , reducinThe model estimated that the greatest number of averted HPV-related cancer deaths were for head and neck cancer  and cervical cancer (594 [5.8%]) after 100 years of catch-up GNV relative to FOV with the 9vHPV vaccine .In accordance with the projected reductions in HPV-related disease burdens associated with the catch-up GNV relative to FOV with the 9vHPV vaccine, the model projected a 7.1% reduction  in HPV 6/11/16/18/31/33/45/52/58-related disease costs over a 100-year period . IncludiCatch-up GNV with the 9v HPV vaccine is a cost-effective strategy versus FOV with the 9vHPV vaccine, with an ICER of €6,127 per QALY. Sensitivity analyses projected that decreasing the VCR improves ICER, with a 5% VCR leading to an ICER of €2,866 per QALY, whereas boosting the VCR to 70% increases the ICER to €8,990 per QALY . DecreasThis analysis included evaluation of the health impact  and cost-effectiveness of HPV GNV with 9vHPV vaccine on HPV-related diseases based on recommendations from the Superior Health Council of Belgium as well as other health technology assessment agencies . Using aAlthough there is no official cost-effectiveness threshold in Belgium,  a threshin situ], vaginal cancer [including VaIN 1/2/3], vulvar cancer [including VIN 1/2/3], anal cancer, head and neck cancer, and penile cancer), although sensitivity analyses show that GNV with the 9vHPV is still cost-effective relative to FOV with either the 2vHPV or the 9vHPV vaccines when only considering indications in the label. As per label, HPV vaccines are not indicated for the prevention of RRP, head and neck cancer, and penile cancer  of penile intraepithelial neoplasia occurred among males in the 9vHPV arm versus four cases  in the control arm .The public health impact and cost-effectiveness of GNV with the 9vHPV vaccine has been previously assessed using different transmission dynamic models calibrated to other European settings, including Austria, Italy, Germany, Spain, and France . SimilarSeveral limitations are associated with this modeling approach. The model did not assess possible changes to cervical cancer screening methods over the course of the 100 years. The model also did not assess indirect costs, the impact on fertility, as well as neonatal morbidity and mortality due to cervical lesions. In addition, we did not conduct multivariate deterministic sensitivity analyses or probabilistic sensitivity analyses. Currently, disease transmission among populations at high risk, such as MSM and HIV populations, is not included in the model, which may result in underestimating the added benefit of male vaccination compared with an FOV strategy. The presence of groups at high risk can decrease the sensitivity to parameter values, which can lead to relatively small changes in the prevalence of infection despite large changes in transmission rates and infectious periods . NeverthCompared with an FOV strategy, a GNV strategy with the 9vHPV vaccine is projected to provide substantial public health  and economic benefits in Belgium when incorporated into the national catch-up program, as well as independent regional vaccination programs in Flanders and Wallonia-Brussels. As a result, a GNV strategy will provide greater benefit in the prevention of HPV-related diseases in the entire male population and additional benefits in females. GNV with the 9vHPV vaccine may also be considered a cost-effective strategy relative to FOV with a broad range of vaccine coverage rates in the regional and national vaccination programs. Future research will look to expand analyses to include populations at high risk, such as MSM, which will truly capture the full impact of a GNV strategy.


However, there is scant published literature on the interconnection of patient related factors that impact on optimal management of diabetes in resource-limited settings such as Kenya.a hospital based cross-sectional survey involving 270 freely consenting adult diabetics investigated the interconnection between diabetes knowledge, medication adherence and glycemic control. Data on the patient´s knowledge and medication adherence was collected using validated tools whilst glycemic control was evaluated using the patients' glycated haemoglobin values. Data analysis was carried out using STATA version 13 statistical software, employing the chi square test for association and simple linear regression for prediction, with p ≤0.05 considered significant.participants´ level of knowledge on diabetes was significantly associated with the academic achievement (p=0.001), while their medication adherence was significantly associated with family support (p=0.001) and duration of disease since diagnosis (p=0.019). On linear regression, family support occasioned by the nature of participants’ household setup, had a strong positive correlation [r=0.99 (CI 0.60-1.00)] with optimal glycemic control.family support is an important determinant of medication adherence and optimal glycemic control among diabetic patients. Clinicians should team up with family members of diabetic patients for optimal glycemic control. Scientific background: the optimal management of chronic illnesses such as diabetes is influenced by environmental, patient-related, health system and disease-related factors  with optimal glycemic control. The disease duration had a weak negative correlation [r=-0.22 (CI -0.97-0.94)] to optimal glycemic control. Notably, the nature of household setup posted a strong positive correlation [r=0.99 (CI 0.60-1.00)] to optimal glycemic control among study participants.et al. [The number of participants with optimal glycemic control was higher than that observed in similar studies; for instance a study done in Kenyatta National Hospital, the largest referral hospital in Kenya, where optimal glycemic control was recorded by 36.9% of the sample population . This obet al.  revealedThe participants´ level of income and disease duration were positively correlated with medication adherence. High-income earners (KES+50000) and those with longer disease duration 11-15 years) dominated the MMAS-8 score > 75% bracket. Participants living with their families recorded higher adherence scores than those living alone. These findings are consistent with those reported from related studies -15 years. ProbablThe weak correlation of participants´ education level with optimal glycemic control, implied that whilst patient knowledge is essential, there may be other intervening factors affecting actual glycemic control. Factors that promote self-monitoring, diet and exercise adherence must also be taken into account . The weaLimitations: while we appreciate that the strength of the present study is use of validated data collection tools, a potential limitation is the inclusion of patients primarily from a referral hospital in the coastal region of Kenya as a representation of the general Kenyan population. Additionally, as common with cross-sectional studies involving direct interviews, patients could have understated or overstated their experiences.Generalisability: the generalisability of the study findings elsewhere in Kenya warrants further investigation. Notably, the study population has unique cultural practices and norms compared to the rest of the country.Patients´ literacy level, a determinant for knowledge showed a weak positive correlation to optimal glycemic control among the study participants. Living within a family setup was significantly associated with better treatment adherence and positively correlated to optimal glycemic control. Involvement of family members in the diabetic patients´ hospital visits and treatment plans should be encouraged.Successful management of diabetes mellitus is influenced by a multitude of factors; environmental, health system, patient-related and disease-related factors;Patient related factors form the backbone for optimal management of diabetes mellitus;Among these patient related factors are diabetes knowledge, medication adherence, economic and psychosocial factors.The current study elucidates the interplay of factors between patient knowledge, medication adherence and glycemic control among adult diabetics in a low resource setting;Education level, a determinant for patient knowledge, showed a weak positive correlation to optimal glycemic control;Family support, a determinant for medication adherence, showed a strong positive correlation to optimal glycemic control.


INK4a immunostaining (p16) in triaging HPV-positive self-samples, focusing on HPV-positive, cytology-negative (HPCN) women.Self-sampling for human papillomavirus (HPV) testing is a feasible option to improve the cervical screening coverage. However, an ideal triage method for HPV-positive self-samples does not yet exist. The aim of this study was to explore the utility of HPV genotyping and p16A total of 73,699 women were screened in a cervical screening project in China via SeqHPV assay on self-samples. HPV-positive women were called-back and collected cervical sample for p16 immunostaining and liquid-based cytology, those who met any result of HPV16/18+ or visual inspection with acetic acid (VIA) + or p16+ were referred for colposcopy, and HPCN women with adequate data on p16 and pathology were analyzed. A triage strategy was considered acceptable if the negative predictive value (NPV) for cervical intraepithelial neoplasia 3 or worse (CIN3+) was 98% or more, combined with an improvement of sensitivity and specificity for CIN2+/CIN3+ in reference to the comparator, being HPV16/18 + .A total of 2731 HPCN women aged 30–64 years were enrolled, 136 (5.0%) CIN2+ and 53 (1.9%) CIN3+ were detected. Five triage strategies met the criteria: p16+; HPV16/33+; ‘HPV16+ or HPV33/58/31/35+&p16+’; ‘HPV16/33+ or HPV58/31/35+&p16+’; HPV16/18/31/33/45/52/58 + & p16+. These strategies required less or similar colposcopy referrals, and less colposcopies to detected one case of CIN2+/CIN3+, achieving favorable false positive (negative) rates to the comparator. Among them, p16 staining detected 83.1% (79.2%) of underlying CIN2 + (CIN3+) in HPCN women. Moreover, three triage strategies were favorable in sensitivity and/or specificity to the ‘HPV16/33+’ strategy: p16+; ‘HPV16+ or HPV33/58/31/35 + &p16+’; HPV16/18/31/33/45/52/58 + &p16 + .Genotyping for HPV16/33 could be utilized to optimize the management of HPCN women. Moreover, p16 immunostaining, either alone or combined with extended genotypes, is more effective than HPV genotypes alone in the triage of HPCN women.The online version contains supplementary material available at 10.1186/s12879-021-06109-4. High-risk human papillomavirus (hrHPV) is assumed to be an indispensable cause of cervical cancers , thus HPThe optimal management of HPCN women remains a huge challenge. Triage options for HPCN women including immediate referral to colposcopy, repeat cytology/HPV within 12 months, or triaging with genotypes, viral loads, HPV E6/E7 protein, or other biomarkers . HoweverHPV genotype is a potent predictor of the risk for oncogenic progression . HoweverINK4a(p16) immunostaining indicates HPV-induced cell-cycle deregulation and predicts the transformation process and future progression of HPV infection [Genotypes can stratify risk for cervical diseases among HPCN results , but cannfection , thus manfection . p16 immDue to the convenience and easy-to-operate of HPV-based self-sampling, it could serve as a tool to increase the coverage of cervical screening in China. Moreover, both our and other clinical trials have confirmed that PCR-based HPV testing has similar accuracy on self-samples and clinician-samples , 19–21. This prospective observational study was nested into a large population-based cervical cancer screening program from Nov 2018 to Dec 2019, which was organized by a prefecture city in Henan Province, China. No large-scale cervical screening program was organized previously in the city. A total of 73,699 non-pregnant women aged 30–64 years, without history of cervical neoplasia or cancer, without uterine or cervical resection were screened via SeqHPV assay as the primary screening on self-collected samples. Women with HPV-negative result were advised to regular screening by HPV assay after 3 years. While HPV-positive women were called-back for collection of a cervical sample by local gynecologists prior to their colposcopy or visual inspection under acetic acid (VIA). The cervical sample was used for p16 immunostaining and liquid-based cytology (LBC), and LBC was used for research purpose but not for patient care Fig. . HPV-posAfter registration for participation via the mobile device, each participant offered a cervicovaginal specimen collected with a nylon conical brush by herself, following user instructions on a printed guide. Cervicovaginal samples were collected by participants themselves and placed on the solid media transport card . Subsequently, self-collected samples were prepared for Seq HPV assay , an HPV genotyping assay using multiplex PCR and next generation sequencing . Seq HPVINK4a immunostaining, which was carried out by PathCIN®p16INK4a automatic immunocytochemistry system  via using a p16INK4a monoclonal antibody (clone sy-a01) at a dilution of 1:400. Moreover, ten samples can be detected on one slide at the same time, which saves cost and qualifies p16 immunostaining with a high-throughput capability. The residual specimens after p16 staining were used for LBC test. p16 staining and LBC slides were independently read by two senior cytologists in the Peking University Shenzhen Hospital blinded to other data except HPV positivity, and discordant results were resolved via consensus review. One or more cervical epithelial cell with nuclear and/or cytoplasmic stained brownish-yellow were classified as positive regardless of their morphology [When HPV-positive women returned for their secondary visit, the local clinicians, who were trained by the research team, used a cyto-brush to collect a cervical specimen. The sample was placed in the preservation solution . Thin-layer cell slides were prepared for p16rphology . Slides rphology , and atyWomen that underwent colposcopy received direct and random biopsies by quadrant , or endoThe following triage algorithms were evaluated:(i) Single test: HPV16+; HPV16/18+; HPV16/33+; HPV16/33/58+; HPV16/33/58/31+; HPV16/33/58/31/35+; HPV16/18/31/33/45/52/58+; p16 + .(ii) HPV genotypes or p16 staining: HPV16/18+ or p16+; HPV16+ or p16+; HPV16/33+ or p16+; HPV16/33/58+ or p16+; HPV16/33/58/31+ or p16+; HPV16/33/58/31/35+ or p16+; HPV16/18/31/33/45/52/58+ or p16 + .(iii) HPV genotypes & p16 staining: ‘HPV16/33+ or HPV58/31/35+&p16+’; ‘HPV16+ or HPV33/58/31/35+&p16+’; HPV16/33/58/31/35 + &p16+; HPV16/18/31/33/45/52/58 + &p16 + .P < 0.05 was assumed to be statistically significant.CIN2+/CIN3+ were the main outcomes. For individual genotype, a hierarchical ordering was developed based on sequentially maximizing the positive predictive value (PPV) for the next HPV genotype after excluding women with multiple infections with types higher in the hierarchy , 26. To n = 243), and those without p16 (n = 1402) or cytology result (n = 2) were excluded. Moving forward, those who had abnormal cytology (n = 1048) and those without pathologic results (n = 417) were excluded, leaving a pool of 2731 HPCN women included in the final analysis , HPV52 (16.7%), HPV58 (14.0%), HPV51 (11.1%), and HPV18 (10.5%). The prevalence of multiple infections was 21.5% (588/2731). In the CIN2+ group, the most prevalent hrHPV genotypes were HPV16 (62.5%), HPV58(15.4%), HPV52(13.2%), HPV33(7.4%), HPV31(6.6%) and HPV18(6.6%); and 27.9% (38/136) of women were infected with multiple genotypes. The genotypes were divided into three strata  according to their hierarchical risks for CIN2+ Table .P < 0.0001; Table The p16 positivity rates among three genotype strata were 40.2%(HPV16/33), 22.9% (HPV58/31/35), and 18.8% (other 9 types) respectively, increasing with the elevation of hierarchical types (Strategy 7 (HPV16/18+) showed a sensitivity of 92.5% (81.8–97.9%) and a specificity of 68.7% (66.9–70.4%) for CIN3+ (Table Strategy 1 (p16+); Strategy 3 (HPV16/33+); Strategy 16 (HPV16+ or HPV33/58/31/35 + &p16+); Strategy 17 (HPV16/33+ or HPV58/31/35 + &p16+); Strategy 15 (HPV16/18/31/33/45/52/58 + &p16+), see Tables Strategy 1 detected 83.1% (79.2%) of underlying CIN2+ (CIN3+) among HPCN women. Strategy 1 and 16 were favorable in specificity and sensitivity for CIN2+/CIN3+. Strategy 3 and Strategy 15 revealed a favorable specificity without a reduction in sensitivity. In addition, Strategy 17 was favorable in sensitivity and similar in specificity for detecting CIN2+/CIN3+ to the comparator (Tables Strategy 2 (HPV16+) showed a lower sensitivity  for CIN2+ to the comparator. Strategy 18 (HPV16/33/58/31/35+ &p16+) was less sensitive to the comparator for CIN3+ . The other strategies showed an improved or comparable sensitivity for detecting CIN2+/CIN3+ at the expense of a lower specificity to the comparator (Tables egy 7 HPV6/18+ shoStrategy 3 (HPV16/33+) to the other four strategies, and found that the Strategy 1, Strategy 16 and Strategy 15 were favorable in sensitivity and/or specificity to Strategy 3. Whereas the Strategy 17 had a higher sensitivity but a lower specificity than Strategy 3 for detecting CIN2+  required a CRR of 32.5%, and NRNDs of 9.5/18.1 for CIN2+/CIN3+, resulting in an FPR/FNR of 30.6%/31.6% for CIN2+  for CIN3+, but a less NRND (8.1 vs 9.5) for CIN2+, leading to a better FNR of 19.9% for CIN2+ to the comparator. Triage with the other four optimal strategies above  provided lower CRRs , less NRNDs , accompanied by better FPRs (range 18.2–25.9% vs 30.6%) and better FNRs than Strategy 7 for CIN2+   p16+; 2) HPV16/33+; 3) HPV16+ or HPV33/58/31/35 + &p16+; 4) HPV16/33+ or HPV58/31/35 + &p16+; 5) HPV16/18/31/33/45/ 52/58+ &p16+. The high NPV and improved sensitivity and/or specificity of these strategies would safely allow less frequent repetitions of screening for low-risk women [Currently, there still lacks an ideal triage option and associated evidences for HPV-positive self-samples. Cytology is a common triage method, the optimal management of HPCN women is controversial. Thus, the performance of multiple strategies based on extended genotyping and p16sk women , but alsA recent meta-analysis showed that an HPCN women had on average a 6.4% risk of CIN3+ within 5 years, but her 1-year risk of CIN3+ was less than 4%, which was proposed as the threshold for immediate referral to colposcopy in the most recent ASCCP guidelines . SimilarInterestingly, HPV assays provide individual genotype information help clarify the type-specific risks , 13, 15,However, we furtherly found that strategies incorporating p16 staining could achieve a preferable accuracy either alone or combined with genotypes than strategies derived from genotyping alone (the ‘HPV16/18+’ and the ‘HPV16/33+’) in screening. The reasons may be that management by genotyping alone is based solely on risk of cervical precancer or cancer without knowledge about what’s occurring within the cells [Although the utility of p16 staining may somewhat increase the costs of screening tests relative to pure genotyping as the triage, it could improve the colposcopy referral and reduce the short-term follow-up burden, leading to the early detection of cervical precancers, therefore it may be a cost-effective and efficient strategy in the long run. Moreover, due to the low-cost of HPV genotyping assay and the high-throughput and easy-to-interpret results of p16 staining utilized in our study, they are affordable and likely to serve as a potential candidate for large cervical screening programs . Notablyp = 0.005) [p16/Ki-67 dual staining might improve the specificity of p16 staining and attracts attention recently . A previ= 0.005) . Herein,INK4a kits, in triaging HPCN women. In addition, recently several studies have analyzed genotype distribution and type-specific risks in HPCN women in China [Although several previous studies have analyzed the role of p16 immunostaining in the early detection of cervical cancer , few robin China , to our in China . Thus thIn summary, genotyping for HPV16/33 could be utilized to optimize the management of HPCN women. Moreover, p16 immunostaining, either alone or combined with extended genotypes, is more effective than HPV genotypes alone in the triage of HPCN women. Hopefully, these findings provide new insights into optimizing the management of HPCN women, and offer a potent evidence for the implementation of HPV-based screening on self-samples.Additional file 1: Table S1. Positive Predictive Value (PPV) for CIN2+ and CIN3+. Table S2. p16 positivity stratified by HPV genotypes and histologic grades . Fig. S1. Management protocol of HPV-positive women. VIA, visual inspection under acetic acid; LBC, liquid-based cytology. Genotyping for HPV16/18, VIA, and p16 immunostaining were used for triage sequentially. Fig. S2. p16 immunostaining, (A) positive, one or more cervical epithelial cells with nuclear and/or cytoplasmic stained brownish or yellow were defined as positive regardless of their morphology. (B) negative.


We developed a stochastic optimization technology based on a COVID-19 transmission dynamics model to determine optimal pathways from lockdown toward reopening with different scales and speeds of mass vaccine rollout in order to maximize social economical activities while not overwhelming the health system capacity in general, hospitalization beds, and intensive care units in particular. We used the Province of Ontario, Canada as a case study to demonstrate the methodology and the optimal decision trees; but our method and algorithm are generic and can be adapted to other settings. Our model framework and optimization strategies take into account the likely range of social contacts during different phases of a gradual reopening process and consider the uncertainties of these contact rates due to variations of individual behaviors and compliance. The results show that, without a mass vaccination rollout, there would be multiple optimal pathways should this strategy be adopted right after the Province's lockdown and stay-at-home order; however, once reopening has started earlier than the timing determined in the optimal pathway, an optimal pathway with similar constraints no longer exists, and sub-optimal pathways with increased demand for intensive care units can be found, but the choice is limited and the pathway is narrow. We also simulated the situation when the reopening starts after the mass vaccination has been rolled out, and we concluded that optimal pathways toward near pre-pandemic activity level is feasible given an accelerated vaccination rollout plan, with the final activity level being determined by the vaccine coverage and the transmissibility of the dominating strain. COVID-19 has had tremendous impacts on public health and the economy globally. Non-pharmaceutical interventions (NPIs), including school and business closures, have been implemented in an effort to reduce transmission of COVID-19 and to protect those most vulnerable to severe disease (The circulation of more transmissible variants of concern (VOCs) has further made the identification of safe reopening strategies more complex. While efficacious vaccines exist and are being administered, most nations are in the situation where the wide rollout of vaccination among the general population has not been achieved; hence, most individuals remain susceptible to infection and controlling virus transmission  while more transmissible variants are actively circulating still poses a real challenge. In this light, the task of identifying smart reopening strategies provides an opportunity for mathematical modeling to assist and inform the reopening decision-making process through means of evidence synthesis.In the Canadian province of Ontario, the first wave of COVID-19 occurred during the Winter and Spring months of 2020 and was controlled through the implementation of NPIs including so-called lockdown and stay-at-home measures . PreviouHere we utilize the established optimization framework , updatedThe province of Ontario has implemented a variety of control measures with varying intensity in response to the circulation of SARS-CoV-2 virus in the population, as well as relaxations to partially resume social and economic activities. A series of physical distancing control measures were implemented in March 2020 to mitigate the first wave in the province. These restrictions began to be lifted in the province's staged reopening process from mid-May until October 10, 2020. At this time on October 10, 2020, control measures to mitigate the second wave were implemented in select hotspots in the province. On November 23, 2020, the enhancement of control measures had commenced, and this date marked the second so-called lockdown of Toronto and Peel. The modeling of these distinct phases of varying control measures (of first two waves) in terms of activity levels was conducted in prior transmission modeling works and the key event timelines discussed , 6. The c per day during this time period.We identified optimal strategies to increase the contact rate in the Ontario population toward a mass vaccination rollout, should the optimal strategy be initiated on February 14, 2021, March 14, 2021, May 16, 2021 and June 14, 2021. We accomplished this by using a stochastic optimization technology, based on a compartmental model of COVID-19 transmission dynamics  (1) are between 14 days and 16 days, the length of de-escalation phase 2 (ϵ2) varies from approximately 24 days to 27 days, depending on the scenario ( (days)] 11. The scenario . Critica0 = 24.47 (days)]. Details of the phase transition dates and the scenario tree are shown in The optimal reopening strategies in terms of the contact rates, projected number of ward beds, ICU beds shown in 0, ϵ1, ϵ2 = 7) despite the modeled uncertainty in the contact rates.The de-escalation phase duration, corresponding activity levels and date of initiation according to each scenario are presented in The contact rate according to each scenario is shown in The results from this simulation are shown in We also considered the emergence of B.1.617 (Delta) in our optimal pathways since June 1, 2021, which has a higher estimated transmission rate shown in We have utilized a disease transmission model and stochastic optimization model to identify reopening strategies in the province of Ontario, Canada from February 14, 2021 or March 14, 2021 that will minimize the loss of social contacts while the health system is not overwhelmed. Third, fourth and fifth sets of reopening strategies were identified from May 16, 2021 and from June 14, 2021 which considered the evolution of the outbreak situation and vaccination program in Ontario as well as the health system capacity. The third, fourth and fifth sets of simulations relaxed the constraint that the ICU beds for COVID-19 patients must be below 500 during cost-evaluation. In each simulation, we have considered a staged reopening approach composed of distinct de-escalation stages.The first simulation shows that if the public health interventions such as contact tracing and case isolation remain at their estimated February 14, 2021-levels, and if this optimal strategy was adopted on February 14, 2021, the relaxation of physical distancing interventions could have been achieved without exceeding the ICU capacity until the vaccine became available to a broader range of individuals in Ontario (Phase 2 of Ontario's vaccination plan) . This reShould this optimal strategy not be adopted at the beginning of lifting the lockdown, as was the case in Ontario, the choice of optimal pathways was much more limited but still feasible. The simulation based on the assumption that Ontario considered adopting this stochastic optimization technology on March 14, 2021 when the Province has advanced from lockdown for almost 1 month. Our simulation shows that there was no pathway toward mass vaccination taking effect in May 2021 if it is required that the activity level not reduced from its current level and if the ICU beds were limited to 350. We have run additional simulations to identify potential sub-optimal pathways when the COVID-19 patients needing ICU beds is increased to 500, and the maximal level of activities is below the estimated activity levels during Modified Stage 2 . Our simAs the ICU capacity for COVID-19 patients was exceeded in early April 2021, we ran a third set of simulations with strategy initiation on May 16, 2021 to reflect these new circumstances, also including the increased vaccine distribution and circulation of VOCs. We identified reopening strategies that enabled the contact rate from those estimated during lockdown to be increased to a final rate between 9.5 and 10 contacts per day per individual by June 7, 2021 . CriticaDespite modeled uncertainties in the contact rates for each of the de-escalation phases, the optimal timings identified in each scenario were similar for time of strategy initiation on February 14, March 14, May 16, and June 14, 2021 . In the The optimization framework established in our prior work  and exteWe have expanded our previously established study to incorporate the circulation of the VOCs circulating in Ontario, and parameterized the transmission model according to the current circumstances (including ongoing vaccination efforts and circulation of VOCs), in order to identify optimal pathways out of the restrictions starting on February 14, 2021, March 14, 2021, May 16, 2021 and June 14, 2021 while considering the health system capacity. We note the generality of this method; the transmission model can be parameterized according to different regions and the contact levels for reopening phases may also be altered based on regional constraints. In this sense, we have developed and illustrated a methodology with the intention that it can be utilized to identify reopening measures in different regions. We further have demonstrated the practical usage of this framework through this retrospective analysis, which allowed for the identification of the updated optimal pathways according to evolving circumstances.The original contributions presented in the study are included in the article/YX, SC, AA, and JW: conceived the experiment. YX, SC, YZ, ZM, AA, and JW: run the experiment and analyzed the data. YX, SC, YZ, ZM, NB, AA, and JW: drafted and revised the manuscript. All authors contributed to the article and approved the submitted version.This project has been partially supported by the Canadian Institute of Health Research (CIHR) 2019 Novel Coronavirus (COVID-19) rapid research program.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.


The most used methodologies for HPV genotyping in Tunisian studies are based on hybridization that are limited to a restricted number of HPV types and to a lack of specificity and sensitivity for same types. Recently, Next-Generation sequencing (NGS) technology has been efficiently used for HPV genotyping. In this work we designed and validated a sensitive genotyping method based on nested PCR followed by NGS. Eighty-six samples were tested for the validation of an HPV genotyping assay based on Nested-PCR followed by NGS. These include, 43 references plasmids and 43 positive HPV clinical cervical specimens previously evaluated with the conventional genotyping method: Reverse Line Hybridization (RLH). Results of genotyping using NGS were compared to those of RLH. The analytical sensitivity of the NGS assay was 1GE/μl per sample. The NGS allowed the detection of all HPV types presented in references plasmids. On the clinical samples, a total of 19 HPV types were detected versus 14 types using RLH. Besides the identification of more HPV types in multiple infection (6 types for NGS versus 4 for RLH), NGS allowed the identification of HPV types that were not detected by RLH. In addition, the NGS assay detected newly HPV types that were not described in Tunisia so far: HPV81, HPV43, HPV74, and HPV62. The high sensitivity and specificity of NGS for HPV genotyping in addition to the identification of new HPV types may justify the use of such technique to provide with high accuracy the profile of circulating types in epidemiological studies. In Tunisia, it is the fourth cause of mortality in women with 185 deaths each year .High risk Human Papillomavirus (HR-HPV) infection is known to be the leading cause of Cervical cancer (CC) , 2. CC rTwo hundred HPV genotypes have been characterized to date and more than 40 types have been reported to infect the female genital tract , 3. TherIn a previous study, and in order to evaluate HPV types distribution in Tunisian women, we developed a nested PCR based on detection of HPV L1 using PGMY and GP5+/GP6+ primers, followed by Reverse Line Hybridization (RLH) targeting 31 genotypes . This teThe study (study reference: (2014/2A/ONMNE)) was approved by the Ethical committee of Institut Pasteur de Tunis, subscribed on the office of human research protections under the reference IORG001040, and conducted with good clinical practice, ensuring confidentiality and anonymity.A total of 86 samples were tested for the validation of the HPV genotyping assay based on Nested-PCR followed by NGS. These include, 43 references plasmids (WHO HPV proficiency panel) and 43 positive HPV clinical cervical specimens.To validate the sensitivity and specificity of the Miseq genotyping approach here reported, 43 samples of the 2014 WHO proficiency study panel were used as reference samples. This panel was composed of purified plasmid DNA in which genomic DNA of different HPV types was cloned and diluted in a background of human placental DNA as previously described [The forty-three HPV positive samples identified in an earlier study that examined the prevalence of genital HPV infection in the Region of Grand Tunis  were useDNA was extracted from a 200μl aliquot of the suspended cell samples. Extraction and quality control by betaglobin PCR, are as previously described . All samAll the clinical samples were typed during a previous study using RLH as previously described . BrieflyFirst PCR amplification. The PGMY primers [ primers  were use primers . Then thLibraries preparation. The PGMY PCR amplicons were used in a second PCR using custom designed primers. These primers contain the GP5+/6+ sequence and additional 10 bases Illumina adapter sequences (P5/P7). The sequences of primers are represented in The PCR reaction was conducted in 25 μl mixture contained 2.5μl of first PCR product, 12.5μl Kapa Mix . PCR cycling parameters were composed of a 3 minutes of initial denaturation at 95°C, followed by 14 amplifications of 30 seconds at 94°C, 1 minute at 55°C and 1 minute at 72, and a final extension step for 5 minutes at 72°C.Different specific indexes were added to the generated amplicons. The size of the PCR products (~200 pb) was evaluated with the 2100 Bio analyzer system . They were purified using the Agent court® Apure® XP beads  following the manufacturer’s instructions. After purification, 96 different primer indexes, using the NextEra XT Index Kit, were added to the end of HPV amplicons. Each amplicon had a different index, which allowed the pooling of all the samples together.Libraries were purified then quantified using Real Time PCR. Pooling was performed at equimolar ratios (4nM) to yield one sequencing sample. In preparation for cluster generation and sequencing, pooled libraries were denatured with NaOH, diluted with hybridization buffer, and then heat denatured before MiSeq sequencing. The sequencing was performed using the Miseq V2 Kit .Data analysis. Specific reads of each sample were identified according to their respective corresponding index. Adaptor sequences and primers were trimmed using Trimmomatic 0.33 tool [http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The cut-off of read depth was fixed to 30 [https://github.com/trinityrnaseq/trinity). We processed only high quality reads having at least 80% of base pairs with base calling accuracy of 99.9%and used stringent assembly parameters in the Trinity assembler..33 tool . Qualityed to 30 . Good seed to 30 . Only unhttp://pave.niaid.nih.gov)   .HPV genotype frequencies were calculated by simple counts and percentages. Association between HPV infections and read numbers was evaluated using chi-squared test. A p-value <0.05 was considered statistically significant.http://www.rstudio.com/).The agreement between RLH and NGS was calculated using the Cohen kappa test, interpreted as perfect agreement (κ > 0.81), substantial agreement (κ = 0.61–0.80), moderate agreement (κ = 0.41–0.60), fair agreement (κ = 0.21–0.40), slight agreement (κ = 0.01–0.20) or poor agreement (κ = 0.21–0.40). Statistical counts and tests were performed using R Studio version 1.2.5033 (−3) , ranging between 137 and 180, while the modal length of the reads was 160 nt but without significant differences among samples with different HPV types, nor between samples with HR-HPV and LR-HPV, nor between single and multiple infections. Meanwhile, the HPV aligned reads in HPV positive samples  was different from those of HPV negative (maximum: 6). The greatest number of reads was obtained in multiple infections (HPV types >1) (p<10−3) .To confirm the detected HPV types, a cut-off of 30 reads was used. HPV types in the WHO panel were correctly detected and typed with NGS at different DNA amounts (5 to 2500 GE/5μL). The negative control of this panel was also identified as negative (Panel C). HPV 18 was detected at the level of 5GE/5μl and HPV16 at the level of 25 GE/5μl. All samples showed complete concordance for all types, even with multiple infections containing up to 4 genotypes. No other HPV types from the plasmids were detected .Forty-three HPV positive clinical samples were genotyped using the designed NGS protocol. These samples were firstly genotyped by RLH protocol in a previous study. The results showed that 12 of the 43 samples (4%) had unknown HPV, and a total of 14 types were identified .Using the designed NGS genotyping assay, a cut-off of 30 reads was used to confirm the identified HPV type in each sample. As results, all samples were typed and a total of 19 types were identified in addition to the detection of new HPV types; HPV81, HPV43, HPV74, and HPV62 . Single Using Cohen’s Kappa test, a perfect agreement between NGS and RLH was observed . Among the 29 cases of single infections, NGS genotyping results were in perfect agreement with the 24 samples genotyped by RLH. Data are summarized in RLH and NGS methodology have both detected HPV16, HPV52, HPV6, HPV40, HPV70, HPV59, HPV55, HPV39, HPV31, HPV53, HPV68 types . NGS allThe table below summarizes the differences between the 2 genotyping assays .In this study, HPV genotyping was designed using a Nested PCR with PGMY and GP5 +/ 6+ followed by Miseq sequencing (Illumina platform). PGMY/GP+ had greater sensitivity and broader coverage of HPV genotypes compared to PYGMY alone, GP+ alone, MY11/09 (MY) alone or MY/GP –22.The choice of the Illumina platform was based on the low number of read errors that can be generated (<0.4%), as well as its ability to generate short-reads that are more appropriate for HPV analysis. In addition, the preparation of the libraries for the Miseq platform did not require a large amount of DNA (4nM) .Setting the NGS cut-off to 30 reads depth was based on recommendations from others studies . For datIn order to increase the throughput and reduce the cost of the NGS, a double indexing method was developed; it consisted in using primers with Miseq adaptor and indexes for libraries construction. This allowed genotyping of 86 samples  in a single sequencing reaction. There are infinite possibilities of multiplexing since several index kits are available to combine indexes during libraries constructions. The study carried out by Xin Yi et al. (2014) [In this study, the results showed that NGS identified higher HPV numbers in the co-infections than RLH. Thus, the detection of multiple HR-HPV infection has become a key issue in the development of cervical lesions and the epidemiological status of the population by efficient genotyping method. In fact, the underestimation of the number of HPV types in patients who developed cervical lesions could underestimate the effect of multiple infections as a risk factor for persistent infections in healthy young women. Different studies have reported that multiple HR-HPV acted synergistically in cervical carcinogenesis , 25, andUsing NGS we identified new LR-HPV types that were not identified by RLH in the previous Tunisian study : HPV81, The advantage of the detection of new types using NGS has been reported in other studies; Barzon L et al.  and ArroNGS identified novel LR-HPV  that are considered as rare HPV types  and limiThis is the first study of HPV genotyping using NGS in Tunisia to our knowledge. This approach was considerably more sensitive than the classical reverse line hybridization method. It was also able to identify novel HPV types that were not detected by RLH or other related methods in Tunisia. This can justify the use of such technique for an accurate assessment of the circulating genotypes for a better epidemiological apprehension. Following on this study, the developed NGS protocol will be used for HPV genotyping of all the clinical samples collected in the frame of an epidemiological study in Tunisia, variants evaluation, and phylogenetic analysis.S1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


CD8+ T cells, which play a vital role in response to adaptive immunity, are closely related to the immunization responses to kill tumor cells. Understanding the effects exerted by tumor-infiltrated CD8+ T cells in HPV+ and HPV- head and neck squamous cell carcinoma (HNSCC) patients is critical for predicting their prognosis as well as their responses towards immunization-related therapy.HNSCC single cell transcriptome was used to screen for differentially expressed genes (DEGs) based on CD8+ T cells. A gene signature associated with CD8+ T cells was built and verified with the cancer genome atlas dataset with a view to predicting the prognosis of HNSCC patients. Risk scores were calculated for HNSCC cases and categorized into either high- or low-risk cohorts. The prognosis-correlated data of the risk scores were analyzed by using Kaplan-Meier survival curves and multi-variate Cox regression plots. In addition, the possibility of using the genetic profiles to predict responses toward immunization-related therapy was explored.From the DEGs screened from the sequencing of single-cell RNA, a gene signature of 4 genes  were identified. It was seen that these genes could predict overall survival in HPV+ HNSCC patients. In addition, high- and low-risk HPV+ HNSCC patients showed marked differences in their CD8+ T-cell infiltration due to immunization when clinical characteristics were taken into consideration. This correlated with their immunization therapy responses.Our work provides insights into explaining the restricted responses of current immunization checkpoint inhibiting substances in HPV+ HNSCC patients. A novel genetic signature to predict the prognosis and immunization-correlated therapeutic responses is presented. This will provide potential new therapeutic opportunities for HPV+ HNSCC patients. Head and neck squamous cell carcinoma (HNSCC) refers to cancer of the oral cavity, oropharynx and larynx, and is the sixth commonest carcinoma globally . More thvia malignant conversion due to HPV infection  or nfection , 8. Thernfection . Howevernfection . Distincnfection . A higheIn the present study, we attempted to elucidate the correlation between the HPV state and immunization environment-related factors by using a multicenter database. A single-cell RNA sequencing dataset was used to assess the various subpopulations of immune cells and particular genes that may differ in HPV+ and HPV- HNSCC patients. Using a combination of RNA-seq data from a large number of HNSCC cases and their corresponding clinical information, a gene signature for tumor-infiltrated CD8+ T cells was established using multiple machine learning algorithms. This risk-associated gene signature was verified using the gene expression profiles and clinically associated information from an independent cancer genome atlas (TCGA) provisional dataset. The genetic signature obtained may provide future targets for increasing our knowledge of the mechanisms that govern HPV+ and HPV- HNSCC. This study may also increase immunization checkpoint blockade therapy efficacy with respect to treatment of this disease.http://timer.comp-genomics.org/) was utilized to explore the relationship between tumor infiltration of CD8+ T cells and the prognosis of HNSCC in patients   (on.html) . The PCAhttps://portal.gdc.carcinoma.gov/), which consists of 279 samples. The validation cohort dataset is TCGA provisional database, consisting of 249 samples. The raw gene expression dataset was processed. Probe IDs received the annotation toward the gene from the corresponding platform annotation profile of the GDC website and the raw matrix data received the quantile normalization and log2 conversion. Samples with missing data were excluded.The RNA sequencing dataset for HNSCC and the corresponding clinically related data originated from the TCGA database (Single-cell data was classified into specific cell types and divided according to their respective tissue sources. The corresponding transcriptome investigation data were compared in order to screen for DEGs. To increase the efficiency of the study the candidate DEGs were taken as min.pct > 0.25 and | Log2 (FC) | > 0.5.The association between HNSCC tumor-infiltration CD8+ T cell-correlated DEGs and overall survival time in TCGA HNSCC cases was studied. Univariate Cox regression analysis was carried out for identifying the genes associated with survival . Subsequently, the significance of candidate genes was selected using variable importance in a randomized survival forest (RSF) algorithm. A risk score model with the selected DEGs was built using multi-variate Cox regression approaches. In addition, the Kaplan-Meier test was employed for a number of gene features and p-values (log) were determined. Receiver operating characteristic (ROC) analysis was carried out for 3- and 5-year overall survival rates and area under the curves (AUCs) were determined for assessing the specificity and sensitivity of the gene signature. In addition, for testing the robustness of the results, the HNSCC tumor-infiltration CD8+ T cell-correlated gene signature was further verified with the TCGA HNSCC dataset.To assess the correlation of risk score distribution and clinically related characteristics, HNSCC patients were grouped according to age and gender as well as their status regarding alcohol consumption and smoking. In addition, risk scores were calculated in order to assess the patients’ prognosis for HNSCC in the presence and absence of HPV by using multi-variate Cox regression correlations.Statistics investigations were carried out with R software (version 3.6.0). Kaplan-Meier tests and ROC analysis were performed with the “survivor” and “survROC” software packages . OptimalFrom the TIMER2.0 website, a number of immunization deconvolution approaches including “XCELL ”, “MCPCOThe RNA-seq based on single cells consisted of 130,721 immunization cells from the tumor infiltrated leukocyte (TIL) samples obtained from HNSCC patients. Using the UMAP algorithm, this mixture of 11 cell types including CD8 T, CD4 T, dendritic, CD8+ T, mast, natural killer (NK) and plasma cells as well as mon/macrophages were unambiguously clustered and annotated .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.


The use of the four-point SDMT cut-off may be useful to assess the clinical meaningfulness of results from cognitive rehabilitation trials.Cognitive impairment (CI) is a remarkable feature in pediatric-onset multiple sclerosis (POMS). The Symbol Digit Modalities Test (SDMT) is increasingly used to explore CI in MS. Recently, a four-point worsening on the SDMT score has been demonstrated to correlate with a clinically meaningful cognitive worsening in adult MS. We conducted a post hoc analysis of a randomized computer-assisted rehabilitation trial for attention impairment in POMS to test the clinical meaningfulness of the changes in SDMT scores at the end of the trial (delta SDMT). A four-point SDMT cut-off was applied. POMS patients exposed to specific computer training (ST) and non-specific training (nST) were compared. Data of 16 POMS  patients were analyzed. At the end of the trial, 25% of patients reported no clinically significant changes (−3 to 3), 12.5% a clinically significant worsening (≤−4) and 62.5% a clinically significant improvement (≥4) in the delta SDMT. The proportion of patients reporting a clinically meaningful improvement was significantly ( There is a growing need to find new and more robust disability outcome measures to be used in multiple sclerosis (MS) randomized clinical trials (RCTs) and in clinical practice. The most common outcomes currently used, annualized relapse rate and sustained Expanded Disability Status Scale (EDSS) progression, miss an important dimension of MS-related disability, namely, the decline in cognitive function. Indeed, cognitive dysfunction is one of the most remarkable features of MS  and partThe percentage of patients with POMS with at least a mild cognitive deficit ranges from 30 to 50% ,3,4,5,6.Currently, the Symbol Digit Modalities Test (SDMT), a simple, brief measure of information processing speed (IPS), is considered the gold standard in screening for cognitive involvement in MS ,11,12,13Due to its short duration and ease of administration, high sensitivity in detecting subtle changes in cognitive functioning in MS, good test–retest reliability and very low practice effects ,11,12,13Recently, a detailed analysis of the psychometric qualities, sensitivity to change and clinical meaningfulness of the SDMT in comparison to the PASAT was performed by the Multiple Sclerosis Outcome Assessments Consortium (MSOAC) . The resIn fact, in our trial, we observed an improvement also in cognitive domains not specifically trained by the program . PatientHere, we present a post hoc analysis aimed to assess the robustness of treatment effects, applying the four-point SDMT cut-off, as proposed by MSOAC , on the A detailed description of the study population, procedures and intervention has been previously reported elsewhere .Briefly, 16 POMS patients diagnosed according to the most recent diagnostic criteria, aged <18 years, with an Expanded Disability Status Scale (EDSS) score ≤5.5 and failing (defined as scores <1.5 standard deviation (SD) of normative values) in at least 2/4 attention tests on a neuropsychological battery, were randomized to ST or nST , performST targets focused, sustained, selective, alternating and divided attention and consists of a group of hierarchically organized tasks that exercise different components of attention, proceeding from sustained to selective, alternating and, finally, divided attention exercises. The sequence of the exercises places increasing demands on complex attention control and working memory systems.A neuropsychological test battery was administered, using alternative versions of the tests, at baseline (T0), and within one week following the end of the three-month training program (T1). The neuropsychological test battery comprised tests which cover different cognitive domains: Selective Reminding Test (SRT), Selective Reminding Test–Delayed (SRT-D), Spatial Recall Test (SPART), Spatial Recall Test–Delayed (SPART-D), SDMT, Trail Making Tests (TMT) A and B, Semantic Verbal Fluency Test (SVFT) and Tower of London Test (TOL). In particular, the SDMT was administered to assess concentration, attention and IPS. A global score, the CII, allowing the evaluation of changes in cognitive performances independently by the number of cognitive tests failed at the neuropsychological evaluation, was obtained using the mean and SD from the normative values for each test. A detailed description of the CII calculation procedures has been extensively reported previously ,21,22,23The primary objective of this post hoc analysis was to test the effects of our cognitive rehabilitation trial by comparing the delta SDMT scores, applying a 4-point SDMT cut-off, in ST and nST groups.Statistical analysis:All analyses were post hoc and not pre-specified. Continuous variables were described as mean and SD, and categorical variables were described as frequency and percentage. Group comparison was performed using Student’s t test, the Mann–Whitney U test and Fisher’s exact test, when appropriate.As previously reported elsewhere , by usinFor each patient, a grading system was applied to individual cognitive tests, based on the number of SDs below the control mean  ,19,21,22− Delta SDMT between −3 and 3 = no clinically significant SDMT change;− Delta SDMT ≤−4 = clinically significant SDMT worsening;− Delta SDMT ≥ 4 = clinically significant SDMT improvement.To quantify the clinical impact of changes over time of the SDMT in the cohorts of patients exposed to ST and to nST, we applied the following categorization of the delta SDMT scores (delta SDMT = SDMT score at T1—SDMT score at T0) by using a 4-point SDMT cut-off:Thereafter, we compared the proportion of patients reporting a clinically significant SDMT improvement at the end of the 3-month training program by using the chi-square test.Finally, to evaluate the relationship between changes in the SDMT scores and in the CII, we compared the delta CII among the groups of patients stratified on the basis of the delta SDMT .Statistical analysis was performed by using SPSS software . The comparisons of baseline demographic and clinical characteristics and of the baseline NP of POMS subgroups who underwent ST and n-ST are reported in At baseline, no differences were found between the two treatment arms regarding sex and age and in terms of NP performances.p < 0.0001) in comparison to those treated with nST.After the 3-month cognitive training, patients exposed to ST showed a significant improvement in SDMT performances (p < 0.0001) [In more detail, a significant effect for time (Baseline (T0) vs. Post-Treatment (T1) comparison) was found for the mean (SD) SDMT values (ST group: 24.5 (4.6) vs. 46.3 (6.7); nST group: 20.5 (3.6) vs. 20.8 (4.1),  0.0001) .By applying the four-point cut-off of the delta SDMT scores, 4 (25%) patients reported no clinically significant changes, 2 (12.5%) patients a clinically significant worsening and 10 (62.5%) patients a clinically significant improvement in the SDMT score at the end of the training program. p = 0.008) higher in patients exposed to ST  in comparison to that in patients exposed to nST   . Among the other patients exposed to nST, 4/8 (50%) reported stable SDMT scores , and 2/8 (25%) had a significant deterioration. p = 0.038)  .Rehabilitation treatment with a computerized cognitive training specifically designed to exercise the attention domain resulted in a significant improvement in overall cognitive performances and, in particular, in the SDMT scores. With this post hoc analysis, we have also demonstrated that a specific attention training is associated with clinically meaningful changes in SDMT scores in the short term.It is noteworthy that all patients exposed to ST exhibited a clinically meaningful improvement in the SDMT scores in comparison to only 2/8 patients exposed to nST at the end of the 3-month cognitive training.To the best of our knowledge, this is the first report of the application of the four-point SDMT cut-off, proposed by MSOAC , to the The research about functional measurers capable of exploring from different perspectives, including non-motor disability, the overall impact of MS on disability has a long-lasting history.In 1996, the MSFC was proposed as a multiple-domain measure to detect and summarize walking impairment , upper extremity dexterity (via the nine-hole peg test) and cognition (via the PAST) abilities in patients with MS .Thereafter, most of the RCTs performed to evaluate the efficacy of disease-modifying therapies included the MSFC as an outcome measure, but due to the difficulty in interpreting the clinical meaning of z-score changes and because it seems that MS patients do not fully accept the tests , the MSFC has not been extensively used in clinical practice ,9.The use of the SDMT as an outcome measure in RCTs and observational studies has progressively gained more attention in recent years ,12,13,14Moreover, SDMT scores are predictive of different patient-related outcomes, such as employment and driving abilities ,32,33, bGiven all these premises and based on its predictive validity, the very high level of sensitivity and specificity and the facility of administration, this cognitive test is often used in clinical practice to perform a basic cognitive screening helping to identify patients at high risk for cognitive impairment who need a more structured neuropsychological evaluation. Recently, the psychometric properties of SDMT and PASAT have been compared and the former proved to be superior to the PASAT in assessing the IPS in patients with MS. Furthermore, a four-point change in the SDMT has been proven to be a cut-off able to discriminate clinically meaningful changes from test score changes due to a practice effect or simply chance . In thisDifferent limitations of this study deserve discussion. First, due to the monocentric nature of the study and due to the absence of external funding resources, the recruitment was limited to 16 POMS patients. POMS is a rare disease; only 5% of MS patients have an onset before 18 years, and only some of them present cognitive deficits which represent the main inclusion criteria of our trial. The internal validity and the consistency of our results are assured by a randomized controlled design and robust statistical analysis.Second, we did not include in the study protocol the collection of measures of clinically meaningful changes such as school performance or parent-reported behaviors. This was a pilot study designed for a fast evaluation of the efficacy of a cognitive training (in treated vs. untreated patients) in POMS patients with attention impairment. Anyhow, we were able to show that a cognitive training specifically addressed to stimulate different attention components resulted in an important transfer effect. In conclusion, in our RCT, the use of the four-point SDMT cut-off allows us to demonstrate the clinical meaningfulness and the robustness of the treatment effects obtained by a home-based computerized program for retraining attention dysfunction in POMS patients with attention impairment. Further studies on larger populations are needed to confirm the clinical validity of this cut-off and its applicability in the routine clinical practice setting.