Brachybacterium conglomeratum and Brevibacterium aureum as well as a decrease in two Lactobacillus iners operational taxonomic units (OTUs). We also identified a positive independent association between HPV-16, the most common HPV subtype linked to SIL, and Brachybacterium conglomeratum. Our work indicates that HPV infection leading to SIL is primarily associated with shifts in vaginal microbiota composition, some of which may be specific to this human population.Cervical cancer is an important health concern worldwide and is one of the leading causes of death in Mexican women. Previous studies have shown changes in the female genital tract microbe community related to human papillomavirus (HPV) infection and cervical cancer; yet, this link remains unexplored in many human populations. This study evaluated the vaginal bacterial community among Mexican women with precancerous squamous intraepithelial lesions (SIL). We sequenced the V3 region of the 16S rRNA gene in cervical samples from 228 Mexican women, including 121 participants with SIL, most of which were HPV positive, and 107 healthy women without HPV infection or SIL. The presence of SIL was associated with changes in composition (beta diversity) and with a higher species richness (Chao1). A comparison of HPV-positive women with and without SIL showed that microbiota changes occurred even in the absence of SIL. Multivariate association with linear models (MaAsLin) analysis yielded independent associations between HPV infection and an increase in the relative abundance of IMPORTANCE Human papillomavirus (HPV) plays a critical role in cervical carcinogenesis but is not sufficient for cervical cancer development, indicating the involvement of other factors. The vaginal microbiota is an important factor in controlling infections caused by HPV, and, depending on its composition, it can modulate the microenvironment in vaginal mucosa against viral infections. Ethnic and sociodemographic factors influence differences in vaginal microbiome composition, which underlies the dysbiotic patterns linked to HPV infection and cervical cancer across different populations of women. Here, we provide evidence for associations between vaginal microbiota patterns and HPV infection linked to ethnic and sociodemographic factors. To our knowledge, this is the first report of the species Brevibacterium aureum and Brachybacterium conglomeratum linked to HPV infection or squamous intraepithelial lesions (SIL). Cervical cancer is one of the most common cancers and one of the leading causes of death in women worldwide , 2. Cerv4–Lactobacillus spp. Lactobacilli provide broad-spectrum protection by producing lactic acid, bacteriocins, and biosurfactants and by adhering to the mucosa that forms barriers against pathogenic infections in the vaginal microenvironment  , and 31 were diagnosed with HPV infection and high squamous intraepithelial lesion (HSIL) (women diagnosed with CIN2 or CIN3). All women were cancer free, but cases exhibited altered results in cytological, histological, and colposcopy analyses, and control, healthy women exhibited normal results.By molecular analysis, the HPV subtype frequency was highest for HPV-16, HPV-58, and HPV-18 types, with 53.72%, 15.7%, and 9.1%, respectively, observed within cases. Within women without SIL, 32.7% were positive for HPV by PCR, and the distribution of HPV subtypes was similar to that observed in cases. Most of the women in both groups did not smoke (75.21% of cases versus 71.96% of controls), had regular menstrual periods (69.42% of cases versus 68.22% of controls), and most did not have intermenstrual bleeding (85.95% of cases versus 88.79% of controls). Statistically significant differences between groups were detected in relation to an active sexual life at the time of the study (73.55% of cases versus 92.53% of controls), use of contraceptives (66.12% of cases versus 52.34% of controls), and genital hygiene, recorded by the frequency of vaginal douching (81.82% of cases versus 52.34% of controls). More details of the characteristics of each group are described in N = 228; Adonis P = 0.002). The presence of SILs was also associated with significantly higher species richness than an absence of SILs  (P = 0.041) . The presence of SILs was associated with changes in bacterial alpha and beta diversity , with no.15E−04)  as well = 0.041) , suggestP = 0.41; Adonis P = 0.11), further strengthening the link between HPV infection, rather than SILs, on the vaginal microbiota. Because of this, we further investigated the differences in vaginal microbiota explained by HPV infection. While results are similar to when data were stratified by SIL  . MaAsLin is a multivariate linear modeling tool with boosting that tests for associations between specific microbial taxa and continuous and/or Boolean metadata. This method reduces the total amount of correlations to be tested, therefore allowing for improvements in the robustness of the additive general linear models. With MaAsLin, we found significant independent associations between HPV-positive status and an increase in the relative abundance of s (OTUs) . This ins (OTUs) . For exanfection .Several factors are known to play a role in cervical carcinogenesis, with HPV infection being one of the most important in the development of the disease . There aOur study also revealed several other factors associated with SIL status, some of which reaffirm previously reported links . FactorsAcinetobacter lwoffii, which has been previously reported in HPV-positive women  secretion, causing proinflammatory activity in the cervix, which may influence the progression of cervical intraepithelial neoplasia  and not SILs, indicating that infection itself may lead to changes in the vaginal microbial community. Among the predominant components of a healthy vaginal microbiome is the presence of  gasseri , 42, whil cancer  and withl cancer , 44. In obacilli –47. For eoplasia . In otheeoplasia .Brevibacterium aureum and Brachybacterium conglomeratum (increased in cases). While these differences were very significant, these species were not uniformly present among either group  and attending the Instituto Mexicano del Seguro Social (IMSS) in Mexico City were invited to participate as volunteers in this study. Written informed consent was obtained from all volunteers after providing detailed information about the study and its characteristics. The clinical research protocol and letter of informed consent were evaluated and approved by the Comité Local de Investigación y de Bioética de la División de Educación e Investigación Médica de la Unidad de Alta Especialidad Médica Pediatría del IMSS. All participants completed a study questionnaire that was used to obtain sociodemographic and risk factor information. Data were registered in a secured database for subsequent statistical analysis.A total of 228 Mexican women over 21 years of age who attended the IMSS from December 2003 to July 2006 were enrolled in the study. These women were divided in two groups. The first group (controls) consisted of 107 healthy women with a mean age of 42.8 years (±7.9) with three previous Papanicolaou (Pap) tests negative for HPV infection for three consecutive years (a fourth negative Pap result occurred at the time participants were invited to join the study) and diagnosed without SIL with normal cytology and colposcopy results by the treating gynecologist. The second group (cases) consisted of 121 patients with a mean age of 37.3 years (±10.9) with different degrees of SILs and were positive for HPV infection based on cytology, histology, and colposcopy examination. This group included women diagnosed with cervical intraepithelial neoplasia from 1 to 3  according to the Bethesda classification . ParticiBesides SIL and HPV status, the following study variables were included in the analysis: age, HPV subtype, smoking (daily number of cigarettes consumed at time of study), regularity of menstrual period, intermenstrual bleeding, current sexual activity status, use and type of contraceptives , vaginal douching, age of sexual debut, number of sexual partners, number of pregnancies, and number of births and miscarriages.Samples of vaginal exudate were taken by swabbing the mucosa using sterile Teflon swabs that were placed in a sterile 15-ml conical plastic tube with sterile 0.9% sodium chloride . The sample was kept at −20°C until its use for microbiome sequencing analysis.Cervical DNA was extracted directly from the brushing done by the gynecologist before the application of acetic acid for the colposcopy study. The brush was stored in 1 ml of saline solution at 4°C for transport. DNA was extracted immediately from a first centrifugation at 8,000 rpm for 5 min, and the pellet was resuspended in 200 μl of extraction buffer (SDS 1%) and proteinase K (200 μg/ml), making a modification in the digestion of the sample (53°C for 5 h) and inactivating the reaction by boiling for 5 min . Once th53–From vaginal DNA samples, the 16S rRNA gene was amplified by PCR in triplicate using bar-coded primer pairs flanking the V3 region as previously described . Each 50For 16S rRNA gene sequencing, each PCR pool was analyzed on an Agilent Bioanalyzer using a high-sensitivity double-stranded DNA (dsDNA) assay to determine approximate library fragment size and verify library integrity. Pooled library concentrations were determined using the TruSeq DNA sample preparation kit, version 2 (Illumina). Library pools were diluted to 4 nM and denatured into single strands using fresh 0.2 N NaOH. The final library loading concentration was 8 pM, with an additional PhiX spike-in of 20%. Sequencing was performed using a Hi-Seq 2000 bidirectional Illumina sequencing and cluster kit, version 4 . PCR products were visualized on E-gels, quantified using an Invitrogen Qubit with PicoGreen, and pooled at equal concentrations, according to a previous report .N of 228. Further filtering was applied to remove mitochondrion and chloroplast sequences and OTUs present in fewer than three samples. The OTU table was included as a supplemental data set in the supplemental material.All sequences were processed using mothur v.1.43.0 according to the standard operating procedure as previously described . Qualityt tests (parametric) were used based on the D´Agostino-Pearson normality test. We assessed vaginal microbial diversity and the relative abundance of bacterial taxa using Phyloseq  and a 95% confidence interval (CI). For numerical variables, Mann-Whitney (nonparametric) or Student Phyloseq  along wiPhyloseq  were usePRJNA766648).Sequences have been deposited to BioProject (accession number


This paper studies optimal treatment allocations for two treatment comparisons when the outcome is ordinal and analyzed by a proportional odds cumulative logits model. The variance of the treatment effect estimator is used as optimality criterion. The optimal design is sought so that this variance is minimal for a given total sample size or a given budget, meaning that the power for the test on treatment effect is maximal, or it is sought so that a required power level is achieved at a minimal total sample size or budget. Results are presented for three, five and seven ordered response categories, three treatment effect sizes and a skewed, bell-shaped or polarized distribution of the response probabilities. The optimal proportion subjects in the intervention condition decreases with the number of response categories and the costs for the intervention relative to those for the control. The relation between the optimal proportion and effect size depends on the distribution of the response probabilities. The widely used balanced design is not always the most efficient; its efficiency as compared to the optimal design decreases with increasing cost ratio. The optimal design is highly robust to misspecification of the response probabilities and treatment effect size. The optimal design methodology is illustrated using two pharmaceutical examples. A Shiny app is available to find the optimal treatment allocation, to evaluate the efficiency of the balanced design and to study the relation between budget or sample size and power. The randomized controlled trial is considered the gold standard for the comparison of multiple treatment conditions ,2. An imOver the past two decades the literature on optimal treatment allocations has grown. For a quantitative outcome variable attention has been paid to two treatment comparisons –7 and cok factorial designs  its efficiency is at least 0.96.Suppose this trial is to be replicated such that the power for any given sample size is maximized. A relevant question is whether a design with an equal number of subjects per treatment condition is the most efficient. The design is locally optimal and the parameter estimates as given above are used to derive the optimal design. β = 1.217 with a power level of 0.8 in a test with type I error rate of 0.05 and a one-sided alternative hypothesis. A relevant question is how large the sample size should be to detect at treatment effect of size n = 210), low dose (n = 190), medium dose (n = 207) and high dose (n = 195).This example uses data from Chuang-Stein and Agresti . Five orp1, p2, p3, p4, p5) = . The estimated probabilities in both conditions are shown in χ2 = 4.86, df = 3, p = 0.18).Suppose this trial has to be replicated and interest is in the comparison of placebo versus medium dose. A proportional odds model shows the estimated treatment effect is of small size and insignificant at cI/cC = 2.5. The estimated treatment effect and thresholds are used to derive the locally optimal design. p* = 0.39 and the efficiency of the balanced design is 0.95. The balanced design is highly efficient but not the best choice. The optimal design is highly robust to an incorrect prior estimate of the treatment effect size. For any population value of the treatment effect in the range OR =  it has an efficiency of at least 0.99.In the derivation of the optimal design we assume differential costs across the two treatments, with a cost ratio β = −0.324 in a one-sided test with a type I error rate of 0.05. The costs are scaled so that c1 = 1; in other words, the budget is expressed in terms of the costs per subject in the control condition. To achieve a power of 0.8 for the test on treatment effect at a type I error rate of 0.05 and a one sided alternative a budget of B = 1262 is needed. The corresponding total sample size is 796, which is almost twice as high as in the original study.This study provides methodology and a Shiny app to calculate the optimal treatment allocation in a trial with two treatment conditions and ordinal outcomes. The optimal design minimizes the variance of the treatment effect estimator, so that the power for the test on treatment effect is maximized. The optimal design is sought under a budgetary constraint, of which a fixed total sample size is a special case.In general the optimal proportion subjects in the intervention condition decreases with the cost ratio. For the polarized distribution the optimal proportion increases with increasing effect size and decreases with increasing number of response categories, while for the other two distributions these relations are decreasing or absent. Furthermore, the optimal proportion depends on the distribution of response probabilities: it is highest for the polarized distribution. However, the optimal design is highly robust with respect to misspecification of the parameters of the ordinal logistic model, hence it is not considered necessary to implement robust optimal designs, such as a maximin optimal design. The widely used balanced design has a high efficiency in the case the total sample size is fixed. However, its efficiency declines when the cost ratio increases. It can therefore be concluded the balanced design is not necessarily the best choice. These findings are also illustrated by the two examples. In the first a fixed total sample size was used as a constraint. The balanced design turned out to be the optimal design. In the second a budgetary constraint was used, resulting in a loss of efficiency of the balanced design.The methodology in this paper can also be used if two naturally existing groups are to be compared on some ordinal outcome, such as males and females or smokers and non-smokers. In some studies the number of subjects in one of the groups may be small, for instance the number of subjects suffering from a rare disease. It may occur fewer of such subjects can be enrolled in the trial than dictated by the optimal design. In that case the thus unused part of the budget may be spend on sampling more subjects in the other group. The Shiny app may be used to evaluate the efficiency of such a sub-optimal design.The optimal designs as presented in this paper are based on a model that uses cumulative logits: the probability of a response category and all previous categories is compared to the probability of all subsequent response categories. There exist other types of logit models for ordinal response variables, see section 3.1 and chapter 4 in Agresti . The adjJ−1, rather than one single, treatment effects should be estimated, and this should be reflected in the optimality criteria. It may be then relevant to focus on a multiple-objective optimal design, where optimality criteria are ordered with respect to their relative importance. For instance, estimating the effect of treatment on the first cumulative logit, that separates the category “death” from all other categories, may be considered more important than the treatment effects on all other cumulative logits. It may also be of interest to study the robustness of the optimal designs as derived under the proportional-odds assumption against violation of this assumption. Other extensions include trials with nested data, such as cluster randomized trials and trials with partially nested data. An example of the latter is a trial with nesting of subjects within therapists in the psychotherapy condition and no nesting in the wait list control. Finally, the effect of predictive covariates on the optimal design should be studied.Future work may focus on the derivation of optimal treatment allocations for extensions to model (1). One such extension are factorial designs, for instance for evaluating pharmaceutical and behavioral interventions simultaneously. Another extension may focus on one treatment factor with more than two levels, for instance a placebo versus different types of drugs. Attention should also be paid to the non-proportional odds model, where the effect of treatment may vary across the cumulative logits. This implies For all these extensions it may turn out the locally optimal design is not robust against misspecification of the model parameters. In such cases one may derive a robust optimal design, such as a maximin design . The optIn summary, this paper has provided optimal allocations for two treatment comparisons when the response variable is ordinal and modelled using a proportional odds cumulative logits model. The Shiny app enables researchers to find the optimal allocation for their trial, and to evaluate the efficiency of alternative designs, including the balanced design.S1 Appendix(DOCX)Click here for additional data file.


What factors are associated with adherence to recommended testing after initial lung cancer screening in the Veterans Health Administration?In this retrospective cohort study of 28 294 veterans, more than one-quarter of veterans received delayed or no follow-up testing. Veterans with higher risk findings and those in high-volume or academic centers were more likely to receive timely follow-up, while Black veterans, veterans with mental health disorders, and veterans with lower income were more likely to have delayed or absent follow-up.These findings suggest that even in an integrated health care system, inequities in adherence to lung cancer screening persisted. This cohort study examines adherence to follow-up testing recommendations after lung cancer screening among US veterans. Lung cancer screening (LCS) can reduce lung cancer mortality with close follow-up and adherence to management recommendations. Little is known about factors associated with adherence to LCS in real-world practice, with data limited to case series from selected LCS programs.To analyze adherence to follow-up based on standardized follow-up recommendations in a national cohort and to identify factors associated with delayed or absent follow-up.This retrospective cohort study was conducted in Veterans Health Administration (VHA) facilities across the US. Veterans were screened for lung cancer between 2015 to 2019 with sufficient follow-up time to receive recommended evaluation. Patient- and facility-level logistic regression analyses were performed. Data were analyzed from November 26, 2019, to December 16, 2020.Receipt of the recommended next step after initial LCS according to Lung CT Screening Reporting & Data System (Lung-RADS) category, as captured in VHA or Medicare claims.Of 28 294 veterans  who had an initial LCS examination, 17 863 veterans (63.1%) underwent recommended follow-up within the expected timeframe, whereas 3696 veterans (13.1%) underwent late evaluation, and 4439 veterans (15.7%) had no apparent evaluation. Facility-level differences were associated with 9.2% of the observed variation in rates of late or absent evaluation. In multivariable-adjusted models, Black veterans , veterans with posttraumatic stress disorder , veterans with substance use disorders , veterans with lower income , and those living at a greater distance from a VHA facility  were more likely to experience delayed or no follow-up; veterans with higher risk findings  and those screened in high LCS volume facilities  or academic facilities  were less likely to experience delayed or no follow-up. In sensitivity analyses, varying how stringently adherence was defined, expected evaluation ranged from 14 486 veterans (49.7%) under stringent definitions to 20 578 veterans (78.8%) under liberal definitions.In this cohort study that captured follow-up care from the integrated VHA health care system and Medicare, less than two-thirds of patients received timely recommended follow-up after initial LCS, with higher risk of delayed or absent follow-up among marginalized populations, such as Black individuals, individuals with mental health disorders, and individuals with low income, that have long experienced disparities in lung cancer outcomes. Future work should focus on identifying facilities that promote high adherence and disseminating successful strategies to promote equity in LCS among marginalized populations. Indeed, 95% of NLST participants were adherent with annual screening, and 93% were adherent with recommended evaluation of screen-detected findings. Inability to achieve such high adherence in nontrial settings is likely to reduce the effectiveness of LCS.2Lung cancer remains the leading cause of cancer mortality, with 1.8 million deaths annually worldwide. In the National Lung Screening Trial (NLST), yearly lung cancer screening (LCS) with low-dose computed tomography (CT) achieved a 20% relative reduction in lung cancer mortality.3 there has been limited evidence on follow-up after initial LCS in real-world settings, with selected institutions reporting adherence ranging from 37% to 86%.9 Barriers to adherence exist at the patient level ,12 clinician level ,15 and system level .16Although the US Preventive Services Task Force has recommended LCS for high-risk individuals since 2013,18 Although the Veterans Health Administration (VHA), the largest national integrated health care system in the US, was an early adopter of LCS, subsequent implementation has been inconsistent across VHA facilities.19 Thus, the VHA provides an ideal real-world setting to study adherence after initial LCS across the US. We sought to determine adherence to recommended next steps  in a national cohort of veterans screened for lung cancer and identify factors associated with delayed or absent follow-up.Veterans are at increased risk of lung cancer, given high rates of tobacco use, with a large population of veterans eligible for LCS.STROBE) reporting guideline for cohort studies.This cohort study was approved by the Bedford VA Healthcare System institutional review board. This was an observational study without any direct patient contact and considered to be of minimal risk; therefore, a waiver of informed consent was obtained from the Bedford VA Healthcare System institutional review board. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology  generated by VHA’s LCS clinical reminder followed by a chest CT within 3 months or a chest CT used for LCS based on Current Procedural Terminology codes or CDW radiology tables , we used the subsequent LCS examination as the index LCS. Veterans were excluded if they had insufficient follow-up time after index LCS to receive expected evaluation according to Lung-RADS category , we identified veterans who had either a health factor for International Classification of Diseases, Ninth Revision (ICD-9)21 and Tenth Revision (ICD-10)22 and Current Procedural Terminology codes  for evidence of expected follow-up tests, including chest CT, positron emission tomography, or invasive procedures, defined by 26 For each veteran, we extracted data from VHA CDW on demographic characteristics and VHA priority status . We used the veteran’s residential zip code to estimate distance to nearest VHA facility, median income, and urbanization using the US Census Bureau’s rural-urban commuting area codes.27 We defined comorbidities based on ICD-9 and ICD-10 codes in VHA CDW or Medicare files and calculated Elixhauser comorbidity index scores.29 As a measure of health care utilization, the number of VHA outpatient visits in the year prior to index LCS was collected for each veteran. We performed log transformation of covariates that did not follow a normal distribution, including outpatient visits, median income, and distance to VHA facility.We a priori selected clinically relevant covariables based on prior studies of adherence to cancer screenings, including self-reported race/ethnicity.31 We used data from a national VHA survey conducted in 2013 to 2014 to determine whether facilities had capacity to perform thoracic surgery.32We assigned veterans to VHA facilities based on where they received the most outpatient visits in the year prior to index LCS. Facility-level covariates included academic status based on graduate medical education affiliations, US census region, and LCS volume.P = .05 as statistically significant. To address potential issues of multicollinearity, we computed variance inflation factors for each patient-level variable in the model. No variables had variance inflation factors greater than 10, a conventional threshold for collinearity concerns, suggesting that collinearity was not problematic.33 To quantify facility-level variation, we calculated an intraclass correlation coefficient on the model with patient fixed effects and random intercepts that varied by site.34 All analyses were performed using SAS statistical software version 9.4 (SAS Institute). Data were analyzed from November 26, 2019, to December 16, 2020.We conducted mixed-effects logistic regression analyses, including patient- and site-level variables, to determine factors associated with delayed or absent follow-up after index LCS compared with early or expected follow-up. Our models assumed that coefficients to patient-level variables were random effects that varied by site. We report the mean patient outcomes, accounting for site-specific random effects, in our 2-sided analytic testing. We a priori selected 35We performed sensitivity analyses to examine different definitions of adherence and to check the validity of our conclusions about factors associated with adherence to Lung-RADS recommendations. First, to explore the potential for error introduced into the model by creating cutoffs for adherence, we created 2 models with varying periods for expected evaluation, one more stringent than that used by NLST and one more liberal. All models defined early evaluation as follow-up testing occurring greater than 2 months before the recommended timeframe, to maintain consistency . In the 36 Because we were unable to capture care provided through private insurers, we performed a sensitivity analysis restricting our analysis to Medicare-eligible veterans , as veterans with VHA or Medicare coverage may be less likely to obtain screening through the private sector.38 Additionally, because Medicare claims data are only available through 2018, we performed a sensitivity analysis restricting our study period to January 1, 2015, to December, 31, 2018.To explore the outcomes associated with combining low-risk findings (Lung-RADS categories 1 and 2) with higher risk findings (Lung-RADS categories 3 and 4) into our model, which may have different mechanisms of adherence, we conducted a sensitivity analysis stratifying our regression model by low and high-risk findings. To account for potential error introduced by lack of evaluation among veterans with limited life expectancy, who are unlikely to benefit from LCS and may have elected to decline evaluation, we performed a sensitivity analysis excluding veterans with Care Assessment Needs score of 95 or greater. The validated Care Assessment Needs score is calculated in the VHA EHR to identify veterans with increased risk of hospitalization and death, and a score of 95 or greater is 89% specific for frailty and correlates with an approximately 20% risk of death within 1 year.To further explore the ability of our claims-based algorithm to detect recommended evaluation, a pulmonologist (E.R.N.) performed detailed EHR reviews on a randomly selected sample of veterans with low-risk  and intermediate risk  findings who received no apparent evaluation; as well as all veterans with Lung RADS category 4B or 4X findings (42 veterans) with no subsequent follow-up captured through VHA or Medicare claims.Overall, 35 699 veterans had initial LCS examinations with Lung-RADS recommendations; 7405 veterans (20.7%) were excluded owing to insufficient time to determine adherence to follow-up testing  had evidence of subsequent testing after the index LCS, with 2296 veterans (8.1%) receiving early testing, 17 863 veterans (63.1%) receiving testing in the expected timeframe, and 3696 veterans (13.1%) receiving delayed testing . Of noteIn our primary multivariable analysis, veterans with high-risk findings were less likely to have delayed or no evaluation  . Black vVeterans classified as severely disabled who had no copays  and those with partial disability with reduced copays  were less likely to have delayed or absent follow-up compared with veterans with copays to access VHA care. Additionally, veterans who had fewer outpatient visits or lived farther from a VHA facility were more likely to experience delayed or absent evaluation . The logA total of 122 facilities performed at least 1 index LCS examination in the study period. Veterans screened in high-LCS volume  facilities were less likely to have delayed or missing follow-up , as were those screened in academic facilities  . Our mulUsing alternate definitions of adherence resulted in expected follow-up ranging from 14 486 veterans (49.7%) in the stringent model to 20 579 veterans (78.8%) in the liberal model, indicating that rates of adherence can vary substantially depending on how adherence is defined . Other sEHR review of randomly selected veterans with low-risk or intermediate-risk findings with no apparent follow-up (based on claims data) confirmed absence of follow-up in 38 of 40 veterans (95.0%) with Lung-RADS category 1 or 2 findings and 36 of 40 veterans (90.0%) with Lung-RADS category 3 or 4 findings. By contrast, of 42 veterans with Lung-RADS category 4B or 4X findings with no apparent follow-up, 16 veterans (38.1%) were found on EHR review to have received evaluation outside of the VHA that our study did not capture . Of the remaining 26 veterans with 4B or 4X findings, the most common reasons for lack of follow up included patient declined follow-up (7 veterans [16.7%]), several attempts but unable to schedule (10 veterans [23.8%]), and no documented follow-up (8 veterans [19.0%]).1 and NELSON trials39 could in part be attributed to high adherence (>90%) to timely, appropriate follow-up after initial screening. In real-world practice, in addition to documented low uptake of initial LCS (estimated at 4%-14% of the eligible US population42), suboptimal adherence to follow-up evaluations could further erode the potential of LCS to reduce lung cancer death.In this national cohort study of 28 294 veterans who underwent initial LCS, we found suboptimal adherence to Lung-RADS recommendations, with approximately 13% receiving late evaluation and approximately 16% receiving no apparent follow-up. Our study, which captured follow-up testing from VHA and Medicare systems, illustrates the challenges of providing timely, recommended next steps after LCS in real-world practice. The mortality benefit of LCS observed in the NLST46 But we identified substantial variation across facilities, suggesting there is room for further improvement in some facilities. Indeed, veterans screened in academically affiliated or high LCS–volume facilities were less likely to have delayed or no follow-up, suggesting lessons could be learned from these LCS programs. Based on the included variables, our model identified a moderate amount of facility-level variation.47 It is likely that additional facility-level LCS infrastructure that we could not capture may be associated with improved adherence. For example, single institution studies suggest LCS coordinators and tracking systems, which are components recommended by the American Thoracic Society and American College of Chest Physicians, may improve adherence and timeliness of evaluation.48 Yet, even when facilities implement LCS coordinators and tracking software, significant interfacility variation in adherence remains.9 Thus, future work should evaluate LCS programs with high vs low rates of adherence to identify and spread best practices for timely, appropriate follow-up.At the patient level, veterans with higher-risk Lung-RADS categories were significantly more like to receive adherent follow-up care. This is reassuring, given that timely evaluation of high-risk findings is essential to identifying cancers at an early, treatable stage, thereby avoiding disease progression and improving lung cancer outcomes.51 which may be owing to the more uniform access to care typically experienced in integrated health systems. Yet, we found disparities in receiving the recommended next steps after LCS among marginalized populations, such as Black veterans and those with lower income, who were more likely to experience delayed or absent evaluation, reflecting the well-established finding of disparities in cancer care among those from underrepresented ethnic and racial backgrounds or lower socioeconomic status.54 We found that veterans with posttraumatic stress disorder and substance use disorders were more likely to have delayed or no follow-up, likely reflecting mental health barriers identified in other cancer screenings.56 Veterans with fewer VHA outpatient visits in the prior year, higher copays, and longer distances to travel to access VHA care were more likely to have delayed or absent follow-up after initial LCS. Prior studies illustrate the importance of health care utilization, insurance, and access to care as critical to ensuring appropriate cancer screening and reducing cancer mortality.61 Unfortunately, LCS access is not evenly distributed across the VHA system.62 Therefore, it is imperative for the VHA to continue to allocate resources to improve LCS access and adherence to reduce lung cancer mortality in high-risk veteran populations, with particular attention to marginalized groups, such as Black veterans, those with mental health conditions, and those with socioeconomic barriers to receiving appropriate and timely care.Disparities in cancer screening and lung cancer outcomes are typically decreased in VHA and military settings compared with the private sector,68 Thus the 63% rate of expected evaluation identified in this study may exceed that anticipated in other settings. Additionally, we were unable to measure patient-centered outcomes, such as lung cancer diagnoses and mortality, and instead focus on adherence to screening, which has been shown to be a key factor in lung cancer mortality.2This study has some limitations. First, we relied on administrative data, which can result in misclassified or missed data if there are errors in coding of LCS examinations or follow-up tests. We were unable to capture care received outside of VHA or Medicare, which may have resulted in an underestimate of adherence if substantial care was received through private insurers. However, our sensitivity analysis excluding veterans younger than 65 years who are more likely to be dual users of VHA and private insurance showed only minor differences in follow-up evaluation. Moreover, our EHR review of randomly selected veterans with Lung-RADS categories 1 through 4A findings, who constituted 97% of our cohort, showed our algorithm missed follow-up testing in less than 10% of veterans. Of note, our results may not be generalizable outside the VHA or among nonveteran populations. In particular, the VHA’s national integrated health care system has well-established processes to facilitate guideline-recommended, multidisciplinary cancer care and pulmonary nodule evaluation, and achieves equivalent or better quality of cancer care and survival than the private sector.To our knowledge, this cohort study represents the first national analysis of adherence to Lung-RADS recommendations among a large cohort with diverse geographic distribution and socioeconomic status. We found that even in an integrated health care system, many patients did not receive timely recommended follow-up after initial LCS. While it is encouraging that veterans with the highest risk findings were more likely to receive recommended follow-up care, disparities in timely follow-up were demonstrated among marginalized populations who have long experienced worse lung cancer outcomes, such as Black veterans and those with low income. To optimize the mortality benefit of LCS, it is clear that further work must be done to improve adherence overall and especially among marginalized populations. Future work should focus on learning effective strategies from LCS centers with high adherence to extend successful program features to the people and locations with greater barriers to care.


M. tuberculosis (TB) promotes HPV infection and increases the risk to develop HPV-associated cancer. Our knowledge of persisting HR HPVs genotypes, and of the factors promoting HR HPV infection in people living with HIV-1 with clinical TB manifestations is sparse. Here, we analyzed 58 women living with HIV-1 with clinical TB manifestations (WLWH with TB) followed up in specialized centers in Russia, a middle income country endemic for HIV-1 and TB, for the presence in cervical smears of DNA of twelve HR HPV genotypes. DNA encoding HPV16 E5, E6/E7 was sequenced. Sociodemographic data of patients was collected by questionnaire. All women were at C2-C3 stages of HIV-infection (by CDC). The majority were over 30 years old, had secondary education, were unemployed, had sexual partners, experienced 2–3 pregnancies and at least one abortion, and were smokers. The most prevalent was HPV16 detected in the cervical smears of 38% of study participants. Altogether 34.5% of study participants were positive for HR HPV types other than HPV16; however, but none of these types was seen in more than 7% of tested samples. Altogether, 20.7% of study participants were positive for several HR HPV types. Infections with HPVs other than HPV16 were common among WLWH with generalized TB receiving combined ART/TB-therapy, and associated with their ability to work, indirectly reflecting both their health and lifestyle. The overall prevalence of HR HPVs was associated with sexual activity of women reflected by the number of pregnancies, and of HPV 16, with young age; none was associated to CD4+-counts, route of HIV-infection, duration of life with HIV, forms of TB-infection, or duration of ART, characterizing the immune status. Thus, WLWH with TB—especially young—were predisposed to infection with HPV16, advancing it as a basis for a therapeutic HPV vaccine. Phylogenetic analysis of HPV16 E5, E6/E7 DNA revealed no common ancestry; sequences were similar to those of the European and American HPV16 strains, indicating that HPV vaccine for WLWH could be the same as HPV16 vaccines developed for the general population. Sociodemographic and health correlates of HR HPV prevalence in WLWH deserve further analysis to develop criteria/recommendations for prophylactic catch-up and therapeutic HPV vaccination of this highly susceptible and vulnerable population group.Women living with HIV-1 are at high risk of infection with human papillomavirus of high carcinogenic risk (HR HPVs).  Up to 4.5% of new cases of cancer registered worldwide—including cervical, anogenital, and head and neck cancers—are associated with human papillomavirus (HPV) infection . CervicaThe highest incidence of cervical cancer is in the countries of South, East, and West Africa, where the standardized rates are 43.1, 40.1, and 29.6 cases per 100,000 population, respectively . Over thStudies have shown that 71% of cervical cancers are caused by persistent HR HPV infection, primarily genotypes 16 and 18 ,13,14,15The disparity in CC incidence between WLWH and HIV-1 negative women is specifically high in the low- and middle-income countries. In Africa, HIV-infected women are six times more likely to develop CC than HIV-negative women. The prevailing hypothesis is that HIV-mediated immune dysfunction leads to longer and more severe cervical HPV infections increasing the likelihood of oncogenic transformations which further develop into cervical cancer . This scM. tuberculosis and develop active tuberculosis (TB) with over 95% of TB deaths occurring in low- and middle-income countries [People living with HIV are 20 to 30 times more likely to be infected with ountries . Over thountries . Still, ountries , and WLWountries .M. tuberculosis is a co-factor promoting HPV infection. TB causes chronic inflammation of organs and tissues and increases the risk of developing malignant neoplasms, including those in the gynecological tract [M. tuberculosis (MTB), specifically drug-resistant strains, induces host metabolic reprogramming, so called “immunometabolism”, modulating the capacity of the body to produce innate and through this, adaptive immune responses [Infection with al tract ,29. Up tal tract ,31. A stal tract . Furtheresponses ,33.M. tuberculosis may strongly predispose to the infection with HR HPVs and consequent development of cervical abnormalities culminating in cervical cancer (more than single HIV-1 and MTB infections). Today, despite the pandemic character of HIV-1 and TB infections and the four-decades-long history of their co-circulation, the actual information on HR HPVs prevalence among women living with HIV-1 with clinical TB manifestations is sparse [Today, 20.1 million women live with HIV-1 and 3.2 million with tuberculosis worldwide . Infectis sparse , and forThe study was approved by the Biomedical Ethics Committee of the N.F. Gamaleja National Research Center for Epidemiology and Microbiology, Moscow, Russia (protocol no. 11 of 3 July 2017). All participants gave written informed consent to take part in the study. According to the General Data Protection Regulation (GDPR) requirements, all participants were de-identified and anonymized by assigning them unique codes expressed as an identifier, based on the name of the individual and year of birth. Anonymization was done by medical doctors responsible for the patients. Patient samples were collected by qualified medical personnel. All materials, data, and study results were stored in an anonymized form.Study group enrolled 58 women living with HIV-1 with clinical manifestations of TB (WLWH with TB) , both nan = 9), fibrous-cavernous (n = 1), disseminated (n = 15), infiltrative TB (n = 21), tuberculosis of the intrathoracic lymph nodes (n = 5), exudative tuberculous pleurisy (n = 2), cirrhotic TB (n = 1), and focal TB (n = 4). Disseminated TB was defined as cases with multiple MTB foci preferentially localized in the lungs; fibrous-cavernous TB was identified as the final stage in the development of the earlier forms of lung tuberculosis, characterized by cavities with a strong fibrous capsule internal caseous-purulent layer and common lung damage . The study was initiated early after hospitalization, with anti-tuberculosis (anti-TB) therapy administered to 14 patients together with antiretroviral therapy, the remaining patients participated in the study before the initiation of TB treatment.All women in the study group were at C2–C3 stage of HIV-infection (CDC): C2—53.4% and C3—43.1%. TB diagnoses were based on the clinical symptoms, sputum microscopy and radiological analyses. TB forms were classified according to TB classification accepted in Russia . According to this classification, patients were diagnosed with generalized ; employment status (work/no work/disability pension); feeling of security (yes/no); marital status (single/married/widow); number of sexual partners during six months preceding the study; number of pregnancies; number of abortions; probable route of HIV infection ; health damaging habits .p = 0.016), duration of ART (in years)  and nadir CD4+ T cell counts , respectively. Control group was different from the study group in higher proporion of WLWH on ART, higher percentage of women with CD4 + T cell counts >399, and lower percentage of women with CD4+ T cell counts ≤67 by the time of analysis  matching in age the study group  represen® HR HPV genotype-EPh kit  with electrophoretic detection of HR HPV types 16, 18, 31, 33, 35, 39, 45, 59, 52, 56, 58, and 66. DNA isolation and PCR analysis were performed according to the protocols of the manufacturers.Smears from the cervical canal, transition zone and exocervix of the patients in the study, and in the control groups, were taken using a disposable brush. The brush together with the material was placed in a container with a stabilizing solution . Isolation of DNA from the collected material was done manually using “Isolation of DNA/RNA on MPs of the SileksMagNA ™ type” kit . HPV typing was performed by PCR using the AmpliSens® 45113D™; ATCC, Manassas, Virginia, USA) served as a positive control. Reaction mixtures were analyzed on a 1.5% agarose gel. DNA fragments of the size corresponding to DNA encoding E5, E6, and E7 were purified from the gel and sequenced on ABI 3500 sequencer using The BigDye® Terminator v3.1 Cycle Sequencing Kit .DNA extracted from cervical smears of WLWH co-infected MTB found positive for HPV 16 was amplified with primers specific for genomic regions encoding oncoproteins E5, E6 and E7. The following primers were used: E5-3745 TGC-AAT-TGT-TAC-ACT-TAC-ATA-TG (forward), E5-4318 ATG-TAC-CTG-CCT-GTT-TGC-ATG (reverse), E6-53 GAA-ACC-GGT-TAG-TAT-AAA-AGC-AGA-C (forward), E6-533 AGC-TGG-GTT-TCT-CTA-CGT-GTT-CT (reverse), E7-478 CCA-TAA-TAT-AAG-GGG-TCG-GTG-GA (forward), E7-923 TTT-TTC-CAC-TAC-AGC-CTC-TAC-AT (reverse); primers were described earlier by van Duin et al. . The conGenBank was searched for the complete genomes of HPV 16 strains from Europe and USA deposited in the period 2017–2020, and 3320 sequences were identified. The reference sequences from the ICTV classification for alphapapillomavirus 9 were used as a comparison group. All sequences had >98% of identity to the ones obtained in the current study. GeneBank sequences were cropped leaving only E5 and E6–E7 encoding regions, and subpopulations of sequences were selected that differed from each other by at least one encoded amino acid residue (cut-off at at least 0.3% divergence). Phylogenetic trees were constructed for these E5 and E6/E7 encoding sequences and the ones obtained in this study using the maximum likelihood (ML) method realized in the PhyML 3.0 package. The optimal model was chosen using jModelTest2 employing the nucleotide evolution model HKY85, with a heuristic search through tree space NNI, and aLRT SH-like tree quality assessment. The resulting tree was visualized using FigTree v1.4.3.https://bethesda.soc.wisc.edu/index.htm) (30 March 2021). Inflammatory cervical smears without epithelial cell abnormality and reactive cellular changes were also reported. Slides were scanned using Vision Pro Cell Imaging Analyzer  and the gallery of full slide images viewed by Vision Slide Viewer program (West Medica) was re-evaluated by an independent cytopathologist with confirmation of the initial results. LSIL type was detected in two women. One, infected with HPV 33 and 52, had infiltrating TB and a concomitant diagnosis of candidal esophagitis and CD4+ counts of 400 cells/μL. The other, infected with HPV 16, had tuberculosis of the thoracic lymph nodes and concomitant diagnosis of chronic hepatitis C and CD4+ counts of 55 cells/μL. The remaining eight were negative for intraepithelial lesions or malignancies.Another independent collection of smears from endo- and exocervix was performed at a later occasion if patients agreed (10 of 58). The material was taken using a standard cervical brush, applied on the glass in a thin layer and transported to the laboratory, where the smear was stained after the Romanovsky–Giemsa method. The dried cytological preparations were fixed in methanol for three to five minutes, then kept in a ready-made solution of the Romanovsky–Giemsa dye for five to seven minutes, after which they were washed with water, dried, and microscoped. Results were reported by the local pathologist according to The Bethesda System 2014: negative for intraepithelial lesion or malignancy (NILM); atypical squamous cells undetermined significance (ASC-US); low grade squamous intraepithelial lesion (LSIL); atypical squamous cells cannot exclude HSIL (ASC-H); high grade squamous intraepithelial lesion (HSIL); AGC ; and squamous cell carcinoma (SCC) .Data analysis was performed using the IBM SPSS Statistics 20.0 software and in part, using nonparametric Mann–Whitney, Kruskal–Wallis, and Spearman rank correlation tests . Analysis of the level of significance for the difference between two proportions from two independent samples and for the means of two independent populations was done using two-sided difference tests for independent samples (n = 1) who were assembled into Group 1 (n = 10). The rest of patients with other TB forms were assembled into Group 2 (n = 48).The study enrolled 58 women living with HIV with an established diagnosis of tuberculosis (WLWH with TB). Most were 31–39 years old 58.6%; . For 96.p < 0.05). Patients with severe clinical manifestations of TB had significantly lower CD4+ counts compared with other patient groups (p < 0.05). CD4+ T cell counts significantly correlated with the severity of clinical TB manifestations  were not treated. Of these, 19 (32.7% of the study group) did not know that they were HIV-1 infected and 16 (27.6%) refused treatment. All patients admitted to the clinics were offered to start/continue ART.p > 0.05). Thus, by the time of HPV study, 31 patients were considered as ART-treated, and 27 as untreated (p > 0.05).The HPV study, including the survey and collection of cervical smears, was performed shortly after hospitalization. By the time of the study, 23 patients continued ART; 8 have started and were on treatment >1 month; 2 started and stopped ART due to drug intolerance; 14 have not yet started or started ART ≤1 month ago, and 5 refused treatment . For thentreated . The medp value = 0.6697; and 2* sided exact p value = 0.6993. In patients treated for TB .By the time of HPV study, TB therapy was administered to 5/10 patients (50%) in Group 1 with severe clinical manifestations of TB. Most of the patients in Group 2 with relatively mild forms of TB such as exudative pleurisy, cirrhotic and focal TB had not yet started either TB-therapy, or ART . Nine (18.8%), including two with disseminated and five with infiltrative TB, received TB-therapy and ART . The ave n = 14, , CD4+ T p < 0.05) and tended to be sexually more active than WLWH with TB not receiving ART  (p < 0.1).Most of WLWH with TB participating in the study  was not married; 77.6% reported one sexual partner in 4–6 months preceding sociodemographic survey and HPV testing; 13.8% reported no sexual contacts in the last six month  prior to the survey. According to the survey, 69% (40) women had children; 53.4% (31) had 3 or more pregnancies; 34.5% (20) had 1 or 2 abortions . Compari F-test) . The oppThe majority  of WLWH with TB had secondary or secondary professional education; 5 (8.6%) of women had completed and 3 (5.2%) had incomplete university education . Fewer tp = 0.09). The same was true for the women receiving TB-therapy (TB-therapy+ART): 50% (7/14) receiving ART+TB therapy were working, among untreated women the percentage of working was significantly lower . Altogether, this indicated that women on ART and ART+TB therapy were in a better health condition and/or were feeling better  than treatment-naïve women.With regards to the means of financial support, 43.5% (10/23) women on ART worked, whereas only 22.9% (8/35) were working among treatment-naïve women (Risk factors for acquiring HIV-1 were identified in 98.3% (57) patients. An important risk factor was injection drug use reported by 17.2% of study participants  of the study participants; Overall, the sociodemographic portrait of an HIV/TB co-infected woman was as following: age over 30, with secondary education, unemployed, having a sexual partner without marriage, with two or three pregnancies and at least one abortion, and a smoker. Absence of the knowledge of HIV and TB status, refusal of ART for those knowing their HIV+ status, along with several health-damaging habits, indicated indifference of the majority of study participants to their health.Cervical smears of 41.4% of HPV study participants (24/58) were negative for all 12 HR HPVs. HPV 16 was detected in 38% (22/58), i.e., in the majority of positive samples . Other HR HPVs were detected in 34.5% (20/58) of WLWH. The prevalence of HR HPV genotypes other than HPV 16  among WLWH with TB was low  , but the diversity of HR HPV types was less pronounced than in WLWH with TB.Interestingly, control group of TB-negative WLWH demonstrated higher HR HPV prevalence . Besides < 0.05) . One patOut of 58 women in the study group, only 10 agreed to undergo repeated sampling with cytological examination of the cells in the cervical canal for morphological changes. LSIL was detected in two women, one infected with HPV 16 and the other, with HPV 33 and 52. Other patients  were negative for the intraepithelial lesions or malignancies.The quality of the samples and HPV viral load allowed us to obtain sequences of E5, E6 and E7 encoding DNA from 11 out of 22 HPV16-positive samples of WLWH with TB. The resulting sequences were entered into the international NCBI GenBank database and assigned numbers MT353690-MT353699, MT353701-MT353712. Their analysis revealed a low level of divergence. The phylogenetic trees based on E5 and on E6/E7 demonstrated that they did not have a common ancestry and were similar to E5, E6, and E7 from HPV 16 isolated in other European countries and the USA reflecting diversity of the circulating HPV 16 strains .E5, E6, and E7 oncoproteins sequences were found to be highly conserved, specifically at the amino acid level. The most conserved was the E7 oncoprotein, in which only one substitution on amino acid level was observed, namely N29S in the GeneBank sequence MT353705. The rest of the E7 sequences were identical to the reference HPV 16 variant NC_001526. More polymorphisms were found in the E6 oncoprotein. E6 oncoprotein contained several single amino acid substitutions unique for each of the samples. Besides, 5 out of 11 samples contained substitutions R17G and L90V , and MT353708, single substitution R17G. For the E5 oncoprotein, a single amino acid substitution I65V was recorded in two samples; other sequences were identical to the NC_001526 reference. Thus, main HPV 16 oncoproteins identified in WLWH with TB did not differ from that circulating in other population groups around the world.Firstly, we analyzed prevalence of HR HPVs WLWH with TB in relation to the objective characteristics, such as age, immune status (CD4+ T cell counts), duration of life with HIV, severity of TB infection, and ART and TB therapy.n = 8) than in other age groups . No age dependence of the prevalence of HR HPVs was observed in the control group . Also, in the study group, HPV 16 tended to be more frequent among patients with CD4+ counts <200 cells/mm3 than in those with CD4+ >350 cells/mm3 (p = 0.059), while the prevalence of other HR HPVs was not related to the immune status (3) (Infection with HR HPVs in the study and in the control groups was not correlated with CD4+ counts . Howevere status . In the atus (3) .p > 0.2). The number of infecting HR HPV types detected in cervical smears of WLWH with TB was not correlated to the years of life with HIV-1 . On the contrary, WLWH without TB showed dependence of the number of detected HR HPV types on the duration of life with HIV-1  exhibited relatively low prevalence of HPV 16 (30%) and high prevalence of other HR HPVs (60%), whereas patients with other TB forms specifically cirrhotic, focal and extrapulmonary TB, on contrary, tended to exhibit higher prevalence of HPV 16 (60%) and lower prevalence of other HR HPVs ; ART-naïve WLWH with TB were characterized by significantly higher prevalence of HPV 16 (n = 45) and WLWH with TB receiving ART >3 month  demonstrated similar high prevalence of HR HPVs other than HPV 16; in treatment naïve WLWH with TB it was almost two times lower . This scenario was valid for both WLWH with and without TB , dominance of HPV 16, and frequent (in up-to 20%) positivity for two or more HR HPV types. However, we observed a marked difference in the prevalence of HR HPVs other than HPV 16. In WLWH with TB their prevalence was two-times lower than in TB-negative WLWH. Furthermore, it was positively associated with ART: in ART treated WLWH, prevalence of HR HPV types other than HPV 16 was nearly two-fold higher than in ART-naïve patients, while prevalence of HPV 16 was lower  as well as in more narrow groups  and multiple HR HPV types  and not working (receiving disability pension and/or supported by the relatives). Working women were characterized by higher prevalence of infection with ‘non-16′ types of HR HPVs (p = 0.048). This relationship was not observed for HPV 16 or ‘non-16′ HR HPVs taken separately  during six months preceding the survey and testing  did not differ in the prevalence of infection with HR HPVs, or HPV 16, or ‘non-16′ HPV genotypes (data not shown).Smoking versus not smoking, and smoking >5 cigarettes per day, use of alcohol or drugs, separately or combined, had no effect on the prevalence of HR HPVs or the number of infecting HR HPV types  antigens recruit mesenchymal stem cells which repress Th1 immune response and promote lung cancer metastasis [Like HIV, TB induces immunosuppression and promotes tumorigenesis. tastasis ,43. In atastasis . The pottastasis .Here, we performed a study of HR HPV infection in a cohort of women living with HIV-1 with clinical manifestations of tuberculosis (WLWH with TB) residing in Moscow, Russia. Russia is highly endemic for both HIV-1 and TB. Russia is classified today as a middle-income country according to WHO criteria of the health care spending (5% of GDP) ,45. The HIV-1 co-infection promotes rapid progression of TB after infection or reinfection as well as reactivation of latent TB . While ip < 0.05). The second most common site of extrapulmonary TB, occurring in primary as well as reactivation disease, is pleura [p < 0.05) as in the areas non-endemic for HIV-1. Another common site of extrapulmonary TB is the genitourinary (GU) tract, a primary target of hematogenous TB infections; GU TB comprises 2–14 % of TB cases in the developed and up to 20% cases in the developing countries [p = 0.02) [Lymph node TB is the most common extrapulmonary TB form in the immunosuppressed individuals including PLWH. It represents 16–30% of extrapulmonary tuberculosis cases ,56,57, as pleura . In non-s pleura ,60. We fountries ,62. GU Tountries . HIV/AIDountries . In our  = 0.02) .p < 0.05). Overall, the clinical features of TB in WLWH of the Moscow cohort, with its significant proportion of pulmonary TB forms, followed the clinical features of TB (mono)infection in the middle/high-middle-income countries [We identified 15 cases of disseminated TB (25.9%) and 9 cases of generalized TB (15.5%) classified as cases with multiple caseous MTB foci preferentially in the lungs or in the lungs and other organs, respectively . HIV-assountries ,67. Impoountries . Our datountries . This arNext, we performed a systematic analysis of the effect(s) of the clinical manifestations of TB infection in WLWH on the infection with high risk HPV types. MTB-induced immunosuppression/immunomodulation is thought to be limited to the infected organs . In view3.Low CD4+ T cell counts are thought to predispose to HPV infection and reactivation of HPV infection due to incomplete viral clearance, whereas sustained HIV-1 suppression and higher CD4+ T cell counts are expected to reduce the risk of persistent HR HPV infection ,70,71. HThe effect of ART was puzzling: it did not influence the prevalence (infection/persistence) of HPV 16, but increased the cumulative prevalence of other HR HPV types in ART-treated compared to untreated WLWH regardless of their TB status .Certain role in HR HPV competition could have been played by varying levels of sex hormones. Estrogen increases the expression of HPV 16 genes  and provides a growth advantage to HPV 16 positive cervical cell lines protecting them from apoptosis . With thTo find an explanation, we compared the sociodemographic portrait of our cohort of WLWH with TB with a portrait of WLWH made in an earlier study of 7000 patients, including 49% women, followed up in 27 Russian AIDS centers during 2014  and withSmoking is a known risk factors for HPV ,90 and fp < 0.05). Interestingly, women who have had three or more pregnancies were earlier reported to have an increased risk of developing cervical cancer [In this unique cohort, we found only two sociodemographic parameters that were associated with the prevalence of infection with HR HPV. One was the number of pregnancies . HR HPVsl cancer ,95. Alsol cancer . The leal cancer . The stel cancer ,99, and l cancer . Our finp < 0.01). We also observed a tendency to a higher prevalence of ‘non-16′ HR HPVs among WLWH with TB who had sex with partners during six months preceding HPV testing.The number of sexual partners is a well-known risk factor for acquiring HPV infection ,102. Prep = 0.039) and were more often infected by ≥2 HR HPV types (p = 0.02). Both features appeared to be connected to ART or combined ART+TB-treatment: women on ART had significantly more pregnancies than untreated women, and worked more often than treatment-naïve individuals . IntereHaving this in mind, we returned to our findings of ART/TB-treatment increasing the prevalence of HR HPVs other than HPV 16, and assessed if ART/TB-treatment (in ward) could have modified the lifestyle of the study participants. Indeed, by analysis of patient histories, we found that due to the success of ART and TB-treatments, and improved health of the patients, they were released home on the weekends. In conversations with the doctors, patients admitted that they felt fit to return to their former lifestyle including their sex life. Most of the study participants were under 40, single and led an active life before hospitalization, and could have met their sexual partners during this leave. Several patients met their future husbands while staying at the hospital. This may have resulted in de novo HPV infections and re-infections. This scenario is supported by the latest evaluations of the positive effects of TB treatment on the physical, emotional and psychological health of TB patients . Thus, sRemaining to address (and explain) was exclusion from this scenario of HPV 16, as its representation among HR HPV positive WLWH on ART was, on contrary, reduced. We hypothesize that women >29 years old may have natural immunity against HPV 16, the infectious agent of sexual debut, restricting re-infections , especiaThus, we have characterized Moscow cohort of women living with HIV-1 with clinical manifestations of TB as a unique population with a complex socio-behavioral profile which translates into a unique pattern of infection with high risk HPV types. Absence of the knowledge of HIV and TB status, and refusal to be ART-treated for those knowing their HIV+ status, along with drug use, smoking and other potentially harmful behaviors, indicated the lack of attention of these women to their health. At the same time, our data speak for the importance of timely provision to ART/TB-treatment and adherence to treatment regimen in this unique category of the population allowing to improve the health and quality of life of this vulnerable group of population. As a backlash, this may lead to enhanced infections with HR HPVs other than HPV 16, which emphasizes the need for efficient measures ensuring durable prevention of HR HPV infections, cervical abnormalities, and cancer. Standard treatment of HSIL to prevent progression to cancer in the settings of HIV infection is hard to accomplish: in our cohort, only 10/58 (17%) agreed to undergo examination of the cervix to detect eventual morphological abnormalities. Different approaches are needed to overcome health ignorance prevailing in this group of population.Significant progress was lately achieved in the development of treatment(s) of HR HPV-associated precancerous lesions and cancer using specific immunotherapy/therapeutic HPV vaccines ,109. DecWLWH with clinical TB manifestations are characterized by the high prevalence of infection with HPV 16 and other HR-HPV genotypes. We found that infections with HR HPVs did not depend on the form of TB infection (pulmonary versus extrapulmonary forms), but noted frequent infections with HR HPVs other than HPV 16 among WLWH with generalized TB, and/or low CD4+ T cell counts; specifically, those receiving combined ART/TB therapy, and in general, in WLWH on ART irrespective of their TB status. Prevalence in WLWH with TB of HR HPV types other than HPV 16 was not associated with their CD4+ T cell counts, duration of life with HIV, route of HIV infection, or risk behaviors, but depended on their sociodemographic characteristics, specifically, number of pregnancies reflecting sexual activity, and capacity to work, jointly reflecting their physical and emotional health. Associations between sociodemographic and clinical factors will be further delineated by multivariate analysis performed on a larger cohort of WLWH with and without TB. High prevalence in this cohort of HPV 16 strains close to those circulating in other population groups outside of Russia, specifically in the structure of E6/E7 oncoproteins, speaks in favor of the development and application of a universal therapeutic vaccine against HPV 16 as an additional therapeutic measure to prevent HPV-related cancers and preserve health of people living with HIV-1 and TB.


HPV genotypes were determined in 63 vaginal intraepithelial neoplasia (VaIN) and 7 vaginal squamous cell carcinomas (VaSCC). Of these, 37 cases had VaIN alone, and 26 cases had both VaIN and cervical intraepithelial neoplasia (CIN) or condyloma. HPV typing was performed in scraped cells by Genosearch-31 and in the archived tissues by uniplex E6/E7 PCR. In a total of 49 VaIN1, 17 VaIN2/3, and 7 VaSCC tissues, the prevalence of HPV was 91.2% in VaIN and 85.7% in VaSCC. Comparing HPV results in scraped cell and tissue, 46.2% of high-risk (HR) types and 68.1% of any HPV types that had been identified in cell samples were not present in corresponding tissues. HPV types in VaIN and CIN lesions differed in 92.3% of cases with multiple lesions. These results suggest that there are many preclinical HPV infections in the vagina or the cervix, and VaIN and CIN are independently developed. The manual microdissection procedure of tissue revealed one HPV type in one lesion. The vagina appears to be the reservoir for any mucosal HPV type, and HR- or pHR-HPV types are causative agents for vaginal malignancies.HPV genotypes were determined in 63 vaginal intraepithelial neoplasia (VaIN) and 7 vaginal squamous cell carcinomas (VaSCC). Of these, 37 cases had VaIN alone, and 26 cases had both VaIN and cervical intraepithelial neoplasia (CIN) or condyloma. HPV typing was performed in scraped cells by Genosearch-31 (GS-31) and in the archived tissues by uniplex E6/E7 PCR. In a total of 49 VaIN1, 17 VaIN2/3, and 7 VaSCC tissues, the prevalence of HPV was 91.2% in VaIN  and 85.7% in VaSCC. Comparing HPV results in scraped cell and tissue, 46.2% of high-risk (HR) types and 68.1% of any HPV types that had been identified in cell samples were not present in corresponding tissues. HPV types in VaIN and CIN lesions differed in 92.3% (24/26) of cases with multiple lesions. These results suggest that there are many preclinical HPV infections in the vagina or the cervix, and VaIN and CIN are independently developed. The manual microdissection procedure of tissue revealed one HPV type in one lesion. Seventeen HPV types, including high-risk (HR), possible high-risk (pHR), and low-risk (LR), were identified in 43 VaIN1 lesions. In higher grade lesions, six HR , one pHR (HPV66), and one LR (HPV42) HPV types were identified in 17 VaIN2/3, and six HPV types, including HPV16, 45, 58, and 68 (HR), and HPV53 and 67 (pHR), were detected in each case of VaSCC. The vagina appears to be the reservoir for any mucosal HPV type, and HR- or pHR-HPV types are causative agents for vaginal malignancies. Human papillomavirus (HPV) has been proven to be the causative agent of cervical cancer and its precancerous lesions, this virus belongs to the Papillomaviridae family, which is a small, non-enveloped type, double-strand DNA (dsDNA) virus group . To date16 However, it has been reported that one HPV type is found in one lesion, including cervical cancer and high-grade SILs (CIN2 or CIN3) [Although the causal relationship between HPV infection and cervical SIL and cancer development has been confirmed by many studies, the role of HPV in the vagina is not well understood. One reason for this is that vaginal squamous cell carcinoma is a rare disease, and HPV-positivity in the cancer (60–70%) is less than that in cervical squamous cell cancer (more than 95%) ,14. Anotor CIN3) . This suor CIN3) . Alternaor CIN3)  also intor CIN3) . The aim of this study was to determine the HPV genotype responsible for VaIN using molecular mapping of target lesions in FFPE tissue specimens and the uniplex E6/E7 HPV-PCR test. We attempted to clarify the clinical–pathological classification of the HPV type in VaIN. Primary HPV test screening has been recently launched in some Western countries , and VaIIn the present study, for convenience the former term, VaIN is used for vaginal squamous intraepithelial lesion (VaSIL), and CIN is used for cervical SIL. One hundred and five patients diagnosed with VaIN and VaSCC, who visited the department of Obstetrics and Gynecology of the Kanazawa Medical University Hospital from 2011 to 2020, were analyzed in this study . All samTwo cases with vaginal condyloma, one with vaginitis, one normal epithelium, and one vaginal cancer case were excluded from further analysis . The oriLBC samples were obtained from the cervices of most subjects at their first visit to the out-patient clinic in Kanazawa Medical University Hospital. The samples were stored at 4 °C until the HPV genotyping test, which was performed within 2 weeks of the collection by a commercial laboratory . The HPV genotype was determined using the Genosearch-31+4 (GS-31+4) method  in the LSI laboratory. This is a new genotyping system based on the PCR-rSSO-Luminex method. This system (GS-31) detects a total of 31 HPV genotypes, including both high-risk (HR), possible high-risk (pHR), and low-risk (LR) HPV types: HPV-6, 11, 16, 18, 26, 31, 33, 35, 39, 42,44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 66, 68,70, 71, 73, 82, 84, 89, and 90. An additional four pHR-HPV types  were also detected with the GS-31+4 system. The GS-31+4 method uses the multiplex primers to amplify different genomic sites of the L1 region on 35 different HPV types, and uses 35 independent probes for detection. It has been confirmed that there is no cross-reaction to other HPV types among these 35 types, and the guaranteed detection limit is 1000 copies of any HPV type in a test .The FFPE blocks for each case were sectioned according to the following procedure: three slices of 4 μM sections were used for histopathological diagnosis after haematoxylin and eosin (H&E) staining, followed by expression of p16 and Ki67 after immunohistochemical (IHC) staining; five slices of 10 μM sections were used for DNA extraction and HPV genotyping; and the final 4 μM section was used for histological evaluation with H&E staining to confirm if the target lesion remained. The microtome was cleaned with 70% alcohol and the blade was replaced for cutting following each tissue block to avoid contamination of HPV in other samples . The tis® Histology Kit  according to the manufacturer’s instructions. Briefly, the 4 µm thick FFPE tissue sections were deparaffinized in xylene and rehydrated in graded alcohols. Antigen retrieval was twice performed in a microwave in Epitope Retrieval Solution  for 5 min at 500 W. The slides were cooled to room temperature before equilibration in phosphate buffered saline (PBS), and incubated with Peroxidase-Blocking Reagent to block endogenous peroxidase activity. Subsequently, the slides were incubated with the ready-to-use primary anti-p16 antibody (clone E6H4) for 30 min at room temperature. The slides were washed in PBS, then incubated with the secondary antibody  for 30 min at room temperature. The slides were incubated in the Visualization Reagent, which contains a polymer reagent conjugated to horseradish peroxidase (HRP), for 30 min at room temperature. After washing in PBS, incubation with Substrate-Chromogen Solution (1:40 dilution of DAB chromogen in DAB buffer) for 10 min led to p16 brown staining. Mayer’s Hematoxylin was used as a counterstain. Slides were washed in deionized water, dehydrated in graded alcohols, and cleared in xylene. A non-aqueous mounting medium was applied to each section before they were mounted with coverslips. For the detection of the Ki67 antigen, we used the monoclonal mouse antibody , and the specific procedure was the same as that described above.p16 immunohistochemical staining was performed using the CINtecOne normal squamous cell tissue without HPV infection was used as the negative control for p16 and Ki67 staining, and two VaIN3 slides with p16 and Ki67 staining were used as the positive control . p16 wasAll slides stained with H&E (the control slides) were reviewed by a surgical pathologist, and the areas of abnormal lesion, and normal squamous or glandular epithelium, were marked on each slide. By viewing the marked areas of the control slide, an abnormal area or a normal tissue on the tissue slides stained with H&E was independently dissected by hand using a sterilized 21G needle under an inverted microscope . The numThe uniplex E6/E7 PCR test was designed to amplify E6 and E7 genes of 39 common HPV types using type-specific primer pairs. This assay is able to individually detect 15 LR-HPV types ; 11 pHR-HPV types ; 13 HR-HPV types ; and the beta-globin gene as a positive control [® Green I  staining in a lane under a UV light , PCR was performed for each HPV type in a T100™ Thermal Cycler  using the following program: 10 min at 94 °C; 40 cycles of 30 s at 94 °C, 30 s at 60 °C, and 30 s at 72 °C; and a final 5 min extension step. A quantity of 5.0 µL of each reaction solution for each HPV type was applied to a 2.5% agarose gel (Bio-Rad) and electrophoresed in 1× TBE buffer for 8 min. HPV type-specific bands were visualized with SYBRUV light .If the beta-globin gene was negative, it was considered to be a poor quality sample or to have contained an insufficient amount of extracted DNA. These samples were observed in 12 of 30 cases, and were excluded from further analysis .HPV typing results obtained with the uniplex E6-E7 PCR system were compared with the previous results determined by GS-31+4. The latter HPV test could not detect HPV40, 74, 81, and 85, and among these HPV types, only HPV81 was identified in some samples in the present study.p < 0.05 was considered to be statistically significant. All analysis was performed with JSP version 14 .We used Mann–Whitney’s U test to undertake analysis of continuous variables (patient’s age), and the chi-square test with Yates’ correction was used to compare the categorical variables . We used Fisher’s exact test when the total sample size was less than 10. A probability value of We used p16 and Ki67 immunohistochemical staining to identify VaIN lesions in tissue specimens using the microdissection procedure. It was shown that 88.2% (15/17) of VaIN2/3 and 28.6% (14/49) of VaIN1 were positive for p16 expression, whereas this was seen in only 9.5% of normal glandular or metaplastic cells, and no expression was found in normal squamous epithelium adjacent to VaIN or CIN. Two VaIN2/3 cases negative for p16 were infected with HPV51 (HR-HPV) and HPV42 (LR-HPV). In contrast, most VaIN1 cases  positive for p16 expression were infected with HR or pHR-HPV types, although only one p16-positive VaIN1 was infected with an LR-HPV type, namely, HPV71. All VaIN2/3 and 81.6% (40/49) of VaIN1 lesions were positive for Ki67. Of six VaIN1 lesions found to be negative for HPV, four lesions were found to be positive for Ki67 but negative for p16.We examined the prevalence of HPV infection in 63 eligible selected cases with VaIN and seven cases with VaSCC. All of these subjects visited our out-patient clinic to undertake further examination for abnormal Pap test worse than atypical squamous cells undetermined significance (ASC-US). All subjects were classified into two groups: the single lesion group, comprising cases with VaIN or VaSCC alone; and the multiple lesions group, comprising cases with VaIN combined with CIN or condyloma. The former group was composed of 23 VaIN1, 14 VaIN2/3, and 7 VaSCC cases, whereas the latter group comprised 7 VaIN1+CIN1 or condyloma, 16 VaIN1+CIN2/3, and 3 VaIN2/3+CIN1 or condyloma . Therefop > 0.1). Seven cases with VaSCC were older (av. 81.7 y) than the other VaIN cases (av. 45.5 y) .The comparison of the ages of cases  showed nHPV genotyping in tissue specimens was performed in all the subjects, whereas HPV typing in scraped cell samples was performed in 36 cases with a single VaIN lesion, 24 cases with multiple lesions, and 3 VaSCC cases. Regarding tissue results, overall HPV prevalence in all subjects was 95.7% .p > 0.1). As for the multiple lesions group, the prevalence of HPV types was calculated separately between VaIN and CIN (Condyloma) groups .When we compared HPV genotype results in tissue and scraped cell samples in the single lesion group , eight cThe same analysis was performed for 24 eligible cases with multiple lesions who undertook HPV testing using both cell and tissue samples. Various HPV types that had been detected in cell samples were not present in 70.8% (17/24) of tissue specimens. Regarding HR-HPV type, these preclinical infections were recognized in 54.2% (13/24) of the multiple lesion group. Conversely, the opposite result, in which the HPV genotype was not identified in cell samples but was positive in tissues, was seen in two cases . p > 0.1). In all subjects, 68.3% (41/60) had preclinical infection with any HPV type, and 46.7% (28/60) were found to have a preclinical HR-HPV-type infection. Moreover, HPV genotypes in VaIN and CIN lesions differed in 92.3% (24/26) of cases with multiple lesions  VaIN tissues were positive for HPV . One VaIIn 49 VaIN1 lesions, 17 HPV types were detected in 43 lesions , including HR-HPV in 6 lesions, pHR-HPV in 18 lesions, and LR-HPV in 19 lesions. The most common type was HPV53, which was found in six VaIN1 lesions, followed by HPV66/81/90 in five lesions, HPV34/67/71 in three lesions, HPV42/61/84 in two lesions, and HPV16/31/52/56/58/68/73 in one lesion. In higher-grade lesions, eight HPV types were identified in VaIN2/3, whereas six HPV types were found in VaSCC. The most common type in VaIN2/3 was HPV16 (HR-HPV) , followed by HPV51/52/58 (HR-HPV) , HPV42 (LR-HPV) , and HPV18/56 (HR-HPV) and HPV66 (pHR-HPV) in one case each. In contrast, HPV16/45/58/68 (HR-HPV) and HPV53/67 (pHR-HPV) were identified as one HPV type in each case of VaSCC, and a negative result was detected in one case. Among the 13 internationally defined HR-HPV types, four HPV types  were not detected in any VaIN lesions in the present study, although HPV39 was identified in two CIN2 cases .p < 0.001), whereas possible HR (pHR) HPV types were more common in VaIN1 (34.8%) than VaIN2/3 (5.9%) (p = 0.015). The LR-HPV type was marginally more prevalent in VaIN1 than VaIN2/3 (p = 0.084)  than in VaIN1 (17.4%) (= 0.084) .n = 1374) was 85.2%, with the prevalence of each study ranging from 20% to 100% [The present study showed the prevalence of HPV was 95.7% in low-grade vaginal squamous intraepithelial lesions (VaIN1s), 100% in high-grade lesions (VaIN2 and VaIN3), and 85.7% in cancer (VaSCC). Different studies have shown different prevalence results of HPV in VaINs. A meta-analysis showed that the overall prevalence of HPV in VaIN  with VaIN alone, and 26 (40.0%) cases with VaIN and CIN or condyloma. In the latter cases, 24 cases (92.3%) were infected with different HPV types in each lesion of VaIN and CIN; this result was not expected. This suggests that VaIN and CIN were independently developed. Moreover, HPV genotyping results from the cell samples showed additional HPV types that were not identified. Okodo M et al.  confirmeIn patients with multiple lesions, 92.3% (24/26) had completely different HPV types in the lesions of CIN and VaIN . In thesWe used p16 and Ki67 immunohistochemical staining to identify VaIN lesions in tissue specimens using the microdissection procedure. It was shown that most of VaIN2/3 (15/17) and 28.6% (14/49) of VaIN1 were positive for p16 expression, and no expression was found in normal squamous epithelium adjacent to VaIN or CIN. Two VaIN2/3 lesions negative for p16 were infected with HPV51 (HR-HPV) and HPV42 (LR-HPV), whereas the other cases that were positive for p16 were infected with HR or pHR HPV types. Moreover, most VaIN1 cases  positive for p16 expression were infected with HR- or pHR-HPV types, although only one p16-positive VaIN1 was infected with HPV71 (LR-HPV). In contrast, all VaIN2/3 and most VaIN1  were positive for Ki67. Despite some exceptions, Ki67 expression is useful to discriminate any VaIN lesions from normal squamous epithelium, and p16 expression is associated with HR-HPV or pHR-HPV infection, regardless of the grades of vaginal lesion. Similar conclusions were reported by Chao et al. . In the The present study showed that HR-HPV is more common in VaIN2/3, whereas pHR- and LR-HPV are more common in VaIN1. VaIN1 were positive for any type of HPV. Castle et al. reported that LR-HPV types belonging to Alpha-3 and -15 are more prevalent in the vagina than in the cervix . Most LRp = 0.019), although further study is needed to warrant it.The ages of VaSCC patients were greater than those of VaIN cases in the present study , suggesting long latency is needed for malignant progression. It was noted that six different HR- and pHR-HPV types were identified in each of the VaSCC cases, suggesting that various HPV types are responsible for VaSCC development. In particular, two of the six (33.3%) cancers were infected with pHR types , suggesting that the prevalence of pHR types appears to be greater in VaSCC than in CxSCC . SakamotThe present study also demonstrated that the manual microdissection procedure, which is a cost-effective method, is as effective as LCM for the acquisition of target tissue for HPV genotyping . From a This study has some limitations. The most important of these is the small size of the samples of VaIN and, in particular, VaSCC, which comprised only seven cases. Thus, similar studies are needed to increase the sample size for further research.Many HPV types, including high-risk (HR), possible high-risk (pHR), and low-risk (LR) types, were identified in low-grade vaginal intraepithelial lesion (VaIN1), whereas HR and pHR-HPV types were identified in high-grade vaginal lesions (VaIN2/3). The vagina appears to be the reservoir for any mucosal HPV type, and HR or pHR HPV types are causative agents for vaginal malignancies. Different HPV type distribution between the vagina and the cervix suggests that CIN and VaIN are independently developed in most cases.