Cervical cancer screening services are however nonexistent and the prevalence of high-risk human papillomavirus (hr-HPV) and cervical precancer is undocumented. We aimed to screen and treat inmates for cervical precancer and determine the prevalence of hr-HPV using the novel AmpFire HPV detection system combined with colposcopy by trained nurses using a mobile colposcope  system).Across Ghana, females comprise 1.2% of the entire prison population  after which mobile colposcopy with the EVA system was performed. EVA images were reviewed by a gynaecologist. Frequencies and percentages were used to describe categorical data, while means, standard deviations, medians and interquartile ranges (IQRs) were used to describe continuous data.75% of the women were convicts with a median sentence of 5 years (IQR: 2–10 years). Their mean age was 41.1 years . The self-reported prevalence rate of HIV was 13.1% : 7.5%–21.9%), all of whom were receiving treatment. The hr-HPV prevalence rate was 47.6% (CI: 36.9%–58.3%) in the general population of imprisoned women and 63.6% (CI: 35.4%–84.8%) among HIV positive women. Six percent (6%) had lesions on the cervix, of which 3.6% were treated with thermal coagulation and 2.4% were treated with loop electrosurgical excision procedure. The average age of hr-HPV positive women was 37.8 years.There is a high prevalence of hr-HPV infection among women in custody at the Nsawam Medium Security Prison. These women will benefit from structured cervical cancer prevention services, including treatment for abnormalities that are picked up during such screening. Cervical cancer was the leading cause of cancer-related death in women in Sub-Saharan Africa in a 2018 report [Knowledge that infection with high-risk Human Papilloma Virus (hr-HPV) is a necessary cause of nearly all cervical cancers has resulted in major changes in cervical cancer prevention methods . There iIn LMICs such as Ghana, it has been suggested that a mix of innovative screening methods tailored to the patient’s unique circumstances need to be employed for cervical cancer screening, if any gains are to be made in the fight to reduce the burden of cervical cancer , 5. ImprIt has been reported that imprisoned women are at increased risk of cervical cancer but are also less likely to have been screened for cervical cancer . These wThere is a paucity of data in HPV testing among incarcerated women in low resource countries. Most studies describing the epidemiology of hr-HPV infection among incarcerated women have emanated from relatively higher-income settings –16. In lWe aimed to screen and treat incarcerated women for cervical precancer and determine the prevalence of hr-HPV using the novel AmpFire HPV detection system. This is in line with a previous study that showed that the sensitivity for CIN2 or worse was similar between AmpFire and Cobas tests. The sensitivity for AmpFire ranged between 95.74% and 96.81% compared to that of Cobas which ranged from 92.55% to 95.74%. Specificity was significantly better for AmpFire (ranged from 89.8% to 90.8) than Cobas (88.5% to 91%) . This woDesign: We conducted a descriptive cross-sectional study involving the screening of 85 incarcerated women at the Nsawam Medium Security Prison in Ghana on 27–28 September 2019 to assess the prevalence of hr-HPV among them. At the time of the study, 90 women were incarcerated at the prison and 85 of them agreed to participate in the study.Setting and participants: All incarcerated women at the Nsawam Medium Security Prison, Ghana on 27–28 September 2019.After counselling and informed consent, women underwent a structured interview based on a questionnaire and their responses were recorded directly into a Microsoft Excel spreadsheet database developed specifically for recording all data associated with cervical screening and treatment. Both the questionnaire and electronic data capture tool were developed at the Cervical Cancer Prevention and Training Centre (CCPTC) at the Catholic Hospital, Battor. These tools have been in use since 2017 for capturing all data on cervical screening and treatment conducted by the team from CCPTC.To ensure the utmost privacy of clients, all clients were identified by unique alphanumeric identifiers and none of their personal identification data such as names and previous addresses were obtained during the screening.For all the women, a speculum was inserted in the vagina to expose the cervix and samples were collected from the cervix with a sterile brush which was put in a collection tube and submitted to the laboratory technologists for testing.J).Ethical clearance was obtained from the Ethical Review Committee of the Ghana Prison Service. All women whose images were used in the manuscript gave us consent to use the images . It is an isothermal polymerase chain reaction (PCR) assay that uses minimum instrumentation and detects 15 hr-HPV  in a single tube reaction and simultaneously identifies specifically the presence of types 16 and 18. Dry brush samples were submitted immediately at room temperature to the laboratory for testing. Samples could be processed individually or batched (1 to 94 samples per run). DNA extraction was not required and sample processing to final results took less than 2 hours .The AmpFire HPV screen kit contained the reaction mix , primer mix (with primers and probes), positive control and negative control. Twelve microlitres of reaction mix was mixed with 11 µL of primer mix in a 0.2 mL reaction single tube, 8-well strip, or 96-well plate. One millilitre of lyse buffer was added to the dry sample brush in a 5 mL empty tube and left to sit at room temperature for 20 minutes after it had been vortexed. After this, 2 µL processed samples were added to the reaction tube to bring the total volume to 25 µL. The reaction tubes were incubated at 60°C for 60 minutes in the Atila Power 96 real-time PCR system . The EVA System consists of a mobile colposcope built around a smartphone, and an online image portal for storing images. A smartphone app is used to control the mobile colposcope, and upload pictures to the image portal or to store images on the phone for review . The adeType 1 (TZ1): The entire circumference of squamocolumnar junction is visible; fully ectocervical.Type 2 (TZ2): The entire circumference of squamocolumnar junction is visible; partly or fully endocervical.Type 3 (TZ3): The entire circumference of the squamocolumnar junction is not visible; partly or fully endocervical.Self-reported data such as HIV status may be under-estimated since we did not conduct HIV testing as part of the screening exercise. This could lead to an over-estimate of the HPV prevalence among HIV positive incarcerated women.t-test was used to compare the means of two populations in order to explore further the relationship between hr-HPV positivity and the age of incarcerated women.Percentages were calculated for categorical data and means, and standard deviations were used to describe continuous data. Skewed continuous data (such as period of incarceration) were summarised using medians and interquartile ranges (IQRs). For our primary objective of estimating the prevalence of hr-HPV among incarcerated women, we used simple percentages and reported 95% confidence intervals (CIs). For the secondary objectives, we explored the association between age of incarcerated women and period of incarceration and hr-HPV positivity using grouped box plots. In addition, we compared the period of incarceration between those with hr-HPV positivity and those without using a Wilcoxon rank-sum test. This was done because the period of incarceration was highly right skewed. A student’s p-values for all our hypothesis tests.We reported 95% CIs for all key estimates obtained. All statistical analyses were performed using STATA version 15 . We considered a statistical significance level of 5% and reported all Informed consent was sought directly from all 90 incarcerated women at this prison for cervical screening.Five of the women did not consent to be screened for the following reasons: Two of them had undergone total abdominal hysterectomy and so did not have a cervix, one said she was too old and did not expect to live much longer and so did not need cervical screening, one had recently undergone cervical screening prior to our visit and one was uncomfortable with strangers observing her genitalia. Of the 85 women who agreed to be screened, 1 of them did not have a cervix ; thus, 84 of the women were screened. Majority  of the women were convicts with a median sentence of 5 years (IQR: 2–10 years). Those on remand comprised 14.3% (12 women), 6% (5 women) were condemned and the rest (4.8%) had been sentenced to life imprisonment.The median period of incarceration as at the time of screening of the women was about 1 year (IQR: 0.5–2.5 years) and 13.1% had been in jail for at least 5 years. The mean age of the participants was 41.1 years . In terms of marital status, 25% of the women were married, 22.6% were divorced, 19.1% were single and 17.9% were widowed. The remaining women were in some form of steady relationship with a partner. The highest education level was varied with 14.3% having attained tertiary education, 52.4% having attained secondary education and 15.5%having attained elementary education. The rest (17.8%) had had no formal education. An overwhelming majority (84.5%) were Christian, 13.10% were Muslim and the rest (2.4%) were practitioners of African traditional religion. None of them earned an income.About 11% were current or past smokers. Most of the women had had multiple births in the past, with 17.9% having had no children, 21.4% having had one child, 14.3% having had two children and the remaining 46.4% having had at least three children.Fourteen percent (12/84) reported having undergone cervical screening in the past, most (9/12) of them having been screened only once ). None of them had been treated in the past following cervical screening. One had been vaccinated for HPV in the past.The self-reported prevalence rate of HIV among the women was13.1% (95% CI: 7.5%–21.9%) and all the HIV positive incarcerated women were receiving treatment for that.Of the 84 screened, 40 had hr-HPV infections, resulting in a hr-HPV prevalence rate of 47.6% (95% CI: 36.9%–58.3%). Of the 40 women infected with hr-HPV, 4 were infected with HPV 16 alone, 8 with HPV 18 alone and 23 with hr-HPV types other than HPV 16 and HPV 18. Some patients were infected with multiple hr-HPV types. Two women had HPV 16 and other hr-HPV types and three had HPV 18 and other hr-HPV types.The positivity rate of hr-HPV infection among the HIV positive women was 63.6% (95% CI: 35.4%–84.8%).Colposcopy using the EVA system was adequate for all 84 incarcerated women; 13.1% had TZ1, 24.1% had TZ2 and 65.5% had TZ3. Five (6%) had lesions on the cervix (aceto-whitening). Three were treated with thermal coagulation and two with loop electrosurgical excision procedure (LEEP).t-test p-value = 0.0686). Similarly, women with hr-HPV infection had spent less time (median period of incarceration = 0.80 years) in prison than women without hr-HPV infection (median period of incarceration = 1.25 years); however, the difference was not statistically significant . The distributions of age and period of incarceration for incarcerated women are shown in Women with hr-HPV infection were younger (mean age = 37.8 years) than those without (mean age = 44.0 years); however, this difference was not statistically significant  had TZ3 (entire circumference of the squamocolumnar junction not visible). The management per our protocol  is folloFive women were treated. Three of them were treated by thermal coagulation on the same day of screening , while taRemand prisoner: A person confined in prison whose case is awaiting hearing or pronouncement of sentence in a lower court . Life prisoner: A person sentenced to spend the whole of his or her life in prison custody by a court of competent jurisdiction. Condemned prisoner: Any person who is found guilty by the court of justice and sentenced to death by hanging or firing squad by a court of competent jurisdiction. Convict prisoner: A person who has committed a crime and has been tried and found guilty by a court of competent jurisdiction and has been sentenced to serve a specific term in prison.This work was not done in the context of a research study. The incarcerated women were offered the same services as clients of the CCPTC at Catholic Hospital, Battor. The same prescreening forms were filled before the incarcerated women were screened. Therefore, questions like history of intravenous drug abuse, sharing needles, illicit drug use, exchanging sex for drugs or money, among others, which are all risk factors for acquiring HIV and predisposing incarcerated women to hr-HPV infection, were not directly asked. The AmpFire HPV detection system does not offer full genotyping and lumps 13 hr-HPV infections together. It was, therefore, impossible to assess the most common hr-HPV types among the incarcerated women, and also impossible to detect multiple HPV infections especially when the multiple infections were due to hr-HPV types other than HPV 16 and 18. Also, it is likely that the true incidence of HIV infection among the incarcerated women is higher than documented here as the figure presented here was based on self-report among incarcerated women with no onsite HIV testing performed.We present the first results of the prevalence of hr-HPV among incarcerated women in Ghana. With this high prevalence of hr-HPV (47.6%), and 13.1% having been in prison for at least 5 years, many incarcerated women are at an elevated risk of developing cervical cancer if cervical cancer prevention services are not offered in the prisons.We have demonstrated that it is possible to offer a high level of onsite screening and treatment of precancerous lesions of the cervix even for incarcerated women in a prison using current technologies such as HPV DNA testing by the AmpFire HPV detection system , mobile colposcopy with the EVA system  and thermal coagulation and LEEP with the Cure/Liger Medical thermal coagulator and electrosurgical unit .None.Conceptualisation: KE, ET and KAL. Participant screening, treatment and data collection: ET, CMW, IG, SK, ACK, PAD, NOME, MA, DA, KAL and KE. Data collation: ET, CMW, IG, SK, ACK, PAD, NOME, MA and DA. Formal analysis: KE and JEA. Writing – original draft: NOME, PKA, JEA, KAL and KE. All the authors read and approved the manuscript in its current form.


Given that only a small percentage of human papillomavirus (HPV)-positive women develop cancer, HPV is necessary but insufficient for carcinogenesis. Mucosally transmitted viral cofactors appear to contribute to HPV-related cervical cancer, such as Epstein-Barr virus (EBV), but previous studies have shown inconsistent outcomes. The exact role of EBV in cervical cancer remains unclear, and more studies are needed to determine its involvement. In this study, we describe the prevalence of EBV and HPV coinfection in HIV-positive women and explore how abnormal host immune status induced by viral coinfections modulates epithelial gene expression. We found a significant correlation between EBV-HPV coinfection and the incidence of high-grade cervical intraepithelial neoplasia (CIN2+). RNA sequencing indicated that CIN tissues coinfected with EBV and HPV led to significant changes in the gene expression of epithelial differentiation and development compared to normal tissues with HPV infection alone. In particular, several differentially expressed genes (DEGs) are closely associated with cancer, such as CACNG4, which was confirmed to be upregulated at both the mRNA and protein levels. Therefore, these findings provide some evidence that EBV may act as a cofactor or mediator in HPV-related cervical cancer. Specific genes or proteins, such as CACNG4, may serve as biomarkers that can risk stratify patients based on pathological changes in the cervix. Sexually transmitted infections (STIs) can cause cancer or have serious reproductive health consequences , especiaSimilar to HPV, Epstein-Barr virus (EBV) is a mucosally transmitted pathogen that is widespread in the human population . This viIn contrast to people with a normal immune system, women living with HIV are at high risk of acquiring oncogenic HPV and are also susceptible to persistent and reactivated HPV infection and to developing cancer . In addiParticipants were recruited from the AIDS Antiretroviral Therapy Department of the Third People’s Hospital of Kunming, Yunnan Province, China, between February and November 2019. The inclusion criteria were as follows: 1) HIV-positive women with ongoing antiretroviral therapy, 2) age of 18-64 years, 3) sexually active, 4) ability to both physically and mentally undergo cervical sampling and colposcopy examinations, and 5) intact cervix and no current pregnancy. The study protocol was approved by the Ethical Review Committees of National Cancer Center/Cancer Hospital Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. All participants provided informed consent. A summary of the study procedure is shown in Trained gynecologists inserted cytology brushes and collected exfoliated cervical cells after a pelvic examination, and cervical samples were collected into a prepared liquid medium . All collected samples were placed in a 4°C refrigerator and transferred to a third-party laboratory, KingMed Diagnostics, Kunming branch, for Cobas 4800 HPV testing  and Sansure PCR HPV test . Sansure PCR HPV test  is a PCRTotal DNA was extracted from exfoliated cervical cells from liquid medium (ThinPrep) using a QIAamp DNA Mini Kit  according to the protocol recommended by the manufacturer. To detect EBV, 5 µl of DNA was then used for subsequent RT-PCR  with a CFX96 Real-Time PCR Detection System . In addition, β-globin primers were used to verify the quality of the DNA and exclude false negative results, and a positive control was included in each RT-PCR assay to ensure the success of the reaction. RT-PCR was performed for 45 cycles of amplification, and samples were considered negative if the cycle threshold (Ct) value was greater than 40. The primers and probes used in this study are identical to those in previous studies .in situ (AIS), or adenocarcinoma (ADC). Women with negative results by all tests (cytology <ASCUS and without HPV detection) were considered to be negative for the outcome of CIN2 or worse (CIN2+). Women with confirmed CIN2+ were recommended for treatment.Women positive for HPV on Cobas or Sansure PCR testing or with a cytological diagnosis of atypical squamous cells of undetermined significance (ASCUS) or higher (ASCUS+) were referred for a colposcopy examination. A directed biopsy was collected if any abnormality was identified under colposcopy. Endocervical curettage was performed if the squamocolumnar junction was invisible. Biopsy tissues were immediately immersed in 10% buffered formalin and transported to KingMed Diagnostics, Kunming branch for processing  staining was conducted according to standard protocols) and diagnosis by experienced pathologists blinded to other screening results. Pathology results were reported as negative, cervical intraepithelial neoplasia grade 1 (CIN1), grade 2 (CIN2), grade 3 (CIN3), microinvasive carcinoma (MIC), squamous cell carcinoma (SCC), adenocarcinoma via exonuclease/polymerase activities. The 3’end adenylated and adaptor-ligated cDNA were then purified with AMPure XP system  to select the fragments with the correct target size (370-420 bp). Finally, the cDNA libraries were enriched by PCR amplification with Phusion High-Fidelity DNA polymerase, universal PCR primers and Index (X) Primer. The library quality was assessed on an Agilent Bioanalyzer 2100 system.Total RNA was extracted from formalin-fixed paraffin embedded (FFPE) biopsy tissues using the RNeasy FFPE Kit  according to the manufacturer’s instructions. RNA purity was evaluated using a NanoPhotometer spectrophotometer , and integrity was assessed using an RNA Nano 6000 Assay Kit with a Bioanalyzer 2100 system . Next, the RNA concentration was measured using a Qubit RNA Assay Kit with a Qubit 2.0 Fluorometer . A total amount of 1 µg of RNA per sample was used to generate sequencing libraries. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations at an elevated temperature in First Strand Synthesis Reaction Buffer. Then, a random hexamer primer and mRNA fragments as templates were used to synthesize the first-strand cDNA. Second-strand cDNA fragments were obtained using DNA Polymerase I and dNTP. Remaining overhangs were converted into blunt ends Before sequencing, clustering of the index-coded samples was performed on a cBot Cluster Generation System using a TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer’s instructions. Then, the library preparations were sequenced on an Illumina HiSeq platform, and 150 bp paired-end reads were generated.ftp://ftp.ensembl.org/pub/release-98/fasta/homo_sapiens/) with HISAT2 v2.0.5.Clean reads were obtained by removing reads containing adapters, reads containing poly-N and low-quality reads from the raw data. All subsequent analyses were based on filtered reads. Paired-end filtered reads were mapped to the homo sapiens sequence (https://www.ncbi.nlm.nih.gov/sra/PRJNA701124).FeatureCounts v1.5.0-p3 was used to count the read numbers mapped to each gene. To normalize the read count, fragments per kilobase of transcript per million mapped fragments (FPKM) were calculated for each gene. Then, the DESeq R package (1.16.1) was utilized to perform differential expression analysis of the two groups. The resulting P values were adjusted using Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes with an adjusted P value <0.05 or | log2-fold change | >0 were considered differentially expressed. Gene Ontology (GO) enrichment analysis of differentially expressed genes (DEGs) was implemented by the GOseq R package. GO terms with corrected P values less than 0.05 were considered significantly enriched by DEGs. Subsequently, we used the clusterProfiler R package to test the statistical enrichment of DEGs in KEGG pathways. The mRNA-seq data for this study can be found in the Sequence Read Archive (SRA)  program and normal tissues from the Genotype-Tissue Expression (GTEx) project.IF was performed on FFPE sections after heat-induced antigen retrieval. Briefly, sections were blocked for 1 h, incubated overnight at 4°C with a rabbit primary antibody against human CACNG4 , and subsequently incubated with a secondary Alexa Fluor 568-conjugated goat anti-rabbit IgG antibody  for 1 h. Slides were mounted with mounting medium containing DAPI and imaged by a Leica SP8 laser scanning confocal microscope system. For WB, samples were pooled and extracted using the FFPE Total Protein Extraction Kit  and then incubated with primary antibody against CACNG4 or β-actin . The proteins were visualized using HRP-conjugated secondary antibody  and chemiluminescent HRP substrate (Bio-Rad).p values less than 0.05 (two-sided) were considered statistically significant. Data analyses were performed using SPSS 20.0.Descriptive statistical analysis was performed to summarize the prevalence of HPV and EBV infections and coinfections. Chi-square tests were used to compare the distribution of CIN2+ between the HPV infection and HPV-EBV coinfection groups. Student t-test was performed to evaluate the mRNA expression difference between two groups. 6 copies/ml) that reflected severe immunosuppression were excluded to avoid interference. Of 176 women, the median age was 40 (34–46) years. Among them, 61.9% have been married, 68.8% had education at junior high school or below, 45.4% were unemployed and 44.3% were farmer or rural migrant worker. The median age at sexual debut and the first delivery were 20 (19–22) and 24 (21–27) years, respectively. The median CD4 count was 540 (IQR 398–656) cells/µL, and the median HIV viral load was <50 (IQR <50–<50) copies/ml. Valid HPV test results were obtained from 176 women. The prevalence of HPV and EBV in HIV-positive women was 33.5% and 15.9%, respectively. In addition, the coinfection rate of EBV-HPV was 9.1%. Eleven participants were missing from the histopathological analyses due to loss to follow-up. Thus, a total of 165 women were involved in the distribution analysis of CIN2+ among different coinfection statuses, and p=0.017) than those without coinfection.A total of 180 women were enrolled in this study; of these, four women with extremely high HIV viral load  gene cluster members LCE3D and LCE3E, and a number of keratin (KRT) protein-coding genes, such as KRT17, KRT23 and KRT24, were downregulated  family, which regulates multiple physiological processes, including cell survival, differentiation, proliferation, invasion, metabolism, and DNA damage repair, thus contributing to the initiation and development of some cancer types . Indeed, a consistent upregulation of CACNG4 was found in cervical tissues  γ4 subunit. One gene expression analysis revealed upregulation of the CACNG4 gene in human colon cancer  can be found below: The studies involving human participants were reviewed and approved by Ethical Review Committees of National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College. The patients/participants provided their written informed consent to participate in this study.MF, RD, and YG performed the experiments and analyzed the data. HZ analyzed the data and assisted with writing the manuscript. YQ assisted with the project design and revised the manuscript. QL and FZ designed the project. MF, RD, QL, and FZ wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the Science and Technology Major Project of Yunnan Province (2017ZF006), the CAMS Initiative for Innovative Medicine (2016-I2M-1-019), and the National Natural Sciences Foundation of China (81761128006 and 31670173).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.


In deep-sea mining, the coupling dynamic response between the mining vessel and the lifting pipe is a significant problem, which directly affects the structural design of the lifting system and the safety of field operation. The characteristics of coupled motion model have not been fully considered in the existing research. Therefore, this paper uses time-domain coupled numerical model as the research object, considering ocean current, surface wave, pipe dynamics and vessel-pipe contact mechanics, to study the dynamic behavior of the lifting pipe and mining vessel during the process of deep-sea mining using AQWA and OrcaFlex softwares. The response amplitude operator (RAO) is used to compare the measured and simulations dynamic response of the mining vessel. There is a very good agreement in RAO between the experiments and simulations. The coupling simulation results show that the coupling effect has a significant effect on the time domain dynamic response of the lifting pipe, but has little effect on the average effective tension and longitudinal amplitude along the pipe length. The research results of this paper are of great significance to the safety design of deep-sea mining lifting system and the planning of deep-sea operation activities. At present, the proven potential deep-sea mineral resources include polymetallic nodules, cobalt-rich crusts, and polymetallic sulfides, among which deep-sea manganese nodules and cobalt-rich iron-manganese crusts are abundant2. Marine solid mineral resources, such as the most common iron-manganese nodules, are usually found in the 4000–7000 m deep sea3. In order to extract these minerals from the deep sea bed, there are usually three methods, namely, seabed mechanical transportation, air lift system with ejector and hydraulic lift system with pump station4. Since 1970s, many research groups from the United States, Canada, China, France, Germany, Russia, Japan and India have studied the core technology of deep sea mining6. Among them, the pipeline transportation mining system is recognized as the most practical deep-sea mining system, and it is also the only deep-sea mining system that has been verified by deep-sea experiments7. The system typically consists of surface mining vessel, lifting pipe, lifting pump sets, buffer, lifting hose and subsea collector9. In this system, the lifting pipe must be equipped with pump sets for lifting the mineral resources to the vessel, and with a buffer to regulate the density of the ore-fluid mixture. On the other hand, the pump sets and the buffer also increase the stability of the lifting pipe10.Deep sea contains a lot of resources, which can meet the needs of mankind for a long time in the future11, and in this process, a large dynamic load is added to the lifting system12. At the same time, the vibration of the lifting pipe in turn affects the vessel motion. So, a complex coupling dynamic response system is formed between the pipe and the vessel. With the increase of the length of the pipeline, the nonlinear effect and damping effect of pipeline gradually increase, the top tension of pipeline gradually increases, and the coupling effect between mining vessel and pipeline becomes more obvious. For the vessel-pipe coupling system in the deep sea, Zan et al.13 proposed a coupled time-domain numerical model of pipelines and pipe-laying vessel motions. The model was solved by Newmark method and verified with OrcaFlex software. The results showed that there is a significant connection between the dynamic responses of pipelines and the pipe laying vessel motions. Wang et al.14 established the coupled dynamic model of deep-water flexible pipeline, carried out the hydrodynamic analysis of pipe laying vessel by using AQWA software, and completed the numerical simulation of flexible pipeline by using OrcaFlex software, and analyzed the factors affecting the dynamic characteristics of the pipeline. Dai and Liu15 combined the single body dynamic model of the mining vessel with the multi-body discrete element dynamic model of the lifting pipe, considered the heave motion and longitudinal and transverse towing motion caused by the sea wave, and established the overall dynamic model of the deep-sea mining system. Using multi-body dynamics simulation method and finite element method, Oh et al.16 designed a coupling device for a deep-seabed mining system, and analyzed the kinematic characteristics between the coupling device and the buffer system, flexible pipe and mining robot. Based on Morison’s equations, lumped mass method and three-dimensional potential flow theory, Sun et al.17 proposed a method of coupled vessel/riser/body system in deep sea mining combined with dynamic positioning (DP), and obtained the time domain simulation result of the vessel operated in two DP modes. Chung18 carried out offshore tests of deep-sea mining systems to measure the response of full-scale pipelines and the motion of mining vessel in the deep sea. The measurement results show that the axial stress may be one order of magnitude larger than the bending stress for a 5000 m long pipe, and the amplitude of the axial stress is coupled with the motion of the mining vessel. In order to improve the analysis efficiency, based on the subsystem synthesis method of deep-sea integrated mining system, Kim et al.19 established the motion equations of vessel-lifting pipe subsystem and the flexible pipe-mining robot subsystem respectively, and extended them to the comprehensive analysis of multiple mining robots.Due to the motion of the mining vessel, the lifting pipe is prone to longitudinal vibration, transverse vibration and torsional vibration4. Therefore, the dynamic characteristics of the lifting pipe has always been a research hotspot. Erol20 established the dynamic model of the stepped lifting pipe, and studied the longitudinal vibration characteristics of the lifting system with and without dynamic vibration absorber (DVA) under the over damping and under damping modes respectively by using the method of separating variables. The exact analytical solutions of the free response and forced response of the lifting pipe under the heave motion of the mining vessel were obtained. Cheng et al.21 proposed a time-domain hybrid finite element–boundary element method to investigate nonlinear interaction between vessel waves and slender structures and obtained the dynamic characteristics of multi-component mooring lines and deep water steel catenary risers under wave action. Using the finite element and Wilson-θ direct integral methods, Liu and Xiao et al.23 simplified the mining pipeline as beam element, and established the physical model and mathematical model of the mining pipeline system. Based on the Lagrangian method and the lumped element method, Song et al.24 built a dynamic model of the rigid space stepped-pipe strings system and obtained the longitudinal vibration characteristic of the stepped lifting pipe with and without dynamic vibration absorbers.The lifting pipe with a length of several kilometers is the weakest and most technically difficult part in the mining vessel-pipe coupling system. The safety and success of offshore mining mainly depends on the reliable design of the lifting pipe structure25 proposed that wave parameters have a significant effect on hydrodynamic response. Therefore, the main goal of this paper is to offer a qualitative evaluation of the influence of coupling effect on the dynamic characteristics of mining vessel and lifting pipe during deep-sea mining. Thus, a comprehensive vessel-pipe time-domain coupled model is established using the software OrcaFlex to simulate the dynamic responses of mining vessel and lifting pipe under different waves. Meanwhile, a 1:80 scale experimental model is used to carry out relevant tests in a wave pool to verify the coupling dynamic behavior. On the other hand, the influence of coupling force on dynamic characteristics of mining vessel and lifting pipe is sufficiently discussed using an illustrative example of a MAFUTA mining vessel.As can be seen from the brief review of the most advanced research, the current research on deep-sea mining system mainly focuses on the modeling and simulation analysis of the whole deep-sea mining system cooperative operation, the dynamic characteristics analysis of the lifting pipe. However, there are few researches on the dynamic coupling model of the mining vessel and stepped lifting pipe as well as the effect of wave parameters on dynamic response of lifting pipe. Zhao et al.3. According to the measured data of sea state survey and referring to the Beaufort scale of winds26, the layout and recovery of the lifting pipe are considered as level 4 sea state, and the lifting operation is considered as level 6 sea state. The actual sea state parameters are shown in Table The East Pacific Ocean seabed polymetallic nodules mining area is selected as the deep-sea mining operation area of this paper. The actual operation depth of deep sea mining is 5000–5300 m, the density of sea water is 1028 kg/m27, the current velocity can be calculated asz is the depth below ocean surface.The dynamic analysis of deep sea mining system involves current, waves, Reynolds number (Re), coupling effect and other factors. Among them, the current velocity changes obviously with the change of sea water depth. Using the computer fitting method28, that ism and s represent model and entity respectively.In the hydrodynamic model experiment of deep-sea mining vessel, the motion and force under the action of waves are mainly considered. Therefore, the Froude similarity rule of model and entity should be satisfiedT. So the Strouhal number of the model and the prototype should be equal29, and given by an equation of the formAt the same time, the motion and force of the mining vessel are periodic, and the period is The coupling experiment of deep-sea mining vessel and lifting pipe was carried out at the National Offshore Technology Center . The experimental pool is 130 m long, 18 m wide and 6 m deep and equipped with a moving platform and track, which can simulate various marine environments such as wind and waves. According to the size of experimental pool, the experimental model of deep-sea mining system is a 1:80-scale prototype. This paper takes the MAFUTA mining vessel as an example to analyze and determine the relevant parameters of the mining vessel model, as shown in Table 30 was used to build the lifting pipe model. The lifting pipe model is truncated according to the static similarity principle. In the truncation design, the truncation depth model and the full depth model should have the same layout and similar physical characteristics. Therefore, for a 5000 m mining lifting pipe, the corresponding truncated depth is 320 m, as shown in Fig. The lifting pipe is a slender structure with engineering application depth of 5000 m and pipe diameter of about 224 mm. According to the common model scale, it is difficult to establish the model of lifting pipe. Therefore, the mixed model test technology based on the equivalent truncation design principleThe lifting pipe consists of four parts, each of which has different physical characteristics. It is assumed that there is a buffer at the bottom of the lifting pipe with a concentrated mass of 30ton. Based on the similarity principle and truncation design criteria, the truncation model parameters and test model parameters of the lifting pipe are calculated as shown in Table 31. Meanwhile, the information collection and analysis process was made by self-developed programs.In the light of similarity criterion, the coupling test model of deep sea mining vessel and lifting pipe to be dealt with in the present study is shown in Fig. In this paper, the vessel-pipe coupling system is studied numerically in time domain, and the motion equation of mining vessel is established by using the indirect time domain method from frequency domain to time domain, and the vibration equation of lifting pipe is established by using D'Alembert principle. Based on the analysis of mining vessel motion and the lifting pipe vibration, the elastic pipe vibration theory is used to calculate the vessel-pipe coupling force, and then the mathematical model of the vessel-pipe coupling motion is obtained.27:The mining vessel is rigid body, the hull is stable and balanced in still water, only considering the heave and pitch motion of the vessel.Waves acting on the vessel hull are deep-water small-amplitude waves, so the influence of shallow water waves and high-order nonlinear waves are ignored.The stiffness of the hose connected at the bottom of the lifting pipe is very small, so its influence on the lifting pipe can be ignored, and the bottom of the lifting pipe is regarded as free end.In the dynamic analysis, the bending and torsion deformation of the lifting pipe are ignored, and the lifting pipe is assumed to be a stepped-pipe string having four continuous parts with uniform and isotropic material.In actual engineering, the coupling motion between the mining vessel and the lifting pipe is very complicated. At the coupling interface, the coupling effect between them can be regarded as the interaction between the vibration response of the lifting pipe and the vessel's motion response. Among them, the lifting pipe vibration is affected by inertial force, elastic restoring force and damping, and the mining vessel motion is affected by radiation force, static water restoring force, and wave force. In order to simplify the model, the following assumptions are proposedX–Y–Z is introduced, where X–Y plane coincides with the still water surface, and X, Y and Z are the rotation axes of the vessel’s roll, pitch, and yaw, respectively.Based on the above assumptions, the mechanical model of vessel-pipe coupling system is established, as shown in Fig. M denotes the inertia matrix, X(t) represents the displacement vector, and t represents time.In this paper, the potential flow theory is used to calculate the additional mass, damping and wave force of the mining vessel, and the frequency domain analysis of the coupled motion response between the vessel and the pipe has been made for the test model of the system when the vessel heaves and pitches. The frequency domain hydrodynamic parameters are transformed into the corresponding time domain hydrodynamic parameters by fast Fourier transform (FFT), and the dynamic characteristics of the vessel-pipe coupling motion are obtained. According to the principle of force balance, the dynamic equation of the system can be obtained as:Under the action of waves, the movement of mining vessels mainly includes surge, roll, sway, pitch, heave and yaw30, among them, vessel roll, swell and yaw can be eliminated by dynamic positioning. On the premise that roll can be completely compensated by heave compensation device, this paper studies the longitudinal coupling dynamic characteristics of deep sea mining vessel and lifting pipe.Based on the potential flow theory, the section static water restoring force caused by the displacement change of mining vessel s given by32, which givesThe wave force The impulse response function The radiating force of the fluid is generated by the change of the surrounding fluid due to the motion of the vessel. In view of the linear wave approximation, the dynamic radiation of the ideal fluid in the time domain can be expressed as:According to Cummins pulse principle, the time delay function After transforming the hydrodynamic coefficients of the mining vessel in frequency domain into the time domain, Eq.  takes thIn numerical calculation, the term unrelated to the current velocity  and Eq.  is writt1 and a buffer at the end, as they are modeled by concentrated masses The stepped lifting pipe is composed of four-stage pipes, the top of which is hinged with the mining vessel and synchronized with the movement of the mining vessel, and the bottom of which is generally considered free, as shown in Fig. 35, we have thatFor small diameter and slender lifting pipe, the hydrodynamic force per unit length of the lifting pipe after offset can be calculated by Morrison equationw of the i-th pipe is a function of position and time, denoted as The displacement of any section The vibration differential Eq.  is solveSubstituting  into Eq.Set bi are introduced variables,A harmonic motion with amplitude According to the condition of force continuity, the axial force of the second stage lifting pipe is equal to the sum of the first stage’s axial force and the pump set’s inertia force. At the junction of the third pipe and the fourth pipe, the axial force of the two pipes is equal, and the sum of the axial force and inertia force at the end of the fourth pipe is zero. This means thatSubstituting the modal function Eq.  and the ions Eq.  and contions Eq. , the folMi, Ei, Si,Note that the coefficients In the environment of Matlab R2020, the program (code) is written to model the longitudinal vibration of the pipe of deep-sea mining system, and the complex constants w can be obtained by the following equation:The total axial load of the pipe is composed of the gravity load caused by its own weight and the dynamic load caused by the vibration of the mining vessel. The axial dynamic load of any section Then we can get the dynamic coupling load between the mining vessel and the pipeline as followTherefore, the coupling force It can be seen from the coupling force Eq.  that theThe coupling between the mining vessel and the lifting pipe system is realized by setting boundary conditions at their connecting positions. Because the vessel and the pipe are hinged, there is the same displacement and force in their connection position, and no bending moment occurs. The calculation process of coupling model is shown in Fig. 14. The two numbers refer to the amplitude which relates the amplitude of vessel motion to the amplitude of wave, and the phase which defines the timing of vessel motion relative to the wave, respectively36. During the simulation analysis of mining vessel motion, the wave frequency range is 0.05 Hz ~ 0.5 Hz, and the wave direction range is − 180° ~ 180°. RAO is calculated once every 45° in the wave direction range. The simulation results are shown in Fig. In this paper, the MAFUTA mining vessel is taken as an example, and the boundary element method AQWA is used to analyze the motion of mining vessel, and to predict the response of the mining vessel motion under waves. The software covers the full range of fluid analysis, including transition analysis and response amplitude operators (RAOs) for the calculation of coupling dynamics. Each displacement RAO consists of a pair of numbers that define the vessel response for one particular wave direction and period s, the pitch RAO reaches the maximum of 1.19°/m. For heave and pitch motions, the RAO curves of the mining vessel under the action of the symmetrical wave directions are basically the same, indicating that symmetric mining vessel does not significantly affect motion in horizontal directions. Therefore, the RAO curves of 90°, 135° and 180° wave directions can be used to describe the heave and pitch motions of the mining vessel at the above nine different directions. Meanwhile, the RAO curves at 135° and 180° wave directions are similar, so we choose the wave directions at 90° and 180° as the representative for the dynamic analysis of deep sea mining system.As can be seen from Fig. i-th degree of freedom  and Several frequency values are selected near the resonance frequencies of heave and pitch motions of mining vessels, and the RAO regular wave experiments are carried out at 90° and 180° wave directions. The motion parameters of the vessel model are measured in real time by self-developed programs, and the RAO value under the experimental conditions can be calculated by the following formulaIt is assumed that the mining vessel is considered to be dynamically stable under the action of waves, the comparison between the experimental RAO and the simulated RAO is conducted on aspects of regular waves. As shown in Fig. Assuming that the lifting system is operating under the waves, the motion of the mining vessel affects the vibration displacement and dynamic load of the lifting pipe. During lifting operation, the sea state generally does not exceed level 6. The longitudinal motion of the mining vessel under waves is simulated by applying sinusoidal excitation on the upper end of the lifting pipe model. Through the simulation analysis, the vibration displacement and top tension of lifting pipe under level 6 sea state are obtained without considering the vessel-pipe coupling factor, as shown in Figs.  m and 0.81 m respectively, with a difference of 83.4%. The maximum steady-state top tension value is 9947.26kN and 8579.77kN respectively, with a difference of 13.75%.It can be seen from Figs. The time domain simulation of mining vessel-lifting pipe coupling motion was carried out by using the AQWA software and OrcaFlex software. In Fig. We first time make experiment on the coupling action of the lifting pipe on the vessel motion. When the mining vessel is equipped with a lifting pipe, the current force, pipeline restoring force, damping force and inertia force on the lifting pipe will affect the motion of the mining vessel. By using OrcaFlex software, the heave and pitch motion of the mining vessel with lifting pipe under specific sea states and wave directions are simulated and analyzed. Meanwhile, the 6-DOF motion experiments of the mining vessel model with lifting pipe are carried out. The results of experiment and simulation are shown in Fig. Figure By comparing the motion amplitude of the mining vessel before and after the additional lifting pipe in Table Secondly, we make experiment on the coupling action of the vessel motion on the dynamic response of the lifting pipe. The coupling action of mining vessel motion on the lifting pipe is mainly reflected in the top tension and vibration displacement of the lifting pipe. Orcaflex software is used to simulate the coupling tension and vibration displacement under the condition of level 6 sea state with 90°and 180° wave directions, and the coupling tension is verified by experiments, as shown in Figs. It can be seen from Fig. Considering Figs. As shown in Fig. 2 is 0ton, 30ton and 50ton respectively, and the navigation speed V0 is 0 m/s, 0.8 m/s and 1.5 m/s respectively. OrcaFlex software is used to simulate the coupling tension under the above conditions in time domain. As shown in Fig. Finally, we make simulation on the influence of the buffer mass and the vessel speed on the coupling tension. It is assumed that the wave direction is 90°, the buffer mass MThe mean effective tension and longitudinal amplitude of the lifting pipe at different wave directions are simulated to illustrate the influence of coupling action on the dynamic responses of the lifting pipe along the pipe length. The results are compared with those of the pipeline without considering coupling action, as shown in Fig. As can be seen from Fig. The wave direction and sea state level have a great influence on the dynamic response of vessel-pipe coupling system. At 180° wave direction, the dynamic response is small, and the coupling action time is longer up to 300 s; at 90° wave direction, the dynamic response is significant, and the coupling action time is about 100 s. The difference of heave amplitude, maximum coupling tension, buffer amplitude and vessel-pipe coupling action time are 82.78%, 19.31%, 83.08% and 66.67% respectively under two wave directions of level 6 sea state. The motion response of the mining vessel is positively correlated with the sea state level, and the heave amplitude can reach 3.02 m at level 6 sea state. When the wave direction is 180°, the motion responses of the mining vessel are more significant under different sea states, and the differences of heave amplitude and pitch amplitude are 76.15% and 84.7% respectively. Therefore, the dynamic response of the coupling system can be effectively reduced by reasonably selecting the forward direction of the mining vessel and the operating sea state level in practical engineering.The lifting pipe has a certain influence on the mining vessel motion, and the degree of influence is related to the wave direction. When the wave direction is 90°, the lifting pipe has little effect on the amplitude of the mining vessel motion, and the amplitude of the mining vessel motion with or without the lifting pipe is basically unchanged; When the wave direction is 180°, the heave amplitude and pitch amplitude of the mining vessel with lifting pipe are 31.11%, 33.33%, 33.85% and 1.4% lower than those of the mining vessel without lifting pipe under the level 4 and level 6 sea states, respectively. Therefore, when operating under the level 6 sea state and 90° wave direction, compensation technology should be considered to weaken the heave and pitch motion of the mining vessel, thereby reducing the dynamic response of the system.The coupling effect has a significant impact on the time-domain dynamic response of the lifting pipe, especially in the initial movement stage, the axial tension and longitudinal vibration displacement of the lifting pipe have irregular oscillations, and the system coupling effect is significant. The maximum axial tension occurs at the top of the pipe, and the maximum vibration displacement occurs at the bottom of the pipe. When the sea state is level 6 and the wave directions are 90° and 180° respectively, the difference between the maximum coupling tension and the maximum steady-state tension is 8.6% and 3.33% respectively, and the difference between the maximum coupling vibration displacement and the maximum steady-state vibration displacement of the lifting pipe is 27.4% and 31.3% respectively. The results clearly show the necessity of coupling analysis, which can provide a very intuitive basis for the safety design of key parts of the system.In this paper, a vessel-pipe coupling dynamic model of the deep-sea mining system, considering the ocean current, the motion of the mining vessel, the vibration of the lifting pipe and the coupling between the vessel and the pipe, is established. The hydrodynamic analysis of the mining vessel is carried out by using the finite element software AQWA, and the dynamic performances of the coupling system under different sea states and wave directions are simulated by software OrcaFlex. The vessel-pipe coupling model experiments are carried out, and the simulation results are in good agreement with the experimental results. The main conclusions are as follows:


To evaluate the clinical performance of liquid‐based cytology (LBC), HPV tests and visual inspections with acetic acid or Lugol's iodine (VIA/VILI) as primary screening and triage strategies among Chinese women living with HIV (WLHIV).WLHIV aged 18 years and older were recruited from HIV/AIDS treatment clinic in Yunnan, China from 2019 to 2020. Women were screened with self‐ and physician‐sampling for HPV tests, LBC, and VIA/VILI. Women positive for any HPV or with cytological abnormalities were recalled for colposcopy examination and biopsy when necessary. Clinical performance of primary and triage strategies for detecting cervical intraepithelial neoplasia grade 2 or worse (CIN2+) was evaluated.For primary screening, sensitivity of physician‐HPV tests was 100%, 89.5%, and 100% for hybrid capture 2 (HC2), cobas, and Sansure HPV, and specificity was 80.4%, 85.1%, and 72.0%, respectively. Self‐HPV test achieved considerable performance with physician‐HPV. Sensitivity and specificity were 61.1% and 96.3% for LBC , 40.0% and 77.3% for VIA/VILI. For triaging HPV‐positive women, LBC (ASCUS+), HPV‐16/18 genotyping, and VIA/VILI‐elevated specificity with sensitivity declined 30%–50% compared with HPV screening alone. Restricted HPV genotyping triage (HPV‐16/18/31/33/45/52/58) demonstrated the optimal accuracy , and was similar to HPV‐16/18 with reflex LBC (ASCUS+). Combination antiretroviral therapies (cARTs) <2 years were associated with decreased specificity of HC2  and Sansure HPV .Self‐HPV with restricted genotyping triage is highly recommended for cervical cancer screening for WLHIV in China. Feasible triage to increase HPV specificity among women with short duration of cART is needed. Cytology, HPV test, and visual inspections with acetic acid (VIA) have been recommended for cervical cancer primary screening among general women.Thus, we conducted a study to evaluate the clinical performance of primary screening with HPV tests, cytology, and VIA or visual inspections with Lugol's iodine (VIA/VILI) and several triage strategies for HPV‐positive individuals among WLHIV in a high HIV epidemic and low‐resource area in China. Clinical performance of primary screening was further evaluated by the stratification on CD4 count, cART duration, and age.22.1Participants were recruited from the Department of AIDS antiretroviral therapy at the Third People's Hospital of Kunming, Yunnan province of China form January 2019 to August 2020. Inclusion criteria were: (1) HIV seropositive and with ongoing cART, (2) aged 18 years and older, (3) had initiated sexual activity, (4) able to both physically and mentally undergo cervical sampling, and (5) had an intact cervix and were no currently pregnant. After informed consent, participants were interviewed individually by a trained health worker for demographic characteristics. HIV‐related characteristics were extracted from medical records. The study design is illustrated in Figure 2.2Women collected one vaginal self‐sample with vaginal brush (Qiagen) under the instruction of healthcare providers, and placed at PreservCyt solution . Two exfoliated cervical cell samples were then collected by a trained gynecologist and placed at PreservCyt solution and hybrid capture 2 (HC2) liquid medium (Qiagen), respectively. VIA/VILI was performed on 180 women after sample collection by the trained gynecologist following the International Agency for Research on Cancer (IARC) guidelines.2.3® (Sansure Biotech) and cobas 4800 HPV testing® (Roche Diagnostics). The second physician‐collected samples were prepared for Digene HC2 HPV testing® (Qiagen). Residuals of the first physician‐collected samples were processed for cytology slides and interpreted by experienced cytologists.All collected samples were placed at 4° refrigerator and transferred to KingMed diagnostic laboratory. Self‐collected and the first physician‐collected samples were prepared for Sansure HPV testing® is PCR‐based pioneered One‐step Fast Release technology, using real‐time fluorescent quantitative PCR to target 15 HPV types, including 13 high‐risk types: HPV‐16/18/31/33/35/39/45/51/52/56/58/59/68, and 2 possibly high‐risk types: HPV‐53 and 66, and has been approved by the European Union Certificate. HPV status is measured by the cycle numbers observed (Ct) when the fluorescent signal reaches the set type‐specific threshold. A Ct ≤39 is considered HPV‐positive and a Ct >39 is considered negative.Sansure HPV testCytology results were reported according to the Bethesda 2014 classification system: negative for intraepithelial lesion or malignancy (NILM), atypical squamous cells of undetermined significance (ASC‐US), atypical squamous cells cannot exclude HSIL (ASC‐H), atypical glandular cells, low‐grade squamous intraepithelial lesion (LSIL), high‐grade squamous intraepithelial lesion (HSIL), squamous cell carcinoma (SCC), adenocarcinoma in situ (AIS), and adenocarcinoma (ADC).Women positive for any HPV or with cytological atypical squamous cells of undetermined significance or worse (ASCUS+) or unsatisfactory were recalled for colposcopy examination, and biopsied if any abnormality was identified. Endocervical curettage was performed if the squamocolumnar junction was invisible. Biopsy tissues were immediately immersed in 10% buffered formalin and transported to KingMed diagnostics for processing and diagnosis by experienced pathologists who were blinded to other screening results. Pathology results were reported as normal, cervical intraepithelial neoplasia grade 1 (CIN1), grade 2 (CIN2), grade 3 (CIN3), microinvasive carcinoma (MIC), SCC, AIS, and ADC. Women with CIN2 or worse (CIN2+) were recommended for treatment according to clinical guidelines. Women who were negative for all tests (cytology < ASCUS and no HPV detected) were considered to be negative for the outcome of CIN2+.2.4LBC1.1LBC (ASCUS+)1.1LBC (LSIL+)HPV tests2.1HC22.2Triage of HC2‐positive women 2.2.1LBC (ASCUS+)2.2.2LBC (LSIL+)2.2.3VIA/VILI2.3Physician‐cobas HPV2.4Triage of physician‐cobas HPV‐positive women2.4.1LBC (ASCUS+)2.4.2LBC (LSIL+)2.4.3HPV‐16/18 genotyping2.4.4HPV‐16/18 genotyping with reflex LBC (ASCUS+) for those without HPV‐16/182.4.5VIA/VILI2.5Self‐cobas HPV2.6Triage of self‐cobas HPV‐positive women2.6.1HPV‐16/18 genotyping2.7Physician‐Sansure HPV2.8Triage of physician‐Sansure HPV‐positive women2.8.1LBC (ASCUS+)2.8.2LBC (LSIL+)2.8.3HPV‐16/18 genotyping2.8.4HPV‐16/18/31/33/45/52/58 genotyping2.8.5HPV‐16/18 genotyping with reflex LBC (ASCUS+) for those without HPV‐16/182.8.6VIA/VILI2.9Self‐Sansure HPV2.10Triage of self‐Sansure HPV‐positive women2.10.1HPV‐16/18 genotyping2.10.2HPV‐16/18/31/33/45/52/58 genotypingVIA/VILIWe evaluated the following primary screening and triage strategies:Clinical performance of primary screening and triage strategies was evaluated with sensitivity, specificity, positive predictive value (PPV), negative predictive value, area under the curve (AUC), detection rate, and colposcopy referral. Relative sensitivity, specificity, detection rate, and colposcopy referral  were also calculated with 95% confidence interval (95% CI). Significant difference was considered if the 95% CI of the relative values was entirely above or below one. Sensitivity and specificity were further stratified by duration of cART, CD4 count, and age and were compared using Chi‐square tests and logistic regression. Sensitivity or specificity found to be statistically different for varying strata in univariate analysis was included into multivariate analysis. Data were analyzed on SPSS 20.0 and R software 3.6.2.33.1Of 409 recruited women, 37 women lost to follow‐ups for colposcopy. Three hundred and seventy‐two women with adequate outcomes were included into the final analysis. The median age was 40 (35–47) years, 60.2% have been married, 71.2% had education at junior high school or below, 48.1% were unemployed, and 42.7% were farmer or rural migrant worker. Median age at sexual debut and first delivery was 20 (19–22) and 24 (22–27) years, respectively. The median years living with HIV and duration on cART was 5 (2–9) and 4 (2–8) years. The median values of nadir (at cART initiation) and current CD4 count were 237 (IQR 137–326) and 550 (IQR 401–704) cells/µl, the initial (at cART initiation) and current HIV viral loads were 50  and <50 (IQR <50 to <50) copies/ml, respectively. Data are shown in Table 3.23.2.1Positivity of LBC (ASCUS+), LBC (LSIL+), HC2, cobas, Sansure HPV, and VIA/VILI was 6.5%, 4.1%, 23.7%, 18.7%, 31.7%, and 23.2%, respectively Table . In comp3.2.2Positivity of self‐HPV was 31.7% for cobas and 36.6% for Sansure HPV. For CIN2+ detection, cobas self‐HPV demonstrated a slightly higher sensitivity  but lower specificity  with similar AUC but higher colposcopy referral rate  compared to physician‐HPV Table . For San3.2.3Compared with cobas, Sansure HPV had a marginally increased sensitivity , lower specificity , and similar AUC, with higher colposcopy referral  in detecting CIN2+ Table . While S3.33.3.1Among HPV‐positive women, the test positivity of triage strategies is shown in Table 3.3.2Compared to HPV test without triage, 8%–32% specificity was elevated  with insignificant change on PPV and AUC by VIA/VILI triage, while the sensitivity declined more than 50% (ratio: 0.40–0.50), with largely decreased colposcopy referral (ratio: 0.22–0.38) Table .3.3.3Compared with HPV primary screening, triage with HPV‐16/18 genotyping demonstrated 13%–32% increased specificity  and elevated PPV , with approximately 30% reduced sensitivity and a mild decrease on AUC at CIN2+ threshold Table . HPV‐16/3.4p < 0.05) and HC2  was decreased among women with lower current CD4 count (<350 cells/µl) (Table p < 0.001), cobas , and Sansure HPV  was observed (Table p < 0.05) was observed among women aged <40 years   demonstrated inferior sensitivity while highest specificity, VIA/VILI showed the lowest sensitivity and specificity. For triage strategies for HPV‐positive women, VIA/VILI triage showed unsatisfactory performance, HPV‐16/18 genotyping demonstrated comparable performance to LBC (ASCUS+) triage, while HPV‐16/18/31/33/45/52/58 and HPV‐16/18 with reflex LBC achieved optimal clinical accuracy. Specificity of HPV tests was declined among women with short duration of cART.Successful cervical cancer control has been achieved by organized cytology screening in high‐income countries. In China, cytology is still the main primary screening method for national cervical cancer screening since 2009 for general women. LBC (ASCUS+) in this study showed inferior sensitivity and similar specificity compared to Chinese general women (80.7% and 94.0%),HPV test in this study showed similar sensitivity but lower specificity compared to that of in Chinese general women ,Immunosuppression may affect the screening accuracy among WLHIV. Similar with African WLHIV,To the best of our knowledge, this study is first of its kind evaluating the clinical performance of multiple cervical cancer screening strategies concurrently among Chinese WLHIV, including LBC, hybrid capture‐ and PCR‐based HPV tests, and visual inspections, as single or consecutive triage algorithms, by physician‐ and self‐sampling. In addition, stratification analyses of clinical accuracy on CD4 count, cART duration, and age were conducted to provide the evidence for specific screening guidance. Furthermore, our study has demonstrated the potentiality of novel self‐Sansure PCR HPV test serving as a low‐cost alternative for future “point‐of‐care” algorithm among WLHIV. The major limitation of the study is the limited sample size, and not all participants were provided with VIA/VILI inspections.In conclusion, HPV test is highly recommended for primary cervical cancer screening among WLHIV in China, while high‐quality cytology is recommended if HPV test is not available. For HPV‐positive triage, HPV‐16/18/31/33/45/52/58 was highly recommended, and HPV‐16/18 with reflex LBC cytology triage is recommended if HPV genotyping is not feasible. The affordable one‐time self‐HPV test with the restricted genotyping triage would be the most feasible and optimal cervical cancer screening strategy for WLHIV in China, especially in high HIV epidemic settings. Large‐scale prospective study is suggested to supplement evidence on clinical performance of screening strategies, and address the initiated age of screening and screening intervals for WLHIV.The authors have no conflicts of interest to declare.The study protocol was approved by the Ethical Review Committees of National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.Data availability statement The data that support the findings of this study are available from the corresponding author upon reasonable request.Tables S1–S9Click here for additional data file.


Among the over 200 human papillomavirus (HPV) genotypes identified, approximately 15 of them can cause human cancers. In this review, we provided an updated overview of the distribution of cancer-causing HPV genotypes by countries in East, Southeast and South Asia. Besides the standard screening and treatment methods employed in these regions, we unravel HPV detection methods and therapeutics utilised in certain countries that differ from other part of the world. The discrepancies may be partly due to health infrastructure, socio-economy and cultural diversities. Additionally, we highlighted the area lack of study, particularly on the oncogenicity of HPV genotype variants of high prevalence in these regions.Human papillomavirus (HPV) infection remains one of the most prominent cancer-causing DNA viruses, contributing to approximately 5% of human cancers. While association between HPV and cervical cancers has been well-established, evidence on the attribution of head and neck cancers (HNC) to HPV have been increasing in recent years. Among the cancer-causing HPV genotypes, HPV16 and 18 remain the major contributors to cancers across the globe. Nonetheless, the distribution of HPV genotypes in ethnically, geographically, and socio-economically diverse East, Southeast, and South Asia may differ from other parts of the world. In this review, we garner and provide updated insight into various aspects of HPV reported in recent years (2015–2021) in these regions. We included: (i) the HPV genotypes detected in normal cancers of the uterine cervix and head and neck, as well as the distribution of the HPV genotypes by geography and age groups; (ii) the laboratory diagnostic methods and treatment regimens used within these regions; and (iii) the oncogenic properties of HPV prototypes and their variants contributing to carcinogenesis. More importantly, we also unveil the similarities and discrepancies between these aspects, the areas lacking study, and the challenges faced in HPV studies. Papillomaviruses, belong to the family of Papillomaviridae, are small and non-enveloped viruses of 52–55 nm in diameter. The virion contains a double stranded circular DNA genome that generally encodes six “Early” (E) genes, two “Late” (L) genes, and a long control region (LCR) or upper regulatory region (URR).The human papillomavirus (HPV) “Early” open reading frames (ORFs) contain E1, E2, E4, E5, E6, and E7, all of which play pivotal roles during viral replication and tumourigenesis. E1 functions as an ATP-dependent viral DNA helicase ,4. HPV eAlpha, Beta, Gamma, Mu, and Nu [Alpha-PV group. The high-risk HPVs (hrHPVs) contribute to cancers of the uterine cervix, head and neck, vulva, anus, and penis. Meanwhile, skin and oral cavity swabs harbour abundant Beta- and Gamma-PVs, which suggests that the divergence of HPVs followed prior adaptation in specific niches [The HPV “Late” (L) ORFs encode for the major (L1) and minor (L2) capsid proteins. Since L1 ORF is the most conserved PV gene, it is used for phylogenetic classification and for HPV vaccine production . L2 play, and Nu ,13. The c niches ,15,16.East, Southeast, and South Asia encompass individuals of different ethnicities and genetic backgrounds who live in environmentally and socio-economically diverse countries. Due to these diversities and the discrepancies in study sample size and detection methods used in different studies, the reported range of HPV-positive rates remains large. The overall HPV-positive detection rate in normal women ranges from 7.2%  to 41.7% (Hong Kong). The HPV genotypes commonly detected in these regions are HPV16, 18, 52, 58, 31, 33, 35, 39, 45, 51, 56, 59, 68, 6, 11, 42, 43, and 81 ,58,59,60Globally, the most prevalent hrHPV genotypes detected in human cancers are HPV16 15.56–83.78%) and 18 (3.4–41.1%). The other most prevalent hrHPV genotypes include HPV31 (1.37–8.89%), HPV33 0.74–9.1%), HPV35 (0.5–3.2%), HPV39 (0.7–13.33%), HPV45 (0.8–9.1%), HPV51 (0.3–18.8%), HPV52 (1.08–40.74%), HPV56 (0.2–9%), HPV58 (1.9–15.6%), HPV59 (0.6–4.4%), and HPV68 (0.4–11.11%) [–83.78% a–9.1%, HPThe HPV genotypes detected in head and neck squamous cell carcinoma (HNSCC) might differ from those in cervical cancers. HPV16 remains the most prevalent detected type (1.6–60.7%), followed by HPV31 (1.6–79.2%), HPV18 (0.7–15.1%), HPV56 (0.5–15.1%), HPV52 (2%), HPV33 (1.2%), and HPV35 (0.5%) ,101,102.As China is a geographically wide country, we collated studies on the distribution of HPV genotypes according to traditional geographical regions: East, South, Southwest, Northwest, Central, North, and Northeast China. In general, the most prevalent hrHPV genotypes detected in normal uterine cervixes were found to be HPV16, 52, and 58, followed by HPV18, 31, 33, 35, 39, 45, 51, 56, 59, and 68 ,44,45,63The hrHPV genotypes that are most frequently detected in cervical lesions are HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 ,70,71. AIn Japan, South Korea, and Mongolia, the three most prevalent hrHPV types detected in normal cervical samples were shown to be HPV16, 18, and 31 ,49,50,51In normal cervical samples, HPV16 and 18 are the most prevalent types detected, followed by HPV52 and 58 ,55,56,57In cervical lesions, the detection of HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 has been common ,76. IntrThe pattern of HPV genotypes detected in South Asia differs from East and Southeast Asia. The ranking of HPV genotypes detected in normal cervical samples was revealed to be HPV16, HPV39, 58, 33, and 18 ,60,105. For HNSCC samples with a single HPV infection, most of the samples have been found to be positive for HPV16. Other detected hrHPV types include HPV31, 18, 56, 52, 33, and 35 ,101,102.Screening is an essential way to detect and control HPV infection. To date, various guidelines on the screening of HPV have been published, including the World Health Organization (WHO) HPV Laboratory Manual . However® polyester swabs [Sample collection methods are clearly described in the WHO HPV Laboratory Manual, and they have been widely adopted by many countries within the region. However, the sampling tools used by health practitioners may depend on anatomical sites, the availability of medical supplies, and resources. In general, exfoliated tissues from uterine cervix, skin, and other anogenital areas are collected using swabs/brushes made of nylon, polyester, or cotton. The samples are then stored in preservation transport media. Example of used swabs/brushes include iCleanhcy flocked swabs , FLOQSwab R100 and FLOQSwab U80  ,116, ander swabs . Sampleser swabs , Preserver swabs , LiquiPrer swabs , and Speer swabs . For theer swabs .In recent years, self-sampling has become popular, partly due to convenience and cultural conservation. Another benefit to self-collection is that the HPV-positive detection rate in tissue, ranging from dysplasia to high grade lesions, is comparable to that collected by medical professionals, as shown by studies conducted in Thailand, Hong Kong, and mainland China ,122,123.The detection of abnormal cervical tissues using the Pap test, a multichromatic cytological stain, has been widely adopted by diagnostic laboratories for decades. Nonetheless, the Pap test does not discern whether the changes in cell morphology and proliferation pattern are due to HPV infection. A cost-effective way to make this discernment is by performing the triage HPV test .HPV has adopted an intelligent way to replicate, along with host cell differentiation and maturation, without eliciting host cell lysis and robust immune response. In addition, the transient HPV infection makes HPV serological test inaccurate. Therefore, a sturdy way to detect HPV is by detecting viral nucleic acids, mainly through targeted polymerase chain reaction (PCR) using consensus or type specific primer pairs. Commonly used primer pairs include the standard PGMY09/11 or GP5+/GP6+ L1 consensus primers ,130,131 ®, CapitalBio Corporation, Beijing, China) [® HPV Genotyping Diagnosis Kit  [China the country that produces most of the commercial HPV nucleic acid test kits in the world . In gene, China) ,160, HPV, China) , HPV Gen, China) , PCR-RBD, China) ,161,162,, China) ,164, SNI, China) , and Gen, China) . In addi, China) . This laIn Hong Kong, our team established a joint effort with a research team in Macao to study HPV prevalence. We utilised modified GP5+/6+ L1, designated as GP5+/GP6+_52HK primers, for the better detection of HPV52 variants of high prevalence in East Asia. We also performed HPV genotyping in oral rinse samples using the cutting-edge next generation sequencing (NGS) . Other u®  [™ Kit  [In South Korea, high-throughput HPV genotyping using chip-based technology appears to be preferred in recent years. For instance, the MyHPV Chip Kit, Korea) , HPV Gen, Korea) , and Che, Korea)  were dev, Korea)  and an H, Korea) .In Japan, Genosearch-31 (GS-31) , a high-resolution PCR-SSOP-Luminex-based method that combines PCR and a sequence-specific oligonucleotide probe (SSOP) with a Luminex xMAP system has been used to discriminate 31 HPV genotypes in various studies ,171,172.® Human Papillomavirus Genotyping Detection Kit from Amoy Diagnostics, Xiamen, China) [® E6, E7 mRNA Kit, incellDx, CA, USA) [Commercial kits run on different detection platforms, including the real-time PCR  , gene ch, China) ,156, floCA, USA) , PCR-ResCA, USA) , microplCA, USA) ,179, GenCA, USA) , and AnyCA, USA) .In Thailand, prior to using commercial kits for HPV genotyping, Nopmaneepaisarn and colleagues used a DNA ISH HPV III Family Probe  to identify hrHPV. This method can detect episomal and integrated viral genome expression . In-housIn addition to following the standard and validated assays for HPV screening, various laboratories also use in-house PCR-based ,185 and Some of the non-validated commercial HPV detection kits assess the sensitivity and specificity of their products via comparisons to validated assays. For instance, when comparing PapType HPV assay to Hybrid Capture 2 (HC2) and Linear Array (LA) HPV tests, LA (91.6%) was found to be more sensitive than PapType (90.3%) and HC2 (79.8%), while the specificity of HC2 (55.3%) was found to be higher than PapType (52.5%) and LA (51.7%) . The HPVIn the advanced medical world, various anti-cancer drugs are available to treat cancers, including cancers caused by HPV. Unfortunately, none of these anti-cancer drugs are tailored for HPV-associated cancers. In this part, we describe the treatment regimens for HPV-associated cancers employed within South, East, and Southeast Asia, most of which are standardized and well-established treatment options, including HPV vaccines, surgical ablation, photodynamic therapy, radiotherapy, chemotherapy, immunotherapy, and anti-viral agents. However, certain countries may use locally produced drugs to tackle the disease.®9, that been shown to be plausibly effective in lowering the risk of HPV infection and providing immune protection in both women and men [4-adjuvant vaccine  in providing immune protection for HPV16/18, as well as at least 65% cross-protection for HPV31 and 45 [Escherichia coli provided economical and safe alternative immune-protection against HPV infection [The implementation of HPV vaccination programs has curtailed the incidence of HPV-associated cancers successfully, particularly in HPV-naïve individuals. The US Food and Drug Administration (FDA) has approved HPV prophylactic vaccines, including Gardasil, Cervarix, and Gardasil and men . Results1 and 45 . In Chinnfection ,195. A Snfection .The exploration of HPVE6 and/or E7 DNA therapeutic vaccines has become increasingly popular. These vaccines possess several advantages over prophylactic vaccines. Phase I and II clinical trials have shown that they are able to stimulate a broad range of immune response in immunocompetent hosts, as well have possessing simplicity and low-cost production . In an iFor precancerous cervical lesions, as recommended in WHO guidelines , many coStaphylococcus sp. infection. Remedies to this are treatment with topical fusidic acid or mupirocin [Photodynamic therapy (PDT) serves as a treatment option when surgery is not recommended. PDT is an emerging non-invasive curative measure for HPV-associated cancers. Prior to PDT, the treatment of cervical warts or high-grade lesions with 5-aminolevulinic acid (ALA)  or polyhupirocin .HPV-bearing cancer cells are sensitive to radiation and prone to cell death stimuli. These issues make radiotherapy and chemotherapy effective in both HPV-associated cervical cancers and HNSCC. Generally, these treatment modalities have better impacts on the early stages of ICC. The common chemotherapeutics for both localized cervical cancer and HNSCC include docetaxel, cisplatin, and 5-fluorouracil ,214.Patients with locally advanced cervical cancer treated with an extended field of external-beam radiotherapy covering whole pelvis and extending to para-aortic lymph nodes may have an added advantage. This approach was found to better reduce mortality and para-aortic lymph node recurrence than pelvic radiotherapy/chemotherapy . MeanwhiFor HPV-related HNSCC, the prognosis and survival of patients at early stages are generally good, so chemotherapy may be not be required. Rather, transoral surgery or radiotherapy could be sufficient to curb disease progression and offer an elevated quality of life. In advanced stages of HPV-associated oropharyngeal squamous cell carcinoma (OPSCC), induction chemotherapy and chemoradiotherapy with cisplatin tends to be the choice of treatment . A JapanImmunotherapy has emerged as a pivotal treatment strategy for cancers, including HPV-associated cancers. The prime choices include immune checkpoint inhibitors targeting programmed cell death 1 (PD1)/ligand 1 (PDL1), like pembrolizumab and nivolumab. While HPV infection alone also promotes the infiltration of T cells into tumour sites , immunotLactobacillus casei, a probiotic, with HPV16E7 expressed on its surface. Intriguingly, the treatment appeared to be safe and effective in 75% of patients, and it was able to provoke HPVE7-specific immune IgG production [The efficacy of other immune modulators has also been explored. In South Korea, women were recruited to study the safety and efficacy of poly-gamma glutamic acid (γ-PGA) in treating CIN I cervical cancer and clearing HPV infection. This Phase IIb clinical trial revealed that the treatment enhanced the clearance of HPV . In anotoduction .® cream  onto intravaginal lesions could effectively suppress HPV oncogenes expression [To date, the choice of HPV-specific antiviral agents is limited. Several studies have shown the clinical efficacy of antimicrobial agents in dampening the HPV viral life cycle and stimulating the host HPV-specific immune response. In a Phase I/IIa clinical trial, Jiang and colleagues showed that 3-hydroxyphthalic anhydride-modified bovine beta-lactoglobulin (JB01) topical treatment resulted in a marked reduction of HPV viral load in women with hrHPV infection . Meanwhipression .For treating an HPV-attributable benign condition, like recalcitrant warts and laryngeal papillomatosis, FIT039  inhibitor and an antiviral agent ) togetheRepurposing anti-cancer drugs can fast-tract the use of these drugs to treat HPV-associated cancers in clinics, e.g., ormeloxifene  and gefiIn China, traditional medicines have been used to treat benign hyperplasia caused by HPV. For instance, a four-month treatment with a topical cream composed of paiteling  showed a 92% efficacy in reducing the abundancy of HPV40, 35, and 25 in condyloma acuminatum without recurrence . The BaoJuglans mandshurica , juglone [Cudrania tricuspidata stem [Pleurotus ferulae polysaccharides (PFPS), a traditional Chinese medicine, works as an adjuvant in HPV dendritic cell-based vaccines and showed a great efficiency in reducing tumour volume in an in vivo model [Curcuma longa), neem (Azadirachta indica), tulasi or holy basil (Occumum sanctum), and ginger  can instigate the activation of the apoptotic pathway in an in vitro system [Some preclinical studies have unravelled the anti-tumour properties of natural compounds and traditional medicines using cell-based and animal models. Extracts from  juglone , and Cudata stem   , micro-RThe vast majority of studies have focused on the oncogenic properties of HPV16 and 18, as well as 31 E6 and E7 prototypes. Various studies have also focused on identifying commonly circulating HPV variants in their locality. Through our search, laboratory evidence connecting the link between natural occurring amino acid changes of HPV oncoproteins to their mechanistic roles in promoting cancer progression is lacking. In East Asia, our team identified several HPV52 and 58 variants. Strikingly, we figured out that these variants carry amino acid mutations within E6 for HPV52 and within E7 in HPV58. In the following sections, we describe the impact of the amino acid mutations of HPV52 E6 and HPV58 E7 variants on their oncogenic properties.Though HPV52 is the seventh most commonly detected HPV type worldwide ,250, itsHPV58 is the second or third most prevalent HPV genotype detected in cervical cancer in East Asia ,250. StrIn low- and middle-income countries, the general population may have some grasp on cervical cancer. Their understanding about proper hygiene, the causal link between HPV and cancer, remains poor, and their knowledge about the HPV vaccine as a preventive measure is lacking ,262. In To date, HPV-targeted therapeutics are still lacking. Treatment modalities for HPV-associated cancers are either broad-spectrum antimicrobial or antiviral agents, or they are non-HPV cell-specific killing agents. Despite the high efficacy of vaccines, radiotherapy, chemotherapy, immune modulators, and antiviral agents in treating HPV-related cancers, it is unclear whether the treatment modalities can offer a complete HPV clearance, dampening viral persistency and latency. The downside of radiotherapy and chemotherapy is that these treatment regimens are non-cell type selective and may not eliminate all HPV-bearing cells. HPV DNA has been shown to be detectable in 21% of patients post-treatment with radiotherapy alone or with docetaxel, carboplatin, cisplatin and 5-fluorouracil (5-FU). This has led to recurrence, disease progression to distant metastasis, and a decreased patient survival rate ,264,265.The databases used for the search of primary literature were PubMed, MEDLINE (EBSCOhost), Scopus, and Google Scholar. To ensure all relevant influential factors were included in the search strategy, the MeSH terms “human papillomavirus in Asia,” “human papillomavirus in (name of country),” “diagnosis of human papillomavirus,” “human papillomavirus screening,” “prevalence of human papillomavirus in Asia,” “HPV treatment,” “therapy for human papillomavirus cancers,” and “human papillomavirus carcinogenesis” were used in search engines.n = 722). All the non-relevant and duplicate studies were rejected (n = 472), and the remaining 250 studies were moved to the third stage. In the third stage, we scrutinized the full content of the articles (n = 250), particularly the methodology and results sections. All data were arranged, grouped, and compared for their similarities and discrepancies.We reviewed all relevant accepted publications within the recent 5 years (2015–2020). Articles that were deemed eligible and appropriate for inclusion in this review included: (1) topics covering HPV genotypes of high importance in South, Southeast, and East Asia, particularly on prevalence, HPV-associated cancers, diagnostic methods, treatment modalities, and oncogenic potential of E6- and E7-encoded by HPV; (2) articles written in English; and (3) articles with their full text accessible. There were three stages of the selection process. In the first stage, we assessed a total of 9320 articles on HPV in South, Southeast, and East Asia that were deemed appropriate for this review. Inappropriate articles (n = 8598), including articles that did not include regions and countries of interest, review articles, and meta-analysis articles, were excluded. In the second stage, we assessed the abstracts of the appropriate articles (