A tablet-based questionnaire was used to collect sociodemographic and behavioral characteristics. An anal brush sample was collected for HPV testing and genotyping. Participants were followed up 12 months after enrolment. (3) Results: A total of 196 participants completed two HPV tests with a median age of 27.3 (interquartile range (IQR) 24.0–32.8) years. Rate of incidence, persistence, and clearance for HPV among MSM were 31.3  24.7–39.2), 47.9 (36.8–61.3), and 122.5 (104.3–143.0) per 1000 person months (pm), respectively. HPV 16 (4.1/1000 pm) had the highest incidence rate, and HPV 6 (47.4/1000 pm) had the highest persistence rate. Having lower education and engaging in receptive anal intercourse were potential risk factors of HPV incidence. A higher incidence rate was observed among younger MSM. (4) Conclusions: The high incidence and low clearance of anal HPV highlight the necessity of HPV vaccination among MSM. Further studies are needed to clarify the HPV dynamics at multiple anatomical sites and the burden of HPV-related diseases among MSM. Anal cancer, with increased incidence in recent decades in many countries ,3,4,5,6,It was estimated that the incidence of anal cancer was 85 and 19 per 100,000 person years for HIV-positive and HIV-negative MSM, respectively . MSM alsHPV vaccination is effective in preventing HPV-related diseases, including anal cancer . While aIncidence, persistence, and clearance of HPV among MSM has been reported in several studies conducted in high-income countries, including Spain and The Netherlands ,20. Howep < 0.001), more likely to be recruited from Shenzhen, and less likely to be recruited from Wuxi than men who were only tested at baseline. All other measured sociodemographic characteristics and sexual health behaviors did not differ significantly between the two groups.A total of 196 participants completed two HPV tests, accounting for 36.5% (196/537) of MSM tested for HPV at baseline. Of the 196 men who completed two HPV tests, 14 (7.1%) were HIV-positive at baseline, and the median time interval between two HPV tests was 12.4 months. More than half were aged 20–29 years, enrolled from Guangzhou, white-collar workers, and unmarried, and had a monthly salary ≥CNY 5000, ≥6 sexual partners in lifetime, and ≥3 years of sexual experience. Sociodemographic and behavioral characteristics comparing MSM who completed two HPV tests and MSM only tested at baseline are presented in Genotype-specific rates of anal HPV incidence, clearance, and persistence are shown in Seventy-seven incident infections were observed. Rates of incidence for any HPV, any high-risk HPV, and any nonavalent vaccine-preventable HPV among MSM were 31.3 (95% CI 24.7–39.2), 17.1 (12.3–23.1), and 14.2 (9.9–19.8) per 1000 person months (pm), respectively. The five HPV genotypes that caused the most incident infections were HPV 16 (4.1/1000 pm), 52 (3.1/1000 pm), 18 (2.1/1000 pm), 58 (1.8/1000 pm), and 11 (1.8/1000 pm).Sixty-three persistent infections were observed. Rates of persistence for any HPV, any high-risk HPV, and any nonavalent vaccine-preventable HPV among MSM were 47.9 (95% CI 36.8–61.3), 41.2 (29.2–56.6), and 41.0 (28.9–56.5) per 1000 pm, respectively. The five HPV genotypes responsible for the most persistent infections were HPV 6 (47.4/1000 pm), 39 (43.0/1000 pm), 58 (43.0/1000 pm), 33 (41.7/1000 pm), and 51 (37.3/1000 pm).A total of 161 cleared infections were observed. Rates of clearance for any HPV, any high-risk HPV, and any nonavalent vaccine-preventable HPV among MSM were 122.5 (95% CI 104.3–143.0), 87.9 (69.8–109.2), and 80.9 (63.4–101.8) per 1000 pm, respectively. The five HPV genotypes that were most frequently cleared during the 12-month follow-up period were HPV 26 (90.9/1000 pm), 67 (90.9/1000 pm), 54 (90.9/1000 pm), 66 (88.2/1000 pm), and 31 (87.0/1000 pm).Among all participants, 60 (30.6%) men had persistent infection with any HPV genotype, 45 (23.0%) men infected with HPV at baseline cleared all infections at follow-up, 20 (10.2%) men with no anal HPV infection at baseline had acquired HPV at follow-up, and 71 (36.2%) men had no evidence of anal HPV infection at baseline and follow-up. More than half  of all men were infected with a high-risk HPV genotype at one or both timepoints. HPV 16 (9/174), 52 (7/178), and 18 (5/188) were the most common incident genotypes. HPV 6 (10/18), 16 (8/22), and 58 (8/16) were the most common persistent genotypes. HPV 16 (14/22), 52 (14/18), 11 (12/16), and 84 (11/11) were the most common cleared genotypes. HPV18 and HPV84 infections at baseline achieved 100% clearance, while only 63.6%, 44.4%, and 75.0% of infections with HPV16, HPV6, and HPV11 were cleared, respectively. Distribution of incident, persistent, and cleared infections for each HPV genotype among all participants is shown in Age at recruitment, educational level, and profession were associated with HPV incidence in the univariate model. In the multivariable model, those who had at least a university education  were less likely to acquire anal HPV compared to men who had not attended university. Men who engaged in receptive anal intercourse  were more likely to have incident anal HPV compared to men who did not engage in receptive anal intercourse. Age at recruitment, site of enrollment, and profession were associated with HPV clearance in the univariate model. In the multivariable model, men aged ≥ 33 years  were less likely to clear HPV infection compared to men aged < 23 years. Men enrolled from Wuxi  and men had a profession of teacher/institution staff  were more likely to clear HPV infection compared to men enrolled from Guangzhou and men with white collar employment, respectively. Results of univariate and multivariable analyses on incidence and clearance of anal infection with any HPV genotype among MSM are shown in Incident anal HPV infection was highest in men aged < 23 years (45.0/1000 pm), followed by men aged ≥ 33 years (35.8/1000 pm), and lowest in men aged 23–27 years (23.1/1000 pm). A similar trend was observed in incident infection with low-risk HPV genotypes, with the highest incidence rate (26.0/1000 pm) among men < 23 years and the lowest rate (6.1/1000 pm) among men aged 28–32 years. Men aged 28–32 years had the highest rate of incidence anal HPV infection with the nine genotypes prevented by the nonavalent HPV vaccine (20.2/1000 pm), while men aged 23–27 years had the lowest rate of incident infections with these genotypes (10.5/1000 pm). Men aged 28–32 years had the highest clearance rate (146.5/1000 pm) and men aged ≥ 33 years had the lowest clearance rate for infection with any HPV genotype (98.6/1000 pm). Rates of anal HPV incidence, persistence, and clearance across age groups are shown in This longitudinal study of 196 MSM in China attending sexual health clinics and community-based organizations is the first study to investigate the incidence, persistence, and clearance of anal HPV among MSM in China. We found high rates of incident anal HPV in this population, particularly infection with HPV16 and HPV18 genotypes. Persistence of infection with HPV6 and HPV16 genotypes was common. Over half of MSM were detected with any high-risk HPV genotype at one or both visits during our study. Incidence rates of anal HPV were higher among MSM aged < 23 years and clearance rates of anal HPV were lower among MSM aged ≥ 33 years.We found the incidence of any HPV, high-risk HPV, and HPV16 at the anal site among MSM in China was 31.3, 17.1, and 4.1 per 1000 pm, respectively. Since no global systematic review and meta-analysis has been published on HPV incidence and clearance among MSM, it is hard to compare the results of our study with global averages. We can compare rates to those reported in other countries, although there are significant differences in study design, source population, and laboratory method between ours and previous studies. Incidence rates of infection with anal HPV among general MSM in this study are higher than or similar to those among HIV-negative and even HIV-positive MSM in some countries ,22,23,24Similar to most countries, HPV16, which is the main causative agent of anal cancer, had the highest incidence rate of anal infection among MSM in China. We also found HPV16 was the most prevalent genotype among MSM in a previous cross-sectional study . MSM in Multiple studies in other countries have shown that HPV 16 and 18 are genotypes with the lowest rates of clearance ,25,30,31We found a lower level of education was associated with higher incidence of HPV infection. This risk factor for HPV infection has not been reported in previous studies. A recent systematic review and meta-analysis of HPV prevalence among MSM in China also showed MSM with a lower level of education had higher HPV prevalence . MSM whoOur study has several limitations. First of all, only two time points of measuring were included in this study, which could not exhibit a continuous dynamic change of HPV infection among MSM and could only provide a rough estimation of epidemiological indicators. The limited number of HIV-positive MSM impeded a precise estimation of incidence and clearance rate in HIV-positive individuals. Some data were collected through self-report in questionnaires and, therefore, may be impacted by recall or social desirability biases. We did not swab the urethra or oropharynx of men in this study for HPV testing and, therefore, rates of total HPV infection among MSM were likely underestimated. Only one follow-up visit 12 months after enrollment was performed, so some transient incident infections might be missed during the follow-up. Participants willing to provide samples at two timepoints across a long follow-up period were limited, which may have affected a precise assessment of epidemiological indicators to some extent. However, the results of comparing sociodemographic and sexual behavioral characteristics between MSM tested twice and MSM tested once showed that participants retained in the follow-up might be able to represent the whole sample. In addition, this study enrolled MSM who had anal sex with two or more men in their lifetime or who had a sexually transmitted infection (STI) history, in spite of performing multiple recruitment methods, which might not represent general MSM in China. Though HPV vaccination strategies exclude men in China, some MSM might be vaccinated through other methods. HPV vaccination status was not collected in our survey, which should be involved in future studies. Moreover, beta-globin analyses were not run prior to HPV tests; therefore, it was unknown to us the number of samples providing inadequate materials for HPV testing, which might impact the estimation of HPV indicators in some degree.This study is a sub-analysis of the Text To Test (T2T) study, which investigated the impact of automated text message reminders on testing of HIV and other STIs among MSM in China . The T2TAt each visit, participants completed a self-administered questionnaire that collected data on sociodemographic characteristics , sexual behaviors , and drug use status. A sample of venous blood (5 mL) was collected to test for HIV. An anal brush of exfoliated cells was collected at each visit and was sent for HPV DNA testing and genotyping. Anal HPV testing was performed with a soft cytology brush  inserted 3 cm into the anal canal and rotated ten times clockwise and ten times counterclockwise. Anal swabs were stored in 3 mL of sample transport medium for the Hybribio 37 HPV GenoArray Diagnostic Kit . Blood samples and exfoliated cell samples were stored at −20 °C and 2–8 °C, respectively, before further processing.HPV DNA testing and genotyping were performed using the 37 HPV GenoArray Diagnostic Kit  in accordance with manufacturer instructions. Amplification of HPV L1 gene fragments was performed using the Life ECO Gene Amplification Instrument . HPV genotyping was performed using HPV Hybridization Reagents and the Nucleic Acid Hybridization System for Medical Use  to detect 37 HPV genotypes, including 13 high-risk genotypes  and 24 low-risk genotypes  for carcinogenicity .HPV positivity of a specimen was defined as detection of ≥1 HPV genotype. High-risk genotype positivity of a specimen was defined as detection of ≥1 of any high-risk genotype, and low-risk genotype positivity of a specimen was defined as detection of ≥1 of any low-risk genotype. Incident HPV positivity was defined as the same HPV type detected at 12 months in men who had a negative HPV test at baseline. HPV clearance was defined as the absence of the same HPV type at 12 months in men who had a positive HPV test at baseline. Persistent infection of individual HPV genotypes in this study was identified as the same HPV genotype detected at both baseline and follow-up visits, and persistent infection of any HPV genotype was identified as one or more HPV genotypes detected at both baseline and follow-up visits ,35.Rates of HPV incidence, clearance, and persistence were calculated as the respective number of incident, cleared, and persistent infections divided by total person-time at risk. Corresponding 95% confidence intervals (CI) were calculated presuming a Poisson distribution. The events of incidence and clearance in this study were calculated based on infections rather than participants. Person months (pm) were calculated as the period from the baseline visit during which a participant was recruited to the date of the follow-up visit conducted 12 or more months after recruitment. For HPV incident infection of any/any high-risk/any low-risk/9V/4V/6 or 11 genotypes, no individual was infected with all the genotypes at baseline; therefore, one individual was likely to acquire incident infections with other genotypes included in these categories, and all the participants were included in the calculation of person months. For HPV incident infection of individual genotypes, the period of follow-up of one participant was included only when he was not infected with one specific genotype at baseline. For HPV clearance, individuals infected with one or more genotypes of any/any HR/any LR/9V/4V/2V/6 or 11 at baseline supply person months for corresponding indicators, and individuals infected with one specific genotype at baseline supply person months for corresponding genotype.p-value < 0.2 in univariate analyses, entered into the multivariable models to estimate the adjusted odds ratios (OR), as well as their 95% CIs were as follows: age at recruitment, marriage status, educational level, profession, number of sexual partners during follow-up, time since first anal sex, sexual role in anal sex, and HIV status for HPV incidence; and age at recruitment, site of enrollment, marriage status, profession, number of sexual partners during follow-up, time since first anal sex, sexual role in anal sex, and HIV status for HPV clearance. Rates of HPV incidence, clearance, and persistence were calculated across age groups. Age was divided into 4 groups , using cutoff points close to quartiles, and at least 30 individuals were contained in each group to assure a relatively accurate estimation for HPV indicators. All statistical analyses were performed using Stata SE version 15  and R 3.6.3 .In order to evaluate potential bias due to loss to follow-up, sociodemographic and behavioral characteristics of MSM who completed two HPV tests and MSM only tested at baseline were compared using simple descriptive statistics and Fisher’s exact test. Median and interquartile range (IQR) were used to describe continuous variables, and proportions were used to describe categorical variables. Generalized estimating equations (GEE) logistic regression with robust standard errors and an exchangeable correlation structure was used to investigate the correlates for incident and cleared anal HPV infection. Variables found statistically significantly associated with outcomes in the previous literature or with a This study provides an assessment of HPV incidence, persistence, and clearance for 37 genotypes among MSM, employing multiple methods to recruit participants in three metropolitan cities in China. It is important in informing HPV transmission modelling among MSM and supporting policy making against HPV epidemic. Our study builds on previous prevalence studies of HPV infection among MSM in China. We have found incident HPV infection is common among MSM in China. HPV16 and HPV18 have high rates of incident infection, and HPV6 and HPV16 have high rates of persistent infection, which suggests the need for HPV vaccination among this population. Young MSM are at disproportionately high risk for HPV infection and should be given the priority in HPV vaccination campaigns. Further studies are needed to clarify incidence, persistence, and clearance of HPV infection at other anatomical sites and the burden of HPV-related diseases among MSM, using larger sample size and long-term follow-up.


As the leading sexually transmitted infection worldwide, human papillomavirus (HPV) may disproportionately affect transwomen. We aimed to estimate anal HPV prevalence, especially focusing on high‐risk (hr)‐HPV types and evaluate their associated factors among transwomen living in Rio de Janeiro, Brazil.Transwomen enrolled in a respondent‐driven sampling (RDS)‐based survey conducted between August 2015 and January 2016 self‐collected anal samples, which were promptly stored at minus 80°C. After DNA extraction, HPV detection and genotyping were performed using the PapilloCheck test. We estimated HPV prevalences and evaluated the correlates of anal hr‐HPV infection using a regression logistic model.Out of 345 transwomen, 272 (78.8%) were included in this analysis (122 [44.9%] HIV‐positive). No participant had ever received HPV vaccination. Among participants enrolled, 212 (77.9%) were positive for any anal HPV type and 165 (60.7%) for hr‐HPV. Most common hr‐HPV were as follows: HPV16 (17.6%), HPV68 (14.7%), HPV39 (14.3%), HPV56 (12.5%), HPV51 (11.4%) and HPV52 (11.0%). HIV‐positive transwomen had three times the odds of having an hr‐HPV compared to HIV‐negative transwomen. Participants who had a current rectal Neisseria gonorrhoeae infection had 3.7 times the odds of being coinfected with hr‐HPV. Among HIV‐positive transwomen, neither antiretroviral therapy use, undetectable viral load, current and nadir CD4 counts were associated with anal hr‐HPV infection.Brazilian transwomen in our study exhibit some of the highest population‐specific rates of HPV and hr‐HPV. There is an urgent need to elucidate the burden of HPV infection, prevalence of HPV‐related diseases and access to and uptake of HPV vaccination among transwomen, especially from low‐ and middle‐income settings. Most sexually active people become infected with HPV at some point in their lives , 2, 3. HBesides a necessary factor to cervical oncogenesis , hr‐HPV Transgender women (henceforth “transwomen”) experience numerous socio‐economic factors that increase their vulnerability to HIV/STIs . However2Transcender, a cross‐sectional study that enrolled 345 transwomen using respondent‐driven sampling (RDS) at the Evandro Chagas National Institute of Infectious Diseases (INI)‐FIOCRUZ, Rio de Janeiro, Brazil, between August 2015 and January 2016. RDS has been used to obtain robust and diverse samples of hard‐to‐reach populations [This is a secondary analysis of ulations . ProceduInstituto de Medicina Tropical da Universidade de São Paulo. Analysis methods have been described elsewhere [We offered HIV/STIs testing to all participants. A trained staff member provided anal self‐collection materials and explained how to perform the procedure using illustrated instructions . In a prlsewhere . In summTreponema pallidum. We detected rectal Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) infection by using the Abbott Real Time platform and the Amplification Reagent Kit  in INI‐FIOCRUZ Laboratory. Indeterminate results were repeated with the same tests on the same samples. All participants living with HIV collected CD4+ count (Becton Dickinson FACScan) and HIV viral load (VL). Previous CD4+ counts were retrieved from the Brazilian National Registry, which records most measures performed in the country. Participants self‐reported antiretroviral therapy (ART) use.Syphilis screening used Venereal Disease Research Laboratory (VDRL) tests, and positive results were confirmed by a microhaemagglutination assay for We estimated prevalence of any anal HPV, at least one HPV type in the 2‐valent , 4‐valent  and 9‐valent  vaccines, hr‐HPV (positive for 1+ hour‐HPV detected by the test) and individual HPV types.T. pallidum. The nadir CD4+ count was defined as the participant’s lowest level ever recorded.Continuous variables were reclassified as categorical. We defined active or recent syphilis as VDRL titers of at least 1/8 plus a positive microhaemagglutination assay for p < 0.2 in the univariable logistic regression analysis and potential confounders were included in the initial multivariable model. A backward elimination procedure guided the removal of non‐significant variables until we reached a final multivariable model where all variables had a p ≤ 0.05 or modified the effect of other variables. Statistical analysis was performed using R software version 3.4.3.We used RDS‐unadjusted data. Descriptive analysis used median and interquartile range (IQR) and absolute and relative frequencies. We compared participants according to HIV status using chi‐square tests. We used a regression logistic model to evaluate the correlates of anal hr‐HPV infection. Variables with This study was approved by the INI‐FIOCRUZ Institutional Review Board. All participants provided an informed consent form. This study received grants of the Brazilian Research Council (CNPq) and the National Institute of Allergy and Infectious Diseases (NIAID‐NIH).3Transcender study, 341 (98.8%) agreed to and conducted anal specimen collection, 69 (20.0%) had invalid anal samples due to insufficient material and 272 (78.8%) had samples with valid results and were included in the present analysis. HIV‐negative status was more common among participants with invalid samples than those with valid samples (72.9% vs. 55.1% respectively). Overall, participants were aged 30 years (IQR 24 to 37), 76.8% were non‐white, and 48.9% were currently on hormones. Only 16 (5.9%) participants had gender‐affirming surgery. Ever and currently doing sex work were reported by 79.0% and 48.9% respectively. No participant had received HPV vaccine. HIV‐infection was present in 122 individuals (44.9%). Among those living with HIV, 83 were on ART, 43.7% were virologically suppressed, and median current and nadir CD4 count were, respectively, 631 cells/mm3 (IQR 384 to 833) and 380 cells/mm3 (IQR 196 to 576).Among 345 transwomen enrolled in the Out of 272 participants tested for anal HPV, 212 (77.9%) and 165 (60.7%) were positive for any HPV and hr‐HPV respectively (Table The most common hr‐HPV was HPV16 (17.6%). Only three people had both HPV16 and 18 (1.1%), and none were positive for all 4‐ and 9‐valent vaccine types. Transwomen living with HIV had a significantly higher prevalence of most types of anal HPV than those HIV negative. HPV6 and HPV43 had decreasing prevalences according to age Table . TranswoYounger participants had significantly higher odds of anal hr‐HPV infection Table . TranswoThese data add to the limited research on anal HPV infection among transwomen. In the United States, unvaccinated transwomen had a significantly higher anal HPV prevalence than unvaccinated MSM (88.6% vs. 70.9%), even after stratifying by HIV status . ArgentiRates of anal HPV and hr‐HPV in our sample of transwomen are higher than previously described among individuals with diverse gender identities and sexual orientations. A meta‐analysis found that anal HPV prevalences varied widely between cisgender men (mostly MSM) and women . Their dAnal hr‐HPV detection was significantly higher among younger transwomen. There is a strong relationship between age and cervical HPV prevalence . NeverthTranswomen living with HIV had a higher HPV and hr‐HPV prevalences compared to HIV‐negative participants, consistent with previous findings. MSM have the highest population‐specific HPV rates reported to date, especially among those living with HIV , 33, 34.The 4‐valent HPV vaccine, available at no cost in Brazil since 2014, is currently offered to girls aged 9 to 14 years, boys aged 9 to 14 years and people living with HIV aged 9 to 26 years (regardless of gender identity) . NeverthThis study has several limitations. First, although RDS survey is commonly used for hard‐to‐reach populations, its results may not be generalizable. In addition, the cross‐sectional design does not allow us to establish causal inference. We also did not evaluate anal cytology or histopathology data, which could ultimately reinforce the burden of anal HPV infection among transwomen. Syphilis definition only included laboratory results and did not consider clinical data. Finally, the high proportion of invalid samples may be due to user error. Two rectal samples had to be collected (for HPV and CT/NG), which may create burden on participants and interfered with the process. Although self‐collection has been described as an acceptable tool, including for anal samples, the proportion of invalid samples varied from 5% up to 37% across studies , 42, 43.4Our extremely high rates of HPV and hr‐HPV among transwomen show that this group is disproportionately affected by HPV and may be at a high risk for cancer. HPV16 was the most prevalent anal hr‐HPV type. Prevalence of hr‐HPV was higher among younger participants, those living with HIV and concomitant rectal NG infection. There is an urgent need to elucidate the burden of HPV infection and to increase access to and uptake of HPV vaccination among transwomen, especially from low‐ and middle‐income settings.The authors declare no conflict of interests.BG, EMJ, ECW and VGV conceived the analysis and interpreted the findings. BG, EMJ, ECW and VGV drafted the manuscript. LSV and EMJ performed the statistical analyses. JEL supervised the biological analysis, interpreted the results and provided biological inputs. EMJ, LM, LSV and ACFG helped with data acquisition, interpretation of the results and drafting the manuscript. All authors read and approved the final manuscript.


Tabular output conversion to visualizations entailed 1–2 keystrokes. Biodiversity analysis between low- (LSIL) and high-grade squamous intraepithelial lesions (HSIL) revealed loss of species richness and gain of dominance by HPV-16 in HSIL. Integrating clinically relevant, taxonomized HPV reference genomes within automated workflows proved to be an ultra-fast method of virome profiling. The entire process named “HPV DeepSeq” provides a simple, accurate and practical means of NGS data analysis for a broad range of applications in viral research.Next-generation sequencing (NGS) has actualized the human papillomavirus (HPV) virome profiling for in-depth investigation of viral evolution and pathogenesis. However, viral computational analysis remains a bottleneck due to semantic discrepancies between computational tools and curated reference genomes. To address this, we developed and tested automated workflows for HPV taxonomic profiling and visualization using a customized papillomavirus database in the CLC Microbial Genomics Module. HPV genomes from Papilloma Virus Episteme were customized and incorporated into CLC “ready-to-use” workflows for stepwise data processing to include: (1) Taxonomic Analysis, (2) Estimate Alpha/Beta Diversities, and (3) Map Reads to Reference. Low-grade ( Hippocrates was the first to describe cervical cancer and its destructive nature around 400 BCE . Two thoThe papillomavirus (PV) is a small 8000 base pair (bp) double-stranded, circular DNA virus that co-evolved with an ancestral host over 400 million years . The PV Recent advancements in next-generation sequencing (NGS) have actualized HPV virome profiling . Since tn = 95) and high-grade squamous intraepithelial lesions (HSIL) (n = 60). The “Data quality control (QC) and taxonomic profiling” workflow computational runtime on this dataset was 23:33 and 12:50 min for the LSIL and HSIL samples, respectively  . A merged abundance table inclusive of multiple samples also displays summary statistics  . AdditioThe abundance table may be visualized as a stacked bar chart or sunburst plot with 1-click A–C. Metan = 1), (2) low-quality Sanger sequence (BLAST max id < 90% and max bit score <531) (n = 2), (3) L1 and E6/E7 primer bias (n = 3), or (4) E6 sequencing primer bias (n = 3). The complete Sanger/BLAST table with 9 discordant results (bold) are shown in Both Sanger and NGS sequencing were used to detect HPV genotypes and sub-lineages within each sample. Sanger sequencing resolved the single dominant HPV genotype within each sample. Compared to Sanger sequencing, NGS achieved a higher resolution in detection of mixed genotypes (cut-off at six genotypes) and low-abundance genotypes  . Compari10  were perfectly, linearly anti-correlated, and (2) BLAST max bit score and maximum identity (%) were curvilinearly correlated (10 -150) which were used as the threshold for quality genotyping results. BLAST values less than this threshold were considered as “uncertain” HPV genotyping result.By convention, sequences must possess ≥ 90% homology with the closest known type for HPV genotyping assignment . Correlarrelated . Taken tThe “Merge and Estimate Alpha and Beta Diversities” workflow runtime on this dataset was 5 s . The divThe alpha diversity metric is calculated by sub-sampling the abundances at different depths (number of reads) in each sample. The rarefaction analysis parameters define the granularity of the alpha diversity curve as presented in The workflow output for beta diversity analysis was a Bray-Curtis distance matrix between samples and PCoA plots in 2D or 3D. The 3D PCoA plot visually displayed the dissimilarities in HPV composition between all samples A. After p-value; Bonferroni and FDR corrected p-values).The “Convert Abundance Table to Experiment” and “Proportion-based Statistical Analysis” runtimes on this dataset were 2 s and < 1 s, respectively . The outThe “Create Heat Map for Abundance Table” runtime on this dataset was < 1 s. The resultant heat maps revealed hierarchal clustering of HPV genotypes on the 155 LSIL/HSIL samples. HSIL and LSIL samples with HPV genotypes, such as, HPV-16 and -39 with similarly high abundances (~100% of reads) are noted in the blue-red spectrum versus dissimilar abundance in the red spectrum A. The agn = 155) was 45:24 min with a mean of 18 s per sample  than ever possible with conventional sequencing.The strength of the methods developed herein is the integration of a taxonomized database with automated workflows for viral metagenomic analysis. The desired end state for the software user is a single, user-friendly, multi-functional tool, analogous to the “Swiss Army knife” . We alsoAnother strength of this study is the corroboration of NGS/taxonomic profiling results by Sanger/BLAST. For the 9/155 (5.81%) discordant results, the quality of Sanger sequences was suboptimal in three samples, and primer or sequencing bias was the probable cause in six others. By convention, NGS results necessitate validation by Sanger sequencing as the reference standard. However, recently the necessity of Sanger validation has been called into question for human genome sequencing by several studies ,39. Arten = 95) and HSIL (n = 60) cervical cytology samples were randomly selected from a larger (3000+ samples) Congressionally Directed Medical Research Programs (CDMRP) grant-funded project to test our workflow-based methods described herein. The residual liquid-based cervical cytology samples were procured consecutively from the Department of Pathology after completion of cytological diagnosis. Demographic and cytological data were abstracted from the electronic health record (AHLTA) as metadata for association with taxonomic profiling results.A subset of HPV-positive LSIL  sequencing for validation of deep-sequenced results. Briefly, amplicons (~200 ng DNA/sample) were sequenced using primer GP-E6-3F at Eurofins Genomics . The resulting sequences were BLAST aligned for HPV genotyping as described above.n = 219) and variant genomes (n = 136) from the collection of NIAID PaVE (https://pave.niaid.nih.gov accessed on (11 December 2020) [HPV reference n = 9 and varCLC Genomics Workbench 21.0.4 with CLC Microbial Genomics Module 21.0  were installed on an HP notebook computer  for use throughout this project. The MGM has various pre-built or “ready-to-use” workflows and tools as shown in The pre-built “Data QC and Taxonomic Profiling” workflow with data input/output, individual elements (processing steps) and flow directions (arrows) are presented in n = 95) and HSIL (n = 60) groups of samples were merged into one (n = 155) named “LSIL_HSIL abundance table” and aggregated by feature i.e., “Family” using the “Aggregate feature” option of the table  sequences, and saved as a clean subtable. Second, the merged (filtered) taxon table was joined by clinical metadata (.xlsx format) with the “Add Metadata to Abundance Table” tool. Third, the two  tables from LSIL . For this study, β-diversity or simply the change in HPV genotype composition (proportions) from the LSIL to HSIL group was measured by Bray-Curtis distances or compositional “dissimilarity” between samples [n is the number of operational taxonomic unit (OTU) i and i in samples A and B. The beta diversity workflow produced a Bray-Curtis distance matrix quantifying the dissimilarity of HPV genotype composition between samples. Also, Principal coordinate analysis (PCoA) was performed to determine and 3D-plot the correlative relationship between variables (HPV genotypes) in the LSIL or HSIL groups. The permutational multivariate analysis of variance (PERMANOVA) tool was used separately to test for statistical differences in the centroids and dispersion of the groups.The pre-built “Merge and Estimate Alpha and Beta Diversities” workflow C was use entropy : H = ∑1n samples : (1)Ɓ = For differential abundance analysis of HPV communities between LSIL and HSIL groups, the “Convert Abundance Table to Experiment” and “Proportion-based Statistical Analysis” tools were used in succession. First, the merged LSIL and HSIL abundance table with appended metadata was chosen as input for conversion. The metadata group named “PAP” with two categorical variables “LSIL” and “HSIL” was selected as the factor. The output produced a table labeled “experiment” which was entered into the “Proportion-based Statistical Analysis” tool as input. The weighted proportion test of Baggerly  was chosThe “Map Reads to Reference” workflow C maps eaCLC workflows with integrated, customized HPV reference genomes proved to be an ultra-fast and accurate method of HPV virome genotyping and profiling. By forging a path through the bioinformatics pipeline of a user-friendly software, the impasses to HPV discovery and beyond have been overcome.


Lower prevalence HPV infection has been previously reported in Thai population when compared with Western countries. p16 expression indicates HPV-associated oropharyngeal squamous cell carcinoma (OPSCC), but not non-OPSCC. We therefore evaluated the characteristic and association of p16 and HPV in Thai patients with HNSCC.We used immunohistochemistry and qPCR, respectively, to detect p16 and HPV DNA in archrival formalin-fixed paraffin-embedded HNSCC tissues. Patient characteristics and survival were analyzed.p = 0.049), and non-OPSCC (p = 0.003).p16 expression was detected in tumors of 72 of 662 (10.9%) patients with HNSCC and was significantly associated with higher-grade histology, advanced nodal stage, and oropharynx. p16 was expressed in 28 and 6.5% of patients with OPSCC or non-OPSCC, respectively, and HPV DNA was detected in 15.6 and 1% of patients, respectively. Using p16 as a surrogate for HPV status, sensitivities were 80 and 25% in OPSCC and non-OPSCC, respectively. Positive and negative predictive rates of OPSCC were 38 and 95%. Discordance rates between HPV and p16 were 23 and 7% in OPSCC and non-OPSCC, respectively. Overall survival (OS) were significantly longer in both p16-positive OPSCC (Low prevalence of p16 and HPV associated OPSCC and non-OPSCC were confirmed in Thai patients. High discordance and low positive predictive rates of p16 were observed in HPV-associated OPSCC. p16 was a significant prognostic factor for OS for patients with OPSCC or non-OPSCC. Therefore, HPV testing should be performed to assess the association of HPV with HNSCC regardless of p16 expression.The online version contains supplementary material available at 10.1186/s12885-021-08213-9. Human papilloma virus (HPV), which plays a major role in the pathogenesis and progression of head and neck squamous cell carcinoma (HNSCC), is more commonly associated with oropharyngeal squamous cell carcinoma (OPSCC) . HPV infExpression of p16 significantly correlates with the HPV status of OPSCC and serves as an independent prognostic factor for survival of patients with OPSCC treated with concurrent chemoradiation (CRT) –10. ThusThe frequencies of detection of HPV and p16 status of HNSCC vary because of differences among detection methods and cut-off values, different ethnicities, and the primary site of HNSCC , 18–23. Patients with histology confirmed HNSCC of oral cavity, oropharynx, larynx, hypopharynx, paranasal sinus, and known primary who treated at the Ramathibodi Cancer Center between January 2007 and December 2018 were identified through the Ramathibodi Cancer Registry. We analyzed available archival FFPE tumors using immunohistochemistry (IHC) to detect p16 expression, and polymerase chain reaction (PCR) for HPV DNA detection. Patients with nasopharyngeal carcinoma, cutaneous squamous cell carcinoma, squamous cell carcinoma of external ear canal, and insufficient/unavailable FFPE samples for p16 evaluation were excluded. Eligible patients’ medical records were reviewed for demographic information such as age, sex, Eastern Cooperative Oncology Group (ECOG) performance status, American Joint Committee on Cancer TNM staging (AJCC 7th ed.), smoking status, primary site of tumor, histological grade, and survival outcomes. Overall survival (OS) was defined as the time from date of tissue diagnosis to date of death from any cause or to the last follow-up. The status (living or dead) of each patient was cross-checked with the National Security Death Index of Thailand. The Ramathibodi Ethics Committee approved the study.INK4a (clone E6H4) using a Ventana Benchmark Ultra instrument. According to the most recent College of American Pathologists Guidelines, IHC detection of p16 is scored positive or negative if nuclear and cytoplasmic staining of > 70% or < 70% of cancer cells, respectively, is observed [We used IHC  to analyze FFPE samples of patients with HNSCC for p16 expression. The sections were probed with a mouse monoclonal antibody against p16observed , 12. OroTumor tissues were microdissected from selected tissue blocks after matching with histology slides. DNA was extracted using a QIAamp FFPE tissue extraction kit  following the manufacturer’s protocol and then stored at − 20 °C. DNA samples (250 ng) were measured using a Nanodrop  and subjected to HPV genotyping using an AmoyDx High-risk Human Papillomavirus (HPV) Detection Kit  following the manufacturer’s protocol. The kit has been designed to detect of the conserved L1 region in the HPV DNA of the high-risk HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, and 82. The positive and negative controls for the PCR reaction were used according to the manufacturer instruction. The PCR results were reported as HPV types 16/18, other high-risk HPV types, or negative for detection. The limit of detection of the kit ranges from 50 to 1000 copies of HPV DNA per reaction, depending on different types of HPV. An internal control of the assay was provided for assessment of sample quality and the presence of PCR inhibitors  version 20.0.Categorical data are expressed as numerals and percentages. Differences in proportions were analyzed and compared using the chi-square or Fisher exact test, as appropriate. Continuous variables were summarized using descriptive statistics and compared using the Student p < 0.001) and advanced LN stage (p = 0.049). The oropharynx was the primary site of tumors that were significantly associated with p16 expression (p < 0.001). Patients with p16-positive tumors underwent definitive CRT more than p16-negative patients (55% vs 31%), whereas surgery alone was more preferred in p16-negative patients . The percentage of p16-positive tumors according to each primary tumor site is shown in Fig. We detected p16 expression in 72 of 662 (10.9%) tissues of patients with HNSCC. An example of p16 positive case was shown in the Fig. p < 0.001) and OPSCC (p = 0.001) patients, but not patients with non-OPSCC (p = 0.243) (Table Adequate tumor DNA were obtained from 348 of 662 patients. HPV DNA was detected in 14 of 348 patients (4.0%) (10 OPSCC and 4 non-OPSCC patients). Ten of 64 patients (15.6%) with OPSCC were positive for HPV DNA . On the other hand, 4 of 284 (1%) patients with non-OPSCC were positive for HPV DNA            Fig. a. There 12) Fig. b, and no23) Fig. c. In non01) Fig. d.Fig. 4Low prevalence of p16 expression in HPV-associated HNSCC among Thai patients with OPSCC and non-OPSCC were observed in our study  and the College of American Pathologists do not recommend routine testing of patients with non-OPSCC because of conflicting data on prognosis as well as no significant differences in the outcomes of therapy , 12. HowThough p16/HPV status is known as a prognostic factor for survival of patients with OPSCC, predictive value of p16/HPV for therapeutic guidance remains unclear , 17, 19.Our study observed that Thai patients with OPSCC and non-OPSCC infrequently expressed p16 and had low prevalence of HPV infection. Furthermore, p16 expression was a significant prognostic factor for OS of patients with both OPSCC or non-OPSCC. p16 expression was acceptable as a surrogate marker only for HPV associated OPSCC.However, higher discordant and lower positive predictive rates of p16 expression were observed in patients with HPV-associated OPSCC in our study compared with studies conducted in Western countries with much higher prevalence of HPV-associated OPSCC. In future clinical trials that strictly address HPV status in HNSCC, HPV testing should be performed in all cases to precisely identify an association of HPV with OPSCC and non-OPSCC regardless of p16 expression, especially in areas with low prevalence of HPV infection.Additional file 1: Supplement 1. Real-time PCR typing of HPV with AmoyDx High-risk Human Papillomavirus (HPV) Detection Kit. One reaction with three fluorescents  were mixed in each reaction tube for identification of the 19 possible HPV high risk types in a real-time PCR setting, as described in the Material and Methods section . Supplement 2. Baseline characteristics of non-oropharyngeal HNSCC and p16 status.


To evaluate the risk factors for recurrence of high‐grade disease after cervical excision in women living with HIV (WLWH), with a specific interest in the role of high‐risk (HR‐) HPV positivity.Multicentric retrospective study conducted on WLWH who underwent cervical excision between January 1987 and June 2017 in six Italian institutions. The rate of high‐grade recurrence was determined. Risk factors for recurrence and HR‐HPV positivity were determined with the Log‐rank test and Cox proportional hazards regression models.P < 0.001) and 5.18 , respectively. Age 41 years or more  resulted as a risk factor for HR‐HPV positivity during follow up.A total of 271 WLWH were included in the final analysis. A high‐grade recurrence was found in 58 (21.4%) patients. Age 41 years or more at inclusion and HR‐HPV positivity during follow up were independently associated with a higher risk of disease recurrence with relative risks of 4.15 (95% confidence interval [CI] 2.01–8.58, HR‐HPV positivity is a risk factor for recurrence after cervical excision in WLWH. Women older than 41 years may benefit from a long‐term yearly follow up. Future studies regarding HPV vaccination after treatment in WLWH may be useful, considering the protective role of the higher probability of HPV negativity in vaccinated women. High‐risk‐HPV positivity during long‐term follow up is a risk factor for high‐grade disease recurrence after cervical excisional treatment in women living with HIV. Risk factors for acquiring HPV infection include multiple sexual partners, immunodeficiency status, early sexual debut, parity, use of contraceptives, and smoking.To date, because primary prevention with HPV vaccination in WLWH, although promising, still does not have strong scientific validation and further studies are undoubtedly necessary,Evidence suggests that several factors may be associated with increased rates of persistent and recurrent disease after treatment, including smoking, positivity of cone margins, persistent HR‐HPV infection, and immunosuppression.The aim of the present study was, therefore, to evaluate the risk factors for recurrence of high‐grade lesions after cervical excision in WLWH, with a specific interest in the role of HR‐HPV positivity during follow‐up.2This was a multicentric, retrospective, cohort study that involved six Italian institutions. All WLWH who underwent cervical excision between January 1987 and June 2017 in the six institutions were retrospectively identified by searching the clinical databases and included in the present study.The cervical treatment was performed because of high‐grade squamous intraepithelial lesions  or persistent (>2 years) low‐grade squamous intraepithelial lesions  diagnosis. Only women with a definitive diagnosis of high‐grade intraepithelial lesion  at the cone specimen were included in the final analysis. Women who reported previous treatment for cervical pathology were excluded. Likewise, patients with missing data and with a diagnosis of cancer on the first visit or after conization were not included in the analysis.We collected pertinent sociodemographic and clinical data such as age, menopausal status, age at first sexual intercourse (≤16 years or ≥17 years), number of sexual partners (up to five or six or more), tobacco use, parity (nulliparous or parous), and reported route of transmission of HIV infection. Moreover, we collected data regarding the referral cytology, the colposcopic examination at inclusion, the histopathologic findings on pre‐operative biopsy, the final histopathologic diagnosis at the cone specimen, and the HR‐HPV status before cervical treatment and during follow up. Abnormalities on referral cervical cytology were classified according to the most recent Bethesda system terminology.+ T‐cell counts at inclusion and during follow up (at recurrence or the last negative follow up). CD4+ T‐lymphocyte count was recorded according to the Centers for Disease Control revised Surveillance Case Definition for HIV infection.+ T‐cell count of 500 cells/µl or more, mild for a CD4+ T‐cell count of 350–499 cells/µl, advanced for a CD4+ T‐cell count of 200–349 cells/µl, and severe for a CD4+ T‐cell count below 200 cells/µl.We also collected CD4The use and type of antiretroviral therapy (ART) were recorded on the basis of the medications listed in the patients’ chart. HAART was defined according to the contemporary definition of two nucleoside reverse‐transcriptase inhibitors and at least one of the following: a protease inhibitor, a non‐nucleoside reverse‐transcriptase inhibitor, or an additional nucleoside reverse‐transcriptase inhibitors. All other types of ART used before 1996 were defined as pre‐HAART.The post‐treatment follow‐up strategy was the following: first control with cervical cytology and colposcopy at 6 months from the procedure; after that, these patients underwent cervical cytology and colposcopy every 6 months until 24 months from treatment. After that, women underwent cervical cytology every 12 months with colposcopy in the case of abnormalities at cervical cytology. HR‐HPV testing was also added after 2002, when available, at the time of treatment and at yearly intervals. Biopsies were prompted by abnormal cytology or the presence of an abnormal transformation zone at colposcopy.Disease recurrence was defined as a histopathologic diagnosis of high‐grade lesions  or worse (HSIL+) during follow up. These women were followed from the date of first follow up after treatment until the date of recurrence or until the date of the last registered follow up if no recurrence took place.The primary outcome of the study was the rate of high‐grade recurrence or cancer (HSIL+) among WLWH.All continuous variables were tested for normality with the D’Agostino‐Pearson test. Normally distributed variables were expressed as mean ±standard deviation, while skewed variables were reported as median and interquartile range (IQR). Categorical variables were reported as numbers and percentages. A Kaplan‐Meier analysis at 180 months of follow up for the whole population was performed to evaluate the probability of disease recurrence.P value <0.05 was considered statistically significant. Patients with missing data were excluded and a complete case analysis was performed. Calculations were done using IBM SPSS version 27.0 . As this was a retrospective analysis of routinely collected data, there was no need for ethical approval. Patients signed informed consent for data collection and procedure at the time of intervention.All sociodemographic, clinical, and HIV‐specific variables were evaluated as risk factors for disease recurrence with the log‐rank test. Factors that were associated with recurrence were considered as covariates in a subsequent Cox proportional‐hazards regression model. Risk factors for HPV‐positivity during follow up were evaluated with the same methodology. A 3+ T‐cell count at inclusion was 365 (240–500) cells/µl. According to the WHO immunologic classification, 67 (21.5%) of these women presented a severe immunodeficiency, 66 (21.2%) an advanced immunodeficiency, 86 (27.7%) a mild immunodeficiency, and 92 (29.6%) a not significant immunodeficiency. At the time of treatment, 73 (23.5%) women were not receiving ART, 81 (26.0%) were receiving mono‐ or dual‐nucleoside reverse‐transcriptase inhibitors (pre‐HAART) therapy, and 157 (50.5%) were receiving HAART.During the study period, 345 WLWH underwent cervical excision in the six institutions involved. Thirty‐four women with incomplete data were excluded, leaving 311 for analysis. The majority of patients  were white, with a mean age ±standard deviation at inclusion of 35.5 ± 7.9 years. Fourteen (4.5%) women were in menopause at the time of treatment. The reported age at first intercourse was 16 years or less in 150 (48.2%) women, and 148 (47.6%) women reported a having five or fewer partners. Tobacco use was reported by 209 (67.2%) women. One hundred and sixty (51.5%) women were nulliparous at inclusion. Regarding HIV‐related characteristics, 171 (55.0%) had been infected by HIV via heterosexual contact. The median (IQR) CD4In the 165 women in whom an HR‐HPV test was performed at inclusion, the result was positive in 148 (89.7%). The preoperative diagnosis was HSIL (CIN2/3) at punch‐directed biopsy in 246 (79.1%) patients, high‐grade cervical cytology  in 7 (2.3%), persistent LSIL (CIN1) in 53 (17.0%), and persistent low‐grade cervical cytology  in the remaining 5 (1.6%) patients.At the preoperative colposcopy, the squamocolumnar junction was visible in 206 (66.2%) patients, and major colposcopic changes or signs of suspected invasion were observed in 167 (53.7%).2‐laser in 40 (12.9%), and by cold knife in the remaining 16 (5.1%). At the histopathologic analysis of the cone specimens, the cone result was negative in 3 (1.0%) cases, LSIL (CIN1) in 23 (7.4%) patients, HSIL (CIN2) in 137 (44.1%), HSIL (CIN3) in 140 (45.0%), and invasive cancer in eight (2.5%) patients. The endocervical margin was negative in 272 (87.5%) patients.Treatment was performed by loop electrical excision procedure in 255 (82.0%) patients, by COAmong the 277 WLWH diagnosed with HSIL (CIN2/3) at the cone specimen, six patients with no documented follow up were excluded, leaving 271 patients diagnosed with HSIL (CIN2/3) for the analysis of disease recurrence. Figure The median (IQR) duration of follow up was 44 (17–88) months. Among the 271 women retained in follow up, 213 (78.6%) women had no sign of recurrent disease. A recurrence of HSIL+ was found in 58 (21.4%) women. The median (IQR) time to recurrence was 18 (7–43) months. Among those patients, there were six cases (2.2%) of invasive cancer diagnosed: two at 12 months, two at 24–36 months, and two more than 60 months after treatment. The overall rate of subsequent invasive cervical cancer was 2.2% (6/271 patients). All cases of recurrence underwent a second excisional procedure.The Kaplan‐Meier analysis at 180 months of follow up showed a probability of negative follow up at 24 months of 85.5% (standard error [SE] 2.0%), at 60 months of 78.3% (SE 2.9%), at 120 months of 72.3% (SE 3.6%), and at 180 months of 63.9% (SE 5.2%).P value <0.05: age 41 years or more and HR‐HPV positivity at follow up; those factors were considered as covariates in the subsequent Cox proportional hazards regression model. Both included factors that were significantly and independently associated with a higher risk of disease recurrence: age 41 years or more with a relative risk (RR) of 4.15  and HR‐HPV positivity during follow up with an RR of 5.18 .We evaluated the influence of all sociodemographic, clinical, and HIV‐specific variables as risk factors for disease recurrence with the log‐rank test Table . Two facFigure The HR‐HPV positivity showed a sensitivity of 82.9% (95% CI 66.4%–93.4%), a specificity of 46.9% (95% CI 37.5%–56.5%), a positive predictive value of 32.6% (95% CI 27.8%–37.8%), and a negative predictive value of 89.8% (95% CI 80.6%–94.9%) with regard to disease recurrence.P = 0.047).The log‐rank test of risk factors for HR‐HPV positivity during follow up is reported in Table 4The results from the present study showed that cervical excision is an effective procedure for high‐grade CIN treatment in WLWH, considering that 78.6% of women presented a long‐term negative follow up. However, the risk of recurrent high‐grade lesion or invasive cancer seems to last for many years. In our cohort, women aged 41 years or more had a four times higher risk of recurrence of HSIL+, and women with HR‐HPV positivity during follow up had a five times higher risk. Moreover, age 41 years or more was found to be a risk factor for HR‐HPV positivity during follow up.The 21.4% HSIL+recurrence rate found in the present study is in agreement with those reported for WLWH, summarized in the recent meta‐analysis from Debeaudrap et al.,High‐risk HPV positivity during follow up is a recognized risk factor for both short‐term and long‐term recurrence of cervical lesions after treatment in HIV‐negative women.+ T‐cell count, is considered one of the most significant risk factors for disease recurrence in WLWH.Immunosuppression, expressed as low CD4+ T‐cell count at the time of cervical treatment or at the time of the last control was not associated with a higher risk of recurrence. Literature data about the most appropriate prognostic CD4+ T‐cell count are conflicting. Clifford et al.+ T‐cell count was a more discriminant measure of risk for CIN2+ than CD4+ T‐cell count at diagnosis. Other studies found that CD4+ T‐cell count at the time of treatment is a better predictor of persistent disease or recurrence than nadir CD4+ T‐cell count.In our study, CD4+ T‐cell count and used over longer durations by adherent patients.The use of HAART allows for prolonged suppression of HIV replication and improved immune status, as manifested in rising CD4 cell counts. It seems to be associated with a reduction of the incidence, progression, and recurrence of cervical lesions, especially if started at higher CD4In the WLWH included in our cohort, HR‐HPV test during follow up after cervical excisional treatment presented a high negative predictive value , allowing reassurance to negative patients about the low risk of recurrence, but the expected low positive predictive value  implies the need for execution of further diagnostic tests  to exclude disease recurrence.Being HPV negative appears to be the most important protective factor against disease recurrence. HPV vaccination could therefore represent an additional protective factor after cervical excision, allowing a higher percentage of women to maintain the status of HPV negativity. HPV vaccination could also prevent new HPV infections, which in a long‐term follow up could be confusing regarding the true incidence of high‐grade disease persistence of new‐onset cervical lesions. Future studies are undoubtedly needed regarding HPV vaccination after treatment in WLWH; at present, there are already ongoing study protocols for HPV vaccination after treatment in HIV‐negative women.The increased risk of high‐grade recurrence for patients aged 41 years or older that emerged from our data could be explained by the age‐related immunosenescence of older WLWH that is reported to occur earlier than in HIV‐negative women of the same age.As also reported in a recent meta‐analysis,The major strengths of our study are the large cohort of WLWH subjected to cervical treatment considered for the analysis, the extensive look at clinical data and pre/postoperative variables, the long‐term follow up (up to 5 years from treatment for most patients), and its multicentric nature. Moreover, the outcome chosen in the study for recurrence (HSIL+) might have reduced the probability of detecting transient low‐grade lesions. In addition, the long period of time covered by our study also allowed us to evaluate the potential effect of changes in ART, up to the current therapeutic regimens, that can provide longer lives for patients and reduce HIV transmission. The successes of ART have reduced HIV to a chronic condition in many parts of the world as progression to AIDS has become rare.+ count at the time of treatment may not have the same clinical significance for each included WLWH, and generalization from these data may be limited from the heterogeneous immunologic management of the included women because of temporal changes in HAART use.Limitations of our study include its retrospective nature, the absence of HPV genotyping, and the fact that HR‐HPV testing was not performed in all the included women because it only entered clinical practice from 2002. In addition, it was not possible to discriminate if high‐grade recurrence occurring many years after treatment could be the result of a new HR‐HPV infection. However, the presented data about long‐term follow up are necessary to deepen the history of these patients. Moreover, it also needs to be acknowledged that CD4In conclusion, surgical excision of CIN seems to be an effective procedure in WLWH, with a high rate of negative follow up. However, a careful, intensive, and prolonged follow up after treatment is necessary. As the main risk factor for long‐term recurrence seems to be HR‐HPV positivity during follow up, the implementation of this test is needed in WLWH with a history of cervical excisional treatment with more systematic use of co‐testing.In our opinion, the interval of follow up after treatment could be individualized according to the patient's age. Whereas in young women it is possible to adopt a follow‐up interval similar to that used in HIV‐negative women, older women should be followed at yearly intervals after 24 months from treatment.The authors have no conflicts of interest.AA and CA contributed to the conception of the work; DCG, SF, GL, GB, SM, FMG, and PA contributed to the design of the work; DCG, SM, MM, DFA, FMM, and MC contributed to acquisition and analysis of data; AA, CA, DCG, and SF contributed to interpretation of data; DCG, SM, and GL drafted the work; AA and CA revised it critically for important intellectual content; all authors approved the final version to be published; and all authors agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.


Information processing speed (IPS) is a marker for cognitive function. It is associated with neural maturation and increases during development. Traditionally, IPS is measured using paper and pencil tasks requiring fine motor skills. Such skills are often impaired in patients with neurological conditions. Therefore, an alternative that does not need motor dexterity is desirable. One option is the computerized symbol digit modalities test (c-SDMT), which requires the patient to verbally associate numbers with symbols.Eighty-six participants  were examined, 38 healthy and 48 hospitalized for a non-neurological disease. All participants performed the written SDMT, c-SDMT, and the Test of Non-verbal Intelligence Fourth Edition (TONI-4). Statistical analyses included a multivariate analysis of covariance (MANCOVA) for the effects of intelligence (IQ) and hospitalization on the performance of the SDMT and c-SDMT. A repeated measures analysis of variance (repeated measures ANOVA) was used to compare performance across c-SDMT trials between inpatients and outpatients.p = 0.04) but not with the c-SDMT (p = 0.68), while IQ (p = 0.92) had no effect on IPS. Age (p < 0.001) was the best predictor of performance of both tests. The repeated measures ANOVA revealed no significant difference in within-test performance (p = 0.06) between outpatient and inpatient participants in the c-SDMT.The MANCOVA showed that hospitalization had a significant effect on IPS when measured with the SDMT (Performance of the c-SDMT is not confounded by hospitalization and gives within-test information. As a valid and reliable measure of IPS for children and adolescents, it is suitable for use in both inpatient and outpatient populations. Information processing speed (IPS) is the brain’s potential to process information within a certain time period. One possible operationalization of IPS is the time it takes to react to a verbal, visual or auditory stimulus. This process includes the encoding, integration and retrieval of information, the decision to react, and finally, a response. Being a basic cognitive quality it can be used as a marker for cognitive dysfunction in patients with neurological disease . NeuroloOne of the first standardized tests developed to assess cognitive function is the coding task, which was first documented in the early 1900s . Most veIn an attempt to further minimize the requirement for fine motor skills, the computerized symbol digit modalities test (c-SDMT) was developed and implemented in Canada . It has A study applying the c-SDMT in a cohort of adolescent multiple sclerosis patients revealed that their speed did not consistently increase from trial one to eight as it did in the control group . This shBased on increases in myelination  and neurA rarely examined factor influencing IPS is hospitalization. Although both the SDMT and the c-SDMT are rapid and easy to administer bedside tools for the assessment of IPS, little is known about how hospitalization may influence performance of these tests. It is well recognized that hospitalization can cause a variety of reactions in children, which include distress, pain, the feeling of not being in control, being overwhelmed, discomfort  and fearNervous or emotional conditions have been found to be significantly associated with lower SDMT scores, similarly to other non-specific health conditions . ChildreInformation processing speed is not only linked to everyday activities , but is The overall purpose of this study was to examine the clinical implementation of the c-SDMT in direct comparison to the original written SDMT.Our main hypotheses were that IPS, measured by the SDMT or the c-SDMT, differs between neurologically healthy inpatient and outpatient children and adolescents (H1a). Furthermore, we predicted that the c-SDMT would be less prone to hospitalization effects than the SDMT (H1b). We also did not expect an effect of hospitalization on trial-to-trial behavior in the c-SDMT. Our second hypothesis was that IPS, when measured with the SDMT or the c-SDMT, is associated with intelligence (H2).The study conducted was a single-center pilot study with both cross-sectional and short-term longitudinal components. It took place at the University Children’s Hospital of the University of Bern in Switzerland. The study was approved by the ethics board of the canton of Bern (project ID 2018-00540) and was conducted in accordance with the ethical principles of the Declaration of Helsinki. Recruitment and testing of participants started in July 2018 and ended in December 2018. Data collection and analysis took place between January and June 2019.Eighty-six neurologically healthy pediatric participants were recruited in the region of Bern, Switzerland: 48 of them were inpatients of the surgical and medical ward at the University Children’s Hospital Bern, with a wide range of reasons for admission. The “outpatient” group had no obvious medical issues. Inclusion criteria: sufficient level of German, informed consent, age between 8 and 16 years. Exclusion criteria: medication with known psychotropic effects, diagnosis of psychiatric disorders , visual impairment that was not corrigible with glasses, or history of traumatic brain injury treated by a physician. Written informed consent was obtained prior to participation. Some of the participants returned for retest; however, retest data is beyond the scope of this article.All participants underwent three tests administered in a strict order, by trained examiners : (1) Test of Non-verbal Intelligence ; (2) stAll analyses were done using IBM SPSS Statistics 25.0. To examine the effect of hospitalization on the performance of IPS and the interaction between “groups” (inpatient vs. outpatient) and “age groups” (8–11 years vs. 12–16 years), we used a multivariate analysis of covariance (MANCOVA). “IQ” was set as covariate, and “group” and “age group” as fixed factors. The dependent variables were “SDMT”  and “c-SDMT” .A repeated measures analysis of variance (repeated measures ANOVA) was performed to examine within-trial variation in reaction time between the eight trials of the c-SDMT for inpatient and outpatient participants. In this analysis, the eight single trial times were set as within-subject variables and “group” as the between-subjects factor in order to compare the two groups. Contrasts were adjusted to polynomial in order to compare all the eight trials with one another.p = 0.329), homogeneity of variances (Levene’s test p = 0.807)] for the SDMT, but normality and homogeneity of variances were violated for the c-SDMT. Since non-parametric alternatives are rare for MANCOVA and both MANCOVA and ANOVA are considered fairly robust procedures if cell sizes are >20, we chose to stick to parametric testing.All assumptions were met  nor by the interaction effect (“age group” × “group”) . The main effects of “age group”  and “group”  on IPS were significant. This means that IQ scores did not correlate with IPS and that the effect of hospitalization did not differ between the age groups, whereas both age and hospitalization per se were associated with differences in IPS.A MANCOVA  was usedF = 82.56, p < 0.001] and for the c-SDMT . By contrast, univariate effects of “group” were significant for the SDMT , but not for the c-SDMT . This showed that outpatient and inpatient participants performed differently in the SDMT and, thus, hospitalization affects the results of this test. Univariate analyses were used to further examine the relationship between age group and group and the SDMT and c-SDMT, after adjusting for the non-significant effect of the covariate (IQ). The effect of age group was still significant in univariate tests for the SDMT  and degrees of freedom were therefore corrected using Greenhouse-Geisser estimates of sphericity (ε = 0.65). There was a significant group effect , showing that IPS performance in the c-SDMT across trials one to eight significantly differs between inpatient and outpatient groups. In tests of within-subject effects with the corrected Greenhouse-Geisser, c-SDMT performance over the eight trials was also significant . In general, the participants got faster from trial one to trial eight  = 2.19, p = 0.06]. In other words, the outpatient and inpatient groups did not significantly differ in their changes of performance speed from trial one to trial eight.A one-way repeated measures ANOVA was conducted to compare the effect of “group” (outpatient vs. inpatient) on IPS performance from trial one to trial eight of the c-SDMT. Mauchly’s test indicated that the assumption of sphericity had been violated [χal eight . HoweverThe SDMT and the c-SDMT are two screening tools for IPS. For the SDMT, associations with sex , age Ar, psycholFirst and foremost, our results showed a significant effect of hospitalization on IPS performance as assessed in the written SDMT. Inpatient participants performed more slowly than outpatient ones. This could be interpreted as impairment of IPS performance by hospitalization through its environmental, psychological, and physical impacts . No suchFurthermore, the c-SDMT provides more detailed information about the within-test variation and therefore probably about attention, procedural learning and cognitive fatigability during the test . ConcernSecondly, our results indicated no association between SDMT or c-SDMT scores and the IQ score, i.e., faster IPS did not correlate with higher IQ scores. This is in contrast to the findings of Additionally, our results revealed that age is the best predictor of IPS performance in the c-SDMT as well as in the SDMT. These findings are consistent with the results of previous studies . Hale 1. Bigi etOur results showed no significant difference in the effect of age between the outpatient and inpatient group. In other words, both outpatient and inpatient participants demonstrated improvement in IPS performance with increasing age.The differences between the SDMT and the c-SDMT are crucial. In contrast to classical paper-and-pencil tests, the c-SDMT could more accurately reflect real impairment, because it is not influenced by the effect of hospitalization. The results of paper-and-pencil tests could be influenced by both motor impairment and hospitalization and could therefore be misleading. Therefore, in a clinical setting, the c-SDMT has benefits compared to classical paper-and-pencil tests of IPS: it is fairer for an inpatient population and no motor dexterity is needed making it suitable, for example, for paralyzed patients or patients with a broken arm. In addition, it provides a measure of trial-to-trial variation of IPS, which may be valuable information in some neurological conditions. Finally, all aspects are digitally recorded, the test can be administered anywhere, and is thus an easy-to-use bedside tool.Our study is not without limitations. To reduce bias due to different settings, all participants were tested at the University Children’s Hospital Bern. Therefore, the sample size was relatively small, as the participants came from around the area of Bern and did not represent the whole country with its cultural and linguistic differences. In our current validation studies, we continue to sample healthy participants to pool our data for re-examination and confirmation of the issues discussed in this paper. Furthermore, unlike previous studies , we did Our findings suggest that performance in both SDMT and c-SDMT is independent of intelligence. The c-SDMT is a valid alternative to the classical paper-and-pencil tests of IPS, with the benefits of not requiring motor dexterity, not being influenced by hospitalization, and delivering more detailed information about within-test variation of IPS.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Gesundheits- und Fürsorgedirektion des Kantons Bern, Kantonale Ethikkommission für die Forschung, Bern, Switzerland. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.M-NK and UJ-R performed the acquisition of the data, analysis and interpretation of the data, and draft of the manuscript. SR, CH, MS, and LC were involved in the data acquisition. SB and KL drafted and supervised the study and critically revised the manuscript for intellectual content. All authors were involved in the conceptualization and design of the study, and read and approved the manuscript.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.